WO2002057424A2 - Preparations de liberation d'acide nucleique - Google Patents

Preparations de liberation d'acide nucleique Download PDF

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Publication number
WO2002057424A2
WO2002057424A2 PCT/US2002/001379 US0201379W WO02057424A2 WO 2002057424 A2 WO2002057424 A2 WO 2002057424A2 US 0201379 W US0201379 W US 0201379W WO 02057424 A2 WO02057424 A2 WO 02057424A2
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WIPO (PCT)
Prior art keywords
formulation
nucleic acid
components
reactive
component
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PCT/US2002/001379
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English (en)
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WO2002057424A3 (fr
Inventor
Shikha P. Barman
Krishnendu Roy
Mary Lynne Hedley
Daqing Wang
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Zycos Inc.
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Priority to EP02713428A priority Critical patent/EP1352072A4/fr
Priority to JP2002558478A priority patent/JP2004521109A/ja
Priority to CA002435287A priority patent/CA2435287A1/fr
Priority to US10/466,289 priority patent/US20040147466A1/en
Publication of WO2002057424A2 publication Critical patent/WO2002057424A2/fr
Publication of WO2002057424A3 publication Critical patent/WO2002057424A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Definitions

  • This invention relates to methods and compositions for delivering nucleic acids to cells.
  • the invention relates to delivery of nucleic acids for the purpose of gene expression from a bioabsorbable polymeric network structurally and functionally designed to induce gene expression.
  • exogenous DNA molecules hold considerable potential for the treatment of hereditary diseases, e.g. cystic fibrosis. These techniques can also be used when expression of gene products from genes not naturally found in the host cells is desired, for example, from genes encoding cytotoxic proteins targeted for expression in cancer cells.
  • individuals can be treated with an exogenous DNA that can express a therapeutic polypeptide for some duration (e.g., days, weeks, a month, or several months) as needed for the particular treatment.
  • DNA vaccines can be delivered in these formulations.
  • gene expression technology has focused primarily on the use of viral vectors that provide highly efficient transduction and high levels of gene expression in vivo-
  • the most well-studied vectors are adenoviral vectors, particularly those from replication-defective viruses. These vectors can efficiently transduce non-dividing cells, generally do not integrate into the host cell genome, and can result in high levels of transient gene expression.
  • the use of viral vectors has raised safety issues relating to, for example, host response to the virus, and oncogenic and inflammatory effects.
  • Other, non- viral gene transfer techniques that have been employed include biolistic transfer, injection of "naked" DNA (US Patent No. 5,580,859), delivery via cationic liposomes (U.S. Patent No.
  • the invention is based on the discovery that injectable and nucleic acid- compatible polymeric compositions and formulations can be structurally designed to regulate gene expression f n v v0 , for example, by controlling the bioavailability of the nucleic acid v ⁇ modulation of the biodegradability and crosslink density of the network formed by the components of the formulation.
  • the polymeric network encases the nucleic acid, not only controlling the release of the DNA, but also providing protection from degradation.
  • the invention described herein improves upon prior modes of gene delivery, in that gene expression can be regulated by modulation of a polymeric network formed by combination of at least two water-soluble components capable of reacting with one another.
  • the nucleic acid of interest is incorporated into the network to be released in a sustained manner to achieve the level and duration of expression needed.
  • the invention features an injectable aqueous formulation that contains: (a) a nucleic acid; (b) a first non-nucleic acid, water-soluble component; and (c) a second non-nucleic acid, water-soluble component, wherein the first and second components each include two or more reactive groups, the reactive groups of the first component being reactive with the reactive groups of the second component.
  • the first and second components of the formulation can react with one another to form a branched or a crosslinked polymeric network.
  • the first and/or second components can include one or more succinimidyl, chloroformate, acrylate, amino, alcohol, thiol epoxide, sulfhydryl, or hydrazidyl groups.
  • At least one of the first and second components is a functionalized multi-armed poly(alkylene oxide) (i.e., a branched poly(alkylene oxide, or a poly(alkylene oxide) having more than one arm (e.g., having eight or 16 arms emanating from a center) such as poly(ethylene oxide), poly(ethylene oxide)-co-poly(propylene oxide)-co-poly(ethylene oxide), poly(propylene oxide)-co-poly(ethylene oxide)-co-poly(propylene oxide).
  • at least one of the first and second components is a polyethylene glycol tetraamine.
  • At least one of the first and second components is a polyethylene glycol tetrasuccinimidyl glutarate. In another example, at least one of the first and second components is a polyethylene glycol tetra-sulfhydryl. In another example, at least one of the first and second components is a functionalized poly(alkylene oxide) with at least two reactive functional groups, e.g., an epoxide, aldehyde, pyrophosphate, or any other functional group.
  • At least one of the first and second components is a polyamidoamine having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more (e.g., 4 to 8 or 10 to 15) reactive functional groups, e.g., amino groups.
  • at least one of the first and second components is a polyethylimine or polylysine derivative.
  • at least one of the first and second components is a functionalized chitosan, cyclodextrin, or poly(vinyl alcohol) with at least two reactive functional groups.
  • one or both of the first and second components includes three or more reactive groups, the reactive groups of the first component being reactive with the reactive groups of the second component.
  • a formulation of the invention can further include a third non- nucleic acid, water-soluble component.
  • the third component can optionally include at least one reactive group.
  • the reactive group(s) of the third component can be reactive with at least one reactive group of the first component or the second component, with both the first and second components, with a product formed by reaction of the first and second components, or with neither the first nor the second component.
  • the third component is methoxy-polyethylene glycol-di- stearoyl-phosphatidylethanolamine (PEG-DSPE).
  • a formulation of the invention can further include an excipient.
  • the excipient is a neutral, anionic, or cationic lipid.
  • the excipient is a sugar (e.g., sucrose, dextrose, or trehelose), polyethylene glycol, chitosan, hyaluronic acid, chondroitin sulfate, heparan sulfate, phosphatidyl inositol, glucosamine, polyvinyl alcohol, Pluronics® (BASF, Inc., Mount Olive, North Carolina, U.S.A.), derivatized Pluronics®, or derivatized polyethylene glycol.
  • Pluronics® BASF, Inc., Mount Olive, North Carolina, U.S.A.
  • an excipient includes a permeation enhancer.
  • permeation enhancers include pluronics (e.g., poloxamers), polyethylene glycol, polypropylene glycol, propylene glycol-based molecules, sodium dodecyl sulfate (SDS), poly-vinyl pyrrolidone (PVP), Vitamin E and Vitamin E-tocopherol acetate (e.g., Vitamin E-TPGS®, Eastman Kodak, Inc., Kingsport, Tennessee, U.S.A.), lauroyl and oleoyl macrogol glycerides (e.g., Labrafils® and Gattefosse®, both available from Gattefosse, Westwood, New Jersey, U.S.A.), lipids, glycerol, polyoxyethylene sorbitan monoesters, Tween® 20 and 80, Span® 80, fatty acids, fatty acid esters, bile salts (e.g., tauroc), e.g
  • the excipient includes a bioavailability enhancer.
  • bioavailability enhancers include propylene glycol and macrogol-based enhancers (e.g., Gelucire® (Gattefosse), Labrafil® (Gattefosse), Capryol® (Gattefosse), Labrasol® (Gattefosse), Plurol® (Gattefosse)), Bioperine® (Sabinsa Corporation, New Jersey, U.S.A.), Vitamin E (Sigma, Inc.)and Vitamin E- TPGS® (Eastman Kodak), poloxamers such as Pluronics® (BASF, Inc.), and polyethylene glycol (Sigma, Inc.).
  • the excipient is a protein (e.g., contains a cytokine).
  • the excipient contains a small molecule drug, e.g., an anti- tumor agent, anti-neoplastic, anti-inflammatory, or antibiotic.
  • the excipient is an adjuvant (e.g., a CpG oligonucleotide, oil, lipid, monophosphorolipid (MPL; Sigma, Inc.), lipopolysaccharide(LPS; Sigma, Inc.), or carbohydrate).
  • the excipient is chemically bound to the crosslinked polymeric network or branched polymer, e.g., methoxy-PEG-monoamine, distearoylethanolamine, stearylamine, spermine, spermidine, laurylamine, urea, dioleylethanolamine, or aminocaproic acid. All of these excipients are reactive with the network, forming covalent bonds.
  • the excipient contains a component that stabilizes a nucleic acid, e.g., sodium, calcium, zinc, or magnesium salts of bicarbonates.
  • a reactive excipient is phosphatidyl ethanolamine, which can react with poly(ethylene oxide)-tetrasuccinimidyl glutarate (P4-SG).
  • P4-SG poly(ethylene oxide)-tetrasuccinimidyl glutarate
  • Another example of a reactive excipient is a poly(amino acid) containing multiple cysteines in its backbone
  • cysteines e.g., poly(cysteine) or a peptide susch as poly(arg-lys-cys-guanine-arg-cys-cys-lys-cys).
  • the free -SH of the cysteines can would react easily with P4-SG.
  • Another example is poly(lysine), with the pendant amino groups of which can reacting easily with P4-SG.
  • P4-SG and/or poly(ethylene glycol)-tetraamine (P4-AM) are components of the new formulations, Just cysteine or lysine can also be used as an excipient.
  • the invention features an injectable aqueous formulation that contains: (a) a nucleic acid; (b) a first non-nucleic acid, water-soluble component; (c) a second non-nucleic acid, water-soluble component, and (d) a third non-nucleic acid, water soluble component, wherein the first, second and third components each include two or more reactive groups, the reactive groups of the third component being reactive with the reactive groups of the first component or the second component.
  • a formulation can include more than one species of nucleic acid, e.g., two or more species of nucleic acids, each encoding a different polypeptide or a nucleic acid encoding a polypeptide and an oligonucleotide.
  • a nucleic acid can be an oligonucleotide (e.g. with a phosphodiester or phosphorothioate backbone).
  • a nucleic acid encodes a therapeutic protein or a protein that induces an immune response.
  • a "therapeutic protein” is a protein that when administered to an individual confers a therapeutic benefit upon the individual.
  • protein that induces an immune response is meant a pathogenic protein (e.g., a viral or bacterial protein) or portion thereof, a tumor-associated antigen or portion thereof, or another protein that is involved in disease (e.g., a neurodegenerative (e.g., Alzhiemer's), cardiac, immunologic, autoimmune, or gerontologic disease).
  • a nucleic acid of a formulation described herein can be in any form, e.g., a solution, dispersion, powder, precipitated, condensed, micronized, or emulsion.
  • a nucleic acid can optionally be encapsulated in or associated with a biodegradable polymeric microparticle. Examples of useful microparticles are described in U.S.
  • nucleic acid is released from the branched or crosslinked polymeric network by biodegradation or by simple diffusion.
  • a formulation described herein forms a hydrogel at a temperature between about 20°C and about 40°C within about 20 minutes after the formulation is prepared. In other embodiments, a formulation described herein forms a hydrogel at a temperature between about 25, 30, 35, or 37°C and about 40°C, within about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 1 minute after the formulation is prepared. In one example, a formulation described herein remains injectable for at least fifteen seconds, e.g., at least 15, 30, 45, 60, 90, 120, 180, 240, 300, or 600 seconds, or 15 minutes or 20 minutes after the formulation is prepared. In another embodiment, the network of a formulation described herein forms a viscous liquid.
  • the nucleic acid is protected from serum nucleases by incorporation into the network.
  • the nucleic acid is expressed following injection of the network (e.g., into muscle).
  • an immune response is generated to the nucleic acid encoded antigen following injection of the network/nucleic acid formulation.
  • the release of the nucleic acid following injection is controlled by the cross-linking density of the network.
  • the expression of the nucleic acid following injection is controlled by the cross-linking density of the network.
  • the first and/or second components of a formulation can be biodegradable, e.g., by a hydrolytic or proteolytic mechanism.
  • the network of formulation can be biodegradable, e.g., by a hydrolytic or proteolytic mechanism.
  • the branched or crosslinked polymeric network, e.g., fully or partially crosslinked, of a composition can include linkages selected from the group consisting of ester, carbonate, imino, hydrazone, acetal, orthoester, peptide, amide, urethane, urea, amino, oligonucleotide, and sulfonamidyl bonds.
  • partially means that the stoichiometry of the components can be adjusted, so that some of the functional groups remain unreacted, e.g., to obtain a loose network.
  • “fully” means that the stoichiometry of the components is equimolar, e.g., essentially all available functional groups have been reacted in the network.
  • the first and/or second components of a formulation can include one or more sulfhydryl, amine, epoxide, phosphoroamidates, chloroformate, acrylate, carboxylic acid, aldehyde, succinimide ester, succinimide carbonate, maleimide, iodoacetyl, carbohydrate, isocyanate, and/or isothiocyanate groups.
  • At least one of the first and second components of a formulation described herein can include a biodegradable linkage such as a lactate, caproate, methylene carbonate, glycolate, ester-amide, ester-carbonate, or a combination thereof.
  • the formulations described herein can be in the form of microparticles (e.g., microparticles, nanoparticles, microspheres, or nanospheres).
  • microparticles can be essentially "solid,” meaning that the cross-linked polymer (e.g., the hydrogel) formed by reaction of the first and second non-nucleic acid components can be distributed, evenly or unevenly, throughout each microparticle, with the nucleic acid distributed within the three-dimensional structure of the polymer.
  • the microparticles can have outer shells made up of the cross-linked polymer, and the nucleic acid can be either within the polymeric structure or else in the core of the microparticle.
  • the invention also features a method for making such microparticles.
  • the method includes introducing the nucleic acid and the first and second non-nucleic acid components of any of the formulations described herein into an emulsifying bath (e.g., a homogenizer or blender, or other device capable of emulsifying a mixture), either separately or after combining, and then emulsifying (e.g., by homogenizing or blending) the resulting mixture in the emulsifying bath for at least part of the time that the first and second non-nucleic acid, water-soluble components are reacting with each other.
  • an emulsifying bath e.g., a homogenizer or blender, or other device capable of emulsifying a mixture
  • emulsifying bath e.g.,
  • microparticles By adjusting the concentrations, ratios, emulsification speed, and identities of the components, the size, structure, and other physical properties of the microparticles can be controlled. For example, microparticles smaller than about 500 microns, 250 microns, 100 microns, 50 microns, 20 microns, 15 microns, 10 microns, 5 microns, 2 microns, 1 micron, or still smaller can be prepared. Generally, higher homogenization rates result in smaller microparticles.
  • the invention includes a method of making a polypeptide by applying a formulation described herein to a cell.
  • the nucleic acid contained within the formulation can code for expression of the polypeptide.
  • the formulation is applied to a cell within an animal, e.g., administered to the animal by injection, extrusion, or spraying.
  • the invention includes a method of making a polypeptide by injecting into an animal, e.g., a mouse, rat, pig, non-human primate, or human, a formulation described herein.
  • the nucleic acid contained within the formulation can code for expression of the polypeptide.
  • the formulation is injected in, on, or adjacent to a tumor.
  • the formulation is injected intramuscularly, subcutaneously, or intra-joint.
  • the formulation can be injected into the animal once or more than once.
  • the formulation can be delivered, for example, via an aerosolizer or nebulizer.
  • the formulation can alternatively be applied to the skin, delivered in a patch, or placed on a wound.
  • the formulation is also suitable for delivery via needle-free devices.
  • the formulation can be delivered by any of these mechanisms and then followed by an electrical pulse. Electrical pulses are known to enhance uptake of macromolecules post injection as described in U.S. Patent No. 5,993,434.
  • the formulation can be premixed before injection.
  • the invention includes a method of producing a polypeptide by:
  • the nucleic acid codes for expression of a polypeptide
  • the cell produces the polypeptide following the culturing of the cell vitro-
  • the invention features a method of making a dried nucleic acid formulation by: (a) preparing a mixture by mixing in an aqueous solution (i) a nucleic acid, (ii) a first non-nucleic acid, water-soluble component, (iii) a second non-nucleic acid, water-soluble component, and (iv) a third non-nucleic acid, water-soluble component, wherein the first and second components each include two or more reactive groups, the reactive groups of the first component being reactive with the reactive groups of the second component at a pH greater than 7.0.
  • the third component can include at least one reactive group that is reactive at a pH greater than 7.0 with at least one reactive group of the first component, the second component, of both the first and the second components, of a product formed by reaction of the first and second components, or with neither the first nor the second component.
  • the aqueous solution has a pH and/or temperature that prevents the first and second components from reacting to form a crosslinked network (i.e., at a pH lower than 7.0); and (b) drying the mixture to thereby create a dried formulation (e.g., drying in a lab dryer under vaccum, or in a lyohilizer).
  • the mixing step can be performed at a pH less than about 7.0, e.g., less than about 6.0. In one example, the mixing step is performed at a pH of about 5.5. The mixing step can be performed at or below about 4°C, e.g. between 0°C and 4°C. In one example, the mixture is dried. In another example the mixture is lyophilized.
  • both components can be individually dried (optionally with nucleic acid and/or excipient in one or both of the components), and then a buffer is added to reconstitute the formulation.
  • the first non-nucleic acid, water-soluble component is polyethylene glycol amine.
  • the second non-nucleic acid, water- soluble component is polyethylene glycol succinimidyl glutarate.
  • the first non-nucleic acid, water soluble component is polyethylene glycol sulfliydryl.
  • the third non-nucleic acid, water-soluble component is methoxy-polyethylene glycol-di-stearoyl-phosphatidylemanolamine (PEG-DSPE).
  • PEG-DSPE methoxy-polyethylene glycol-di-stearoyl-phosphatidylemanolamine
  • the method entails adding a buffer having a pH greater than 7.0 to a dried nucleic acid formulation of the invention.
  • the addition of the buffer results in the formation of a crosslinked network between the first and second components.
  • the buffer can be a phosphate buffer with a pH of about 7.5.
  • the buffer can include nucleic acid and/or excipients (e.g., sucrose, Tris, EDTA).
  • the adding step can be performed at or above 20°C, e.g., at or above 37°C.
  • the invention features a dried nucleic acid formulation that contains: (a) a nucleic acid; (b) a first non-nucleic acid, water-soluble component; (c) a second non-nucleic acid, water-soluble component; and (d) a third non-nucleic acid, water-soluble component.
  • the three components are each in an unreacted state, and the nucleic acid and the three components are not in solution.
  • the formulation can be lyophilized.
  • the first non-nucleic acid, water- soluble component is polyethylene glycol amine. In one embodiment, the first non- nucleic acid, water-soluble component is polyethylene glycol sulfliydryl . In still another example, the second non-nucleic acid, water-soluble component is polyethylene glycol succinimidyl glutarate. In another embodiment, the third non-nucleic acid, water-soluble component is methoxy-polyethylene glycol-di-stearoyl-phosphatidylethanolamine (PEG- DSPE).
  • PEG- DSPE methoxy-polyethylene glycol-di-stearoyl-phosphatidylethanolamine
  • the invention also features a kit containing a dried formulation described herein; and a buffer having a pH of at least 7.0.
  • the invention also includes a method of administering a nucleic acid to an individual by: preparing a mixture by adding a buffer having a pH of at least 7.0 to a dried formulation described herein; incubating the mixture to permit the formation of a crosslinked network; and administering the mixture to the individual.
  • a "nucleic acid” can be either RNA or DNA, including, for example, cDNA, genomic DNA, oligonucleotides, mRNA, viral DNA, bacterial DNA, plasmid DNA, triplex nucleic acid, peptide-nucleic acid (PNA) formulations, or condensed DNA.
  • the nucleic acid is plasmid DNA.
  • the nucleic acid is an oligonucleotide.
  • the oligonucleotide can include stabilizing features such as base or backbone modifications (e.g., phosphorothioate backbone).
  • the oligonucleotide can be an antisense oligonucleotide, utilized to treat various diseases.
  • the oligonucleotide can have anti- tumor activity.
  • the oligonucleotide can be used as an adjuvant, e.g., as described in EP 01005368 and WO 99/61056.
  • bioavailability of the nucleic acid is meant that the delivery formulation prolongs availability of the nucleic acid.
  • altering the polymer formulation one can increase or decrease the rate of release of nucleic acid from the polymer network, in turn affecting activity or expression levels.
  • the polymeric network is biodegradable, i.e., it breaks down into components that are readily cleared from the body.
  • modulation of the polymeric network it is meant, for example, that the hydrolytically labile linkages can be varied in length and type to affect the degradation time of the network.
  • a fast-degrading polymeric network would provide a higher bioavailability of the nucleic acid to the target cells than would a slower degrading network.
  • excipients can be added to enhance breakdown of the network.
  • succinimidyl propionates, succinimidyl caproate or succinimidyl carbonates can be substituted for succinimidyl glutarate in a PEG component to lower the rate of hydrolytic degradation of the network.
  • sulfhydryls can be substituted for amines in a PEG component to increase the rate of hydrolytic degradation of the network.
  • concentration of the gel-forming components can be varied to change the nature of the network.
  • a higher concentration of these components results in longer degradation times and increased branching and/or cross-linking, leading to a lower availability of the nucleic acid to the target cells and, consequently, a lower expression level or a lower level of therapeutic nucleic acid.
  • the molecular weight of the gel-forming components can be selected to vary the nature of network, particularly the molecular weight between cross-links.
  • a tighter network can result from the use of lower molecular weight components, causing greater retention of the DNA at the site and, consequently, sustained release for a longer duration.
  • sustained release it is meant that the nucleic acid is available to the target cells for uptake for a longer period of time than would be achieved if the administration of the nucleic acid were in, for example, saline, from which fast dissipation of the DNA from the site would occur.
  • addition of a third polymer to one of the pre-formulation components, to form either an interpenetrating network or a semi-interpenetrating network can vary the nature of the network to control release of DNA at the site to control level and duration of expression.
  • suitable "third polymers” include methoxypolyethylene oxide-monoamine, polyethylene glycol, poloxamers, and methoxypolyethylene oxide-distearoyl ethanolamine and 8-, 16-, and 32-arm derivatized and non-derivatized polyethylene oxide.
  • the invention features a method of delivering a particle to an individual.
  • the method includes administering to the individual a formulation that includes: (1) the particle; (2) a first non-nucleic acid, water soluble component; and (3) a second non-nucleic acid, water soluble component.
  • the first and second components each include two or more reactive groups, the reactive groups of the first component being reactive with the reactive groups of the second component.
  • the particle can be, for example, a virus or viral particle or a virus-like particle (VLP) (e.g., adenovirus or adenoviral particle such as an aviadenovirus or mastadenovirus or a penton, hexon, capsid, or other fragment thereof, or VLP made of hepatitis, or papillomavirus components).
  • VLP virus-like particle
  • excipient is meant a molecule added for the purpose of enhancing or sustainmg DNA uptake, activity, or expression, or to further enhance DNA stability, or to modulate release of DNA or degradation of the network.
  • the excipient is a bioavailability enhancer.
  • bioavailability enhancer is meant an excipient that improves or enhances bioavailability of the DNA to the target cells by its retention at the cell site.
  • the new methods and formulations feature an injectable polymer-based slow release system that can afford sustained systemic protein expression (e.g., by delivering genes into skeletal muscles).
  • sustained systemic protein expression e.g., by delivering genes into skeletal muscles.
  • Such a system can be used in the treatment of "chronic" diseases where multiple administrations are necessary to maintain therapeutic levels of bioactive proteins and peptides.
  • Sustained gene delivery can in turn allow for long-term protein expression.
  • j n vitro release experiments described herein indicate that plasmid DNA is slowly released over time from the crosslinked network formulations, with higher crosslinked hydrogels releasing the DNA more slowly.
  • the formulated plasmid DNA generated longer-term protein expression compared to unformulated "naked" DNA in both immunocompetent and complement deficient animals.
  • the new formulations can also provide protection to the entrapped DNA. Combined with plasmid stability, the new formulations can significantly increase the duration of protein expression following a single administration of DNA; genetic approaches can generate longer protein expression since the intracellular half-life of plasmid DNA is generally much longer than the serum half-life of recombinant proteins. The expression kinetics can be further prolonged by slowly releasing the plasmid over time so that the source of the protein is bio-available for several weeks.
  • Another advantage of the new formulations of the invention is that they are injectable.
  • Polyethylene glycols are considered to be biomimetic and hence highly biocompatible. They have also been shown to generate minimal inflammation and immune response.
  • the new formulations are injectable following reconstitution and do not require surgical implantation procedures.
  • the crosslinked networks of the invention are readily biodegradable due to the presence of, for example, hydrolytic ester linkages on the P4-SG component.
  • the network components can have a molecular weight on the order of about ⁇ 10,000 Daltons; upon degradation the components can be cleared from the body quite readily.
  • FIG. 1 is a chemical structure of certain network components and schematic representation of the crosslinking reaction.
  • the amine and succinimidyl groups react to generate amide linkages between the polymer species thereby forming the network structure.
  • the hydrolytically labile ester linkages in the P4-SG render the network biodegradable.
  • FIGS. 2A to 2C are graphs depicting network characterization by gel permeation chromatography (GPC) and viscometry.
  • FIG 2A is a graph of GPC analysis of formulation A. Individual PEG components (P4-SG and P4-AM) are indicated by arrows as is the resulting network
  • FIG 2B is a graph of formulations A, B, C and D that were analyzed by viscometry.
  • FIG 2C is a graph of gelation time (y-axis) plotted as a function of gel concentration (x-axis).
  • FIG. 3 is a table summarizing the physico-chemical characteristics of network formulations A (2% w/v P4-AM/P4-SG), B (3% w/v P4-AM/P4-SG), C (4% w/v P4-AM/P4-SG) and D (5%w/v P4-AM/P4-SG), detailing appearance of gels, gel swelling and gelation times determined at 25°C and 37°C.
  • FIG. 4 is a picture of a gel showing chemical compatibility of pDNA with network components.
  • lane 1 is a secreted embryonic alkaline phosphatase (SEAP) plasmid
  • in lane 2 is 1 ⁇ g/ml of DNA incubated with 2% w/v (P4-AM+P4-SG);and in lane 3 is 1 ⁇ g/ml of DNA incubated with 5% w/v (P4-AM+P4-SG).
  • SEAP embryonic alkaline phosphatase
  • FIGS. 5 A and 5B are two plots of the swelling properties of 5% (grey), 8% (black), and 10% (white) PEG hydrogels formulated at different intervals following stock solution preparation to examine solution stability. Overnight swelling (percent increase in weight) was performed at 37°C in phosphate-buffered saline (PBS) with blank PEG- hydrogels (A) or with hydrogels containing plasmid and mPEG-DSPE (B).
  • PBS phosphate-buffered saline
  • A blank PEG- hydrogels
  • B hydrogels containing plasmid and mPEG-DSPE
  • FIG. 6 is a table depicting the injectability of P4-AM P4-SG formulations. Maximun time for injection (min) after reconstitution is shown for formulations A (2% w/v P4-AM/P4-SG), B (3% w/v P4-AM/P4-SG), C (4% w/v P4-AM/P4-SG) and D (5% w/v P4-AM/P4-SG).
  • FIG. 7A is a graph showingin vitro release of plasmid from network formulations as measured by HPLC analysis. Depicted is a typical HPLC trace of DNA released from formulation C. The first peak represents polyethylene glycol, the second set of peaks (triplet) represents different isoforms of plasmid (supercoiled plasmid is represented by the second peak).
  • FIG. 7B is a graph depicting cumulative release of DNA from formulations B, C, and D at different time intervals (days post administration). Day 1 is represented by the white bar, day 3 by the black bar, day 7 by the stippled bar, and day 14 by the grey bar.
  • FIG. 8 is a picture of a gel depicting protection of network entrapped DNA from serum digestion.
  • FIG. 9 is a graph depicting the swelling properties of 10% w/v P4-SG/0.5% w/v poly(amidoamine) (PAMAM) hydrogels.
  • FIG. 10 is graph depicting the analysis of gel times for P4-SG/poly(ethylene oxide)-sulfydryl (P4-SH) networks. Viscosity was measured at 25°C for 3%, 4%, and 10%) w/v PEGs formulations. Symbols for each gel formulation are indicated. Y-axis represents viscosity (cp) and the x-axis represents time (minutes). Data were collected at different intervals after mixing the two PEG components using the Brookefield WingatherTM software.
  • FIGS. 11A and 1 IB are graphs depicting the expression of SEAP in mice injected with network containing SEAP DNA.
  • FIG 11 A shows serum SEAP level indicated on the y-axis (ng/ml) and the formulation is indicated on the x-axis. Time points are indicated by different filled bars.
  • FIG 1 IB shows the percent of animals within each group that express more than 300 pg/ml serum SEAP (y-axis) at days 10 (black bars), 33 (striped bars), and 92 (white bars), as indicated for each formulation (x-axis).
  • FIGS. 12A and 12B are graphs depicting the SEAP expression in complement deficient DBA/2 mice.
  • FIG 12 A show the percent of SEAP expressing animals (animals expressing >300 pg ml at a given time point) as indicated on the (y-axis). Mice were injected with the SEAP DNA containing formulation groups indicated on the (x-axis). Timepoints are as indicated (days 7, 35, 81).
  • FIG 12B shows expression of serum SEAP in RAG2 immunocompromised mice injected with P4-AM/P4-SG networks containing SEAP plasmid DNA. Percent of SEAP expressing animals (animals expressing >300 pg/ml at a given time point) is indicated on the (y-axis). Mice were injected with the
  • FIG. 13 is a graph depicting the effect of electroporation on serum SEAP levels. Mice were injected with a GT20 P4-AM/P4-SG formulation. Half the animals received electroporation treatment ("+EP") as depicted on x-axis. Serum SEAP level is indicated on the y-axis (ng/ml). Serum samples were tested 7 days post administration in mouse muscle.
  • FIGS. 14A and 14B are graphs depicting how serum SEAP expression can be influenced by network containing excipients.
  • FIG 14A shows serum SEAP levels (ngs/ml) as indicated on the y-axis.
  • Excipients formulated with the GT20 network are indicated on the x-axis. The bars represent the following excipients formulated with GT20 networks, respectively: GT20 + 0.1% SDS, GT20 + 0.1% L62, GT20 + 0.15% PAMAM.
  • FIG 14B is a graph showing GT20 + 0.025% w/v Streptolysin, GT20 + 250mg/ml, Magainin I, GT20 with no excipient. Serum samples were tested 7 days post administration in mouse muscle.
  • FIG. 15 is a graph depicting how SEAP expression is mediated by DNA in P4-SH/P4-SG networks. Serum SEAP levels (ngs/ml) are indicated on the y axis and the formulation is indicated on the x-axis (3.5 %w/v, formulation A; 5 %w/v, formulation B). Serum samples were tested 7 days post administration.
  • SI the stimulation index
  • FIG. 18 is a graph depicting interferon gamma Elispot analysis of T cells in mice immunized with network formulated DNA.
  • Response to H-2Ld restricted, ⁇ -gal 876- 884 peptide (filled bars) or HBV peptide (hatched bar) or media (open bar) is indicated.
  • the number of IFN- ⁇ + spot forming cells/10 6 T cells is indicated on the y-axis.
  • the relevant formulation (A or B) and untreated control are indicated on the x-axis.
  • the data are presented as the mean + SE of four mice performed in triplicate.
  • FIG. 19 is a table depicting protection of mice immunized with network formulated DNA.
  • BALB/c mice were challenged by i.v injection of either 5 x 10 6 CT26.WT or ⁇ -gal expressing CT26.CL25 tumor cells, three weeks post immunization. The number of tumor nodules is indicated in each group.
  • FIG. 20 is a schematic depicting a method of preparing a lyophilized formulation that can be reconstituted in a single vial prior to use.
  • FIG. 21 is a graph depicting how lyophilization does not effect gel time. Viscosity of a 10% w/v P4-SH/P4-SG network formulation that was not-lyophilized compared to a reconstituted lyophilized formulation. Symbols for each formulation are indicated. The y-axis represents viscosity (cp) and x-axis represents time (minutes).
  • FIG. 22 is a graph depicting interferon gamma Elispot analysis of T cells in mice immunized with lyophilized or non-lyophilized formulations.
  • Formulations included 2% w/v P4-AM/P4-SG and 3% w/v P4-AM/P4-SG.
  • Mean responses to H-2Ld restricted, ⁇ -gal 876-884 peptide (filled bars) or HBV peptide (hatched bar) or media (open bar) are indicated.
  • the number of IFN- ⁇ spot forming cells/10 6 T cells is indicated on the y-axis.
  • FIGS. 24A and 24B are graphs depicting how viscosity of a 10 % w/v P4-SH/P4- SG network formulation varies with temperature and pH.
  • the y-axis represents viscosity (cp) and the x-axis represents time (minutes).
  • viscosity was performed at 25°C or 37°C as indicated.
  • viscosity measurements were performed at various pHs as indicated. Data were collected at different intervals after mixing the two PEG components using the Brookefield WingatherTM software.
  • FIGS. 25A and 25B are graphs depicting oligonucleotide release from P4-SH P4- SG gels.
  • the y-axis indicates percent oligo released and the x-axis represents the time frame.
  • cumulative oligonucleotide release (y- axis) is plotted versus time (x-axis). Release was performed on 10, 20 and 30% gels as indicated.
  • FIG. 26 is a graph depicting oligonucleotide release from 10% w/v P4- SG PAMAM, GO gels.
  • the y-axis indicates percent oligo released and the x-axis represents the time frame.
  • This invention relates to methods and compositions for delivering nucleic acids to cells. These methods and compositions can be used for a variety of functions including but not limited to the induction of cell activation, the regulation of gene expression, or the induction of gene expression.
  • a nucleic acid is released from a bioabsorbable polymeric network structurally and functionally designed to enhance and optimize the level and duration of the released nucleic acid activity or expression.
  • composition of the delivery system includes a polymeric network formed by the chemical combination of at least two injectable non-nucleic acid polymeric components, containing one or more nucleic acids and one or more excipients.
  • the components (1, 2, and optionally 3) are water-soluble and are composed of polymeric backbones modified to have end functional groups capable of reacting with one another.
  • the reactive functional groups of component 1 can be, for example, chloroformates, acrylates, amines, alcohols, tetrasulfydryls, epoxides, sulfhydryls, hydrazides, or combinations thereof, in the same molecule.
  • the reactive functional groups of component 2 can be, for example, chloroformates, acrylates, carboxylic acids, aldehydes, maleimides, iodoacetyl, carbohydrates, isocyanates, or isothiocyanates.
  • the polymeric network can include linkages such as esters, carbonates, imines, hydrazones, acetals, orthoesters, peptides, amides, urethanes, ureas, amines, oligonucleotides, or sulfonamides.
  • the components can be modified to include biodegradable linkages such as lactates, caproates, methylene carbonates, glycolates, ester-amides, ester-carbonates, or combinations thereof.
  • Example 1 ⁇ n .Situ Formation of Polyethylene Oxide-Polyethylene Oxide Networks via Formation of Amide Linkages Reacting Polymers
  • Component 1 Polyethylene oxide-tetraamine (P4-AM), (SunBio Systems, Korea)
  • Component 2 Poly(ethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG), (SunBio Systems) Polymer Characterization
  • the degree of substitution (d.s.) of amines on the terra-armed polyethylene oxide backbone was calculated to be 3.91 by ⁇ -NMR; d.s. of succinimidyl glutarate was 3.85, also by ⁇ -MMR.
  • FIG. 1 shows the chemical structure of the network components (P4-AM/P4-SG) and a schematic representation of the cross-linking reaction by formation of amide linkages. The network is rendered biodegradable by the presence of ester linkages in one of the components, P4-SG.
  • Formulation of 2%-15% Polymeric Networks Solutions of the gel-forming components were prepared (2%, 3%, 4%, 5%, 8%,
  • 5% P4-AM was prepared by dissolving 50 mg P4-AM in 1 ml potassium mono-di phosphate buffer (pH 8.0).
  • 5% w/v P4-SG was prepared by dissolving 50 mg of P4-SG in milliQ de-ionized water. This solution was stored on ice until use.
  • the networks were created following addition of a solution of P4-AM with P4- SG.
  • Formulation A contained 2% w/v solids.
  • Formulation A cross-linked into a viscous branched polymer
  • Formulations B-G included equimolar amounts of the same components as in Formulation A, but at higher concentrations (B, 3% w/v total polymer; C, 4%; D, 5%; E, 8%, F, 10% and G, 15%).
  • Formulations B-G cross-linked into tissue-conforming hydrogels j n s jt u , post-injection into muscle. Incorporation of Plasmid DNA into the Network, and Effect on Gel Time
  • DNA was added to the solution containing P4-SG. 11.1 ⁇ l of a 9mg/ml stock solution of the nucleic acid (e.g., plasmid DNA or oligonucleotide) was added to 38.8 ⁇ l of 6.4% solution of P4-SG, to obtain a 5% P4-SG solution containing 100 ⁇ g nucleic acid in 100 ⁇ l. 50 ⁇ l of this P4-SG/DNA solution was then added to 50 ⁇ l of 5% w/v P4-AM to formulate the desired gel of final concentration (5% total PEGs). The gel time of this formulation was approximately the same as a formulation that did not contain nucleic acid (5-6 minutes at 25°C).
  • the nucleic acid e.g., plasmid DNA or oligonucleotide
  • a third non-reacting, non-nucleic acid polymeric component was added to the formulation.
  • Methoxy-PEG2K-di-stearoyl-phosphatidylethanolamine (mPEG-DSPE, Genzyme) was selected as a polymeric excipient and added to the solution of P4-SG.
  • FIG 2A shows Gel permeation chromatograms of network formulation A (2% PEGs) and the individual PEG components (P4-SG and P4-AM)
  • Crosslink densities can control the ability of a molecule to diffuse through the network.
  • Compatibility of Plasmid DNA with Gel Forming Components A compatibility experiment was performed to ensure that pre- ixing components 1 and 2 did not decrease the integrity (e.g., supercoiling) of plasmid DNA.
  • Plasmid DNA (pDNA) (10 ⁇ g/ml) was mixed with either of the reacting polymers (2% w/v P4-AM or P4-SG, 5% w/v P4-AM or P4-SG) and incubated at room temperature for 30 minutes.
  • FIG 4 demonstrates percent supercoiling of the pDNA as subsequently determined by agarose gel electrophoresis.
  • DNA supercoiling was found to not be affected by either of the non-gelled components.
  • plasmid DNA was incorporated into 2% w/v and 3% w/v hydrogels, and then extracted into phosphate buffered saline to test if the supercoiling of the plasmid was compromised by the crosslinking reaction.
  • the supercoiling of the network-extracted DNA was compared with control DNA that had not been incorporated into networks. No loss in DNA supercoiling was observed by incorporation of plasmid in networks.
  • FIGS.5A and 5B the data demonstrates that DNA integrity was maintained in the presence of a cross-linked formulation.
  • Network Characterization j n ⁇ n ro Equilibrium Swelling Equilibrium swelling can be used to characterize hydrogels. This method, when developed as a method of analysis, can be utilized effectively to determine reproducibility of a formulation. Networks containing plasmid DNA (with, without mPEG-DSPE) were prepared as described above except that the mixing was performed in a 96 well plate. Samples were incubated at 37°C for 1 hour to allow complete gelation. The gels were removed from the wells and placed into scintillation vials. The vials were weighed, and 5 ml of Dulbecco's phosphate-buffered saline (PBS) was added.
  • PBS Dulbecco's phosphate-buffered saline
  • % Swelling ((Final weight of Gel) 3 3 7 7 volatile°C, 2 2 4 4 h h r r s s - w weci i ggh i t o ufi G j ecl i )j 37O> 24 hrs x lOO.
  • In-vitro release of DNA from hydrogels B-D was measured by incubation of plasmid-containing gels in phosphate buffered saline at 37°C (200 ⁇ l hydrogel containing 200 ⁇ g of plasmid in a scintillation vial was incubated in 2 ml of PBS). At defined time points, the supernatant was removed and transferred to a new tube. An additional 2 ml of PBS was then added to each vial and the samples were returned to the incubator.
  • Percent DNA release from hydrogels was quantified using a DNA-NPR® (Tosoh- Biosep Inc.) anion exchange column using a gradient elution (HPLC Method: Buffer A: 0.56M sodium chloride in 50 mM Tris, pH 9.0; Buffer B: 1.2M sodium chloride in 50 mM Tris, pH 9.0; 0-30% Buffer B in 15 minute gradient elution).
  • a standard curve was constructed with control unformulated DNA diluted in PBS at various concentrations and analyzed by HPLC. Relaxed and supercoiled plasmid peaks were identified in comparison with the retention time of the standards.
  • FIG 7 A and 7B demonstrate f n v if ro release data.
  • FIG 7 A shows a representative HPLC trace from DNA released from formulation C (4% w/v P4-AM/P4-SG); the second peak in the triplet set of peaks is supercoiled DNA.
  • FIG 7B shows cumulative release data from formulations B (3% w/v P4-AM/P4-SG), C (4% w/v P4-AM/P4-SG), and D (5% w/v P4-AM/P4-SG). The data indicate that plasmid can be released from the gels and that release is faster with gels containing a lower percentage of P4-AM and P4-SG.
  • PEG-PEG Networks Protect Plasmid DNA from Serum Endonucleases
  • FIG 8 demonstrates that both network formulations protected the plasmid DNA from serum endonucleases (lanes 5-6, 8-9), whereas unformulated DNA showed a loss of supercoiling after 30 minutes of incubation in both serum dilutions (lanes 4 and 7).
  • Network Characterization Injectability
  • the injectability of the formulations was determined in the following manner: P4-AM and P4-SG were loaded into separate 0.3 ml syringes, which were then joined v f a a syringe connector. Solutions of the components were mixed rapidly, and then retrieved into a single syringe. The mixed formulation was extruded through a 26g needle at various time points post-mixing. The time within which a formulation could be injected was recorded in minutes. FIG 6 demonstrates that formulations at higher concentrations gelled faster, lowering the time interval within which injection could occur.
  • Evans Blue dye was added to formulations made up of 2%, 3%, 4%, 5%, 8%, or 10% w/v of total PEGs (P4-AM/P4-
  • the formulations were injected into the muscle tissue of mice.
  • cysteine-HCl and 2.915g EDTA were weighed and added to a 100 ml volumetric flask, then filled to the 100 ml mark.
  • the pH of the solution was adjusted to 6.25.
  • the solution was bubbled with an inert gas such as argon to remove oxygen, and then stored at -20°C until use.
  • 1125 ⁇ l of the papain solution was added to a 15 ml centrifuge tube containing the tissue explant (weight of the explant should be between 30 and 600mgs).
  • 1125 ⁇ l of the collagenase solution and 750 ⁇ l of 10% calcium chloride was added to the centrifuge tube and mixed.
  • the centrifuge tube containing the explant and the digestion cocktail was equilibrated for 8-12 hours in a water bath maintained at 37°C. This step resulted in the digestion of the tissue.
  • the pH of the digested dispersion was adjusted to 11.5 with 30 ⁇ l of 50% NaOH, and then the tube was placed in the 37°C bath for an additional 8-12 hours.
  • the rate of -vivo bioabsorption of these polyethylene glycol-based networks was determined by quantification of total PEGs remaining at the injected muscle site over time.
  • the results demonstrate that ⁇ 40% of total injected PEGs for formulation B had cleared from the site 33 days post-injection, and that 60-80% of the total injected PEG polymer was lost approximately 90 days post injection.
  • the study demonstrated that the network delivery systems can be usedin n v ivo applications.
  • Example 2 Poly(ethylene oxide)-Poly(amidoamine) Networks v ⁇ a formation of amide linkages
  • P4-SG 100 mg in 1 ml
  • Equimolar concentrations of poly(amidoamine) GO (10 mM or 0.5%) and Gl (lOmM or 0.5%) solutions were prepared by diluting the respective stock solutions in phosphate buffer (pH 8.0).
  • the P4-SG solution when mixed in equal volumes with either of the GO or Gl solutions, formed a transparent soft gel. Gels with different crosslinking density could be formed by varying the concentrations of P4-SG and poly(amidoamine) GO or Gl.
  • Different compositions (2%, 5%, 10%) containing PEG-DSPE and nucleic acid were also formulated, and all were found to be injectable.
  • Network Characterization Determination of j n ⁇ uro Equilibrium Swelling
  • FIG 9 demonstrates that the amount of swelling is much lower for these gels than for the PEG hydrogels, and the addition of lipid was found to decrease the swelling.
  • Example 3 Poly(ethylene oxide)-poly(ethyleneimine) networks v ⁇ formation of amide linkages
  • PEI Polyethyleneimine
  • In situ crosslinking gels were formulated using poly(ethylenimine) and P4-SG.
  • a 10% w/v solution of P4-SG was prepared in milliQ water.
  • 100 mM of PEI (0.15 % w/v) was prepared in phosphate buffer, pH 8.0.
  • 100 ⁇ l of this solution (10 times molar excess) was added to the P4-SG solution and quick gelation was observed ( ⁇ 1 minute).
  • Gelation time can be controlled by altering the PEI or P4-SG concentration, and/or the pH of the solutions in which the individual polymers are resuspended.
  • a formulation containing 0.075% w/v PEI and 10% w/v P4-SG reconstituted at pH 8.0 has a gel time of 6 minutes at 25°C.
  • plasmids that encodes a secreted protein permits serum sampling and analysis for expressed protein without sacrificing the animal.
  • plasmids encoding secreted embryonic alkaline phosphatase gene, Factor VIII, Factor LX, erythropoetin (EPO), endostatin, various cytokines, insulin, and bone morphogenic protein (BMP) have been used for this purpose.
  • a plasmid encoding the human secreted embryonic alkaline phosphatase gene (pgWizTM SEAP, henceforth referred as "SEAP”) was used to monitor systemic expression.
  • SEAP a secreted form of the membrane bound placental alkaline phosphatase, has a half-life of from minutes to a few days in serum. A protein with a short half-life is especially useful to reliably determine expression kinetics.
  • Materials pgWiz-SEAP (Gene Therapy Systems Inc., San Diego, California, USA).
  • P4-AM Polyethylene oxide-tetraamine
  • All formulations were prepared by mixing of two solutions, one containing a pre- weighed amount of P4-AM dissolved in 0.1M potassium phosphate buffer, pH 8.0, and the other containing an equimolar amount of P4-SG dissolved in cold deionized water containing SEAP plasmid DNA (100 ⁇ g 100 ⁇ l final volume of formulation) and mPEG- DSPE (10 ⁇ g/100 ⁇ l final volume of formulation).
  • Formulation A included 2% w/v P4-SG/P4-AM cross-linked into a viscous branched polymer
  • Formulations B-D (3, 4, and 5% w/v P4-SG/P4-AM, respectively) included equimolar amounts of the same components as A, but at higher concentrations. These formulations cross-linked into tissue-conforming hydrogels f n S u , post-injection into muscle.
  • the solutions were freshly prepared and injected into mouse muscle immediately after mixing all formulation components.
  • mice were mildly anesthetized using isofluorane and injected with different cross- linked network formulations or with unformulated plasmid DNA (in saline) bilaterally into the anterior tibialis muscles. All animals were injected with 100 ⁇ g of plasmid DNA in an injection volume of 50 ⁇ l per muscle.
  • mice were anesthetized and blood was collected retro-orbitally. Serum was separated from red blood cells by centrifugation and stored at -80°C until assays were performed. SEAP assay
  • Luminescence measurements were performed using a Topcount® plate reader (Packard Instruments, Illinois) following 40 minutes of incubation in the reaction buffer. Serum SEAP levels at each timepoint were expressed in nanograms/ml using the standard curve generated from the positive control (purified human placental alkaline phosphatase) supplied with the assay kit. The data were further analyzed using a Thompson-Tau outlier analysis as described in Wheeler and Ganji, "Introduction to Engineering Experimentation,” Prentice Hall, pp. 142-145 (1996) and plotted as averages and standard deviations.
  • FIG 11 A shows that all networks with higher crosslink densities (i.e., formulations C and D) produced significant serum levels of SEAP expression compared to lightly crosslinked networks (i.e., formulations A and B).
  • percent positive animals as measured by animals expressing more than 300 pg ml of serum SEAP, a level that is 2-3 fold higher than background serum SEAP levels in saline injected mice) were plotted for each formulation FIG.
  • IB demonstrates that DNA delivery from the networks resulted in long-term expression of the encoded protein in serum, whereas protein levels in animals injected with unformulated DNA dropped precipitously after 3-4 weeks.
  • One hypothesis for transient expression of proteins following intramuscular injections of plasmids is antibody-directed complement-mediated cytotoxicity (ADCC).
  • ADCC antibody-directed complement-mediated cytotoxicity
  • DBA/2J mice deficient in a component of complement, were injected with unformulated DNA or DNA in network formulations.
  • FIG 12A show that in complement-deficient animals, network injection produced more sustained expression of SEAP compared to that produced by unformulated DNA.
  • FIG 12B shows that serum SEAP levels from animals injected with network associated DNA were sustained longer than those from the groups injected with unformulated DNA.
  • P4-AM Polyethylene oxide-tetraamine
  • 3% w/v P4-AM/P4-SG was formulated with mPEG-DSPE (10 ⁇ g/100 ⁇ l) and (100 ⁇ g 100 ⁇ l) SEAP DNA.
  • the gel was identified as a GT20 gel.
  • GT20 denotes a gel time of 20 minutes post reconstitution with buffer at pH 8 as measured by viscometry at 25°C.
  • mice were mildly anesthetized using isofluorane and injected with different crosslinked network formulations or with unformulated plasmid DNA (in saline) bilaterally into the anterior tibialis muscles. All animals were injected with 100 ⁇ g of plasmid DNA in an injection volume of 50 ⁇ l per muscle.
  • mice muscles were electroporated immediately post-injection of the formulations with 200 V/cm, 8 pulses, 20 ms pulse width at 1 second intervals (Genetronics electroporator, ECM 830; BTX Inc., San Diego, California, USA).
  • Serum collection, SEAP assays, and data analysis using Thompson-Tau Outlier analysis were performed as in example 5.
  • the data shown in FIG. 13 demonstrates enhancement of SEAP expression in network formulation by electroporation.
  • 3 % w/v P4-AM/P4-SG was formulated with mPEG-DSPE (10 ⁇ g/100 ⁇ l) and (100 ⁇ g/100 ⁇ l) SEAP DNA. The gel was also identified as a GT20 gel.
  • Various excipients were added to the DNA-containing P4-SG solution, before mixing with the P4-AM solution. The final concentrations of these excipients in the formulation were: sodium lauryl sulfate (SDS, 0.1% w/v)(Sigma), pluronic L62 (0.1% w/v)(BASF)
  • Magainin I (0.025% w/v) (Sigma), and poly(amidoamine) (PAMAM; Dentritech) GO (0.15% w/v). More specifically, sodium lauryl sulfate is classified as a anionic lipid, pluronic L62 as a non-ionic surfactant, Magainin I as a cationic peptide, and PAMAM GO as a cationic 4-armed polymer.
  • FIGS 14A and 14B demonstrate that SEAP expression was found to be enhanced by the addition of these excipients to the network formulations.
  • DNA was amplified and purified using a Qiagen Endo-free® kit (Qiagen Inc., Valencia, California) or was purchased from Aldevron LLC (Fargo, ND).
  • All formulations were prepared by mixing of two solutions, one containing a pre- weighed amount of P4-SH dissolved in 0.1M potassium phosphate buffer, pH 8.0, and the other containing an equimolar amount of P4-SG dissolved in cold deionized water containing SEAP plasmid DNA (100 ⁇ g/100 ⁇ l final volume of formulation) and mPEG- DSPE (10 ⁇ g/100 ⁇ l final volume of formulation).
  • Two formulations were tested: Formulation A: 3.5% w/v of each P4-SH/P4-SG gelled after 20 minutes at 25°C; and Formulation B: 5% w/v of each P4-SH/P4-SG gelled after 10 minutes at 25°C.
  • mice were mildly anesthetized using isofluorane and injected with different cross- linked network formulations or with unformulated plasmid DNA (in saline) bilaterally into the anterior tibialis muscles. All animals were injected with 100 ⁇ g of plasmid DNA in an injection volume of 50 ⁇ l per muscle. There were 8 animals per group.
  • mice were anesthetized blood was collected, serum prepared and analyzed as in Example 5. As shown in FIG 15, formulations A and B both induced high levels of gene expression in mice.
  • Example 9 Network-Mediated (P4AM-P4-SG) Gene Expression in Mouse Mucosa Materials pgWiz-SEAP, (Gene Therapy Systems Inc., San Diego, California). Polyethylene oxide)-tetraamine (P4-AM) (SunBio Systems) Poly(ethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems) mPEG-DSPE (Genzyme) 5-6 week old female C57B16 mice (Jackson Laboratories)
  • DNA was amplified and purified using a Qiagen Endo-free® kit (Qiagen Inc., Valencia, California, USA) or was purchased from Aldevron LLC (Fargo, ND, USA).
  • All formulations were prepared by mixing of two solutions, one containing a pre- weighed amount of P4-AM dissolved in 0.1M potassium phosphate buffer, pH 8.0, and the other containing an equimolar amount of P4-SG dissolved in cold deionized water containing SEAP plasmid DNA (100 ⁇ g/100 ⁇ l final volume of formulation) and mPEG- DSPE (10 ⁇ g 100 ⁇ l final volume of formulation).
  • GT5 5% w/v of each of P4-AM and P4-SG gelled after 5 minutes at 25°C.
  • the solutions were freshly prepared and injected into mouse rectum immediately post mixing, of all formulation components. Animal experiments
  • mice were mildly anesthetized using isofluorane and injected with different crosslinked network formulations or with unformulated plasmid DNA (in saline) into the rectum 3.5 cm from the anus. All animals were injected with 100 ⁇ g of plasmid DNA in an injection volume of 50 ⁇ l. There were 5 animals per group.
  • mice were anesthetized blood was collected, serum prepared and analyzed as in example 5. Animals receiving unformulated DNA did not show SEAP expression. GT5 formulations induced significant levels of gene expression in 3 of 5 mice.
  • pCMV/ ⁇ -gal encoding Escherichia coli ⁇ -gal driven by the human CMV intermediate early promoter was used as the reporter gene for all immunizations.
  • CT26.WT is a clone of CT26, a BALB/c
  • CT26.CL25 is a CT26.WT clone stably transfected with the ⁇ ac gene.
  • Cell lines were maintained in RPMI 1640, 10% heat- inactivated fetal calf serum (FCS; Life Technologies, Grand Island, NY), 2mM L- glutamine, 100 ⁇ g/ml streptomycin, and 100 U/ml penicillin (Life Technologies, Grand Island, NY).
  • CT26.CL25 was maintained in the presence of 400 ⁇ g ml G418 sulfate (Life Technologies, Grand Island, NY).
  • All formulations were prepared by mixing of two solutions, one containing a pre- weighed amount of P4-AM dissolved in 0.1M potassium phosphate buffer, pH 8.0, and the other containing a pre-weighed amount of P4-SG dissolved in cold deionized water containing ⁇ -gal DNA (100 ⁇ g 100 ⁇ l of formulation) and mPEG-DSPE (10 ⁇ g/100 ⁇ l of formulation).
  • Formulation A included 2 % w/v P4-AM/P4-SG and created a viscous branched polymeric network post-mixing of the components.
  • Formulation B included 3% w/v P4-AM/P4-SG and formed a hydrogel post-mixing.
  • formulation A The molecular weight and size distribution profile of formulation A was determined to be one million by aqueous gel permeation chromatography using a TSK Gel Mixed Bed column with 0.02M phosphate buffer, pH 7.5, as the mobile phase.
  • the network had a fluid viscosity of ⁇ 5 cp, as measured by Brookefield rheometry.
  • the gel point of formulation B was ⁇ 11 minutes at 37°C as measured by Brookefield rheometry. Immunizations
  • mice were mildly anesthetized using isoflurane and injected with different crosslinked network formulations or saline bilaterally into the anterior tibialis muscles. All animals were injected a single time with 30 ⁇ g of plasmid DNA in an injection volume of 50 ⁇ l per muscle.
  • dissection of the muscle site approximately an hour post injection of formulation B demonstrated presence of a hydrogel conformed to tissue. Examination of the muscle site an hour post-injection of formulation A demonstrated formation of a thick, viscous gelatinous material.
  • Sera was collected from mice by retro-orbital bleeding at 12 weeks post- immunization. Titers of ⁇ -gal specific antibodies at 12 weeks were measured by a standard ELISA protocol, ⁇ -gal titers were defined as the highest serum dilution that resulted in an absorbance (OD 405) value twice than that of non-immune sera at that dilution.
  • FIG 16 demonstrates that administration of DNA in networks derived from both formulations stimulated robust ⁇ -gal antibody responses measured 12 weeks post injection. Similar results were obtained in two separate experiments with identical formulation groups.
  • T cell proliferation assays were performed by incubating purified T cells and syngeneic irradiated splenocytes (2 x 10 5 each) in the presence of 30 ⁇ g/ml of ⁇ -gal or chicken ovalbumin protein at 37°C for
  • FIG 17 shows that delivery of DNA in both network formulations induced ⁇ -gal specific proliferative T cell responses. This type of response is usually associated with a T helper restricted T cell population. Similar results were obtained in two separate experiments with identical formulation groups.
  • Purified T cells (2 x 10 5 ) were stimulated with 2 x 10 5 irradiated ⁇ -gal or HBV peptide pulsed syngeneic spleen cells for 24 hrs.
  • the MHC Class I restricted T cell response elicited by these formulations was measured in a gamma-interferon ( ⁇ -IFN) enzyme-linked immunospot (ELISpot) assay according to the manufacturer's directions (R&D Systems, Cat# EL485, Minneapolis, MN, USA).
  • ⁇ -IFN gamma-interferon
  • ELISpot enzyme-linked immunospot
  • FIG 18 demonstrates that responses were detected at both the 12 week time points and were higher in mice given formulation A in comparison to those of mice receiving formulation B.
  • Tumor Protection Studies Mice were challenged intravenously with 5xl0 5 CT26.WT or CT25.CL25 cells post immunization with formulated DNA or saline control. Mice were sacrificed on day 13, lungs were isolated and stained with 0.2% X-gal solution after fixing with 0.25% glutaradehyde/0.01% formalin in PBS. Tumor nodules could then be visualized and enumerated. The protective response to this tumor is dependent on the class I restricted T cell response.
  • Example 11 Preparation of A Lyophilized Formulation
  • a schematic of a method for formulating a "one vial" lyophilized product that contains an excipient(s) such as a lipid, unreacted PEG-amine, unreacted PEG- succinimidyl glutarate, and a nucleic acid is provided in FIG. 20.
  • an excipient(s) such as a lipid, unreacted PEG-amine, unreacted PEG- succinimidyl glutarate, and a nucleic acid.
  • pHs greater than 7.0 the two PEG components mutually react to form a crosslinked network. Therefore, the pH of the solution containing the two PEG components was maintained below this threshold (e.g., the pH is maintained at 5.5 by the dissolution of the components in deionized water).
  • FIG 21 shows a schematic for characterization of gels at lower temperature. After the mixing of the components, vials containing the DNA were filled with the solution and then lyophilized. The lyophile was reconstituted with phosphate buffered saline, pH 8.0, and gelation times (onset of gelation) were measured. A 3% w/v gel formed in approximately 25 minutes at 25 °C and did not vary from the gel time of a non- lyophilized formulation.
  • Lyophilization was also performed by mixing solutions of the reactive polymers (e.g., P4-SG and P4-SH), maintaining a pH of below 7, and lyophilizing in the absence of nucleic acid.
  • the nucleic acid was added to the formulation upon reconstitution.
  • gel times for formulations prepared in this way did not vary by the lyophilization procedure.
  • Example 12 Generation of Networks (P4-AM/P4-SG) containing oligonucleotides
  • Formulations at concentrations of 5 and 10%) w/v PEGs (P4-AM andP4-SG) with and without oligonucleotide were also prepared, and the formation of gels was noted in all cases.
  • FIGS 23 A and 23B show the results of i n - v itro release assays that were performed for 5% and 10% hydrogels containing 1 ⁇ g/ml of oligo.
  • FIG 25A and 25B show that in 14 days,the total % ODN released was -98% for 10% gels, - 85% for 20% gels, and -78% for 30% gels.
  • Example 15 Micronized Calcium Phosphate Oligonucleotide In P4-AM/P4- SG Networks
  • Oligonucleotides with phosphorothioate or phosphodiester backbones (Oligos, etc.) Formulations
  • the white precipitate was dialyzed by centrifugation/filtration using a 1.5 ml Centricon Filtrion® centrifuge tube.
  • the white precipitate was reconstituted in a 3% w/v solution of P4-AM.
  • 50 ⁇ l of the P4-AM OligoCaP dispersion was added to 50 ⁇ l of a 3% w/v P4-SG solution to form a 3% total PEGs formulation.
  • Gel time of a 3% PEGs gel with micronized CaP-ODN was -10 minutes at 37°C, and 19 minutes at 25 °C. The gel was characterized as a "hard" gel.
  • Example 16 Networks Containing Microparticles in Hydrogel
  • DNA-containing microparticles were added to 50 ⁇ l solutions of 10%) w/v P4-SH (A, B, C, respectively) made up in phosphate buffer, pH 8.0. 50 ⁇ l of a solution of 10% w/v P4-SG made up in DI water was added to solutions A, B and C to make formulations A, B and C. Gel times and gel characteristics were determined.
  • Network Characterization Formulations A, B and C all gelled after between 2-3 minutes at room temperature, demonstrating no inhibition of gelation by addition of microparticles.
  • Hydrogels fabricated from formulation C were found to be hard and brittle. Hydrogels from A and B were hard, but pliable. This study demonstrates the feasibility of incorporation of microparticles into hydrogels for the purpose of applying drug delivery devices to rounded tissues and surfaces. The hydrogel in this case would hold the microparticles "in place.”
  • Network Characterization Gel Time, Hardness/Softness
  • the formulation gelled instantaneously at 25°C, forming a hard gel.
  • This formulation demonstrates the feasibility of a proteolytically degradable network (e.g., a network degradable by lysozymes).
  • Example 18 Poly(lysine)/P4-SG Networks (PL/P4-SG)
  • a solution containing 1.0% w/v poly(lysine) hydrobromide (PL) was prepared in phosphate buffer, pH 8.0. 50 ⁇ l of this solution was added to 50 ⁇ l of a solution containing 5% w/v P4-SG and 1 ⁇ g/ ⁇ l DNA in DI water.
  • This formulation is another variation of a network formulation that can be used for nucleic acid delivery.
  • Example 19 (PEO-PPO-PEO-tetra-SH)/P4-SG Networks (PEO-PPO- PEO/P4-SG)
  • a solution containing 10% w/v PEO-PPO-PEO-tetra-SH was prepared in phosphate buffer, pH 8.0. 50 ⁇ l of this solution was added to 50 ⁇ l of a solution of 10% w/v P4-SG and 1 ⁇ g/ ⁇ l DNA in DI water to form a 10% w/v gel.
  • Network Characterization Gel Time, Hardness/Softness The formulation gelled in 6-7 minutes, and formed a hard, oily gel. This formulation is yet another variation of a network formulation that can be used for nucleic acid delivery.

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Abstract

Cette invention est fondée sur la découverte selon laquelle des compositions et des préparations polymériques injectables et compatibles avec de l'acide nucléique peuvent être mises au point structurellement pour réguler in vivo l'activité de l'acide nucléique ou l'expression génique, par exemple, par régulation de la biodisponibilité de l'acide nucléique par modulation de la biodégradabilité et de la densité de réticulation du réseau formé par les constituants de la préparation. Le réseau polymérique encapsule l'acide nucléique, et non seulement régule la libération de l'ADN, mais le protège également de la dégradation. L'invention constitue une amélioration par rapport au modes de libération génique antérieurs, en ce que l'expression génique peut être régulée par modulation d'un réseau polymérique formé par combinaison d'au moins deux constituants solubles dans l'eau pouvant réagir l'un avec l'autre. L'acide nucléique d'intérêt est incorporé dans le réseau pour être libéré de manière constante, en fonction du niveau et de la durée d'activité ou d'expression voulus.
PCT/US2002/001379 2001-01-17 2002-01-17 Preparations de liberation d'acide nucleique WO2002057424A2 (fr)

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CA002435287A CA2435287A1 (fr) 2001-01-17 2002-01-17 Preparations de liberation d'acide nucleique
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EP1482974A2 (fr) * 2002-02-15 2004-12-08 Zycos Inc. Procedes d'electroporation pour introduire des agents bioactifs dans des cellules
WO2005042702A2 (fr) * 2003-10-23 2005-05-12 Alza Corporation Compositions d'adn stabilise pour microprojections de revetement
WO2006053836A1 (fr) * 2004-11-16 2006-05-26 Universite De Liege Dispositif d’administration d’une substance active comprenant une matrice d’hydrogel et des microporteurs
US7375096B1 (en) 1998-12-04 2008-05-20 California Institute Of Technology Method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network
US8252276B2 (en) 2002-09-06 2012-08-28 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8497365B2 (en) 2007-01-24 2013-07-30 Mark E. Davis Cyclodextrin-based polymers for therapeutics delivery
EP3313451A4 (fr) * 2015-09-30 2018-07-04 Sunbio Inc. Injectable d'hydrogel de polyéthylène glycol
CN109134855A (zh) * 2018-07-23 2019-01-04 安徽大学 一种酸敏感的聚酰胺胺的阳离子聚合物poeamam及其制备方法和应用
EP3412313A4 (fr) * 2016-02-05 2019-09-11 Pharmaresearch Products Co., Ltd. Composition d'hydrogel sensible à la température comprenant un acide nucléique et du chitosane
CN111333878A (zh) * 2019-05-23 2020-06-26 吾奇生物医疗科技(镇江)有限公司 一种双交联壳聚糖水凝胶及其制备方法和应用
CN113683780A (zh) * 2021-09-15 2021-11-23 广州医科大学 具有穿膜活性和细胞核定位功能的抗血清、低细胞毒性的聚氨基酸类基因递送载体材料
US11452759B2 (en) * 2015-01-19 2022-09-27 Technion Research & Development Foundation Limited Ubiquitin ligase KPC1 promotes processing of P105 NF-κB1 to p50, eliciting strong tumor suppression
US11464871B2 (en) 2012-10-02 2022-10-11 Novartis Ag Methods and systems for polymer precipitation and generation of particles

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Cited By (30)

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US7375096B1 (en) 1998-12-04 2008-05-20 California Institute Of Technology Method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network
EP1482974A4 (fr) * 2002-02-15 2006-08-02 Zycos Inc Procedes d'electroporation pour introduire des agents bioactifs dans des cellules
EP1482974A2 (fr) * 2002-02-15 2004-12-08 Zycos Inc. Procedes d'electroporation pour introduire des agents bioactifs dans des cellules
US8580242B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8580244B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US9550860B2 (en) 2002-09-06 2017-01-24 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8252276B2 (en) 2002-09-06 2012-08-28 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8314230B2 (en) 2002-09-06 2012-11-20 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8389499B2 (en) 2002-09-06 2013-03-05 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8399431B2 (en) 2002-09-06 2013-03-19 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8404662B2 (en) 2002-09-06 2013-03-26 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8475781B2 (en) 2002-09-06 2013-07-02 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8680202B2 (en) 2002-09-06 2014-03-25 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8518388B2 (en) 2002-09-06 2013-08-27 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8609081B2 (en) 2002-09-06 2013-12-17 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8580243B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8603454B2 (en) 2002-09-06 2013-12-10 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
WO2005042702A2 (fr) * 2003-10-23 2005-05-12 Alza Corporation Compositions d'adn stabilise pour microprojections de revetement
WO2005042702A3 (fr) * 2003-10-23 2009-03-26 Alza Corp Compositions d'adn stabilise pour microprojections de revetement
WO2006053836A1 (fr) * 2004-11-16 2006-05-26 Universite De Liege Dispositif d’administration d’une substance active comprenant une matrice d’hydrogel et des microporteurs
US8497365B2 (en) 2007-01-24 2013-07-30 Mark E. Davis Cyclodextrin-based polymers for therapeutics delivery
US9610360B2 (en) 2007-01-24 2017-04-04 Ceruliean Pharma Inc. Polymer drug conjugates with tether groups for controlled drug delivery
US11464871B2 (en) 2012-10-02 2022-10-11 Novartis Ag Methods and systems for polymer precipitation and generation of particles
US11452759B2 (en) * 2015-01-19 2022-09-27 Technion Research & Development Foundation Limited Ubiquitin ligase KPC1 promotes processing of P105 NF-κB1 to p50, eliciting strong tumor suppression
EP3313451A4 (fr) * 2015-09-30 2018-07-04 Sunbio Inc. Injectable d'hydrogel de polyéthylène glycol
EP3412313A4 (fr) * 2016-02-05 2019-09-11 Pharmaresearch Products Co., Ltd. Composition d'hydrogel sensible à la température comprenant un acide nucléique et du chitosane
US11672756B2 (en) 2016-02-05 2023-06-13 Pharmaresearch Co., Ltd. Temperature sensitive hydrogel composition including nucleic acid and chitosan
CN109134855A (zh) * 2018-07-23 2019-01-04 安徽大学 一种酸敏感的聚酰胺胺的阳离子聚合物poeamam及其制备方法和应用
CN111333878A (zh) * 2019-05-23 2020-06-26 吾奇生物医疗科技(镇江)有限公司 一种双交联壳聚糖水凝胶及其制备方法和应用
CN113683780A (zh) * 2021-09-15 2021-11-23 广州医科大学 具有穿膜活性和细胞核定位功能的抗血清、低细胞毒性的聚氨基酸类基因递送载体材料

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WO2002057424A3 (fr) 2002-10-03

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