U.S. patent application number 12/032292 was filed with the patent office on 2008-08-21 for nanoparticle delivery vehicle.
Invention is credited to Miles F. Anderson, Daniel L. Feldheim, Stefan Franzen, Marisha L. Godek, Joseph A. Ryan, Alexander G. Tkachenko.
Application Number | 20080199529 12/032292 |
Document ID | / |
Family ID | 23175645 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199529 |
Kind Code |
A1 |
Franzen; Stefan ; et
al. |
August 21, 2008 |
NANOPARTICLE DELIVERY VEHICLE
Abstract
A nanoparticle delivery vehicle, comprising a nanoparticle, an
active agent and a nuclear localization signal and methods of
modulating gene expression and protein expression employing the
nanoparticle delivery vehicle. A representative method includes
providing a nanoparticle delivery vehicle comprising a nanoparticle
having a diameter of about 30 nm or less, an active agent and a
nuclear localization signal; and contacting a target cell with the
nanoparticle delivery vehicle, whereby an active agent is delivered
to the nucleus of a target cell. Another representative method
includes providing a nanoparticle delivery vehicle comprising a
nanoparticle having a diameter greater than or equal to about 30
nm, an active agent and a nuclear localization signal; and
contacting a target cell with the nanoparticle delivery vehicle,
whereby an active agent is delivered to the cytoplasm of a
cell.
Inventors: |
Franzen; Stefan; (Apex,
NC) ; Feldheim; Daniel L.; (Cary, NC) ;
Tkachenko; Alexander G.; (Raleigh, NC) ; Godek;
Marisha L.; (Fort Collins, CO) ; Ryan; Joseph A.;
(Raleigh, NC) ; Anderson; Miles F.; (Raleigh,
NC) |
Correspondence
Address: |
JENKINS, WILSON, TAYLOR & HUNT, P. A.
Suite 1200 UNIVERSITY TOWER, 3100 TOWER BLVD.,
DURHAM
NC
27707
US
|
Family ID: |
23175645 |
Appl. No.: |
12/032292 |
Filed: |
February 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10192393 |
Jul 10, 2002 |
7332586 |
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12032292 |
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60304236 |
Jul 10, 2001 |
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Current U.S.
Class: |
424/497 ;
424/490; 514/1.1; 514/44A |
Current CPC
Class: |
Y10S 977/773 20130101;
A61K 9/5115 20130101; Y02A 50/30 20180101; A61P 43/00 20180101;
C12N 15/88 20130101; A61K 48/0025 20130101; A61K 49/0047
20130101 |
Class at
Publication: |
424/497 ; 514/44;
424/490; 514/12; 514/2 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61K 9/51 20060101 A61K009/51; A61K 38/16 20060101
A61K038/16; A61K 38/02 20060101 A61K038/02; A61P 43/00 20060101
A61P043/00 |
Goverment Interests
GRANT STATEMENT
[0002] This work was supported by NSF grants NSF-DMR (9900073) and
NSF-MDB (9874895). Thus, the U.S. Government has certain rights in
the invention.
Claims
1-49. (canceled)
50. A method of delivering an active agent to a cell, the method
comprising: (a) providing a nanoparticle delivery vehicle
comprising a nanoparticle scaffold, an active agent and a plurality
of targeting agents, the plurality of targeting agents comprising a
nuclear localization signal and one or more extracellular targeting
agents; and (b) contacting a target cell with the nanoparticle
delivery vehicle, whereby an active agent is delivered to the
target cell.
51. (canceled)
52. (canceled)
53. The method of claim 50, wherein the active agent is capable of
interacting with a target nucleic acid sequence whose expression is
to be modulated; the target cell comprises the target nucleic acid
sequence whose expression is to be modulated and the method further
comprises modulating the expression of the target nucleic acid
sequence.
54. The method of claim 53, further comprising determining a degree
to which the target nucleic acid sequence is expressed.
55. The method of claim 54, wherein the determining is by a
technique selected from the group consisting of SDS-PAGE, enzyme
activity assay, ELISA-based assay, spectroscopic assay, northern
blot, Southern blot and radiological-based assay.
56-58. (canceled)
59. The method of claim 50, wherein the active agent comprises a
single stranded antisense nucleic acid sequence complementary to a
nucleic acid sequence encoding a target protein; the target cell
comprises the nucleic acid sequence encoding the target protein and
the method further comprises modulating the expression of the
target protein.
60. The method of claim 59, further comprising determining a degree
to which the target protein is expressed.
61. The method of claim 59, wherein the determining is by a
technique selected from the group consisting of SDS-PAGE, enzyme
activity assay, ELISA-based assay, spectroscopic assay, northern
blot, Southern blot and radiological-based assay.
62-64. (canceled)
65. The method of claim 50, wherein the active agent comprises a
ligand for which a wild-type transcription component has greater
affinity than a natural ligand of the wild-type transcription
component; the cell comprises the wild-type transcription component
and the method further comprises modulating transcription in the
cell.
66. The method of claim 65, wherein the ligand for which a
wild-type transcription component has greater affinity than a
natural ligand of the wild-type transcription component comprises a
morpholino oligonucleotide.
67. The method of claim 65, wherein ligand for which a wild-type
transcription component has greater affinity than a natural ligand
of the wild-type transcription component comprises modified
phosphodiester bonds.
68. The method of claim 65, wherein the ligand for which a
wild-type transcription component has greater affinity than a
natural ligand of the wild-type transcription component has the
ability to interact with a nucleic acid sequence encoding a
regulatory protein to thereby form at least one of (a) an
untranscribable three-dimensional structure and (b) untranslatable
three-dimensional structure.
69. The method of claim 65, wherein the nanoparticle delivery
vehicle further comprises a tether sequence attached to, and
disposed between, the ligand for which a wild-type transcription
component has greater affinity than a natural ligand of the
wild-type transcription component and the nanoparticle
scaffold.
70. The method of claim 65, further comprising determining a degree
to which transcription is modulated.
71. The method of claim 70, wherein the determining is by a
technique selected from the group consisting of SDS-PAGE, enzyme
activity assay, ELISA-based assay, spectroscopic assay, northern
blot, Southern blot and radiological-based assay.
72-78. (canceled)
79. The method of claim 50, wherein the active agent comprises a
nucleic acid sequence known or suspected to alter the splicing
pattern for a target gene; the cell comprises the target gene and
the method further comprises modulating RNA splicing in the
cell.
80. The method of claim 79, wherein the nucleic acid sequence known
or suspected to alter the splicing pattern for a target gene
comprises a morpholino oligonucleotide.
81. The method of claim 79, wherein the nucleic acid sequence known
or suspected to alter the splicing pattern for a target gene
comprises modified phosphodiester bonds.
82. The method of claim 79, wherein the nanoparticle delivery
vehicle further comprises a tether sequence attached to, and
disposed between, the nucleic acid sequence known or suspected to
alter the splicing pattern for a target gene and the nanoparticle
scaffold.
83. The method of claim 79, further comprising determining a degree
to which the RNA splicing in a sample is modulated.
84. The method of claim 83, wherein the determining is by a
technique selected from the group consisting of SDS-PAGE, enzyme
activity assay, ELISA-based assay, spectroscopic assay, northern
blot, Southern blot and radiological-based assay.
85-90. (canceled)
91. The method of claim 50, wherein the active agent is a single
stranded nucleic acid sequence complementary to a nucleic acid
sequence of an mRNA sequence encoding a protein of interest; the
cell comprises the mRNA sequence encoding a protein of interest and
the method further comprises modulating the translation of the mRNA
sequence encoding a protein of interest in the cell.
92. The method of claim 91, wherein the nanoparticle scaffold has a
diameter of less than or equal to 1,000 nm.
93. The method of claim 91, wherein the nanoparticle scaffold
ranges in diameter from about 30 nm to about 1,000 nm.
94. The method of claim 91, wherein the single stranded nucleic
acid sequence complementary to the nucleic acid sequence of the
mRNA sequence encoding a protein of interest comprises a morpholino
oligonucleotide.
95. The method of claim 91, wherein the single stranded nucleic
acid sequence complementary to the nucleic acid sequence of the
mRNA sequence encoding a protein of interest comprises modified
phosphodiester bonds.
96. The method of claim 91, wherein the single stranded nucleic
acid sequence complementary to the nucleic acid sequence of the
mRNA sequence encoding a protein of interest has the ability to
interact with the nucleic acid sequence of the mRNA sequence
encoding a protein of interest to thereby form at least one of (a)
an untranscribable three-dimensional structure and (b)
untranslatable three-dimensional structure.
97. The method of claim 91, wherein the nanoparticle delivery
vehicle further comprises a tether sequence attached to, and
disposed between, the single stranded nucleic acid sequence
complementary to the nucleic acid sequence of the mRNA sequence
encoding a protein of interest and the nanoparticle scaffold.
98. The method of claim 91, further comprising determining a degree
to which the concentration of a regulatory protein in solution is
modulated.
99. The method of claim 98, wherein the determining is by a
technique selected from the group consisting of SDS-PAGE, enzyme
activity assay, ELISA-based assay, spectroscopic assay, northern
blot, Southern blot and radiological-based assay.
100-108. (canceled)
109. The method of claim 50, wherein the nanoparticle delivery
vehicle has a diameter that facilitates entry into the nucleus via
a nuclear pore.
110. The method of claim 50, wherein the nanoparticle scaffold has
a diameter of about 30 nm or less.
111. The method of claim 50, wherein the active agent is selected
from the group consisting of a nucleic acid sequence, a nucleotide,
a protein, a peptide sequence, and a small molecule.
112. The method of claim 111, wherein the nucleic acid sequence is
selected from the group consisting of a RNA, a DNA, a peptide
nucleic acid sequence, and a chemically modified nucleic acid
sequence.
113. The method of claim 111, wherein the nucleic acid sequence is
a full length gene or fragment thereof.
114. The method of claim 111, wherein the nucleic acid sequence is
an oligonucleotide.
115. The method of claim 111, wherein the nucleic acid sequence has
a length between about 20 and about 50 nucleotides.
116. The method of claim 111, wherein the nucleic acid sequence is
selected from a single stranded nucleic acid sequence and a double
stranded nucleic acid sequence.
117. The method of claim 50, wherein the active agent is selected
from a chemotherapeutic; a toxin; a radiotherapeutic; a
radiosensitizing agent; an imaging agent; a diagnostic agent; a
gene therapy vector; an antisense nucleic acid construct; a
transcription factor decoy; and an agent known to interact with one
of an intracellular protein, a nucleic acid, a soluble ligand, and
an insoluble ligand.
118. The method of claim 117, wherein the antisense nucleic acid
construct is an antisense oligodeoxynucleotide.
119. The method of claim 117, wherein the active agent is an
antisense nucleic acid construct and delivering an active agent to
the cell prevents or delays one of infection, inflammation and
tumor formation.
120. The method of claim 50, wherein the nanoparticle delivery
vehicle comprises two or more different active agents.
121. The method of claim 50, wherein the nanoparticle delivery
vehicle is disposed in a pharmaceutically acceptable diluent.
122. The method of claim 50, wherein the nanoparticle delivery
vehicle is in a pharmaceutical composition for administration to a
subject or a sample.
123. The method of claim 122, wherein the pharmaceutical
composition is administered to the subject via parenteral
administration, intravenous administration, or infusion directly
into a desired target tissue.
124. The method of claim 123, wherein the desired target tissue is
a solid tumor or other neoplastic tissue.
125. The method of claim 50, wherein the cell is suspended in a
cell culture and the nanoparticle delivery vehicle is added to a
cell culture medium.
126. The method of claim 50, wherein the nanoparticle scaffold
comprises a material selected from the group consisting of cadmium
selenide, titanium, titanium dioxide, tin, tin oxide, silicon,
silicon dioxide, iron, iron.sup.III oxide, silver, nickel, gold,
copper, aluminum, steel, cobalt-chrome alloy, titanium alloy,
brushite, tricalcium phosphate, alumina, silica, zirconia, diamond,
polystyrene, silicone rubber, a polypeptide, polycarbonate,
polyurethanes, polypropylenes, polymethylmethacrylate, polyvinyl
chloride, polyesters, polyethers, and polyethylene.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is based on and claims priority to U.S.
Provisional Patent Application Ser. No. 60/304,236, filed Jul. 10,
2001, herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to compositions for and
methods of delivering an active agent to and into cells. More
particularly, the method employs a nanoparticle delivery vehicle as
a vehicle for carrying proteins, nucleic acids, protein and nucleic
acid analogs, small molecules and other compounds to the surface of
a cell, into the cytoplasm of a cell or into a cell's nucleus.
TABLE-US-00001 Table of Abbreviations ATP adenosine triphosphate
ADP adenosine diphosphate AS antisense AS-ODN antisense
oligodeoxynucleotides bipy bipyridine cDNA complementary DNA DNA
deoxyribonucleic acid dsDNA double stranded DNA EDTA
ethylenediaminetetraacetic acid HEPES
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid ITO indium tin
oxide IV intravenous kDa kilodalton(s) LB Luria broth MES
2-[N-Morpholino]ethanesulfonic acid mRNA messenger RNA NDP
nucleotide diphosphate NLS nuclear localization signal nt
nucleotide NTP nucleotide triphosphate ODN oligodeoxynucleotide
PACVD plasma-assisted chemical vapor deposition PAGE polyacrylamide
gel electrophoresis PBS phosphate buffered saline PCR polymerase
chain reaction pl isoelectric point PNA peptide nucleic acid analog
RES reticuloendothelial system RME receptor mediated endocytosis
RNA ribonucleic acid SDS sodium dodecyl sulfate SDS-PAGE sodium
dodecyl sulfate polyacrylamide gel electrophoresis ssDNA single
stranded DNA TEM transmission electron microscopy
TABLE-US-00002 Amino Acid Abbreviations Single-Letter Code
Three-Letter Code Name A Ala Alanine V Val Valine L Leu Leucine I
Ile Isoleucine P Pro Proline F Phe Phenylalanine W Trp Tryptophan M
Met Methionine G Gly Glycine S Ser Serine T Thr Threonine C Cys
Cysteine Y Tyr Tyrosine N Asn Asparagine Q Gln Glutamine D Asp
Aspartic Acid E Glu Glutamic Acid K Lys Lysine R Arg Arginine H His
Histidine
BACKGROUND ART
[0004] The development of new forms of therapeutics that use
macromolecules such as proteins or nucleic acids as therapeutic
agents has created a need to develop new and effective approaches
of delivering such macromolecules to their appropriate cellular
targets. Therapeutics based on either the use of specific
polypeptide growth factors or specific genes to replace or
supplement absent or defective genes are examples of therapeutics
that might require such new delivery systems. Therapeutics
involving oligonucleotides that interact with DNA to modulate the
expression of a gene or other segment of DNA might also require a
new delivery system. Clinical application of such therapies depends
not only on the reliability and efficiency of new delivery systems
but also on their safety and on the ease with which the
technologies underlying these systems can be adapted for
large-scale pharmaceutical production, storage, and distribution of
the therapeutic formulations.
[0005] Gene therapy has become an increasingly important mode of
treating various genetic disorders. The potential for providing
effective treatments, has stimulated an intense effort to apply
this technology to diseases for which there have been no effective
treatments. Recent progress in this area has indicated that gene
therapy can have a significant impact not only on the treatment of
single gene disorders, but also on other more complex diseases such
as cancer. However, a significant obstacle in the attainment of
efficient gene therapy regime has been the difficulty of designing
new and effective approaches for delivering therapeutic nucleic
acids to cells and intracellular targets. Indeed, an ideal vehicle
for the delivery of nucleic acids or proteins into cells and
tissues should be highly efficient, safe to use, easy to produce in
large quantity and have sufficient stability to be practicable as a
pharmaceutical delivery vehicle.
[0006] When nucleic acids are used as "active agents" in a gene
therapy regime, there are essentially two systems based on viral
vectors or nonviral vectors that are described in the art: (1)
retro, adeno and herpes viruses (or their recombinants) are
presently being studied in vivo as viral vectors; and (2) liposomes
and ligands of cell surface-specific receptors are being researched
in vivo as nonviral vectors (Wu & Wu, (1991) Biotherapy 3:
87-95; Ledley, (1993) Clin. Invest Med. 16: 78-88). Nanocrystalline
particles are also being investigated (U.S. Pat. No. 5,460,831 to
Kossovsky). All of these approaches suffer from a variety of
disadvantages, including undesired in vivo degradation and a lack
of specificity for a given target structure, for example, the
nucleus of a cell or the surface of a cell expressing a particular
type of structure.
[0007] Nanoparticle technology has found application in a variety
of disciplines, but has found minimal application in pharmacology
and drug delivery. The development of therapeutic nanoparticles was
first attempted around 1970, and the proposed nanoparticles were
intended to function as carriers of anticancer and other drugs
(Couvreur et al., (1982) J. Pharm. Sci., 71: 790-92). Attempts were
also made to elucidate methods by which the uptake of the
nanoparticles by the cells of the reticuloendothelial system (RES)
would be minimized (Couvreur et al., (1986) in Polymeric
Nanoparticles and Microspheres, (Guiot & Couvreur, eds.), CRC
Press, Boca Raton, pp. 27-93). Other attempts pursued the use of
nanoparticles for treatment of specific disorders. See, e.g.,
Labhasetwar et al., (1997) Adv. Drug. Del. Rev., 24: 63-85.
[0008] Although nanoparticles have shown promise as useful tools
for drug delivery systems, many problems remain. Some unsolved
problems relate to the control, selection, and behavior of various
particle sizes, as well as problems surrounding the loading of
particles with therapeutics. Additionally, the targeting of the
nanoparticle to the appropriate cellular site has remained
problematic. The design and provision of a nanoparticle delivery
vehicle that addresses these problems thus represents and ongoing
and long-felt need in the art.
SUMMARY OF THE INVENTION
[0009] A nanoparticle delivery vehicle is disclosed. In one
embodiment, the nanoparticle delivery vehicle comprises: (a) a
nanoparticle; (b) an active agent; and (c) a nuclear localization
signal. In another embodiment, the nanoparticle delivery vehicle
comprises (a) a plurality of targeting agents; (b) a nanoparticle
scaffold; and (c) an active agent.
[0010] Preferably, the active agent is selected from the group
consisting of double stranded nucleic acids, single stranded
nucleic acids, chemically modified nucleic acids, peptide nucleic
acids, proteins and small molecules. Preferably, the nanoparticle
delivery vehicle further comprises a tether sequence attached to,
and disposed between, the active agent and the nanoparticle.
Preferably, the nanoparticle delivery vehicle further comprises a
cell surface recognition sequence. Preferably, the nanoparticle
delivery vehicle is disposed in a pharmaceutically acceptable
diluent. Preferably, the nanoparticle delivery vehicle further
comprises a detectable moiety.
[0011] Optionally, the nanoparticle delivery vehicle can further
comprise two or more different active agents. Also optionally, the
nanoparticle delivery vehicle can further comprise a
biocompatibility-enhancing agent. As a further option, the
nanoparticle delivery vehicle can further comprise a protective
coating covering at least part of the delivery vehicle. In one
embodiment, the protective coating can cover the entire delivery
vehicle, including the active agent(s) and targeting agent(s). The
protective coating can comprise a polymer. The protective coating
can also comprise a biological material. The biological material
can be a protein, lipid, carbohydrate, or combination thereof.
[0012] A method of delivering an active agent to the nucleus of a
cell is disclosed. The method comprises: (a) providing a
nanoparticle delivery vehicle of the present invention comprising a
nanoparticle having a diameter of about 30 nm or less; and (b)
contacting a target cell with the nanoparticle delivery vehicle,
whereby an active agent is delivered to the nucleus of a target
cell. Preferably, the active agent is selected from the group
consisting of double stranded nucleic acids, single stranded
nucleic acids, chemically modified nucleic acids, peptide nucleic
acids, proteins and small molecules. Preferably, the nanoparticle
delivery vehicle further comprises a tether sequence attached to,
and disposed between, the active agent and the nanoparticle.
Preferably, the nanoparticle delivery vehicle further comprises a
cell surface recognition sequence. Preferably, the nanoparticle
delivery vehicle is disposed in a pharmaceutically acceptable
diluent. Preferably, the nanoparticle delivery vehicle further
comprises a detectable moiety.
[0013] A method of delivering an active agent to the cytoplasm of a
cell is disposed. The method comprises: (a) providing a
nanoparticle delivery vehicle of the present invention comprising a
nanoparticle having a diameter greater than or equal to about 30
nm; and (b) contacting a target cell with the nanoparticle delivery
vehicle. Preferably, the active agent is selected from the group
consisting of double stranded nucleic acids, single stranded
nucleic acids, chemically modified nucleic acids, peptide nucleic
acids, proteins and small molecules. Preferably, the nanoparticle
delivery vehicle further comprises a tether sequence attached to,
and disposed between, the active agent and the nanoparticle.
Preferably, the nanoparticle delivery vehicle further comprises a
cell surface recognition sequence. Preferably, the nanoparticle
delivery vehicle is disposed in a pharmaceutically acceptable
diluent. Preferably, the nanoparticle delivery vehicle further
comprises a detectable moiety.
[0014] A method of modulating the expression of a target nucleic
acid sequence is disclosed. The method comprises: (a) providing a
nanoparticle delivery vehicle of the present invention comprising
an active agent capable of interacting with a target nucleic acid
sequence whose expression is to be modulated; (b) contacting a
target cell comprising a target nucleic acid sequence with the
nanoparticle delivery vehicle; and (c) modulating the expression of
the target nucleic acid sequence through the contacting of step
(b). Preferably, the nanoparticle delivery vehicle further
comprises a tether sequence attached to, and disposed between, the
active agent and the nanoparticle. Preferably, the nanoparticle
delivery vehicle further comprises a cell surface recognition
sequence. Preferably, the nanoparticle delivery vehicle is disposed
in a pharmaceutically acceptable diluent. Preferably, the
nanoparticle delivery vehicle further comprises a detectable
moiety.
[0015] A method of modulating the expression of a target protein is
disclosed. The method comprises: (a) providing a nanoparticle
delivery vehicle of the present invention comprising a single
stranded antisense nucleic acid sequence complementary to a nucleic
acid sequence encoding a target protein; (b) contacting a target
cell comprising a nucleic acid sequence encoding a target protein
with the nanoparticle delivery vehicle; and (c) modulating the
expression of the target protein through the contacting of step
(b). Preferably, the nanoparticle delivery vehicle further
comprises a tether sequence attached to, and disposed between, the
active agent and the nanoparticle. Preferably, the nanoparticle
delivery vehicle further comprises a cell surface recognition
sequence. Preferably, the nanoparticle delivery vehicle is disposed
in a pharmaceutically acceptable diluent. Preferably, the
nanoparticle delivery vehicle further comprises a detectable
moiety.
[0016] A method of modulating transcription in a sample is
disclosed. The method comprises: (a) providing a nanoparticle
delivery vehicle of the present invention comprising an active
agent comprising a ligand for which a wild-type transcription
component has greater affinity than a natural ligand of the
wild-type transcription component; (b) contacting a sample
comprising the wild-type transcription component with the
nanoparticle delivery vehicle; and (c) modulating transcription in
the sample through the contacting of step (b). Preferably, the
nanoparticle delivery vehicle further comprises a tether sequence
attached to, and disposed between, the active agent and the
nanoparticle. Preferably, the nanoparticle delivery vehicle further
comprises a cell surface recognition sequence. Preferably, the
nanoparticle delivery vehicle is disposed in a pharmaceutically
acceptable diluent. Preferably, the nanoparticle delivery vehicle
further comprises a detectable moiety.
[0017] A method of modulating RNA splicing in a sample is
disclosed. The method comprises: (a) providing a nanoparticle
delivery vehicle of the present invention comprising a nucleic acid
sequence known or suspected to alter the splicing pattern for a
target gene; and (b) contacting a sample comprising the target gene
with the nanoparticle delivery vehicle; and (c) modulating RNA
splicing in a sample through the contacting of step (b).
Preferably, the nanoparticle delivery vehicle further comprises a
tether sequence attached to, and disposed between, the active agent
and the nanoparticle. Preferably, the nanoparticle delivery vehicle
further comprises a cell surface recognition sequence. Preferably,
the nanoparticle delivery vehicle is disposed in a pharmaceutically
acceptable diluent. Preferably, the nanoparticle delivery vehicle
further comprises a detectable moiety.
[0018] A method of modulating the translation of an mRNA sequence
encoding a protein of interest is disclosed. The method comprises:
(a) providing a nanoparticle delivery vehicle of the present
invention comprising a single stranded nucleic acid sequence
complementary to a nucleic acid sequence of an mRNA sequence
encoding a protein of interest; (b) contacting a sample comprising
the mRNA sequence encoding a protein of interest with the
nanoparticle delivery vehicle; and (c) modulating the translation
of an mRNA sequence encoding a protein of interest through the
contacting of step (b). Preferably, the nanoparticle delivery
vehicle further comprises a tether sequence attached to, and
disposed between, the active agent and the nanoparticle.
Preferably, the nanoparticle delivery vehicle further comprises a
cell surface recognition sequence. Preferably, the nanoparticle
delivery vehicle is disposed in a pharmaceutically acceptable
diluent. Preferably, the nanoparticle delivery vehicle further
comprises a detectable moiety.
[0019] Accordingly, it is an object of the present invention to
provide a nanoparticle delivery vehicle. This and other objects are
achieved in whole or in part by the present invention.
[0020] Some of the objects of the invention having been stated
hereinabove, other objects will be evident as the description
proceeds, when taken in connection with the accompanying Drawings
as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A is a transmission electron micrograph of hepatocytes
grown in a medium comprising 5 nm nanoparticles. A region of the
nucleus is highlighted in the inset.
[0022] FIG. 1B is a transmission electron micrograph of hepatocytes
grown in a medium comprising 30 nm nanoparticles. A region of the
cytoplasm including a vacuole is highlighted in the inset.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides a nanoparticle delivery
vehicle as a vehicle for carrying proteins, nucleic acids, protein
and nucleic acid analogs, small molecules and other compounds to
the surface of a cell, into the cytoplasm of a cell and/or into a
cell's nucleus. A plurality of sequences can be associated with a
nanoparticle delivery vehicle, preferably a plurality of different
sequences, such as RME sequences and NLS sequences. These sequences
can aid in the translocation of a vehicle across various membranes,
such as the nuclear membrane of a cell or the outer membrane of a
cell. Thus, if membranes and other structures that generally
inhibit translocation of a vehicle to a given location in or on a
cell are analogized as "locks", NLS and RME sequences can be
analogized to be "keys". Thus, in a preferred embodiment, a
nanoparticle delivery vehicle of the present invention can comprise
a plurality of different sequences or "keys," which can enable a
given nanoparticle delivery vehicle to pass through various
potential barriers to translocation in a variety of different cell
types.
I. Definitions
[0024] Following long-standing patent law convention, the terms "a"
and "an" mean "one or more" when used in this application,
including the claims.
[0025] As used herein, the term "active agent" means a therapeutic
agent, including but not limited to chemotherapeutic agents,
radiotherapeutics, or radiosensitizing agents; an imaging agent; a
diagnostic agent; or other agent known to interact with an
intracellular protein, a nucleic acid or a soluble or insoluble
ligand.
[0026] As used herein, the term "amino acid sequence" means an
oligopeptide, peptide, polypeptide, or protein sequence, and
fragments thereof, and naturally occurring or synthetic molecules.
Where "amino acid sequence" is recited herein to refer to an amino
acid sequence of a synthetic peptide or a naturally occurring
protein molecule, amino acid sequence, and the like. The term is
not meant to limit the amino acid sequence to a complete, native
amino acid sequence associated with a recited protein molecule, but
is intended to encompass variations on the native amino acid
sequence as well.
[0027] As used herein, the term "biodegradable" means any
structure, including but not limited to a nanoparticle, which
decomposes or otherwise disintegrates after prolonged exposure to
physiological conditions. To be biodegradable, the structure should
be substantially disintegrated within a few weeks after
introduction into the body. Brushite is a preferred biodegradable
nanoparticle material.
[0028] As used herein, the terms "extracellular targeting agent"
and "cell surface recognition sequence" are used interchangeably
and refer to a small molecule or protein sequence that is
recognized and bound by one or more receptors present on the
surface of a particular cell. Cell surface recognition sequences
can include HIV coat proteins (gp160, 41, 120) corona virus coat
proteins, EBV coat proteins (gp350) and peptides. Other
representative, but non-limiting cell surface recognition sequences
can comprise carbohydrate and lipid, carbohydrates, peptide nucleic
acids, morpholino oligonucleotides and polymers. It is intended
that the term "cell surface recognition sequence" encompass any
sequence or molecule recognized and/or bound by a cell surface
receptor. It is preferable, but not required, that a "cell surface
recognition sequence" that is recognized and/or bound by a cell
surface receptor leads to receptor-mediated endocytosis (RME). A
list of representative moieties that can be employed as targeting
agents for internalization by RME is presented in Table 1.
[0029] As used herein, the term "chemical modification" means
alteration of a first moiety by covalently, noncovalently or
ionically binding a second moiety to the first moiety. Chemical
modification can involve the addition of a detectable moiety to a
peptide or protein.
[0030] As used herein, the term "detecting" means confirming the
presence of a target entity by observing the occurrence of a
detectable signal, such as an electrical, radiological or
spectroscopic signal that will appear exclusively in the presence
of the target entity. The term encompasses the use of
electrophoresis techniques and blotting techniques, including
northern, Southern, western and far western blots. The term
"detecting" also includes the use of microscopy techniques, such as
transmission electron microscopy. "Detecting" an event or the
presence of a compound can be done directly or indirectly, for
example, by monitoring the rate of transcription to detect the
presence of transcription factors. Thus, the term "detecting"
broadly means identifying the presence or absence of an event,
compound, molecule, etc.
[0031] As used herein, the term "gene" is used for simplicity and
means a functional protein, polypeptide or peptide encoding unit.
As will be understood by those in the art, this functional term
includes both genomic sequences and cDNA sequences.
[0032] As used herein, the term "gold" means element 79, which has
the chemical symbol Au; the term specifically excludes any
connotation related to color or other calorimetric properties.
[0033] As used herein, the term "homology" means a degree of
complementarity. There can be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid can be considered "substantially homologous".
The inhibition of hybridization of the completely complementary
sequence to the target sequence can be examined using a
hybridization assay (Southern or northern blot, solution
hybridization and the like) under conditions of low stringency. A
substantially homologous sequence or hybridization probe will
compete for and inhibit the binding of a completely homologous
sequence to the target sequence under conditions of low stringency.
This is not to say that conditions of low stringency are such that
non-specific binding is permitted; low stringency conditions
require that the binding of two sequences to one another be a
specific (i.e., selective) interaction. The absence of non-specific
binding can be tested by the use of a second target sequence that
lacks even a partial degree of complementarity (e.g., less than
about 30% identity). In the absence of non-specific binding, the
probe will not hybridize to the second non-complementary target
sequence.
[0034] As used herein, the term "hybridization" means the binding
of a probe sample to a target sample. The probe sample can comprise
a molecule to which a detectable moiety has been bound, thereby
making it possible to detect the presence or absence of a probe
sample.
[0035] As used herein, the term "interact" means detectable
interactions between molecules, such as can be detected using, for
example, transmission electron microscopy or fluorescence
microscopy. The term "interact" is also meant to include "binding"
interactions between molecules. Interactions can be, for example,
nucleic acid-nucleic acid, protein-protein or protein-nucleic acid
in nature.
[0036] As used herein, the term "isolated" means oligonucleotides
substantially free of other nucleic acids, proteins, lipids,
carbohydrates or other materials with which they can be associated,
such association being either in cellular material or in a
synthesis medium. The term can also be applied to polypeptides, in
which case the polypeptide will be substantially free of nucleic
acids, carbohydrates, lipids and other undesired polypeptides.
[0037] As used herein, the term "labeled" means the covalent,
noncovalent or ionic attachment of a moiety capable of detection by
electrochemical, spectroscopic, radiologic or other methods to a
probe molecule.
[0038] As used herein, the term "modified" means an alteration from
an entity's normally occurring state. An entity can be modified by
removing discrete chemical units or by adding discrete chemical
units. The term "modified" encompasses detectable labels as well as
those entities added as aids in purification. Any variation from
the normally occurring state, regardless of degree, is encompassed
by the term "modified".
[0039] As used herein, the term "modulate" means an increase,
decrease, or other alteration of any, or all, chemical and
biological activities or properties of a sample which are mediated
by a nucleic acid sequence, a peptide or a small molecule. The term
"modulation" as used herein refers to both upregulation (i.e.,
activation or stimulation) and downregulation (i.e. inhibition or
suppression) of a response or property.
[0040] As used herein, the term "mutation" carries its traditional
connotation and means a change, inherited, naturally occurring or
introduced, in a nucleic acid or polypeptide sequence, and is used
in its sense as generally known to those of skill in the art.
[0041] As used herein, the terms "nano", "nanoscopic"
"nanometer-sized", 4"nanostructured", "nanoscale",
"DNA-nanoparticle complexes" and grammatical derivatives thereof
are used synonymously and interchangeably and mean nanoparticles,
nanoparticle composites and hollow nanocapsules less than or equal
to about 1000 nanometers (nm) in diameter, preferably less than
about 30 nanometers in diameter and more preferably less than about
10 nanometers in diameter. A nanoparticle can be fashioned from any
material. A preferred nanoparticle is fashioned of a semiconductor
material or metal, and more preferably of gold, TiO.sub.2 or gold
or TiO.sub.2-containing materials. Biodegradable materials are also
preferred, e.g. polypeptides. The terms can refer not only to the
metal component of a nanoparticle, but the composite of metal and
other component parts as well.
[0042] The term "nanoparticle" as used herein denotes a carrier
structure which is biocompatible with and sufficiently resistant to
chemical and/or physical destruction by the environment of use such
that a sufficient amount of the nanoparticles remain substantially
intact after injection into the blood stream, given
intraperitoneally or orally or incubated with an in vitro sample so
as to be able to reach the nucleus of a cell or some other cellular
structure. If the drug can enter the cell in the form whereby it is
adsorbed to the nanoparticles, the nanoparticles must also remain
sufficiently intact to enter the cell. Biodegradation of the
nanoparticle is permissible upon entry of a cell's nucleus.
Nanoparticles can be solid colloidal particles ranging in size from
1 to 1000 nm. Nanoparticle can have any diameter less than or equal
to 1000 nm, including 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750
nm. Drugs, active agents, bioactive or other relevant materials can
be incubated with the nanoparticles, and thereby be adsorbed or
attached to the nanoparticle:
[0043] As used herein, the term "nanoparticle metal component"
means a component of a nanoparticle delivery vehicle of the present
invention to which a nuclear localization signal, drugs, bioactive
and other relevant materials are bound. Typically, but not
necessarily, the nanoparticle metal component comprises an
approximately spherical metal atom-comprising entity. Preferably
the nanoparticle metal component is an elemental metal or
semiconductor material, such as a gold or TiO.sub.2 particle.
[0044] As used herein, the term "nuclear localization signal" means
an amino acid sequence known to, in vivo, direct a protein disposed
in the cytoplasm of a cell across the nuclear membrane and into the
nucleus of the cell. A nuclear localization signal can also target
the exterior surface of a cell. Thus, a single nuclear localization
signal can direct the entity with which it is associated to the
exterior of a cell and to the nucleus of a cell. Such sequences can
be of any size and composition, for example more than 25, 25, 15,
12, 10, 8, 7, 6, 5 or 4 amino acids, but will preferably comprise
at least a four to eight amino acid sequence known to function as a
nuclear localization signal (NLS).
[0045] As used herein, the term "pharmaceutically acceptable" and
grammatical variations thereof, as it refers to compositions,
carriers, diluents and reagents, means that the materials are
capable of administration to or upon a vertebrate subject without
the production of undesirable physiological effects such as nausea,
dizziness, gastric upset, fever and the like.
[0046] As used herein, the terms "polypeptide", "protein", "gene
product" and "peptide" are used interchangeably and mean any
polymer comprising any of the 20 protein amino acids, regardless of
its size. Although "protein" is often used in reference to
relatively large polypeptides, and "peptide" is often used in
reference to small polypeptides, usage of these terms in the art
overlaps and varies. The term "polypeptide" as used herein refers
to peptides, polypeptides and proteins, unless otherwise noted. As
used herein, the terms "protein", "polypeptide" and "peptide" are
used interchangeably herein when referring to a gene product.
[0047] As used herein, the term "sequencing" means determining the
ordered linear sequence of nucleic acids or amino acids of a DNA or
peptide (or protein) target sample, using manual or automated
laboratory techniques known in the art.
[0048] As used herein, the term "small molecule" means a molecule
that has a molecular weight of less than or equal to 5000
daltons.
[0049] As used herein, the term "substantially pure" means that the
polynucleotide or polypeptide is substantially free of the
sequences and molecules with which it is associated in its natural
state, and those molecules used in the isolation procedure. The
term "substantially free" means that the sample is at least 50%,
preferably at least 70%, more preferably 80% and most preferably
90% free of the materials and compounds with which is it associated
in nature.
[0050] As used herein, the term "targeting agent" means any agent
having the ability to direct a moiety associated with the targeting
agent to the surface of a cell, to the surface of a particular type
of cell, or to the nucleus of a cell. A targeting agent can
comprise, but is not limited to, proteins, peptides, small
molecules, oligonucleotides, morpholino oligonucleotides and
peptide nucleic acids. A targeting can be of any size, as long as
it retains its ability to direct a moiety associated with the
targeting agent to the surface of a cell or to the surface of a
particular type of cell.
[0051] As used herein, the term "therapeutic agent" means any agent
having a therapeutic effect, including but not limited to
chemotherapeutics, toxins, radiotherapeutics, or radiosensitizing
agents. Also encompassed by the term are gene therapy vectors,
antisense nucleic acid constructs and transcription factor
decoys.
[0052] As used herein, the term "transcription factor" means a
polypeptide that is involved in the transcription of DNA.
Transcription factors can, but are not required to bind DNA. A
transcription factor can function in response to an external
stimulus, or a transcription factor's action can be
constitutive.
[0053] As used herein, the terms "transcription factor decoy" and
"decoy" are used interchangeably and mean molecules that bind to or
interact with transcription factors and/or prevent their binding to
native enhancer sequences. Decoys include nucleic acid sequences,
including, but not limited to, oligonucleotides that correspond to
(i.e., are identical to or essentially identical to) the native
enhancer. Such oligonucleotides include, but are not limited to:
single stranded palindromic oligonucleotides comprising one or more
repeats of the enhancer sequence; sense and antisense
oligonucleotides comprising one or more repeats of the enhancer
sequence; oligonucleotides that form hairpin structures such that a
duplex binding site for the transcription factor is generated; and
one or more oligonucleotides that form a cruciform structure such
that one or more binding sites for the transcription factor are
generated; and double stranded DNA sequences that have a higher
affinity for a genomic binding site of a transcription factor than
does the natural DNA sequence.
[0054] As used herein, the term "wild-type" means the naturally
occurring form of a protein or nucleic acid sequence. The term is
not used to denote a baseline from which a mutation is established.
The term "wild-type" is meant to describe the form of a protein or
nucleic acid sequence as it is most commonly found in nature.
II. General Considerations
[0055] The present invention pertains in part to the regulation and
modulation of gene expression. Gene expression can be regulated by
placing a foreign DNA or RNA oligonucleotide (or an analog such as
phosphorothioate DNA/RNA) in the cell for the purpose of (a)
incorporation into the genome; (b) expression of a gene (which is
sometimes considered "transient transfection"); (c) altering
regulatory protein concentrations (wherein a vehicle can act as a
transcription factor decoy); (d) altering RNA splicing; (e) binding
to messenger RNA in the cytoplasm, (f) RNA interference or (g)
altering the expression of a segment of DNA by inducing the
formation of untranscribable structures, such as a triple helix.
The strategy for (a), (b) and (g) generally involves the delivery
of a relatively long double stranded DNA oligomer to the nucleus.
The strategy for (c) through (g) can involve a short DNA oligomer
that is an operator (i.e. sequence of DNA known to act as a binding
site) for a particular regulatory protein.
[0056] The strategy for (d) and (e) can involve RNA, DNA or analogs
such as peptide nucleic acids and morpholino DNA/RNA
oligonucleotides as well as the use of antisense oligonucleotides.
However, there has been a great deal of difficulty implementing
antisense strategies. At the present time, it is thought that
perhaps this is related to a delivery problem or, alternatively,
that the cell has a mechanism for overcoming the reduction in
concentration of a particular message. In either case, delivery of
oligonucleotides to the nucleus is still a requirement for
strategies (a) through (d).
[0057] For strategies (e) and (f), it might only be necessary to
deliver an oligonucleotide to the cytoplasm, but this will depend
on how the oligonucleotide is to be intercepted. Consequently, the
controlled delivery of oligonucleotides is a key to understanding
the mechanism and effecting control of gene expression. The present
invention directly addresses this historic antisense problem.
[0058] A goal of the present invention is the regulation and/or
perturbation of gene expression. This is of use both for research
purposes and also in therapeutic applications. Delivery is a major
obstacle in the use of oligonucleotides and chemically modified
oligonucleotides for these applications. Nanoparticles of various
compositions can be used to achieve a desired result. Materials
such as titanium, titanium dioxide, tin, tin oxide, silicon,
silicon dioxide, iron, iron.sup.III oxide, silver, nickel, gold,
copper, aluminum and other materials can be used, however gold is a
preferred material. Gold nanoparticles possess several advantages.
First, gold nanoparticles offer the ability to easily regulate
nanoparticle size and, as explained below, subcellular
localization. Additionally, synthesis of such nanoparticles is
facile, and many art-recognized techniques are available.
[0059] Nanoparticles can be conveniently produced by known methods,
including emulsion polymerization in a continuous aqueous phase,
emulsion polymerization in continuous organic phase, interfacial
polymerization, solvent deposition, solvent evaporation,
dissolution of an organic polymer solution, cross-linking of
water-soluble polymers in emulsion, dissolution of macromolecules,
and carbohydrate cross-linking. These fabrication methods can be
performed with a wide range of materials. Metal atoms, and
structures comprising metal atoms, can also serve as effective
nanoparticles. Nanoparticles can be solid or can comprise a hollow
structure that can contain a material.
[0060] A delivery vehicle of the present invention can comprise one
or more appropriate oligonucleotides associated with a
nanoparticle. Next, a nuclear localization signal or other
localization peptides that will help with transport and direct the
nanoparticle to the nucleus are associated with the nanoparticle.
The size of the nanoparticle can be used to prevent transport to
the nucleus when this is desirable Finally, appropriate proteins
can also be localized on the surface of the particle. Appropriate
proteins can comprise ligases, restriction enzymes or other DNA
processing enzymes useful for a given application. The localization
and effect of the delivery vehicle can then be identified using
transmission electron microscopy, Raman microscopy, confocal
microscopy and other analytical techniques for determining the
concentration and localization of the active nanoparticles in the
cell. Even delivery vehicles comprising nanoparticles as small as 5
nm can be identified using one or more of the above analytical
techniques.
[0061] Thus, the present invention provides a novel approach to
solving the problems of nanoparticles as drug delivery vehicles
encountered in the art. Specifically, the present invention
discloses nanoparticles that do not necessarily encapsulate a
biologically active structure, but rather serve as a scaffold for
the biologically active structure to be attached to the surface of
the nanoparticle. Significantly, the nanoparticles of the present
invention can also comprise a nuclear localization signal, which
can target a therapeutic agent to the nucleus of a cell. Until the
disclosure of the present invention, nuclear localization signals
have not been used to direct a nanoparticle across both the plasma
membrane and the nuclear membrane.
[0062] In another aspect of the present invention, a plurality of
sequences can be associated with a nanoparticle delivery vehicle.
Various sequences, such as RME sequences, can also be associated
with a vehicle. These additional sequences can aid in the
translocation of a vehicle across various membranes, such as the
nuclear membrane of a cell or the outer membrane of a cell. Thus,
if membranes and other structures that generally inhibit
translocation of a vehicle to a given location in or on a cell are
analogized as "locks", NLS and RME sequences can be analogized to
be "keys". Thus, in a preferred embodiment, a nanoparticle delivery
vehicle of the present invention can comprise a plurality of
different sequences or "keys," which can enable a given
nanoparticle delivery vehicle to pass through various potential
barriers to translocation and can provide for the targeting of a
variety of different cell types.
[0063] The present invention describes a nanoparticle delivery
vehicle that can be used with a variety of subjects including warm
blooded animals, particularly mammals, including humans, dogs, cats
and other small animals, and farm animals. Additionally, the
nanoparticles of the present invention can be used with prokaryotic
and eukaryotic microorganisms and with in vitro cultures. The
nanoparticle delivery vehicle of the present invention can be used
as a diagnostic agent in all of the above subjects, as well as in
the capacity of a therapeutic agent. There is no limitation on the
type of biologically active structure the subject to which a
nanoparticle of the present invention can be introduced. See, e.g.,
U.S. Pat. Nos. 5,783,263 and 6,106,798. See also, Colloidal Drug
Delivery Systems, (1994) (Kreuter, ed.), Marcel Dekker, Inc., New
York, pp 219-342; Kreuter, (1994) Eur. J. Drug Metab. Ph. 3:
253-56.
IV. Selection and Preparation of a Targetable Nanoparticle Delivery
Vehicle
[0064] A targetable nanoparticle delivery vehicle of the present
invention preferably comprises at least three components: a
nanoparticle, one or more targeting agents (e.g. "keys" such as
nuclear localization signals and cell surface targeting signals)
and one or more active agents. The active agent can be one or more
of any chemical entity, for example, a peptide sequence, a single
stranded nucleic acid oligomer, a double stranded nucleic acid
oligomer, a peptide nucleic acid or a small molecule. These three
components are prepared and joined together to function as a
delivery vehicle, which can be targeted a cell's nucleus via the
nuclear localization signal. Active agents and nuclear localization
signals can be synthesized using primers or templates previously
associated with the nanoparticle. The nuclear localization signal
directs the translocation of the delivery vehicle to the nucleus of
a cell, whereupon the active agent can interact with one or more
proteins or nucleic acids to facilitate a desired effect. If a
nanoparticle of larger size is selected, for example greater than
or equal to about 30 nm, the delivery vehicle will be translocated
to a cell's cytoplasm.
[0065] A targetable nanoparticle delivery vehicle of the present
invention can further comprise an extracellular targeting agent. In
this embodiment, a nanoparticle delivery vehicle can comprise two
targeting signals. First, a targeting agent can be selected which
can direct a delivery vehicle to the surface of a target structure
that recognizes the selected targeting agent. Second, a nuclear
localization sequence can be included, which will direct a delivery
vehicle to the nucleus of a target structure.
[0066] IV.A. Selection and Preparation of a Nanoparticle
[0067] There are no limits on the physical parameters of a
nanoparticle component of the present invention, although the
design of a delivery vehicle should take into account the
biocompatibility of the nanoparticle vehicle, where appropriate.
The physical parameters of a nanoparticle vehicle can be optimized,
with the desired effect governing the choice of size, shape and
material. Preferred particle sizes for transport to a cell's
nucleus are on the order of 5 nm although, as discussed below,
larger particles might be desired for a given application.
Additionally, particles smaller than about 25 nm in diameter are
preferred for use in nuclear targeting to facilitate entry into the
nucleus via a nuclear pore. (Feldherr & Akin, (1990) Electron
Microsc. Rev. 3(1):73-86; Feldherr et al., (1992) Proc. Natl. Acad.
Sci. U.S.A. 89:11002-5; Feldherr & Akin, (1999) J. Cell Sci.
112:2043-48; Feldherr & Akin, (1994) Exp. Cell Res.
215:206-10.)
[0068] The nanoparticle, which can also be referred to as a
scaffold, of a nanoparticle delivery vehicle can comprise a variety
of inorganic materials including, but not limited to, metals,
semi-conductor materials or ceramics. Preferred metal-based
compounds for the manufacture of nanoparticles include titanium,
titanium dioxide, tin, tin oxide, silicon, silicon dioxide, iron,
iron.sup.III oxide, silver, gold, copper, nickel, aluminum, steel,
cobalt-chrome alloys, cadmium (preferably cadmium selenide) and
titanium alloys. Preferred ceramic materials include brushite,
tricalcium phosphate, alumina, silica, and zirconia. The
nanoparticle can be made from organic materials including carbon
(diamond). Preferred polymers include polystyrene, silicone rubber,
polycarbonate, polyurethanes, polypropylenes,
polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers,
and polyethylene. Biodegradable, biopolymer (e.g. polypeptides such
as BSA, polysaccharides, etc.), other biological materials (e.g.
carbohydrates), and/or polymeric compounds are also suitable for
use as a nanoparticle scaffold. Gold is especially preferred due to
its well-known reactivity profiles and biological inertness.
[0069] Nanoparticles comprising the above materials and having
diameters less than 1,000 nanometers are available commercially or
they can be produced from progressive nucleation in solution (e.g.,
by colloid reaction), or by various physical and chemical vapor
deposition processes, such as sputter deposition. See, e.g.,
Hayashi, (1987) Vac. Sci. Technol. July/August 1987, A5(4):1375-84;
Hayashi, (1987) Physics Today, December 1987, pp. 44-60; MRS
Bulletin, January 1990, pgs. 16-47.
[0070] Alternatively, nanoparticles can be produced using
HAuCl.sub.4 and a citrate-reducing agent, using methods known in
the art. See, e.g., Marinakos et al., (1999) Adv. Mater. 11: 34-37;
Marinakos et al., (1998) Chem. Mater. 10: 1214-19; Enustun &
Turkevich, (1963) J. Am. Chem. Soc. 85: 3317. Tin oxide
nanoparticles having a dispersed (in H.sub.2O) aggregate particle
size of about 140 nm are available commercially from Vacuum
Metallurgical Co., Ltd. of Chiba, Japan. Other commercially
available nanoparticles of various compositions and size ranges are
available, for example, from Vector Laboratories, Inc. of
Burlingame, Calif. Biodegradable, ceramic and polymeric
nanoparticle materials will be known to those of skill in the art
and can comprise a biodegradable composition.
[0071] Besides sputter deposition, plasma-assisted chemical vapor
deposition (PACVD) is another technique that can be used to prepare
suitable nanoparticles. PACVD functions in relatively high
atmospheric pressures (on the order of one torr and greater) and is
useful for generating particles having diameters of about 1000
nanometers and smaller. For example, aluminum nitride particles
having diameters of less than 1000 nanometer can be synthesized by
PACVD using Al(CH.sub.3).sub.3 and NH.sub.3 as reactants. The PACVD
system typically includes a horizontally mounted quartz tube with
associated pumping and gas feed systems. A susceptor is located at
the center of the quartz tube and heated using a 60 KHz radio
frequency source. The synthesized aluminum nitride particles are
collected on the walls of the quartz tube. Nitrogen gas is commonly
used as the carrier of the Al(CH.sub.3).sub.3. The ratio of
Al(CH.sub.3).sub.3:NH.sub.3 in the reaction chamber is controlled
by varying the flow rates of the N.sub.2/Al(CH.sub.3).sub.3 and
NH.sub.3 gas into the chamber. A constant pressure in the reaction
chamber of 10 torr is generally maintained to provide deposition
and formation of the ultrafine aluminum nitride nanoparticles.
PACVD can be used to prepare a variety of other suitable
biodegradable nanoparticles.
[0072] The size of a nanoparticle can be an important
consideration. Larger nanoparticles, on the order of greater than
or equal to about 30 nm, are seen to enter cells, but are not
translocated across the nuclear membrane into the nucleus of a
cell, as seen in FIG. 1B. Presumably, this effect is directly
related to the size of the nanoparticle, since 5 nm nanoparticles
do cross the nuclear membrane and are translocated into the
nucleus, as seen in FIG. 1A. Thus, selection of the appropriate
delivery vehicle size will be important, but also offers an
additional level of targetability and facilitates the design and
employment of nanoparticles carrying active agents that need to be
located in the cytoplasm and not the nucleus.
[0073] IV.B. Selection and Preparation of an Active Agent
[0074] Having selected and prepared a nanoparticle to which at
least a nuclear localization signal and preferably another,
different targeting agent are attached, a desired active agent is
selected and prepared. Appropriate active agents can comprise any
small molecule, protein or nucleic acid sequence, and the selection
is governed by the intended application of the delivery vehicle.
Various applications of nanoparticle delivery vehicles of the
present invention are discussed in depth below, and include gene
therapy, modulation of gene expression, altering RNA splicing and
modulation of protein-protein interactions. Appropriate active
agents will be apparent to one of skill in the art upon review of
the present disclosure and will be selected with to regard to a
desired experimental or clinical goal.
[0075] It is also an aspect of the present invention to provide a
nanoparticle delivery vehicle comprising two or more different
active agents, e.g. 2, 3, 4, 5, or other desired number of
different active agents. Thus, delivery of the different active
agents can be accomplished in the same cellular or tissue location,
or in two or more different cellular or tissue locations, depending
on the targeting sequences that are employed.
[0076] Any combination of any of the active agents disclosed herein
can be provided. For example, a nanoparticle delivery vehicle
comprising a therapeutic agent and an imaging agent can be
provided, for use in for example, delivery to a tumor. As another
example, a nanoparticle delivery vehicle comprising a
chemotherapeutic agent and a radiosensitizing agent can be
provided. As yet another example, a nanoparticle delivery vehicle
comprising different polynucleotide sequences for use in modulation
of transcription and/or translation of the same or different genes
can be provided.
[0077] IV.B.1. Selection of an Active Agent
[0078] Generally, a single stranded nucleic acid sequence
appropriate for use as an active agent in the present invention can
be selected on the basis of the context in which the present
invention is employed. In one embodiment, appropriate single
stranded DNA are complementary to a nucleic acid sequence known or
suspected to be present in a disease condition. In another
embodiment, appropriate single stranded DNA is complementary to an
overexpressed gene. Functional equivalents of known sequences can
also be used as active agents and are considered to be an aspect of
the present invention. Nucleic acid sequences of any manageable
length can be used as an active agent. Typically, such agents range
between about 20 and about 50 nucleotides in length, although
longer sequences can be used. In yet another embodiment, a nucleic
acid sequence corresponding to a full-length gene, or a fragment
thereof, can be used as an active agent.
[0079] Double stranded DNA of various lengths and compositions is
suitable for use as an active agent in the present invention.
Double stranded DNA can be of any length, from a few base pairs up
to the length of a full-length gene. As discussed below, long
lengths of DNA, and notably full-length genes, find utility in gene
therapy applications. In this embodiment, full-length genes can be
incorporated into a host cell's genome, or can be transiently
expressed within the cell. In this embodiment, then, a cell is
lacking a particular gene and an appropriate double stranded DNA
sequence selected as an active agent is the gene absent from the
cell's genome.
[0080] Nucleic acid analogs can also be used as active agents in
the present invention. In one aspect of the present invention,
peptide nucleic acid analogs (PNAs) can be used as active agents. A
peptide nucleic acid analog is a DNA analog wherein the backbone of
the analog, normally a sugar backbone in DNA, is a pseudopeptide. A
PNA backbone can comprise a sequence of repeated
N-(2-amino-ethyl)-glycine units. Peptide nucleic acid analogs react
as DNA would react in a given environment, and can additionally
bind complementary nucleic acid sequences. Peptide nucleic acid
analogs offer the potential advantage over unmodified DNA of the
formation of stronger bonds, due to the neutrally charged peptide
backbone of the analogs, and can impart a higher degree of
specificity than is achievable by unmodified DNA.
[0081] PNAs have been employed in a wide array of biochemical
roles, which is applicable to the present invention, including
sequence mapping. In vitro studies indicate that PNA could inhibit
both transcription and translation of genes to which it has been
engineered with a complementary sequence. This suggests that PNAs
could be useful in antigene and antisense therapy. e.g., Norden et
al., (2000) FASEB J. 14(9): 1041-60. To date, however, researchers
have been unable to reproducibly target such a sequence to the cell
nucleus from outside the plasma membrane.
[0082] The present invention addresses this problem and offers
potential for heretofore unattainable applications of PNAs. PNAs
suitable for use as active agents in the present invention will,
therefore, comprise a sequence complementary to a sequence of
interest. Other nucleic acid analogs useful in the context of the
present invention include morpholino nucleic acid analogs.
Morpholino analogs can be substituted for a nucleic acid sequence
and has the benefits of both complementarity and a unique chemical
reaction and binding profile not found in native nucleic acid
sequences. See, e.g., Chakhmakhcheva et al., (1999) Nucleos.
Nucleot. 18: 1427-28.
[0083] Additionally, proteins are appropriate for use as an active
agent in the present invention. In one embodiment, appropriate
proteins comprise proteins known to interact with proteins
associated with DNA replication and expression, for example,
ligases. In this embodiment, a nanoparticle-bound protein can be a
protein that interacts with DNA or RNA sequences, possibly as an
up- or downregulator of the transcription process, the translation
process or both. In an alternative embodiment, the present
invention can be used to prove or disprove a putative
protein-protein interaction. In this case, the nanoparticle-bound
sequence is a probe protein and the application of the invention
can give data similar to that achievable using the
well-characterized yeast two-hybrid system and other analytical
systems, although the present invention affords the opportunity to
examine such an interaction in situ. More specifically, proteins
capable of interacting with receptors on nuclear regulatory
proteins can be employed as active agents.
[0084] Finally, small molecules attached to a nanoparticle can be
used as active agents in the present invention. Appropriate small
molecules will have the ability to interact with enzymes,
cofactors, nucleic acids and other intracellular structures. Small
molecules can be those identified as natural ligands, inhibitors
(competitive, uncompetitive and noncompetitive) or designed
modulators. Chemotherapeutic agents, radiotherapeutics, or
radiosensitizing agents; an imaging agent; a diagnostic agent; or
other agent known to interact with an intracellular protein, a
nucleic acid or a soluble or insoluble ligand can also be used as
active agents.
[0085] It should be noted that the use of one of the above active
agents does not preclude the binding of a different active agent to
the nanoparticle and several active agents can be joined to a
single nanoparticle. Moreover, active agents can be multivalent
and/or multifunctional.
[0086] IV.B.2. Preparation of an Active Agent
[0087] Nucleic acid sequences useful as active agents in the
context of the present invention can be prepared in a variety of
ways and will be apparent to one of skill in the art upon review of
the present disclosure. For example, an appropriate DNA sequence
can be excised from a larger DNA sample using restriction
endonucleases, which sever nucleic acid sequences at known
sequences. Excised nucleic acid sequences can be excised and
purified using methods known in the art. See, e.g., Sambrook et
al., (1992) Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, N.Y. for a general discussion of cloning strategies.
Alternatively, and more preferably, nucleic acid sequences can be
synthesized using well-known manual and automated nucleic acid
synthesis methods. All nucleic acid sequences that are used as
active agents, whether they are excised, synthesized or otherwise
prepared, should be substantially pure. Synthesis of nucleic acid
or protein active agents can proceed using a template previously
associated with a nanoparticle.
[0088] Isolation and purification of proteins will correspond with
techniques established for preparation of a given protein; those
proteins of interest that have not been purified can be isolated
using methods known to those of skill in the art and are not
discussed here. Similarly, strategies of synthesizing and purifying
small molecules can be found in the art and will be evident to one
of skill in the art of organic chemistry or other chemical
discipline.
[0089] IV.C. Selection and Preparation of a Nuclear Localization
Signal
[0090] The inclusion of a nuclear localization signal (NLS) as a
delivery vehicle component is an aspect of; the present invention.
A representative nuclear localization signal is a peptide sequence
that directs the protein to the nucleus of the cell in which the
sequence is expressed. A nuclear localization signal is
predominantly basic, can be positioned almost anywhere in a
protein's amino acid sequence, generally comprises a short sequence
of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem.
273: 14731-37) to eight amino acids, and is typically rich in
lysine and arginine residues (Magin et al., (2000) Virology 274:
11-16). Nuclear localization signals often comprise proline
residues. A variety of nuclear localization signals have been
identified and have been used to effect transport of biological
molecules from the cytoplasm to the nucleus of a cell. See, e.g.,
Tinland et al., (1992) Proc. Natl. Acad. Sci U.S.A. 89:7442-46;
Moede et al., (1999) FEBS Lett. 461:229-34. Translocation is
currently thought to involve nuclear pore proteins.
[0091] In a preferred embodiment of the present invention, a
nuclear localization signal be attached to the nanoparticle. The
nuclear localization signal can be synthesized or excised from a
larger sequence. As noted, a variety of nuclear localization
signals are known and selection of an appropriate sequence can be
made based on the known properties of these various sequences.
Representative NLS's include the monopartite sequence PKKKRKV (SEQ
ID NO: 1) and the bipartite sequence KRPAAIKKAGQAKKKK (SEQ ID NO:
2).
[0092] Nuclear localization signals appear at various points in the
amino acid sequences of proteins. NLS's have been identified at the
N-terminus, the C-terminus and in the central region of proteins.
Thus, a selected sequence can serve as the functional component of
a longer peptide sequence. The residues of a longer sequence that
do not function as component NLS residues should be selected so as
not to interfere, for example ionically or sterically, with the
nuclear localization signal itself. Therefore, although there are
no strict limits on the composition of an NLS-comprising sequence,
in practice, such a sequence can be functionally limited in length
and composition.
[0093] In another aspect of the present invention, a plurality of
sequences can be associated with a nanoparticle delivery vehicle.
Various sequences, such as RME sequences, can also be associated
with a vehicle. These additional sequences can aid in the
translocation of a vehicle across various membranes, such as the
nuclear membrane of a cell or the outer membrane of a cell. Thus,
if membranes and other structures that generally inhibit
translocation of a vehicle to a given location in or on a cell are
analogized as "locks", NLS and RME sequences can be analogized to
be "keys". Thus, in a preferred embodiment, a nanoparticle delivery
vehicle of the present invention can comprise a plurality of
different sequences or "keys," which can enable a given
nanoparticle delivery vehicle to pass through various potential
barriers to translocation.
[0094] IV.D. Selection and Preparation of a Cell Surface Receptor
Recognition Moiety
[0095] In one aspect of the present invention, a moiety that
imparts the ability to be recognized and/or bound by a cell surface
receptor is bound to the nanoparticle. This moiety will generally
comprise a protein sequence known to be recognized by a cell
surface receptor. Preferably, the cell surface receptor recognition
moiety can further comprise a nucleic acid sequence, either alone
or as part of a nucleic acid-protein hybrid, or peptide analog. A
vast number of cell surface receptors are known and can be useful
in the present invention, including the macrophage mannose receptor
and its various homologs, and those associated with retroviruses
such as HIV.
[0096] A representative, but non-limiting, list of moieties that
can be employed as targeting agents in the present invention is
presented in Table 1. Homologs of the presented moieties can also
be employed. These targeting agents can be associated with a
nanoparticle and used to direct the nanoparticle to a target
structure, where it can subsequently be internalized. There is no
requirement that the entire moiety be used as a targeting agent.
Smaller fragments of these moieties known to interact with a
specific receptor or other structure can also be used as a
targeting agent. The targeting agents of Table 1 can function to
internalize (e.g., by receptor mediated endocytosis) a delivery
agent interacting with a targeting agent.
TABLE-US-00003 TABLE 1 Diptheria Toxin Pseudomonas toxin Cholera
toxin Ricin Concanavalin A Rous sarcoma virus Semliki forest virus
Vesicular stomatitis virus Adenovirus Transferrin Low Density
Lipoprotein Transcobalamin Yolk Proteins IgE Polymeric IgA Maternal
IgG IgG, via Fc receptors Insulin Epidermal Growth Factor Growth
Hormone Thyroid Stimulating Hormone Nerve Growth Factor Calcitonin
Glucagon Prolactin Luteinizing Hormone Thyroid hormone Platelet
Derived Growth Factor Interferon Catecholamines Nuclear
Localization Signal
[0097] The recognition moiety can further comprise a sequence that
is subject to enzymatic or electrochemical cleavage. The
recognition moiety can thus comprise a sequence that is susceptible
to cleavage by enzymes present at various locations inside a cell,
such as proteases or restriction endonucleases (e.g. DNAse or
RNAse).
[0098] It must be emphasized that a cell surface recognition
sequence is not an absolute requirement for the present invention.
Indeed, as shown in FIG. 1A, hepatocytes grown in media containing
nanoparticles lacking a cell surface recognition sequence were
translocated across the cell membrane, in the absence of such a
sequence. Thus, although a cell surface receptor sequence can be
useful for targeting a given cell type, or for inducing the
association of a nanoparticle with a cell surface, there is no
requirement that a cell surface recognition sequence be present on
the surface of a nanoparticle in order to practice the present
invention.
[0099] The presence of a cell surface receptor unique to a given
type of cell can assist in the selection and delivery of an active
agent to that cell type. For example, macrophages express a cell
surface receptor (the macrophage mannose receptor) that mediates
pinocytosis of particles that comprise mannose through carbohydrate
recognition domains. See, Mullin et al., (1997) J. Biol. Chem. 272:
5668-81. Thus, the selection of an appropriate cell surface
receptor can facilitate cell specific selection by a nanoparticle
delivery vehicle and consequently, cell specific interaction.
[0100] IV. E. Selection and Preparation of a Tether Sequence
[0101] In another aspect of the present invention, a short tether
sequence can be disposed between the nanoparticle and a cell
surface recognition sequence of a nanoparticle delivery vehicle of
the present invention. The tether can be a protein sequence, a
nucleic acid sequence or any other composition that is compatible
with an intracellular environment. In the present invention,
protein and nucleic acid sequences are preferred due to their
enzymatic cleavability. For example, a nucleic acid tether that
comprises a known cut site for a restriction endonuclease found in
the targeted cell can be employed. Alternatively, a protein tether
can be employed that comprises a cut site for a protease commonly
found in the targeted cell type. Finally, a tether can be designed
that can be chemically or electrochemically cleaved.
[0102] Cleavage of a tether is one method by which the
nanoparticle, which will comprise an active agent and a nuclear
localization signal, can be freed to translocate across the nuclear
membrane and into the nucleus of a cell. In one example, upon
association of a cell surface receptor with a cell surface
recognition sequence disposed on the surface of a nanoparticle of a
delivery vehicle of the present invention, the nanoparticle
delivery vehicle is, in effect, bound to the cell surface. Upon
translocation of the delivery vehicle to the interior of a cell, by
endocytosis or other mechanism, the nanoparticle delivery vehicle
remains bound to the receptor. In order to free the nanoparticle
delivery vehicle and its associated active agent, the tether can be
cleave by endogenous proteases, nucleases or other chemical or
electrochemical techniques. Cleavage of the tether sequence frees
the nanoparticle delivery vehicle and enables subsequent direction
of the delivery vehicle to the nucleus, via the nuclear
localization signal disposed on the nanoparticle surface.
[0103] IV.F. Assembly of a Nanoparticle Delivery Vehicle
[0104] Having selected and prepared the various individual
components of a nanoparticle delivery vehicle of the present
invention, the agent itself is then assembled. The order of
assembly is not critical and can be governed primarily by the
requirements of a desired chemical reaction. The chemical
properties of an active agent, an NLS, a nanoparticle, as well as
physiological and various other considerations, should also be
weighed. Thus, the assembly procedure described hereinbelow is
presented in an arbitrary order. Further, the materials described,
i.e. composition of the nanoparticle, etc., are presented only as
examples and are not meant to be limiting in any way. Suitable
materials and sequences will be known to those of skill in the art
when evaluated in light of the present disclosure and the knowledge
and resources available to researchers in the applicable
fields.
[0105] IV.F.1. Association of an Active Agent with a
Nanoparticle
[0106] Following selection and preparation of a nanoparticle and a
suitable active agent, the two components are joined to form a
complex. In one embodiment, the nanoparticle is fashioned of gold.
Gold is particularly useful in the present invention due to its
well-known reactivity profile and its relatively inertness in the
context of biological systems. Colloidal gold can be used, although
the overall negative charge of common preparations of gold imparts
the quality that colloidal gold has a high non-specific affinity
for certain proteins. The negative charge of a preparation can be
imparted by association of gold molecules with negatively-charged
ligands such as citrate or bis-sulfonatotriphenylphosphine. These
negatively-charged ligands, and thus the overall negative charge
associated with a colloidal gold preparation, are preferably
reduced or eliminated by exchanging any negatively-charged ligands
with neutral ligands, such as polyethylene glycol, or
positively-charged ligands, such as amines.
[0107] Alternatively, the use of colloidal gold can be accompanied
by additional treatments such that it can be coated at some point
in the preparation process with another protein, such as BSA, in
order to block undesired nonspecific protein binding. Small
molecule and peptide coatings can also be used to avoid specific
interactions with cellular proteins. For example, colloidal gold
has been prepared with a coating of glutathione. Since glutathione,
a natural antioxidant, is one of the most abundant peptides in the
cell, this coating may impart to the nanoparticle a camouflage-like
effect. Alternatively, some gold clusters and particles are
commercially available and can be used in the present invention.
For example, NANOGOLD.RTM. gold particles are available from
Nanoprobes, Inc., Yaphank, N.Y. Also preferable for use in the
present invention are biodegradable particles, which can be
fashioned of an appropriate polymer or other material. Some
commercially available nanoparticles are prepared for labeling
prior to shipping and are convenient for attaching entities to the
nanoparticles.
[0108] In one embodiment, using a gold nanoparticle as the
nanoparticle and a single or double stranded nucleic acid as an
active agent, a thiolation reaction can be performed to add a thiol
group to the 5' end of the nucleic acid oligomer. Alternatively, an
amination reaction can be performed and will proceed mutatis
mutandis to the thiolation reaction described herein. The general
purpose of the reaction is to introduce a nucleophilic center,
which can subsequently be functionalized with a desired moiety. A
representative thiol modifier phosphoramidite reagent is presented
as Compound 1, which is available from Glen Research, Inc. of
Sterling, Va.
##STR00001##
[0109] Nucleic acid oligomers are incubated with a thiol modifier
phosphoramidite under conditions that permit attachment of the
phosphine to the 5' end of the probe DNA. The reaction can be
carried out in a DNA synthesizer using standard conditions.
Compound 1 can be added as a step in automated DNA synthesis using
an automated apparatus, such as the ABIT.TM. 3900 high-throughput
DNA synthesizer (Applied Biosystems, Foster City, Calif.). The
thiol modifier is added in the last step of synthesis of an
oligonucleotide. The phosphine is oxidized using iodine and the
purification is exactly the same as that used for unlabeled
oligonucleotides. The purification process is easier for labeled
oligonucleotides since labeled oligonucleotides are significantly
more hydrophobic and therefore tend to elute much more slowly under
typical HPLC conditions. The phosphoramidite reacts spontaneously
with the 5' hydroxyl of DNA, which can be disposed in acetonitrile.
In this reaction, the thiol group is protected by a protecting
trityl or acetic thioester group and is separated from the
5'-phosphodiester by a variable length carbon linker. A six-carbon
linker is present in Compound 1.
[0110] The nucleic acid complex is then subjected to thiol
deprotection to remove the trityl group. Specifically, the
protecting trityl group is removed by treatment with silver nitrate
and dithiothreitol (DTT). The nucleic acid complex is incubated
with a nanoparticle metal component. The two entities are joined at
the thiol exposed by the removal of the trityl group during the
deprotection reaction. The formed active agent-nanoparticle
complexes (in this embodiment nucleic acid-nanoparticle complexes)
can be maintained in the reaction vessel until use.
[0111] IV.F.2. Association of a Nuclear Localization Signal with a
Nanoparticle
[0112] A suitable nuclear localization signal is joined to the
active agent-nanoparticle complex. Nuclear localization signals can
be synthesized using standard peptide chemistry techniques, or can
be isolated by proteolytic cleavage from a larger protein. Isolated
or synthesized nuclear localization signals can be of any size,
with the only requirement that the sequence comprise at least a
known NLS, which are typically four to eight amino acids in length.
Protein and peptide purification methods suitable for preparing
nuclear localization signals, which are isolated from larger
proteins, are known in the art. See generally, Protein Purification
Applications: A Practical Approach, (1989) (Harris & Angal,
eds.) IRL Press; Protein Purification: Principles, High Resolution
Methods, Applications, (1989) (Janson & Ryden, eds.) VCH
Publishers.
[0113] The present invention also encompasses the preparation of
and association of a protein-peptide conjugate with a nanoparticle.
Such a conjugate can comprise, for example, a comparatively large
protein and a comparatively small NLS. Although both entities
comprise amino acids, the distinction of protein and peptide is
made based on, among other criteria, the functionality of each
entity. In a specific example, a protein-peptide conjugate can
comprise BSA and an NLS. Protein-peptide conjugates can
subsequently be bound to gold nanoparticles.
[0114] The chemistry of attaching proteins and peptides to gold
nanoparticles is similar to the chemistry required for attaching
nucleic acids to gold nanoparticles. In one aspect, a thiol
reaction is performed. The reaction can involve a thiol group
disposed on the nuclear localization signal, which can take the
form of a terminal cysteine or methionine residue, or on the
nanoparticle. The thiol group can be convenient reacted with a
primary amine on the alternate entity. The primary amine can
conveniently take the form of a terminal lysine or arginine residue
in the nuclear localization signal, but can also be disposed on the
surface of the nanoparticle. See, e.g., Hainfeld & Furuya,
(1992) J. Histochem. Cytochem. 40: 177-84; Hainfeld, (1992)
Ultramicroscopy 46: 135-44.
[0115] IV.F.3. Association of a Cell Surface Recognition Sequence
with a Nanoparticle
[0116] An appropriate cell surface recognition sequence can be
selected (for example, one selected from or based on those
presented in Table 1) and prepared as described above in Section
IV.D above. The sequence can be, essentially, a protein or a
peptide known to bind to a receptor expressed on the surface of a
given cell type. Thus, the same chemical reactions can be performed
to associate a cell surface recognition sequence with a
nanoparticle as are performed to associate a nuclear localization
signal or a protein active agent with a nanoparticle. Continuing
with the example from Section IV.F.2 above, a thiol-amine reaction
can be performed to associate a thiol disposed on either the
nanoparticle or the cell surface recognition sequence with a
primary amine disposed on the alternate member of a thiol-amine
reaction pair. Depending on the reactivity profiles and the order
in which the various component parts of a nanoparticle delivery
vehicle are bound to a nanoparticle, a scheme of site blocking can
be developed. Such a scheme can prevent binding of an entity to
undesired sites on the nanoparticle or on the component parts
themselves.
[0117] IV. F.4. Association of a Tether Sequence with a
Nanoparticle
[0118] A tether sequence can also be bound to a nanoparticle. Such
a sequence can be disposed between the nanoparticle and a cell
surface recognition sequence or other entity associated with the
nanoparticle. In order to serve its purpose, the tether sequence
preferably comprises a site at which chemical, electrochemical or
enzymatic cleavage can take place. When a tether sequence comprises
a single or double stranded nucleic acid sequence, the sequence can
comprise a cut site for a nuclease known to be present in the cells
to which the delivery vehicle is being introduced. When the tether
is a protein, it can comprise a proteolytic site. Finally, when it
is desired that the tether be cleaved photolytically, it can
comprise a material amenable to photocleavage. Commercially
available photocleavable tether sequences include various spacer
phosphoramidites, available from Glen Research of Sterling, Va.
[0119] IV.F.5. Biocompatibility and Protection of the Delivery
Vehicle
[0120] If the nanoparticle comprises a metal component such as
gold, it is desirable to assure biocompatibility between the
nanoparticle and a subject to which the delivery vehicle is being
administered. Gold is relatively inert and less physiologically
intrusive than other metals, but the detrimental effects of any
metal or polymer nanoparticle material can be minimized by coating
or otherwise wholly or partly covering the nanoparticle with a
biocompatible substance. Compounds that can be used to achieve
biocompatibility include polymers (such as polyethylene
glycol-PEG), proteins (such as BSA), lipids (including membrane
envelopes) and carbohydrates. Addition of these biocompatibility
compounds can be performed following the addition of the other
delivery vehicle components and can serve as the final synthetic
step before introduction of the delivery vehicle to a subject or
system.
[0121] These materials can also protective or masking agents for
the delivery vehicle and the active agent(s) and targeting agent(s)
attached thereto to prevent recognition by the immune system or
other biological systems (e.g. proteases, nucleases (e.g. DNAse or
RNAse), or other enzymes or biological entities associated with
undesired degradation). Thus, the protective coating or shell
provides cloaking or stealth features to facilitate that the
delivery vehicle reaches a desired cell or tissue with the active
agent(s) and targeting agent(s) intact.
[0122] IV.F.6. Associating Multiple Sequences with a Delivery
Vehicle
[0123] Multiple sequences can be associated with a delivery vehicle
of the present invention. By associating multiple sequences with a
single vehicle, the vehicle can be adapted to pass through various
cellular barriers, such as the cell membrane or a nuclear membrane.
For example, a nanoparticle vehicle can comprise an NLS and a RME
sequence. The RME sequence can assist in the translocation of a
vehicle across the membrane of a cell. Once inside a cell, the NLS
can target the vehicle to the nucleus of the cell.
[0124] Preferably any sequences associated with a nanoparticle
delivery vehicle are independently associated with the vehicle,
rather than forming components of a single long sequence. As
indicated by the results disclosed in Laboratory Example 1,
independent association of multiple sequences (preferably multiple
different sequences) is a more efficient method targeting a
nanoparticle delivery vehicle to a desired cellular structure.
However, sequential association of multiple sequences can also be
an effective method of directing a nanoparticle delivery vehicle to
a given site, and this approach forms another aspect of the present
invention.
V. Introduction of a Nanoparticle Delivery Vehicle to a Subject or
Sample
[0125] After a sufficiently pure nanoparticle delivery vehicle
(preferably comprising a nanoparticle, an active agent and an NLS)
has been prepared, it might be desirable to prepare the vehicle in
a pharmaceutical composition that can be administered to a subject
or sample. Preferred administration techniques include parenteral
administration, intravenous administration and infusion directly
into any desired target tissue, including but not limited to a
solid tumor or other neoplastic tissue. This can be achieved by
employing a final purification step, which disposes the vehicle in
a medium comprising a suitable pharmaceutical composition.
[0126] Suitable pharmaceutical compositions in accordance with the
invention generally comprise an amount of the desired delivery
vehicle-active agent in accordance with the dosage information
(which is determined on a case-by-case basis), admixed with an
acceptable pharmaceutical diluent or excipient, such as a sterile
aqueous solution, to give an appropriate final concentration in
accordance with the dosage information set forth above with respect
to the active agent. Such formulations will typically include
buffers such as phosphate buffered saline (PBS), or additional
additives such as pharmaceutical excipients, stabilizing agents
such as BSA or HSA, or salts such as sodium chloride.
[0127] For parenteral administration it is generally desirable to
further render such compositions pharmaceutically acceptable by
insuring their sterility, non-immunogenicity and non-pyrogenicity.
Such techniques are generally well known in the art as exemplified
by Remington's Pharmaceutical Sciences, (1980) (Osol, ed.) 16th
Ed., Mack Publishing Company, Easton, Pa., incorporated herein by
reference. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biological Standards.
[0128] When delivery vehicles are being introduced into cells
suspended in a cell culture, it is sufficient to incubate the cells
together with the nanoparticle delivery vehicles an appropriate
growth media, for example Luria broth (LB) or a suitable cell
culture medium. Although other introduction methods are possible,
these introduction treatments are preferable and can be performed
without regard for the entities present on the surface of a
delivery vehicle.
[0129] When in vitro experiments are to be performed, delivery
vehicles can be added to directly to a selected cell growth medium
before cells are introduced into the medium. Such a medium must,
obviously, be compatible not only with the physiological
requirements of the cells, but also with the chemical and
reactivity profile of the delivery vehicle. The delivery vehicle's
profile will be apparent to one of skill in the art upon review of
the present disclosure and in view of the moieties bound to the
nanoparticle.
[0130] V.A. Receptor Mediated Endocytosis of a Delivery Vehicle
[0131] Recognition and binding of a cell surface recognition
sequence disposed on a nanoparticle delivery vehicle of the present
invention is an aspect of the present invention. The present
invention takes advantage of the understanding that a cell surface
binding event is often the initiating step in a cellular cascade
leading to a range of events, notably receptor-mediated
endocytosis.
[0132] The above methods describe methods by which a delivery
vehicle can be introduced into a sample or subject. These agents
are translocated across the cell membrane in a variety of ways.
However, when a cell recognition sequence is bound to a
nanoparticle, a different type of internalization can occur, namely
receptor mediated endocytosis.
[0133] The term "receptor mediated endocytosis" ("RME") generally
describes a mechanism by which, catalyzed by the binding of a
ligand to a receptor disposed on the surface of a cell, a
receptor-bound ligand is internalized within a cell. Many proteins
and other structures enter cells via receptor mediated endocytosis,
including insulin, epidermal growth factor, growth hormone, thyroid
stimulating hormone, nerve growth factor, calcitonin, glucagon and
many others, including those presented in Table 1. In the context
of the present invention, receptor mediated endocytosis affords a
convenient mechanism for transporting a nanoparticle to the
interior of a cell.
[0134] In RME, the binding of a ligand by a receptor disposed on
the surface of a cell can initiate an intracellular signal, which
can include an endocytosis response. Thus, an agent that is bound
on the surface of a cell is invaginated and internalized within the
cell. Subsequently, any tether sequence present on the nanoparticle
can be cleaved by the cell's endogenous enzymes, thereby freeing
the agent to deliver its active agent to the appropriate
structure.
[0135] It must be reemphasized that RME is not the exclusive method
by which a delivery vehicle can be translocated into a cell. Other
methods of uptake that can be exploited by attaching the
appropriate entity to a nanoparticle include the advantageous use
of membrane pores. Phagocytotic and pinocytotic mechanisms also
offer advantageous mechanisms by which a nanoparticle delivery
vehicle can be internalized.
VI. Detection of a Nanoparticle Delivery Vehicle
[0136] Nanoparticle delivery vehicles of the present invention can
be detected on both the interior and exterior of cells in a variety
of ways. Indeed, the ability to select one of several techniques
for detection is an aspect of the present invention. One method of
detecting the presence of a nanoparticle delivery vehicle is by
monitoring a sample for the homeostatic change the nanoparticle
delivery vehicle is designed to produce. For some applications,
however, it might be desirable to monitor the presence of a
nanoparticle delivery vehicle by a different approach. Several, but
not all, methods of detecting the presence of nanoparticle delivery
agents can include the use of transmission electron, fluorescence
and other microscopy techniques; spectroscopic-based detection; and
detection methods involving proteins, such as immunological
methods. Other methods are possible and will depend on the specific
circumstances of the experiment or treatment protocol.
[0137] VI.A. Transmission Electron Microscopy Detection of a
Nanoparticle Delivery Vehicle
[0138] Transmission electron microscopy (TEM) can be used to
determine the presence of a nanoparticle delivery vehicle.
Nanoparticle delivery vehicles comprising nanoparticles of 5 nm and
larger can be clearly visualized by TEM, as evidenced by the TEM
images presented in FIGS. 1A and 1B. FIG. 1A depicts nanoparticle
delivery vehicles comprising 5 nm nanoparticles. FIG. 1B depicts
nanoparticle delivery vehicles comprising 30 nm nanoparticles. In
both figures, the nanoparticle delivery vehicles comprise a nuclear
localization signal. FIGS. 1A and 1B indicate that TEM is a useful
method of detecting the presence and subcellular localization of
nanoparticle delivery vehicles. Nanoparticle delivery vehicles
comprising nanoparticles as small as 5 nm in size are visible, as
depicted in FIG. 1A.
[0139] FIGS. 1A and 1B demonstrate that TEM can be used to detect
the presence of a nanoparticle delivery vehicle in the nucleus of a
cell. Nanoparticle delivery vehicles comprising 5 nm nanoparticles
locate to the cell nucleus, as shown in FIG. 1A. Nanoparticle
delivery vehicles comprising 30 nm nanoparticles remain in the
cell's cytoplasm, as shown in FIG. 1B. Thus, TEM facilitates the
detection of nanoparticle delivery vehicles and the subcellular
localization of the delivery vehicles.
[0140] TEM can also be used to estimate the density of nanoparticle
delivery vehicles in a region. A density calculation can be
performed by counting the number of observed particles in a given
area scanned by TEM. An understanding of the density of
nanoparticle delivery vehicles in a defined region, such as a
cell's nucleus or cytoplasm, can provide information regarding the
size requirements for a nanoparticle, the effectiveness of a given
nuclear localization signal and other parameters.
[0141] VI.B. Spectroscopic Detection of a Nanoparticle Delivery
Vehicle
[0142] Nanoparticle delivery vehicles of the present invention can
also be detected spectroscopically. UV, visible and IR
spectroscopic methods can be employed in the present invention. The
choice of detection method will typically depend on the
experimental design. In one embodiment, nanoparticle delivery
vehicles of the present invention can be indirectly detected using
fluorescence spectroscopy.
[0143] Expression of GFP and other fluorescent marker proteins
provided by an active agent of a nanoparticle delivery vehicle of
the present invention can be detected by fluorescence and can act
as an indicator of the presence of a nanoparticle delivery vehicle.
Alternatively, a fluorescent moiety can be associated with the
nanoparticle component of a nanoparticle delivery vehicle and in
this way, the presence of the nanoparticle delivery vehicle itself
can be identified.
[0144] VI.C. Microscopy-Based Detection of a Nanoparticle Delivery
Vehicle
[0145] As noted in section VI.A above, TEM is one form of
microscopy useful for detecting delivery vehicles. Other forms of
microscopy, however, can also be employed. Microscopy techniques
such as bright field microscopy, phase contrast microscopy,
confocal microscopy and other techniques can be employed to detect
the presence of delivery vehicles.
[0146] Phase contrast microscopy is typically used for the
visualization of cellular organelles, and can be employed to detect
the presence of delivery vehicles. Confocal microscopy can also be
useful for detecting delivery vehicles. The resolution of any of
the above microscopy techniques can be enhanced by the introduction
of various contrast enhancement or other agents known to refine
images and increase resolution.
[0147] VI.D. Protein-Based Detection of a Nanoparticle Delivery
Vehicle
[0148] Protein-based detection of a nanoparticle delivery vehicle
is also possible. For example, a second protein known to associate
with a first protein bound to a nanoparticle can be labeled and
used as a probe. Suitable labels include fluorescent moieties and
other labels. Upon association of the first and second proteins,
and therefore association of the labeled second protein and the
nanoparticle delivery vehicle, the presence of the nanoparticle
delivery vehicle is detectable by detecting the presence of the
probe. Any suitable protein pair can be used to detect a
nanoparticle delivery vehicle of the present invention; preferably,
a first protein is associated with a nanoparticle and a second
protein is labeled with a detectable label, and the two proteins
are known to associate.
VII. Applications of the Nanoparticle Delivery Vehicles of the
Present Invention
[0149] The nanoparticle delivery vehicles of the present invention
can be employed to deliver a variety of active agents to a variety
of different cellular and subcellular locations. As described more
fully below, the present invention is useful for analysis of gene
expression; incorporating a nucleic acid sequence or a sequence
comprising nucleic acid analogs into a cell's genome; altering the
concentration of a regulatory protein in a cell; altering an RNA
splicing pattern; and interacting with mRNA in the cytoplasm of a
cell.
[0150] As a general rule, when nucleic acid sequences are being
selected and manipulated, care should be taken wherever possible to
minimize the potential for the formation of self-annealed
structures. Sequences of any chemical composition that are
predicted to give rise to self-annealing structures should be
avoided when practicing the present invention.
[0151] Additionally, the stringency of hybridization conditions can
be varied, with the general rule that the temperature should remain
within approximately 10.degree. C. of the duplex's predicted
T.sub.m, which is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe. An example of stringent hybridization conditions for
analysis of complementary nucleic acids having more than about 100
complementary residues is overnight incubation in 50% formamide
with 1 mg of heparin at 42.degree. C. A high stringency wash can be
preceded by a low stringency wash to remove background probe
signal. An example of medium stringency wash conditions for a
duplex of more than about 100 nucleotides is incubation for 15
minutes in 1.times.SSC at 45.degree. C. An example of low
stringency wash for a duplex of more than about 100 nucleotides is
incubation for 15 minutes in 4-6.times.SSC at 40.degree. C. For
short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve incubation in salt concentrations of
less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other ion) concentration, at pH 7.0-8.3, at a
temperature of at least about 30.degree. C. Stringent conditions
can also be achieved with the addition of destabilizing agents such
as formamide. In general, a signal to noise ratio of 2-fold (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization.
[0152] VII.A. Modulation and Analysis of Gene Expression
[0153] The nanoparticle delivery vehicles of the present invention
can be used to modulate and analyze gene expression in a model
system. It is fundamental that the expression of a gene of interest
correlates with the production of mRNA transcribed from the gene's
DNA sequence. Transcribed mRNA is subject to standard Watson-Crick
base pairing rules. Thus, in one embodiment a nanoparticle delivery
vehicle can be used to directly modulate expression of a gene by
selecting an active agent that will remove any
expression-inhibiting structures or will introduce
expression-enhancing structures. In another embodiment, a delivery
vehicle of the present invention is employed to mimic a component
part of a cell's natural second messenger system and thereby
modulate the transcription and translation of a given gene.
[0154] In one embodiment, a transcription factor or other protein
having the ability to modulate protein expression, which has been
presented to the cytoplasm or nucleus of a cell by a nanoparticle
delivery vehicle of the present invention, modulates gene
expression. Modulation encompasses both up- and downregulation of a
gene. In this embodiment, a nanoparticle delivery vehicle comprises
either a competent transcription or translation modulator (e.g., a
transcription factor) or a nucleic acid sequence encoding a
transcription or translation modulator in the role of active agent.
Competent modulators can be active in the form in which they are
bound to a nanoparticle, or they can be in a form that is activated
by proteolytic or other enzymatic or chemical treatment in the
cytoplasm or nucleus of a target cell. Similarly, nucleic acid
sequences encoding a transcription or translation modulator can be
translated by the translation machinery of the target cell, which
can be used in a method analogous to a feedback inhibition loop:
the target cell's expression machinery can be used to express the
nucleic acid introduced by a delivery vehicle, which will then
produce a protein can inhibit further protein expression.
[0155] In another embodiment, gene expression is modulated in a
cell having protein whose expression is dependent on a given
splicing pattern. In this embodiment, a nanoparticle delivery
vehicle of the present invention comprising a morpholino
oligonucleotide, PNA or other modified oligonucleotide of
appropriate sequence that alters splicing, is introduced to target
cells that comprise a gene whose expression is dependent on a
splicing event. When a gene encoding green fluorescent protein
("GFP") is employed to detectably demonstrate this embodiment of
the invention, in the absence of such a morpholino oligonucleotide,
there is no GFP expression. In cells where the morpholino
oligonucleotide delivered by a delivery vehicle of the present
invention is present, alternate splicing events occur and the GFP
gene is expressed and can be detected spectrophotometrically. This
example can be extended to any gene that can be spliced to generate
a functional protein, with an appropriate nucleotide serving as an
active agent bound on a delivery vehicle.
[0156] VII.B. Modulating Translation of a Protein
[0157] A delivery vehicle of the present invention can be used to
deliver a nucleic acid sequence for incorporation into the genome
of a target cell. This concept is sometimes referred to as
"antisense" or "gene therapy". Breakthroughs in molecular biology
and the Human Genome Project have opened previously unforeseen
possibilities for targeted intervention with gene expression. These
include permanent approaches such as transgenic overexpression or
recombinant disruption of specific genes, as well as novel
approaches for transient suppression of gene function. Short
synthetic antisense (AS) oligodeoxynucleotides (ODN) designed to
hybridize with specific sequences within a targeted mRNA belong to
the latter class. Integration of a lacking gene into a host's
genome has also been of significant interest.
[0158] AS intervention in the expression of specific genes can be
achieved by the use of synthetic antisense oligodeoxynucleotides
(AS-ODNs). See generally, Agrawal, (1996) Trends Biotechnol.
14(10): 376-87; Lev-Lehman et al., (1997) in Antisense
Therapeutics, (Cohen & Smisek, eds.), Plenum Press, New York;
and Lefebvre-D'Hellencourt et al., (1995) Eur. Cytokine Netw., 6:
7-19; Oligonucleotide & Gene Therapy-Base Antisense
Therapeutics, (1997), (Mori, ed.), Drug & Market Development
Publications, Westborough, Mass.; Antisense Therapeutics, (1996)
(Agrawal, ed.), Humana Press, Totowa, N.J. for general antisense
reviews. AS-ODNs are short sequences of DNA, typically 15 to 25
bases in length, and are designed to complement a target mRNA of
interest and to form an RNA:ODN duplex. This duplex formation can
prevent processing, splicing, transport or translation of the
relevant mRNA. Moreover, certain AS-ODNs can elicit cellular RNase
H activity when hybridized with their target mRNA, resulting in
mRNA degradation. Calabretta et al., (1996) Semin. Oncol., 23:
78-87. In that case, RNase H will cleave the RNA component of the
duplex and can potentially release the AS-ODN to further hybridize
with additional molecules of the target RNA. An additional mode of
action results from the interaction of AS-ODNs with genomic DNA to
form a triple helix that might be transcriptionally inactive.
[0159] The nanoparticle delivery vehicles of the present invention
can be used to vary a target cell's expression profile using the
above discussion as a general guide. In one embodiment, a
nanoparticle delivery vehicle can be prepared that comprises at
least one AS sequence, an ODN sequence, an AS-ODN sequence or other
nucleic acid or modified nucleic acid sequence. This sequence can
serve as an active agent in a nanoparticle delivery vehicle. The
nanoparticle delivery vehicle also comprises a nuclear localization
signal, which targets the delivery vehicle to the nucleus of a
cell. Alternatively, if it is desired that the nanoparticle
delivery vehicle remain in the cytoplasm, nanoparticles of larger
size (e.g., 30 nm) can be employed. The precise sequence and/or
composition of an active agent reflects the role or roles of the
nanoparticle delivery vehicle. For example, an active agent can be
complementary to a gene of interest.
[0160] In practice, a nanoparticle delivery vehicle designed to
vary a target cell's protein translation profile can be
administered to a subject by injection or, if the target cells are
present in an in vitro environment, the nanoparticle delivery
vehicle can be added directly to the cell's growth medium. Both
introduction procedures are described herein above, and others will
be apparent to one of skill in the art upon review of the present
disclosure.
[0161] VII.C. Modulating Regulatory Protein Concentration
[0162] The nanoparticle delivery vehicles of the present invention
can be used to modulated the concentration of various cellular
regulatory proteins. A specific example of regulation is known as
using a transcription factor decoy. As stated herein above, the
terms "decoy" and "transcription factor decoy" refer to molecules
that bind to or interact with transcription factors and prevent
their binding to native enhancer sequences. It is possible to
design transcription factor decoys that specifically interact with
transcription factors and mimic or resemble the natural genomic
binding site for the particular transcription factor. Some
transcription factor decoys can bind the transcription factor with
an affinity near or exceeding its affinity for the natural genomic
binding site.
[0163] Some transcription factors, in addition to binding an
endogenous genomic binding site, can also bind to intracellular
soluble ligands. Binding of such a transcription factor to an
appropriate ligand subsequently alters the binding profile of the
transcription factor to its genomic binding site or sites.
Restated, ligand binding by a transcription factor can modulate the
ability of the transcription factor to bind its intended genomic
site. Such transcription factors are referred to in the art as
intracellular or nuclear receptors for soluble ligands.
[0164] Transcription factor decoys can function in a variety of
ways and thus can comprise a variety of elements. For example,
nucleic acid sequences can compete with cellular target DNA for
binding to one or more transcription factors. In this example,
nucleic acid sequences can form a duplex with a target sequence and
effectively inactivate the sequence. Nucleic acid sequences can be
introduced that will form other duplex-type structures such as
hairpins, cruciforms or other structures that will effectively
inactivate cellular target DNA.
[0165] There is no requirement that a sequence introduced to
cellular target DNA necessarily comprise unmodified nucleic acids.
Sequences can comprise nucleic acid molecules that contain modified
phosphodiester bonds. Modified phosphodiester bonds can include
phosphorothioate, phosphoramidite, and methyl phosphate
derivatives, for example.
[0166] The nanoparticle delivery vehicles of the present invention
are able to facilitate a modulation in regulatory protein
concentration. Modulation can be achieved by associating an
appropriate active agent with a nanoparticle. For example, a short
sequence of double stranded DNA, for which a given regulatory
protein (e.g., transcription factor) has a high affinity, can be
used as a transcription factor decoy, as described herein above.
Additional regulatory protein-modulating applications for a
delivery vehicle of the present invention will be apparent to one
of skill in the art when considered in view of the present
disclosure.
[0167] VII. D. Modulating RNA Splicing
[0168] Generally, the expression of a specific gene can be
regulated at any step in the process of producing an active
protein. Modulation of total protein activity can occur via
transcriptional, transcript-processing, translational or
post-translational mechanisms. One role of a nanoparticle delivery
vehicle of the present invention is to modulate transcription of a
nucleic acid sequence.
[0169] Transcription means a cellular process involving the
interaction of an RNA polymerase with a gene that directs the
expression as RNA of the structural information present in the
coding sequences of the gene. The process includes, but is not
limited to the following steps: (1) transcription initiation, (2)
transcript elongation, (3) transcript splicing, (4) transcript
capping, (5) transcript termination, (6) transcript
polyadenylation, (7) nuclear export of the transcript, (8)
transcript editing, and (9) stabilizing the transcript.
Transcription can be modulated by altering the rate of
transcriptional initiation or the progression of RNA polymerase
(Maniatis et al., (1987) Science, 236: 1237-45).
Transcript-processing can be influenced by circumstances such as
the pattern of RNA splicing (the splicing of the RNA to yield one
or more mRNA species), the rate of mRNA transport to the cytoplasm
or mRNA stability.
[0170] Additionally, although some eukaryotic mRNA transcripts are
directly translated, many contain one or more regions, known as
"lintrons," which are excised from a transcript before it is
translated. The remaining (and therefore translated) regions are
known as "exons" and are spliced together to form a continuous mRNA
sequence. mRNA splice sites, i.e., intron-exon junctions, can be
target regions for an active agent of the present invention, and
can be particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
mRNA splice product is implicated in disease. Aberrant fusion
junctions due to rearrangements or deletions can also be
targets.
[0171] The present invention can be employed to modulate RNA
splicing. This aspect of the present invention can be accomplished
by selecting an appropriate active agent, such as a nucleic acid
sequence known to alter the splicing pattern for a given gene. The
use of an appropriate sequence (e.g., DNA, RNA, morpholino
nucleotide, etc.) can influence the splicing pattern and
consequently the protein expression profile for a cell. Delivery of
the appropriate sequence is the major obstacle in therapeutic
applications of RNA splicing that can be overcome by employing
nanoparticle delivery vehicles comprising a nuclear targeting
capability.
[0172] VII.E. Interaction with mRNA in the Cytoplasm
[0173] The present invention can be used to interact with mRNA
transcript for a given protein while the transcript is in the
cytoplasm. Interaction can take a variety of forms, including
modulation of the amount of a given protein produced by a cell. In
one aspect of the present invention, a nanoparticle delivery
vehicle of the present invention can employ an antisense nucleotide
to interact with mRNA which has been exported to the cytoplasm.
See, e.g., Bassell et al., (1999) FASEB J. 13: 447-54.
[0174] A nanoparticle delivery vehicle of the present invention can
be designed to interact with mRNA in the cytoplasm of a cell. The
specific hybridization of an oligomeric compound with mRNA can
interfere with the normal function of the mRNA. This modulation of
function of a nucleic acid by compounds that specifically hybridize
to it is generally referred to as "antisense" and is discussed
herein above. Antisense compounds can interrupt various functions
of RNA can include all vital functions such as, for example,
translocation of the RNA to the site of protein translation,
translation of protein from the RNA, splicing of the RNA to yield
one or more mRNA species, and catalytic activity which might be
engaged in or facilitated by the RNA. The overall effect of such
interference with mRNA function is the modulation of the expression
of the protein for which the mRNA codes.
[0175] Association of an antisense compound with, for example,
mRNA, can be utilized for diagnostics, therapeutics, prophylaxis
and as research reagents and kits. When used as a therapeutic, a
subject suspected of having a disease or disorder that can be
treated by modulating the expression of a given protein can be
treated by administering a nanoparticle delivery vehicle of the
present invention comprising an antisense active agent. Use of the
nanoparticle delivery vehicles of the present invention, when
antisense compounds are included as the active agent, can also be
used prophylactically, e.g., to prevent or delay infection,
inflammation or tumor formation, for example.
[0176] Antisense-bearing delivery vehicles of the present invention
are useful for research and diagnostics, because the antisense
component can hybridize with nucleic acids encoding a particular
protein of interest, enabling sandwich and other assays to easily
be constructed to exploit this fact.
[0177] Additionally, the nanoparticle delivery vehicles of the
present invention can be localized subcellularly through the
selection of an appropriately sized nanoparticle. As shown in FIG.
1B, delivery vehicles comprising nanoparticles of 30 nm or greater
remain localized to the cytoplasm of a cell and are not localized
in the cell's nucleus, regardless of the presence or absence of a
nuclear localization signal. This observation indicates that larger
nanoparticles can be targeted to the cytoplasm, wherein
untranslated mRNA is localized. Smaller nanoparticles can be used
to target pre-mRNA in the nucleus. Thus, a delivery vehicle of the
present invention can be targeted to the cytoplasm of a cell, where
it can interact with RNA sequences disposed therein.
VIII. Advantages of the Delivery Vehicles of the Present
Invention
[0178] There are a number of advantages of the present invention
over the methods for delivery presently known in the art. First,
there is a distinct advantage in the use of a nuclear localization
signal. The inclusion of an NLS as a component part of a
nanoparticle delivery vehicle assures that, assuming optimization
of other variables, the nanoparticle delivery vehicle is targeted
directly to the nucleus of a cell. This advantage greatly increases
the efficacy of an active agent designed to interact with nuclear
structures by increasing the amount of material delivered to the
nucleus of a target cell; inclusion of the NLS results in less
material disposed in the cytoplasm for shorter periods of time and
ultimately less degradation of material. The NLS additionally
offers the ability to effectively target cellular processes that
occur in the nucleus, such as DNA replication, transcription, and
various splicing events.
[0179] The association of additional targeting agents can aid in
the translocation of a vehicle across various membranes, such as
the nuclear membrane of a cell or the outer membrane of a cell,
thus providing another advantage. If membranes and other structures
that generally inhibit translocation of a vehicle to a given
location in or on a cell are analogized as "locks", NLS and RME
sequences can be analogized to be "keys". Thus, in a preferred
embodiment, a nanoparticle delivery vehicle of the present
invention can comprise a plurality of different sequences or
"keys," which can enable a given nanoparticle delivery vehicle to
pass through various potential barriers to translocation.
[0180] Another advantage of a preferred embodiment of the
nanoparticle delivery vehicles of the present invention is the
ability to create nanoparticles that comprise a material that is
biologically inert. For example, it is possible to fashion
nanoparticles from gold and other materials. Gold, unlike some
other materials, is biologically inert and can be physiologically
tolerated without significant adverse effects by biological
systems. Further, a nanoparticle can comprise biodegradable
material, which upon breakdown, can yield the nanoparticle delivery
vehicle's component parts, all of which are themselves
biodegradable.
[0181] Yet another advantage of a preferred embodiment of the
nanoparticle delivery vehicles of the present invention is their
ability to function in any of a variety of roles, due to the lack
of restriction on the active agent. A nanoparticle delivery vehicle
of the present invention can therefore fill a variety of roles by
simply changing the active agent to suit the need. Thus, a
nanoparticle delivery vehicle designed to modulate gene expression
by delivering an antisense strand to the nucleus of a cell can also
function as a transcription factor decoy by replacing the antisense
strand active agent with a double stranded sequence of DNA.
[0182] Finally, the size of the nanoparticle can be varied, which
can provide for differential targeting of a nanoparticle delivery
vehicle. Nanoparticle size can influence the targeting of a
delivery vehicle. A nanoparticle delivery vehicle comprising a
nanoparticle of about 30 nm or larger will not be transported into
the nucleus of a cell and will remain in the cytoplasm of the cell,
even if an NLS is present. However, although such a nanoparticle
delivery vehicle might not be transported to the nucleus of a cell,
the nanoparticle delivery vehicle can be internalized by the cell
and remain localized in the cytoplasm. Thus, such vehicles can be
useful for modulating processes occurring in the cytoplasm, such as
translation and translocation.
LABORATORY EXAMPLE
[0183] The following Laboratory Example has been included to
illustrate preferred modes of the invention. Certain aspects of the
following Laboratory Example are described in terms of techniques
and procedures found or contemplated by the present inventors to
work well in the practice of the invention. This Laboratory Example
are exemplified through the use of standard laboratory practices of
the inventors. In light of the present disclosure and the general
level of skill in the art, those of skill will appreciate that the
following Laboratory Example is intended to be exemplary only and
that numerous changes, modifications and alterations can be
employed without departing from the spirit and scope of the present
invention.
LABORATORY EXAMPLE
[0184] Targeted entry into cells is an increasingly important area
of research. The nucleus is a desirable target since the genetic
information of the cell and transcription machinery resides there.
The diagnoses of disease phenotype, the identification of potential
drug candidates, and the treatment of disease by novel methods such
as antisense therapy would be enhanced greatly by the efficient
transport of materials to living cell nuclei (Kole & Sazani,
(2001) Curr. Opin. Mol. Ther 3: 229-234). The intracellular fate of
gold nanoparticles chemically designed to transit from outside a
living cell into the nucleus is reported in the present Laboratory
Example.
[0185] Although metal, semiconductor, polymer, and magnetic
particles have been introduced into cells previously (Liu et al.,
(2001) Biomacromolecules 2: 362-368; Marinakos et al., (2001) J.
Phys. Chem. B. 105: 8872-8876; West & Halas, (2000) Curr. Opin.
Biotech. 11: 215-217; Hogemann et al., (2002) Bioconjugate Chem.
13: 116-121), there is no comprehensive cytochemical approach to
targeting the nucleus from outside the plasma membrane of living
cells. The development of an approach that permits the transport of
nanometer-sized particles into cells has important applications in
cell biology as a tool for the study of cell development and
differentiation.
[0186] A number of techniques have been used previously to
determine cellular trajectories of particles. Indeed, the use of
electron microscopy with colloidal gold stains was perhaps the
first modern method of cell structure characterization (Hayat
(Ed.), (1998) Colloidal Gold, Principles, Methods and Applications;
Academic Press, Inc.: San Diego, Vol. 1). More recently,
fluorescence microscopy has been used to locate fluorophores,
including luminescent CdSe nanoparticles in cells (Bruchez et al.,
(1998) Science 281: 2013-2016; Nie & Chan, (1998) Science 281:
2016-2018). However, prior studies of nuclear translocation of
nanoparticles were performed using microinjection or chemically
modified cells, thus bypassing cellular membrane entry. The
combination of targeted endocytosis coupled with nuclear uptake has
not been demonstrated in a nanoparticle vector using intact cells,
prior to the present disclosure.
[0187] Targeted nuclear delivery is a challenging task, as any
cell-specific nuclear probe must satisfy minimally the following
requirements (Hallenbeck & Stevenson, (2000) Targetable Gene
Delivery Vectors (Habib, Ed.), Kluwer Academic/Plenum. Publishers:
New York, pp 37-46): it must (i) be small enough to enter the cell
and cross the nuclear membrane; (ii) bind to cell-specific plasma
membrane receptors by receptor-mediated endocytosis (RME), for
example; (iii) escape endosomal/lysosomal pathways; (iv) pass
through the nuclear pore complex, and (v) have low toxicity. In the
present Laboratory Example, results of intracellular trafficking
studies of nanoparticles designed to perform these and other
functions are reported.
[0188] A nanoparticle vector of the present Laboratory Example
comprises a core of a 20 nm gold particle and a shell of bovine
serum albumin (BSA) conjugated to various cellular targeting
peptides, which are presented in the following Table of
Representative Peptide Sequences:
TABLE-US-00004 Table of Representative Peptide Sequences SEQ
Peptide/ Peptide Sequence ID NO Source BSA N0 CGGGPKKKRKVGG 3 SV40
large 7 .+-. 1 T NLS N1 CGGFSTSLRARKA 4 Adenoviral 8 .+-. 1 NLS N2
CKKKKKKSEDEYPYVPN 5 Adenoviral 9 .+-. 2 RME N3 CKKKKKKKSEDEYPYVP 6
Adenoviral 6 .+-. 2 NFSTSLRARKA Fiber Protein
When preparing the peptides disclosed in the Table of
Representative Peptide Sequences, peptides were conjugated to BSA
with a 3-maleimido benzoic acid N-hydroxysuccinimide ester linker.
Gel electrophoresis (SDS-PAGE and IEF) was used to quantify
peptide:BSA ratio. Each peptide was chosen to perform a certain
task (e.g., RME). Individual peptides have been explored previously
as therapeutic delivery vectors (Morris et al., (2000) Curr. Opin.
Biotech. 11: 461-466). However, highly efficient nuclear targeting
in biology is accomplished by viruses, which utilize different
peptides for each barrier mentioned above. A significant
observation of the present Laboratory Example is that viral
peptides conjugated to proteins on the surface of a nanoparticle
retain their function of promoting cell entry and nuclear
targeting. Moreover, separate short peptides on a single particle
lead to more efficient nuclear targeting than a single long
peptide. Together the gold core and multifunctional peptide shell
provides a flexible scaffold that can be tuned to target specific
cells for intranuclear assays or therapeutic delivery.
[0189] Gold was chosen as an intracellular targeting vector
primarily for three reasons. First, gold can be routinely
synthesized in sizes varying continuously from 0.8 nm to 200 nm
with <5% size dispersity. Secondly, gold can be modified with a
large collection of small molecules, peptides, proteins, DNA, and
polymers. Moreover, all of these functional elements can be
combined on a single particle, often via simple one-pot procedures.
Finally, gold particles have strong visible light extinctions that
can be used to monitor their trajectories inside cells under
polarized light conditions. These properties were advantageously
employed in a novel combination of video-enhanced color (VEC)
microscopy and differential interference contrast microscopy (DIC),
which facilitated the observation of the trajectory of 20 nm gold
nanoparticles inside cells.
[0190] Dynamic light scattering and transmission electron
microscopy revealed that BSA-peptide conjugates add <2 nm to the
radius of the nanoparticle complex. The fact that BSA does not add
greatly to the size of the gold particle is important in its use in
constructing nuclear targeting vectors because the diameter of the
nuclear pore complex is 20-50 nm depending on the cell line
(Feldherr & Akin, (1990) J. Cell Biol. 111:1-8). The 20 nm gold
particles used in the present Laboratory Example have a maximum
diameter of 25 nm when complexed with any of the BSA-peptide
conjugates studied (see Supporting Information).
[0191] Nuclear translocation through the nuclear pore complex has
previously been studied using gold nanoparticles labeled with an
NLS from SV-40 virus (large T antigen). In the classic studies
nuclear targeting was observed by transmission electron microscopy
(TEM) following microinjection into the cell (Feldherr et al.,
(1992) Proc. Nat Acad. Sci. U.S.A. 89: 11002-11005). As a test
case, nanoparticle complexes comprising peptide N0 were introduced
into the growth medium of HepG2 cells. Surprisingly, N0 complexes
were observed inside the cytoplasm of HepG2 cells, however N0 did
not enter the nucleus. Experiments at 4.degree. C. indicated that
cell entry was via an energy-dependent pathway. This observation
suggests that N0 entered the cell by receptor-mediated endocytosis,
but was unable to escape the endosome and target the nucleus (TEM
and confocal fluorescence microscopy confirmed that nanoparticles
were confined to endosomes). These results highlight the challenges
associated with nuclear targeting: although a known NLS peptide is
able to enter HepG2 cells, it cannot target the nucleus unless it
is capable of endosomal escape.
[0192] In an effort to enhance nuclear targeting efficiency in
HepG2 cells, peptides from the adenovirus were explored. The
adenovirus is widely used in gene delivery and there is a great
deal of interest in replacing the whole virus, which is potentially
infectious and immunogenic, with peptide sequences derived from the
adenovirus fiber protein (Seth, (2000) Adenoviral Vectors; (Habib,
Ed.) Kluwer Academic/Plenum Publishers: New York, pp 13-22; Bilbao
et al., (1998) Targeted Adenoviral vectors for Cancer Gene Therapy,
Plenum Press: New York, Vol. 57, pp 365-374). This protein is known
to contain both RME and NLS sequences (N1 and N2, in the Table of
Representative Peptide Sequences). The full length fiber containing
both the RME and NLS is peptide N3 in the Table of Representative
Peptide Sequences. A comparison of the functions of these targeting
peptides when complexed to a gold nanoparticle is as follows. Ni
does not enter the cell. N2 enters the cell, but remains trapped in
endosomes and does not reach the nucleus. N3 targets the nucleus,
however, NI/N2 has a greater propensity for nuclear targeting than
N1, N2, or N3. These results are interpreted as follows. N1
presents only the adenovirus NLS (Table of Representative Peptide
Sequences). This peptide does not function as an RME and has no
other chemical moiety that permits cell entry. N2 presents the RME
(NPXY (SEQ ID NO: 7) motif) and it enters the cell, however, it is
not capable of nuclear targeting (Chen et al., (1990) J. Biol Chem.
265: 3116-3123).
[0193] These results show that the nanoparticle complex must
present both RME and NLS in order to both enter the cell and
achieve nuclear localization. The VEC-DIC results clearly show
significant numbers of N3 in the nucleus in agreement with a gene
delivery study using this peptide (Zhang et al., (1999) Gene Ther.
6: 171-181). The N1/N2-labeled nanoparticle is even more efficient,
as seen by VEC-DIC microscopy.
[0194] Another comparison to be made is between a multi-functional
nanoparticle N1/N2 that presents the RME and NLS on separate BSA
bioconjugates and N3, which presents the full-length adenoviral
fiber peptide. N1N2 was present in the nucleus in greater numbers
than N3. The origin of the higher nuclear targeting efficiency in
particles carrying two short peptides versus one long sequence
could be structural or spatial. Infrared spectroscopy indicates
that all peptides employed in this Exa,[;e adopt an extended
confirmation when attached to nanoparticles. However, when one long
peptide is synthesized with two consecutive signals, it is likely
that one of the signals will be less accessible to cellular
receptors. This is important for NPXY (SEQ ID NO: 7) motifs, for
example, since tandem interaction of two NPXY (SEQ ID NO: 7)
regions has been shown to facilitate RME (Hussain, (2001) Front
Biosci. 6: 417-428). Attaching the two-peptide signals to a
nanoparticle as separate, shorter pieces likely gives them equal
access to cellular receptors.
[0195] The methods used here provide an approach for rapidly
assessing the efficacy of various combinations of targeting
peptides using nanoparticle complexes for nuclear targeting. The
VEC-DIC combination microscopy permits examination of hundreds of
samples per day, an improvement over costly and time consuming
electron and confocal microscopy techniques.
[0196] The multifunctional approach demonstrated using adenoviral
targeting sequences provides a test of the function of individual
peptide sequences that will permit effective and cell-specific
targeting for a range of scientific and medical applications.
REFERENCES
[0197] The references listed below as well as all references cited
in the specification are incorporated herein by reference to the
extent that they supplement, explain, provide a background for or
teach methodology, techniques and/or compositions employed herein.
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Antisense Therapeutics, (1996) (Agrawal, ed.), Humana Press,
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[0257] It will be understood that various details of the invention
can be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
Sequence CWU 1
1
717PRTHomo sapiens 1Pro Lys Lys Lys Arg Lys Val1 5216PRTHomo
sapiens 2Lys Arg Pro Ala Ala Ile Lys Lys Ala Gly Gln Ala Lys Lys
Lys Lys1 5 10 15313PRTSimian virus 40 3Cys Gly Gly Gly Pro Lys Lys
Lys Arg Lys Val Gly Gly1 5 10413PRTUnknownUnknown adenovirus 4Cys
Gly Gly Phe Ser Thr Ser Leu Arg Ala Arg Lys Ala1 5
10517PRTUnknownUnknown adenovirus 5Cys Lys Lys Lys Lys Lys Lys Ser
Glu Asp Glu Tyr Pro Tyr Val Pro1 5 10 15Asn628PRTUnknownUnknown
adenovirus 6Cys Lys Lys Lys Lys Lys Lys Lys Ser Glu Asp Glu Tyr Pro
Tyr Val1 5 10 15Pro Asn Phe Ser Thr Ser Leu Arg Ala Arg Lys Ala 20
2574PRTUnknownUnknown adenovirus 7Asn Pro Xaa Tyr1
* * * * *