GENE EXPRESSION

CROSS-REFERENCE OF RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No.

62/449,884 filed January 24, 2017, the entire contents of which are hereby incorporated by reference.

BACKGROUND

1. Technical Field

[0002] The field of the currently claimed embodiments of this invention relates to systems and methods for tailored gene expression in a tissue using a vehicle that is therapeutic.

2. Discussion of Related Art

[0003] Gene therapy has incredible potential to treat a variety of health impairments. ¹ Whether conditions exist from birth ^{2 3} or develop with age, ^{4 5}

supplementing key genes involved in critical biological functions could improve the quality of life of many individuals. Wound healing is an important process that is impaired in the elderly and diabetics, among others. In fact, over three million Americans suffer from non-healing wounds. ⁶ Wound healing is dynamic and genetically complex as there are a variety of factors expressed throughout the stages of inflammation, proliferation, and maturation. ⁷

[0004] Many genes involved in wound healing have been shown to be

downregulated with age or the onset of diseases, leading to compromised healing. ^{8 9} An ongoing clinical trial at the Ohio State University, expected to end in 2020, aims to identify genes that are expressed differently in the elderly, diabetics, and others with nonhealing wounds compared to those with normal-healing wounds. ¹⁰ One study was able to identify specific periods of time within the first week after the formation of a wound when certain classes of proinflammatory and angiogenic genes were expressed. ¹¹ Mimicking the expression of key wound healing genes ^{12 15} that occur in normal healing may result in accelerated wound closure for those with non-healing wounds.

[0005] In the past, viral vectors have been the most utilized system for gene delivery due to their singular purpose of transferring genetic material to cells. ¹⁶ However, they evoke immune responses in the body, and present the risk of long-term expression and insertional mutagenesis, which can lead to cancer. ¹⁷ Naked DNA plasmids do not have these problems, but achieving transfection efficiency with them is difficult. ¹⁸ Non- viral strategies for improving transfection efficiency in vivo include electroporation, which has been successful. ^{8 9} Currently, attention has focused on nanoparticle carriers, specifically in the realm of polymers. ^{19 21}

[0006] Polyethylenimine (PEI) and polylysine are two popular polymers used for nanoparticles that have been successful in gene delivery. ²² However, as their efficiency increases so too does their toxicity. ²³ Additionally, these polymers only offer a single, short-term course of gene expression and rely on supplementary systems for controlled release. ²⁴ Recently, polymeric scaffolds have been incorporated into gene delivery to obtain slow release of plasmid-loaded nanoparticles. ²⁵ To avoid the toxicity that frequently accompanies polymeric nanoparticles, scaffolds have been used to deliver naked plasmids encoding factors to assist in wound healing. ^{26 27} However, this method does not facilitate cellular uptake or protection of the DNA once it transitions to the skin. ²⁸ Furthermore, to create the scaffold, many polymers require the addition of chemical crosslinking reagents which cause concern for toxicity. ²⁹

[0007] Although there exists an overarching goal of optimized delivery efficiency and biocompatibility of gene delivery materials, ^{30 31} not much attention has focused on choosing a polymer that contributes to the treatment at hand and allows for different patterns of expression.

SUMMARY

[0008] Some embodiments of the current invention relate to a composition for tailored, non- viral polynucleotide delivery to a cell having a plurality of nanoparticles and a polynucleotide attached to an exterior surface of at least one of the plurality of nanoparticles. Each of the plurality of nanoparticles includes a natural or synthetic cationic polymer electrostatically crosslinked with an anionic electrostatic crosslinking agent, each of the plurality of nanoparticles have a positive zeta potential, and each of the plurality of nanoparticles are between 1 nm - 1000 nm in diameter.

[0009] Some embodiments of the current invention relate to a method for making a composition for tailored, non-viral polynucleotide delivery to a cell that includes preparing a first suspension that includes a first plurality of nanoparticles of a first diameter, centrifuging the first suspension under conditions sufficient to separate a second plurality of nanoparticles of a second diameter, extracting a supernatant from the centrifuged first suspension, centrifuging the extracted supernatant under conditions sufficient to separate a third plurality of nanoparticles of a third diameter, preparing a second suspension that includes a predetermined concentration of the third plurality of nanoparticles, and contacting the third plurality of nanoparticles in the second suspension with a polynucleotide such that the polynucleotide attaches to an exterior surface of at least one of the third plurality of nanoparticles. Preparing the first suspension includes crosslinking a polymer with a crosslinking agent. The first diameter is greater than the second diameter and the second diameter is greater than the third diameter. Each of the first, second and third plurality of nanoparticles includes a plurality of nanoparticles, each having a positive zeta potential.

[0010] Some embodiments of the current invention relate to a method for tailored, non-viral polynucleotide delivery to a cell or tissue that includes obtaining a composition that includes a polynucleotide and a plurality of nanoparticles, and contacting the cell or tissue with the composition. The polynucleotide is attached to an exterior surface of at least one of the plurality of nanoparticles. The plurality of nanoparticles are present at a predetermined concentration, the plurality of nanoparticles include a polymer crosslinked with a crosslinking agent, the plurality of nanoparticles have a positive zeta potential, and the plurality of nanoparticles are between lnm - lOOOnm, or between 50 nm - 500 nm in diameter. Contacting results in internalization of the composition by the cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1A is a graph showing the average nanoparticle diameter as a function of centrifugation steps according to an embodiment of the current invention; [0012] FIG. IB is a graph showing the average zeta potential of the nanoparticles as a function of centrifugation steps according to an embodiment of the current invention;

[0013] FIG. 1C is a scanning electron microscopy (SEM) image of nanoparticles of a variety of sizes (scale bar is Ιμιη) according to an embodiment of the current invention;

[0014] FIG. ID is a scanning electron microscopy (SEM) image of nanoparticles of a variety of sizes (scale bar is 250nm) according to an embodiment of the current invention;

[0015] FIG. 2A shows an atomic force microscopy image of plasmid DNA with a

1% nanoparticle solution (top panel) and an illustration of the like (bottom panel) (scale bar is lOOnm) according to an embodiment of the current invention;

[0016] FIG. IB shows an atomic force microscopy image of plasmid DNA with a

5% nanoparticle solution (top panel) and an illustration of the like (bottom panel) (scale bar is 50nm) according to an embodiment of the current invention;

[0017] FIG. 2C shows an atomic force microscopy image of plasmid DNA with a

10% nanoparticle solution (top panel) and an illustration of the like (bottom panel) (scale bar is lOOnm) according to an embodiment of the current invention;

[0018] FIG. 2D shows an atomic force microscopy image of plasmid DNA with a

50% nanoparticle solution (top panel) and an illustration of the like (bottom panel) (scale bar is 50nm) according to an embodiment of the current invention;

[0019] FIG. 3A is a graph showing average transfection efficiency of various concentrations of nanoparticles according to an embodiment of the current invention as measured as a function of average luminescence over the course of 6 days;

[0020] FIG. 3B is a graph showing the total transfection efficiency of various concentrations of nanoparticles according to an embodiment of the current invention as measured a function of average luminescence;

[0021] FIG. 3C is a panel of images showing the location of gene expression according to an embodiment of the current invention along a wound over the course of 3 days;

[0022] FIG. 4A is a graph showing average transfection efficiency of various concentrations of nanoparticles as measured as a function of average luminescence over the course of 7 days; [0023] FIG. 4B is a graph showing the total transfection efficiency of various concentrations of nanoparticles as measured as a function of average luminescence;

[0024] FIG. 4C is a panel of images showing the location of gene expression along a wound over the course of 4 days;

[0025] FIG. 5A is a graph showing average transfection efficiency of naked DNA as measured as a function of average luminescence over the course of 7 days;

[0026] FIG. 5B is a graph showing average transfection efficiency of a 5% concentration of nanoparticles as measured as a function of average luminescence over the course of 7 days;

[0027] FIG. 5C is a graph showing average transfection efficiency of a 50% concentration of nanoparticles as measured as a function of average luminescence over the course of 7 days;

[0028] FIG. 6A is a graph showing accelerated wound closure over the course of

7 days as a result of treating a wound with a 50% nanoparticle solution;

[0029] FIG. 6B is a graph showing accelerated wound closure over the course of

28 days as a result of treating a wound with a 50% nanoparticle solution;

[0030] FIG. 6C shows the results of Kaplan-Meier analysis of wound healing as a result of treating a wound with a 50% nanoparticle solution

[0031] FIG. 6D is a graph showing peak force measured during a tensiometry test of healed tissue; and

[0032] FIG. 6E is a graph showing amount of work measured during a tensiometry test of healed tissue.

DETAILED DESCRIPTION

[0033] Some embodiments of the current invention are discussed in detail below.

In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated. In particular, the previously filed U. S. patent Application No. 15/097,904, filed April 13, 2016 is hereby incorporated by reference.

[0034] Chitosan is a cationic polymer created by the deacetylation of chitin, a derivative of glucose found in the exoskeleton of many crustacean. It has been used as a non-viral vehicle for the encapsulation of nucleic acids in many gene delivery studies. ³² Others who have used polymer-coated gold nanoparticles have even started substituting chitosan for PEI due to chitosan' s biocompatibility and biodegradability. ³³ In addition to the safety of this natural polymer, it has also shown beneficial wound healing properties through its involvement in the process of inflammation and by the stimulation of cell proliferation and maturation. ^{34 39} Wound dressings composed of chitosan are constantly being developed, ^{40 42} and some products are already used clinically.

[0035] Some embodiments of the invention relate to a system for tailored gene expression in a wound using a vehicle that is therapeutic. This method differs from traditional polymeric nanoparticle approaches in that nanoparticles are first formed via electrostatic interactions with a polyanion rather than encapsulate nucleic acids during the nanoparticles' formation. The nanoparticles are then used as tangential adjuncts to plasmid DNA for obtaining controlled gene expression. This ensures that all plasmid DNA, or any nucleic acid, is being used for a therapeutic purpose compared to other methods where unincorporated plasmid DNA is discarded. Both intensity and duration of gene expression in the skin of a wound healing model in vivo are assessed while varying the concentration of nanoparticles in a solution with plasmid DNA. The effect of the nanoparticles on wound healing when administered around a wound is also explored.

[0036] An embodiment of the current invention relates to a composition for tailored, non- viral polynucleotide delivery to a cell having a plurality of nanoparticles and a polynucleotide attached tangentially to an exterior surface of at least one of the plurality of nanoparticles. In such embodiments, each of the plurality of nanoparticles includes a natural or synthetic cationic polymer electrostatically crosslinked with an anionic electrostatic crosslinking agent. Also, each of the plurality of nanoparticles have a positive zeta potential, and are between lnm - lOOOnm, or between 50 nm - 500 nm in diameter.

[0037] Some embodiments of the invention relate to the composition described above, where the natural biopolymer is chitosan and wherein the crosslinking agent is tripolyphosphate. [0038] Some embodiments of the invention relate to the composition above wherein the chitosan has a molecular weight of at least 1 kDa.

[0039] Some embodiments of the invention relate to the composition described above, where the chitosan has a molecular weight of at least lkDa or of at least 150kDa.

[0040] Some embodiments of the invention relate to the composition described above, where the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0041] Some embodiments of the invention relate to the composition described above, where the positive zeta potential is at least 5mV or between 5mV to 20mV.

[0042] Some embodiments of the invention relate to the composition described above, where the plurality of nanoparticles have a concentration of at least 3x10 ⁶ particles/ml or of at least 3x10 ⁸ particles/ml.

[0043] Some embodiments of the invention relate to the composition described above, where the polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell.

[0044] An embodiment of the current invention relates to a method for making a composition for tailored, non-viral polynucleotide delivery to a cell including preparing a first suspension comprising a first plurality of nanoparticles of a first diameter, centrifuging the first suspension under conditions sufficient to separate a second plurality of nanoparticles of a second diameter, extracting a supernatant from the centrifuged first suspension, centrifuging the extracted supernatant under conditions sufficient to separate a third plurality of nanoparticles of a third diameter, preparing a second suspension comprising a predetermined concentration of the third plurality of nanoparticles, and contacting the third plurality of nanoparticles in the second suspension with a polynucleotide such that the polynucleotide attaches tangentially to an exterior surface of at least one of the third plurality of nanoparticles. In such embodiments, the first suspension comprises crosslinking a natural biopolymer by substituting the natural biopolymer with a crosslinking agent, where the first diameter is greater than the second diameter and the second diameter is greater than the third diameter, and where each of the first, second and third plurality of nanoparticles comprise a plurality of nanoparticles each having a positive zeta potential.

[0045] Some embodiments of the invention relate to the method described above, where the natural biopolymer is chitosan and wherein the crosslinking agent is tripolyphosphate. [0046] Some embodiments of the invention relate to the method described above, where the chitosan has a molecular weight of at least lkDa or of at least 150kDa.

[0047] Some embodiments of the invention relate to the method described above, where the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0048] Some embodiments of the invention relate to the method described above, where the positive zeta potential is at least 5mV or between 5mV to 20mV.

[0049] Some embodiments of the invention relate to the method described above, where the predetermined concentration is at least 3x10 ⁶ particles/ml or at least 3x10 ⁸ particles/ml.

[0050] Some embodiments of the invention relate to the method described above, where the polynucleotide includes a DNA plasmid having a gene, and wherein the DNA plasmid is configured to express the gene within the cell.

[0051] An embodiment of the current invention relates to a method for tailored, non-viral polynucleotide delivery to a cell or tissue including obtaining a composition comprising a polynucleotide and a plurality of nanoparticles, and contacting the cell or tissue with the composition. In such embodiments, the polynucleotide is attached tangentially to an exterior surface of at least one of the plurality of nanoparticles, the plurality of nanoparticles are present at a predetermined concentration, the plurality of nanoparticles comprise a natural biopolymer substituted with a crosslinking agent, the plurality of nanoparticles have a positive zeta potential, the plurality of nanoparticles are between lnm - lOOOnm, or between 50 nm - 500 in diameter, and the contacting results in internalization of the composition by the cell or tissue.

[0052] Some embodiments of the invention relate to the method described above, where the natural biopolymer is chitosan and wherein the crosslinking agent is tripolyphosphate.

[0053] Some embodiments of the invention relate to the method described above, where the chitosan has a molecular weight of at least lkDa or of at least 150kDa.

[0054] Some embodiments of the invention relate to the method described above, where the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0055] Some embodiments of the invention relate to the method described above, where the positive zeta potential is at least 5mV or between 5mV to 20mV. [0056] Some embodiments of the invention relate to the method described above, where the predetermined concentration is at least 3x10 ⁶ particles/ml or at least 3x10 ⁸ particles/ml.

[0057] Some embodiments of the invention relate to the method described above, where the predetermined concentration is between 3xl0 ⁸ particles/ml and 1.7xl0 ¹⁰ particles/ml.

[0058] Some embodiments of the invention relate to the method described above, where the polynucleotide includes a DNA plasmid having a gene, and wherein the DNA plasmid is configured to express the gene within the cell.

[0059] Some embodiments of the invention relate to a composition for tailored, non-viral polynucleotide delivery to a cell having: a plurality of nanoparticles; and a polynucleotide attached to an exterior surface of at least one of the plurality of nanoparticles. Wherein each of the plurality of nanoparticles comprise a natural or synthetic cationic polymer substituted with a crosslinking agent, each of the plurality of nanoparticles have a positive zeta potential, and each of the plurality of nanoparticles are between 1 nm - 1000 nm in diameter.

[0060] Some embodiments of the invention relate to the composition above, wherein the natural or synthetic cationic polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.

[0061] Some embodiments of the invention relate to the composition above wherein the chitosan has a molecular weight of at least 1 kDa.

[0062] Some embodiments of the invention relate to the composition above wherein the chitosan has a molecular weight of at least 150 kDa.

[0063] Some embodiments of the invention relate to the composition above wherein the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0064] Some embodiments of the invention relate to the composition above wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.

[0065] Some embodiments of the invention relate to the composition above wherein the plurality of nanoparticles have a concentration of at least 3x10 ⁶ particles/ml or of at least 3x10 ⁸ particles/ml.

[0066] Some embodiments of the invention relate to the composition above wherein the polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell. [0067] Some embodiments relate to a method for making a composition for tailored, non- viral polynucleotide delivery to a cell including: preparing a first suspension comprising a first plurality of nanoparticles of a first diameter; centrifuging the first suspension under conditions sufficient to separate a second plurality of nanoparticles of a second diameter; extracting a supernatant from the centrifuged first suspension;

centrifuging the extracted supernatant under conditions sufficient to separate a third plurality of nanoparticles of a third diameter; preparing a second suspension comprising a predetermined concentration of the third plurality of nanoparticles; and contacting the third plurality of nanoparticles in the second suspension with a polynucleotide such that the polynucleotide attaches to an exterior surface of at least one of the third plurality of nanoparticles. Wherein the preparing the first suspension comprises crosslinking a polymer by crosslinking the polymer with a crosslinking agent, the first diameter is greater than the second diameter and the second diameter is greater than the third diameter, and each of the first, second and third plurality of nanoparticles comprise a plurality of nanoparticles each having a positive zeta potential.

[0068] Some embodiments relate to the method above, wherein the polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.

[0069] Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least 1 kDa.

[0070] Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least lkDa or of at least 150kDa.

[0071] Some embodiments relate to the method above, wherein the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0072] Some embodiments relate to the method above, wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.

[0073] Some embodiments relate to the method above, wherein the predetermined concentration is at least 3x10 ⁶ particles/ml or at least 3x10 ⁸ particles/ml.

[0074] Some embodiments relate to the method above, wherein the

polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell.

[0075] Some embodiments relate a method for tailored, non-viral polynucleotide delivery to a cell or tissue including: obtaining a composition comprising a polynucleotide and a plurality of nanoparticles; and contacting the cell or tissue with the composition. Wherein the polynucleotide is attached to an exterior surface of at least one of the plurality of nanoparticles, the plurality of nanoparticles are present at a predetermined concentration, the plurality of nanoparticles comprise a polymer crosslinked with a crosslinking agent, the plurality of nanoparticles have a positive zeta potential, the plurality of nanoparticles are between lnm - lOOOnm, or between 50 nm - 500 in diameter, and contacting results in internalization of the composition by the cell or tissue.

[0076] Some embodiments relate to the method above, wherein the polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.

[0077] Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least lkDa or of at least 150kDa.

[0078] Some embodiments relate to the method above, wherein the chitosan has a degree of deacetylation of at least 60% or at least 80%.

[0079] Some embodiments relate to the method above, wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.

[0080] Some embodiments relate to the method above, wherein the predetermined concentration is at least 3x10 ⁶ particles/ml or at least 3x10 ⁸ particles/ml.

[0081] Some embodiments relate to the method above, wherein the

polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell.

[0082] Some embodiments relate to the method above, wherein the gene is expressed for at least 24 hours.

[0083] Some embodiments of the invention relate to systems or compositions for tailored, non-viral polynucleotide delivery to a cell. These systems or compositions include a plurality of nanoparticles and a polynucleotide attached tangentially to an exterior surface of at least one of the plurality of nanoparticles. In such embodiments, the polynucleotide is not contained within an internal space or compartment of the nanoparticles.

[0084] In some embodiments, the nanoparticles are made at least partially from a natural biopolymer. In some embodiments, the nanoparticles are made completely from a natural biopolymer. As used throughout, a natural biopolymer is a polymer produced by living organisms. Alternatively, the natural polymer can be derived, synthesized or processed from a natural compound. One of ordinary skill in the art can envision that any type of natural polymer can be used. In some embodiments, the biopolymer is positively charged.

[0085] In some embodiments, the nanoparticles are made at least partially from chitosan or a chitosan derivative. In some embodiments, the nanoparticles are made completely from a chitosan or a chitosan derivative. In some embodiments, the chitosan has a molecular weight of at least 150 kDa. As an alternative to chitosan, a derivative thereof can also be used, understanding as such a chitosan with a molecular weight at least 150 kDa wherein one or more hydroxy! groups and/or one or more amine groups have been modified, with the aim of increasing the solubility of the chitosan, increasing the target potential towards specific cells or tissues, or increasing the adhesive nature thereof. These derivatives include, among others, acetylated, alkylated or sulfonated chitosans, thiolated derivatives, as is described in Roberts, Chitin Chemistry, Macmillan, 1992, 166. In some embodiments, when a derivative is used it is selected from O-alkyl ethers, O-acyl esters, trimethyl chitosan, chitosans modified with polyethylene glycol, etc. Other possible derivatives are salts, such as citrate, nitrate, lactate, phosphate, glutamate, etc. In any case, a person skilled in the art knows how to identify the modifications which can be made on the chitosan without affecting the stability and commercial feasibility of the formulation.

[0086] In some embodiments, the nanoparticles are chitosan-based compounds.

By "chitosan-based based" is meant any compound having the polysaccharide chemical structure as common to chitosan and chitin. Chitosan is a linear polysaccharide composed of two monosaccharides: N-acetyl-D-glucosamine and D-glucosamine linked together by β(1-4) glycosidic bonds. Chitosan is derived from chitin (poly -N-acetyl-D-glucosamine). Chitin is deacetylated to chitosan by the treatment of strong NaOH at elevated temperatures with the material being kept in the solid phase to gain the highest possible yield. The term "chitosan based compound" includes chitin, chitosan, chitosan oligomers, as well as derivatives or analogues thereof that are capable of forming suitable compositions in combination with a nucleic acid or an oligonucleotide.

[0087] By "analogs" or "derivatives thereof are meant chitosan-based compounds having: (i) specific or non-specific cell targeting moieties that can be covalently attached to chitin, chitosan, and chitosan oligomers or ionically or hydrophobically adhered to a chitosan-based compound complexed with a nucleic acid or an oligonucleotide, and (ii) various derivatives or modifications of chitin, chitosan, and chitosan oligomers which serve to alter their physical, chemical, or physiological properties. Examples of analogs include, but are not limited to, chitosan-based compounds having specific or non-specific targeting ligands, membrane permeabilization agents, sub-cellular localization components, endosomolytic (lytic) agents, nuclear localization signals, colloidal stabilization agents, agents to promote long circulation half- lives in blood, and chemical derivatives such as salts, O-acetylated and N-acetylated derivatives, etc. These analogs can be formed by covalent attachment, derivatization, or modification to the complexing agents directly, adhered to complex particles by ionic or hydrophobic interaction, or simply physically combined with the complexing agents or their complex particles. Examples of such analogs include, but are not limited to, agents such as a lipophilic peptide binding molecule or JTS-1 or a derivative as a lysis agent as described in patent application Ser. No. 08/584,043, entitled "Lipophilic Peptides For Macromolecule Delivery", filed on Jan. 11, 1995, incorporated by reference herein in its entirety including any drawings or figures. In a an embodiment some sites for chemical modification of chitosan include: C ₂ (NH— CO— CH ₃ or NH ₂ ), C ₃ (OH), or C ₆ (CH ₂ OH).

[0088] By "nucleic acid" is meant both RNA and DNA including: cDNA, genomic DNA, plasmid DNA, antisense molecule, polynucleotides or olignucleotides, RNA or mRNA. In an embodiment, the nucleic acid administered is plasmid DNA which comprises a "vector". By "vector" is meant a nucleic acid molecule incorporating sequences encoding polypeptide product(s) as well as, various regulatory elements for transcription, translation, transcript stability, replication, and other functions as are known in the art and as described herein. Vector can include expression vector. An "expression vector" is a vector which allows for production or expressing a product encoded for by a nucleic acid sequence contained in the vector. The product may be a protein or a nucleic acid such as an mRNA which can function as an antisense molecule. A "transcript stabilizer" is a sequence within the vector which contributes to prolonging the half life (slowing the elimination) of a transcript. By "oligonucleotide" is meant a single-stranded polynucleotide chain.

[0089] A "DNA vector" is a vector whose native form is a DNA molecule. By

"non-viral" is meant any vector or composition which does not contain genomic material of a viral particle. An "antisense molecule" can be a mRNA or an oligonucleotide which forms a duplex with a complementary nucleic acid strand and can prevent the

complementary strand from participating in its normal function within a cell. For example, expression of a particular growth factor protein encoded by a particular gene. A "gene product" means products encoded by the vector. Examples of gene products include mRNA templates for translation, ribozymes, antisense RNA (mi RNA, etc.), proteins, glycoproteins, lipoproteins and phosphoproteins. "Post-translational processing" means modifications made to the expressed gene product. These may include addition of side chains such as carbohydrates, lipids, inorganic or organic compounds, the cleavage of targeting signals or propeptide elements, as well as the positioning of the gene product in a particular compartment of the cell such as the mitochondria, nucleus, or membranes. The vector may comprise one or more genes in a linear or circularized configuration. The vector may also comprise a plasmid backbone or other elements involved in the production, manufacture, or analysis of a gene product. The nucleic acid may be associated with a targeting ligand to effect targeted delivery.

[0090] By "administering" is meant the route of introduction of the composition into a body. Administration can be directly to a target tissue or through systemic delivery. In particular, administration may be by direct injection to the cells. Routes of

administration include, but are not limited to, intramuscular, aerosol, oral, topical, systemic, nasal, ocular, intraperitoneal and/or intratracheal, buccal, sublingual, oral, intradermal, subcutaneous, pulmonary, intra-artricular, and intra-arterial. In an embodiment administration is by intravenous administration.

[0091] In yet another aspect, the composition is administered to an organism. By

"administering or administration" is meant the route of introduction of the composition into an organism. Administration can be directly to a target tissue or through systemic delivery. Administration can include but is not limited to: oral, subcutaneous, intradermal, intramuscular, rectal, intravenous, intra tumoral, pulmonary, nasal, intra articular, ocular, topical, and intra-osseous methods of delivery. In particular, the present invention can be used for administering nucleic acid for expression of specific nucleic acid sequence in cells. Routes of administration include intramuscular, aerosol, olfactory, oral, topical, systemic, ocular, intraperitoneal and/or intratracheal.

[0092] In some embodiments, the nanoparticles specifically exclude the presence of hyaluronan (HA). [0093] In contrast to the two commonly used methods of non-viral, polymeric gene delivery— polyplex and nanoparticle encapsulation— some embodiments of the instant invention are directed to the creation of spherical nanoparticles with an electrostatic crosslinker (e.g. a salt), rather than harmful chemical crosslinkers. In some embodiments, the nanoparticles are optimized for "nano" size, and are then adsorbed to a plasmid for protection from nucleases within the skin, assisted delivery into a cell, assisted endosomal escape, and/or ease of DNA unpacking. This ensures that all plasmid DNA, or any nucleic acid, is being used for a therapeutic purpose compared to other methods where unincorporated plasmid DNA is discarded.

[0094] Some embodiments of the instant invention are more efficient than traditional polyplexes. With polyplexes, the polymers adsorb to and essentially entangle themselves around a plasmid. Once in the cell, this entanglement is difficult to undo, resulting in DNA unpacking issues and failed gene expression. In some embodiments, spherical nanoparticles are adsorbed to the plasmid DNA. Because they are spherical in nature, they only tangentially adhere to the plasmid and allow for facilitated DNA unpacking, while still offering protection from nucleases as well as assisting with entry into the cell and endosomal escape.

[0095] Some embodiments of the instant invention are more efficient than encapsulation by nanoparticles. Creating polymeric "nanoparticles" is often accompanied with the formation of larger particles due to the random number of polymers per particle. The larger particles are unable to enter a cell, resulting in an inconsistent dose of DNA with a compromised therapeutic outcome. In some embodiments, no plasmid DNA is wasted during the preparation of the particles. In some embodiments, optimized nanoparticles are directly added to the plasmid solution that are to be administered to a subject.

[0096] In some embodiments, the compositions are suitable for in vivo delivery of a nucleic acid or oligonucleotide, and are "pharmaceutical compositions". Such compositions produce a physiological effect when administered to an organisms, and produce a therapeutic effect. Also, in some embodiments the compositions are suitable for internal administration. Such pharmaceutical compositions include a nucleic acid or oligonucleotide and a chitosan-based compound, and in some embodiments also includes one or more other pharmaceutically acceptable components. Such components can, for example, include pharmaceutically acceptable carriers and solutes. Some examples of pharmaceutical compositions include any liquid composition (i.e. suspension or dispersion of the nanoparticles of the invention) for oral, buccal, sublingual topical, ocular, nasal or vaginal application, or any composition in the form of gel ointment, cream or bairn for its topical, ocular, nasal or vaginal administration.

[0097] In some embodiments, the composition is capable of delivering a nucleic acid or oligonucleotide into a cell. By "delivering the nucleic acid or oligonucleotide into a cell" is meant transporting a complexed and condensed nucleic acid or a complexed oligonucleotide in a stable and condensed state through the membrane of a cell (in vitro or in vivo), thereby transferring the nucleic acid or oligonucleotide from the exterior side of the cell membrane, passing through the lipid bilayer of the cell membrane and subsequently into the interior of the cell on the inner side (i.e., cytosol side) of the cell membrane and releasing the nucleic acid or oligonucleotide once within the cellular interior. The phrase "delivering the nucleic acid or oligonucleotide into a cell" is also meant to exclude the type of transport and/or diffusional loss of DNA.

[0098] In some embodiments, a "100% concentration" of nanoparticles refers to a solution or suspension having a concentration of at least 3.32 x 10 ¹⁰ particles/mL. In some embodiments, a "50% concentration" of nanoparticles refers to a solution or suspension having a concentration of at least 1.66 x 10 ¹⁰ parti cles/mL. In some embodiments, a "10% concentration" of nanoparticles refers to a solution or suspension having a concentration of at least 3.32 x 10 ⁹ particles/mL. In some embodiments, a "5% concentration" of nanoparticles refers to a solution or suspension having a concentration of at least 3.32 x 10 ⁸ parti cles/mL.

[0099] Examples

[00100] The following examples describe some concepts of the current invention with reference to particular embodiments. The general concepts of the current invention are not limited to the examples described. [00101] Synthesis and Characterization of Chitosan-Tripolyphosphate

Nanoparticles

[00102] Chitosan-tripolyphosphate (CS-TPP) nanoparticles used in drug and gene delivery studies, both in vitro and in vivo, are created by the ionic gelation method developed by Calvo, et al.* ³ This method requires that a therapeutic protein or nucleic acid is added to a solution containing tripolyphosphate, an electrostatic crosslinker. When combined with chitosan, the protein or DNA is encapsulated within CS-TPP particles. The particles are isolated via centrifugation and the precipitate is re-suspended for administration. The molecular weight of the chitosan polymer, however, is the limiting factor in the amount of DNA encapsulated within the particles. Full encapsulation occurs when using polymers of very low molecular weights, around lOkDa. When the weight is slightly increased to 80kDa, encapsulation efficiency becomes very variable, ranging from 70%-95%, and the size of the particles increases more than two-fold. ⁴⁴

[00103] Chitosan in many forms, including nanoparticles, has been shown to be advantageous in the wound healing process. ³⁴ Specifically, it improves healing through its antibacterial activity, ³⁵ involvement in the inflammation process, ^{36 37} and promotion of granulation tissue and re-epithelization, ³⁸ among other beneficial properties. Multiple studies, however, have shown that the beneficial properties of chitosan arise when the polymer is of high molecular weight. ^{35 39} Additionally, chitosan polymers with high molecular weights 150kDa-600kDa have shown minimal cytotoxicity. ⁴⁵

[00104] To obtain nanoparticles with inherent wound healing properties that also act as non-viral vehicles for enhanced transfection efficiency, we developed a modified version of the ionic gelation method described hereafter. CS-TPP nanoparticles were synthesized using a high molecular weight chitosan polymer of 310-375kDa with a degree of deacetylation greater than 80%. This specific molecular weight range has shown significantly increased proliferation of keratinocytes in vitro compared to a low molecular weight chitosan of 50-190kDa. ³⁹

[00105] First, a solution of sodium tripoylphosphate, without the addition of DNA, was added to chitosan. Particles of various sizes formed, some of which were visible to the naked eye and obstructed 1mm openings of pipette tips. Had the DNA been added to the tripolyphosphate solution, the largest particles would be physically incapable of undergoing the typical endosomal pathway of a polymeric nanoparticle's entry into a cell. ⁴⁶ [00106]

[00107] The average diameter of the initially formed particles measured 943 ±

43nm (Figure 1A). When taking size measurements, though, many of the particles settled to the bottom of the cuvette, resulting in smaller reported sizes in each consecutive measurement. In reality, the average diameter of the particles was likely greater than the reported value of 943 nm. To prevent larger particles from sequestering DNA when combined with the plasmid solution, the originally prepared solution was centrifuged in 50mL aliquots for 10 minutes at 4,000 rotations per minute (RPM), and the supernatant was extracted for further analysis. The average diameter of the particles in the resulting supernatant was significantly decreased to 392 ± 4.2nm (Figure 1 A). When observed, some larger particles could still be seen moving in solution. When left undisturbed, the solution formed a sediment at the bottom of the conical tube in which it was stored. To further remove these large particles, ImL aliquots of the supernatant were centrifuged for a second time for 5 minutes at 10,000RPM. The average diameter of these particle was decreased again to 255 ± 4.8nm (Figure 1A). These nanoparticles measured 12.11 ± 0.35mV in zeta potential.

[00108] Scanning electron microscopy (SEM) images showed the presence of nanoparticles of various sizes all well below 500nm in diameter in the second retrieved supernatant (Figure 1C). A highly resolved image revealed the topology of CS-TPP nanoparticles (Figure ID). The concentration of these nanoparticles in solution was 3.32 x 10 ¹⁰ particles/mL and there was no statistically significant difference among three independently prepared nanoparticle solutions. This concentration (3.32 x 10 ¹⁰ parti cles/mL) represents a 100% concentration of nanoparticles in a solution. When left undisturbed, this solution did not form a sediment at the bottom of the conical tubing, suggesting a more desired colloidal solution. This solution was our 100% CS-TPP nanoparticle solution for the dilutions performed in subsequent gene delivery and wound healing studies.

[00109] As described above and shown in Figures 1A-1D, large CS-TPP particles were removed from solution through a series of centrifugations to isolate the smallest nanoparticles (*p<0.05; **p<0.00\ versus each prior solution via one-way ANOVA with Holm-Sidak's post-hoc test; n=3 preparations). The first round of centrifugation had the largest effect on decreasing average particle size. The solution was refined further by an additional centrifugation, as shown in Figure 1 A. The zeta potential decreased after the first round of centrifugation but did not exhibit a significant change after the second centrifugation, as shown in Figure IB. SEM images of CS-TPP nanoparticles showed a variety of sizes, as seen in Figure 1C (scale bar=^m) and a magnified image of three nanoparticles, as seen in Figure ID (scale bar=250nm).

[00110] Concentration of CS-TPP Nanoparticles Determines Coverage of Plasmid

[00111] Gene delivery studies that use polymeric vehicles predominantly form polyplexes by combing DNA with their polymer of choice, resulting in spherical or toroidal particles of DNA-polymer entanglements. ^{47 49} Although this offers protection against nucleases and facilitates entry into cells, a limiting factor in gene expression is unpacking the DNA from the polymer coating. ⁵⁰ For this reason, low molecular weights polymers are generally used for polyplexes as polymers of larger weights have shown compromised gene expression ⁵¹ as well as cytotoxicity. ⁵²

[00112] For the gene delivery study, polymeric nanoparticles formed from a modified ionic gelation method were used. The in situ complexation of a 3194bp plasmid (NTC9385) with CS-TPP nanoparticles at various nanoparticle concentrations (1%, 5%, 10%, and 50%) was observed using atomic force microscopy, as shown in Figures 2A- 2D. When a solution containing 1% nanoparticles was allowed to interact with DNA, there was very little coverage of the plasmid. Nanoparticles were seen adhering to random locations along DNA, as shown in Figure 2A. The plasmid started to arrange into a more compact structure when combined with a solution containing 5% nanoparticles. Most of the plasmid was seen concentrated towards the nanoparticle center with loops of DNA extending outward, as seen in Figure 2B. Coverage increased when using 10% nanoparticles. At this concentration, multiple nanoparticles adhered along the plasmid and the DNA started to coil and condense, as shown in Figure 2C. At an even more concentrated 50%, nanoparticles completely covered the plasmid DNA, which led to a coiled DNA structure that was barely visible, as shown in Figure 2D. Unlike traditional nanoparticles that encapsulate plasmids, the instant nanoparticles act as an adjunct to DNA and adhere tangentially.

[00113] Figures 2A-2D show atomic force microscopy images of NTC9385 plasmid DNA with CS-TPP nanoparticle adjuncts. Nanoparticles can be seen randomly associated with plasmid DNA at 1%, as shown in Figure 2A (scale bar=100nm). When the concentration was increased to 5%, more consolidated complexes emerged with DNA protrusions, as shown in Figure 2B (scale bar=50nm). Nanoparticle coverage of DNA increased with 10% and plasmids became more condensed, as shown in Figure 2C (scale bar=100nm). At 50%, there was full coverage of DNA with nanoparticles and plasmids were in their most condensed state, as shown in Figure 2D (scale bar=50nm).

[00114] CS-TPP Nanoparticles Allow for Improved and Tailored Gene Expression

[00115] A purpose of this study was to develop a non-viral, gene delivery system in which the vector itself contributed to the treatment at hand. Therefore, a wound healing model in vivo was used and skin around the wound was transfected. It has been shown that keratinocytes around a wound edge, along with those that migrate to cover the wound surface, increase their production of growth factors involved in angiogenesis after the formation of a wound. ⁵³ Delivering plasmids encoding genes expressed in wound healing to healthy skin around the wound has potential in accelerating rate of closure. Due to the beneficial properties of high molecular weight chitosan in wound healing, ^{34 39} the potential of nanoparticles composed of a 310-375kDa chitosan as a non- viral vehicle for plasmid DNA delivery in the skin was evaluated.

[00116] Wild type, healthy rats were given 8mm circular wounds on their dorsum.

Four intradermal injections of the CS-TPP nanoparticle/plasmid DNA solution containing the firefly luciferase gene controlled by a CMV promoter (NTC9385) were administered to the skin around the wound. These solutions contained the clinically relevant concentration of lmg/ml of plasmid DNA ⁵⁴ along with either 0% (control), 1%, 5%, 10%, or 50% nanoparticles.

[00117] A solution containing 0% nanoparticles showed an average peak expression of 1102 ± 129 counts on day one and returned to baseline (below 100 luminescence counts) by day three. When using 1% or 5% nanoparticles, average peak expression was increased to 1523 ± 167 and 2071 ± 170 luminescence counts, respectively, on day one and returned to baseline by day three. As the concentration of nanoparticles increased to 10%, expression on day one started to decline as it reached only 953 ± 136 luminescence counts; however, characteristics of a more modest, prolonged expression started to emerge as the levels on day one and day two remained similar. With 50% nanoparticles in solution, the average peak expression was delayed until day two with 726 ± 78 luminescence counts. However, expression remained above baseline and statistically significant at day three. Of the five groups tested, the solution containing 5% nanoparticles showed the highest peak expression of any group, while the 50% group had the longest lasting expression. All groups returned to baseline by day six (Figure 3A).

[00118] When observing the total, additive amount of expression over the course of treatment in an injection site, the 1% and 5% group showed a significant increase compared to the 0% group. There was no significant difference between the 10% or 50% group with the 0% group (Figure 3B). Representative images of tailored gene expression can be seen in Figure 3C.

[00119] Interestingly, the course of gene expression was dependent on the concentration of nanoparticles in solution with the plasmid. Lower concentrations followed the same course of expression as the group without nanoparticles but with enhanced transfection, whereas higher concentrations led to expressions with longer durations (Figure 3A). On possible explanation for this is that these nanoparticles, which exhibit positive zeta potentials when formed, adhere to negatively charged plasmid DNA with enough strength for protection within the tissue and endosomal entry into the cell. Once inside the endosome, the nanoparticles assist in endosomal escape using the proton sponge mechanism described by Boussif, et al. ⁴⁶ An increase to a neutral pH within the cytoplasm allows the particles to become decreasingly protonated and lose their affinity for the plasmid. Since the nanoparticles are spherical structures and are thought to adhere to the plasmid in a tangential, rather than encapsulating, manner, they can easily be disassociated from the plasmid. As more nanoparticles adhere to plasmid DNA, the amount of time for unpacking DNA increases, causing a delay in peak expression as well as a longer lasting expression.

[00120] As discussed above and shown in Figures 3A-3C, the percent of chitosan- tripolyphosphate nanoparticles in solution dictated transfection efficiency of a 3194bp plasmid (NTC9385) in the skin (*p<0.05; **p<0.00\ versus 0% nanoparticles via oneway ANOVA with Holm-Sidak's post-hoc test; n=3). The highest expression at any given day occurred with a concentration of 5% nanoparticles. The longest lasting expression took place with the 50% nanoparticles that initially showed lower transfection on day 1, as shown in Figure 3A. The total transfection over the course of the experiment significantly increased when using 1% or 5% nanoparticles. There was not a significant difference when using 10% or 50% nanoparticles, as shown in Figure 5B. The different trends in gene expression based on the concentration of nanoparticles were seen along the wound edge of Sprague-Dawley rats, as shown in Figure 3C.

[00121] CS-TPP Nanoparticles Have a More Significant Effect with a Larger

Plasmid

[00122] The NTC9385 plasmid was optimized by removing unnecessary components of a previous plasmid's (NTC8685) backbone. ⁵⁵ In order to test the CS-TPP nanoparticles' effectiveness as a necessary facilitator for the entrance of larger genes into the cell, a plasmid construct with over 1000 additional base pairs in its 4622bp backbone (NTC8685) was used encoding the same luciferase gene and CMV promoter. New CS- TPP nanoparticles were created to investigate the consistency of this system.

Additionally, to ensure that the nanoparticles, rather than acetic acid or tripolyphosphate in solution were responsible for these unique tailoring capabilities, the process used to create the nanoparticles was repeated but without the addition of chitosan. Concentrations of 0%, 5%, and 50% of both the CS-TPP nanoparticle solutions and chitosan-removed solutions were tested with the NTC8685 plasmid.

[00123] A solution containing 0% nanoparticles had a slightly lower expression of

705 ± 62 luminescence counts (Figure 4A) compared to the 0% NTC9385 (Figure 3A) due to the increase in size of the NTC8695 plasmid. The pattern of gene expression followed the same trend as the previous study. A concentration of 5% nanoparticles resulted in a peak expression of 1561 ± 60 luminescence counts on day one, which was a more significant increase compared to the NTC9385 study. Expression of this group lasted three days compared to two days of the previous study. At a concentration of 50%, peak expression reached 1229 ± 93 luminescence counts and was delayed until day two. This was also a more significant increase compared to the previous study. The 50% group's expression remained above baseline and statistically significant until day four. Expression of all groups ceased by day seven (Figure 4A).

[00124] In comparison, the CS-TPP nanoparticles had its greatest affect with the larger NTC8685 plasmid. In the study with the smaller NTC9385 plasmid, a nanoparticle concentration of 5% and 50% allowed for 1.88x and 1.56x peak expression, respectively, compared to 0% (Figure 3A). In the NTC8685 study, a concentration of 5% and 50% allowed for 2.21x and 1.88x peak expression, respectively, compared to 0% (Figure 4A). The larger plasmid benefited the most from the CS-TPP nanoparticles at both

concentrations.

[00125] At 5% of both the chitosan nanoparticle and chitosan-removed solutions, there was a statistically significant improvement on day one compared to the 0% group. However, expression in the 5% chitosan-removed group quickly diminished and remained below the 0% group each day thereafter. The 50% chitosan-removed group showed no significant difference with the control (Figure 4A). When comparing the total expression in an injection site over the course of the study, there was a significant difference between the 5% and 50% groups with the 0% group, but not between the 5% and 50% chitosan-removed groups with the 0% group (Figure 4B). In two separate cases involving plasmids differing in size by lOOObp, the system showed a consistent partem of gene expression over a four-day course. Representative images of tailored gene expression can be seen in Figure 4C.

[00126] As discussed above and shown in Figures 4A-4C, chitosan- tripolyphosphate nanoparticles exhibited a greater effect with a larger plasmid

(NTC8685) but followed the same trend of transfection as the smaller plasmid

(NTC9385) (*/ 0.05; **/?<0.001 versus 0% nanoparticles via one-way ANOVA with Holm-Sidak's post-hoc test; n=3). The highest expression occurred with 5% nanoparticles which showed a more significant expression on both day 1 and 2. With 50%

nanoparticles, expression was low initially but climbed to much higher levels as expression was significantly improved on days 2, 3, and 4, as shown in Figure 4A. With 5% and 50%, the total expression was much higher. Chitosan-removed solutions, consisting only of dissolved tripolyphosphate and acetic acid, did not have an effect on the total amount of expression over the course of the experiment, suggesting that the nanoparticles were the cause of the unique transfection properties, as shown in Figure 3B. The different trends in gene expression based on the concentration of nanoparticles were seen along the wound edge of Sprague-Dawley rats, as shown in Figure 5C.

[00127] Electroporation Does Not Improve Transfection Efficiency of CS-TPP NP- Coated Plasmids

[00128] In the same study with the NTC8685 plasmid, electroporation was used to further understand the mechanism of entry of our nanoparticle-DNA complexes into the cell. Electroporation has been shown to create pores within the cell membrane through which naked DNA can enter. In contrast, nanoparticles promote entry via endosomes. ⁴⁶ It was previously shown that electroporation significantly increases transfection efficiency when applied over the injection site within two minutes of naked DNA administration. ⁸ ' ⁹

[00129] Therefore, electroporation was done over injection sites containing 0%,

5%, or 50% CS-TPP nanoparticles. As expected, transfection improved on day one while using electroporation over 0% (naked DNA) injections (Figure 5 A). Injections of CS-TPP nanoparticle/plasmid solutions, however, performed no better when electroporated. With the 5% group, there was a significant decrease in transfection efficiency on day one when the injection site was electroporated (Figure 5B). There was no difference in expression between the 50% group when using electroporation (Figure 5C). This further suggests that CS-TPP nanoparticle-coated plasmids are endocytosed versus entering through pore complexes.

[00130] As discussed above and shown in Figures 5A-5C, electroporation improved transfection efficiency of naked DNA (0%) on day 1 (A); however, it did not enhance transfection efficiency of CS-TPP nanoparticle-coated NTC8685 plasmids (*/?<0.05; **p<0.0\ versus group without electroporation via Student's t-test; n=3). In fact, expression of the 5% nanoparticle group with electroporation showed lower transfection efficiency on day 1 (B). The 50% group did not show a difference when using electroporation (C).

[00131] CS-TPP Nanoparticles Improve Wound Healing

[00132] After demonstrating the gene delivery properties of the CS-TPP nanoparticles, their potential for improving wound healing was investigated. At first, the 8mm circular wound healing model in healthy, Sprague-Dawley rats was used. A solution containing 50% CS-TPP nanoparticles via four intradermal injection was administered around each wound's edge and compared to a group without any treatment. Digital planimetry was used to monitor the size of the wound for the first ten days to identify any effects on healing. The 50% CS-TPP nanoparticles group showed an overall significant increase in wound closure compared to the untreated group over the 10-day period (Figure 6A).

[00133] This was re-explored using larger 18mm wounds and with a vehicle injection containing an inactive DNA plasmid in the control group. Nanoparticles were administered around the wound edge once a week for four weeks. Wound sizes were measured with digital planimetry, and again, the 50% CS-TPP nanoparticle group showed more rapid healing over the course of a month (Figure 6B). A Kaplan-Meier analysis confirmed that the 50% CS-TPP nanoparticles accelerated the time at which wounds closed compared to the sham injection group (p<0.05) (Figure 6C).

[00134] A way to determine the quality of a healed wound is by its tensile strength. ⁵⁶ One approach that has been used to determine the physical properties of healed skin in rats, with wounds up to 60mm in diameter, is to allow 28 days of healing before testing for strength. ⁵⁷ In a similar manner, the rats were sacrificed on day 31, and the tensile properties of the healed wounds were tested using tensiometry. There was a significant increase in the peak force (Figure 6D) and work (Figure 6E) required to burst healed wounds of the 50% CS-TPP nanoparticles group compared to the sham injection group. This demonstrates that CS-TPP nanoparticles not only heal wounds faster, but also improve the physical properties of newly formed skin.

[00135] As discussed above and shown in Figures 6A-6E, CS-TPP nanoparticles

(NPs) improved wound healing. An 8mm, circular wound showed improved healing when 50% CS-TPP NPs were administered immediately after wounding compared to a group with no treatment (p<0.05 via two-way ANOVA with Holm-Sidak's post-hoc test; n=4), as shown in Figure 6A. An 18mm, circular wound showed improved healing when 50% CS-TPP NPs were administered once a week compared to corresponding dosages with sham injections (p=0.01 via two-way ANOVA with Holm-Sidak's post-hoc test; n=5), as shown in Figure 6B. Kaplan-Meier analysis showed accelerated closure of 18mm wounds when using 50% CS-TPP NPs compared to the sham injection group (p<0.05 via survival -log rank; n=5), as shown in Figure 6C. The healed 18mm wounds of the 50% CS-TPP NPs group showed a significantly higher peak force and amount of work to rupture healed skin in a tensiometry test (*/?<0.05; **p<0.01 via Student's t-test; n=4), as shown in Figures 6D and 6E, respectively.

[00136] Conclusions

[00137] In the example above, a new chitosan-based nanoparticle gene delivery system for dermal wound healing is described. The system allows for tailored gene expression using therapeutic polymeric nanoparticles. CS-TPP nanoparticles were synthesized using tripolyphoshpate as an electrostatic crosslinker before plasmid DNA was added. Gene expression was tailored by varying the concentration of the nanoparticles in solution with plasmid DNA. Lower concentrations of CS-TPP nanoparticles in solution with plasmid DNA allowed for enhanced, short-term expression whereas high concentrations allowed for a longer expression at more modest level. As the size of the plasmid increased, the CS-TPP nanoparticles had a greater effect on transfection compared to controls without nanoparticles, but still followed the same trend of gene expression as the smaller plasmid. Electroporation decreased the efficiency of transfection with low concentration of CS-TPP nanoparticles, but had no effect on transfection with high concentrations of CS-TPP nanoparticles. CS-TPP nanoparticles of 50% showed improved wound healing properties through accelerated wound closure (p<0.05) and increased tensile properties in healed wounds (p<0.05).

[00138] Materials and Methods

[00139] Preparation of Nanoparticles: Chitosan-tripolyphsphate nanoparticles were created by a modified ionic gelation method and nanoparticle refinement process. A 50mL solution of 0.125g sodium tripolyphosphate (Sigma Aldrich, St. Louis, MO) was added, dropwise, to a 50mL solution of 0.125g of chitosan (310-375kDa, >80% DDA) (Sigma Aldrich, St. Louis, MO) in acetic acid (Sigma Aldrich, St. Louis, MO) that was diluted to 1%. All solutions were prepared using saline (VWR International, Radnor, PA). During the titration process, constant stirring was achieved by a magnetic stir bar set to a speed of four. The two solutions were completely combined after an hour and the chitosan particles were allowed to continue stirring for an additional 30 minutes. The solution was separated into two, 50mL conical tubes and centrifuged for 10 minutes at 4000RPM and 4°C in an Eppendorf™ Centrifuge 5810 R (Hauppauge, NY). The supematant was collected and centrifuged a second time for 5 minutes in ImL Eppendorf tubes at 10,000RPM and room temperature in a Costar® Centrifuge (Corning, Coming, NY). The supematant was collected as the 100% nanoparticle solution.

[00140] Scanning Electron Microscopy: The nanoparticles were imaged with a

Hitachi SU-70 Scanning Electron Microscope (Tokyo, Japan). The sample was deposited at a volume of 5μ1. onto an aluminum imaging mount (Electron Microscopy Sciences, Hatfield, PA) and placed in a desiccator with a vacuum seal until the solution was completely evaporated for imaging.

[00141] Nanoparticle Characterization: A Malvern ZetaSizer ZS90 (Malvern Instrument Ltd, Malvern, United Kingdom) was used to determine the diameter and zeta potential of the chitosan-tripolyphosphate nanoparticles. A Malvern NanoSight LM10 (Malvern Instrument Ltd, Malvern, United Kingdom) was used to measure the concentration of chitosan-tripolyphosphate nanoparticles in solution.

[00142] Nanoparticle/DNA Complexation: Plasmid DNA encoding the luciferase gene was provided by Nature Technology Corporation (Lincoln, NE). The two plasmid constructs used were NTC9385 with a 3194bp backbone and NTC8685 with a 4622bp backbone. Plasmids were stored in saline at -20°C. The order of addition in creating the nanoparticle/plasmid solutions was saline, nanoparticle solution, and then plasmid DNA. Each solution administered in vivo contained lmg/mL plasmid DNA with either 0%, 1%, 5%, 10%, or 50% nanoparticles in solution. Saline was used to adjust the concentration of the stock plasmid solution.

[00143] Atomic Force Microscopy: An MFP-3D Atomic Force Microscope

(Asylum Research, Santa Barbara, CA) was used to image the in situ complexation between the nanoparticles and NTC9385 plasmid DNA at nanoparticle dilutions of 1%, 5%, 10%, and 50%. First, 70μί of a ImM NiCb solution was deposited onto a mica imaging substrate (Ted Pella, Redding, CA) for 5 minutes and then removed. Then, 70μί of a 20μg/mL solution of pDNA was deposited onto the surface for 30 minutes before removing the solution and washing three times with saline. The nanoparticle containing solution was diluted to 1%, 5%, 10% or 50% then added to the surface for 30 mins before washing three times with saline. Finally, 70μί of saline was added to image in aqueous conditions.

[00144] Animals: Retired breeder, Sprague-Dawley rats (400-600g) from Charles

River (Wilmington, MA) were used for all procedures. All procedures involving rats were approved by The Johns Hopkins University Animal Care and Use Committee.

[00145] Wound Healing Models: Rats were anesthetized with 3% isofluorane

(Baxter Healthcare Corporation, Deerfield, IL) and had their entire dorsum shaved. Punch biopsies of either 8mm (Integra, Plainsboro, NJ) or 18mm in diameter were performed on the dorsum of each rat. In the case of two punch biopsies, 8mm wounds were about two inches apart along the spine. Otherwise, the wound was in the center of the dorsum. All rats received lmL of an intraperitoneal injection of 0. lmg/mL buprenophorine (Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA) immediately after wounding for pain management. Wound size was measured using digital planimetry. [00146] Nanoparticle Administration: Nanoparticle/DNA solutions were intradermally injected at a volume of 50μί around the edge of each wound four times in a cross-like pattem. Skin blebs were used to verify that the injection did not escape into the underlying fascia.

[00147] Electroporation: Electroporation protocol was used from Liu et al. ⁹

Briefly, animals were electroporated at the site of injection within 2 minutes after plasmid injection using an ECM 830 square wave electroporator (BTX Genetronics, San Diego, CA). Parameters consisted of: two 10 mm rows of parallel acupuncture needles separated by 5 mm; ten square wave pulses at an amplitude of 400 V for a duration of 20 ms;

interval between pulses of 125 ms.

[00148] Bioluminescence Imagining: An IVIS Xenogen Camera (Caliper Life

Science, Alameda, CA) was used to obtain photographs of rats superimposed with luminescence images. Prior to imaging, rats were anesthetized with 3% isofluorane and given 5mL intraperitoneal injections of 15mg/mL luciferin (Biosynth International, Itasca, IL). After 30 minutes following injections, the rats were imaged. Luminescence was calculated by the Living Image 4.5.2 (PerkinElmer, Waltham, MA) software for equal-sized regions of interest at each injection site.

[00149] Tensiometry: Animals of the 18mm wound model were sacrificed on day

31. Their healed wounds were immediately excised and samples of 2mm x 20mm, along the center of the wound, were used for strength testing. A custom setup consisting of a Check Line FGS-50PXH-L motorized horizontal test stand (Check Line, Birmingham, United Kingdom) and Shimpo FGV-XY digital force gauge (Shimpo Instruments, Cedarhurst, NY) was used for tenstiometry. The 2mm x 20mm sample was placed between two opposite clamps attached to the force gauge. The samples were pulled at a consistent speed. Reading from the force gauge were automatically transcribed to an excel document that allowed us to calculate peak force and work required for rupture.

[00150] Statistical Methods: Data were presented as mean ± SEM. Statistical significance was determined using either two-way ANOVA with Holm-Sidak's post-hoc, one-way ANOVA with Holm-Sidak's post-hoc, or a Student's t-test. A p-value less than 0.05 was considered significant.

[00151] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

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