U.S. patent application number 11/936574 was filed with the patent office on 2008-08-21 for methods and products for delivering biological molecules to cells using multicomponent nanostructures.
This patent application is currently assigned to JOHNS HOPKINS UNIVERSITY. Invention is credited to Kam W. Leong, Aliasger Karimjee Salem, Peter Charles Searson.
Application Number | 20080199531 11/936574 |
Document ID | / |
Family ID | 33563836 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199531 |
Kind Code |
A1 |
Salem; Aliasger Karimjee ;
et al. |
August 21, 2008 |
Methods And Products For Delivering Biological Molecules To Cells
Using Multicomponent Nanostructures
Abstract
This invention is predicated on the present applicants'
discovery that nanostructures comprising discrete regions of
different composition can be used to deliver to a biological cell a
desired combination of molecules in close proximity. Different
molecules can be selectively bonded to discrete regions of
different composition in sufficiently close physical relationship
to enhance delivery or effectiveness within the cell. The preferred
nanostructures are multicomponent nanorods. Important applications
include delivery of missing DNA sequences for gene therapy and
delivery of antigens or DNA encoding antigens for vaccination.
Inventors: |
Salem; Aliasger Karimjee;
(Coralville, IA) ; Leong; Kam W.; (Ellicott City,
MD) ; Searson; Peter Charles; (Baltimore,
MD) |
Correspondence
Address: |
PATENT DOCKET ADMINISTRATOR;LOWENSTEIN SANDLER PC
65 LIVINGSTON AVENUE
ROSELAND
NJ
07068
US
|
Assignee: |
JOHNS HOPKINS UNIVERSITY
Baltimore
MD
|
Family ID: |
33563836 |
Appl. No.: |
11/936574 |
Filed: |
November 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10875543 |
Jun 24, 2004 |
7344887 |
|
|
11936574 |
|
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|
|
60482141 |
Jun 24, 2003 |
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Current U.S.
Class: |
424/499 ;
424/184.1; 514/44R |
Current CPC
Class: |
B82Y 30/00 20130101;
A61K 47/6929 20170801; A61K 47/02 20130101; C12N 15/87 20130101;
A61K 9/5115 20130101; A61K 47/6923 20170801 |
Class at
Publication: |
424/499 ; 514/44;
424/184.1 |
International
Class: |
A61K 9/50 20060101
A61K009/50; A61K 31/70 20060101 A61K031/70; A61K 39/00 20060101
A61K039/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under
DARPA/AFOSR contract number F49620-02-1-0307. The government has
certain rights in this invention.
Claims
1-20. (canceled)
21. A device for delivering biological molecules to a cell
comprising: a nanostructure comprising a plurality of discrete
regions of respectively different materials; and a plurality of
molecules of different composition bound to the respectively
different discrete regions.
22. The device of claim 21 wherein the nanostructure comprises a
nanotube or nanowire having a plurality of axial segments of
respectively different materials.
23. The device of claim 21 wherein the materials comprise different
metals.
24. The device of claim 21 wherein the molecules include at least
one material from the group consisting of DNA, RNA, proteins,
antigens, adjuvants endosomylytic agents and cytokines.
25. The device of claim 21 wherein the different materials include
at least one material from the group consisting of metals and
semiconductors.
26. The device of claim 21 wherein at least one of the different
materials comprises a material selected from the group consisting
of nickel, gold and platinum.
27. The device of claim 21 wherein at least one of the molecules is
bound by a linkage from the group consisting of thiol linkages,
isonitrile linkages and carboxylate linkages.
28. The device of claim 21 wherein at least one of the molecules
comprises compacted DNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/875,543, fled Jun. 24, 2004, which claims the benefit of
U.S. Provisional Patent Application Ser. No. 60/482,141 filed by
Dr. Ali-ager K. Salem et al on Jun. 24, 2003 and entitled
"Multifunctional Nanorods for Gene Delivery", which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0003] This invention relates to methods of delivering biological
molecules to cells and, in particular, to methods of delivering to
cells a desired combination of biological molecules in close
physical proximity. It also includes products for effecting such
delivery.
BACKGROUND OF THE INVENTION
[0004] The capability of delivering biologically active molecules
to plant and animal cells is of great importance to medicine and
genetic research and engineering. In medicine, for example, the
development of effective vaccines requires systems for providing
characteristic portions of infectious biological entities to immune
system cells so that the immune system will recognize and fight an
infection. When such characteristic portions (antigens) of entities
such as viruses, bacteria or even tumors are appropriately
provided, the immune systems identifies the antigens as foreign and
stimulates development of immunological countermeasures. One way to
provide antigens is to deliver them directly into cells. Another
way is to deliver to the cells DNA sequences that encode the
antigens.
[0005] Gene therapy seeks to introduce additional genetic material
(typically DNA) into a cell in such a way that the additional
genetic material will be functionally incorporated into the
existing genetic material of the cell, For example, there are
certain diseases that are caused by the absence in cells of
normally present DNA sequences (genes) needed to make critical
proteins. Gene therapy seeks to alleviate such diseases by
providing the cells with the missing DNA sequences so that the
cells themselves can provide the critical proteins. To achieve this
goal, the missing DNA sequences need to be introduced into cells in
such a fashion that they are functionally incorporated into the
genetic material and mechanisms of the cells.
[0006] The effectiveness of an active biological molecule in a cell
often can be enhanced by the presence of one or more additional
different molecules. For example, there are molecules, called
adjuvants, that will increase the likelihood that an antigen will
be recognized as an appropriate target for immunological
countermeasures. As another example, there are also molecules that
will interact with cell receptors and increase the likelihood of
incorporation into the cell. Such enhancing molecules, however,
typically must be close to the active molecule in order to enhance
its effectiveness.
[0007] Conventional approaches to delivering biological molecules
to cells leave much to be desired. The common approach to gene
therapy is based on the fact that viruses have evolved to inject
genetic material into a cell and use the cell's genetic machinery
to replicate the viral genetic material. Appropriate modification
of the virus might eliminate its harmful features and redirect a
viral vector to deliver desirable genetic material into the cell.
However virus vectors often generate counterproductive host immune
responses and present a risk of killing infected host cells
(cytotoxicity).
[0008] Other delivery approaches that have been suggested include
the use of carriers comprising liposomes, pollaners and gold
nanoparticles. They have not, however, achieved notable success in
efficiently incorporating new genetic material or in making more
effective vaccines. Accordingly there Is a need for improved
methods and products for delivering biological molecules to
cells.
SUMMARY OF THE INVENTION
[0009] This invention is predicated on the present applicants'
discovery that nanostructures comprising discrete regions of
different composition can be used to deliver to a biological cell a
desired combination of molecules, including at least one biological
molecule, in close proximity. Different molecules can be
selectively bonded to discrete regions of different composition in
sufficiently close physical relationship to enhance delivery or
effectiveness within the cell. The preferred nanostructures are
multicomponent nanorods. Important applications include delivery of
missing DNA sequences for gene therapy and delivery of antigens or
DNA encoding antigens for vaccination, and simultaneous delivery of
interacting medicines in specific proportion and close
proximity,
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0011] The advantages, nature and various additional features of
the invention will appear more fully upon consideration of the
illustrative embodiments now to be described in detail in
connection with the accompanying drawings. In the drawings:
[0012] FIG. 1 is a schematic block diagram of a method of
delivering biological molecules to cells in accordance with the
invention.
[0013] FIG. 2 is a schematic diagram illustrating functionalization
of multicomponent nanostructures. In FIG. 2a nanorods are incubated
with the 3-[(2-aminoethyl)dithio] propionic acid (AEDP) linker. The
carboxylate end group binds to the nickel segment. The disulfide
linkage at the center acts as a cleavable point within the spacer
promoting DNA release within the reducing environment of the cell.
In FIG. 2b plasmids are bound by electrostatic interactions to the
protonated amines presented on the surface of the nickel segment.
In FIG. 2c calcium chloride compacts the plasmids encoding the
luciferase or GFP reporter genes; and in FIG. 2d
rhodamine-conjugated tansferrin presenting sulfhydryl groups is
selectively bound to the gold portion of the nanorods.
[0014] FIG. 3 shows microphoto images pertaining to functionalized
multicomponent nanostructures. FIG. 2a is a visible light image of
dual fictionalized 200 nm long AuNi nanorod. FIG. 3b fluorescence
image of the rhodamine-tagged (543/570 nm) transferrin on the Au
segment. FIG. 3c is a fluorescence image of the Hoechst stained
(350/450 nm) plasmids on the Ni segment; and FIG. 3d is a
fluorescent overlay image combining FIGS. 3b and 3c.
[0015] FIG. 4 shows microphoto images of cells transfected in
accordance with the method of FIG. 1. FIG. 4a presents stacked
laser scanning confocal microscope images of a live HEK293 cell
(red/633 nm, green/543 nm). Rhodamine (633 nm) identifies the
sub-cellular location of the nanorods whilst GFP expression (543
nm) provides confirmation of transfection throughout the cell.
FIGS. 4b and 4c are, orthogonal sections that confirm the nanorods
are within the cell. FIG. 4d shows confocal microscope stacked
images, of a live HEK 293 cell stained with Lysotracker Green
identifying the location of the nanorods (Rhodamine) in relation to
acidic organelles in both orthogonal sections (FIGS. 4e and
4f).
[0016] FIG. 5 presents scanning electron microscope images of cells
transfected in accordance with the method of FIG. 1. FIG. 5a is a
SEM image of HEK293 cells after Ih incubation with 200 nm Au/Ni
nanorods. FIG. 5b is a back-scattering SEM image of 200 nm Au/Ni
nanorods after 4 h incubation showing the nanorods beneath the
surface of the cell. FIG. 5c is a TEM cross-sectional image showing
the intra-cellular location of the nanorods after 4 h incubation,
and FIG. 5d is a, SEM image of 200 nm long nanorods after 4 h
incubation.
[0017] FIG. 6 is a set of histograms summarizing results of
transfection experiments. FIG. 6a shows percentage of GFP
expression (area of cells fluorescing/total cell area) and FIG. 6b
shows luciferase expression of: 1. nanorod-plasmid complex, 2.
nanorod-plasmid/transferrin complex, 3. nanorod-plasmid/transferrin
complex incubated with 100 micromoles chloroquine, 4. Lipofectamine
(positive control) and 5. naked DNA (negative control).
[0018] FIG. 7 is a graphical illustration of ovalbumin-specific
antibody responses in C57BL/6 mice immunized with various antigen
or plasmid nanorod and gold particle formulations. C57BL/6 mice
were immunized with control plasmid (no insert) bound to nanorods,
ovalbumin antiegen-nanorod formulation, ovalbumin antigen-gold
particle formulation, pcDNA3-OVA7-nanorod formulation,
pcDNA3-OVA7-gold particle formulation and ovalbumin antigen/control
pcDNA3 (no insert)nanorod formulation via a gene gun. Serum samples
were obtained from immunized mice 21 days after the initial
vaccination. The presence of the ovalbumin-specific antibody was
detected by ELISA using serial dilution of sera. The results from
the 1:1000 dilutions are presented showing the mean absorbance
(A450 nm).+-.SE.
[0019] FIG. 8 graphically illustrates ovalbumin-specific CD8+T-cell
precursors in C57BL/6 mice immunized with various antigen or
plasmid-nanorod and gold particle formulations. C57BL/6 mice were
immunized with control plasmid (no insert) bound to nanorods,
ovalbumin antigen-nanorod formulation, ovalbumin antigen-gold
particle formulation, pcDNA3-OVA7-nanorod formulation,
pcDNA3-OVA7-gold particle formulation and ovalbumin antigen/control
pcDNA3 (no insert)-nanorod formulation via a gene gun. For
vaccinated mice, 2 .mu.g of DNA or antigen/mouse were given twice.
Splenocytes were harvested 7 days after the last DNA /antigen
vaccination. Flow cytometry analysis: Splenocytes from vaccinated
mice were cultured in vitro with the ovalbumin antigen overnight
and were stained for both CD8 and intracellular IFN-. The number of
IFN- secreting (CD8+ T-cell precursors in mice immunized with
antigen or plasmid-nanorod and gold particle formulations were
analyzed by flow cytometry. The number of CD8+IFN-+double-positive
T cells in 3.times.10.sup.5 splenocytes are represented by the
quadrant in the upper right corner.
[0020] FIG. 9 schematically illustrates formation of three
component nanowires.
[0021] FIG. 10 schematically shows a general approach for selective
derivatisation of Au/Ni/Pt nanowires; and
[0022] FIG. 11 is a set of micrographs illustrating 3 component
nanostructures. FIG. 11a is a back-scattering SEM image of
nanowires showing integrity of Au, Ni and Pt segments. The image
confirms platinum segments are longer than Ni and Au segments.
FIGS. 11b and 11c light and fluorescence microscope images of
Au/Ni/Pt nanowires functionalized with BIC and Rhodamine
Red-12-dodecanoic acid (Ex 570, Em 590). Confirmation of selective
derivatisation of Au/Ni/Pt nanowires with BIC, Rhodamine
Red-12-dodecanoic acid and Marina Blue-1-undecane-thiol is observed
by light microscope images (FIG. 11d) and fluorescence microscope
images FIGS. 11e-11g. FIG. 11d is a light microscope image of a
functionalized Au/Ni/Pt nanowire. FIG. 11e shows the fluorescence
from the Marina Blue-1-undecane-thiol (Ex 365, Em 460) bound to the
gold segment. FIG. 10e shows the fluorescence from the Rhodamine
Red-12-dodecanoic acid (Ex 570, Em 590) from the nickel segment,
and FIG. 11g is a fluorescent overlay image combining 10e and
10f.
[0023] It is to be understood that these drawings are for
illustrating the concepts of the invention and, except for the
graphs, are not to scale.
DETAILED DESCRIPTION
[0024] Referring to the drawings, FIG. 1 is a schematic block
diagram of a method for delivering biological molecules to cells in
accordance with the invention. As shown in Block A, an initial step
is lo provide a multicomponent nanostructure comprising at least
two discrete regions of respectively different materials. The term
"nanostructure" as used herein refers to structures having maximum
dimensions in at least two dimensions that are substantially
smaller than the diameter of a cell so that the structures may
enter a cell without destroying its functionality. Typically the
nanostructure has two maximum dimensions of less than about 500
nanometers and preferably less than about 200 nanometers. The
maximum third dimension is also preferably less than the diameter
of a cell so that the nanostructure can be incorporated in the
cell, but it can be greater (into the micrometer range) and still
transfect a cell. Useful multicomponent nanostructures have at
least two discrete regions large enough to bind respective
biological molecules but positioned sufficiently close together
that both molecules can be delivered into the same cell at the same
time.
[0025] The inventive method can use multicomponent nanostructures
in a wide variety of sizes and shapes including multicomponent
nanorods, nanowires, nanotubes, nanoscale bars, nanodisks,
nanoscale ovals, nanoscale parallelpipeds and multicomponent
nanoparticles of regular or irregular shape. Multicomponent
nanostructures with any one of a wide variety of shapes, sizes and
material combinations can be fabricated by techniques well known in
the art, as by depositing successive nanolayers on a removable
substrate, patterning the layers by nanoimprint lithography, and
removing the substrate. Further details concerning nanoimprint
lithography can be found, for example, in U.S. Pat. No. 6,309,580
issued to Stephen Chou on Oct. 30, 2001, which is incorporated
herein by reference.
[0026] The preferred multi component nanostructures are nanorods or
nanowires comprised of discrete segments of respectively different
materials (See FIG. 2). Such segmented rods or tubes can be
fabricated, for example, by electro-depositing successive layers of
different metals in a nanoporous matrix material and removing the
matrix material, as by selectively dissolving in acid or base.
[0027] The next step shown in Block B is to attach one or more
molecules of different materials to the respectively different
discrete regions of the nanostructure. In essence, each different
molecule is provided with a chemical group that selectively bonds
to a respectively different material of the multicomponent
nanostructure. The preferred molecules for attachment are
biological molecules. The term "biological molecules" as used
herein includes, without limitation, molecules of genetic material
(DNA and RNA), molecules of materials that activate cell receptors
(external or internal), antigens or their genetic material, and
materials that enhance the incorporation of genetic material or
stimulate the immune response. The term also includes molecules of
medications that are active at the cellular level, and especially
different medications that have a synergistic effect when delivered
together. Thus, for example, molecules to stimulate cell receptors
can be selectively bonded to a first material segment of a nanorod
and a DNA sequence can be selectively bonded to a second material
segment. As another example, DNA encoding an antigen can be
selectively bonded to a first segment and an immune system
stimulating adjuvant molecule can be bonded to a second segment,
and an antigen can be bonded to a yet third segment. Exemplary of
useful RNA biological molecules is siRNA that can be used to
silence undesirable genes. Thus a multicomponent nanostructure
could contain RNA to silence a defective gene and DNA to provide
the correct gene. An example of synergistic medications that could
be simultaneously delivered by multicomponent nanostructures
include Taxol and Discodermolide.
[0028] The third step, Block C, is to deliver the nanostructure and
its bonded molecules to biological cells. The method of delivery
may depend on the location and type of cells. For delivery to
somatic cells, the preferred approach is to use a nanostructure
including a bonded biological molecule to stimulate cell receptors
that will take the structure into the cell. The nanostructures can
be introduced, as by pneumatic injection, into desired tissues and
stimulated cell receptors will facilitate their intake into cells.
For dendritic cells located near the surface of the body, the
nanostructures may be injected directly into the cells as by
pneumatic pressure. Deeper penetration into somatic cells may be
achieved by orienting nanotubes so that their cylindrical axes are
aligned approximately perpendicular to the target tissue at the
point of injection.
[0029] As will be illustrated in the exemplary embodiments
described herein below, a major advantage of this method is the
ability to simultaneously provide specific combinations of
biological molecules in close adjacency where they can interact to
produce more effective biological results, e.g. more effective
incorporation in the cell, an enhanced immune response, or a more
effective combination of medicines.
[0030] The invention can now be more clearly understood by
consideration of the following examples.
EXAMPLE 1
[0031] Delivery of Genetic material For Gene Therapy
[0032] The goal of gene therapy is to introduce foreign genes into
somatic cells to supplement the defective genes or to provide
additional biological functions. Gene transfer ("transfection") can
be achieved using either viral or synthetic non-viral delivery
systems ("vectors"). While viral vectors exhibit high efficiency,
synthetic transfection systems provide several advantages including
ease of production and reduced risk of cytotoxicity and immune
response. Much of the poor transfection efficiency of non-viral
vector stems from the difficulty of controlling their properties at
the nanoscale. One aspect of the present invention is a novel
non-viral delivery system based on nanostructures that can
simultaneously bind compacted DNA plasmids and target cell
receptors for enhanced internalization. The present example
demonstrates the potential of this system to deliver genetic
material with precise composition and size control.
[0033] Achieving efficient gene delivery into a target cell
population or tissue without causing associated toxicity is
critical to the success of gene therapy. To this end, both viral
and non-viral vectors have been extensively investigate. Although
viral vectors such as adenovirus, lentil virus, influenza virus,
and adeno-associated virus are efficient in transfecting cells,
their toxicity and immunogenicity remain severe limitations.
[0034] As alternatives to viruses non-viral vectors such as
liposomes and polymers have been increasingly studied to overcome
this long-term safety issue. In contrast, inorganic gene carriers
have received limited attention in the gene therapy community. Gold
nanoparticles with bound DNA are used in particle
bombardment-mediated gene transfer ("gene gun technology"). While
this gene gun technology may be effective in transfecting cells in
the skin for genetic immunization, it has limited utility in
general gene transfer applications involving internal organ
transfection.
[0035] To be effective, non-viral vectors must gain entry into the
target cells and then release the condensed plasmid into the
cytoplasm for translocation into the nucleus. To date,
particle-based vectors' have been formulated by using polycationic
polymers or lipids to condense DNA into nano-complexes that can be
internalized by cells. The size of these nano-complexes is
typically difficult to control and widely dispersed. Targeting
ligands can be conjugated to the carrier or complexes either pre-
or post- complexation with the DNA from the complexes may also
become a rate-limiting step. To optimize these different aspects in
designing an effective non-viral gene delivery system has been a
major challenge in the field.
[0036] The possibility of achieving control of size and composition
by inorganic synthesis has prompted us to evaluate the potential of
multi-segment metallic carriers in gene delivery. In this example,
we demonstrate the novel properties of bi-functional Au/Ni nanorods
in gene transfer. Deposition of the AuNi nanorods was achieved by
template synthesis. This technique involves electrochemical
deposition into a non-conducting membrane having an array of
cylindrical pores and has been used for the synthesis of a wide
range of materials and structures. Template synthesis is preferred
over other techniques because it is easily adapted for the
deposition of multiple sub-micron segments. Furthermore, template
synthesis can produce large quantities of monodisperse nanorods,
and properties such as aspect ratio can be controlled in a
systematic way.
[0037] Referring to FIG. 2, the nanorods 20 were fabricated by
electrodeposition into an Al.sub.2O.sub.3 template (Anodisc,
Whatman) with a pore diameter of 100 nm. An evaporated silver film
on one side of the template served as the working electrode in a
three-electrode configuration. Ak thin layer of silver was
electrodeposited into the template from 50 mM KAg(CN).sub.2, 0.25 M
Na.sub.2CO.sub.3 buffered to pH 13 at a potential of -1.0V
(Ag/AgCl) and Ni segments 21 were deposited from a solution of 20 g
L.sup.-1 NiCl.sub.26H.sub.2O, 515 g L.sup.-1
Ni(H.sub.2NSO.sub.3).sub.24 H.sub.2O), 20 gL.sup.-1 H.sub.3BO.sub.3
buffered to pH 3.4 at a potential of -1.0 V (Ag/AgCl) to ensure
easy release of the nanorods from the template. The Au segments 22
were deposited from a commercial gold plating solution (Technic
Inc.) at a potential of -1.0V (Ag/AgCl) and the Ni segments 21 were
deposited from a solution of 20 gL.sup.-1 NiCl.sub.26 H.sub.2O, 515
gL.sup.-1 Ni(H.sub.2NSO.sub.3).sub.24 H.sub.2O), 20
gL.sup.-1H.sub.3BO.sub.3 buffered to pH 3.4 at a potential of -1.0
V (Ag/AgCl). The silver layers were dissolved in 70 vol % nitric
acid and the alumina template was then dissolved in 2 M potassium
hydroxide. The nanorods 20 were washed repeatedly using 2 M
potassium hydroxide, de-ionized water and ethanol. The nanorods
were 100 nm in diameter and 200 nm in length with 100 nm gold
segments and 100 nm nickel segments.
[0038] Using molecular linkages that bind selectively to either
gold or nickel, we have attached DNA 23 and a cell-targeting
protein 24, transferrin, to the different segments, as shown
schematically in FIG. 2. Transferrin was one of the first proteins
to be exploited for receptor-mediated endocytosis of the
transferrin-iron complex. The transferrin 24 was bound to the gold
segments 22 of the nanorods 20 through a thiolate linkage (not
shown), by converting a small proportion of the primary amine
groups of transferrin to sulfhydryl groups. A rhodamine tag (not
shown) on the transferrin provided a mechanism for confirmation of
internalization and intracellular tracking of the nanorods.
[0039] DNA 23 was bound to the nickel segments 21 by suspending the
dual component nanorods in a 0.1 M solution of
3-[2-aminoethyl)dithio] propionic acid (AEDP). The carboxylic acid
terminus of AEDP binds to the native oxide on the nickel segments
This resulted in the surface presentation of primary amine groups
spaced by a reducible disulfide linkage 25. Plasmids encoding the
firefly luciferase (pCMV-luciferase VR1255_C) with 6.413 kbt driven
by the cytomegalovirus (CMV) promoter/enhancer (luciferase-plasmid)
or plasmids encoding the GFPmut1 variant (PEGFP-C1) with 4.7 kb
driven by the SV40 early promoter (GFP-plasmid) were conjugated to
the AEDP bound to the nickel segments 21 of the nanorods 20 at pH
5.7. The plasmid concentration, determined from absorbance
spectroscopy, was about 4.times.10.sup.12 molecules cm.sup.-2.
[0040] To further compact: the DNA bound to the nanorods for more
efficient cell entry and protection of the DNA from enzyiatic
degradation, the nanorods were incubated in 2M CaCl.sub.2 after
excess non-bound plasmids had been removed. Ca.sup.2+ has a high
affinity to DNA (K.sub.d of 1.1.times.10.sup.-3M.sup.-1), forming
CaPO.sub.4 complexes with the nucleic backbone to provide
stabilization and compaction to the DNA structure.
[0041] Confirmation of the selective binding of transferrin and
plasmid was obtained by fluorescence microscopy. Since the 200 nm
long nanorods cannot be seen by optical microscopy, these
experiments were performed on 20 micron long and 100 nm diameter
nanorods with Ni and Au segments of equal length.
[0042] FIG. 3 shows uniform red fluorescence from the
rhodamine-tagged transferrin on the gold segments and uniform blue
fluorescence from the Hoechst, which selectively binds to the DNA
conjugated to the nickel segments.
[0043] To evaluate the gene delivery potential of these dual
functionalized Au/Ni nanorods, in vitro transfection experiments
were performed on the Human Embryonic Kidney (HEK293) mammalian
cell line with the GFP and luciferase reporter genes, respectively.
For transfection, the nanorods were incubated with HEK293 cells at
a dosing level (4.4.times.10.sup.-5 mg mL.sup.-1) significantly
below the cytotoxicity (LD50 ) value for 4 hours in Opti-MEM cell
culture medium (Gibco BRL, Rockville, Md.). Following washing,
cells were further incubated in serum-containing media for two
days.
[0044] FIG. 4 shows confocal microscopy sections of cells following
transfection. FIG. 4a shows the characteristic green fluorescence
from the GFP expressed by the cells as a result of tranfection.
Superimposed on the GFP emission is the red emission from the
rhodamine conjugated to the Au segments of the nanorods. The
orthogonal sections show clearly that the nanorods are located in
the cell. FIG. 4d shows fluorescence images from cells after 4 h
incubation that have been stained with Lysotracker green revealing
that the nanorods are located in or around acidic organelles.
[0045] The uptake of the nanorods by HEK293 cells is shown in the
scanning electron microscope images in FIGS. 5a and 5b, after 1 and
4 hours incubation, respectively. Transmission electron microscope
images (FIG. 5c) showed that nanorods were located in vesicles or
the cytoplasm but not the nucleus. This suggests that transfection
is due to plasmids released or cleaved from the nanorods prior to
nuclear entry. In contrast, 20 .mu.m long nanorods were found only
partially internalized after 4 hours (FIG. 5d) presumably because
of size constraints.
[0046] To further understand the transfection mechanism, a series
of experiments were undertaken to compare the two-component
nanorods with and without transferrin and chloroquine. Chloroquine
is an endosomolytic agent widely used to promote escape of the
sequestered complexes from endosomal into cytoplasmic
compartments.
[0047] FIG. 6 summarizes the transfection experiments. A
significantly higher fraction of cells expressed GFP when
transfected with plasmid-nanorods than with naked DNA, which was
<3%. Comparing with the luciferase plasmid, transfection by
nanorods shows a 255-fold higher expression than naked DNA.
Nanorods with transferrin produced 22% of GFP-positive cells, 2
times higher than those with transferring; the enhancement is 3.4
times for luciferase expression level. Addition of chloroquine to
nanorods with transferrin further improved GFP expression to 27% of
positive cells, and increased the luciferease expression level by a
factor of 1.9. The fact that chloroquine enhances
transferrin-mediated transfection suggests that receptor-mediated
endocytosis is involved. Chloroquine may also enhance transfection
by protecting against DNA degradation.
[0048] To confirm that transfection was due to intracellular rather
than extracellular release of plasmids, nanorods complexed with the
luciferase-plasmid were incubated in serum-containing media. The
supernatant was removed at various time points from 15 minutes to 4
hours and used to transfect the HEK293 cells. In all cases no
significant transfection above background could be detected in
these samples. These data confirm that the transfection detected is
a result of the intracellular released plasmids from the 200 nm
nanorods. Further details concerning the methods and materials of
Example 1 are set forth in Appendix A attached hereto.
[0049] In summary, this example demonstrates a new approach for
gene delivery using multi-segment nanorods. Using molecules with
end-groups that selectively bind to different metals, specific
functionalities can be introduced to individual segments in the
nanorod. Here we have used differential binding to attach plasmids
and a cell-targeting protein to spatially separated regions of the
delivery system. This approach can be extended to include other
components that allow additional functionalities to be introduced.
For example, an additional segment could be used to bind an
endosomolytic agent. In addition to components that allow selective
binding, other functions can also be exploited. For example, an
external magnetic field can be used to manipulate nanorods with
magnetic segments. In addition, the introduction of segments of
semiconductor materials can be used to trick individual nanorods
through their characteristic absorbance or photoluminescence. The
ability to configure different segments in varying combinations and
with different segment lengths can also be used to barcode
individual nanorods. These properties can be exploited to
externally control gene delivery in vivo. Thus, this versatile
synthetic gene delivery system may help realize the potential of
non-viral gene therapy.
EXAMPLE 2
Vaccinations
[0050] The goal in genetic vaccinations is to encode cells to
transiently manufacture antigens that are subsequently taken up by
macrophages or dendritic cells (key antigen presenting cells or
APCs). APCs process these antigens via class I or class II pathways
where they bind to major histocompatibility complexes that present
the antigen on the surface of the APCs. These APCs then move to the
lymphoid organs where T lymphocytes that scavenge the surfaces of
the APCs become stimulated to respond against the antigen
presented. When, for example, the encoded antigen is tumor specific
a strong CD8+ and CD4+ T-cell and antibody response can be
generated for protection and prevention against that tumor The
inorganic nanorod vectors described herein can generate strong but
transient transgene expression when bombarded into skin, which has
natural abundance of antigen presenting cells. These nanorods
therefore have potential for vaccination applications. In contrast
to other inorganic non-viral vectors, these nanorods can be
engineered with different functionalities in spatially defined
regions, which lead to the potential for precise control of
antigen: adjuvant ratios and the possibility of stimulating
multiple immune responses. However, before these unique nanorod
properties can be exploited for further development, it is
essential to ensure that the nanorods can generate a strong
versatile immune response in vivo.
[0051] In this example, we evaluate the CD4+ antibody and CD8+
T-Cell responses from particle bombardment of nanorods delivering
the model antigen ovalbumin or plasmids encoding ovalbumin.
Ovalbumin is involved in a number of conditions related to
children. For example, children with cystic fibrosis display higher
anti-ovalbumin antibodies. Ovalbumin antibodies are also observed
in kidney diseases such as nephropathy. Children with insulin
dependent diabetes mellitus show elevated immune responses to both
.beta.-lactoglobulin and ovalbumin, which may be associated with
the progression of the disease.
[0052] The nanorods were fabricated by electrodeposition into an
Al.sub.2O.sub.3 template (Anodisc, Whatman) with a nominal pore
diameter of 100 nm. An evaporated silver film on one side of the
template served as the working electrode in a three-electrode
configuration. A thin layer of silver was electrodeposited into the
template to ensure easy release of the nanorods from the template.
Au segments were deposited prior to nickel segments to prevent
erosion of the nickel layers during silver removal. The silver
layers were dissolved in 70 vol % nitric acid and the alumina
template was then dissolved in 2 M potassium hydroxide. The
nanorods were 1.6 .mu.m in length by 170 nm in diameter with 800 nm
length gold segments and 800 nm length nickel segments.
[0053] Confirmation of deposition of the nickel and gold segments
was seen by back-scattering SEM. Using chemical moieties that bind
selectively to either gold or nickel, we attached plasmids of the
antigen ovalbumin, to the different segments as described
previously. A small proportion of the primary amine groups of
ovalbumin were converted to sulfhydryl groups. The ovalbumin was
then bound to the gold segments of the nanorods through a thiolate
linkage. Electrostatic interactions were used to bind DNA to the
nickel segments by suspending the dual component nanorods in a 0.1
M solution 3-[(2-aminoethyl)dithio] propionic acid (AEDP). The
carboxylic acid terminum of AEDP binds to the native oxide on the
nickel segments. This results in the surface presentation of
primary amine groups spaced by a reducible disulfide linkage. In
the reducing environment of the cell, the disulfide linkage between
the plasmid and the nanowire is cleavable, enhancing release of the
plasmid. In this example, plasmids encoding ovalbumin (pcDNA3-OVA7)
or control plasmids with blank inserts (pcDNA3) were utilized.
Previous UV-visible spectroscopy calibration measurements (260 nm)
of DNA binding to the nanowires provided an average surface
coverage of 4.times.10.sup.12 molecules/cm.sup.2. For condensation
of the plasmids bound to the nanowires, a CaCl.sub.2 solution was
added to the n-nanowire-plasmid formulations. Ca.sup.2+ has a high
affinity to DNA (K.sub.d of 1.1.times.10.sup.-3M-.sup.-1, forming
CaPO.sub.4 complexes with the nucleic backbone to provide
stabilization and compaction to the DNA structure.
[0054] To evaluate the genetic vaccination potential of these
nanorods, CD4+ antibody responses from the bloodstream and CD8+
T-cell responses from the spleen were measured from C57BL/6 mice
vaccinated with the nanorod/plasmid or nanorod/antigen
formulations. In addition, we compared these responses to the
industrially optimized gold particle formulations as analogous
responses are essential for the future development of these
nanorods in clinical applications. For antigen/microcarrier
formulations, the gold particles generated a 7-fold higher CD8+
T-cell response that the nanorods. In contrast, for the CD4+
antibody response, the nanorods produced a 7-fold higher response
in comparison with the 1.6 .mu.m gold particles (FIGS. 7 and 8). To
evaluate the benefit of the nanorods multifunctionality, pcDNA3 was
bound to the nickel segments of the nanorods in conjunction with
the ovalbumin-SH antigen on the gold segments. In control
experiments, pcDNA3 bound to the nanorods alone generated very low
or no CD4+ antibody and CD8+ T-cell responses. However, co-addition
of pcDNA3 and the ovalbumin antigen on the nanorods generated a
significant 8-fold increase in the CD8 response in comparison to
the nanorods bound to the ovalbumin alone. This increase is likely
to be due to a role of the CpG motif in the pcDNA3 acting as a
strong immunostimulatory adjuvant to the ovalbumin antigen thus
enhancing the overall CTL immune response. The nanorods ability to
deliver the CpG motif and the antigen to the same cell is essential
for generating a stronger immune response. For example, Babuik and
colleagues have shown that in pigs, administration of CpG ODN and
HBsAg vaccine in separate sites of the sante muscle did not show an
enhanced antibody response compared to administration of the HBsAg
vaccine alone, whereas administration of CpG ODN with the HBsAg
vaccine significantly enhanced the antibody responses.
[0055] Delivering plasmics encoding ovalbumin by both nanorods and
gold particles generated stronger CD4 and CD8 responses than the
ovalbumin antigen alone Gene gun delivery of antigens can directly
enter and prime dendritic cells, but the delivery of plasmids
encoding the antigen probably enhances the overall response because
in addition to direct priming of dendritic cells, keratinocytes
also become transfected. The keratinocytes then produce antigens
that, once released, cross-prime more dendritic cells thereby
enhancing overall immune response. Further details concerning the
methods and materials of Example 2 are set forth in Appendix B
hereto.
[0056] In summary, this example that nanorod based vaccines
generate strong CD4+ antibody and CD8+ T-cell responses and
therefore have significant potential for further development in
vaccination applications. We contemplate that aligning the nanorods
within the cartridges to produce "arrow" like delivery will allow
us to achieve greater depths of penetration in particle bombardment
than the gold particles. Advantages to this would include
transfecting both skin and the subcutaneous tissues for pressure
modulated control over sustained or transient expression of genes
and greater depths of penetration at lower pressures. The ability
to add new components to the nanorods such as adjuvants and/or
cytokines in controlled ratios will allow us to generate stronger
immune responses than single component particles as demonstrated in
this example using the CpG motif from the pcDNA3 as an
immunostimulatory adjuvant to the antigen. In addition, the ability
to engineer and add extra segments to the nanorods will allow for
the possibility of delivering multiple agents such as RNA, antigens
and DNA to the same cell for the stimulation of multiple immune
responses.
EXAMPLE 3
Delivery of Multiple Active Molecules
[0057] This example demonstrates the selective derivatization of
three segment Au/Ni/Pt nanowires using metal specific ligands. By
taking advantage of the individual metal segments' affinity to
unique functional groups, we show that Au/Ni/Pt nanowires can be
functionalised with a thiol linkage on the gold segments, an
isonitrile linkage on the platinum segment and a carboxylate
linkage on the nickel segment. Selective functionalisation of the
Au, Ni and Pt segments is achieved by first functionalizing the Ni
segment with carboxylic acid terminated ligands and the Au and Pt
segments with an isonitrile terminated ligand. Carboxylic acids
have been found to bind to nickel surfaces at an adduct formation
constant of 6.+-.5.times.10.sup.6 M.sup.-1. Isonitrile groups are
reported to form monolayers on both Au and Pt surfaces. The
isonitrile groups on the Au surface can then be selectively
substituted with thiol terminated ligands.
[0058] The formation of three component nanowires is shown in FIG.
9, Au/Ni/Pt nanowires 90 are fabricated by electrodeposition into
an Al.sub.2O.sub.3 template 91 (Anodisc, Whatman) with a nominal
pore diameter of 100 nm. An evaporated silver film 88 on one side
of the template serves as the working electrode in a
three-electrode configuration. A thin layer of silver 89 is first
electrodeposited from 50 mm KAg(CN)2 and 0.25 M Na.sub.2CO.sub.3
buffered to pH 13 at -1.0 V (Ag/AgCl) in order to ensure easy
release of the nanorods from the template. The Au segments 92 of
the nanowires are deposited from a commercial gold plating solution
(Technic) at -1.0 V (Ag/AgCl), and the Ni segments 92 are deposited
from a solution of 20 g L.sup.-1 NiCl.sub.26H.sub.2O, 515 g
L.sup.-1 Ni(H.sub.2NSO.sub.3).sub.24 H.sub.2O), 20 gL.sup.-1
H.sub.3BO.sub.3 buffered to pH 3.4 at a potential of -1.0 V
(Ag/AgCl). The Pt segments 94 are deposited from a solution of
0.015M of (NH.sub.4).sub.2PtCl.sub.6 and 0.2M
Na.sub.2HPO.sub.47H.sub.2O at -0.4 V (Ag/AgCl). The gold segments
92 are deposited before the nickel segments 93 in order to ensure
that the nickel segments 93 are not etched by the nitric acid
during removal of the silver, and the platinum segments 94 are
deposited after the nickel and with longer length segments to
clearly differentiate the segment from the gold. The silver layers
(88, 89) are dissolved in 70 vol. % nitric acid and the alumina
template 91 is then dissolved in 2 M KOH. The nanowires 90 are
washed repeatedly using 2 M KOH, de-ionized water, and ethanol. The
nanowires 90 are on average 170 nm +/- 23 nm in diameter and 8-10
.mu.m in length.
[0059] Confirmation of the integrity of the three segments is
observed by back-scattering SEM (FIG. 11a). Collection of the
nanowires by centrifugation at 8000 rpm often results in bending of
the nanowires, in particular at the junctions of the segments.
Magnetic collection of the nanowires results in significantly
reduced bending but the remnant magnetized state of the nickel
segments produces aggregated nanowires that reduce the efficiency
of selective derivatization and leads to greater difficulty in
subsequent imaging of single nanowires.
[0060] FIG. 10 schematically illustrates the functionalization of
the nanowires 90. In the first step of the functionalization (FIG.
10a), Au/Ni/Pt nanowires (.apprxeq.10.sup.9 mL.sup.-1) are
suspended in 2 ml of ethanol containing 2 mM 12-amino-dodecanoic
acid (Aldrich) and 2 mM 1-butane isocyanide (BIC). The suspension
is agitated using rotation for 24 hours. The nanowires 90 are then
washed using repeat centrifugation and resuspension cycles using
ethanol. The nanowires are then reacted with 3.84 mg Rhodamine Red
succinimidyl ester (Molecular Probes) in 5 mL of a 50:50 mixture of
pH 8.5 sodium tetrahorate buffer and dimethylsulfoxide (DMSO)
overnight under an argon blanket at room temperature (FIG. 10b).
The succinimidyl ester reacts rapidly in the presence of primary
amine groups producing a strong amide bond between the
self-assembled monolayer molecules and the fluorophore. The
nanowire suspension is then sonicated for 1 hour, followed by
washes with DMSO, water and ethanol. For microscopic imaging,
nanowires are spin coated onto a glass coverslip rotating at 2500
rpm for 15 secs.
[0061] FIGS. 11b and 11c show light microscope and fluorescence
microscope images of the functionalized nanowires. Fluorescence
from the rhodamine (Ex 570, Em 590) is predominantly localized to
the Ni segment. Significantly weaker fluorescence is also observed
on the Au and Pt sections. This is most probably due to
physisorption between the hydrophobic rhodamire red flurophore and
the hydrophobic BIC functionalised Au and Pt sections. Carboxylic
acids have been reported to bind weakly to Au surfaces. The
contrast between the weak fluorescence on the Au/Pt segments and
the strong fluorescence on the Ni segment indicates that the BIC
has preferentially bound to the Au/Pt surfaces significantly
blocking carboxylic acid binding. Note that whilst the quenching of
fluorescence molecules proximate to metal surfaces has been
previously reported fluorophores bound to nanowires remain
sufficiently detectable to identify selective
functionalization.
[0062] Referring back to FIG. 10 the nanorods are next suspended in
2 ml of 2 mM 11-amino-1-undecanethiol (Dojindo) in ethanol and
mixed using rotation for 24 hours (FIG. 10c). The nanowires are
washed with ethanol using repeat centrifugation and resuspension
cycles. The nanowires are then reacted with 3.67 mg Marina Blue
succinimidyl ester (Molecular Probes) in 5 mL of a 50:50 mixture of
pH 8.5 sodium tetraborate buffer and DMSO overnight under an argon
blanket at room temperature (FIG. 10d). The nanowire suspension is
then sonicated for 1 hour, followed by washes with DMSO water and
ethanol
[0063] FIG. 11d-11g show light microscope and fluorescent
microscope images of the tri-functionalized nanowires. FIG. 11e
shows that fluorescence observed from the Marina
Blue-1-undecanethiol (Ex 365, Em 460) is specifically from the gold
segment.
[0064] FIG. 11f shows that fluorescence from the rhodamine (Ex 570,
Em 590) is still emanating from the Ni segment. Either very weak
fluorescence (Ex 365, Em 460) or no fluorescence at all is observed
from the longer Pt sections functionalised with BIC. This suggests
that the carboxylic acid has retained its binding affinity to the
Ni, whilst the thiol terminated molecules have displaced isonitril
groups on the Au segment but not the Pt section. Surface
engineering Au and Pt with BIC first followed by thiol displacement
on the Au segment is preferential because of the reported ability
of the BIC molecules to prop up the thiol terminated molecules in
the upright orientation.
[0065] In control experiments, Au/Ni/Pt nanowires are
functionalized with BIC and 12-amino-dodecanoic acid followed by
treatment with Rhodamine Red succinimidyl ester. when the wires are
then exposed to 1-decanethiol, fluorescence is observed only on Ni
segments. Similarly, when the nanowires are functionalized with BIC
and palmitic acid, followed by exposure to
11-amino-1-undecanethiol, subsequent treatment with Marina blue
succinimidyl ester results in fluorescence predominantly observed
on the Au sections.
[0066] In summary, this example demonstrates selective
derivatization of three component Au/Ni/Pt nanowires using metal
specific surface chemistries. The ability to direct unique
fluorescent, biological or chemical molecules to individual
segments in three or more component nanowires has potential for
further advances in gene/drug delivery, chemical sensing and
self-assembly.
[0067] It is understood that the above-described embodiments are
illustrative of only a few of the many possible specific
embodiments, which can represent applications of the invention.
Numerous and varied other arrangements can be made by those skilled
in the art without departing from the spirit and scope of the
invention.
* * * * *