U.S. patent application number 12/322366 was filed with the patent office on 2010-09-16 for tubular nanostructure targeted to cell membrane.
This patent application is currently assigned to Searete LLC, a limited liability corporation of the State of Delaware.. Invention is credited to Mahalaxmi Gita Bangera, Ed Harlow, Roderick A. Hyde, Muriel Y. Ishikawa, Edward K.Y. Jung, Eric C. Leuthardt, Nathan P. Myhrvold, Dennis J. Rivet, Elizabeth A. Sweeney, Clarence T. Tegreene, Lowell L. Wood, JR., Victoria Y.H. Wood.
Application Number | 20100233781 12/322366 |
Document ID | / |
Family ID | 42005401 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100233781 |
Kind Code |
A1 |
Bangera; Mahalaxmi Gita ; et
al. |
September 16, 2010 |
Tubular nanostructure targeted to cell membrane
Abstract
Devices, compositions, and methods are described which provide a
tubular nanostructure targeted to a lipid bilayer membrane. The
targeted tubular nanostructure can have a surface region configured
to pass through a lipid bilayer membrane of a cell, a hydrophobic
surface region flanked by two hydrophilic surface regions
configured to form a pore in a lipid bilayer membrane of a cellular
organelle, and at least one ligand configured to bind one or more
cognates on the lipid bilayer membrane of the cellular organelle.
The target cell can be, for example, a tumor cell, an infected
cell, or a diseased cell in a subject. The tubular nanostructure
can form a pore in the lipid bilayer membrane of the cellular
organelle, e.g., mitochondria, which can permit transit or
translocation of at least one compound across the membrane and
cause cell death of the target cell.
Inventors: |
Bangera; Mahalaxmi Gita;
(Renton, WA) ; Harlow; Ed; (Boston, MA) ;
Hyde; Roderick A.; (Redmond, WA) ; Ishikawa; Muriel
Y.; (Livermore, CA) ; Jung; Edward K.Y.;
(Bellevue, WA) ; Leuthardt; Eric C.; (St. Louis,
MO) ; Myhrvold; Nathan P.; (Medina, WA) ;
Rivet; Dennis J.; (Portsmouth, VA) ; Sweeney;
Elizabeth A.; (Seattle, WA) ; Tegreene; Clarence
T.; (Bellevue, WA) ; Wood, JR.; Lowell L.;
(Bellevue, WA) ; Wood; Victoria Y.H.; (Livermore,
CA) |
Correspondence
Address: |
THE INVENTION SCIENCE FUND;CLARENCE T. TEGREENE
11235 SE 6TH STREET, SUITE 200
BELLEVUE
WA
98004
US
|
Assignee: |
Searete LLC, a limited liability
corporation of the State of Delaware.
|
Family ID: |
42005401 |
Appl. No.: |
12/322366 |
Filed: |
January 30, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12283908 |
Sep 15, 2008 |
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12322366 |
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12283907 |
Sep 15, 2008 |
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12283908 |
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Current U.S.
Class: |
435/193 ;
423/447.2; 525/50; 530/317; 530/350; 530/363; 530/370; 530/387.1;
530/395; 536/1.11; 536/23.1; 548/545; 549/346; 560/1; 562/553;
564/1; 568/303; 568/420; 568/579; 568/700; 977/742; 977/906 |
Current CPC
Class: |
A61K 38/1703 20130101;
Y10S 977/915 20130101; Y10S 977/848 20130101; A61K 47/60 20170801;
C01B 2202/34 20130101; B82Y 30/00 20130101; A61P 43/00 20180101;
C01B 32/174 20170801; B82Y 5/00 20130101; C01P 2004/13 20130101;
Y10S 977/75 20130101; Y02A 50/30 20180101; Y10S 977/762 20130101;
C01B 32/168 20170801; C01B 2202/02 20130101; Y10S 977/743 20130101;
Y10S 977/788 20130101; A61K 47/6871 20170801; A61K 38/1732
20130101; C01B 2202/36 20130101; Y10S 977/896 20130101; Y10S
977/795 20130101; A61K 47/6901 20170801; B82Y 15/00 20130101; B82Y
40/00 20130101; C01B 21/0648 20130101; C07K 16/32 20130101 |
Class at
Publication: |
435/193 ; 560/1;
568/303; 568/579; 562/553; 568/420; 564/1; 568/700; 530/387.1;
530/350; 530/317; 549/346; 536/23.1; 530/363; 530/395; 536/1.11;
548/545; 530/370; 423/447.2; 525/50; 977/906; 977/742 |
International
Class: |
C07C 49/00 20060101
C07C049/00; C07C 69/00 20060101 C07C069/00; C07C 43/00 20060101
C07C043/00; C07C 229/00 20060101 C07C229/00; C07C 47/00 20060101
C07C047/00; C07C 211/00 20060101 C07C211/00; C07C 35/00 20060101
C07C035/00; C07K 16/00 20060101 C07K016/00; C07K 14/00 20060101
C07K014/00; C12N 9/10 20060101 C12N009/10; C07K 2/00 20060101
C07K002/00; C07D 323/00 20060101 C07D323/00; C07H 21/04 20060101
C07H021/04; C07K 14/765 20060101 C07K014/765; C07H 1/00 20060101
C07H001/00; C07D 207/40 20060101 C07D207/40; C07K 14/415 20060101
C07K014/415; D01F 9/12 20060101 D01F009/12; C08G 63/91 20060101
C08G063/91 |
Claims
1.-58. (canceled)
59. A composite tubular nanostructure comprising: two or more
nanotubes wherein at least one nanotube includes, a surface region
configured to pass through a lipid bilayer membrane of a cell, and
a hydrophobic surface region flanked by two hydrophilic surface
regions configured to form a pore in a lipid bilayer membrane of a
cellular organelle.
60. The composite tubular nanostructure of claim 59, further
comprising at least one ligand configured to bind one or more
cognates on the lipid bilayer membrane of the cellular
organelle.
61. The composite tubular nanostructure of claim 60, further
comprising at least one second ligand configured to bind one or
more cognates on the lipid bilayer membrane of the cell.
62. The composite tubular nanostructure of claim 60, wherein two or
more ligands are configured to bind to the one or more cognates on
the lipid bilayer membrane of the cellular organelle.
63. The composite tubular nanostructure of claim 59, wherein the
composite tubular nanostructure induces cell death.
64. The composite tubular nanostructure of claim 59, wherein the
surface region configured to pass through a lipid bilayer membrane
of the cell is a hydrophobic surface region or a structured
amphiphilic surface region.
65. The composite tubular nanostructure of claim 59, wherein the
surface region configured to pass through a lipid bilayer membrane
of the cell is configured to interact with a cellular component to
produce the at least one tubular nanostructure including the
hydrophobic surface region flanked by two hydrophilic surface
regions.
66. The composite tubular nanostructure of claim 65, wherein the
cellular component is a cytoplasmic component.
67. The composite tubular nanostructure of claim 66, wherein the
cytoplasmic component is an esterase which removes a hydrophobic
ester from the surface region configured to pass through a lipid
bilayer membrane of the cell to produce the at least one tubular
nanostructure including the hydrophobic surface region flanked by
two hydrophilic surface regions.
68. The composite tubular nanostructure of claim 59, wherein the
cell is a neoplastic cell or an infected cell.
69. The composite tubular nanostructure of claim 59, including
three or more nanotubes.
70. The composite tubular nanostructure of claim 69, wherein at
least one nanotube includes a completely hydrophobic surface
region.
71. The composite tubular nanostructure of claim 70, wherein the at
least one nanotube including the completely hydrophobic surface
region is surrounded by at least six nanotubes including the
hydrophobic surface region flanked by two hydrophilic surface
regions configured to form the pore in the lipid bilayer
membrane.
72. The composite tubular nanostructure of claim 59, wherein at
least two of the nanotubes have different diameters.
73. The composite tubular nanostructure of claim 59, wherein at
least two of the nanotubes have different lengths.
74. The composite tubular nanostructure of claim 59, wherein the
nanotubes are substantially parallel.
75. The composite tubular nanostructure of claim 59, wherein the
nanotubes are substantially orthogonal.
76. The composite tubular nanostructure of claim 59, wherein at
least one of the two or more nanotubes includes a carbon nanotube,
cyclic peptide nanotube, crown ether nanotube, polymer nanotube,
polymer/carbon nanotube, DNA nanotube, or inorganic nanotube.
77. The composite tubular nanostructure of claim 59, wherein the
hydrophobic surface region includes a single wall carbon nanotube
surface region.
78. The composite tubular nanostructure of claim 59, wherein the
hydrophilic surface region includes one or more of amines, amides,
charged or polar amino acids, alcohols, carboxylic groups, oxides,
ester groups, ether groups, ester-ether groups, ketones, aldehydes,
or derivatives thereof.
79.-101. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to and claims the benefit
of the earliest available effective filing date(s) from the
following listed application(s) (the "Related Applications") (e.g.,
claims earliest available priority dates for other than provisional
patent applications or claims benefits under 35 USC .sctn.119(e)
for provisional patent applications, for any and all parent,
grandparent, great-grandparent, etc. applications of the Related
Application(s)). All subject matter of the Related Applications and
of any and all parent, grandparent, great-grandparent, etc.
applications of the Related Applications is incorporated herein by
reference to the extent such subject matter is not inconsistent
herewith.
RELATED APPLICATIONS
[0002] For purposes of the USPTO extra-statutory requirements, the
present application constitutes a continuation-in-part of U.S.
patent application Ser. No. To Be Assigned, entitled TUBULAR
NANOSTRUCTURE TARGETED TO CELL MEMBRANE, naming Mahalaxmi Gita
Bangera, Ed Harlow, Roderick A. Hyde, Muriel Y. Ishikawa, Edward K.
Y. Jung, Jordin T. Kare, Eric C. Leuthardt, Nathan P. Myhrvold,
Dennis J. Rivet, Elizabeth A. Sweeney, Clarence T. Tegreene, Lowell
L. Wood, Jr. and Victoria Y. H. Wood as inventors, filed 15 Sep.
2008, which is currently co-pending, or is an application of which
a currently co-pending application is entitled to the benefit of
the filing date.
[0003] The United States Patent Office (USPTO) has published a
notice to the effect that the USPTO's computer programs require
that patent applicants reference both a serial number and indicate
whether an application is a continuation or continuation-in-part.
Stephen G. Kunin, Benefit of Prior-Filed Application, USPTO
Official Gazette Mar. 18, 2003, available at
http://www.uspto.gov/web/offices/com/sol/og/2003/week11/patbene.htm.
The present Applicant Entity (hereinafter "Applicant") has provided
above a specific reference to the application(s) from which
priority is being claimed as recited by statute. Applicant
understands that the statute is unambiguous in its specific
reference language and does not require either a serial number or
any characterization, such as "continuation" or
"continuation-in-part," for claiming priority to U.S. patent
applications. Notwithstanding the foregoing, Applicant understands
that the USPTO's computer programs have certain data entry
requirements, and hence Applicant is designating the present
application as a continuation-in-part of its parent applications as
set forth above, but expressly points out that such designations
are not to be construed in any way as any type of commentary and/or
admission as to whether or not the present application contains any
new matter in addition to the matter of its parent
application(s).
[0004] All subject matter of the Related Applications and of any
and all parent, grandparent, great-grandparent, etc. applications
of the Related Applications is incorporated herein by reference to
the extent such subject matter is not inconsistent herewith.
SUMMARY
[0005] Devices, compositions, and methods described herein provide
a tubular nanostructure targeted to a lipid bilayer membrane. The
targeted tubular nanostructure can have a surface region configured
to pass through a lipid bilayer membrane of a cell, a hydrophobic
surface region flanked by two hydrophilic surface regions
configured to form a pore in a lipid bilayer membrane of a cellular
organelle, and at least one ligand configured to bind one or more
cognates on the lipid bilayer membrane of the cellular organelle.
The target cell can be, for example, a tumor cell, an infected
cell, or a diseased cell in a subject. The tubular nanostructure
can form a pore in the lipid bilayer membrane of the cellular
organelle, e.g., mitochondria, which can permit transit or
translocation of at least one compound across the membrane and
cause cell death of the target cell.
[0006] A tubular nanostructure is provided which includes a surface
region configured to pass through a lipid bilayer membrane of a
cell, and a hydrophobic surface region flanked by two hydrophilic
surface regions configured to form a pore in a lipid bilayer
membrane of a cellular organelle. The nanostructure may further
comprise at least one ligand configured to bind one or more
cognates on the lipid bilayer membrane of the cellular organelle.
The at least one second ligand may be configured to bind one or
more cognates on the lipid bilayer membrane of the cell. The two or
more ligands may be configured to bind to the one or more cognates
on the lipid bilayer membrane of the cellular organelle. The
tubular nanostructure can induce cell death. The surface region
configured to pass through a lipid bilayer membrane of the cell may
be a hydrophobic surface region or a structured amphiphilic surface
region or may be configured to interact with a cellular component
to produce the at least one tubular nanostructure including the
hydrophobic surface region flanked by two hydrophilic surface
regions. The cellular component may be a cytoplasmic component. The
cytoplasmic component may be an esterase which removes a
hydrophobic ester from the surface region configured to pass
through a lipid bilayer membrane of the cell to produce the at
least one tubular nanostructure including the hydrophobic surface
region flanked by two hydrophilic surface regions. In one aspect,
the nanostructure is a multi-walled nanotube including one or more
outer walls configured as the surface region to pass through a
lipid bilayer membrane of the cell, and one or more inner walls
including the hydrophobic surface region flanked by two hydrophilic
surface regions configured to form a pore in a lipid bilayer
membrane of a cellular organelle. The surface region to pass
through a lipid bilayer membrane of the cell may be a hydrophobic
surface region or a structured amphiphilic surface region. The cell
includes, but is not limited to, a neoplastic cell or an infected
cell. The nanostructure includes, but is not limited to, one or
more of a carbon nanotube, cyclic peptide nanotube, crown ether
nanotube, polymer nanotube, polymer/carbon nanotube, DNA nanotube,
or inorganic nanotube. The inorganic nanotube further includes, but
is not limited to, a boron nitride nanotube. The polymer nanotube
includes, but is not limited to, polystyrene,
polytetrafluoroethylene, polymethylmethacrylate, polyaniline, or
poly-L-lactide/palladium acetate. The polymer/carbon nanotube
includes, but is not limited to, a polyaniline/carbon nanotube. The
hydrophobic surface region includes, but is not limited to, a
single wall carbon nanotube surface region. The hydrophilic surface
region includes, but is not limited to, one or more of amines,
amides, charged or polar amino acids, alcohols, carboxylic groups,
oxides, ester groups, ether groups, or ester-ether groups, ketones,
aldehydes, or derivatives thereof. The one or more cognates
include, but are not limited to, one or more cell surface receptors
or cell surface markers in the lipid bilayer membrane. The one or
more cognates include, but are not limited to, at least one of a
protein, a carbohydrate, a glycoprotein, a glycolipid, a
sphingolipid, a glycerolipid or a metabolite thereof. One or both
of the hydrophilic surface regions may be at the end of the
nanostructure. The nanostructure may have a length of about 1 nm to
about 1500 nm, or a length of about 20 .ANG. to about 40 .ANG.. The
nanostructure may have a diameter of about 0.5 nm to about 5 nm or
a diameter of about 5 .ANG. to about 20 .ANG.. The at least one
ligand includes, but is not limited to, at least a portion of an
antibody, antibody-coated liposome, polynucleotide, polypeptide,
receptor, viral plasmid, polymer, protein, small chemical compound,
carbohydrate, lipid, toxin, pore-forming toxin, or lectin. The at
least one ligand includes a therapeutic compound configured to
affect a cell or process, or to treat at least one of a disease,
condition, or symptom. The nanostructure may further include at
least one second ligand configured to bind one or more cognates on
the lipid bilayer membrane. The tubular nanostructure induces cell
death. In one aspect, the pore permits transit or translocation of
at least one compound across the membrane of the cellular
organelle. The nanostructure may anchor in the membrane of the cell
organelle. The surface region may be configured to match a
configuration of the lipid bilayer membrane of the cell organelle.
In one aspect, the organelle is a mitochondria.
[0007] The nanostructure may further include one or more elements
to control transport of molecules through the tubular
nanostructure. In one aspect, the one or more elements control
transport of molecules into the cellular organelle. In a further
aspect, the one or more elements control transport of molecules out
of the cellular organelle. The one or more elements may include a
hydrophilic inner liner of the tubular nanostructure. The one or
more elements further includes at least one ligand configured to
reversibly bind a cognate of interest, wherein the cognate of
interest passes through the pore. The at least one ligand includes,
but is not limited to, a monospecific antibody or a bispecific
antibody. The one or more elements may reversibly blocks the pore.
The one or more elements includes, but is not limited to, a
magnetic entity or a molecular entity. The molecular entity
includes, but is not limited to, at least a portion of a carbon
nanostructure, polynucleotide, polypeptide, antibody, receptor,
glycoprotein, lipid, polysaccharide, or polymer. The one or more
elements may include a charged group. The one or more elements may
be passive or active. In one aspect, the pore permits transit or
translocation of at least one compound across the membrane. The one
or more active elements includes, but is not limited to, at least
one of an ATPase transport element, Na.sup.+K.sup.+ ATPase,
H.sup.+K.sup.+ ATPase, or Ca.sup.2+ ATPase. The one or more active
elements further includes, but is not limited to, at least one of
an ABC transporter element, CFTR transporter, TAP transporter, or
liver cell transporter. The one or more active elements further
includes, but is not limited to, at least one of a symport pump,
Na.sup.+/iodide transporter, E. coli permease, or an antiport
pump.
[0008] The nanostructure may further include a marker attached to
the nanostructure. The marker includes, but is not limited to, a
fluorescent marker, a radioactive marker, quantum dot, metal, or
magnetic resonance imaging marker. The marker may be activated by
anchoring in the membrane of the cellular organelle. The marker may
be activated by a ligand reaction. The marker may be activated by
interaction with a hydrophobic medium. The hydrophobic surface
region may be extended in diameter. The hydrophobic surface region
may be extended in diameter by a disk, a stub, or a graphene
sheet.
[0009] A composite tubular nanostructure is provided which includes
two or more nanotubes wherein at least one nanotube includes a
surface region configured to pass through a lipid bilayer membrane
of a cell, and a hydrophobic surface region flanked by two
hydrophilic surface regions configured to form a pore in a lipid
bilayer membrane of a cellular organelle. The composite tubular
nanostructure may further include at least one ligand configured to
bind one or more cognates on the lipid bilayer membrane of the
cellular organelle. The two or more ligands may be configured to
bind to the one or more cognates on the lipid bilayer membrane of
the cellular organelle. The composite tubular nanostructure may
further include at least one second ligand configured to bind one
or more cognates on the lipid bilayer membrane. The two or more
ligands may be configured to bind to the one or more cognates on
the lipid bilayer membrane. The composite tubular nanostructure may
induce cell death. In one aspect, the surface region configured to
pass through a lipid bilayer membrane of the cell is a hydrophobic
surface region or a structured amphiphilic surface region. In a
further aspect, the surface region configured to pass through a
lipid bilayer membrane of the cell is configured to interact with a
cellular component to produce the at least one tubular
nanostructure including the hydrophobic surface region flanked by
two hydrophilic surface regions. The cellular component may be a
cytoplasmic component. In a further aspect, the cytoplasmic
component may be an esterase which removes a hydrophobic ester from
the surface region configured to pass through a lipid bilayer
membrane of the cell to produce the at least one tubular
nanostructure including the hydrophobic surface region flanked by
two hydrophilic surface regions. The cell includes, but is not
limited to, a neoplastic cell or an infected cell.
[0010] The composite tubular nanostructure may further include
three or more nanotubes. The composite tubular nanostructure may
further include at least one nanotube includes a completely
hydrophobic surface region. The at least one nanotube including the
completely hydrophobic surface region may be surrounded by at least
six nanotubes including the hydrophobic surface region flanked by
two hydrophilic surface regions configured to form the pore in the
lipid bilayer membrane. The at least two of the nanotubes may have
different diameters. The at least two of the nanotubes may have
different lengths. The nanotubes may be substantially parallel. The
nanotubes may be substantially orthogonal. The composite tubular
nanostructure includes, but is not limited to, at least one of the
two or more nanotubes is a carbon nanotube, cyclic peptide
nanotube, crown ether nanotube, polymer nanotube, polymer/carbon
nanotube, DNA nanotube, or inorganic nanotube. The inorganic
nanotube further includes, but is not limited to, a boron nitride
nanotube. The polymer nanotube includes, but is not limited to,
polystyrene, polytetrafluoroethylene, polymethylmethacrylate,
polyaniline, or poly-L-lactide/palladium acetate. The
polymer/carbon nanotube includes, but is not limited to, a
polyaniline/carbon nanotube. The hydrophobic surface region
includes, but is not limited to, a single wall carbon nanotube
surface region. The hydrophilic surface region includes, but is not
limited to, one or more of amines, amides, charged or polar amino
acids, alcohols, carboxylic groups, oxides, ester groups, ether
groups, or ester-ether groups, ketones, aldehydes, or derivatives
thereof. The one or more cognates include, but are not limited to,
one or more cell surface receptors or cell surface markers in the
lipid bilayer membrane. The one or more cognates include, but are
not limited to, at least one of a protein, a carbohydrate, a
glycoprotein, a glycolipid, a sphingolipid, a glycerolipid or a
metabolite thereof. One or both of the hydrophilic surface regions
may be at the end of the nanostructure. The nanostructure may have
a length of about 1 nm to about 1500 nm, or a length of about 20
.ANG. to about 40 .ANG.. The nanostructure may have a diameter of
about 0.5 nm to about 5 nm or a diameter of about 5 .ANG. to about
20 .ANG.. The at least one ligand includes, but is not limited to,
at least a portion of an antibody, antibody-coated liposome,
polynucleotide, polypeptide, receptor, viral plasmid, polymer,
protein, small chemical compound, carbohydrate, lipid, toxin,
pore-forming toxin, or lectin. The at least one ligand includes a
therapeutic compound configured to affect a cell or process, or to
treat at least one of a disease, condition, or symptom.
[0011] A method for disrupting a lipid bilayer membrane of a
cellular organelle in a cell is provided which includes contacting
the cell with at least one tubular nanostructure including a
surface region configured to pass through a lipid bilayer membrane
of the cell, the at least one tubular nanostructure including a
hydrophobic surface region flanked by two hydrophilic surface
regions configured to form a pore in the lipid bilayer membrane of
the cellular organelle, and at least one ligand bound to the at
least one tubular nanostructure, the ligand configured to bind one
or more cognates on the lipid bilayer membrane of the cellular
organelle. The at least one tubular nanostructure may further
include one or more elements to control transport of molecules
through the tubular nanostructure. In one aspect, the one or more
elements control transport of molecules into the cellular
organelle. In a further aspect, the one or more elements control
transport of molecules out of the cellular organelle. The one or
more elements may include a hydrophilic inner liner of the tubular
nanostructure. The one or more elements further includes at least
one second ligand configured to reversibly bind a cognate of
interest, wherein the cognate of interest passes through the pore.
The at least one second ligand includes, but is not limited to, a
monospecific antibody or a bispecific antibody. The one or more
elements may reversibly blocks the pore. The one or more elements
includes, but is not limited to, a magnetic entity or a molecular
entity. The one or more elements may include a charged group. The
one or more elements may be passive or active. The nanostructure
may further include a marker attached to the nanostructure. The
marker includes, but is not limited to, a fluorescent marker, a
radioactive marker, quantum dot, metal, or magnetic resonance
imaging marker.
[0012] The foregoing summary is illustrative only and is not
intended to be in any way limiting. In addition to the illustrative
aspects, embodiments, and features described above, further
aspects, embodiments, and features will become apparent by
reference to the drawings and the following detailed
description.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A, 1B, 1C, and 1D depict a diagrammatic view of one
aspect of an exemplary embodiment of a tubular nanostructure and a
method for inserting a tubular nanostructure into a lipid bilayer
membrane of a cell.
[0014] FIGS. 2A, 2B, 2C, 2D, and 2E depict a diagrammatic view of
one aspect of an exemplary embodiment of a tubular nanostructure
and a method for inserting a tubular nanostructure into a lipid
bilayer membrane of a cellular organelle.
[0015] FIGS. 3A, 3B, and 3C depict a logic flowchart of a method
for disrupting a lipid bilayer membrane of a cellular organelle in
a cell.
DETAILED DESCRIPTION
[0016] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented here.
[0017] The present application uses formal outline headings for
clarity of presentation. However, it is to be understood that the
outline headings are for presentation purposes, and that different
types of subject matter may be discussed throughout the application
(e.g., method(s) may be described under composition heading(s)
and/or kit headings; and/or descriptions of single topics may span
two or more topic headings). Hence, the use of the formal outline
headings is not intended to be in any way limiting.
[0018] Devices, compositions, and methods described herein provide
a tubular nanostructure targeted to a lipid bilayer membrane. The
targeted tubular nanostructure can have a surface region configured
to pass through a lipid bilayer membrane of a cell, a hydrophobic
surface region flanked by two hydrophilic surface regions
configured to form a pore in a lipid bilayer membrane of a cellular
organelle, and at least one ligand configured to bind one or more
cognates on the lipid bilayer membrane of the cellular organelle.
The target cell can be, for example, a tumor cell, an infected
cell, or a diseased cell in a subject. The tubular nanostructure
can form a pore in the lipid bilayer membrane of the cellular
organelle, e.g., mitochondria, which can permit transit or
translocation of at least one compound across the membrane and
cause cell death of the target cell. The targeted tubular
nanostructure can further include at least one second ligand
configured to bind one or more cognates on the lipid bilayer
membrane of the cell.
[0019] At least one ligand includes a compound that binds a cognate
and can be at least a portion of an antibody, antibody-coated
liposome, polynucleotide, polypeptide, receptor, viral plasmid,
polymer, protein, carbohydrate, lipid, toxin, lectin, pore-forming
toxin, small chemical compound, or any combination thereof. In one
aspect, the ligand can be a therapeutic compound configured to
affect a cell or process or to treat at least one of a disease,
condition, or symptom
[0020] One or more cognates can be associated with a target cell or
organelle and may include, but is not limited to, at least one of a
protein, a carbohydrate, a glycoprotein, a glycolipid, a
sphingolipid, a glycerolipid, or metabolites thereof. The cognate
can be a cell surface receptor or a cell surface marker on the
lipid bilayer membrane of a target cell, for example, on a tumor
cell, an infected cell, or a diseased cell in a subject, or on a
bacterial cell or a parasite cell.
[0021] Ligands can be targeted to cognates which are associated
with lipid bilayer membranes of target cells and/or organelles. A
target cell may include a tumor cell and/or other diseased cell
type in a mammalian subject. A target cell may also include a
pathogen, e.g., bacteria, fungi, and/or parasites. In some
instances, the tubular nanostructures may be designed to target a
specific cellular organelle, e.g., the mitochondria. The tubular
nanostructure can include a surface region configured to pass
through a lipid bilayer membrane of a cell, a hydrophobic surface
region flanked by two hydrophilic surface regions configured to
form a pore in a lipid bilayer membrane of a cellular organelle,
and at least one ligand configured to bind one or more cognates on
the lipid bilayer membrane of the cellular organelle.
[0022] The tubular nanostructures may be modified in such a manner
as to allow transit of the nanotubes through the plasma membrane
with subsequent targeting and insertion into the lipid bilayer of
one or more internal organelles, e.g., mitochondria. Once targeted
to the lipid bilayer of the organelle membrane, the tubular
nanostructure may form pores that enable active transport,
facilitated transport, or passive transport of contents into or out
of the organelle. In certain organelles, disruption of the lipid
bilayer may lead to cell death. In one example, tubular
nanostructures may be selectively directed to the outer membrane of
mitochondria in target cells where they insert into and disrupt the
outer mitochondrial membrane leading to target cell death. The
tubular nanostructures having hydrophobic surface region flanked by
two hydrophilic surface regions for insertion and retention in a
lipid bilayer may be modified in such a manner as to mask the
hydrophilic ends and allow transit through the plasma membrane. In
one embodiment, the hydrophilic ends of the tubular nanostructure
are modified with a hydrophobic moiety through a chemical bond that
may be cleaved once the nanotube has passed into the cell.
[0023] The tubular nanostructure includes, but is not limited to,
one or more of a carbon nanotube, cyclic peptide nanotube, crown
ether nanotube, polymer nanotube, polymer/carbon nanotube, DNA
nanotube, or inorganic nanotube. The inorganic nanotube can include
a boron nitride nanotube. The polymer nanotube can include
polystyrene, polytetrafluoroethylene, polymethylmethacrylate,
polyaniline, or poly-L-lactide/palladium acetate. The
polymer/carbon nanotube can include a polyaniline/carbon nanotube.
A single wall carbon nanotube can have a hydrophobic surface region
at least a portion of, or all of, the surface structure of the
tubular nanostructure.
[0024] A hydrophobic surface region of a tubular nanostructure
includes a tubular nanostructure with a carbon surface structure
and/or a linker molecule having a hydrophobic portion adsorbed onto
the tubular nanostructure, e.g., a phospholipid. A hydrophobic
polymer refers to any polymer resistant to wetting, or not readily
wet, by water, i.e., having a lack of affinity for water. A
hydrophobic polymer typically will have a surface free energy of
about 40 dynes/cm (10.sup.-5 Newtons/cm or N/cm) or less. Examples
of hydrophobic polymers include, by way of illustration only,
polylactide, polylactic acid, polyolefins, such as polyethylene,
poly(isobutene), poly(isoprene), poly(4-methyl-1-pentene),
polypropylene, ethylene-propylene copolymers, and
ethylenepropylene-hexadiene copolymers; ethylene-vinyl acetate
copolymers; styrene polymers, such as poly(styrene),
poly(2-methylstyrene), styrene-acrylonitrile copolymers having less
than about 20 mole-percent acrylonitrile, and
styrene-2,2,3,3,-tetrafluoro-propyl methacrylate copolymers.
Further examples are given in U.S. Pat. No. 6,673,447, hereby
incorporated by reference.
[0025] A hydrophilic surface region of a tubular nanostructure
includes a tubular nanostructure with a surface structure, e.g., a
carbon surface structure, and/or a linker molecule having a
hydrophilic portion adsorbed onto the tubular nanostructure, e.g.,
polyethylene glycol (PEG). The hydrophilic surface region may
include one or more of amines, amides, charged or polar amino
acids, alcohols, carboxylic groups, oxides, ester groups, ether
groups, or ester-ether groups, ketones, aldehydes, or derivatives
thereof. In one aspect, the hydrophilic surface region includes
PEG, which refers to a polymer with the structure
(--CH.sub.2CH.sub.2O--).sub.n that is synthesized normally by ring
opening polymerization of ethylene oxide. The PEG will impart water
(and serum) solubility to the hydrophobic nanoparticle and lipid
portion of the polar lipid. The polymer is usually linear at
molecular weights (MWs) less than or equal to 10 kD. The PEG will
have an MW below 5,400, preferably below 2,000, or about 45
repeating ethylene oxide units. However, the higher MW PEGs (higher
"n" repeating units) may have some degree of branching.
Polyethylene glycols of different MWs have already been used in
pharmaceutical products for different reasons (e.g., increase in
solubility of drugs). Therefore, from the regulatory standpoint,
they are very attractive for further development as drug or protein
carriers. The PEG used here should be attached to the nanoparticles
at a density adjusted for the PEG length. For example, with PL-PEG
2000, we have an estimate of 4 nm spacing between PEG chain along
the tube. At this spacing, PEG5400 is too long and starts to block
interaction with cell surface. For PEG at approximately 1 nm
distance, the PEG MW should be less than about 200, to allow
hydrophobicity.
[0026] In some instances, the one or more tubular nanostructures
may be functionalized with one or more ligands, therapeutic
compounds, toxin, marker, or combinations thereof. The
functionalized component may be a small chemical compound. Small
chemical compounds that might be added to a tubular nanostructure
include, but are not limited to, targeting biomolecules, e.g.,
receptor binding ligands; therapeutic biomolecules, e.g.,
therapeutic small chemical compound drugs; toxins, e.g.,
chemotherapy agents; and markers, e.g., fluorescent dyes and/or
radioactive compounds. Any of a number of homobifunctional,
heterofunctional, and/or photoreactive cross linking agents may be
used to bind biomolecules to tubular nanostructures. Examples of
homobifunctional cross linkers include, but are not limited to,
primary amine/primary amine linkers. Examples of heterofunctional
cross linkers include, but are not limited to, primary
amine/sulfhydryl linkers.
[0027] The one or more tubular nanostructures may be further
functionalized with ligands as therapeutic agents, including but
not limited to, anti-cancer therapeutic agents, anti-microbial
therapeutic agents. The one or more tubular nanostructures may be
further functionalized with markers to identify a cell target,
e.g., a fluorescent marker, a radioactive marker, a quantum dot, a
contrast agent for magnetic resonance imaging (MRI) marker, a
ligand reaction activated marker, lipid membrane reactive marker,
cell environment reactive marker, or combinations thereof.
[0028] A composite tubular nanostructure may comprise two or more
tubular nanostructures each including a hydrophobic surface region,
each hydrophobic region flanked by two hydrophilic surface regions
configured to form a pore in a lipid bilayer membrane. For example,
the composite tubular nanostructure can include 3 tubular
nanostructures or 7 tubular nanostructures. Composite tubular
nanostructures may be used to create multiple pores at one or more
sites in the targeted lipid bilayer. Tubular nanostructures or
composite tubular nanostructures may be modified to facilitate one
or more elements to control transport of molecules through the
tubular nanostructure. In one aspect, the one or more elements
includes at least one second ligand configured to reversibly bind a
cognate of interest, wherein the cognate of interest passes through
the pore. In another aspect, the one or more elements can
reversibly block the pore. Tubular nanostructures or composite
tubular nanostructures may be further modified to facilitate active
transport, facilitated transport, or passive transport of
biomolecules through the pores formed by the nanotubes in the lipid
bilayer. Active transport requires an external energy source, e.g.,
the hydrolysis of ATP to transport biomolecules such as ions
against a concentration gradient, the biomolecules moving, for
example, from low to high concentration.
[0029] With reference to the figures, and with reference now to
FIGS. 1, 2, and 3, depicted is one aspect of a system that may
serve as an illustrative environment of and/or for subject matter
technologies, for example, a tubular nanostructure which comprises
a hydrophobic surface region flanked by two hydrophilic surface
regions configured to form a pore in a lipid bilayer membrane, and
at least one ligand configured to bind one or more cognates on the
membrane, or for example, a tubular nanostructure which comprises a
surface region configured to pass through a lipid bilayer membrane
of a cell, and a hydrophobic surface region flanked by two
hydrophilic surface regions configured to form a pore in a lipid
bilayer membrane of a cellular organelle. Accordingly, the present
application first describes certain specific exemplary methods of
FIGS. 1, 2, and 3; thereafter, the present application illustrates
certain specific exemplary methods. Those having skill in the art
will appreciate that the specific methods described herein are
intended as merely illustrative of their more general
counterparts.
[0030] Continuing to refer to FIG. 1, depicted is a partial
diagrammatic view of an illustrative embodiment of a tubular
nanostructure or a composite tubular nanostructure and a method for
inserting a tubular nanostructure or a composite tubular
nanostructure into a lipid bilayer membrane. In FIG. 1A, a tubular
nanostructure 100 includes a hydrophobic surface region 110 flanked
by two hydrophilic surface regions 120 is configured to form a pore
170 in a lipid bilayer membrane 150, 160. The tubular nanostructure
100 further includes at least one ligand 130 configured to bind one
or more cognates 140 on the lipid bilayer membrane 150, 160. In
FIG. 1B, the tubular nanostructure 100 includes the at least one
ligand 130 configured to bind to the one or more cognates 140 on
the membrane 150, 160. The one or more cognates 140 may be in
various positions relative to the extracellular side 160 of the
membrane and the intracellular side 150 of the membrane. In FIG.
1C, the tubular nanostructure 100 including the hydrophobic surface
region 110 flanked by two hydrophilic surface regions 120 is
integrated into the lipid bilayer membrane 150, 160 of the cell.
The tubular nanostructure is configured to form a pore 170 in the
lipid bilayer membrane 150, 160. In FIG. 1D, the tubular
nanostructure 100 includes the at least one ligand 130 configured
to bind to the one or more cognates 140 on the membrane 150, 160.
In this aspect, the at least one ligand 130 is configured to bind
to the one or more cognates 140 on the intracellular side 150 of
the membrane. The tubular nanostructure is configured to form a
pore 170 in the lipid bilayer membrane 150, 160 of the cell.
[0031] Continuing to refer to FIG. 2, depicted is a partial
diagrammatic view of an illustrative embodiment of a tubular
nanostructure or a composite tubular nanostructure and a method for
inserting a tubular nanostructure or a composite tubular
nanostructure into a lipid bilayer membrane of a cellular
organelle. In FIGS. 2A and 2B, a tubular nanostructure 200 which
comprises a surface region 210 is configured to pass through a
lipid bilayer membrane 250, 260 of a cell. The lipid bilayer
membrane of the cell has an extracellular side 260 of the membrane
and an intracellular side 250 of the membrane In FIG. 2C, the
tubular nanostructure further includes a hydrophobic surface region
220 flanked by two hydrophilic surface regions 225 configured to
form a pore 290 in a lipid bilayer membrane 270, 280 of a cellular
organelle. The tubular nanostructure 200 which comprises a surface
region 210 is configured to pass through a lipid bilayer membrane
250, 260 of the cell. The tubular nanostructure 200 is configured
to interact with a cellular component 285 to produce the at least
one tubular nanostructure including the hydrophobic surface region
220 flanked by two hydrophilic surface regions 225. The tubular
nanostructure 200 may further include at least one ligand 230
configured to bind one or more cognates 240 on the lipid bilayer
membrane 270, 280 of the cellular organelle. The one or more
cognates 240 may be in various positions relative to the
cytoplasmic side 280 of the lipid bilayer membrane or the
intraorganellar side 270 of the lipid bilayer membrane of the
cellular organelle. In FIG. 2D, the tubular nanostructure 200
including the hydrophobic surface region 220 flanked by two
hydrophilic surface regions 225 is integrated into the lipid
bilayer membrane 270, 280 of the cellular organelle. The tubular
nanostructure is configured to form a pore 290 in the lipid bilayer
membrane 270, 280. In FIG. 2E, the tubular nanostructure 200 may
include the at least one ligand 230 configured to bind to the one
or more cognates 240 on the membrane 270, 280 of a cellular
organelle. In this aspect, the at least one ligand 230 is
configured to bind to the one or more cognates 240 on the
intraorganellar side 270 of the membrane. The tubular nanostructure
is configured to form a pore 290 in the lipid bilayer membrane 270,
280 of the cellular organelle.
[0032] FIGS. 3A, 3B, and 3C illustrate some exemplary aspects of a
method as that described in FIGS. 1 and 2. FIGS. 3A, 3B, and 3C
illustrate an exemplary method 300 for disrupting a lipid bilayer
membrane of a cellular organelle in a cell 301 comprising
contacting the cell with at least one tubular nanostructure
including a surface region configured to pass through a lipid
bilayer membrane of the cell, the at least one tubular
nanostructure including a hydrophobic surface region flanked by two
hydrophilic surface regions configured to form a pore in the lipid
bilayer membrane of the cellular organelle, and at least one ligand
bound to the at least one tubular nanostructure, the ligand
configured to bind one or more cognates on the lipid bilayer
membrane of the cellular organelle 302. The method further provides
wherein the at least one tubular nanostructure includes at least
one second ligand configured to bind one or more cognates on the
lipid bilayer membrane of the cell 303. The method further provides
wherein the at least one tubular nanostructure includes a marker
attached to the nanostructure 308. The method further provides the
at least one tubular nanostructure including the surface region
configured to pass through the lipid bilayer membrane of the cell
is configured to interact with a cellular component to produce the
at least one tubular nanostructure including the hydrophobic
surface region flanked by two hydrophilic surface regions 309. The
method further provides the at least one tubular nanostructure
includes one or more elements to control transport of molecules
through the tubular nanostructure 310. The method further provides
the at least one tubular nanostructure includes one or more
elements to control transport of molecules through the tubular
nanostructure 311.
Tubular Nanostructure
[0033] Tubular nanostructures as described herein may be made from
a wide variety of materials, for example, organic, inorganic,
polymeric, biodegradable, biocompatible and combinations thereof.
Non-limiting examples of inorganic materials to make tubular
nanostructures as described herein include iron oxide, silicon
oxide, titanium oxide and the like. Examples of biodegradable
monomers formed into tubular nanostructures include
polysaccharides, cellulose, chitosan, carboxymethylated cellulose,
polyamino-acids, polylactides and polyglycolides and their
copolymers, copolymers of lactides and lactones, polypeptides,
poly-(ortho)esters, polydioxanone, poly-.beta.-aminoketones,
polyphosphazenes, polyanhydrides, polyalkyl(cyano)acrylates,
poly(trimethylene carbonate) and copolymers,
poly(.epsilon.-caprolactone) homopolymers and copolymers,
polyhydroxybutyrate and polyhydroxyvalerate, poly(ester)urethanes
and copolymers, polymethyl-methacrylate and combinations thereof.
The carrier may even include or made from polyglutamic or
polyaspartic acid derivatives and their copolymers with other
amino-acids.
[0034] The tubular nanostructure as described herein may be a
carbon nanotube. Carbon nanotubes are all-carbon hollow graphitic
tubes with nanoscale diameter. They can be classified by structure
into two main types: single walled CNTs (SWNTs), which consist of a
single layer of graphene sheet seamlessly rolled into a cylindrical
tube, and multiwalled CNTs (MWNTs), which consist of multiple
layers of concentric cylinders. Carbon sources for use in
generating carbon nanotubes include, but are not limited to, carbon
monoxide and hydrocarbons, including aromatic hydrocarbons, e.g.,
benzene, toluene, xylene, cumene, ethylbenzene, naphthalene,
phenanthrene, anthracene or mixtures thereof, non-aromic
hydrocarbons, e.g., methane, ethane, propane, ethylene, propylene,
acetylene or mixtures thereof; and oxygen-containing hydrocarbons,
e.g., formaldehyde, acetaldehyde, acetone, methanol, ethanol or
mixtures thereof.
[0035] Carbon nanotubes may be synthesized from one or more carbon
sources using a variety of methods, e.g., arc-discharge, laser
ablation, or chemical vapor deposition (CVD; see, e.g., Bianco, et
al., in Nanomaterials for Medical Diagnosis and Therapy. pp.
85-142. Nanotechnologies for the Live Sciences Vol. 10 Edited by
Challa S. S. R. Kumar, WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim, 2007, which is incorporated herein by reference).
[0036] Carbon nanotubes may be synthesized using the arc discharge
method which creates nanotubes through arc-vaporization of two
carbon rods placed end to end, separated by approximately 1 mm, in
an enclosure that is filled, for example, with inert gas (e.g.,
helium, argon) at low pressure (between 50 and 700 mbar). A direct
current of 50 to 100 amperes driven by approximately 20 volts
creates a high temperature discharge between the two electrodes.
The discharge vaporizes one of the carbon rods and forms a small
rod shaped deposit on the other rod.
[0037] Alternatively, carbon nanotubes may be synthesized using
laser ablation in which a pulsed or continuous laser energy source
is used to vaporize a graphite target in an oven at 1200.degree. C.
The oven is filled with an inert gas such as helium or argon, for
example, in order to keep the pressure at 500 Ton. A hot vapor
plume forms, expands, and cools rapidly. As the vaporized species
cool, small carbon molecules and atoms quickly condense to form
larger clusters. The catalysts also begin to condense and attach to
carbon clusters from which the tubular molecules grow into
single-wall carbon nanotubes. The single-walled carbon nanotubes
formed in this case are bundled together by van der Waals
forces.
[0038] Carbon nanotubes may also be synthesized using chemical
vapor deposition (CVD). CVD synthesis is achieved by applying
energy to a gas phase carbon source such as methane or carbon
monoxide, for example. The energy source is used to "crack" the gas
molecules into reactive atomic carbon. The atomic carbon diffuses
towards a substrate, which is heated and coated with a catalyst,
e.g., Ni, Fe or Co where it will bind. The catalyst is generally
prepared by sputtering one or more transition metals onto a
substrate and then using either chemical etching or thermal
annealing to induce catalyst particle nucleation. Thermal annealing
results in cluster formation on the substrate, from which the
nanotubes will grow. Ammonia may be used as the etchant. The
temperatures for the synthesis of nanotubes by CVD are generally
within the 650-900.degree. C. range. A number of different CVD
techniques for synthesis of carbon nanotubes have been developed,
such as plasma enhanced CVD, thermal chemical CVD, alcohol
catalytic CVD, vapor phase growth, aero gel-supported CVD and
laser-assisted thermal CVD, and high pressure CO disproportionation
process (HiPCO). Additional methods describing the synthesis of
carbon nanotubes may be found, for example, in U.S. Pat. Nos.
5,227,038; 5,482,601; 6,692,717; 7,354,881 which are incorporated
herein by reference.
[0039] Carbon nanotubes may be synthesized as closed at one or both
ends. As such, forming a hollow tube may necessitate cutting the
carbon nanotubes. Carbon nanotubes may be cut into smaller
fragments using a variety of methods including but not limited to
irradiation with high mass ions, intentional introduction of
defects into the carbon nanotube during synthesis, sonication in
the presence of liquid or molten hydrocarbon, lithography,
oxidative etching with strong oxidating agents, mechanical grinding
with diamond balls, or physical cutting with an ultra microtome
(see, e.g., U.S. Pat. No. 7,008,604; Wang et al, Nanotechnol.
18:055301, 2007, which are incorporated herein by reference). For
irradiation with high mass ions, for example, the carbon nanotubes
are subjected to a fast ion beam, e.g., from a cyclotron, at
energies of from about 0.1 to 10 giga-electron volts. Suitable high
mass ions include those over about 150 AMU's such as bismuth, gold,
uranium and the like. To generate defects that are susceptible to
cleavage, the carbon nanotubes may be synthesized in the presence
of a small amount of boron, for example. For sonication, carbon
nanotubes may be sonicated in the presence of 1,2-dichloroethane,
for example, using a sonicator with sufficient acoustic energy over
a period ranging from 10 minutes to 24 hours, for example. For
oxidative etching, carbon nanotubes may be incubated in a solution
containing 3:1 concentrated sulfuric acid:nitric acid for 1 to 2
days at 70.degree. C. For cutting with an ultra microtome, the
carbon nanotubes are magnetically aligned, frozen to a temperature
of about -60.degree. C., and cut using an ultra-thin cryo-diamond
knife.
[0040] Once synthesized, carbon nanotubes may be further purified
to eliminate contaminating impurities, e.g., amorphous carbon and
catalyst particles. Methods for further purification include, but
are not limited to, acid oxidation, microfiltration,
chromatographic procedures, microwave irradiation, and
polymer-assisted purification (see, e.g., U.S. Pat. No. 7,357,906,
which is incorporated herein by reference). Chromatography and
microfiltration may also be used to isolate a uniformed population
of carbon nanotubes with similar size and diameter, for example
(see, e.g., Bianco, et al., in Nanomaterials for Medical Diagnosis
and Therapy. pp. 85-142. Nanotechnologies for the Live Sciences
Vol. 10 Edited by Challa S. S. R. Kumar, WILEY-VCH Verlag GmbH
& Co. KGaA, Weinheim, 2007, which is incorporated herein by
reference). Alternatively, purified carbon nanotubes may be
purchased from a commercial source (from, e.g., Carbon
Nanotechologies, Houston, Tex.; Sigma-Aldrich, St. Louis, Mo.).
[0041] Alternatively, a tubular nanostructure as described herein
may be a peptide nanotube. Peptide nanotubes are extended tubular
beta-sheet-like structures and are constructed by the self-assembly
of flat, ring-shaped peptide subunits made up of alternating D- and
L-amino acid residues as described in U.S. Pat. Nos. 6,613,875 and
7,288,623, and in Hartgerink, et al., J. Am. Chem. Soc. 118:43-50,
1996, which are incorporated herein by reference. For example,
gramicidin is a pentadecapeptide which forms a n-helix with a
hydrophilic interior and a lipophilic exterior bearing amino acid
side chains in membranes and nonpolar solvents. In this instance,
the helix length is approximately half of the thickness of a lipid
bilayer and as such, two gramicidin molecules form an end-to-end
dimer stabilized by hydrogen bonds that spans the lipid bilayer.
Peptide nanotubes are constructed by highly convergent noncovalent
processes by which cyclic peptides rapidly self-assemble and
organize into ultra large, well ordered three-dimensional
structures, upon an appropriate chemical- or medium-induced
triggering. The properties of the outer surface and the internal
diameter of peptide nanotubes may be adjusted by the choice of the
amino acid side chain functionalities and the ring size of the
peptide subunit employed.
[0042] Alternatively, a tubular nanostructure as described herein
may be a lipid nanotube. Lipid nanotubes are typically formed using
self-assembling microtubule-forming diacetylenic lipids, such as
complex chiral phosphatidylcholines, and mixtures of these
diacetylenic lipids as described in U.S. Pat. Nos. 4,877,501,
4,911,981 and 4,990,291, which are incorporated herein by
reference. The synthesis of self-assembling lipid nanotubes may be
accomplished by combining the appropriate lipids with an alcohol
and a water phase which leads to the production of lipid
microcylinders by direct crystallization. The formation of the
lipid tubules may be modulated by the choice of alcohol and/or
combination of alcohols, the ratio of alcohol to water, and
variations in the reaction temperature (see, e.g., U.S. Pat. No.
6,013,206, which is incorporated herein by reference). A simple
method for generating uniform lipid nanotubes from single-chain
diacetylene secondary amine salts has been described in Lee, et
al., J. Am. Chem. Soc. 126:13400-13405, 2004, which is incorporated
herein by reference.
Functionalization of Tubular Nanostructures for Targeting and
Insertion into a Cellular Membrane
[0043] Tubular nanostructures as described herein may be
functionalized to include hydrophilic surface regions at one or
both ends of the tubular nanostructure to facilitate insertion and
retention of the tubular nanostructure into a lipid bilayer
membrane associated with a target cell or organelle (see, e.g.,
U.S. Patent Application 2004/0023372 A1, which is incorporated
herein by reference). The hydrophilic surface region may include
one or more of amines, amides, charged or polar amino acids,
alcohols, carboxylic groups, oxides, ester groups, ether groups, or
ester-ether groups, ketones, aldehydes, or derivatives thereof.
Tubular nanostructures may be further functionalized to include one
or more ligand, one or more therapeutic compounds, one or more
toxins, one or more markers, or combinations thereof. A tubular
nanostructure may be functionalized using non-covalent and covalent
methodologies.
[0044] Non-covalent functionalization of carbon nanotubes, for
example, may be accomplished using .pi.-.pi. stacking interactions
between conjugated molecules and the graphitic sidewall of the
tubular nanostructure. For example, compounds with a pyrene moiety,
e.g., N-succinimidyl-1-pyrenebutanoate may be irreversibly absorbed
onto the surface of a carbon nanotube through .pi.-.pi. stacking
interaction. In this instance, the succinimidyl ester group
associated with the pyrenebutonaote may be used to link to primary
or secondary amines and as such may be used to couple biomolecules,
e.g., proteins and nucleic acids to the tubular nanostructure.
Other molecules that may be linked to a tubular nanostructure via
.pi.-.pi. stacking interactions include the photosensitizers
phthalocyanines and porphyrins (see, e.g., Bianco, et al., in
Nanomaterials for Medical Diagnosis and Therapy. pp. 85-142.
Nanotechnologies for the Live Sciences Vol. 10 Edited by Challa S.
S. R. Kumar, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007,
which is incorporated herein by reference).
[0045] Alternatively, non-covalent functionalization may be
accomplished using hydrophobic interactions with amphiphilic
molecules. In this instance, the hydrophobic surface of the
amphiphilic molecules interact noncovalently with the aromatic
surface of the carbon nanotube while exposing their hydrophilic
parts to the aqueous medium, allowing for solubilization of
hydrophobic tubular nanostructures in aqueous solutions. Examples
of molecules that may be used for this purpose include, but are not
limited to, water-soluble polymers, e.g., polyvinylpyrrolidone and
polystyrenesulfonate; surfactants, e.g., anionic, nonionic, and
cationic surfactants including, for example, deoxycholic acid,
taurodeoxycholic acid, sodium dodecylbenzene sulfonate, and sodium
dodecyl sulfate; amphiphilic peptides, and single stranded DNA. In
addition, a biomolecule may be attached indirectly to a tubular
nanotube, e.g., a carbon nanotube through an amphiphilic
bifunctional linker, e.g., phospholipid (PL)-poly(ethylene glycol)
(PEG) chains and terminal amine (PL-PEG-NH.sub.2) in which the PL
alkyl chains interact noncovalently with the carbon nanotube and
the amine group may be used to link to biomolecules. Other examples
of functionalized PEG lipids include, but are not limited to,
phospholipid-PEG-carboxylic acid, phospholipid-PEG-maleimide, and
phospholipid-PEG-biotin, for example. For example, the
phospholipid-PEG-biotin derivative
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene
glycol] 2000] (DSPE-PEG(2000)-biotin) may be added to carbon
nanotubes by sonication, followed by centrifugation to isolate the
functionalized nanotubes (see, e.g., Chakravarty, et al., Proc.
Natl. Acad. Sci. USA 105:8697-8702, 2008, which is incorporated
herein by reference). Similarly, DNA or RNA may be linked to a
carbon nanotube using a heterofunctional crosslinker, e.g.,
sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate
(sulfo-LC-SPDP). (see, e.g., Bianco, et al., in Nanomaterials for
Medical Diagnosis and Therapy. pp. 85-142. Nanotechnologies for the
Live Sciences Vol. 10 Edited by Challa S. S. R. Kumar, WILEY-VCH
Verlag GmbH & Co. KGaA, Weinheim, 2007, which is incorporated
herein by reference).
[0046] Tubular nanostructures may also be functionalized using
covalent interactions. Covalent functionalization of carbon
nanotubes, for example, may involve defect functionalization and/or
side-wall functionalization. Defect functionalization takes
advantage of defects in the carbon nanotube structure characterized
by disruptions in the six-membered rings of the graphene sheets
such as might be found at the cut ends of carbon nanotubes. Defect
functionalization may also be present on the side-walls,
characterized by the presence of five- and seven-membered rings
within the graphene sheet of six-membered rings. Treatment of
carbon nanotubes with strong oxidizing agents, e.g., nitric acid,
KMnO.sub.4/H.sub.2SO.sub.4, O.sub.2,
K.sub.2Cr.sub.2O.sub.7/H.sub.2SO.sub.4 or OsO.sub.4 may be used to
cut carbon nanotubes, generating open ends and creating a hollow
tube (see, e.g., U.S. Pat. No. 7,008,604, which is incorporated
herein by reference). Oxidation may also be used to add functional
groups, e.g., carboxylic acid, ketone, alcohol and ester groups to
the ends and defect sites on the side-walls and as such may be used
to create hydrophilic surface regions.
[0047] The functional groups added to carbon nanotubes by
oxidation, for example, may be used to further modify the ends
and/or the side walls of the nanotubes. For example, carboxylic
acid moieties on the nanotube may be used to form amide and ester
linkages. In this instance, reactive intermediates are formed by
treating the carboxylic acid groups with thionyl chloride,
carbodiimide, or N-hydroxysuccinimide (NHS). The reactive
intermediates are then able to form covalent linkages with
biomolecules, e.g., polymers such
poly-propionyl-ethylenimine-co-ethylenimine (PPEI-EI),
poly-n-vinylcarbazole (PVK-PS) and polyethylene glycol (PEG),
poly-n-butyl methacrylate (PnBMA), poly-methyl methacrylate (PMMA),
and PMMA-b-poly-hydroxyethyl methacrylate (PHEMA); proteins such as
bovine serum albumin; DNA molecules; and other biomolecules, e.g.,
biotin.
[0048] End and/or side-wall functionalization of a tubular
nanostructure may be accomplished using various chemical reactions
including but not limited to fluorination, radical addition,
nucleophilic addition, electrophilic addition, and cycloaddition,
for example (see, e.g., Bianco, et al., in Nanomaterials for
Medical Diagnosis and Therapy. pp. 85-142. Nanotechnologies for the
Live Sciences Vol. 10 Edited by Challa S. S. R. Kumar, WILEY-VCH
Verlag GmbH & Co. KGaA, Weinheim, 2007, which is incorporated
herein by reference). Fluorine may be added to the surface of a
carbon nanotube, for example, by heating the nanotube in the
presence of elemental fluorine at temperatures ranging from 150 to
600.degree. C. (see, e.g., U.S. Pat. No. 6,841,139, which is
incorporated herein by reference). The fluorine group on the carbon
nanotube may be further substituted with strong nucleophilic
reagents, e.g., Grignard, alkyllithium reagents and/or metal
alkoxides. Alternatively, a tubular nanostructure, e.g., a carbon
nanotube, may be functionalized by cycloaddition with, for example,
dichlorocarbene, nitrenes, bromomalonates, o-quinodimethane, azido
group, alkyne/azide, and/or azomethine ylides. For example,
protected amino groups may be introduced onto the surface of carbon
nanotubes using 1,3-dipolar cycloaddition of azomethine ylides. The
N-protected amino acid may then be used to link biomolecules, e.g.,
bioactive peptides (see, e.g., Pantorotto, et al., J. Am. Chem.
Soc. 125:6160-6164, 2003, which is incorporated herein by
reference).
[0049] In some instances, it may be beneficial to selectively
functionalize one portion or portions of the ends and/or sidewalls
of a tubular nanostructure. Asymmetric functionalization of carbon
nanotubes may be accomplished using a masking technique. For
example, carbon nanotubes may be partially embedded in a polymer
matrix, including, but not limited to, poly(dimethylsiloxane),
polystyrene, poly(methyl methacrylate), or polydiene rubber or a
combination thereof and the non-embedded or exposed portion
functionalized (see, e.g., Qu & Dai, Chem. Commun. 37:
3829-3861, 2007, which is incorporated herein by reference). An
organic solvent, e.g., toluene, may be used to wash away the
masking polymer. Asymmetric functionalization of the ends of carbon
nanotubes may be accomplished using a lithographic procedure to cut
the nanotubes followed by chemical modification of the exposed tube
ends via plasma treatment while the tube side-walls remain
protected by a resist layer (see, e.g., Burghard Small 1:1148-1140,
2005, which is incorporated herein by reference).
[0050] Alternatively, asymmetric functionalization of carbon
nanotubes may be accomplished by floating the nanotubes on a
photoreactive solution with only one side of the nanotube in
contact with the solution and exposing the solution to UV light
(see, e.g., U.S. Patent Application 2006/0257556 A1, which is
incorporated herein by reference). Photoreactive reagents are
chemically inert reagents that become reactive when exposed to
ultraviolet or visible light and are exemplified by derivatives of
aryl azides. When an aryl azide is exposed to UV light, it forms a
nitrene group that can initiate addition reactions with double
bonds, insertion into C--H and N--H sites, or subsequent ring
expansion to react with a nucleophile (e.g., primary amines).
Examples of photoreactive cross linkers include, but are not
limited, to primary amine linkers such as
ANB-NOS(N-5-azido-2-nitrobenzyloxysuccinimide), NHS-ASA
(N-hydroxy-succinimidyl-4-azidosalicyclic acid), Sulfo HSAB
(N-hydroxysulfosuccinimidyl-4-azidobenzoate), Sulfo SAED
(sulfosuccinimidyl
2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate),
Sulfo SAND (sulfosuccinimidyl
2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-propionate), Sulfo SANPAH
(sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate),
Sulfo SADP (sulfosuccinimidyl (4-azidophenyldithio) propionate, and
Sulfo SASD
(sulfosuccinimidyl-2-(rho-azidosalicylamido)ethyl-1,3-dithiopropionate;
carbohydrate linkers such as ABH (azidobenzoyl hydrazide); arginine
linkers such as APG (azidophenyl glyoxal monohydrate), sulfhydryl
linkers such as APDP
(N-(4-[rho-azidosalicylamido]butyl)-3'-(2'-pyridyldithio)
propionamide); non selective linkers such as BASED
(bis(beta-[4-azidosalicylamido]-ethyl) disulfide).
[0051] Tubular nanostructures may be functionalized to include one
or more ligand, therapeutic compound, toxin, marker, or
combinations thereof. In some instances the one or more ligand,
therapeutic compound, toxin and/or marker is a protein biomolecule.
Protein biomolecules that might be added to a tubular nanostructure
include, but are not limited to, targeting biomolecules, e.g.,
antibodies, receptor ligands, and lectins; therapeutic
biomolecules, e.g., therapeutic proteins or peptides; transporter
biomolecules, e.g., components of the ATP-binding cassette (ABC)
transporters; pore-forming agents such as antimicrobial peptides;
and toxic biomolecules such as protein-based plant and bacterial
toxins. The tubular nanostructure may be functionalized with
amines, carboxylic acids, thiols, aldehydes and combinations
thereof to facilitate linkage to protein biomolecules. For example,
attachment of one or more protein molecules to a carbon nanotube
may be performed using heterobifunctional crosslinkers. For
example, a heterobifunctional crosslinker may be added covalently
to a carbon nanotube by adding amino groups to the nanotube via
azomethine ylide cycloaddition or alkyne azide cycloaddition,
followed by derivatization of the amino groups with a
heterobifunctional crosslinker, e.g.,
succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)
(LC-SMCC). The nanotube functionalized in this manner is combined
with a protein into which reactive sulfhydryl groups have been
introduced with 2-iminothiolane-HCl (see, e.g., McDevitt, et al.,
J. Nucl. Med. 48:1180-1189, 2007, which is incorporated herein by
reference). Alternatively, a protein biomolecule may be added to a
tubular nanostructure such as a carbon nanotube, for example, by
non-covalent attachment of phospholipid-PEG-NH.sub.2 to the
nanotube and covalent interaction of the associated amine group
with thiolated protein (see, e.g., Welsher, et al., Nano Lett.
8:586-590, 2008, which is incorporated herein by reference).
Alternatively, a protein biomolecule may be added to a tubular
nanostructure using a biotin/avidin linkage in which the carbon
nanotubes are functionalized with biotin using a
phospholipid-PEG-biotin as described herein and combined with
avidin- or streptavidin-modified protein. A protein may be modified
with avidin, for example, by activating the avidin with
m-maleimidobenzoyl-N-hydroxysuccinimide ester and linking it to
thiolated target protein (see, e,g, Chakravarty, et al., Proc.
Natl. Acad. Sci. USA, 105: 8697-8702, 2008, which is incorporated
herein by reference).
[0052] Biomolecules such as antibodies, for example, may also be
attached to peptide nanotubes and boron nitride nanotubes (see,
e.g., Zhao & Matsui Small 3:1390-1393, 2007; U.S. Patent
Application 2006/0067941, which are incorporated herein by
reference). For example, boron nitride nanotubes may be chemically
modified with primary amines such as methylamine and ethanolamine
that may be used for additional functionalization of the nanotubes
(Wu, et al., J. Am. Chem. Soc. 128:12001-12006, 2006, which is
incorporated herein by reference).
[0053] One or more tubular nanostructures may be functionalized
with one or more peptides. In some instances, one or more peptides
may be linked to a tubular nanostructure using the methods
described above for proteins. Alternatively, one or more peptides
may be linked to a tubular nanostructure using fragment
condensation of fully protected peptides and/or selective chemical
ligation (see, e.g., U.S. Patent Application 20060199770;
Pantarotto, et al., J. Am. Chem. Soc. 125:6160-6164, 2003, which
are incorporated herein by reference). For selective chemical
ligation, for example, carbon nanotubes may be functionalized with
primary amines and N-succinimidyl 3-maleimidopropionate and reacted
with N-terminal acetylated peptide to form peptide-carbon nanotube
conjugates. Alternatively, peptides may be designed using phage
display methodologies that selectively recognize and bind carbon
nanotubes as described in U.S. Pat. No. 7,304,128, which is
incorporated herein by reference.
[0054] In some instances, the one or more tubular nanostructures
may be functionalized with one or more ligand, therapeutic
compound, toxin, marker, or combination thereof that is a
polynucleotide biomolecule. Polynucleotide biomolecules that might
be added to a tubular nanostructure include, but are not limited
to, aptamers, antisense RNA, RNAi, DNA, or combinations thereof.
For example, DNA may be added to a tubular nanostructure such as a
carbon nanotube using a streptavidin-biotin linkage. In this
instance, streptavidin may be non-covalently associated with the
carbon nanotube and combined with biotin modified DNA.
Alternatively, single strand DNA may be bound to a carbon nanotube
by direct non-covalent interaction forming a coil around the
nanotube. Alternatively, a small oligonucleotide such as an
aptamer, for example, may be linked to a carbon nanotube using
carbodiimidazole (CDI)-Tween (see, e.g., So, et al., J. Am. Chem.
Soc. 127:11906-11907, 2005, which is incorporated herein by
reference). Alternatively, a DNA or RNA aptamer may be linked to a
carbon nanotube via a streptavidin-biotin linkage. In this
instance, biotin may be introduced into the DNA or RNA aptamer
during synthesis of the aptamer and then bound to streptavidin
associated with the carbon nanotube. Alternatively, a DNA or RNA
aptamer may be conjugated to a tubular nanotube using amine- or
sulfhydryl-reactive crosslinkers (e.g., from Pierce-Thermo
Scientific, Rockford, Ill., USA) using the methods described
herein. As such, the aptamer may be synthesized in the presence of
specific bases modified with primary amines or thiols.
[0055] In some instances, the one or more tubular nanostructures
may be functionalized with one or more ligand, therapeutic
compound, toxin, marker, or combinations thereof as a small
chemical compound. Small chemical compounds that might be added to
a tubular nanostructure include, but are not limited to, targeting
biomolecules, e.g., receptor binding ligands; therapeutic
biomolecules, e.g., therapeutic small chemical compound drugs;
toxins, e.g., chemotherapy agents; and markers, e.g., fluorescent
dyes and/or radioactive compounds. For example, reversible
attachment of a platinum based chemotherapy to a carbon nanotube
can be used in which the platinum compound was modified with a
linker arm and an N-succinimidyl ester group which readily formed
amide linkages with PEG-tethered primary amines on the surface of
carbon nanotubes (Feazell, et al., J. Am. Chem. Soc. 129:8438-8439,
2007, which is incorporated herein by reference).
[0056] In general, any of a number of homobifunctional,
heterofunctional, and/or photoreactive cross linking agents may be
used to bind biomolecules to tubular nanostructures. Examples of
homobifunctional cross linkers include, but are not limited to,
primary amine/primary amine linkers such as BSOCES
((bis(2-[succinimidooxy-carbonyloxy]ethyl) sulfone), DMS (dimethyl
suberimidate), DMP (dimethyl pimelimidate), DMA (dimethyl
adipimidate), DSS (disuccinimidyl suberate), DST (disuccinimidyl
tartate), Sulfo DST (sulfodisuccinimidyl tartate), DSP
(dithiobis(succinimidyl propionate), DTSSP
(3,3'-dithiobis(succinimidyl propionate), EGS (ethylene glycol
bis(succinimidyl succinate)) and sulfhydryl/sulfhydryl linkers such
as DPDPB (1,4-di-(3'-[2' pyridyldithio]-propionamido) butane).
Examples of heterofunctional cross linkers include, but are not
limited to, primary amine/sulfhydryl linkers such as MBS
(m-maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo MBS
(m-maleimidobenzoyl-N-hydroxysulfosuccinimide), GMBS
(N-gamma-maleimidobutyryl-oxysuccinimide ester), Sulfo GMBS
(N-gamma-maleimidobutyryloxysulfosuccinimide ester), EMCS
(N-(epsilon-maleimidocaproyloxy) succinimide ester), Sulfo EMCS
(N-(epsilon-maleimidocaproyloxy) sulfo succinimide), SIAB
(N-succinimidyl (4-iodoacetyl)aminobenzoate), SMCC (succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SMPB
(succinimidyl 4-(rho-maleimidophenyl) butyrate), Sulfo SIAB
(N-sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), Sulfo SMCC
(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
Sulfo SMPB (sulfosuccinimidyl 4-(rho-maleimidophenyl) butyrate),
and MAL-PEG-NHS (maleimide PEG N-hydroxysuccinimide ester);
sulfhydryl/hydroxyl linkers such as PMPI (N-rho-maleimidophenyl)
isocyanate; sulfhydryl/carbohydrate linkers such as EMCH
(N-(epsilon-maleimidocaproic acid) hydrazide); and amine/carboxyl
linkers such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride).
Ligands Targeted to Cognates which are Associated with Target Cells
and/or Organelles
[0057] The tubular nanostructures as described herein may include
one or more ligands that are configured to bind to one or more
cognates associated with the lipid bilayer membrane of a target
cell or organelle. A target cell may include a tumor cell and/or
other diseased cell type in a mammalian subject. A target cell may
also include a pathogen, e.g., bacteria, fungi, and/or parasites.
In some instances, the tubular nanostructures may be designed to
target a specific cellular organelle, e.g., the mitochondria. One
or more cognates associated with a target cell or organelle may
include at least one of a protein, a carbohydrate, a glycoprotein,
a glycolipid, a sphingolipid, a glycerolipid, or metabolites
thereof.
[0058] Tumor Markers
[0059] One or more tubular nanostructures may include one or more
ligands that bind one or more cognates associated with a tumor
cell. In this instance, the cognate may be a cell surface receptor
or cell surface marker on a tumor cell. Examples of cognates
associated with tumor cells may include, but are not limited to,
BLyS receptor, carcinoembryonic antigen (CA-125), CD25, CD34, CD33
and CD123 (acute myeloid leukemia), CD20 (chronic lymphocytic
leukemia), CD19 and CD22 (acute lymphoblastic leukemia), CD30,
CD40, CD70, CD133, 57 kD cytokeratin, epithelial specific antigen,
extracellular matrix glycoprotein tenascin, Fas/CD95,
gastrin-releasing peptide-like receptors, hepatocyte specific
antigen, human gastric mucin, human milk fat globule, lymphatic
endothelial cell marker, matrix metalloproteinase 9, melan A,
melanoma marker, mesothelin, mucin glycoproteins (e.g., MUC1, MUC2,
MUC4, MUC5AC, MUC6), prostate specific antigen, prostatic acid
phosphatase, PTEN, renal cell carcinoma marker, RGD-peptide binding
integrins, sialyl Lewis A, six-transmembrane epithelial antigen of
the prostate (STEAP), TNF receptor, TRAIL receptor, tyrosinase,
villin. Other tumor associated antigens include, but are not
limited to, alpha fetoprotein, apolipoprotein D, clusterin,
chromogranin A, myeloperoxidase, MyoD1 myoglobin placental alkaline
phosphatase c-fos, homeobox genes, or aberrantly glycosylated
antigens.
[0060] Bacterial Cognates
[0061] One or more tubular nanostructures may include one or more
ligands that bind one or more cognates associated with bacteria. A
cognate on bacteria may be a component of the bacterial outer
membrane, cell wall, and/or cytoplasmic membrane, for example.
Examples of cognates associated with the bacterial outer membrane
of Gram-negative bacteria include, but are not limited to,
lipopolysaccharide and OMP (outer membrane protein) porins, the
latter of which are exemplified by OmpC, OmpF and PhoP of E. coli.
Examples of cognates associated with the bacterial cell wall of
both Gram-positive and Gram-negative bacterial include, but are not
limited to, peptidoglycans polymers composed of an alternating
sequence of N-acetylglucoamine and N-acetyl-muraminic acid and
crosslinked by amino acids and amino acid derivatives. Examples of
cognates associated with the bacterial cytoplasmic membrane
include, but are not limited to, the MPA1-C (also called
polysaccharide copolymerase, PCP2a) family of proteins, the MPA2
family of proteins, and the ABC bacteriocin exporter accessory
protein (BEA) family of proteins. Other examples of cognates
associated with bacteria include, but are not limited to,
transporters, e.g., sugar porter (major facilitator superfamily),
amino-acid/polyamine/organocation (APC) superfamily, cation
diffusion facilitator, resistance-nodulation-division type
transporter, SecDF, calcium:cation antiporter, inorganic phosphate
transporter, monovalent cation:proton antiporter-1, monovalent
cation:proton antiporter-2, potassium transporter,
nucleobase:cation symporter-2, formate-nitrite transporter,
divalent anion:sodium symporter, ammonium transporter, and
multi-antimicrobial extrusion; channels, e.g., major intrinsic
protein, chloride channel, and metal ion transporter; and primary
active transporters, e.g., P-type ATPase, arsenite-antimonite
efflux, Type II secretory pathway (SecY), and sodium-transporting
carboxylic acid decarboxylase. A number of other potential cognates
associated with bacteria have been described in Chung, et al., J.
Bacteriology 183: 1012-1021, 2001, which is incorporated herein by
reference.
[0062] Mitochondrial Cognates
[0063] One or more tubular nanostructures may include one or more
ligands that bind one or more cognates associated with an
organelle, e.g., mitochondria within a tumor cell and/or other
targeted cell. Examples of cognates associated with the
mitochondrial outer membrane include, but are not limited to,
camitine palmitoyl transferase 2, translocase of outer membrane
(TOM70), sorting/assembly machinery, ANT, voltage dependent anion
channel (VDAC/Porin), and monoamine oxidase. In some instances, one
or more tubular nanostructures as described herein may include one
or more ligands that bind to one or more cognates on the inner
mitochondrial membrane. A cognate of the inner mitochondrial
membrane may be a membrane associated receptor or protein, e.g.,
one or more proteins associated with the carnitine acyltransferase
II transporter, NADH dehydrogenase complex (Complex I), succinate
dehydrogenase (Complex II), cytochrome bcl complex (Complex III),
cytochrome c oxidase complex (Complex IV), ATP synthase, or
uncoupling protein (UCP).
Functionalization of Tubular Nanotubes with Various Ligands that
Bind to Cognates
[0064] A tubular nanostructure as described herein may include one
or more ligands that bind one or more cognates on a target cell or
organelle. A ligand that binds a cognate may include, but is not
limited to, at least a portion of an antibody, antibody-coated
liposome, polynucleotide, polypeptide, receptor, viral plasmid,
polymer, protein, carbohydrate, lipid, pore-forming toxin, lectin,
or any combination thereof. As such, the tubular nanostructure
containing one or more ligands may be selectively directed towards
target cells expressing the corresponding one or more cognates. In
one aspect, a protein cognate may bind to a compound having a lipid
or carbohydrate moiety, e.g., a saccharide, a glycoprotein or a
lipoprotein/proteolipid. The one or more ligands may be attached to
the side-walls of the tubular nanostructure. Alternatively, one or
more ligands may be attached to either and/or both ends of the
tubular nanostructure. Increased tissue or cell specificity may be
garnered by multi functionalization of the tubular nanostructure
with two or more ligands directed towards two or more distinct
cognates on the target tissue. In the instance where the end
targets are mitochondria in specific cells, the tubular
nanostructure may be multifunctionalized, e.g., with a first ligand
directed to a first cognate on the cell membrane of the target cell
and with a second ligand directed to a second cognate on the
membrane of the mitochondria.
[0065] Antibody Ligands
[0066] In some instances, tubular nanostructures may be modified
with one or more ligands that are antibodies. Antibodies or
fragments thereof for use in functionalizing a tubular
nanostructure may include, but are not limited to, monoclonal
antibodies, polyclonal antibodies, Fab fragments of monoclonal
antibodies, Fab fragments of polyclonal antibodies, Fab.sub.2
fragments of monoclonal antibodies, and Fab.sub.2 fragments of
polyclonal antibodies, among others. Single chain or multiple chain
antigen-recognition sites can be used. Multiple chain
antigen-recognition sites can be fused or unfused. Antibodies or
fragments thereof may be generated using standard methods as
described by Harlow & Lane (Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press; 1.sup.st edition 1988), which
is incorporated herein by reference). In another embodiment, the
functional group is an antigen-binding moiety, e.g., a moiety
comprising the antigen-recognition site of an antibody.
Alternatively, an antibody or fragment thereof directed against a
cognate may be generated using phage display technology (see, e.g.,
Kupper, et al. BMC Biotechnology 5:4, 2005, which is incorporated
herein by reference). A single chain antibody, for example, may
also incorporate streptavidin as part of a fusion protein to
facilitate attachment of the antibody to the tubular nanostructure
via a biotin-streptavidin linkage, for example (see, e.g., Koo, et
al. Appl. Environ. Microbiol. 64:2497-2502, 1998). An antibody or
fragment thereof could also be prepared using in silico design
(Knappik et al., J. Mol. Biol. 296: 57-86, 2000, which is
incorporated herein by reference). In addition or instead of an
antibody, the assay may employ another type of recognition element,
such as a receptor or ligand binding molecule. Such a recognition
element may be a synthetic element like an artificial antibody or
other mimetic. U.S. Pat. Nos. 6,255,461; 5,804,563; 6,797,522;
6,670,427; and 5,831,012; and U.S. Patent Application 20040018508;
and Ye and Haupt, Anal Bioanal Chem. 378: 1887-1897, 2004; Peppas
and Huang, Pharm Res. 19: 578-587 2002, provide examples of such
synthetic elements and are incorporated herein by reference. In
some instances, antibodies, recognition elements, or synthetic
molecules that recognize a cognate may be available from a
commercial source, e.g., Affibody.RTM. affinity ligands (Abeam,
Inc. Cambridge, Mass. 02139-1517; U.S. Pat. No. 5,831,012,
incorporated here in by reference).
[0067] Polypeptide Ligands
[0068] In some instances, tubular nanostructures may be modified
with one or more ligands that are cellular receptors that recognize
and/or bind to bacteria. For example, CD14, which is normally
associated with monocyte/macrophages is known to bind
lipopolysaccharide associated with gram negative bacteria as well
as lipoteichoic acid associated with the gram positive bacteria
Bacillus subtilis (see, e.g., Fan, et al. (1999) Infect. Immun. 67:
2964-2968). Other examples of cellular receptors include, but are
not limited to, adenylate cyclase (Bordatella pertussis), Gal alpha
1-4Gal-containing isoreceptors (E. coli), glycoconjugate receptors
(enteric bacteria), Lewis(b) blood group antigen receptor
(Heliobacter pylori), CR3 receptor, protein kinase receptor,
galactose N-acetylgalactosamine-inhibitable lectin receptor, and
chemokine receptor (Legionella), annexin I (Leishmania mexicana),
ActA protein (Listeria monocytogenes), meningococcal virulence
associated Opa receptors (Meningococcus), alpha5beta3 integrin
(Mycobacterium avium-M), heparin sulphate proteoglycan receptor,
CD66 receptor, integrin receptor, membrane cofactor protein, CD46,
GM1, GM2, GM3, and CD3 (Neisseria gonorrhoeae), KDEL receptor
(Pseudomonas), epidermal growth factor receptor (Samonella
typhiurium), alpha5beta1 integrin (Shigella), and nonglycosylated
3774 receptor (Streptococci) (see, e.g., U.S. Patent Application
2004/0033584 A1). In some instances the pathogen specific
receptor/ligand may be bound to the surface of the modified red
blood cell through an antibody linkage (see, e.g., U.S. Patent
Application 2006/0018912 A1, each incorporated herein by
reference).
[0069] In some instances, tubular nanostructures may be modified
with one or more ligands that are peptide hormones which interact
with specific cognates, for example, cell surface receptors on
target cells. Examples of peptide hormones that may be used to
modify tubular nanostructures include, but are not limited to,
neuropeptides, for example, enkephalins, neuropeptide Y,
somatostatin, corticotropin-releasing hormone,
gonadotropin-releasing hormone, adrenocorticotropic hormone,
melanocyte-stimulating hormones, bradykinins, tachykinins,
cholecystokinin, vasoactive intestinal peptide (VIP), substance P,
neurotensin, vasopressin, and calcitonin; cytokines, for example,
interleukins (e.g., IL-1 through IL-35), erythropoietin,
thrombopoietin, interferon (IFN), granulocyte monocyte
colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF),
and others; chemokines, e.g., RANTES, TARC, MIP-1, MCP, and others;
growth factors, for example, platelet derived growth factor (PDGF),
transforming growth factor beta (TGF.beta.), nerve growth factor
(NGF), epidermal growth factor (EGF), insulin-like growth factor
(IGF), basic fibroblast growth factor (bFGF); other peptide
hormones, for example, atrial natriuretic factor, insulin,
glucagon, angiotensin, prolactin, oxyocin, and others. In one
aspect, Mattson, et al., describe functionalizing carbon nanotubes
with nerve growth factor (see U.S. Pat. No. 6,670,179, which is
incorporated herein by reference). Similarly, Liu, et al., describe
functionalizing carbon nanotubes with cyclic
arginine-glycine-aspartic acid (RGD) peptide, the latter of which
is a ligand for integrin alpha.sub.v-beta.sub.3receptors
up-regulated in a wide range of solid tumors (Liu, et. al., ACS
Nano 1:50-56, 2007, which is incorporated herein by reference).
Alternatively, novel peptides that bind selective target, for
example, tumor cells may be generated using phage display
methodologies (see, e.g., Spear, et al., Cancer Gene Ther.
8:506-511, 2001, which is incorporated herein by reference).
[0070] Small Chemical Compound Ligands
[0071] In some aspects, the tubular nanostructure may be configured
to include one or more small chemical compound ligands. As such, a
tubular nanostructure may be modified with a small chemical
compound ligand that interacts with a cognate on a target cell,
such as a receptor. Examples of small chemical compound ligands
include, but are not limited to, acetylcholine, adenosine
triphosphate (ATP), adenosine, androgens, dopamine,
endocannabinoids, epinephrine, folic acid, gamma-aminobutyric acid
(GABA), glucocorticoids, glutamate, histamine, leukotrienes,
mineralocorticoids, norepinephrine, prostaglandins, serotonin,
thromoxanes, or vitamins. For example, the modification of carbon
nanotubes with folic acid provides the modified nanotubes which can
bind to folate receptors overexpressed on some tumor cells (see Kam
et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005, which is
incorporated herein by reference).
[0072] Aptamer Ligands
[0073] In some instances, tubular nanostructures may be modified
with one or more ligands that are aptamers. Aptamers are artificial
oligonucleotides (DNA or RNA) that can bind to a wide variety of
entities (e.g., metal ions, small organic molecules, proteins, and
cells) with high selectivity, specificity, and affinity. Aptamers
may be isolated from a large library of 10.sup.14 to 10.sup.15
random oligonucleotide sequences using an iterative in vitro
selection procedure often termed "systematic evolution of ligands
by exponential enrichment" (SELEX; see, e.g., Cao, et al., Current
Proteomics 2:31-40, 2005; Proske, et al., Appl. Microbiol.
Biotechnol. 69:367-374, 2005, which are incorporated herein by
reference). For example, an RNA aptamer May be generated against
leukemia cells using a cell based SELEX method (see, e.g.,
Shangguan, et al., Proc. Natl. Acad. Sci. USA 103:11838-11843,
2006, which is incorporated herein by reference). Similarly, an
aptamer that recognizes bacteria may be generated using the SELEX
method against whole bacteria (see, e.g., Chen, et al., Biochem.
Biophys. Res. Commun. 357:743-748, 2007, which is incorporated
herein by reference).
[0074] Lectin Ligands
[0075] In some embodiments, tubular nanostructures may be modified
with one or more ligands that are lectins. The term "lectin" was
originally used to define agglutinins which could discriminate
among types of red blood cells and cause agglutination. Currently,
the term "lectin" is used more generally and includes sugar-binding
proteins from many sources regardless of their ability to
agglutinate cells. Lectins have been found in plants, viruses,
microorganisms and animals. Because of the specificity that each
lectin has toward a particular carbohydrate structure, even
oligosaccharides with identical sugar compositions can be
distinguished or separated. Some lectins will bind only to
structures with mannose or glucose residues, while others may
recognize only galactose residues. Some lectins require that the
particular sugar is in a terminal non-reducing position in the
oligosaccharide, while others can bind to sugars within the
oligosaccharide chain. Some lectins do not discriminate between a
and b anomers, while others require not only the correct anomeric
structure but a specific sequence of sugars for binding. Examples
of lectins include, but are not limited to, algal lectins, e.g.,
b-prism lectin; animal lectins, e.g., tachylectin-2, C-type
lectins, C-type lectin-like proteins, calnexin-calreticulin, capsid
protein, chitin-binding protein, ficolins, fucolectin, H-type
lectins, 1-type lectins, sialoadhesin, siglec-5, siglec-7,
micronemal protein, P-type lectins, pentrxin, b-trefoil, galectins,
congerins, selenocosmia huwena lectin-I, Hcgp-39, Ym1; bacterial
lectins, e.g., Pseudomonas PA-IL, Burkholderia lectins,
chromobacterium CV-IIL, Pseudomonas PA IIL, Ralsonia RS-ILL,
ADP-ribosylating toxin, Ralstonia lectin, Clostridium
hemagglutinin, botulinum toxin, tetanus toxin, cyanobacterial
lectins, FimH, GafD, PapG, Staphylococcal enterotoxin B, toxin
SSL11, toxin SSL5; fungal and yeast lectins, e.g., Aleuria aurantia
lectin, integrin-like lectin, Agaricus lectin, Sclerotium lectin,
Xerocomus lectin, Laetiporus lectin, Marasmius oreades agglutinin,
agrocybe galectin, coprinus galectin-2, Ig-like lectins, L-type
lectins; plant lectins, e.g., alpha-D-mannose-specific plant
lectins, amaranthus antimicrobial peptide, hevein, pokeweed lectin,
Urtica dioica UD, wheat germ agglutinins (WGA-1, WGA-2, WGA-3),
artocarpin, artocarpus hirsute AHL, banana lectin, Calsepa,
heltuba, jacalin, Maclura pomifera MPA, MornigaM, Parkia lectins,
abrin-a, abrus agglutinin, amaranthin, castor bean ricin B, ebulin,
mistletoe lectin, TKL-1, cyanovirin-N homolog, and various legume
lectins; and viral lectins, e.g., capsid protein, coat protein,
fiber knob, hemagglutinin, and tailspike protein (see, e.g., E.
Bettler, R. Loris, A. Imberty "3D-Lectin database: A web site for
images and structural information on lectins" 3rd Electronic
Glycoscience Conference, The interne and World Wide Web, 6-17 Oct.
1997; http://www.cermay.cnrs.fr/lectines/
[0076] Pore Forming Ligands
[0077] In some aspects, tubular nanostructures may be modified with
one or more ligands that are pore-forming toxins. Examples of
pore-forming toxins include, but are not limited to,
beta-pore-forming toxins, e.g., hemolysin, Panton-Valentine
leukocidin S, aerolysin, Clostridial epsilon-toxin; binary toxins,
e.g., anthrax, C. perfringens Iota toxin, C. difficile cytolethal
toxins; cholesterol-dependent cytolysins; pneumolysin; small
pore-forming toxins; and gramicidin A
[0078] In some aspects, tubular nanostructures may be modified with
one or more ligands that are pore-forming antimicrobial peptides.
Antimicrobial peptides represent an abundant and diverse group of
molecules that are naturally produced by many tissues and cell
types in a variety of invertebrate, plant and animal species. The
amino acid composition, amphipathicity, cationic charge and size of
antimicrobial peptides allow them to attach to and insert into
microbial membrane bilayers to form pores leading to cellular
disruption and death. More than 800 different antimicrobial
peptides have been identified or predicted from nucleic acid
sequences, a subset of which have are available in a public
database (see, e.g., Wang & Wang Nucleic Acids Res.
32:D590-D592, 2004); http://aps.uninc.edu/AP/main.php, which is
incorporated herein by reference). More specific examples of
antimicrobial peptides include, but are not limited to, anionic
peptides, e.g., maximin H5 from amphibians, small anionic peptides
rich in glutamic and aspartic acids from sheep, cattle and humans,
and dermcidin from humans; linear cationic alpha-helical peptides,
e.g., cecropins (A), andropin, moricin, ceratotoxin, and melittin
from insects, cecropin P1 from Ascaris nematodes, magainin (2),
dermaseptin, bombinin, brevinin-1, esculentins and buforin II from
amphibians, pleurocidin from skin mucous secretions of the winter
flounder, seminalplasmin, BMAP, SMAP (SMAP29, ovispirin), PMAP from
cattle, sheep and pigs, CAP18 from rabbits and LL37 from humans;
cationic peptides enriched for specific amino acids, e.g.,
praline-containing peptides including abaecin from honeybees,
praline- and arginine-containing peptides including apidaecins from
honeybees, drosocin from Drosophila, pyrrhocoricin from European
sap-sucking bug, bactenicins from cattle (Bac7), sheep and goats
and PR-39 from pigs, praline- and phenylalanine-containing peptides
including prophenin from pigs, glycine-containing peptides
including hymenoptaecin from honeybees, glycine- and
praline-containing peptides including coleoptericin and holotricin
from beetles, tryptophan-containing peptides including indolicidin
from cattle, and small histidine-rich salivary polypeptides,
including histatins from humans and higher primates; anionic and
cationic peptides that contain cysteine and from disulfide bonds,
e.g., peptides with one disulphide bond including brevinins,
peptides with two disulfide bonds including alpha-defensins from
humans (HNP-1, HNP-2, cryptidins), rabbits (NP-1) and rats,
beta-defensins from humans (HBD1, DEFB118), cattle, mice, rats,
pigs, goats and poultry, and rhesus theta-defensin (RTD-1) from
rhesus monkey, insect defensins (defensin A); and anionic and
cationic peptide fragments of larger proteins, e.g., lactoferricin
from lactoferrin, casocidin 1 from human casein, and antimicrobial
domains from bovine alpha-lactalbumin, human hemoglobin, lysozyme,
and ovalbumin (see, e.g., Brogden, Nat. Rev. Microbiol. 3:238-250,
2005, which is incorporated herein by reference).
Ligands as Therapeutic Agents
[0079] In some instances, the tubular nanostructure as described
herein may be configured to include one or more ligands that is a
therapeutic agent. As such, the one or more therapeutic agent may
contribute to disruption and/or death of the targeted cell in
addition to the disruptive pore-forming capability of the tubular
nanostructure. Examples of therapeutic agents that might be
incorporated into the tubular nanostructure to aide in disrupting
and/or killing cancer cells or microbes include anti-cancer
therapeutic agents and/or antimicrobial therapeutic agents.
[0080] Antimicrobial therapeutic agents may include, but are not
limited to, antibacterial, antifungal and antiparasital agents.
[0081] Anti-Cancer Therapeutic Agents
[0082] In one aspect, the therapeutic agent is an anti-cancer drug.
The anti-cancer drug may be selected from a variety of known small
chemical compound pharmaceuticals. Alternatively, the chemotherapy
agent may include, but is not limited to, an inactivating peptide
nuclei acid (PNA), an RNA or DNA oligonucleotide aptamer, short
double-stranded RNA (e.g., interfering RNA, microRNA), a peptide,
or a protein. Examples of chemotherapy agents include, but are not
limited to, antimetabolites such as capecitabine, cladribine,
cytarabine, fludarabine, 5-fluorouracil, gemcitabine,
6-mercaptopurine, methotrexate, pemetrexed, and 6-thioguanine;
antitumor antibiotics such as bleomycin, epipodophyllotoxins such
as etoposide and teniposide; taxanes such as docetaxel and
paclitaxel; vinca alkaloids such as vinblasine, vinfristine, and
vinorelbine; alkylating agents such as busulfan, carmustine,
cyclophosphamide, dacarbazine, ifosfamide, lomustine,
mechlorethamine, melphalan, temozolomide, and thiotepa;
anthracyclines such as daunorubucin, doxorubicin, epirubicin,
idarubicin, and mitoxantrope; antitumor antibiotics such as
dactinomycin and mitomycin; camptothecins such as irinotecan and
topotecan; and platinum analogs such as carboplatin, cisplatin, and
oxaliplatin; hormonally active agents such as flutamide,
bicalutamide, nilutamide, tamoxifen, megestrol acetate,
hydrocortisone, prednisone, goserelin acetate, leuprolide,
aminoglutethimide, anastrozole, exemestane, and letrozole; and
miscellaneous drugs used for cancer chemotherapy such as arsenic
trioxide, erlotinib, gefitinib, imatinib, bortezomib, hydroxyurea,
mitoxantrone, retinoic acid derivatives, estramustine, leucovorin
and the photosensitizer Photofrin.
[0083] The anti-cancer drug may be a biological agent, e.g., a
peptide, a protein, an enzyme, a receptor and/or an antibody.
Examples of biological agents currently used to treat cancer
include, but are not limited to, cytokines such as
interferon-.alpha., interferon-.gamma., and interleukin-2, an
enzyme such as asparaginase, and monoclonal antibodies such as
alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, and
trastuzumab.
[0084] Novel biological agents for the treatment of cancer may be
generated by screening a peptide phage library, for example, in
proliferation assays against cancerous cells, e.g., cultured
transformed cells lines and/or against primary tumors from patients
with various cancers (see, e.g., Spear, et al. Cancer Gene Therapy
8:506-511, 2001; Krag, et al. Cancer Res. 66:7724-7733, 2006, which
are incorporated herein by reference).
[0085] Antimicrobial Therapeutic Agents
[0086] In another aspect, the therapeutic agent is an antibacterial
drug. Examples of antibacterial drugs include, but are not limited
to, beta-lactam compounds such as penicillin, methicillin,
nafcillin, oxacillin, cloxacillin, dicloxacilin, ampicillin,
ticarcillin, amoxicillin, carbenicillin, and piperacillin;
cephalosporins and cephamycins such as cefadroxil, cefazolin,
cephalexin, cephalothin, cephapirin, cephradine, cefaclor,
cefamandole, cefonicid, cefuroxime, cefprozil, loracarbef,
ceforanide, cefoxitin, cefmetazole, cefotetan, cefoperazone,
cefotaxime, ceftazidine, ceftizoxine, ceftriaxone, cefixime,
cefpodoxime, proxetil, cefdinir, cefditoren, pivoxil, ceftibuten,
moxalactam, and cefepime; other beta-lactam drugs such as
aztreonam, clavulanic acid, sulbactam, tazobactam, ertapenem,
imipenem, and meropenem; other cell wall membrane active agents
such as vancomycin, teicoplanin, daptomycin, fosfomycin,
bacitracin, and cycloserine; tetracyclines such as tetracycline,
chlortetracycline, oxytetracycline, demeclocycline, methacycline,
doxycycline, minocycline, and tigecycline; macrolides such as
erythromycin, clarithromycin, azithromycin, and telithromycin;
aminoglycosides such as streptomycin, neomycin, kanamycin,
amikacin, gentamicin, tobramycin, sisomicin, and netilmicin;
sulfonamides such as sulfacytine, sulfisoxazole, silfamethizole,
sulfadiazine, sulfamethoxazole, sulfapyridine, and sulfadoxine;
fluoroquinolones such as ciprofloxacin, gatifloxacin, gemifloxacin,
levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, and
ofloxacin; antimycobacteria drugs such as isoniazid, rifampin,
rifabutin, rifapentine, pyrazinamide, ethambutol, ethionamide,
capreomycin, clofazimine, and dapsone; and miscellaneous
antimicrobials such as colistimethate sodium, methenamine
hippurate, methenamine mandelate, metronidazole, mupirocin,
nitrofurantoin, polymyxin B, clindamycin, choramphenicol,
quinupristin-dalfopristin, linezolid, spectrinomycin, trimethoprim,
pyrimethamine, and trimethoprim-sulfamethoxazole.
[0087] In another aspect, the therapeutic agent is an antifungal
agent. Examples of antifungal agents include, but are not limited
to, anidulafungin, amphotericin B, butaconazole, butenafine,
caspofungin, clotrimazole, econazole, fluconazole, flucytosine
griseofulvin, itraconazole, ketoconazole, miconazole, micafungin,
naftifine, natamycin, nystatin, oxiconazole, sulconazole,
terbinafine, terconazole, tioconazole, tolnaftate, and/or
voriconazole.
[0088] In another aspect, the therapeutic agent is an anti-parasite
agent. Examples of anti-parasite agents include, but are not
limited to, antimalaria drugs such as chloroquine, amodiaquine,
quinine, quinidine, mefloquine, primaquine,
sulfadoxine-pyrimethamine, atovaquone-proguanil,
chlorproguanil-dapsone, proguanil, doxycycline, halofantrine,
lumefantrine, and artemisinins; treatments for amebiasis such as
metronidazole, iodoquinol, paromomycin, diloxanide furoate,
pentamidine, sodium stibogluconate, emetine, and dehydroemetine;
and other anti-parasite agents such as pentamidine, nitazoxanide,
suramin, melarsoprol, eflornithine, nifurtimox, clindamycin,
albendazole, and timidazole.
[0089] In some instances, the antimicrobial agent may be an
antimicrobial peptide. A number of naturally occurring
antimicrobial peptides have been described herein and amino acid
sequence information for a subset of these may be found as part of
a public database (see, e.g., Wang & Wang Nucleic Acids Res.
32:D590-D592, 2004); http://aps.unmc.edu/AP/main.php, which is
incorporated herein by reference). Alternatively, a phage library
of random peptides may be used to screen for peptides with
antimicrobial properties against live bacteria, fungi and/or
parasites. The DNA sequence corresponding to an antimicrobial
peptide may be generated ex vivo using standard recombinant DNA and
protein purification techniques and subsequently attached to
tubular nanostructures using the methods described herein.
Markers on Tubular Nanostructures
[0090] In some instances, the tubular nanostructure as described
herein may be configured to include one or more marker. The one or
more marker may include, e.g., a fluorescent marker, a radioactive
marker, a quantum dot, a contrast agent for magnetic resonance
imaging (MRI) marker, or combinations thereof. One or more markers
may be used to facilitate imaging of the tubular nanostructure in
association with target cells or organelles.
[0091] Fluorescent Markers
[0092] In one aspect, the tubular nanostructure may include one or
more markers capable of fluorescence in response to appropriate
wavelengths of electromagnetic energy. The one or more fluorescent
marker associated with the tubular nanostructure may include one or
more of the fluorescent compounds currently approved by the United
States Food and Drug Administration (FDA) for use in human mammals
including, but not limited to, fluorescein (FITC), indocyanine
green, and rhodamine B. FITC, for example, may be readily added to
a carbon nanotube functionalized with PL-PEG-NH.sub.2 as described
in Kam, et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005,
which is incorporated herein by reference. Alternatively, the one
or more fluorescent marker associated with the tubular
nanostructure may include one or more of a number of other
fluorescent compounds including, but not limited to, cyanine dyes
such as Cy5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway,
N.J., USA) and/or a variety of Alexa Fluor dyes including Alexa
Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa
Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 (Molecular
Probes-Invitrogen, Carlsbad, Calif., USA; see, e.g., U.S. Pat. App.
No. 2005/0171434 A1). Additional fluorophores include IRD41 and
IRD700 (LI-COR, Lincoln, Nebr., USA), NIR-1 and 105-OSu (Dejindo,
Kumamotot, Japan), LaJolla Blue (Diatron, Miami, Fla., USA),
FAR-Blue, FAR-Green One, and FAR-Green Two (Innosense, Giacosa,
Italy), ADS 790-NS and ADS 821-NS (American Dye Source, Montreal,
Calif.) and VivoTag 680 (VT680; VisEn Medical, Woburn, Mass., USA).
Many of these fluorophores are available from commercial sources
either attached to primary or secondary antibodies or as
amine-reactive succinimidyl or monosuccinimidyl esters, for
example, ready for conjugation to appropriately functionalized
tubular nanostructures using the methods described herein.
Alternatively, the fluorophore may be added to a small
single-stranded DNA and the fluorophore/DNA conjugate attached to
the tubular nanostructure via non-covalent interaction between the
DNA and nanotube (see, e.g., Kam, et al., Proc. Natl. Acad. Sci.
USA 102:11600-11605, 2005, which is incorporated herein by
reference).
[0093] In one aspect, the tubular nanostructure may include one or
more markers that are quantum dots (Q-dots). Q-dots are nanocrystal
semiconductors with unique optical properties, fluorescing at
various excitation wavelengths depending upon composition and size.
A variety of Q-dots are available from a number of commercial
sources and may be added to tubular nanostructures through, e.g.,
amines, carboxyl groups, biotin, streptavidin, secondary
antibodies, and phopholipid-PEG (from, e.g., Evident Technologies,
Troy, N.Y.; Invitrogen, Carlsbad, Calif.). For example, Chen et
al., describe adding Q-dots conjugated to streptavidin to nanotubes
modified with biotin through pyrene bound to the nanotube side-wall
via .pi.-.pi. stacking (see Chen et al., Proc. Natl. Acad. Sci. USA
104:8218-8222, 2007, which is incorporated herein by reference.
Similarly, Didenko and Baskin describe using an enzymatic process
with horseradish peroxidase to add streptavidin conjugated Q-dots
to nanotubes (BioTechniques 40:295-302, 2006, which is incorporated
herein by reference).
[0094] In a further embodiment, the tubular nanostructures
themselves may be inherently fluorescent at specific wavelengths of
electromagnetic energy. For example, single-walled carbon nanotubes
have been shown to exhibit photoluminescence in the near infrared
when excited by a diode laser at 785 nm (see, e.g., Welsher, et
al., Nano Lett 8: 586-590, 2008, which is incorporated herein by
reference).
[0095] Fluorescence associated with tubular nanostructures may be
monitored using invasive and non-invasive methods. Invasive methods
are exemplified by insertion of an endoscope or a catheter
containing optical fibers for fluorescence excitation and
measurement into body cavities or vessels (see, e.g., U.S. Pat.
Nos. 7,341,557; 6,389,307, which are incorporated herein by
reference). Non-invasive methods are exemplified by fluorescence
mediated molecular tomography. For example, non-invasive monitoring
of near infrared (NIR) fluorescence may be performed using
fluorescence mediated molecular tomography as described in U.S.
Pat. No. 6,615,063, which is incorporated herein by reference.
Additional information regarding NIR imaging in human subjects is
described in Frangioni Curr. Op. Chem. Biol. 7:626-634, 2003, which
is incorporated herein by reference. In some instances, a wireless
system may be used in which light sources such as light emitting
diodes (LEDs) of appropriate wavelength as well as detectors such
as charge-coupled devices (CCDs) are housed along with a power
supply and a wireless communication circuit to create a device that
may be placed on the skin of a subject to monitor NIR signal as
described by Muehlemann, et al., Optics Express, 16:10323, 2008,
which is incorporated herein by reference.
[0096] Radioactive Markers
[0097] In another embodiment, the tubular nanostructure may include
one or more markers that are radioactive. Tubular nanostructures
modified with one or more radioisotopes may be monitored using a
gamma camera, positron emission tomography (PET), other gamma ray
probe. Examples of radioactive molecular that might be used for
this purpose include, but are not limited to, carbon-11,
nitrogen-13, oxygen-15, fluorine-18, rubidium-82, yttrium-86,
technetium-99, iodine-123, indium-111, thallium-201. For example,
indium-111 may be added to carbon nanotubes using bifunctional
metal chelating agents such as
2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraa-
cetic acid (DOTA-NCS) or diethylentriaminepentaacetic (DTPA) (see,
e.g., McDevitt, et al., J. Nucl. Med. 48:1180-1189, 2007; Singh, et
al., Proc. Natl. Acad. Sci. USA 103:3357-3362, 2006, which are
incorporated herein by reference). Similar methods are described
for adding yttrium-86 to carbon nanotubes (McDevitt et al., PLoS
ONE 2:e907, 2007, which is incorporated herein by reference).
[0098] Contrast Agent Markers
[0099] In another aspect, the tubular nanostructures may include
one or more markers that are contrast agents used in magnetic
resonance imaging (MRI). For example, tubular nanostructures, e.g.,
carbon nanotubes may be combined with the high-spin paramagnetic
gadolinium (Gd.sup.3+) metal ions to form an effective contrast
agent for MRI (see, e.g., Sitharaman & Wilson Int. J. Nanomed.
1:291-295, 2006, which is incorporated herein by reference).
Alternatively, tubular nanostructures may be functionalized with a
combination of iron and cobalt salts to form MRI and near infrared
imaging agents (see, e.g., Seo, et al., Nat. Mater. 5:971-976,
2006, which is incorporated herein by reference). Other divalent
metal ions that might be included in tubular nanostructures for MRI
detection include, but are not limited to, cobalt, nickel, zinc,
magnesium, and manganese (see, e.g., U.S. Patent Application
2008/0124281, which is incorporated herein by reference).
Alternatively, bacterial derived magnetic nanocrystals may be
absorbed onto the tubular nanostructure as described in U.S. Patent
Application 2007/0200085, which is incorporated herein by
reference.
Activated Markers on Tubular Nanostructures
[0100] The tubular nanostructure as described herein may include
one or more markers that may be activated. One or more markers
associated with the tubular nanostructure may be activated by a
ligand reaction, anchoring in the membrane and interaction with a
hydrophobic medium, and/or change in the cellular environment
(e.g., changes in pH). One or more marker associated with the
tubular nanostructure may be activated upon reaching the intended
target. Alternatively, one or more marker associated with the
tubular nanostructure may be activated upon disruption and/or death
of the target cell. Alternatively, one or more marker associated
with the tubular nanostructure may be activated upon passage of the
tubular nanostructure from one cellular compartment to another.
[0101] Ligand Reaction Activated Markers
[0102] The one or more activatable marker associated with the
tubular nanostructure may be activated by a ligand reaction. The
marker may be activated when the marker or a component associated
with the marker binds to, comes in close contact with, or otherwise
interacts with a ligand associated with the target cell or
organelle. The marker may include a donor and an acceptor molecule
that undergo fluorescence resonance energy transfer (FRET) in
response to interaction of the marker with the ligand. FRET is a
distance-dependent interaction between the electronic excited
states of two dye molecules in which excitation is transferred from
a donor molecule to an acceptor molecule without emission of a
photon. In some instances, interaction of a donor molecule with an
acceptor molecule may lead to a shift in the emission wavelength
associated with excitation of the acceptor molecule. In other
instances, interaction of a donor molecule with an acceptor
molecule may lead to quenching of the donor emission.
[0103] The donor and acceptor molecules of the marker may be
conjugated to the same biomolecule such that changes in the
conformation of the biomolecule in response to ligand interaction
move the donor and acceptor molecules relative to one another.
Examples of biomolecules that might be used in this manner include,
but are not limited to, polynucleotides, e.g., aptamers or
polypeptides, e.g., antibodies. In this instance, the aptamers or
antibodies associated with the marker may be the same aptamer or
antibody used to bind the tubular nanostructure to cognates on a
target cell or organelle. Alternatively, the aptamers or antibodies
associated with the marker may be distinct, interacting with
different components on the target cell or organelle. Other
biomolecules that change conformation in response to binding a
ligand may be used for this purpose.
[0104] Alternatively, the donor and acceptor molecules may be
conjugated to separate biomolecules such that changes in proximity
of the separate biomolecules moves the donor and acceptor molecules
relative to one another. In this instance, the target cell or
organelle may be modified with either a donor or acceptor molecule
while the tubular nanostructure may be modified with the
corresponding donor or acceptor molecule. In either instance, the
interaction of the tubular nanostructure with the target cell or
organelle triggers a measurable response.
[0105] Tubular nanostructures may be modified with one or more
activatable marker, for example, an aptamer-based molecular beacon.
Molecular beacons are dual labeled aptamer probes with a donor
fluorophore at one end and an acceptor fluorophore or quencher at
the other end. Upon binding of a specific target, the aptamer
undergoes a conformational shift such that the distance between the
donor fluorophore and the acceptor fluorophore or quencher is
altered, leading to a change in measurable fluorescence through the
phenomenon of FRET, as discussed above (see, e.g. Cao, et al.,
Current Proteomics, 2:31-40, 2005, which is incorporated herein by
reference). In some instances, the fluorescence associated with
aptamer may be quenched until the tubular nanostructure reaches its
intended target. Alternatively, the fluorescence associated with
the aptamer may be shifted in wavelength when the tubular
nanostructure reaches its intended target.
[0106] Tubular nanostructures may be modified with one or more
activatable marker that is an antibody-based molecular beacon. In
this instance, the antibody may be labeled with a donor or acceptor
molecule and a secondary protein associated with the antibody such
as Protein A, Protein G, or a F.sub.ab fragment, for example, may
be labeled with the corresponding donor or acceptor molecule (see,
e.g., Lichlyter, et al., Biosens. Bioelectron. 19:219-226, 2003,
which is incorporated herein by reference). Interaction of the
labeled antibody/secondary protein complex with the appropriate
ligand shifts the antibody and the secondary protein relative to
one another and induces a FRET signal. Alternatively, the one or
more marker may be an antibody labeled near the antigen-binding
site with a donor or acceptor molecule and a flexible arm attached
to an analog of the antigen recognized by the antibody which itself
includes the corresponding donor or acceptor molecule (see, e.g.
U.S. Patent Application 2006/0172318 A1). Competition for the
antigen-binding site by the analog and the actual ligand on the
target cell or organelle results in measurable changes in the
spatial relationship between the donor and acceptor molecules. In
some instances, the tubular nanostructures may be modified with a
marker that is an antibody that is labeled with a solvent sensitive
fluorophore, e.g., dansyl chloride
(5-dimethylaminonaphthalene-1-sulfonyl chloride), and exhibits a
shift in fluorescent signal in response to interaction with a
ligand associated with the target cell or organelle antigen (see,
e.g., Brennan J. Fluor. 9:295-312, 1999, which is incorporated
herein by reference). An antibody of this type may be labeled such
that interaction of the ligand with the antibody shields the
solvent sensitive fluorescent in the active binding site from the
solvent water, in a measurable change fluorescence intensity (see,
e.g., Bright, et al. Anal. Chem. 62:1065-1069, 1990, which is
incorporated herein by reference).
[0107] The donor and acceptor fluorophore pairs associated with the
marker may include, but are not limited to, fluorescein and
tetramethylrhodamine; IAEDANS and fluorescein; fluorescein and
fluorescein; and BODIPY FL and BODIPY FL. Alternatively, the marker
may include any of a number of Alexa Fluor (AF) fluorophores (from,
e.g., Invitrogen, Carlsbad, Calif.) paired with other AF
fluorophores for use in FRET. Some examples include AF 350 with AF
488; AF 488 with AF 546, AF 555, AF 568, or AF 647; AF 546 with AF
568, AF 594, or AF 647; AF 555 with AF594 or AF647; AF 568 with
AF6456; and AF594 with AF 647.
[0108] Alternatively, the donor and acceptor fluorophore pairs
associated with the marker may include cyanine dyes. The cyanine
dyes Cy3, Cy5, Cy5.5 and Cy7, which emit in the red and far red
wavelength range (>550 nm), offer a number of advantages for
FRET-based detection systems. Their emission range is such that
background fluorescence is often reduced and relatively large
distances (>100 .ANG.) can be measured as a result of the high
extinction coefficients and good quantum yields. For example, Cy3,
which emits maximally at 570 nm and Cy5, which emits at 670 nm, may
be used as a donor-acceptor pair. When the Cy3 and Cy5 are not
proximal to one another, excitation at 540 nm results only in the
emission of light by Cy3 at 590 nm. In contrast, when Cy3 and Cy5
are brought into proximity by a conformation change in an aptamer,
for example, excitation at 540 nm results in an emission at 680
nm.
[0109] Alternatively, the donor or acceptor molecular of the marker
may include one or more semiconductor quantum dots (Q-dots) paired
with an appropriate organic dye donor or acceptor molecule as
described by Bawendi, et al., in U.S. Pat. No. 6,306,610, which is
incorporated herein by reference.
[0110] In some instances, the donor molecule of the marker may be a
quenching dye that quenches the fluorescence of visible
light-excited fluorophores when in close proximity. Examples
include DABCYL, the non-fluorescing diarylrhodamine derivative dyes
QSY 7, QSY 9 and QSY 21 (from, e.g., Invitrogen, Carlsbad, Calif.),
the non-fluorescing Black Hole Quenchers BHQ0, BHQ1, BHQ2, and BHQ3
(from, e.g., Biosearch Technologies, Inc., Novato, Calif., USA) and
Eclipse (from, e.g., Applera Corp., Norwalk, Conn., USA). A variety
of donor fluorophore and quencher pairs may be considered for FRET
including but not limited to fluorescein with DABCYL; EDANS with
DABCYL; or fluorescein with QSY 7 and QSY 9. In general, QSY 7 and
QSY 9 dyes efficiently quench the fluorescence emission of donor
dyes including blue-fluorescent coumarins, green- or
orange-fluorescent dyes, and conjugates of the Texas Red and Alexa
Fluor 594 dyes. QSY 21 dye efficiently quenches all red-fluorescent
dyes. A number of the Alexa Fluor (AF) fluorophores (from, e.g.,
Invitrogen, Carlsbad, Calif.) may be paired with quenching
molecules as follows: AF 350 with QSY 35 or DABCYL; AF 488 with QSY
35, DABCYL, QSY7 or QSY9; AF 546 with QSY 35, DABCYL, QSY7 or QSY9;
AF 555 with QSY7 or QSY9; AF 568 with QSY7, QSY9 or QSY21; AF 594
with QSY21; and AF 647 with QSY 21.
[0111] In some instances, the tubular nanostructure itself may act
as a quencher. Carbon nanotubes, for example, can act collectively
as quenchers of covalently tethered and/or .pi. stacked pyrenes and
chromophores. This phenomenon is attributed to electron transfer or
energy transfer from the photoactive compound to the carbon
nanotubes if sufficiently close in proximity. As such, fluorescence
emitted by chromophores bound to carbon nanotubes may be quenched
by the association. For example, lysophospholipid
1,2-dipalmitoyl-sn-glycero-3-lysophosphoethanolamine-N-(Liss-amine
rhodamine B sulfonyl), or Rd-LPE may be added to carbon nanotubes
as described by Lin et al. (Appl. Phys. Lett. 89:143118, 2006,
which is incorporated herein by reference). In this instance,
Rd-LPE solubilizes carbon nanotubes in aqueous solution via pure
hydrophobic interactions and these self-assembled supramolecular
complexes, once excited, readily undergo fluorescence energy
transfer from the Rd-LPE to the carbon nanotubes, quenching the
rhodamine associated fluorescence. This energy transfer may be used
to detect membrane translocation of modified carbon nanotubes and
dissociation of Rd-LPE in cells, for example. During translocation
through the plasma membrane, the lipid-rhodamine moiety may be
transferred off the carbon nanotubes and as such the quenching is
removed and the rhodamine associated fluorescence is detected.
Alternatively, the lipid rhodamine moiety is stripped from the
carbon nanotube during entry into the cell, quenching is removed
and rhodamine associated fluorescence is detected.
[0112] Lipid Membrane Reactive Markers
[0113] The one or more activatable marker associated with the
tubular nanostructure may be activated by a lipid versus aqueous
environment. As such, incorporation of the tubular nanostructure
modified with an activatable marker that is lipid sensitive into
the lipid bilayer of a target tissue or organelle may result in a
measurable response. For example, the marker may be a fluorescent
dye such as one of several aminonaphthlethenyl-pyridinium (ANEP)
dyes which are essentially non-fluorescent in an aqueous
environment but fluoresce within a lipid environment. Examples of
lipid sensitive fluorescent ANEP dyes include, but are not limited
to, di-4ANEPPS and di-8-ANEPPS. When bound to phospholipid
vesicles, di-8-ANEPPS has excitation/emission maxima of
.about.467/631 nm. The fluorescence excitation/emission maxima of
di-4-ANEPPS bound to neuronal membranes, for example, are
.about.475/617 nm.
[0114] Alternatively, the marker may be a derivative of
nitrobenzoxadiazole (NBD) which is almost non-fluorescent in
aqueous solvents. The NBD fluorophore is moderately polar and both
its homologous 6-carbon and 12-carbon fatty acid analogs and the
phospholipids derived from these probes may be used to sense the
lipid.about.water interface region of membranes.
[0115] The marker may be fluorescent phospholipid analog .beta.-DPH
HPC which comprises diphenylhexatriene propionic acid coupled to
glycerophosphocholine at the sn-2 position. DPH and its derivatives
exhibit strong fluorescence enhancement when incorporated into
membranes, as well as sensitive fluorescence polarization
(anisotropy) responses to lipid ordering. .beta.-DPH HPC may be
used to specifically label one leaflet of a lipid bilayer, thus
facilitating analysis of membrane asymmetry.
[0116] A number of phospholipid analogs with pyrene-labeled sn-2
acyl chains, e.g.,
4-hydroxy-N,N,N-trimethyl-10-oxo-7-((1-oxo-10-(1-pyrenyl)decyl)oxy)-hydro-
xide are also non-fluorescent in aqueous solution but become
fluorescent in a lipid environment. Various pyrenedecanoyl-labeled
glycerophospholipids may be used for this purpose including but not
limited to those with phosphocholine, phosphoglycerol, and
phosphomethanol head groups.
[0117] Alternatively, the marker may be a derivative of the
polyunsaturated fatty acid cis-parinaric acid which offers several
experimentally advantageous optical properties, including a very
large fluorescence Stokes shift (.about.100 nm) and an almost
complete lack of fluorescence in water.
[0118] Cell Environment Reactive Markers
[0119] The one or more activatable marker associated with the
tubular nanostructure may be activated in response to the cellular
environment. For example, the marker may be activated by changes pH
and/or by enzymatic reactions associated with lipid bilayer and/or
components of the cytoplasm.
[0120] The tubular nanostructures may include a marker that is
sensitive to pH changes in the cellular environment. For example,
the marker may be a pH sensitive fluorescent dye such as LysoSensor
Yellow/Blue DND-160 (Invitrogen, Carlsbad, Calif.) which undergoes
a pH dependent emission and excitation shift to longer wavelengths
in acidic environments. Examples of pH sensitive dyes include, but
are not limited to, other LysoSensor probes, e.g., LysoSensor Blue
DND-167 and LysoSensor Green DND-189 which are almost
nonfluorescent except when inside acidic compartments; and
fluorescein containing dyes such as dichlorofluorescein,
carboxydichlorofluorescein, carboxydifluorofluorescein, and BCECF;
and Oregon Green 514 carboxylic acid, Oregon Green 488 carboxylic
acid, 5-(and 6-)carboxy-2',7'-, 9-amino-6-chloro-2-methoxyacridine
(ACMA) (e.g., from Invitrogen, Carlsbad, Calif.).
[0121] The tubular nanostructures may include a marker that is
activated by a chemical process. For example, the marker may be a
bis-BODIPY FL C.sub.11-PC which has BODIPY FL dye-labeled sn-1 and
sn-2 acyl groups, resulting in partially quenched fluorescence that
increases when one of the acyl groups is hydrolyzed by
phospholipase A.sub.1 or A.sub.2. The phospholipase may be
associated with either the membrane or the cytoplasm. The
hydrolysis products are BODIPY FL undecanoic acid and BODIPY FL
dye-labeled lysophosphatidylcholine. Other examples include markers
that are linked to the tubular nanostructures through a cleavable
disulfide bond, ester linkage, or ortho carboxy phenol derived
acetal linkage (see, e.g., U.S. Pat. Nos. 7,087,770 and 7,348,453,
which are incorporated herein by reference). For example, Q-dots
linked to carbon nanotubes by disulfide bond may be cleaved from
the nanotubes upon entry into the cell (see, e.g., Chen, et al.,
Proc. Natl. Acad. Sci. USA 104:8218-8222, 2007, which is
incorporated herein by reference). As such, donor and acceptor
molecules associated with the marker may be separated from one
another by breaking a cleavable bond, resulting in a measurable
signal.
Assemblies of Tubular Nanostructures
[0122] The one or more tubular nanostructures as described herein
may be individual, discrete nanotubes. Alternatively, tubular
nanostructures may form higher order assemblies or compositive
tubular nanostructures. A composite tubular nanostructure may
comprise two or more tubular nanostructures each including a
hydrophobic surface region, each hydrophobic region flanked by two
hydrophilic surface regions configured to form a pore in a lipid
bilayer membrane. Composite tubular nanostructures may be used to
create multiple pores at one or more sites in the targeted lipid
bilayer.
[0123] In general, carbon nanotubes, for example, have a tendency
to form large, insoluble aggregates due to substantial van der
Waals interactions. As such, solubilization techniques may be used
to break up these aggregates into smaller bundles and/or individual
nanotubes. The nanotubes may be solubilized by acid oxidation, by
surfactants, by polymer wrapping and/or by chemical
functionalization, for example. Solubilization in acid or
surfactant or other solubilizing agent such as polyoxometalates,
for example, may be carried out in the presence of sonication and
may be monitored using scanning and/or transmission electron
microscopy (see, e.g., Fei, et al., Nanotechnol. 17:1589-1593,
2006, which is incorporated herein by reference). Alternatively,
Raman spectroscopy may be used to monitor disaggregation of carbon
nanotubes. For example, Raman signals at 266 cm.sup.-1 correspond
to aggregated nanotube bundles whereas a broad photoluminescence
peak observed at approximately 3,200 cm.sup.-1 (1,050 nm)
corresponds to individual tubes (see, e.g., Kam, et al., Proc.
Natl. Acad. Sci. USA 102:11600-11605, 2005, which is incorporated
herein by reference). There Is evidence to suggest that electron
and ion irradiation of nanotubes give rise to covalent bonds
between tubes in bundles (see, e.g., Sammalkorpi, et al., Nucl.
Instr. Methods Phys. Res. B 228:142-145, 2005; Szabados, et al.,
Phys. Rev. 73:195404, 2006, which are incorporated herein by
reference).
[0124] In a further aspect, bundles of two or more tubular
nanostructures may be formed by modification of the nanotube
sidewall that confers attraction between individual nanotubes. For
example, bundles of two or more tubular nanostructures may be
formed by combining an appropriate ratio of nanotubes modified with
biotin and nanotubes modified with streptavidin. Other biomolecule
binding interactions that might be used to construct composite
tubular nanostructures include, but are not limited to,
protein-protein interactions, antibody-antigen interactions,
sense-antisense DNA or RNA interactions, aptamer-target
interaction, peptide-nucleic acid (PNA)-DNA or RNA interactions.
Biomolecules for use in forming higher ordered bundles of tubular
nanostructures may be added to the nanotubes using one or more of
the methods described herein. Optionally, asymmetric sidewall
functionalization in which one surface or portion of a surface is
masked during the functionalization process may be used to
selectively place biomolecules on the surface of tubular
nanostructures as described herein. As such, the compatible
surfaces are expected to come together to form composite tubular
nanostructures.
[0125] Two or more tubular nanostructures may be bundled together
through the interaction of biomolecules associated with the
nanotubes that normally oligomerize into higher order complexes.
Tubular nanostructures may be modified with a protein or proteins
that naturally form a triplex, for example, and as such would bring
together three associated nanotubes. An example is the ATP
responsive, cation-selective ion channels P2X1, P2X2, and P2X3
which have been shown by various means including atomic force
microscopy to form trimeric structures (see, e.g., Barrera, et al.,
J. Biol. Chem. 280:10759-10765, 2005, which is incorporated herein
by reference). Alternatively, tubular nanostructures may be
modified with a protein or proteins that naturally form a heptamer
and as such would bring together seven associated nanotubes. An
example is the pore-forming toxin hemolysin which forms a
heptameric beta-barrel structure in biological membranes.
Assembly of Tubular Nanostructures Enabling Active Transport,
Facilitated Transport, or Passive Transport
[0126] Tubular nanostructures as described herein may be further
modified to control flow of biomolecules through the pores formed
by the nanotubes in the lipid bilayer. For example, tubular
nanostructures may be modified with one or more proteins or
peptides that facilitate active and or passive transport across the
pore. Active transport requires an external energy source, e.g.,
the hydrolysis of ATP to transport biomolecules such as ions
against a concentration gradient, the biomolecules moving, for
example, from low to high concentration. In contrast, passive
transport is driven by the concentration gradient of the
biomolecule across an open pore, the biomolecules moving from high
to low concentration to establish equilibrium. Facilitated
transport is a form of passive transport in which materials are
moved across the plasma membrane by a transport protein down their
concentration gradient; hence, it does not require energy.
Biomolecules that are involved in active transport, facilitated
transport, or passive transport of molecules across the lipid
bilayer may be incorporated into the tubular nanostructures.
[0127] Tubular nanostructures may be include one or more components
of an ATP-binding cassette transporters (ABC transporters). ABC
transporters are composed of transmembrane domains connected to one
or more ligand binding domains on either the intracellular or
extracellular side of the lipid bilayer and one or more ATP binding
domains on the intracellular surface. ATP transporters may be
classified as half or full transporters. Full transporters may
contain two transmembrane domains and two ATP binding domains and
are fully functional. Half transporters contain one transdomain and
one ATP binding domain and must combine with another half
transporter to be fully functional. As such, a tubular
nanostructure may include all or part of a full transporter
sufficient to confer functionality. Alternatively, a tubular
nanostructure may include half of a full transporter or all of part
of a half transporter which upon interacting with one or more
similarly modified tubular nanostructure generates a functional ABC
transporter.
[0128] One or more tubular nanostructures may include all or part
of an ABC transporter, for example, the cystic fibrosis
transmembrane conductance regulator (CFTR), the transporter
associated with antigen processing (TAP), or the multidrug
resistance efflux pump (MDR). There are seven distinct gene
families of ABC transporters found in humans including, but not
limited to, ABCA, ABCB, ABCD, ABCE, ABCF, and ABCG, with each
family consisting of 1 to 12 members. Examples of ABC transporter
genes found in prokaryotes include, but are not limited to,
transporters such as Carbohydrate Uptake Transporter-1 (CUT1),
Carbohydrate Uptake Transporter-2 (CUT2), Polar Amino Acid Uptake
Transporter (PAAT), Peptide/Opine/Nickel Uptake Transporter (PepT),
Hydrophobic Amino Acid Uptake Transporter (HAAT), Sulfate/Tungstate
Uptake Transporter (SulT), Phosphate Uptake Transporter (PhoT),
Molybdate Uptake Transporter (MolT), Phosphonate Uptake Transporter
(PhnT), Ferric Iron Uptake Transporter (FeT),
Polyamine/Opine/Phosphonate Uptake Transporter (POPT), Quaternary
Amine Uptake Transporter (QAT), Vitamin B12 Uptake Transporter
(B12T), Iron Chelate Uptake Transporter (FeCT), Manganese/Zinc/Iron
Chelate Uptake Transporter (MZT), Nitrate/Nitrite/Cyanate Uptake
Transporter (NitT), Taurine Uptake Transporter (TauT), Cobalt
Uptake Transporter (CoT), Thiamin Uptake Transporter (ThiT).
Brachyspira Iron Transporter (BIT), Siderophore-Fe3+ Uptake
Transporter (SLUT), Nickel Uptake Transporter (NiT), Nickel/Cobalt
Uptake Transporter (NiCoT), and Methionine Uptake Transporter
(MUT); and exporters such as Lipid Exporter (LipidE), Capsular
Polysaccharide Exporter (CPSE), Lipooligosaccharide Exporter
(LOSE), Lipopolysaccharide Exporter (LPSE), Teichoic Acid Exporter
(TAE), Drug Exporter-1 (DrugE1), Lipid Exporter (LipidE), Putative
Heme Exporter (HemeE), .beta.-Glucan Exporter (GlucanE), Protein-1
Exporter (Prot1E), Protein-2 Exporter (Prot2E), Peptide-1 Exporter
(Pep1E), Peptide-2 Exporter (Pep2E), Peptide-3 Exporter (Pep3E),
Probable Glycolipid Exporter (DevE), Na.sup.+ Exporter (NatE),
Microcin B17 Exporter (McbE), Drug Exporter-2 (DrugE2), Microcin
J25 Exporter (McjD), Drug/Siderophore Exporter-3 (DrugE3),
(Putative) Drug Resistance ATPase-1 (Drug RA 1), (Putative) Drug
Resistance ATPase-2 (Drug RA2), Macrolide Exporter (MacB),
Peptide-4 Exporter (Pep4E),3-component Peptide-5 Exporter (Pep5E),
Lipoprotein Translocase (LPT), .beta.-Exotoxin I Exporter (PETE),
AmfS Peptide Exporter (AmfS-E), SkfA Peptide Exporter (SkfA-E), and
CydDC Cysteine Exporter (CydDC-E).
[0129] Alternatively, the tubular nanostructures may include one or
more components of an ion channel. Ion channels are integral
membrane proteins that regulate the flow of ions across the cell
membrane and often include a circular arrangement of identical or
homologous proteins closely packed around a water-filled pore
through the plane of the lipid bilayer. The pore-forming subunit(s)
are called the .alpha. subunit, while the auxiliary subunits are
denoted .beta., .gamma., and so on. In some ion channels, passage
through the pore is governed by a "gate," which may be opened or
closed by chemical or electrical signals, temperature, or
mechanical force, depending on the variety of channel. Examples of
ion channels that might be incorporated into one or more tubular
nanostructures include, but are not limited to, voltage-gated
sodium, calcium and potassium channels, voltage gated proton
channels, transient receptor potential channels (TRP), cyclic
nucleotide-gated channels, light gated channels, inward-rectifier
potassium channels, calcium-activated potassium channels, and
ligand gated channels, e.g., ionotropic glutamate-gated receptors,
ATP-gated P2X receptors, and anion-permeable gamma-aminobutyric
acid-gated GABA receptors.
[0130] In some instances, the tubular nanostructures may include
one or more components that alone or in combination form a
synthetic ion channel. Compounds that might be used to form
synthetic ion channels include, but are not limited to, crown
ethers, octiphenyl derivatives, octa- and decapeptides, and
bolaamphiphiles (two-headed amphiphiles; see, e.g., Fyles Chem.
Soc. Rev. 36: 335-347, 2007, which is incorporated herein by
reference).
[0131] In some instances, opening or closing of the pore associated
with the tubular nanostructure may be controlled by a component of
the tubular nanostructure that reversibly covers and or uncovers
the one or more pore openings. For example, the tubular
nanostructures may include one or more components at one or both
pore openings that change in conformation in response stimuli such
as, for example, pH, temperature, electric field, light, and or
ligand binding. Conformational changes in proteins, for example, in
response to stimuli may modulate activity of the protein and or
play a role in signal transduction. An example is the glutamate
receptor family of glutamate binding proteins in which the
glutamate binding domain is in a clam-shell like hinge region which
opens in the absence of glutamate and closes in the presense of
glutamate (see, e.g., Dinglehine, et al., Pharmacol. Rev. 51:7-62,
1999, which is incorporated herein by reference). Similarly, DNA
and RNA biomolecules such as aptamers, for example, may be designed
to change in conformation in response to ligand binding (see, e.g.,
Ha, et al., PNAS 96:9077-9082, 1999, which is incorporated herein
by reference). As such, the tubular nanostructure may be modified
with a biomolecule such as a protein or an aptamer at one or both
pore openings that is able to open and close in response to ligand
binding and as such can control the flow of other biomolecules
through the pore.
[0132] Alternatively, the tubular nanostructures may include one or
more components at one or both pore openings that is responsive to
light or electromagnetic energy. Electromagnetic energy may include
gamma rays, x-rays, ultraviolet, visible, infrared, microwave and
or radio waves. In this instance, the one or more component may
contain one or more cleavage sites, for example, that are activated
by electromagnetic energy and results in removal of portion of the
component that may be covering the pore opening. For example, Rock,
et al., describe a number of dithiane adduct derivatives that may
be used with proteins as photolabile linkers (U.S. Pat. No.
5,767,288, which is incorporated herein by reference).
Alternatively, the energy activated component may change
conformation in response to electromagnetic energy and as such
cover or uncover the pore opening.
[0133] In a further aspect, the tubular nanotubes may include
components that are magnetic and allow binding of one or both ends
of the pore to a magnet bead that physically blocks the pore
opening. For example, one or both ends of the tubular nanostructure
may be modified with molecules having magnetic properties. Examples
of molecules having magnetic properties include but are not limited
to the common magnetic metals iron, nickel, and cobalt and their
alloys as well as the rare earth metals and alloys or combinations
thereof such as for example gadolinium, samarium, and europium.
Tubular nanostructures such as carbon nanotubes, for example, may
be functionalized with iron and or gadolinium, for example, using
methods described in Seo, et al., (Nat. Mater. 5:971-976, 2006) and
Sitharaman & Wilson (Int. J. Nanomed. 1:291-295, 2006),
respectively, which are incorporated herein by reference. The
magnetized tubular nanotubes may be administered to a subject to
form pores in targeted lipid membranes, and magnetic beads
administered at a subsequent time point to block the pore opening.
Alternatively, the magnetized tubular structures may be combined
with magnetic beads prior to administration, and an external
magnetic source, for example, may be used to separate the beads
from the nanotubes.
[0134] In some instances, the pore associated with the tubular
nanostructure may be covered by a nanoparticle such as for example
a bead which has been modified with an aptamer or antibody, for
example, that binds to a corresponding ligand at one or both ends
of the tubular nanostructure. Alternatively, the nanoparticle may
include streptavidin or biotin which binds to biotin or
streptavidin, respectively, at the end of the tubular
nanostructure.
[0135] The tubular nanostructures may be further modified to allow
for controlled release of an agent such as, for example, a
therapeutic agent and or toxin in proximity to the pore opening.
For example, the tubular nanotubes may include a binding moiety
such as an aptamer or antibody situated at one or both ends of the
tubular nanostructure to which is reversibly bound an agent. The
affinity of the antibody for the agent is such that the agent
dissociates from the antibody and because of its proximity to the
pore, has a higher probability of passing through the pore.
Alternatively, the tubular nanostructure may include a ligand that
is recognized by a bifunctional binding moiety such as, for
example, a bifunctional antibody. In this instance, the
bifunctional antibody has a component that binds to a ligand on the
tubular nanostructure as well as a component that reversibly binds
to an agent such as, for example, a therapeutic agent and or toxin.
In this instance, the bifunctional antibody carrying an agent may
be administered to the subject at a point in time following
administration of the tubular nanostructures. As such, the tubular
nanostructure embedded into the lipid bilayer, binds the
bifunctional antibody, and over time, the agent is released from
the bifunctional antibody and passes through the lipid bilayer by
way of the proximal tubular nanostructure pore.
Tubular Nanostructure Directed to Specific Organelles
[0136] In some instances, the tubular nanostructures as described
herein may be modified in such a manner as to allow transit of the
nanotubes through the plasma membrane with subsequent targeting and
insertion into the lipid bilayer of one or more internal
organelles. Once targeted to the lipid bilayer of the organelle
membrane, the tubular nanostructure may form pores that enable
active transport, facilitated transport, or passive transport of
contents into or out of the organelle. In certain organelles,
disruption of the lipid bilayer may lead to cell death. In one
example, the membrane target is the outer membrane of mitochondria.
In general, mitochondrial outer membrane permeabilization is
considered the "point of no return" during apoptosis of cells as it
results in the diffusion to the cytosol of numerous proteins that
normally reside in the space between the outer and inner
mitochondrial membranes and initiates a cascade of events leading
to cell death (see, e.g., Chipuk, et al., Cell Death Differ.
13:1396-1402, 2006, which is incorporated herein by reference). As
such, tubular nanostructures may be selectively directed to the
outer membrane of mitochondria in target cells where they insert
into and disrupt the outer mitochondrial membrane leading to target
cell death.
[0137] The tubular nanostructures with hydrophobic surface region
flanked by two hydrophilic surface regions for insertion and
retention in a lipid bilayer may be modified in such a manner as to
mask the hydrophilic ends and allow transit through the plasma
membrane. In one embodiment, the hydrophilic ends of the tubular
nanostructure are modified with a hydrophobic moiety through a
chemical bond that may be cleaved once the nanotube has passed into
the cell. Examples of biologically cleavable bonds include, but are
not limited to, disulfide bonds, diols, diazo bonds, ester bonds,
sulfone bonds, acetals, ketals, enol ethers, enol esters, enamines
and imines (see, e.g., U.S. Pat. Nos. 7,087,770, 7,098,030 and
7,348,453, which are incorporated herein by reference).
[0138] Alternatively, the cleavable bond may be a photolabile bond.
Examples of hydrophobic moieties that might be added to the ends of
the tubular nanostructure include, but are not limited to,
non-polar hydrocarbon chains of various lengths. In one aspect, the
hydrophobic moiety is an ester that can be cleaved by an
intracellular esterase to form a hydrophilic acid moiety and
alcohol moiety. For example, hydrophilic moieties may be masked by
acetoxymethyl esters of phosphates, sulfates, or other compounds
having alcohol moieties or acid moieties which will enhance
permeability of the tubular nanostructure across the lipid bilayer
membrane. Because acetoxymethyl esters are rapidly cleaved
intracellularly, they facilitate the delivery of tubular
nanostructures into the cytoplasm of the cell without puncturing or
disruption of the cell plasma membrane (see, e.g., Schultz et al.,
J. Biol. Chem. 268: 6316-6322, 1993, which are incorporated herein
by reference). Once within the cytoplasm, the tubular
nanostructures having a hydrophobic surface region flanked by two
hydrophilic surface regions is configured to form a pore in the
lipid bilayer membrane of the cellular organelle.
[0139] Alternatively, the tubular nanostructure may be tethered to
a protein transduction domain (PTD) such as human immunodeficiency
virus type 1 (HIV-1) transactivator of transcription (Tat),
Drosophila Antennapedia (Antp), or herpes simplex virus VP22 that
masks the hydrophilic ends and facilitates entry of the nanotubes
into the cell. In one aspect, all or part of the 86 amino acid long
Tat protein may be added to tubular nanostructures through primary
amines associated with the peptide and/or the functionalized
nanotubes using the methods described herein (also see, e.g.,
Santra, et al., Chem. Commun. 24:2810-2811, 2004, which is
incorporated herein by reference). The Tat protein or other protein
transduction domain may be linked to the tubular nanostructure to
the hydrophilic regions on either end of the nanotube through a
cleavable bond such as those described herein and as such removed
from the tubular nanostructure once the latter has entered the
cell, unmasking the hydrophilic regions.
[0140] Under certain conditions, the masked tubular nanostructures
may be actively taken up by the cell through the process of
endocytosis (see, e.g., Kam, et al., Angew. Chem. Int. Ed. 44:1-6,
2005, which is incorporated herein by reference). Endocytosis is
the process whereby cells absorb extracellular material by
engulfing the material with their cell membrane. The engulfed
material is contained in small vesicles that pinch off from the
plasma membrane, enter the cytoplasm and fuse with other
intracellular vesicles, e.g., endosomes or lysosomes.
[0141] Material such as tubular nanostructures may be released from
endosomes by a number of mechanisms. In one aspect, artificial
acceleration of endosomal release may be achieved by
photo-excitation of fluorescent probes associated with the engulfed
material (see, e.g., Matsushita, et al., FEBS Lett. 572:221-226,
2004, which is incorporated herein by reference). Alternatively,
the tubular nanostructure may include a pH sensitive element that
is activated in the low pH environment of the endosome. In a
further aspect, all or part of the influenza virus hemagglutinin-2
subunit (HA-2), a pH-dependent fusogenic peptide that induces lysis
of membranes at low pH, may be used to induce efficient release of
encapsulated material from cellular endosomes (see, e.g.,
Yoshikawa, et al., J. Mol. Biol. 380:777-782, 2008, which is
incorporated herein by reference).
[0142] Alternatively, the masked tubular nanostructures may enter
the cell by passing directly through the cell membrane and into the
cytoplasm. In this instance, the tubular nanostructure may include
moieties on the surface of the nanotubes that confers direct
passage through the lipid bilayer, e.g., an amphiphilic striated
surface on the nanotube. The deposition of a
hydrophilic-hydrophobic striated pattern of molecules, e.g., the
anionic ligand 11-mercapto-1-undecanesulphonate (MUS) and the
hydrophobic ligand 1-octanethiol (OT) on the surface of nanotubes
may facilitate direct passage of the tubular nanostructures into
the cytoplasm (see, e.g., Verma, et al., Nature Materials 7:
588-95, 2008, which is incorporated herein by reference). Once the
masked tubular nanostructures has entered the cytoplasm, it can be
modified to reveal tubular nanostructures with hydrophobic surface
region flanked by two hydrophilic surface regions and at least one
ligand bound to the nanostructure and configured to bind one or
more cognates on an organellar membrane, e.g., a mitochondrial
membrane.
[0143] The one or more tubular nanostructures may include one or
more ligands that binds to one or more cognate on a cellular
organelle, e.g., mitochondria, as well as one or more ligand that
binds to one or more cognates on the cell surface membrane of the
target cell. The one or more ligands may be an antibody,
antibody-coated liposome, polynucleotide, polypeptide, receptor,
viral plasmid, polymer, protein, carbohydrate, lipid, toxin,
lectin, or any combination thereof as described herein. Cognates
associated with a mitochondrial membrane may include at least one
of a protein, a carbohydrate, a glycoprotein, a glycolipid, a
sphingolipid, a glycerolipid, or metabolites thereof. Examples of
cognates associated with the mitochondrial outer membrane, for
example, include, but are not limited to, carnitine palmitoyl
transferase 2, translocase of outer membrane (TOM70),
sorting/assembly machinery, ANT, voltage dependent anion channel
(VDAC/Porin), and monoamine oxidase. In some instances, one or more
tubular nanostructures may include one or more ligands that bind to
one or more cognates on the inner mitochondrial membrane. A cognate
of the inner mitochondrial membrane may be a membrane associated
receptor or protein, e.g., one or more proteins associated with the
carnitine acyltransferase II transporter, NADH dehydrogenase
complex (Complex I), succinate dehydrogenase (Complex II),
cytochrome bcl complex (Complex III), cytochrome c oxidase complex
(Complex IV), ATP synthase, or uncoupling protein (UCP).
Pharmaceutical Formulation of a Tubular Nanostructure and
Administration to a Subject
[0144] The compositions and methods described herein for inserting
a tubular nanostructure into a lipid bilayer membrane are useful
for treatment of a disease or condition, e.g., cancer or infectious
disease, in a mammalian subject in need thereof. A pharmaceutical
formulation including the tubular nanostructures or the composite
tubular nanostructures described herein may be formulated neat or
may be combined with one or more acceptable carriers, diluents,
excipients, and/or vehicles such as, for example, buffers,
surfactants, preservatives, solubilizing agents, isotonicity
agents, and stablilizing agents as appropriate. A "pharmaceutically
acceptable" carrier, for example, may be approved by a regulatory
agency of the state and/or Federal government such as, for example,
the United States Food and Drug Administration (US FDA) or listed
in the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. Conventional
formulation techniques generally known to practitioners are
described in Remington: The Science and Practice of Pharmacy,
20.sup.th Edition, Lippincott Williams & White, Baltimore, Md.
(2000), which is incorporated herein by reference.
[0145] Acceptable pharmaceutical carriers include, but are not
limited to, the following: sugars, such as lactose, glucose and
sucrose; starches, such as corn starch and potato starch;
cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose, cellulose acetate, and
hydroxymethylcellulose; polyvinylpyrrolidone; cyclodextrin and
amylose; powdered tragacanth; malt; gelatin, agar and pectin; talc;
oils, such as mineral oil, polyhydroxyethoxylated castor oil,
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; polysaccharides, such as alginic acid and
acacia; fatty acids and fatty acid derivatives, such as stearic
acid, magnesium and sodium stearate, fatty acid amines,
pentaerythritol fatty acid esters; and fatty acid monoglycerides
and diglycerides; glycols, such as propylene glycol; polyols, such
as glycerin, sorbitol, mannitol and polyethylene glycol; esters,
such as ethyl oleate and ethyl laurate; buffering agents, such as
magnesium hydroxide, aluminum hydroxide and sodium benzoate/benzoic
acid; water; isotonic saline; Ringer's solution; ethyl alcohol;
phosphate buffer solutions; other non-toxic compatible substances
employed in pharmaceutical compositions.
[0146] A pharmaceutical formulation including the tubular
nanostructures or the composite tubular nanostructures described
herein may be formulated in a pharmaceutically acceptable liquid
carrier. The liquid carrier or vehicle may be a solvent or liquid
dispersion medium comprising, for example, water, saline solution,
ethanol, a polyol, vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The solubility of a chemical blocking
agent may be enhanced using solubility enhancers such as, for
example, water; diols, such as propylene glycol and glycerol;
mono-alcohols, such as ethanol, propanol, and higher alcohols; DMSO
(dimethylsulfoxide); dimethylformamide, N,N-dimethylacetamide;
2-pyrrolidone, N-(2-hydroxyethyl)pyrrolidone, N-methylpyrrolidone,
1-dodecylazacycloheptan-2-one and other
n-substituted-alkyl-azacycloalkyl-2-ones and other
n-substituted-alkyl-azacycloalkyl-2-ones (azones). The proper
fluidity may be maintained, for example, by the formation of
liposomes, by the maintenance of the necessary particle size in the
case of dispersions or by the use of surfactants. One or more
antimicrobial agent may be included in the formulation such as, for
example, parabens, chlorobutanol, phenol, sorbic acid, and/or
thimerosal to prevent microbial contamination. In some instances,
it may be preferable to include isotonic agents such as, for
example, sugars, buffers, sodium chloride or combinations
thereof.
[0147] A pharmaceutical formulation including the tubular
nanostructures or the composite tubular nanostructures described
herein may be formulated for transdermal delivery. For example,
water-insoluble, stratum corneum-lipid modifiers such as for
example 1,3-dioxanes, 1,3-dioxolanes and derivatives thereof, 5-,
6-, 7-, or 8-numbered lactams (e.g., butyrolactam, caprolactam),
morpholine, cycloalkylene carbonate have been described for use in
transdermal iontophoresis (see, e.g., U.S. Pat. No. 5,527,797,
which is incorporated herein by reference). Other suitable
penetration-enhancing agents include but are not limited to
ethanol, hexanol, cyclohexanol, polyethylene glycol monolaurate,
azacycloalkan-2-ones, linoleic acid, capric acid, lauric acid,
neodecanoic acid hexane, cyclohexane, isopropylbenzene; aldehydes
and ketones such as cyclohexanone, acetamide; N,N-di(lower
alkyl)acetamides such as N,N-diethylacetamide, N,N-dimethyl
acetamide; N-(2-hydroxyethyl)acetamide; esters such as N,N-di-lower
alkyl sulfoxides; essential oils such as propylene glycol,
glycerine, isopropyl myristate, and ethyl oleate; salicylates; and
mixtures of any of the above (see, e.g., U.S. Patent Publication
2008/0119449).
[0148] In some instances, the pharmaceutical formulation including
the tubular nanostructures or the composite tubular nanostructures
described herein may be formulated in a dispersed or dissolved form
in a hydrogel or polymer associated with, for example, implantable
or a transdermal delivery method. Examples of hydrogels and/or
polymers include but are not limited to gelled and/or cross-linked
water swellable polyolefins, polycarbonates, polyesters,
polyamides, polyethers, polyepoxides and polyurethanes such as, for
example, poly(acrylamide), poly(2-hydroxyethyl acrylate),
poly(2-hydroxypropyl acrylate), poly(N-vinyl-2-pyrrolidone),
poly(n-methylol acrylamide), poly(diacetone acrylamide),
poly(2-hydroxylethyl methacrylate), poly(allyl alcohol). Other
suitable polymers include but are not limited to cellulose ethers,
methyl cellulose ethers, cellulose and hydroxylated cellulose,
methyl cellulose and hydroxylated methyl cellulose, gums such as
guar, locust, karaya, xanthan gelatin, and derivatives thereof. For
iontophoresis, for example, the polymer or polymers may include an
ionizable group such as, for example, (alkyl, aryl or aralkyl)
carboxylic, phosphoric, glycolic or sulfonic acids, (alkyl, aryl or
aralkyl) quaternary ammonium salts and protonated amines and/or
other positively charged species as described in U.S. Pat. No.
5,558,633, which is incorporated herein by reference in its
entirety.
[0149] Information regarding formulation of FDA approved tubular
nanostructures or the composite tubular nanostructures may be found
in the package insert and labeling documentation associated with
each approved agent. A compendium of package inserts and FDA
approved labeling may be found in the Physician's Desk Reference.
Alternatively, formulation information for approved chemical
blocking agents may be found on the internet at websites such as,
for example, www.drugs.com and www.rxlist.com. For those tubular
nanostructures or composite tubular nanostructures described herein
which do not currently have a formulation appropriate for use in
any of the delivery methods described above, an appropriate
formulation may be determined empirically and/or experimentally
using standard practices. The pharmaceutical compositions are
generally formulated as sterile, substantially isotonic and in full
compliance with all Good Manufacturing Practice (GMP) regulations
of the U.S. Food and Drug Administration.
[0150] Pharmaceutical compositions including the tubular
nanostructures or the composite tubular nanostructures described
herein can be administered to an individual by any of a number of
routes including, but not limited to, oral, nasal, pulmonary,
rectal, transdermal, vaginal, or transmucosal routes as well as the
parenteral routes. Suitable parenteral delivery routes for the
pharmaceutical compositions include, but are not limited to,
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections. Examples of microbead and
nanoparticle approaches and materials that would be appropriate for
the delivery of pharmaceutical compositions including the tubular
nanostructures or the composite tubular nanostructures are
described in Nanomaterials for Medical Diagnosis and Therapy,
1.sup.st edition, edited by Challa Kumar (Nanotechnologies for the
Life Sciences Vol. 10, 2007, WILEY-VCH Verlag GmbH & Co. KGaA,
Wienham; Nanomaterials for Cancer Therapy, edited by Challa Kumar
(Nanotechnologies for the Life Sciences, Vol. 6, 2006, WILEY-VCH
Verlag GmbH & Co. KGaA, Wienham, which are incorporated herein
by reference).
[0151] The methods and compositions are further described with
reference to the following examples; however, it is to be
understood that the methods and compositions are not limited to
such examples.
EXEMPLARY ASPECTS
Example 1
Preparation of Tubular Nanostructures for Targeting Cancer
Cells
[0152] One or more tubular nanostructures may be used to
selectively target and kill tumor cells in a subject with cancer.
The one or more tubular nanostructures may be selectively directed
to the tumor cells through a ligand associated with the tubular
nanostructures that recognizes a corresponding cognate on the
membrane of the tumor cells. The one or more ligands may be at
least a portion of an antibody, antibody-coated liposome,
polynucleotide, polypeptide, receptor, viral plasmid, polymer,
protein, carbohydrate, lipid, toxins, pore-forming toxins, or
lectin. The one or more cognates on a membrane of a tumor cell may
be a least one of a protein, a carbohydrate, a glycoprotein, a
glycolipid, a sphingolipid, a glycerolipid, or a metabolite
thereof. For example, one or more carbon nanotubes may be modified
with a ligand that is an antibody or fragment thereof that
specifically binds a cognate that is a cell surface receptor on a
tumor cell. A tumor cell may be a breast cancer cell. An example of
a cell surface receptor on a breast cancer cell may be the
HER2/erb/neu receptor. An antibody to HER2 may be attached to
tubular nanostructures and used to direct interaction of the carbon
nanotube to the breast cancer cells. Once at the targeted tumor
cell, the one or more tubular nanostructures may form pores in the
plasma membrane through which intracellular and extracellular
components may flow. Disruption of the highly controlled barrier
function of the plasma membrane ultimately results in death of the
targeted tumor cell.
[0153] One or more tubular nanostructures for selective targeting
of tumor cells may be derived from one or more carbon nanotubes.
Carbon nanotubes may be generated using one of several methods
including, but not limited to, arc-discharge, laser ablation,
chemical vapor deposition (CVD), or the gas-phase catalytic process
(HiPCO). For example, carbon nanotubes may be generated using an
appropriate carbon source as described herein in the presence of
one or more Group VI and/or Group VIII transition metals, e.g.,
chromium, iron, cobalt, ruthenium, nickel and platinum using laser
vaporization with dual pulsed lasers as described in U.S. Pat. No.
7,008,604, which is incorporated herein by reference.
Alternatively, carbon nanotubes may be purchased from a commercial
source (from, e.g., Unidym, Menlo Park, Calif.; Sigma-Aldrich, St.
Louis, Mo.; Carbolex, Inc., Lexington, Ky.)
[0154] The carbon nanotubes may be used directly for
functionalization. Alternatively, the carbon nanotubes may be cut
to generate more uniform, open-ended nanotubes. Disrupting the
closed ends of the carbon nanotube will also facilitate
functionalization of the ends. Carbon nanotubes may be cut by any
of a number of different methods as described herein. For example,
carbon nanotubes may be cut using an ultra microtome (Wang et al.,
Nanotechnology 18: 055301, 2007, which is incorporated herein by
reference). In this instance, a magnetic field may be used to align
the nanotubes prior to cutting. Pristine nanotubes are dispersed in
water, stabilized in surfactant and passed under pressure through a
nylon filter in the bore of a resistive coil magnetic, e.g., with a
magnet field of 17.3 T. The nanotubes aligned on the filter are
dried under vacuum. The resulting film of aligned nanotubes is cut
and the strips stacked to form a rigid block of nanotubes. The
block of nanotubes may be cut with a cryo-diamond knife at a
temperature of approximately -60.degree. C. using an ultra
microtome, e.g., the Leica EM UC6 or EM FC6 microtome (from, e.g.,
Leica Microsystems, Bannockburn, Ill.).
[0155] The carbon nanotubes may be further treated by oxidation to
facilitate functionalization of the ends and side-walls of the
carbon nanotubes. As such, carbon nanotubes may be oxidized in the
presence of strong oxidizing agents, e.g., nitric acid,
KMnO.sub.4/H.sub.2SO.sub.4, O.sub.2,
K.sub.2Cr.sub.2O.sub.7/H.sub.2SO.sub.4 or OsO.sub.4, to clean the
nanotubes, cut the nanotubes, and/or prepare the nanotubes for
functionalization. Oxidation of carbon nanotubes in nitric acid at
a temperature of 120.degree. C., for example, may be used to
further clean the nanotubes by eliminating amorphous carbon and
other contaminants. Oxidation may be also be used to cut the carbon
nanotubes into shorter lengths and to open up the ends of the
nanotubes. In addition, oxidation creates defects in the carbon
nanotube sidewall which may be used to add moieties to the
otherwise inert sidewall. As such, oxidation may be used to prepare
the carbon nanotubes for functionalization. Following oxidation,
the carbon nanotubes may be treated with neutralizing agents and
further purified by size using electrophoresis, filtration or
chromatography.
[0156] The carbon nanotubes are inherently hydrophobic. To
facilitate improved insertion of the tubular nanostructure into the
plasma membrane of a tumor cell, the carbon nanotubes may be
functionalized at either or both ends with hydrophilic moieties.
Hydrophilic moieties might include one or more of amines, amides,
charged or polar amino acids, alcohols, carboxylic groups, oxides,
ester groups, ether groups, ester-ether groups, ketones, aldehydes,
or derivatives thereof. For example, carboxylic groups may be added
to a carbon nanotube by sonicating the carbon nanotubes in a 3:1
vol/vol solution of concentrated sulfuric acid (98%) and
concentrated nitric acid (70%) for 24 hours at 35-40.degree. C.,
and washed with water, leaving an open hole in the nanotube and
functionalizing the open end with one or more carboxyl group (see,
e.g., Li, et al., Proc. Natl. Acad. Sci. USA 103:19658-19663, 2006,
which is incorporated herein by reference).
[0157] The carbon nanotube may be further modified with a ligand
that is an antibody or fragment thereof that specifically binds a
cognate that is a cell surface receptor on a breast cancer. For
example, the carbon nanotube may be modified with an antibody that
specifically binds to the HER2/neu receptor on certain breast
cancer cells. An example of an antibody that binds HER2/neu
receptors on breast cancer cells is trastuzumab (Genentech, South
San Francisco, Calif.). An antibody such as trastuzumab may be
added to functionalized carbon nanotubes using one or more of the
methods described herein. Alternatively, any of a number of
commercially available antibodies to the HER2/neu receptor may be
used (from, e.g., Novus Biologicals, Littleton, Colo.; Affinity
BioReagents, Inc., Golden Colo.; Genway Biotech, Inc., San Diego,
Calif.). For example, a thiolated antibody may be conjugated to
carbon nanotubes functionalized with primary amines or phospholipid
(PL)-PEG-NH.sub.2 (see, e.g., McDevitt, et al., J. Nucl. Med.
48:1180-1189, 2007; Welsher, et al., Nano Lett. 8:586-590, 2008,
which are incorporated herein by reference). PL-PEG-NH.sub.2 (from,
e.g., Avanti Polar Lipids, Inc., Alabaster, Ala.) at a
concentration of 100-200 .mu.M is mixed with approximately 0.25
mg/ml carbon nanotubes previously functionalized with hydrophilic
ends in water and sonicated for 1 hour. The suspension is
centrifuged at 200,000.times.g for 1 hour and the resulting pellet
discarded. Excess PL-PEG-NH.sub.2 may be removed by filtration
through a filter, e.g., a filter with a 100 kDa molecular weight
cut off (from, e.g., Millipore, Billerica, Mass.). The
PL-PEG-NH.sub.2 modified carbon nanotubes may be conjugated to
thiolated antibody through a sulfo-SMCC linker. Thiolation may be
accomplished using 2-iminothiolane.HCl which reacts with primary
amines on the antibody to introduce sulfhydryl groups. The antibody
(10 mg/ml) is mixed with 10-fold molar excess of
2-iminothiolane.HCl (e.g., 46 .mu.l of a 14 mM stock solution of
2-iminothiolane to each milliliter of antibody solution) in
phosphate buffered saline in the presence of 20 mM EDTA for 2
hours. Unreacted 2-iminothiolane may be removed by filtration
through a 100 kDa filter. To finish conjugation, the
PL-PEG-NH.sub.2 modified carbon nanotubes (400 nM) are treated with
2 mM sulfo-SMCC (Pierce-Thermo Scientific, Rockford, Ill.) for 2
hours in phosphate buffered saline at pH 7.4 and excess sulfo-SMCC
removed by filtration as above. The sulfo-SMCC treated carbon
nanotubes are mixed with the thiolated antibody at a 1:10 molar
ratio and allowed to incubate overnight at 4.degree. C. to generate
the carbon nanotube-antibody conjugate.
[0158] The tubular nanostructure as described herein is further
modified with a ligand that is an antibody or fragment thereof,
e.g., trastuzumab antibody, that specifically binds a cognate that
is a HER2/neu cell surface receptor on certain breast cancer cells.
The tubular nanostructure is targeted to the breast cancer cells,
wherein the tubular nanostructure has a hydrophobic surface region
flanked by two hydrophilic surface regions and is configured to
form a pore in a lipid bilayer membrane of the breast cancer cell,
and thus causing cell death of the breast cancer cell.
Example 2
Tubular Nanostructure with Lectin
[0159] One or more tubular nanostructures modified with a lectin
may be used to selectively target and kill tumor cells in a subject
with cancer. The one or more tubular nanostructures may be
selectively directed to the tumor cells through a ligand, e.g., a
lectin, associated with the tubular nanostructures that recognizes
a corresponding cognate on the membrane of the tumor cells. In some
instances, the binding of the lectin to the cognate on the target
tumor cell may contribute to disruption and death of the targeted
cell. For example, the lectin may be one of several
galactose-binding plant lectins, e.g., Ricinus communis agglutinin
I (RCA.sub.1) or Bandeirae simplicifolia lectin I, which may bind
to abnormally high quantities of galactose moieties found on the
plasma membranes of some tumor cells, such as bladder carcinoma
cells, and thereby weakening the membrane of the tumor cells and
contributing to cell death (see, e.g., U.S. Pat. No. 4,496,539,
which is incorporated herein by reference).
[0160] Tubular nanostructures generated using the methods as
described herein may be modified with a lectin. For example,
RCA.sub.1, which is a 120,000 molecular weight protein, may be
purchased from commercial sources (e.g., from Sigma-Aldrich, St.
Louis, Mo.) and used to functionalize tubular nanostructures.
Alternatively, all or part of RCAI may be generated using standard
recombinant molecular biology techniques and corresponding cDNA
sequences reported in GenBank as part of the National Center for
Biotechnology Information (NCBI) (see, e.g., Benson, et al.,
Nucleic Acids Res. 36:D25-D30, 2008, which is incorporated herein
by reference). RCA.sub.1 may be conjugated to primary amines
associated with tubular nanostructures using the methods described
herein.
[0161] The tubular nanostructures may be further modified with one
or more ligand such as an antibody or an aptamer, for example, that
directs the nanotubes to the target tissue and enhances target
specificity. For example, one or more aptamers specific for one or
more cognates on a tumor cell may be generated using SELEX. In
general, a diverse library of random DNA oligonucleotide sequences
(40 to 55 nucleotides in length) may be amplified using the
polymerase chain reaction (PCR) in the presence of a 5' primer
labeled with a fluorescent tag and a 3' primer labeled with biotin.
After denaturing the DNA under alkaline conditions, the
fluorescently labeled sense single strand DNA (ssDNA) can be
separated from the biotinylated antisense ssDNA using streptavidin
coated Sepharose beads. Aptamers to live cells, for example, may be
isolated by incubating the fluorescently labeled ssDNA with live
cells and monitoring ssDNA binding by flow cytometry. Those ssDNA
sequences that bind to the cells may be subjected to another round
of PCR in the presence of labeled primers as described above. This
cycle may be repeated several times until aptamers of appropriate
binding affinity and selectivity are selected. Once the specific
aptamer sequence for a target has been identified, the
oligonucleotide sequence may be generated using standard
procedures.
Example 3
Tubular Nanostructure with Toxin
[0162] One or more tubular nanostructures modified with one or more
toxins may be used to selectively disrupt and kill target cells.
The one or more toxins may act as a ligand to direct specific
interaction with a cognate on a target cell. Alternatively, the one
or more tubular nanostructures may be further modified with a
ligand that specifically binds to a cognate on a target cell and
brings the associated one or more toxins into proximity with the
target cell.
[0163] The tubular nanostructures may include one or more toxins
that specifically target and kill bacteria. For example, the one or
more toxins may be one or more antimicrobial peptides.
Antimicrobial peptides represent an abundant and diverse group of
molecules that are naturally produced by many tissues and cell
types in a variety of invertebrate, plant and animal species. The
amino acid composition, amphipathicity, cationic charge and size of
antimicrobial peptides allow them to attach to and insert into
microbial membrane bilayers to form pores leading to cellular
disruption and death. Antimicrobial peptides are generated as part
of the host innate immune system and as such are capable of
selectively targeting bacterial cells. For example, magainin 2, an
antimicrobial peptide originally isolated from Xenopus laevis, may
first be attracted to the net negative charges on the surface of
bacteria associated with anionic phospholipids and the phosphate
groups of lipopolysaccharide (LPS) on Gram-negative bacteria and
teichoic acids on Gram-positive cells. Passing through the outer
portions of the bacteria, the magainin 2 reaches the cytoplasmic
membrane where it oligomerizes with other magainin 2 subunits to
form a toroidal pore resulting in the immediate loss of cytoplasmic
potassium and cell death (see, e.g., Brogden Nat. Rev. Microbiol.
3:238-250, 2005, which is incorporated herein by reference). As
such, magainin 2, for example, may be used to target and contribute
to the death of bacteria.
[0164] Antimicrobial peptides, e.g., magainin 2 may be added to a
tubular nanostructure using the methods described herein. Like many
antimicrobial peptides, magainin 2 is a relatively small peptide
with only 23 amino acids and as such is amenable to direct chemical
peptide synthesis using commercial custom peptide synthesis
services (from, e.g., Invitrogen, Carlsbad, Calif.; Sigma-Genosys,
The Woodlands, Tex.; Abgent, San Diego, Calif.). Alternatively,
magainin 2 or other antimicrobial peptides may be generated using
standard recombinant molecular biology techniques and DNA sequence
information available in GenBank as part of the National Center for
Biotechnology Information (NCBI) (Benson, et al., Nucleic Acids
Res. 36:D25-D30, 2008, which is incorporated herein by reference).
The peptide is preferably synthesized with an amino terminal
cysteine residue that enables interaction with a reactive group
associated with the tubular nanostructure such as a succinimidyl
group, for example. Tubular nanostructures such as carbon nanotubes
are synthesized as described herein. The nanotubes are further
functionalized with a primary amine group followed by addition of
N-succinimidyl-3-maleimidopropionate (from, e.g., Pierce-Thermo
Scientific, Rockford, Ill.) in preparation for adding the peptide.
For example, carbon nanotubes (5-10 mg) are suspended in 2
milliliters of dimethylformamide (DMF) and mixed with 2 milliliters
of N-succinimidyl-3-maleimidopropionate in DMF. The reaction is
stirred for 4-8 hours at room temperature and excess
N-succinimidyl-3-maleimidopropionate removed by incubation with a
resin containing a primary amine, e.g., PEGA-NH.sub.2 resin (from,
e.g., Sigma-Aldrich, St. Louis, Mo.). The resin is removed by
filtration. The carbon nanotubes as prepared are added to
approximately 4 mg of purified peptides in 1.5 milliliters of an
aqueous solution, e.g., water. After 4-8 hours, PEGA-NH.sub.2 resin
derivatized with N-succinimidyl-3-maleimidopropionate may be used
to eliminate excess peptide and is removed by filtration.
[0165] In some instances, the tubular nanostructures may
specifically target tumor cells and include one or more toxins. The
one or more toxins may be a pore-forming toxin, e.g., aerolysin.
Aerolysin is a bacterial toxin derived from Aeromonas spp that
binds to glycosylphosphatidylinositol-anchored proteins (GPI-AP) on
mammalian cells and oligomerizes, inserting into the target
membranes and forming channels that cause cell death. Aerolysin may
be generated using standard recombinant molecular biology
techniques and the known polynucleotide sequences of aerolysin
(see, e.g., Howard, et al., J. Bacteriol. 169:2869-2871, 1987,
which is incorporated herein by reference).
[0166] The one or more toxin associated with a tubular
nanostructure may by itself lack sufficient cell type specificity
to selectively target tumor cells, for example. As such, the
tubular nanostructures may further include a ligand that
specifically binds a cognate on tumor cells. For example, the
tubular nanostructures may include the luteinizing
hormone-releasing hormone (LHRH) peptide. LHRH binds to LHRH
receptors that are overexpressed on ovarian tumor cells and to a
lesser extent on breast and prostate tumor cells (see, e.g.,
Khandare, et al., J. Pharmacol. Exp. Ther. 317:929-937, 2006;
Dharap, et al., Proc. Natl. Acad. Sci. USA 102:12962-12967, 2005,
which are incorporated herein by reference). LHRH may be generated
using standard recombinant molecular biology techniques and the
known polynucleotide sequences of LHRH available in GenBank as part
of the National Center for Biotechnology Information (NCBI) (see,
e.g., Benson, et al., Nucleic Acids Res. 36:D25-D30, 2008, which is
incorporated herein by reference). Alternatively, LHRH may be
obtained from commercial sources (from, e.g., Sigma-Aldrich, St.
Louis, Mo.). Alternatively, LHRH may be purified from a nature
source. LHRH may be conjugated to tubular nanostructure through its
primary amines using the methods described herein for peptide
ligands.
Example 4
Tubular Nanostructure with Controlled Flow
[0167] One or more tubular nanostructures targeted to a tumor cell
may be further modified to control flow of biomolecules through the
pores formed by the nanotubes in the lipid bilayer. For example,
the pore associated with the tubular nanostructure may be closed by
physically blocking the pore. The pore may be blocked by
administering an agent to the subject that specifically binds at or
near the pore opening. The agent may be a nanoparticle such as, for
example a bead. The bead may be modified with an antibody, for
example, that recognizes and binds to a ligand associated with one
or both ends of the tubular nanostructure. Alternatively, the bead
may be modified with a ligand that binds to an antibody associated
with one or both ends of the tubular nanostructure. Alternatively,
the bead may be modified with either streptavidin or biotin and as
such binds to biotin or streptavidin, respectively, attached to the
tubular nanostructure. Other biomolecule binding interactions that
might be used to bind a bead to a tubular nanostructure include but
are not limited to protein-protein interactions, sense-antisense
DNA or RNA interactions, aptamer-target interaction,
peptide-nucleic acid (PNA)-DNA or RNA interactions. The beads may
be administered to the subject at some point in time after
administration of the tubular nanostructures to block further
movement of biomolecules through the pore.
[0168] Beads may be modified with an antibody, for example, using a
number of methods. For example, antibodies may be conjugated to
beads using amine or carboxyl derivatized beads (from, e.g.,
Pierce, Rockford, Ill.) using the cross linking methods described
herein. Alternatively, an antibody may be conjugated to beads using
immunoglobulin binding proteins derived from bacteria such as, for
example, Protein A or Protein G. Beads modified with Protein A or
Protein G are available from commercial sources (e.g., .mu.MACS
Protein A or .mu.MACS Protein G MicroBeads, from Miltenyi Biotec,
Auburn, Calif.; Protein A or Protein G sepharose, from Invitrogen,
Carlsbad, Calif.).
[0169] Alternatively beads may be labeled with either streptavidin
or biotin. Beads labeled with streptavidin are available from
commercial sources (from, e.g., Applied Biosystems, Foster City,
Calif.; BD Biosciences, San Jose, Calif.; and Invitrogen, Carlsbad,
Calif.). Beads labeled with biotin are also available from
commercial sources (from, e.g., Polysciences, Inc., Warrington,
Pa.). Biotin and or streptavidin, for example, may be added to a
tubular nanostructure using the methods described herein.
[0170] In some instances, the interaction between the tubular
nanostructure and the bead may be reversible. For example, the
binding affinity of the bead to the tubular nanostructure may be
such that over time the two entities dissociate and the pore is
re-opened. Alternatively, the bead may be dissociated from the
tubular nanostructure by competition with free ligand.
Example 5
Tubular Nanostructure with Controlled Release of an Agent
[0171] One or more tubular nanostructures targeted to a tumor cell
may be further modified to allow delivery of an agent proximal to
the pore through the lipid bilayer formed by the nanotube. The
agent may be a therapeutic agent and or a toxin that contributes to
the death of the tumor cell. The agent may be bound to an antibody
or aptamer that is itself bound to the tubular nanostructure.
Alternatively, the agent may be bound to an antibody or aptamer
that is administered subsequent to administration of the tubular
nanostructures and binds to the membrane associated nanotube. In
either instance, the agent dissociates from the antibody or aptamer
and due to its proximity to the nanotube pore, flows through the
pore and through the associated lipid bilayer.
[0172] An antibody may be generated against a therapeutic agent
using the methods described herein. For example, antibodies to
taxols such as the chemotherapy agent paclitaxel, for example, may
be generated by attaching the taxol to a carrier protein such as
bovine thyroglobin (BTG), immunizing mice, and generating
monoclonal antibodies using standard hybridoma techniques (see,
e.g., U.S. Pat. No. 7,175,993, which is incorporated herein by
reference). Optionally, additional screening may be done to access
binding affinity for the therapeutic agent to identify antibodies
that have sufficient affinity to bind the agent but are able to
dissociate the agent over a given time frame. Antigen/antibody
on-off rates may be assessed using a Biacore 3000, for example
(from Biacore, Inc., Piscataway, N.J.). Alternatively an antibody
to a therapeutic agent may be available from a commercial source.
For example, antibodies to the chemotherapeutic agent doxorubicin
are commercially available (from, e.g., United States Biological,
Swampscott, Mass.).
[0173] An antibody that recognizes and binds a chemotherapy agent,
for example, may be bound to a tubular nanostructure using a
heterofunctional cross linker or using other methods described
herein. The antibody attached to the tubular nanostructure may be
loaded with the chemotherapy agent prior to administering the
tubular nanostructure to a subject. Alternatively, the chemotherapy
agent may be administered before or after administration of the
tubular nanostructure. In this instance, binding of the
chemotherapy to the antibody associated with the tubular
nanostructure would occur in vivo.
[0174] Alternatively, an antibody that recognizes and binds a
chemotherapy agent may be a bifunctional antibody. In addition to
recognizing and binding a chemotherapy agent, the bifunctional
antibody may also recognize and bind to a ligand on the surface of
the tubular nanostructure. The antibodies within the bifunctional
antibody may be two or more intact antibodies and/or two or more
antibody fragments such as, for example, Fab, F(ab).sub.2 and/or
F.sub.v that are linked in some way to one another. The two or more
antibodies may be fused by chemical conjugation, crosslinking
and/or linker moieties. For example, polypeptides may be covalently
bonded to one another through functional groups associated with the
polypeptides such as, for example, carboxylic acid or free amine
groups.
[0175] Alternatively, one or more antibodies may be linked through
disulfide bonds. For example, the antibody that binds the
chemotherapy agent may be reacted with N-succinimidyl
S-acetylthioacetate (SATA) and subsequently deprotected by
treatment with hydroxylamine to generate an SH-antibody with free
sulfhydryl groups (see, e.g., U.S. Pat. App. No. 2003/0215454 A1,
which is incorporated herein by reference). The antibody the binds
the tubular nanostructure may be reacted with sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sSMCC). The two
antibodies treated as such are purified by gel filtration and then
reacted with one another to form a bifunctional antibody complex.
Alternatively, the antibodies may be chemically cross-linked to
form a heteropolymerized complex using, for example, SPDP
[N-succinimidyl-3-(2-pyridyldithio) propionate] (see, e.g., Liu, et
al. PNAS 82:8648-8652, 1985; U.S. Pat. No. 5,470,570, which are
incorporated herein by reference).
[0176] Alternatively, the two antibody binding activities may be
incorporated into a single fusion protein using recombinant DNA
approaches (see, e.g., U.S. Pat. No. 6,132,992, which is
incorporated herein by reference). For example, cDNA encoding the
variable regions (V.sub.L and V.sub.H) of two antibodies directed
against separate and distinct antigens, for example, may be
combined into a linear expression construct from which a bispecific
single-chain antibody may be produced (see, e.g., Haisma, et al.
Cancer Gene Ther. 7:901-904, 2000, which is incorporated herein by
reference).
[0177] As such, cDNA encoding the variable regions (V.sub.L and
V.sub.H) of the antibody that binds the chemotherapy agent and the
antibody that binds the tubular nanostructure, for example, may be
manipulated to form a bispecific single-chain antibody.
[0178] The bifunctional antibody recognizing a chemotherapeutic
agent and a tubular nanostructure may be combined with the tubular
nanostructure prior to administering the nanotubes to a subject.
Alternatively, the bifunctional antibody may be administered before
or after administering the tubular nanostructures. As such, binding
of the bifunctional antibody to the tubular nanostructures would
occur in vivo. The chemotherapy agent may be bound to the
bifunctional antibody when the latter is administered.
Alternatively, the chemotherapy agent may be administered
separately.
Example 6
Tubular Nanostructure with Marker
[0179] One or more tubular nanostructures modified with one or more
marker may be used to selectively mark a target cell, e.g., a tumor
cell. One or more tubular nanostructures may include one or more
marker that is a fluorescent marker, a radioactive marker, a
quantum dot, and/or magnetic resonance imaging marker. The one or
more tubular nanostructures modified with one or more marker may be
selectively directed to tumor cells or other target cells through a
ligand associated with the tubular nanostructures that recognizes a
corresponding cognate on the target cells. Imaging of the one or
more marker may be used to monitor association of the tubular
nanostructures with the targeted cells.
[0180] Tubular nanostructures generated using the methods described
herein may be further modified with one or more markers. For
example, a tubular nanostructure that includes an antibody to the
HER-2 receptor as described herein may be further modified with one
or more fluorescent markers, for example, to enable imaging of
breast cancer cells. The one or more fluorescent markers may be any
of a number of fluorescent dyes some of which are described herein.
For example, fluorescein isothiocyanate (FITC) may be added to a
tubular nanostructure using FITC modified phospholipid-PEG-NH.sub.2
(see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605,
2005, which is incorporated herein by reference). PL-PEG-NH.sub.2
may be purchased from Avanti Polar Lipids (Alabaster, Ala.) and
dissolved in 0.1 M carbonate buffer solution (pH 8.0) to which is
added FITC (from, e.g., Sigma-Aldrich, St. Louis, Mo.). The mixture
may be incubated overnight at room temperature with protection from
light. The PL-PEG-FITC may be isolated from the reaction mix by gel
chromatography on a Sephadex G-25 column, for example. The
PL-PEG-FITC is mixed with carbon nanotubes and sonicated for 45
minutes to 1 hour and centrifuged at 22,000.times.g for 4-8
hours.
[0181] Alternatively, the one or more markers are indirectly linked
to the carbon nanotube, for example, through a fluorescently
labeled protein, antibody, oligonucleotide, aptamer or combinations
thereof. For example, carbon nanotubes may be modified with a
commercially available antibody to the HER-2 receptor that is
itself labeled with a fluorescent marker (from, e.g, BioLegend, San
Diego Calif.; R&D Systems, Inc., Minneapolis, Minn.).
[0182] In some instances, it may be beneficial to modify the
tubular nanostructures with a fluorescent marker that emits at far
red and/or near infrared wavelengths to minimize interference
associated with endogenous cell and tissue autofluorescence.
Examples of near infrared fluorescent markers include, but are not
limited to, IRDye 800CW, IRDye 800RS, and IRDye 700DX (maximum
emission wavelengths equal 794 nm, 786 nm, and 687 nm,
respectively; from LI-COR, Lincoln, Nebr.); Cy5, Cy5.5, and Cy7
(maximum emission wavelengths equal 670 nm, 694 nm, and 760 nm,
respectively; from Amersham Biosciences, Piscataway, N.J.); VivoTag
680 (VT680; VisEn Medical, Woburn, Mass.) and/or a variety of Alexa
Fluor dyes including Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor
660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750 (maximum
emission wavelengths equal 647 nm, 668 nm, 690 nm, 702 nm, 723 nm,
and 775 nm, respectively; from Molecular Probes-Invitrogen,
Carlsbad, Calif., USA; see, e.g., U.S. Pat. App. No. 2005/0171434
A1). For example, IRDye 800CW may be added to functionalized
tubular nanostructures using the methods described herein. IRDye
800CW with a reactive N-hydroxysuccinimide (NHS) group may be
purchased from LI-COR, Lincoln, Nebr. The tubular nanostructures
are appropriately prepared to include free amines such as with
PL-PEG-NH.sub.2 as described above which may react with IRDye
800CW-NHS to conjugate the IRDye to the nanotube.
[0183] In vivo, non-invasive monitoring of near infrared (NIR)
fluorescence, for example, may be performed using fluorescence
mediated molecular tomography as described, for example, in U.S.
Pat. No. 6,615,063, which is incorporated herein by reference.
Additional information regarding NIR imaging in human subjects, for
example, is described in Frangioni Curr. Op. Chem. Biol. 7:626-634,
2003, which is incorporated herein by reference. In some instances,
a wireless system may be used in which light sources such as light
emitting diodes (LEDs) of appropriate wavelength as well as
detectors such as charge-coupled devices (CCDs) are housed along
with a power supply and a wireless communication circuit to create
a device that may be placed on the skin of a subject to monitor NIR
signal as described by Muehlemann, et al., Optics Express,
16:10323, 2008, which is incorporated herein by reference.
Example 7
Tubular Nanostructure with Activatable Marker
[0184] One or more tubular nanostructures modified with one or more
activatable marker may be used to selectively mark a target cell.
One or more markers associated with the tubular nanostructure may
be activated by a ligand reaction, anchoring in the membrane and
interaction with a hydrophobic medium, and/or change in the
cellular environment (e.g., changes in pH). The one or more tubular
nanostructures modified with one or more marker may be selectively
directed to tumor cells or other target cells through a ligand
associated with the tubular nanostructures that recognizes a
corresponding cognate on the target cells. Imaging of the one or
more marker may be used to monitor association of the tubular
nanostructures with the targeted cells.
[0185] In some instances, the marker associated with the tubular
nanostructures may be activated by anchoring in the hydrophobic
lipid membrane. For example, a tubular nanostructure may be labeled
with one or more fluorescent markers that fluoresce in the presence
of a lipid environment. Examples of lipid-sensitive fluorescent
markers include, but are not limited to, nitrobenzoxadiazole (NBD),
diphenylhexatriene propionic acid (DHP), pyrene-labeled sn-2 acyl
chains, and various derivatives thereof. A tubular nanostructure
may be modified with NBD, for example, using commercially available
NBD derivatives ready for conjugation. For example, the NBD
derivatives 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD fluoride),
succinimidyl
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X,
SE), and 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoic
acid (from, e.g., Invitrogen, Carlsbad, Calif.) can be reacted with
primary amines as well as thiols, cysteines and secondary amines on
the tubular nanostructures to conjugate NBD to the surface. NBD-X,
SE, for example, may be added to tubular nanostructures with
primary amines by combining the components together in a slightly
basic buffer lacking primary amines, e.g., 0.1-0.2 M sodium
bicarbonate buffer at pH 8.3 and incubating for 1-2 hours, followed
by size exclusion gel filtration to separate the labeled nanotubes
from the free NBD.
[0186] Alternatively, the maker associated with the tubular
nanostructures may be activated by binding to a specific ligand on
the target cell. For example, the marker may be an aptamer based
molecular beacon. In this instance, the fluorescence associated
with the molecular beacon is quenched until the beacon interacts
with its intended target. Tumor targeting aptamers, for example,
may be generated against whole tumor cells and/or specific tumor
targets using the SELEX method described here.
[0187] In some instances, an aptamer may have a fluorophore in a
region of the molecule known to undergo conformational change upon
binding of a target that leads to an increase in fluorescence
intensity. An aptamer of this sort may be selected for using an in
vitro selection process with fluorescently labeled aptamers (see,
e.g., Jhaveri, et al. Nat. Biotech. 18:1293-1297, 2000, which is
incorporated herein by reference). A pool of RNA molecules is
generated in which the random sequence region (45-60 residues) is
skewed such that one of the residues, uridine, for example, is
disproportionately underrepresented. The three to four randomly
placed uridine residues are substituted with fluorescein-12-UTP,
Cascade Blue-7-UTP, Texas Red-5-UTP, and/or Rhodamine Green-5-UTP
during in vitro transcription. The labeled pool of RNA molecules
are screened against the target cells or a specific target
associated with the cells. Those RNA molecules that bind with high
affinity to the target cells or a specific target associated with
the cells are further screened for their fluorescence signaling
properties in response to binding the target cells or a specific
target associated with the cells. For example, the baseline
fluorescence intensity is measured for RNA aptamer molecules
labeled with fluorescein-12-UTP (excitation maxima 494 nm, emission
maxima 521 nm) or Rhodamine Green-5-UTP (excitation maxima 505 nm,
emission maxima 533 nm), for example, then re-measured in response
to increasing concentrations of target cells or a specific target
associated with the cells. As such, fluorescent aptamers may be
selected that exhibit a 100-200% increase in fluorescence intensity
in response to target binding.
[0188] An aptamer may be labeled either by direct incorporation of
nucleic acids modified with fluorescent dyes or quenchers or by
conjugation of fluorescent dyes or quenchers to appropriately
modified nucleic acids. For example, an aptamer may be labeled
directly with Cy3. The fluorophores may be attached to various
chemical moieties that allow for attachment at various sites within
the aptamer. For example, 3'-DABCYL CPG may be used to place DABCYL
at the 3 prime terminus of the aptamer whereas 5'-DABCYL
phosphoramidite may be used to place DABSYL at the 5 prime terminus
of the aptamer (see, e.g., product information at Glen Research;
http://www.glenres.com/Catalog/labelling.html). DABCYL dT may be
used to place DABCYL within the sequence. Labeling aptamers with
appropriate commercially available fluorophores may be achieved
following instructions provided by the respective manufacturer.
Alternatively, an aptamer-based molecular beacon may be special
ordered from a commercial source (from, e.g., Biosearch
Technologies, Inc., Novato, Calif., USA).
[0189] An aptamer may be attached to a carbon nanotube (So et al,
JACS). Tween may be bound non-covalently to the carbon nanotube
sidewalls through hydrophobic interactions while the
carboiimidazole may be covalently attached to the 3'-amine group of
an RNA or DNA based aptamer.
[0190] In some instance, the tubular nanostructures may be modified
with a marker that is an antibody that emits a signal a shift in
emission wavelength, for example, in response to interacting with a
ligand on the target cell or organelle (see, e.g., Brennan (1999)
J. Fluor. 9:295-312). An antibody that exhibits a shift in
fluorescent signal in response to binding of an antigen may be
generated by labeling the antibody with a solvent-sensitive
fluorophore such as dansyl chloride
(5-dimethylaminonaphthalene-1-sulfonyl chloride), for example (see,
e.g., Brennan (1999) J. Fluor. 9:295-312). The antibody is labeled
such that binding of the antigen to the antibody shields the
solvent sensitive fluorescent label near the active binding site
from the solvent water, resulting in a 3-5 fold increase in
fluorescence intensity (see, e.g., Bright, et al. (1990) Anal.
Chem. 62:1065-1069). As such, an antibody directed against a
specific illicit drug and/or drug of abuse, e.g., methamphetamine
is incubated with methamphetamine (0.10 mg/ml) to block or protect
the antibody/antigen binding site. The antibody/antigen complex is
non-selectively labeled with 0.1 uM dansyl chloride under basic
conditions of pH 8.5. The methamphetamine is removed from the
dansylated antibody. In this instance, for example, subsequent
binding of methamphetamine will result in a measurable increase in
the intensity of the dansyl fluorescence at an emission wavelength
of 420 nm when excited with a wavelength of 325 nm.
[0191] The tubular nanostructures modified with one or more marker
may be further modified with one or more ligand that binds to a
specific cognate on tumor cells. A ligand may be an antibody. An
antibody may be conjugated to tubular nanostructures such as carbon
nanotubes using a sulfo-SMCC linkage as described in Example 1.
Alternatively, an antibody as well as other ligands may be
conjugated to tubular nanostructures via a biotin/avidin
interaction. In this instance, the tubular nanostructures may be
modified with a phospholipid PEG-biotin moiety and interacted with
an avidin labeled antibody. Biotin may be added to carbon nanotubes
by mixing the carbon nanotubes (0.1 to 1 mg) in 1 to 5 ml of 166
.mu.M DSPE-PEG(2000)-biotin (from Avanti Polar Lipids, Inc.,
Alabaster, Ala.) with sonication for 10 minutes. The samples are
washed twice with water by centrifugation at 90,000.times.g for 15
minutes at 4.degree. C. The supernatant may be discarded and the
pellet resuspended in water and further centrifuged for 10 min at
16,000.times.g at room temperature. The top 50% of the supernatant
containing biotinylated carbon nanotubes is taken for further
conjugation. To prepare the antibody other ligand for conjugation,
the antibody is thiolated with 2-iminothiolane as described herein
to add sulfhydryl groups to the protein. The avidin protein is
activated with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
as described by the manufacturer (product # 22311, Pierce
Biotechnology, Rockford, Ill.). The thiolated antibody and the
activated avidin may be conjugated to one another by mixing the two
components at a molar ratio of 1:2 for 2 hours at room temperature
with gentle shaking. The resulting conjugate may be purified by gel
filtration on a Sephacryl S-300 HR column using 0.1 M phosphate
buffered saline, 0.05% Tween-20, at pH 7.4. Carbon nanotubes
modified with antibody are generated by mixing the biotinylated
carbon nanotubes with the avidin labeled antibody in a 1:2 (wt/wt)
ratio and incubated for 35 minutes at room temperature with gentle
rocking. The mixture is centrifuged for 5 minutes at 16,000.times.g
at 4.degree. C., the supernatant disgarded and the pellet used for
treatment. Alternatively, the carbon nanotube may be functionalized
with streptavidin by non-covalent interactions and a biotinylated
antibody or other ligand attached to the carbon nanotube via the
streptavidin-biotin interaction (see, e.g., Lyonnais et al, Small
4:442-446, 2008, which is incorporated herein by reference).
Example 8
Composite Tubular Nanostructure
[0192] Two or more tubular nanostructures may be configured to form
higher order assemblies or composite tubular nanostructures. A
composite tubular nanostructure may comprise two or more tubular
nanostructures each including a hydrophobic surface region, each
hydrophobic region flanked by two hydrophilic surface regions
configured to form a pore in a lipid bilayer membrane. Composite
tubular nanostructures comprised of two or more tubular
nanostructures may be used to create multiple pores at one or more
sites in the targeted lipid bilayer. A composite tubular
nanostructure may be generated by selective oxidation, sonication,
and/or solubilization of carbon nanotube aggregates to generate
smaller bundles of appropriate size and number. Alternatively, a
composite tubular nanostructure may be generated from ordered
assembly of single carbon nanotubes using biomolecule binding
interactions, for example. Biomolecular binding interaction that
might be used to bind a bead to a tubular nanostructure include but
are not limited to streptavidin-biotin interactions,
antigen-antibody interactions, protein-protein interactions,
sense-antisense DNA or RNA interactions, aptamer-target
interaction, peptide-nucleic acid (PNA)-DNA or RNA
interactions.
[0193] Acid oxidation and sonication may be used to generate a
stable aqueous suspension of purified single or small bundles of
shortened nanotubes. Acid oxidation and sonication may also be used
to introduce surface carboxylates on the nanotubes for chemical
derivatization. As such, carbon nanotubes grown by laser ablation,
for example, are refluxed for about 36 hours in 2.5 M HNO3,
subjected to sonication for 30 minutes, and then refluxed again for
another 36 hours. The mixture may be filtered through a
polycarbonate filter with a defined pore size ranging from 10 nm to
100 nm (see, e.g., GE PCTE filters, GE Osmonics Labstore,
Minnetonka, Minn.) to isolate a defined size range of nanotubes.
Optionally, centrifugation at 7000 rpm for 5 min, for example, may
be used to remove larger un-reacted impurities from the solution.
Atomic force microscopy may be used to assess the size and
dispersion of the tubular nanostructures following acid oxidation
and Zeta potential measurements may be used to confirm the
existence of negatively charged acidic groups on the nanotube
sidewalls. (U.S. Patent Application 2006/0275371 A1, which is
incorporated herein by reference). Alternatively, scanning and/or
transmission electron microscopy and/or Raman spectroscopy may be
used to monitor disaggregation of carbon nanotubes.
[0194] In some instances, the composite tubular nanostructure may
be built by combining individual nanotubes that have been
asymmetrically functionalized with compatable binding biomolecules
such as, for example, biotin and streptavidin. For example, a
polymer masking technique may be used to asymmetrically modify the
nanotube sidewall as described by Qu & Dai Chem. Commun.
3859-3861, 2007, which is incorporated herein by reference. In this
instance, one surface of the carbon nanotubes is embedded in a
polystyrene film. The exposed surface is subsequently modified. For
example, carbon nanotubes previously treated with acid and
sonication and containing carboxylate groups as judged by Zeta
potential measurements may be embedded in polystyrene. Carbodiimide
and derivatives thereof may be used to convert the carboxylate
groups to primary amines. These reactive amines are subsequently
available for addition of other biomolecules. Additional
modifications may be made while the nanotubes are embedded.
Alternatively, the masking agent may be removed from the nanotubes
prior to addition of other biomolecules. A masking agent such as
polystyrene, for example, may be removed by treating the nanotubes
with an treated with an organic solvent such as, for example,
toluene.
[0195] The tubular nanotubes which have been asymmetrically
functionalized with primary amine groups may be further modified
with biotin using N-hydroxysuccinimide ester (NHS). Various
NHS-biotin conjugates may be used for this purpose. For example,
NHS-PEG4-Biotin and NHS-PEG12-Biotin (from Pierce-Thermo
Scientific, Rockford, Ill.) may be used for simple and efficient
biotin labeling of primary amine groups associated with, for
example, carbon nanotubes. The associated polyethylene glycol (PEG)
spacer associated with these NHS derivatives may also increase the
solubility of the nanotubes. In some instances, it may be
beneficial to use a biotin linker group with a cleavable disulfide
bound (e.g., EZ Link NHS-SS-Biotin; from Pierce-Thermo Scientific,
Rockford, Ill.), allowing for the disruption of the nanotube bundle
in, for example, the interior of the cell.
[0196] To modify primary amines with NHS-PEG12-Biotin, for example,
1-10 mg of primary amine containing nanotubes are solubilized at a
concentration of 2-10 mg/ml in an aqueous buffer at pH 7.2-8.0. In
this instance, the carbon nanotubes, for example, may be
concentrated in a small volume of dimethylformamide (DMF) or
dimethyl sulfoxide (DMSO) or other water miscible solvent and added
with gentle vortexing to the aqueous buffer. The NHS-PEG12-Biotin
is similarly dissolved in DMF or DMSO other water miscible solvent
and added at 10-20 fold molar excess relative to the carbon
nanotubes. The NHS-PEG12-Biotin is allowed to incubate with the
carbon nanotubes for 2-3 hours on ice or for 30-45 minutes at room
temperature. The unbound NHS-PEG12-Biotin may be removed by
dialysis.
[0197] A second set of tubular nanostructures may be modified with
avidin or streptavidin and used with the biotin modified tubular
nanostructures to form higher order bundles. Avidin or streptavidin
may be non-specifically and non-covalently bound to the tubular
nanostructures as described above. Alternatively, avidin or
streptavidin may be added to tubular nanostructures using one or
more of the various cross-linking agents described herein. For
example, SMCC (succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) may be used to
crosslink the primary amines associated with functionalized carbon
nanotubes with sulfhydryl groups associated with cysteine residues
in avidin or streptavidin.
[0198] The tubular nanostructures modified with streptavidin and
biotin, for example, may be combined to form composite tubular
nanostructures. In some instances, the ratio of asymmetric labeled
nanotubes to symmetric labeled nanotubes may be controlled. For
example, to form a heptamer composite tubular nanostructure
containing seven nanotubes, the ratio of asymmetric to symmetric
nanotubes may be 6:1, for example.
Example 9
Composite Tubular Nanostructure with Ligand
[0199] One or more composite tubular nanostructures may be used to
selectively target and kill tumor cells in a subject with cancer.
One or more composite tubular nanostructure may be generated using
the methods described. The one or more composite tubular
nanostructures may be selectively directed to the tumor cells
through a ligand associated with the composite tubular
nanostructures that recognizes a corresponding cognate on the
membrane of the tumor cells. The one or more ligands may be at
least a portion of an antibody, antibody-coated liposome,
polynucleotide, polypeptide, receptor, viral plasmid, polymer,
protein, carbohydrate, lipid, toxins, pore-forming toxins, or
lectin. Methods for modifying tubular nanostructures with ligands
have been described herein. The one or more cognates on a membrane
of a tumor cell may be a least one of a protein, a carbohydrate, a
glycoprotein, a glycolipid, a sphingolipid, a glycerolipid, or a
metabolite thereof. Once at the targeted tumor cell, the one or
more composite tubular nanostructures may form pores in the plasma
membrane through which intracellular and extracellular components
may flow. Disruption of the highly controlled barrier function of
the plasma membrane ultimately results in death of the targeted
tumor cell.
Example 10
Tubular Nanostructures Targeted to Bacteria
[0200] One or more tubular nanostructures may be used to
selectively target and damage bacterial cells in a subject with a
bacterial infection. The one or more tubular nanostructures may be
selectively directed to bacteria through a ligand associated with
the tubular nanostructures that recognizes a corresponding cognate
on the bacteria.
[0201] An antibody may be added to a tubular nanostructure to
enable targeting of the nanotube to bacteria as described, for
example, by Elkin et al (ChemBioChem 6:640-643, 2005, which is
incorporated herein by reference). Tubular nanostructures, e.g.,
carbon nanotubes are functionalized with bovine serum albumin (BSA)
using a carbodiimide-activated amidation reaction.
Functionalization of the nanotubes with BSA renders the nanotubes
more soluble in physiological buffers. An antibody directed against
one or more bacteria can be non-covalently absorbed by the
nanotube-BSA conjugate. In a typical procedure, a solution of
antibody (10 ug/ml) in phosphate-buffered saline (PBS) or other
physiologically relevant buffer is added to the nanotube-BSA
solution (20 mg/ml). The suspension is mixed by slow rotation at 40
rpm for 20-24 hours at room temperature, for example, and then
subjected to centrifugation at 14000.times.g to remove unbound
antibody. The supernatant is discarded and the pelleted
nanotube-BSA-antibody conjugate is washed repeatedly with
additional PBS and centrifugation. The resulting
nanotube-BSA-antibody conjugate may be passed through a membrane
filter (e.g., 0.2 .mu.m) to eliminate clumped nanotubes. Other
methods for adding an antibody to tubular nanostructure may be
contemplated, some methods of which are described herein.
Example 11
Tubular Nanostructure Targeted to Intracellular Organelle
[0202] One or more tubular nanostructure may be modified to allow
transit of the nanotubes through the plasma membrane of a cell and
subsequent targeting and insertion of the nanotubes into the lipid
bilayer of an internal organelle such as, for example,
mitochondria. In general, mitochondrial outer membrane
permeabilization is considered the "point of no return" during
apoptosis of cells as it results in diffusion to the cytosol of
numerous proteins that normally reside in the space between the
outer and inner mitochondrial membranes and initiates a cascade of
events leading to cell death (see, e.g., Chipuk, et al., Cell Death
Differ. 13:1396-1402, 2006, which is incorporated herein by
reference). As such, one or more tubular nanostructures may be
targeted to the outer membrane of mitochondria for insertion into
and disruption of the outer mitochondrial membrane, leading to cell
death. In some instances, the one or more tubular nanostructures
may be further modified to target only mitochondria in cells of
interest such as, for example, tumor cells. As such, tubular
nanostructures may be first targeted to tumor cells with in a
subject, pass through the tumor cell membrane, and target and
disrupt the tumor cell mitochondria, leading to tumor cell
death.
[0203] The tubular nanostructures as described herein have a
hydrophobic surface region flanked by two hydrophilic surface
regions for insertion and retention in a lipid bilayer. As such,
tubular nanostructures generated as described herein may be
modified in such a manner as to mask the hydrophilic ends and allow
transit through the plasma membrane of a target cell. In one
embodiment, the hydrophilic ends of the tubular nanostructure are
modified with a hydrophobic moiety using a chemical bond that may
be cleaved once the nanotube has passed into the cell. Examples of
biologically cleavable bonds include, but are not limited to,
disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,
acetals, ketals, enol ethers, enol esters, enamines and imines
(see, e.g., U.S. Pat. Nos. 7,087,770, 7,098,030 and 7,348,453,
which are incorporated herein by reference). Alternatively, the
cleavable bond may be a photolabile bond. Examples of hydrophobic
moieties that might be added to the ends of the tubular
nanostructure include, but are not limited to, non-polar
hydrocarbon chains of various lengths. In one aspect, the
hydrophobic moiety is an ester that can be cleaved by an
intracellular esterase to form a hydrophilic acid moiety and
alcohol moiety. For example, ceramides, which are long chain
sphingoid bases linked to fatty acids, may be conjugated to other
compounds through an ester linkage and used to transport compounds
through the lipid bilayer and to release compounds inside the cell
(see, e.g., Yatvin, et al., Cell. Mol. Biol. Lett. 5:119-132, 2000,
which is incorporated herein by reference). As such, tubular
nanostructures may be modified with ceramide or another long-chain
nonpolar compound through an ester linkage at one or both ends of
the nanotube.
[0204] Alternatively, the masked tubular nanostructures may enter
the cell by passing directly through the cell membrane and into the
cytoplasm. In this instance, the tubular nanostructure may include
moieties on the surface of the nanotubes that confers direct
passage through the lipid bilayer, e.g., an amphiphilic striated
surface on the nanotube. The deposition of a
hydrophilic-hydrophobic striated pattern of molecules, e.g., the
anionic ligand 11-mercapto-1-undecanesulphonate (MUS) and the
hydrophobic ligand 1-octanethiol (OT) on the surface of nanotubes
may facilitate direct passage of the tubular nanostructures into
the cytoplasm (see, e.g., Verma, et al., Nature Materials 7:
588-95, 2008, which is incorporated herein by reference). For
example, the hydrophilic ends of the tubular nanostructure may be
modified with an amphipathic or hydrophobic moiety using a chemical
bond that may be cleaved once the nanotube has passed into the
cell. Examples of biologically cleavable bonds are discussed above.
Once the masked tubular nanostructures has entered the cytoplasm,
it can be modified to reveal tubular nanostructures with
hydrophobic surface region flanked by two hydrophilic surface
regions and at least one ligand bound to the nanostructure and
configured to bind one or more cognates on an organellar membrane,
e.g., a mitochondrial membrane.
[0205] In one aspect, hydrophilic moieties may be masked by
acetoxymethyl esters of phosphates, sulfates, or other compounds
having alcohol moieties or acid moieties, which will enhance
permeability of the tubular nanostructure across the lipid bilayer
membrane. Because acetoxymethyl esters are rapidly cleaved
intracellularly, they facilitate the delivery of tubular
nanostructures into the cytoplasm of the cell without puncturing or
disruption of the cell plasma membrane (see, e.g., Schultz et al.,
J. Biol. Chem. 268: 6316-6322, 1993, which are incorporated herein
by reference). Once within the cytoplasm, the tubular
nanostructures having a hydrophobic surface region flanked by two
hydrophilic surface regions is configured to form a pore in the
lipid bilayer membrane of the cellular organelle. The cellular
organelle may be mitochondria. Disruption of the outer membrane of
the mitrochondria by the tubular nanostructures will cause cell
death.
[0206] Under certain conditions, the masked tubular nanostructures
may be actively taken up by the cell through the process of
endocytosis (see, e.g., Kam, et al., Angew. Chem. Int. Ed. 44:1-6,
2005, which is incorporated herein by reference). As such, the
tubular nanostructure may be optionally modified with an element
that facilitates release of the tubular nanostructure from the
endosome. For example, the masked tubular nanostructures may be
modified with all or part of the influenza virus hemagglutinin-2
subunit (HA-2). HA-2 is a pH-dependent fusogenic peptide that
induces lysis of membranes at low pH and may be used to induce
efficient release of encapsulated material from cellular endosomes
(see, e.g., Yoshikawa, et al., J. Mol. Biol. 380:777-782, 2008,
which is incorporated herein by reference). All or part of HA-2 may
be generated using standard recombinant molecular biology
techniques and attached to the tubular nanostructures using methods
described herein.
[0207] The tubular nanostructures are further modified with one or
more ligands that binds to one or more cognates on mitochondria.
The one or more ligands may be an antibody, antibody-coated
liposome, polynucleotide, polypeptide, receptor, viral plasmid,
polymer, protein, carbohydrate, lipid, toxin, lectin, or any
combination thereof as described herein. Cognates associated with a
mitochondrial membrane may include at least one of a protein, a
carbohydrate, a glycoprotein, a glycolipid, a sphingolipid, a
glycerolipid, or metabolites thereof. Examples of cognates
associated with the mitochondrial outer membrane, for example,
include, but are not limited to, carnitine palmitoyl transferase 2,
translocase of outer membrane (TOM70), sorting/assembly machinery,
ANT, voltage dependent anion channel (VDAC/Porin), and monoamine
oxidase.
[0208] The tubular nanostructures may be modified with one or more
ligands that recognize VDAC/Porin, for example, a common protein
expressed on the surface of the mitochondrial outer membrane. The
ligand may be an antibody. Antibodies to VDAC/Porin, for example,
may be generated using standard methods. Alternatively, antibodies
to VDAC/Porin may be available from one or more commercial sources
(from, e.g., GeneTex, Inc., San Antonio, Tex.; Sigma Aldrich, Saint
Louis, Mo.; Genway Biotech Inc., San Diego, Calif.). An antibody to
an outer mitochondrial membrane cognate such as VDAC/Porin may be
attached to a tubular nanostructure using methods described
herein.
[0209] Alternatively, the ligand may be all or part of an
endogenous protein that is binding partner of VDAC/Porin. Examples
of proteins that interact with VDAC/Porin include but are not
limited to hexokinse, glycerol kinase, and Bax (see, e.g.,
Vyssokikh & Brdiczka, Acta Biochimica Polonica 50:389-404,
2003, which is incorporated herein by reference). As such, all or
part of hexokinase, for example, may be generated using standard
recombinant molecular biology techniques and the known
polynucleotide sequences of hexokinase available in GenBank as part
of the National Center for Biotechnology Information (NCBI) (see,
e.g., Benson, et al., Nucleic Acids Res. 36:D25-D30, 2008, which is
incorporated herein by reference). A protein or binding partner
that interacts with one or more outer membrane proteins may be
attached to a tubular nanostructure through amine groups associated
with the protein, for example, using the methods described
herein.
[0210] The tubular nanostructures may be further modified with one
or more ligands that targets the tubular nanostructures
specifically to tumor cells. The one or more ligand may be an
antibody, an aptamer and or a peptide, for example, and attached to
the tubular nanostructures as described here in.
[0211] Each recited range includes all combinations and
sub-combinations of ranges, as well as specific numerals contained
therein.
[0212] All publications and patent applications cited in this
specification are herein incorporated by reference to the extent
not inconsistent with the description herein and for all purposes
as if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference for all purposes.
[0213] Those having ordinary skill in the art will recognize that
the state of the art has progressed to the point where there is
little distinction left between hardware and software
implementations of aspects of systems; the use of hardware or
software is generally (but not always, in that in certain contexts
the choice between hardware and software can become significant) a
design choice representing cost vs. efficiency tradeoffs. Those
having ordinary skill in the art will appreciate that there are
various vehicles by which processes and/or systems and/or other
technologies described herein can be effected (e.g., hardware,
software, and/or firmware), and that the preferred vehicle will
vary with the context in which the processes and/or systems and/or
other technologies are deployed. For example, if an implementer
determines that speed and accuracy are paramount, the implementer
may opt for a mainly hardware and/or firmware vehicle;
alternatively, if flexibility is paramount, the implementer may opt
for a mainly software implementation; or, yet again alternatively,
the implementer may opt for some combination of hardware, software,
and/or firmware. Hence, there are several possible vehicles by
which the processes and/or devices and/or other technologies
described herein may be effected, none of which is inherently
superior to the other in that any vehicle to be utilized is a
choice dependent upon the context in which the vehicle will be
deployed and the specific concerns (e.g., speed, flexibility, or
predictability) of the implementer, any of which may vary. Those
skilled in the art will recognize that optical aspects of
implementations will typically employ optically-oriented hardware,
software, and or firmware.
[0214] In a general sense, those skilled in the art will recognize
that the various aspects described herein which can be implemented,
individually and/or collectively, by a wide range of hardware,
software, firmware, or any combination thereof can be viewed as
being composed of various types of "electrical circuitry."
Consequently, as used herein "electrical circuitry" includes, but
is not limited to, electrical circuitry having at least one
discrete electrical circuit, electrical circuitry having at least
one integrated circuit, electrical circuitry having at least one
application specific integrated circuit, electrical circuitry
forming a general purpose computing device configured by a computer
program (e.g., a general purpose computer configured by a computer
program which at least partially carries out processes and/or
devices described herein, or a microprocessor configured by a
computer program which at least partially carries out processes
and/or devices described herein), electrical circuitry forming a
memory device (e.g., forms of random access memory), and/or
electrical circuitry forming a communications device (e.g., a
modem, communications switch, or optical-electrical equipment).
Those having ordinary skill in the art will recognize that the
subject matter described herein may be implemented in an analog or
digital fashion or some combination thereof.
[0215] The herein described components (e.g., steps), devices, and
objects and the description accompanying them are used as examples
for the sake of conceptual clarity and that various configuration
modifications using the disclosure provided herein are within the
skill of those in the art. Consequently, as used herein, the
specific exemplars set forth and the accompanying description are
intended to be representative of their more general classes. In
general, use of any specific exemplar herein is also intended to be
representative of its class, and the non-inclusion of such specific
components (e.g., steps), devices, and objects herein should not be
taken as indicating that limitation is desired.
[0216] With respect to the use of substantially any plural or
singular terms herein, those having skill in the art can translate
from the plural to the singular or from the singular to the plural
as is appropriate to the context or application. The various
singular/plural permutations are not expressly set forth herein for
sake of clarity.
[0217] The herein described subject matter sometimes illustrates
different components contained within, or connected with, different
other components. It is to be understood that such depicted
architectures are merely exemplary, and that in fact many other
architectures can be implemented which achieve the same
functionality. In a conceptual sense, any arrangement of components
to achieve the same functionality is effectively "associated" such
that the desired functionality is achieved. Hence, any two
components herein combined to achieve a particular functionality
can be seen as "associated with" each other such that the desired
functionality is achieved, irrespective of architectures or
intermedial components. Likewise, any two components so associated
can also be viewed as being "operably connected," or "operably
coupled," to each other to achieve the desired functionality, and
any two components capable of being so associated can also be
viewed as being "operably couplable," to each other to achieve the
desired functionality. Specific examples of operably couplable
include but are not limited to physically mateable or physically
interacting components or wirelessly interactable or wirelessly
interacting components or logically interacting or logically
interactable components.
[0218] While particular aspects of the present subject matter
described herein have been shown and described, it will be apparent
to those skilled in the art that, based upon the teachings herein,
changes and modifications may be made without departing from the
subject matter described herein and its broader aspects and,
therefore, the appended claims are to encompass within their scope
all such changes and modifications as are within the true spirit
and scope of the subject matter described herein. Furthermore, it
is to be understood that the invention is defined by the appended
claims. It will be understood that, in general, terms used herein,
and especially in the appended claims (e.g., bodies of the appended
claims) are generally intended as "open" terms (e.g., the term
"including" should be interpreted as "including but not limited
to," the term "having" should be interpreted as "having at least,"
the term "includes" should be interpreted as "includes but is not
limited to," etc.). It will be further understood that if a
specific number of an introduced claim recitation is intended, such
an intent will be explicitly recited in the claim, and in the
absence of such recitation no such intent is present. For example,
as an aid to understanding, the following appended claims may
contain usage of the introductory phrases "at least one" and "one
or more" to introduce claim recitations. However, the use of such
phrases should not be construed to imply that the introduction of a
claim recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
inventions containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an"; the same holds
true for the use of definite articles used to introduce claim
recitations. In addition, even if a specific number of an
introduced claim recitation is explicitly recited, such recitation
should typically be interpreted to mean at least the recited number
(e.g., the bare recitation of "two recitations," without other
modifiers, typically means at least two recitations, or two or more
recitations). Furthermore, in those instances where a convention
analogous to "at least one of A, B, and C, etc." is used, in
general such a construction is intended in the sense one having
skill in the art would understand the convention (e.g., "a system
having at least one of A, B, and C" would include but not be
limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, or A, B, and C
together, etc.). In those instances where a convention analogous to
"at least one of A, B, or C, etc." is used, in general such a
construction is intended in the sense one having skill in the art
would understand the convention (e.g., "a system having at least
one of A, B, or C" would include but not be limited to systems that
have A alone, B alone, C alone, A and B together, A and C together,
B and C together, or A, B, and C together, etc.). Virtually any
disjunctive word and/or phrase presenting two or more alternative
terms, whether in the description, claims, or drawings, should be
understood to contemplate the possibilities of including one of the
terms, either of the terms, or both terms. For example, the phrase
"A or B" will be understood to include the possibilities of "A" or
"B" or "A and B."
[0219] The various aspects and embodiments disclosed herein are for
purposes of illustration and are not intended to be limiting, with
the true scope and spirit being indicated by the following
claims.
* * * * *
References