WO2018140411A2 - Nanoparticules polymères thérapeutiques pour une expression génique personnalisée - Google Patents

Nanoparticules polymères thérapeutiques pour une expression génique personnalisée Download PDF

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WO2018140411A2
WO2018140411A2 PCT/US2018/014898 US2018014898W WO2018140411A2 WO 2018140411 A2 WO2018140411 A2 WO 2018140411A2 US 2018014898 W US2018014898 W US 2018014898W WO 2018140411 A2 WO2018140411 A2 WO 2018140411A2
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nanoparticles
chitosan
composition
gene
cell
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PCT/US2018/014898
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WO2018140411A3 (fr
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Louis Joseph BORN
John W. Harmon
Frank Lay
Guy MARTI
Christopher Ng
Zahra ALIKHASSY
Amir Ansari
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The Johns Hopkins University
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Publication of WO2018140411A3 publication Critical patent/WO2018140411A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions

Definitions

  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • FIG. 1A is a graph showing the average nanoparticle diameter as a function of centrifugation steps according to an embodiment of the current invention
  • 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
  • FIG. 1C is a scanning electron microscopy (SEM) image of nanoparticles of a variety of sizes (scale bar is ⁇ ) according to an embodiment of the current invention
  • 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
  • FIG. 2A shows an atomic force microscopy image of plasmid DNA with a
  • top panel 1% nanoparticle solution
  • bottom panel scale bar is lOOnm
  • FIG. IB shows an atomic force microscopy image of plasmid DNA with a
  • top panel 5% nanoparticle solution
  • bottom panel scale bar is 50nm
  • FIG. 2C shows an atomic force microscopy image of plasmid DNA with a
  • FIG. 2D shows an atomic force microscopy image of plasmid DNA with a
  • top panel 50% nanoparticle solution
  • bottom panel an illustration of the like
  • scale bar is 50nm
  • 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;
  • 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;
  • 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;
  • 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;
  • FIG. 4B is a graph showing the total transfection efficiency of various concentrations of nanoparticles as measured as a function of average luminescence;
  • FIG. 4C is a panel of images showing the location of gene expression along a wound over the course of 4 days;
  • 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;
  • 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;
  • 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;
  • FIG. 6A is a graph showing accelerated wound closure over the course of
  • FIG. 6B is a graph showing accelerated wound closure over the course of
  • FIG. 6C shows the results of Kaplan-Meier analysis of wound healing as a result of treating a wound with a 50% nanoparticle solution
  • FIG. 6D is a graph showing peak force measured during a tensiometry test of healed tissue.
  • FIG. 6E is a graph showing amount of work measured during a tensiometry test of healed tissue.
  • 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.
  • 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.
  • 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.
  • 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 are between lnm - lOOOnm, or between 50 nm - 500 nm in diameter.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Some embodiments of the invention relate to the method described above, where the natural biopolymer is chitosan and wherein the crosslinking agent is tripolyphosphate.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • compositions 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 comprise a natural or synthetic cationic polymer substituted with a crosslinking agent
  • each of the plurality of nanoparticles have a positive zeta potential
  • each of the plurality of nanoparticles are between 1 nm - 1000 nm in diameter.
  • compositions above wherein the natural or synthetic cationic polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.
  • Some embodiments of the invention relate to the composition above wherein the chitosan has a molecular weight of at least 1 kDa.
  • Some embodiments of the invention relate to the composition above wherein the chitosan has a molecular weight of at least 150 kDa.
  • 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%.
  • Some embodiments of the invention relate to the composition above wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.
  • 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.
  • 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.
  • 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;
  • 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.
  • Some embodiments relate to the method above, wherein the polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.
  • Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least 1 kDa.
  • Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least lkDa or of at least 150kDa.
  • Some embodiments relate to the method above, wherein the chitosan has a degree of deacetylation of at least 60% or at least 80%.
  • Some embodiments relate to the method above, wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.
  • 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.
  • polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell.
  • 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.
  • 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.
  • Some embodiments relate to the method above, wherein the polymer comprises chitosan or a chitosan derivative and wherein the crosslinking agent is tripolyphosphate.
  • Some embodiments relate to the method above, wherein the chitosan has a molecular weight of at least lkDa or of at least 150kDa.
  • Some embodiments relate to the method above, wherein the chitosan has a degree of deacetylation of at least 60% or at least 80%.
  • Some embodiments relate to the method above, wherein the positive zeta potential is at least 5mV or between 5mV to 20mV.
  • 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.
  • polynucleotide comprises a DNA plasmid comprising a gene, and wherein the DNA plasmid is configured to express the gene within the cell.
  • Some embodiments relate to the method above, wherein the gene is expressed for at least 24 hours.
  • 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.
  • the polynucleotide is not contained within an internal space or compartment of the nanoparticles.
  • the nanoparticles are made at least partially from a natural biopolymer. In some embodiments, the nanoparticles are made completely from a natural biopolymer.
  • 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.
  • 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.
  • derivatives include, among others, acetylated, alkylated or sulfonated chitosans, thiolated derivatives, as is described in Roberts, Chitin Chemistry, Macmillan, 1992, 166.
  • a derivative 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.
  • the nanoparticles are chitosan-based compounds.
  • Chitosan-based based 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 ⁇ (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.
  • 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.
  • 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.
  • 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.
  • 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).
  • nucleic acid is meant both RNA and DNA including: cDNA, genomic DNA, plasmid DNA, antisense molecule, polynucleotides or olignucleotides, RNA or mRNA.
  • the nucleic acid administered is plasmid DNA which comprises a "vector".
  • 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.
  • oligonucleotide is meant a single-stranded polynucleotide chain.
  • a "DNA vector” is a vector whose native form is a DNA molecule.
  • 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
  • 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.
  • 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.
  • administration is by intravenous administration.
  • composition is administered to an organism.
  • 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.
  • the nanoparticles specifically exclude the presence of hyaluronan (HA).
  • 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.
  • 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.
  • Some embodiments of the instant invention are more efficient than traditional 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.
  • 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.
  • 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.
  • 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.
  • the composition is capable of delivering a nucleic acid or oligonucleotide into a cell.
  • 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.
  • a "100% concentration" of nanoparticles refers to a solution or suspension having a concentration of at least 3.32 x 10 10 particles/mL.
  • 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.
  • a "10% concentration” of nanoparticles refers to a solution or suspension having a concentration of at least 3.32 x 10 9 particles/mL.
  • 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.
  • CS-TPP Chitosan-tripolyphosphate 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.
  • a therapeutic protein or nucleic acid is added to a solution containing tripolyphosphate, an electrostatic crosslinker.
  • 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 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
  • 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
  • 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.
  • 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.
  • average peak expression was increased to 1523 ⁇ 167 and 2071 ⁇ 170 luminescence counts, respectively, on day one and returned to baseline by day three.
  • 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.
  • 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.
  • 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).
  • 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.
  • NTC9385 plasmid was optimized by removing unnecessary components of a previous plasmid's (NTC8685) backbone. 55
  • 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
  • a way to determine the quality of a healed wound is by its tensile 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.
  • NPs improved wound healing.
  • 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.
  • CS-TPP nanoparticles 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).
  • Hitachi SU-70 Scanning Electron Microscope (Tokyo, Japan). The sample was deposited at a volume of 5 ⁇ 1. 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.
  • an aluminum imaging mount Electric Microscopy Sciences, Hatfield, PA
  • 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.
  • 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.
  • Atomic Force Microscopy An MFP-3D Atomic Force Microscope
  • Nanoparticle/DNA solutions were intradermally injected at a volume of 50 ⁇ 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.
  • Electroporation protocol was used from Liu et al. 9
  • mice 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;
  • Bioluminescence Imagining An IVIS Xenogen Camera (Caliper Life
  • thermo-responsive polymers incorporated with thermo-responsive polymers as novel biomaterials. Macromol. Biosci. 6, 991-1008 (2006).
  • vascular permeability factor vascular endothelial growth factor

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Abstract

L'invention concerne une composition pour une administration personnalisée de polynucléotide non viral à une cellule comprenant une pluralité de nanoparticules et un polynucléotide fixé à une surface extérieure d'au moins une nanoparticule parmi la pluralité de nanoparticules et des méthodes d'utilisation et de fabrication de ladite composition. Chaque nanoparticule parmi la pluralité de nanoparticules a un polymère cationique naturel ou synthétique réticulé électrostatiquement avec un agent de réticulation électrostatique anionique. Chaque nanoparticule parmi la pluralité de nanoparticules a un potentiel zêta positif, et chaque nanoparticule parmi la pluralité de nanoparticules présente un diamètre situé dans la plage allant de 1 nm à 1 000 nm.
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WO2021081495A1 (fr) * 2019-10-25 2021-04-29 The Johns Hopkins University Nanoparticules polymères pour l'administration intracellulaire de protéines
CN114317478A (zh) * 2022-01-05 2022-04-12 北京化工大学 一种蔗糖磷酸化酶的应用及利用其制备2-α-甘油葡萄糖苷的方法

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US20100086613A1 (en) * 2008-10-03 2010-04-08 Chang-Jer Wu Chitosan vehicle and method for making same

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021081495A1 (fr) * 2019-10-25 2021-04-29 The Johns Hopkins University Nanoparticules polymères pour l'administration intracellulaire de protéines
CN114317478A (zh) * 2022-01-05 2022-04-12 北京化工大学 一种蔗糖磷酸化酶的应用及利用其制备2-α-甘油葡萄糖苷的方法
CN114317478B (zh) * 2022-01-05 2023-10-20 北京化工大学 一种蔗糖磷酸化酶的应用及利用其制备2-α-甘油葡萄糖苷的方法

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