WO2010005847A1 - Matériaux polyhydroxylés modifiés dégradables par des acides et par bio-érosion - Google Patents

Matériaux polyhydroxylés modifiés dégradables par des acides et par bio-érosion Download PDF

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WO2010005847A1
WO2010005847A1 PCT/US2009/049415 US2009049415W WO2010005847A1 WO 2010005847 A1 WO2010005847 A1 WO 2010005847A1 US 2009049415 W US2009049415 W US 2009049415W WO 2010005847 A1 WO2010005847 A1 WO 2010005847A1
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modified
composition
polymer
particles
polyhydroxylated
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Jean M. J. Frechet
Eric M. Bachelder
Tristan T. Beaudette
Kyle E. Broaders
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The Regents Of The University Of California
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Publication of WO2010005847A1 publication Critical patent/WO2010005847A1/fr
Priority to US12/987,679 priority Critical patent/US9644039B2/en
Priority to US15/474,827 priority patent/US10995156B2/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
    • A61L17/06—At least partially resorbable materials
    • A61L17/10—At least partially resorbable materials containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
    • A61L17/14—Post-treatment to improve physical properties
    • A61L17/145—Coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/148—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00—Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/12—Degraded, destructured or non-chemically modified starch, e.g. mechanically, enzymatically or by irradiation; Bleaching of starch
    • C08B30/18—Dextrin, e.g. yellow canari, white dextrin, amylodextrin or maltodextrin; Methods of depolymerisation, e.g. by irradiation or mechanically
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/02—Cellulose; Modified cellulose
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/08—Cellulose derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/02—Compositions of unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
    • C08L3/02—Starch; Degradation products thereof, e.g. dextrin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/06—Pectin; Derivatives thereof

Definitions

  • Patent Application No. 60/798,177 filed on May 5, 2006. This application is also related to and incorporates by reference co-pending divisional U.S. Patent Application No. 11/388,924, filed on March 28, 2006.
  • This invention generally relates to the field of acid-degradable and bioerodible materials and polymers for use in delivery of bioactive materials such as antigens, DNA and other therapeutics or as bulk materials such as sutures, scaffolds, and implants.
  • Polyesters, polyorthoesters, and polyanhydrides are widely used materials for biomedical applications due to their biodegradability, biocompatibility and processability (Yolles, S.; Leafe, T. D.; Meyer, F. J., J. Pharm. Sci. 1975, 64, 115-6; Heller, J., Ann. N. Y. Acad. Sci. 1985, 446, 51-66; Rosen, H. B.; Chang, J.; Wnek, G. E.; Linhardt, R. J.; Langer, R., Biomaterials 1983, 4, 131-3). Microparticles made from these polymers have been used as carriers for vaccine applications, gene delivery and chemotherapeutic agents. (Solbrig, C.
  • the encapsulated cargo is typically released over the course of several months via surface erosion and the slow degradation of the polymer.
  • the present invention is directed to bioerodible modified polyhydroxylated polymers for application in the delivery of proteins, vaccines, drugs (such as the anticancer drugs cisplatin, paclitaxel or taxotere), and other bioactive materials.
  • the modified polyhydroxylated polymer comprising a polyhydroxylated polymer with reversibly modified hydroxyl groups, wherein the hydroxyl groups are modified by a one-step reaction to feature a functional group selected from the group consisting of acetals, aromatic acetals, and ketals.
  • the hydroxyl groups in the polyhydroxylated polymers are modified, thereby rendering the modified polyhydroxylated polymer acid- degradable, pH sensitive and insoluble in water.
  • the modified polyhydroxylated polymers are also acid-degradable comprising acid-degradable modified polyhydroxylated polymers that are designed to deliver bioactive materials.
  • the modified polyhydroxylated polymers deliver bioactive materials upon hydrolysis of an acetal or ketal linkage at pH 5 to pH 7.4.
  • the polymer compositions are made using polyhydroxylated polymers resulting in modified polyhydroxylated polymers containing an acid-degradable linkage, which hydrolyzes to release and deliver bioactive material.
  • the modified polyhydroxylated polymers are bioerodible whereby degradation of the polymers allows for slow release of any bioactive material to be delivered.
  • the polymers may be processed to form particles, bulk materials or implants for the pH dependent controlled release of small drug or biotherapeutics. These polymers could also be used as vehicles for drug conjugation or complexation designed to release their drug at mild pH values or scaffolds for tissue engineering purposes.
  • the polymers of the current invention are designed to degrade into natural polyhydroxylated products, releasing their contents in response to the mildly acidic conditions found in lysosomes, tumors, and inflammatory tissues.
  • the present polymers will hydrolyze at a preferred pH range of 4.5 to 6.8, more preferably pH 5.0 to 6.0.
  • the polymers will completely hydrolyze within 24 hours at pH 5.0, or conditions such as in the lysosome, and release their encapsulated or bound contents after entering a cell.
  • the polyhydroxylated polymers are preformed natural polymers or hydroxyl-containing polymers including but not limited to, multiply- hydroxylated polymers, polysaccharides, carbohydrates, polyols, polyvinyl alcohol, poly amino acids such as polyserine, and other polymers such as 2-(hydroxyethyl)methacrylate.
  • the polysaccharides that can be used include but are not limited to, dextran, mannan, pullulan, maltodextrin, starches, cellulose and cellulose derivatives, gums (e.g., xanthan, locust bean, etc.), and pectin.
  • the polysaccharides are dextran or mannan.
  • the modified polysaccharides have pendant acetals, thus providing acetal-derivatized polysaccharides.
  • the modified polyhydroxylated polymers are acetal-derivatized dextran, acetal-derivatized mannan or acetal-derivatized polyvinyl alcohols.
  • the reversible modification of the polyhydroxylated polymer to produce the present acid-degradable and bioerodible modified polyhydroxylated polymers is performed in a one-step modification process.
  • the one-step reversible modification of the hydroxyl groups can be carried out to provide modified hydroxyl groups, wherein at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the hydroxyl groups in the polymer are modified.
  • polyhydroxylated polymers are prepared and reacted with a functionalizing group and result in a variety of pH sensitive and functionalized polyhydroxylated polymers with different solubilities.
  • This class of polymers are simple to prepare and completely degradable. Select polymers were characterized. The degradation of these polymers into small molecules was monitored at pH 7.4 and pH 5 over time along with methods of controlling the rate of degradation, thus making these polymers promising candidates for drug delivery systems.
  • a method of preparing a modified polyhydroxylated acid-degradable composition for delivering a bioactive material to a cell comprising the steps of (a) preparing a mixture which contains a polyhydroxylated polymer and a functional group, wherein a one-step reaction provides a modified polyhydroxylatedpolymer having modified hydroxyl groups containing an acid-degradable linkage; (b) forming particles of the polymer in the presence of a bioactive material; and (c) recovering the resulting polymer particles having bioactive material bound or entrapped thereto.
  • Figure 1 shows previous acid-degradable systems (top and middle) compared to the present system (bottom).
  • Figure 2 shows a general synthetic scheme for the preparation of modified polyhydroxylated polymers.
  • Figure 3 shows an overall synthetic scheme for the preparation of modified polydroxylated polymers.
  • Figure 4 shows the scheme for synthesis of dextran modified to dextran having cyclic and acyclic ketals masking the hydroxyl groups.
  • Figure 5 shows water-soluble dextran modified to organic-soluble dextran having cyclic and acyclic acetals masking the hydroxyl groups and anSEM image of the Ac- DEX particles.
  • Figure 6 shows the synthesis of acetal-modified dextran (Ac-DEX) and particle formation (i) 2-methoxypropene, pyridinium-/?-toluenesulfonate, DMSO (ii) solvent- evaporation-based particle formation (scale bar is 2 ⁇ m).
  • Figure 7 shows the scheme for synthesis of dextran modified to dextran having aliphatic acetals masking the hydroxyl groups.
  • Figure 8 shows the scheme for synthesis of dextran modified to dextran having aromatic acetals masking the hydroxyl groups.
  • Figure 9 shows the scheme for synthesis of pre-functionalized dextran having alkyne functional groups or a dye to prefunctionalized dextran having cyclic and acyclic ketals masking the hydroxyl groups.
  • Figure 10 shows the scheme for the synthesis of mannan modified to mannan having cyclic and acyclic ketals masking the hydroxyl groups and an SEM of particles formed by a solvent-evaporation-based technique (scale bar is 1 ⁇ m).
  • Figure 11 shows the synthetic scheme of acetalated polyvinyl alcohol using 2- methoxypropene, pyridinium-/?-toluenesulfonate, DMSO.
  • Figure 12 Representative SEM image of (A) Ac-DEX particles, (C)single emulsion Ac-DEX particles and (D) single emulsion acetalated mannan particles.
  • Figure 12B shows time-lapse photos of Ac-DEX particles under physiological or acidic conditions [034]
  • Figure 13 dissolution halflife at pH 5 vs dextran reaction time.
  • Figure 14 (a) Dissolution of dextran from Ac-DEX particles in either pH 5 or pH 7.4 buffer at 37 0 C. (b) Normalized 1 H-NMR data from the degradation of Ac-DEX particles at pH 5.5 and 37 0 C showing integrations of signals corresponding to acetone, methanol and acetal groups, (c) Time-lapse photos of Ac-DEX particles under physiological or acidic conditions. [036] Figure 15 shows a graph of the results of the B3Z assay measuring antigen presentation of RAW macrophages pulsed with free OVA or Ac-DEX particles encapsulating OVA.
  • FIG. 18 Stack plot of 1 H NMR spectra of empty Ac-DEX particles incubated in deuterated pH 5.5 buffer over time. Spectra are shown for the first eight days and are normalized with respect to the integration of the TMS peak.
  • FIG. 20 Cell viability of RAW macrophages was measured by MTT assay after overnight culture with (a) Ac-DEX particles or PLGA particles or (b) Ac-DEX particle degradation products.
  • FIG. 21 Particles made from Ac-DEXwere (a) modified at their reducing ends through oxime linkages using cell penetrating peptides (CPP) containing an aminoxy group .
  • CPP cell penetrating peptides
  • bioactive material refers to a composition having a physiological effect on a cell, such as a protein, antigen, polypeptide, polynucleotide, an enzyme or other organic molecule, for example, drugs or chemotherapeutics.
  • nucleotide refers to single- or multi-stranded deoxyribonucleotides (DNA), single- or multi-stranded ribonucleotides (RNA), or single-or multi-stranded peptide nucleic acids (PNA).
  • acetal herein refers to a geminal diether in which both ether oxygens are bound to the same carbon.
  • aryl herein refers to a homocyclic aromatic, whether or not fused, having 6 to 12 carbon atoms optionally substituted with one to three substituents, wherein said substituents are preferably N or O, or unsubstituted.
  • alkyl herein refers to an aliphatic linear or branched chain univalent groups of the general formula C n H 2n+I derived from aliphatic hydrocarbons such as methyl CH 3 , ethyl C 2 H 5 , propyl C 3 Hv 1 2-methyl propyl C 4 Hn, and the like or cyclic aliphatic univalent groups of the general formula C n H 2n -I derived from cyclic aliphatic hydrocarbons, such as cyclypropyl C3H5, cyclopentyl C5H9 and the like , where n is between 2 and 20.
  • loading refers to the amount of bioactive material that is encapsulated per milligram of the drug delivery systems. This may be expressed in terms of ⁇ g material/mg drug delivery system, on average, based on the starting bioactive material/polymers ratio.
  • loading efficiency refers to the percentage of the starting amount of bioactive material that is actually encapsulated.
  • ketal herein refers to an acetal in which the central carbon bound to two oxygen atoms is bound to two alkyl groups.
  • the present invention provides a modified polyhydroxylated polymer comprising a polyhydroxylated polymer having reversibly modified hydroxyl groups, wherein the hydroxyl groups are modified by a one-step reaction to feature a functional group selected from the group consisting of acetals, aromatic acetals, ketals.
  • the hydroxyl groups in the polyhydroxylated polymers are modified, thereby rendering the modified polyhydroxylated polymer acid degradable, pH sensitive and insoluble in water.
  • the present invention describes a system with the flexibility and biocompatibility of polyester materials, but with the additional benefit of a change in rate of hydrolysis or degredation that is sensitive to physiologically relevant acidic conditions.
  • a solubility switching mechanism is used in which a biocompatible, water-soluble (polyhydroxylated) polymer may be reversibly modified to make it insoluble in water, but soluble in organic solvents. Materials made from the modified polyhydroxylated polymer could then be degraded under the specific conditions that reverse the original modification.
  • hydroxyl groups displayed on the polyhydroxylated polymer backbone are modified to display a functional group having an acetal or ketal linkage therein.
  • This group is designed to remain largely stable in plasma at neutral physiological pH (about 7.4), but degrade intracellularly by hydrolysis in the more acidic environment of the endosome or lysosome (about pH 5.0-6.0).
  • the modified polyhydroxylated polymers exhibit hydrolysis and degradation, whereby the resulting degradation products are the polyhydroxylated polymer and the small molecule byproducts.
  • the modified polyhydroxylated polymers are processed to deliver a bioactive material.
  • polymer particles hydrolyze under acidic conditions and release the bioactive material in response to the mildly acidic conditions, found in the body such as in tumors, inflammatory tissues and in cellular compartments such as lysosomes and phagolysosomes of antigen presenting cells.
  • the bioactive material includes but is not limited to, antigens, proteins, polynucleotides, polypeptides, peptoids, small drug molecules and other bioactive material.
  • the polyhydroxylated polymers are preformed natural polymers or hydroxyl-containing polymers including but not limited to, multiply- hydroxylated polymers, polysaccharides, carbohydrates, polyols, polyvinyl alcohol, poly amino acids such as polyserine, and other polymers such as 2-(hydroxyethyl)methacrylate.
  • the polysaccharides that can be used include but are not limited to, dextran, mannan, pullulan, maltodextrin, starches, cellulose and cellulose derivatives, gums (e.g., xanthan, locust bean, etc.), and pectin.
  • the polysaccharides are dextran or mannan.
  • the modified polyhydroxylated polymers are prepared by a single one-step reaction.
  • the hydroxyl groups in the polyhydroxylated polymer are modified to feature a functional group selected from the group consisting of acetals, aromatic acetals, ketals, vinyl ethers, aldehydes and ketones.
  • the modification process involves an acid-catalyzed reaction between a polyhydroxylated polymer and functional molecules such as vinyl ethers, acetals, aldehydes, or ketones
  • the reversible modification of the hydroxyl groups should be carried out to provide modified hydroxyl groups, wherein at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the hydroxyl groups in the polymer are modified. In one embodiment, at least 20-85% of the hydroxyl groups are modified. In another embodiment, at least 75-85% of the hydroxyl groups are modified.
  • the choice of the polyhydroxylated polymer and the degree of modification also reflects such factors as ease of synthesis, solubility, commercially available reagents, the type of acid-degradable polymer desired, the loading efficiency, dispersion of drug delivery systems comprised of the polymers, toxicity and the hydrolysis rates of the acetal linkage.
  • the degradation products are biocompatible and biodegradable.
  • the degradation products are small molecules as well as polymers with a molecular mass of up to 10,000 daltons or lower, more preferably 1000 daltons, and most preferably 400 daltons or lower.
  • the degradation product(s) should be non-immunogenic and non-toxic, for example, with the size and/or toxicity levels preferred by one having skill in the art for approved in vivo use.
  • the modified polyhydroxylated polymers are modified polysaccharides with pendant acetals, thus providing acetal-derivatized polysaccharides.
  • the modified polyhydroxylated polymers are acetal-derivatized dextran, acetal-derivatized mannan or acetal-derivatized polyvinyl alcohols.
  • the modified polyhydroxylated polymers have reversibly modified hydroxyl groups, wherein the hydroxyl groups are modified to feature a functional group selected from the group consisting of acetals, aromatic acetals, ketals.
  • the functional group is an acetal, aromatic acetal or a ketal.
  • the modicification is made by a one-step reaction.
  • the present acid degradable polymers described herein should have a significantly lower rate of degradation in solution at pH 7.4 than at pH 5.
  • the modified polymers having a modified functional (e.g., acetal or ketal) linkage at the modified hydroxyl groups should degrade by acid catalyzed hydrolysis into lower molecular weight compounds that can be completely excretable.
  • the rate of hydrolysis of these polymers can be changed by varying the functional group (e.g., acetal or ketal) linkage from slow degrading to fast degrading, the degree of modification, or the hydrophobicity of the modification, thus providing a wide range of release kinetics for drug delivery.
  • a variety of acid degradable linkages with different acid-sensitivities can be incorporated onto the polymer backbones using this technology, allowing for excellent control of the rate of polymer hydrolysis.
  • Drug delivery systems comprised of the polymers can be hydro lyzed to release their contents in a pH dependent manner.
  • a feature of the present degradable polymers is the pendant modified hydroxyl groups on the main chain of the modified polyhydroxylated polymer hydrolyzes in a pH dependant manner.
  • the polymers should preferably have a degradation half-life at pH 5.0 of 5 minutes to 24 hours at 37°C, but a longer half life at pH 7.4 of at least 12 hours to 250 days.
  • the degradable polymers have degradation rates at pH 5.0 ranging from half-life of 5 minutes to over 26 hours .
  • the polymers may be useful for the polymers to have a half-life at pH
  • the half-life of polymer degradation at pH 5.0, 37°C preferably be 5-30 minutes, and even more preferably be less than 5 minutes and a half-life at pH 7.4, 37°C of about 24 hours in order to quickly release the bioactive materials.
  • the modified functional groups are acetals
  • the acceleration of the hydrolysis kinetics of acetals from pH 7.4 to pH 5.0 is expected because the hydrolysis of the acetal is proportional to the hydronium ion concentration, which should increase between pH 7.4 and pH 5.0.
  • the kinetics of acetal hydrolysis can be easily manipulated by introducing the appropriate electron withdrawing or donating groups and therefore it is possible to engineer degradable polymers that have hydrolysis rates tailor-made for a given application.
  • a kinetic factor that may be taken into account when designing acid degradable linkages on the modified polyhydroxylated polymer is the acid degradable linkage's speed of hydrolysis in solution.
  • the acetal should preferably hydrolyze within 5-30 minutes at pH 5.0 at 37°C.
  • this timescale is chosen because it is approximately the amount of time taken for a phagocytosed drug delivery system to be trafficked to cellular compartments such as lysosomes.
  • these particles will degrade rapidly in the lysosome and cause lysosomal destabilization. Having a particle that degrades too slowly will increase its residence time in the lysosome and provide the lysosomal enzymes an increased chance of hydro lyzing the bioactive material before reaching the cytoplasm through lysosomal disruption. Therefore, in a preferred embodiment, the polymer should hydrolyze fairly rapidly at a preferred range of pH 7.4 to 4.5 and even more preferably between pH 6.8 to 4.5.
  • the present modified polyhydroxylated polymers are largely stable at pH higher than 7.4 but hydrolyze at a pH preferably about 5.
  • the modified polymers are soluble in common organic solvents to facilitate processing into a variety of materials. In another embodiment, these modified polymers are not water soluble. 3. Methods for Polymer Modification [074] Generally, rate of degradation of modified polymers will depend on the degree of modification and the hydrophobicity of the modifying group. For example, in the case of dextran modified with 2-methoxypropene, the degradation rate of the modified polymer will depend on the amount of time that the material is allowed to react. [075] For acetal modification with vinyl ethers, an example of a method can be as follows.
  • the polyhydroxylated polymer is dissolved in an organic solvent such as DMSO and mixed with a vinyl ether and an acid-catalyst such as para-toluene sulfonic acid. Isolation occurs by precipitating the material in water.
  • acetal modification with acetals an example of a method can be as follows.
  • the polyhydroxylated polymer is mixed with an acetal and an acid-catalyst such as para- toluene sulfonic acid over molecular sieves. After reaction, the material is isolated by precipitation into water.
  • an example of a method can be as follows.
  • the polyhydroxylated polymer is mixed with an aldehyde or ketone and an acid- catalyst such as para-toluene sulfonic acid under conditions that remove water (such as azeotropic distillation or molecular sieves). After reaction, the material is isolated by precipitation into water.
  • an acid- catalyst such as para-toluene sulfonic acid under conditions that remove water (such as azeotropic distillation or molecular sieves).
  • the invention contemplates entrapping or conjugation of such bioactive materials including but not limited to, nucleotides, oligonucleotides, polynucleotides, ribonucleotides, amino acids, oligopeptides, polypeptides, peptoids, proteins, antigens, plasmid DNA, growth factors and hormones, interleukins, immunostimulatory agents, drugs, vaccines, neuromodulatory agents such as neurotransmitters, stimulatory and adrenergic agents, enzymes, proteases, anticancer and antitumor agents, imaging agents, diagnostic agents, antiviral agents and antibacterial agents as well as combinations of two or more of these species.
  • bioactive materials including but not limited to, nucleotides, oligonucleotides, polynucleotides, ribonucleotides, amino acids, oligopeptides, polypeptides, peptoids, proteins, antigens, plasmid DNA, growth factors and hormones
  • the bioactive material is selected from the group consisting of: nucleotides, oligonucleotides, polynucleotides, proteins, oligopeptides, polypeptides, immunostimulatory agents, vaccines, antigens, anti-viral agents, protein antigens, anticancer agents and antitumor agents.
  • bioactive materials can be conjugated to the polymer chains.
  • the bioactive materials can be conjugated to the polymer through the pendant hydroxyl groups.
  • materials can be conjugated to the polymer through aldehydes introduced by periodate cleavage of 1,2-diols.
  • the polyhydroxylated polymers are polysaccharides
  • latent aldehydes are present at the reducing ends and can be used for modification.
  • the linkage between the polymer chain and the bioactive molecule can be designed to be cleaved under various physiological conditions.
  • the bioactive material can also be adsorbed onto the surface of drug delivery systems, or reacted to the surface of the drug delivery systems.
  • the bioactive material can also be physically trapped inside the drug delivery systems comprised of the modified polyhydroxylated polymers.
  • the modified polyhydroxylated polymers are made into particles for such applications as vaccine delivery.
  • Typical formulations for therapeutic agents incorporated in these delivery systems are well known to those skilled in the art and include but are not limited to solid particle dispersions, encapsulated agent dispersions, and emulsions, suspensions, liposomes or microparticles, wherein said liposome or microparticle comprise a homogeneous or heterogeneous mixture of the therapeutic agent.
  • the amount of the drug that is present in the device, and that is required to achieve a therapeutic effect depends on many factors, such as the minimum necessary dosage of the particular drug, the condition to be treated, the chosen location of the inserted device, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the modified polyhydroxylated polymers made into particles that are 40 to 2000 nm.
  • particles can be synthesized by various techniques, such as double emulsion or spray drying methods, as is known in the art.
  • the particles can be made according to the procedures described by Liu, R.; Ma, G.; Meng, F.; Su, Z. J. Controlled Release 2005, 103, 31-43 and Witschi, C; Mrsny, J. R. Pharm. Res. 1999, 16, 382-390.
  • the particles are made by double emulsion, single emulsion, or precipitation processes.
  • Single emulsion and the double emulsion method and precipitation processes can be used to produce particles from sub-micrometer to multi-micrometer sizes; a preferable size range is from 30 nm to 5000 nm, more preferably 30 nm to 2000 nm, and most preferably 40 to 200 nm.
  • the polymer is dissolved in organic solvent along with the surfactants. Then, a small amount of aqueous solution containing the bioactive materials is dispersed into the organic/polymer phase by sonication forming a primary water-in-oil emulsion. This primary emulsion is then dispersed into a larger amount of water containing stabilizers to form a secondary water-in-oil-in-water emulsion. After forming the secondary emulsion, the solution is stirred until the organic phase evaporates. When evaporated, the polymer collapses around the aqueous bioactive material solution forming therapeutic-loaded particles.
  • the acid-degradable polymers are processed to form particles comprised of the modified polyhydroxylated polymers having a bioactive material bound to or entrapped within the formed particles.
  • the modified polyhydroxylated polymers are made into drug delivery systems such as a small molecule implant, or time-release device or implant.
  • drug delivery systems such as a small molecule implant, or time-release device or implant.
  • Methods and compositions useful in making or administering an implant or time-release device in vivo are known and used by one having skill in the art. Examples of such methods and compositions are described in U.S. Patent Nos. 3,976,071; 5,876,452; 7,077,859; 5 ,021 ,241 , hereby incorporated by reference.
  • the modified polyhydroxylated polymers of the invention can be prepared in solid form of a needle or bar-like shape or as a bulk shaped material and administered to the body or implanted into the body by injection or an injection- like method and whereby the bioactive material is released at an effective level for a long period of time after administration.
  • Loading efficiency is the amount of bioactive material that is entrapped in or conjugated to within the drug delivery systems comprised of the polymers as compared to the total starting amount of bioactive material placed in the loading reaction.
  • the loading is the amount of bioactive material contained in the polymer particle, it is generally expressed in mass of bioactive material per unit mass of particle.
  • the loading efficiency and the amount of bioactive material entrapped are important aspects in light of such factors as the amount of bioactive material needed to be delivered to the target for an effective dose and the amount of available bioactive material.
  • a major drawback in previous therapeutics and vaccines is there is often difficulty in obtaining large enough amounts of the therapeutic composition of bioactive material for production.
  • the degradable polymer particles should exhibit preferred loading as is known in the art.
  • the polymer particles should exhibit high loading efficiency to allow sufficient drug molecules to be conjugated to the polymer or otherwise retained by the polymer without loss of solubility of the overall formulation.
  • the loadings and efficiencies of the drug delivery systems should be comparable to other microparticle systems which have efficiencies purported to be about 1-2 ⁇ g DNA/mg polymer for 500 nm PLGA particles.
  • the loadings and efficiencies of the drug delivery systems should be comparable to other microparticle systems which have efficiencies purported to be about 1-2 ⁇ g DNA/mg polymer for 500 nm PLGA particles.
  • the loading efficiencies for the amount of DNA material entrapped in degradable particles of the preferred embodiment should preferably be at least 40%, more preferably at least 50% and even more preferably at least 54%. Loadings for bacterial DNA for immunostimulation purposes should be around 1-30 ⁇ g DNA/mg. [092] In a preferred embodiment, wherein the bioactive material loaded is protein, the loading efficiencies for the amount of protein entrapped in particles comprised of the acid degradable polymers of the preferred embodiment should be at least 20%, preferably at least 40%, more preferably around 50%, and most preferably >90%.
  • the target cells should preferably exhibit at least 50% viability after 24 hours of incubation with the polymers of the invention, more preferably at least 70% viability after 24 hours, even more preferably at least 80% viability and most preferably more than 90% viability after 24 hours according to the MTT assay.
  • Polymers with high MW are not easily excreted from the body, therefore another aspect of the invention is to make polymers that are easily and safely excreted by the body after being degraded in the acidic environments. In general it is preferred that the polymers degrade into many small molecules and/or molecules that are non toxic and readily excreted from the body.
  • the degradation products of the present modified polyhydroxylated polymers of the invention should be easily excreted from body due to the small molecule size of the degradation products produced after hydrolysis of the pendant modified groups and the use of a main chain polyhydroxylated polymer that is biocompatible (e.g., a polysaccharide such as dextran).
  • a main chain polyhydroxylated polymer that is biocompatible e.g., a polysaccharide such as dextran.
  • Another aspect of the invention is to make particles that are easily and safely excreted by the body after being degraded in the acidic cellular compartment. In general it is preferred that the particles degrade into degradation products that are linear polymers and/or smaller molecules (e.g., 10,000 daltons or less), and that the degradation products are not toxic to a mammalian subject.
  • This strategy for the synthesis of modified polyhydroxylated polymers has many applications including the delivery of bioactive materials, including but not limited to polynucleotides, polypeptides, proteins, peptides, organic molecules, antibodies, vaccines, antigens, genetic agents, small drugs or therapeutic agents, into the cytoplasm of phagocytic cells, site of inflammation, tumor tissues, endosomes, or other sites of low pH. Thse materials can also be fashioned into bulk materials such as sutures, scaffolds, and implants.
  • the polymers of the present invention would have applications in vaccine therapeutics and disease prevention. Protein loaded particles prepared using these polymers could be injected into a patient, stimulating phagocytosis by macrophages and antigen presenting cells.
  • the acid-degradable modified polyhydroxylated polymer particles are delivered to antigen presenting cells and then phagocytosed and trafficked to the lysosome or phagolysosome of the cells.
  • the mild acidic conditions found in lysosomes and phagolysosomes of APCs should cause the pendant acetal groups along the polymer backbones to be hydro lysed thereby degrading the particles. This acid hydrolysis of the acid- degradable linkage causes degradation of the polymers.
  • the particles comprised of the acid degradable polymers of the invention would be particularly useful in combating infections that need a strong cytotoxic T lymphocyte response, including diseases such as HIV/ AIDS and Hepatitis C infections.
  • antigens which can be used as bioactive material and entrapped in the particles of the present invention, include but are definitely not limited to, the TAT protein from HIV, the ENV protein from HIV, the Hepatitis C Core Protein from the Hepatitis C virus, the prostatic acid phosphatase for prostate cancer and the protein MART-I for melanoma.
  • the modified polyhydroxylated polymers particles enhance CTL activation by dendritic cell (DC)-targeting.
  • OVA is encapsulated in acid-degradable polymeric particles further conjugated with anti-DEC-205 mAbs monoclonal antibody.
  • the particles are taken up by DEC-205 expressing dendritic cells in vivo. After hydrolysis in the acidic lysosome of DCs, encapsulated OVA is released into the cytoplasm.
  • signal peptides are attached to the particle. Any suitable signal peptide can be used in the particles of the invention.
  • the peptide should be able to target (i.e., mediate entry and accumulation) a particle to a subcellular compartment and/or organelle of interest.
  • Signal peptides are typically about about 5 to about 200 amino acids in length.
  • Suitable signal peptides include, e.g., nuclear localization signal peptides, peroxisome-targeting signal peptides, cell membrane-targeting signal peptides, mitochondrial-targeting signal peptides, and endoplasmic reticulum-targeting signal peptides, and trans-Golgi body-targeting signal peptides.
  • Signal peptides may also target the particles to any cell surface receptor including e.g.
  • Nuclear localization signal peptides typically comprise positively charged amino acids.
  • Endoplasmic reticulum targeting signal peptides typically comprise about 5 to about 10 hydrophobic amino acids.
  • Mitochondria targeting signal peptides are typically about 5 to about 10 amino acids in length and comprise a combination of hydrophobic amino acids and postively charged amino acids.
  • Peroxisome targeting signal peptides include PTSl, a 3 amino acid peptide and PTS2, a 26-36 amino acid peptide. Examples of signal peptide sequences include but are not limited to the following sequences in Table 1. Table 1.
  • Signal peptides can be chemically synthesized or recombinantly produced.
  • the nucleic acid sequences encoding signal peptides and related nucleic acid sequence homologues are cloned from cDNA and genomic DNA libraries or isolated using amplification techniques with oligonucleotide primers. Standard techniques are used for nucleic acid and peptide synthesis, cloning, DNA and RNA isolation, amplification and purification. Basic texts disclosing the general methods of use in this invention include Sambrook et ah, Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Rriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).
  • the particles are decorated with a targeting functional group or other cell penetrating peptides to penetrate non-phagocytic cells.
  • targeting functional groups include antibodies, various oligopeptides, or carbohydrate moieties
  • Cell-penetrating peptides can also include oligopeptides such as oligomers of arginine or polymers rich in arginine motifs.
  • immunostimulatory groups are attached to, displayed on, or encapsulated in the particle.
  • immunostimulatory groups include but are not limited to mannose, plasmid DNA, oligonucleotides, ligands for the Toll-like receptors, interleukins and chemokines.
  • T-cells activate B-cells to secrete Interleukin-6 (IL-6) to stimulate B cells into antibody-secreting cells.
  • IL-6 Interleukin-6
  • targeting antibodies are attached to the particle. Any antibody specific for a target in vivo can be attached to the particle to target and allow particle delivery of the bioactive material.
  • the acid-degradable polymers particles enhance CTL activation by dendritic cell (DC)-targeting as described above.
  • DC dendritic cell
  • the polymers of the invention would be used to prepare drug delivery systems for gene therapeutics.
  • Cationic polymers would be especially relevant for this application because polycations can complex with DNA. Since gene therapy involves the delivery of a sequence of DNA to the nucleus of a cell, the particles comprised of these polymers of the invention would be especially suited for this application.
  • the polynucleotide Once a polynucleotide is delivered by the drug delivery systems to the cytoplasm, the polynucleotide can undergo translation into a protein. This has the potential, then, to make proteins that are not normally produced by a cell.
  • the bioactive material is a plasmid that encodes for a protein or antigenic peptide initially.
  • a plasmid that encodes for a protein that would display antigens for cancer are not easy to generate in multi-milligram to gram quantities to be delivered to a patient, therefore using the present particle delivery systems prepared with polymers of the present invention to deliver plasmid DNA encoding these antigens is a preferred alternative.
  • plasmid DNA has the added characteristic of generating an immune response because plasmid DNA is generated from bacteria.
  • Other potential bioactive materials are CpG oligonucleotides that are also derived from bacterial DNA.
  • Bacterial DNA has two major differences compared with vertebrate DNA: 1) bacterial DNA has a higher frequency of CG dinucleotides in the sequence (1/16 dinucleotides in microbial DNA are CG pairs, but only 25% of that is observed in vertebrate DNA); and 2) bacterial DNA is unmethylated as compared to vertebrate DNA which is often methylated. Vertebrate systems will recognize the DNA then as being foreign, and the cell should react as for a bacterial infection. This immune response is manifested in the production of cytokines and interleukins that then go on to activate T cells, B cells, and other cells, proteins, and cellular machinery involved in the immune response.
  • the plasmid DNA used as the bioactive material would have an added interleukin sequence.
  • Interleukins are secreted peptides or proteins that mediate local interactions between white blood cells during immune response (B. Alberts et al, Molecular Biology of the Cell, 4th ed., Garland Science, 2002). Different interleukins (e.g. IL-12, IL-2) will direct the type of immune response that is generated.
  • IL-6, IL-I, IL-8, IL-12, and TNF- ⁇ are secreted by infected macrophages as an immune response and IL-6 serves to activate lymphocytes and increase antibody production.
  • the differentiation of helper T cells into either T H 1 or T H 2 efffector cells determines the nature of the response.
  • a T H 1 response is characterized by a CTL response; a T R 2 response is characterized by antibody production.
  • interleukin-2 or 12 IL-2 or IL- 12
  • IL-2 or IL- 12 interleukin-2 or 12
  • T-cells activate B-cells to secrete Interleukin-6 (IL-6) to stimulate B cells into antibody-secreting cells.
  • bioactive drug molecules may be temporarily attenuated by incorporation into modified polyhydroxylated polymers for applications such as chemotherapy.
  • Drug molecules may be incorporated into the polymers covalently, where the drug molecules are attached to the main polyhydroxylated polymer chain via labile linkages.
  • Water soluble polymer-drug conjugates will preferably be administered intravenously or orally, and biologically active drug molecules will be released from the polymer upon cleavage of the labile polymer-drug linkages.
  • Drug molecules may also be incorporated noncovalently by entrapment of drugs into particles or implant devices fashioned from water insoluble variants of the acid-degradable polymers.
  • Water insoluble polymers will preferably be administered orally or will be implanted in the body, and drug molecules will be released from the polymer upon degradation of the polymer matrix in which the drug is entrapped or conjugated.
  • Drug delivery systems comprised of the invention may be suspended or stored in a conventional nontoxic vehicle, which may be solid or liquid, water, saline, or other means which is suitable for maintaining pH, encapsulation of the bioactive material for an extended period of time, sufficient dispersion or dilution of the delivery systems and the overall viability of the delivery systems for their intended use.
  • a conventional nontoxic vehicle which may be solid or liquid, water, saline, or other means which is suitable for maintaining pH, encapsulation of the bioactive material for an extended period of time, sufficient dispersion or dilution of the delivery systems and the overall viability of the delivery systems for their intended use.
  • the delivery systems comprised of the polymers of the invention are stored in dry state (vacumm dried) and stored at 4°C for several months.
  • the systems may be dispersed in buffer and sonicated or vortexed for a few minutes to resuspend into solution when needed.
  • the polymers of the invention can be used to prepare delivery systems for RNA interference (RNAi) agents such as small interfering RNA (siRNA), long double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • dsRNA long double-stranded RNA
  • shRNA short hairpin RNA
  • siRNA in the form of double stranded RNA molecules less than 40 nucleotides in length can be encapsulated in polymers of the invention.
  • Encapsulation efficiency can be improved using cationic lipids such as DOTAP (N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate) or cationic polymers such as PEI (polyethyleneimine) or poly- ⁇ -aminoesters.
  • DOTAP N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate
  • PEI polyethyleneimine
  • Poly- ⁇ -aminoesters cationic lipids
  • Materials delivering siRNA have the potential to interfere with cellular protein production. This may be therapeutically relevant for treating many different genetic or pathogenic diseases as well as cancer.
  • the loaded drug delivery systems of the invention can be administered by various suitable means to a patient, including but not limited to parenterally, by intramuscular, intravenous, intraperitoneal, or subcutaneous injection, or by inhalation.
  • the delivery of the systems to a patient is preferably administered by injection once but does not preclude the necessity for multiple injections that would be required to illicit the desired response.
  • the delivery system is an implant system, wherein the polymer is implanted into an affected tissue, such as a tumor, and allowed to degrade and release the bioactive material.
  • an affected tissue such as a tumor
  • bioactive material such as a tumor
  • water insoluble degradable polymers are implanted in the body, and drug molecules will be released from the polymer upon degradation of the polymer matrix in which the drug is entrapped.
  • the amount of delivery vehicle needed to deliver a pharmaceutically effective dosage of the bioactive material will vary based on such factors including but not limited to, the polymer solubility, the therapeutic loading capacity and efficiency, the toxicity levels of the polymers, the amount and type of bioactive material needed to effect the desired response, the subject's species, age, weight, and condition, the disease and its severity, the mode of administration, and the like.
  • the amount of bioactive material that could be administered by the delivery systems of the invention is from 1 ng to more than 1 g quantities.
  • Dextran was rendered insoluble in water by modification of its hydroxyl groups through reaction with 2-methoxypropene under acid catalysis ( Figure 4, 6).
  • the high density of pendant acetals makes the new "acetalated-dextran" (Ac-DEX) soluble in organic solvents such as dichloromethane, ethyl acetate or acetone.
  • organic solvents such as dichloromethane, ethyl acetate or acetone.
  • the molecular weight of the dextran increases upon modification from 13 kDa to 29 kDa while the polydispersity remains essentially constant (1.13 to 1.20), suggesting coverage of the hydroxyl groups and minimal polymer cross-linking.
  • a model hydrophilic payload, ovalbumin (OVA) was encapsulated with a protein loading of 3.7 ⁇ 0.4 wt % ( Figure 6).
  • OVA ovalbumin
  • Figure 6 Using a single emulsion technique, we were able to encapsulate a model hydrophobic drug, pyrene, with a loading of 3.6 ⁇ 0.5 wt %.
  • the particles were imaged using scanning electron microscopy (Figure 12A) and particle size was analyzed using dynamic light scattering.
  • the double emulsion particles were found to have an average diameter of 230 ⁇ 13 nm ( Figure 16) and the single emulsion particles had similar shapes and sizes with an average diameter of 258 ⁇ 1 nm.
  • Multiangle light scattering (MALS) experiments were performed with a Waters 510 pump, a 7125 Rheodyne injector, a Wyatt Optilab differential refractive index detector and a Wyatt DAWN-EOS MALS detector. Absolute molecular weights determined from light scattering data were calculated using Astra software from Wyatt assuming a quantitative mass recovery (online method). Columns were thermostatted at 35°C. MALS experiments run with THF as a solvent were performed using two 7.5 x 300 mm PLgel mixed-bed C columns with a 5 micron particle size.
  • MALS experiments run in aqueous conditions were performed using dd-H 2 O with 5% acetic acid as a solvent and Viscotek C-MBMMW-3078 and C-MBHMW-3078 cationic columns (7.8 mm x 300 mm) in series. Fluorescence measurements were obtained on a Fluorolog FL3-22 spectra fluorometer (Horiba Jobin Yvon) or a Spectra Max Gemini XS (Molecular Devices, USA) for microplate -based assays. Fourier transform infrared spectroscopy (FT-IR) was carried out on a 3100 FT-IR spectrometer (Varian, USA).
  • FT-IR Fourier transform infrared spectroscopy
  • UV-Vis spectroscopic measurements were obtained from samples in quartz cuvettes using a Lambda 35 spectrophotometer (Perkin Elmer, USA) or using a Spectra Max 190 (Molecular Devices, USA) for microplate-based assays.
  • RAW 309 and HeLa cells were obtained from ATCC (Manassas, VA) and grown according to ATCCs directions.
  • Acetalated Dextran (Dimethyl Acetal Dextran: Ac-DEX).
  • Anhydrous DMSO (10 mL) was added and the resulting mixture was stirred until complete dissolution of the dextran was observed.
  • Pyridinium/?-toluenesulfonate (15.6 mg, 0.062 mmol) was added followed by 2-methoxypropene (3.4 mL, 37 mmol).
  • the flask was placed under a positive pressure of N 2 , then sealed to prevent evaporation of 2- methoxypropene.
  • Anhydrous DMSO (3 mL) was added and the resulting mixture was stirred until complete dissolution of the dextran was observed.
  • Dihydropyran (3.4 mL, 37 mmol) was added followed by pyridinium/?-toluenesulfonate (4.7 mg, 0.019 mmol). After stirring overnight the modified dextran was precipitated in dd-H 2 O (100 mL, pH 8).
  • Anhydrous DMSO (4 rnL) was added and the resulting mixture was stirred until complete dissolution of the dextran was observed.
  • Benzaldehyde dimethyl acetal (0.35 mL, 2.3 mmol) was added followed by 5 A molecular sieves (5 g) and /?-toluenesulfonic acid monohydrate (15 mg, 0.08 mmol).
  • the resulting double emulsion was immediately poured into a second PVA solution (10 ml, 0.3% w/w in PBS) and stirred for 3 h allowing the organic solvent to evaporate.
  • the particles were isolated by centrifugation (14 800 x g, 15 min) and washed with PBS (50 mL) and dd-H 2 O (2 x 50 mL, pH 8) by vortexing and sonication followed by centrifugation and removal of the supernatant.
  • the washed particles were resuspended in dd-H 2 O (2 mL, pH 8) and lyophilized to yield a white fluffy solid (135 mg).
  • Particles that did not contain protein were made in the same manner as above omitting OVA.
  • Preparation of Empty PLGA Particles Particles prepared from poly(DL-lactide- co-glycolide) (PLGA, 85% lactide, 15% glycolide) were made in the same manner as above substituting PLGA for Ac-DEX.
  • Single emulsion particles encapsulating pyrene were prepared according to a procedure adapted from Jung et al. (Rosen, H. B.; Chang, J.; Wnek, G. E.; Linhardt, R. J.; Langer, R., Biomaterials 1983, 4, 131-3). Briefly, Ac-DEX (49.9 mg) and pyrene (5.5 mg) were dissolved in CH 2 Cl 2 (1 mL). This solution was added to a PVA solution (3 mL, 1% w/w in PBS) and emulsified by sonicating for 30 s on ice using a probe sonicator (Branson Sonif ⁇ er 450) with an output setting of 5 and a duty cycle of 70%.
  • a probe sonicator Branson Sonif ⁇ er 450
  • the resulting emulsion was poured into a second PVA solution (50 ml, 0.3% w/w in PBS) and stirred for 4 h allowing the organic solvent to evaporate.
  • the single emulsion particles were isolated in the same manner as described for the double emulsion particles above.
  • the washed particles were resuspended in dd-H2O (2 mL, pH 8) and lyophilized to yield a white fluffy solid (38 mg).
  • Particle Size Analysis by Dynamic Light Scattering Particle size distributions and average particle diameters were determined by dynamic light scattering using a Nano ZS (Malvern Instruments, United Kingdom). Particles were suspended in dd-F ⁇ O (pH 8) at a concentration of 1 mg/mL and three measurements were taken of the resulting dispersions. Size distribution histograms are presented in Figure 16.
  • Particle Degradation Detection of Soluble Polysaccharides via BCA assay.
  • the collected supernatant samples were analyzed for the presence of reducing polysaccharides using a microplate reductometric bicinchoninic acid based assay according to the manufacturer's protocol (Micro BCA Protein Assay Kit, Pierce, USA; Figure 2a).(Gvili, K.; Benny, O.; Danino, D.; Machluf, M., Biopotymers 2007, 85, 379- 91).
  • Particle Degradation Digital Photography. Empty Ac-DEX particles were suspended at a concentration of 2 mg/mL in either a 0.3 M acetate buffer (pH 5.0) or PBS (pH 7.4) and incubated at 37 0 C under gentle stirring. Digital photographs of the samples were obtained after various time points. The white object visible in some of the vials is a magnetic stir bar.
  • particles prepared from these acid degradable acetal- derivatized dextran polymers are designed to release their bioactive material payload into the cytoplasm of cells upon lysosomal destabilization. Higher loading capacity of the particles may also lead to greater antigen presentation of the encapsulated bioactive material.
  • the LacZ MHC Class I antigen presentation assay as described by Sanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369-376, is performed with degradable polymer particles made according to Example 3 with the modified hydroxylated polymers of Example 2 to test their ability to deliver proteins into APCs for Class I antigen presentation.
  • This experiment uses the LacZ B3Z hybridoma, which is a CTL that recognizes the peptide sequence, SIINFEKL (SEQ ID NO: 8), from ovalbumin, complexed with the MHC Class I molecule H- 2K b .
  • This hybridoma produces ⁇ -galactosidase after encountering APCs that present SIINFEKL as a Class I antigen, thus allowing Class I antigen presentation to be quantified by measuring ⁇ -galactosidase activity.
  • a proper control would be to compare the amount presented by the particles when incubated with the SIINFEKL peptide (SEQ ID NO: 8), which is directly displayed on the antigen presenting cells and not delivered to the cytoplasm of the cells first.
  • the bioactive loading capacity and efficiency should lead to an absorbance of that is equal to the saturation absorbance of the SIINFEKL peptide (SEQ ID NO: 8), control using the antigen presentation assay described by Sanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369-376.
  • Ovalbumin encapsulated in the degradable particles was orders of magnitude more efficient than free ovalbumin at inducing the activation of CTLsThus the acid degradable particles werecapable of delivering protein antigens into APCs for Class I antigen presentation (Figure 15).
  • Control Release 2005, 106, 172-80 were maintained in RPMI 1640 (Invitrogen, USA) supplemented with 10% fetal bovine serum, 2 mM Glutamax, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin.
  • 1x10 4 RAW macrophages were seeded overnight in a 96 well plate and subsequently incubated with OVA-containing Ac-DEX particles or free OVA. After 6 h, the cells were washed and 1x10 5 B3Z cells were added to the macrophages and cocultured for an additional 16 h.
  • OVA ovalbumin
  • This mixture was then emulsified by sonicating for 30 s on ice using a probe sonicator (Branson Sonifier 450) with an output setting of 3 and a duty cycle of 10%.
  • PVA, M w 13000 - 23000 g/mol, 87-89% hydrolyzed
  • the resulting double emulsion was immediately poured into a second PVA solution (10 ml, 0.3% w/w in PBS) and stirred for 3 h allowing the organic solvent to evaporate.
  • the particles were isolated by centrifugation (14800 x g, 15 min) and washed with PBS (50 mL) and dd-H 2 O (2 x 50 mL, pH 8) by vortexing and sonication followed by centrifugation and removal of the supernatant. The washed particles were resuspended in dd- H 2 O (2 mL, pH 8) and lyophilized to yield a white fluffy solid (135 mg).
  • Double emulsion particles containing siRNA were prepared as follows: siRNA (40 nmol) was dissolved in IDTE (pH 7.5, 50 ⁇ L). Ac-Dex (50 mg) was dissolved in CH 2 Cl 2 (0.6 mL). DOTAP (400 ⁇ L of a 25 mg/mL solution in chloroform) was added to the Ac- DEX solution (20% DOTAP). The DOTAP/Ac-DEX solution was added to the siRNA solution. This mixture was then emulsified by sonicating for 30 s on ice using a probe sonicator (Branson Sonifier 450) with an output setting of 5 and a duty cycle of 80%.
  • IDTE pH 7.5, 50 ⁇ L
  • Ac-Dex 50 mg
  • CH 2 Cl 2 0.6 mL
  • DOTAP 400 ⁇ L of a 25 mg/mL solution in chloroform
  • the DOTAP/Ac-DEX solution was added to the siRNA solution. This mixture was then emulsified
  • PVA poly( vinyl alcohol)
  • M w 13000 - 23000 g/mol, 87-89% hydrolyzed
  • the particles were isolated by centrifugation (14800 x g, 15 min) and washed with PBS (50 mL) and dd-H 2 O (2 x 50 mL, pH 8) by vortexing and sonication followed by centrifugation and removal of the supernatant. The washed particles were resuspended in dd-H 2 O (2 mL, pH 8) and lyophilized to yield a white fluffy solid.
  • Microparticles containing both the hydrophilic macromolecule OVA and the hydrophobic small molecule imiquimod were prepared in the same fashion as particles containing only OVA except that the CH 2 Cl 2 was replaced with CHCI 3 containing 2 mg/mL imiquimod.
  • FITC fluorescein isothiocyanate
  • Plasmid- loaded Microparticles were made using a modified double emulsion water/oil/water evaporation method.
  • Ac-DEX 40 mg
  • a poly( ⁇ -aminoester) polymer (10 mg) were dissolved in ice-cold CH 2 Cl 2 (1 mL).
  • Plasmid DNA encoding firefly luciferase reporter protein, gWIZ Luciferase was purchased from Aldevron/Genlantis (USA) and was dissolved in TE buffer (10 rnM Tris, 1 mM EDTA, pH 8.5) at a concentration of 5 mg/mL.
  • the plasmid solution (100 ⁇ L) was added to the polymer solution and the mixture was emulsified by sonicating for 5 s using a Branson Sonifier 450 sonicator with a microtip probe, an output setting of 1, and a continuous duty cycle.
  • This primary emulsion was added to an ice-cold aqueous solution of PVA (20 mL, 3% w/w in PBS) and homogenized for 30 s at 10,000 rpm using an IKA T-25 Ultra-Turrax digital homogenizer with an S25N-10G generator.
  • the resulting double emulsion was immediately poured into a second PVA solution (40 mL, 0.3% w/w in PBS) and stirred for 2 h allowing the organic solvent to evaporate.
  • the particles were isolated by centrifugation (3,000 x g, 5 min) and washed with PBS (1 x 20 mL) and dd-H 2 O (2 x 20 mL, pH 8). The washed particles were resuspended in dd-H 2 O (2 mL, pH 8) and lyophilized to yield a white fluffy solid (34 mg). (2 mL, pH 8) and lyophilized to yield a white fluffy solid (135 mg). 6)
  • Particles made from acetalated mannan were prepared as described above substituting acetalated mannan for acetalated dextran and omitting the OVA solution and the first emulsion step (see Figure 10).
  • Single emulsion particles encapsulating pyrene were prepared according to a procedure adapted from Jung et al. (Jung, J.; Lee, I. H.; Lee, E.; Park, J.; Jon, S., Biomacromolecules 2007, 8, 3401-3407). Briefly, Ac-Dex (49.9 mg) and pyrene (5.5 mg) were dissolved in CH 2 Cl 2 (1 mL). This solution was added to a PVA solution (3 mL, 1% w/w in PBS) and emulsified by sonicating for 30 s on ice using a probe sonicator (Branson Sonifier 450) with an output setting of 5 and a duty cycle of 70%.
  • a probe sonicator Branson Sonifier 450
  • the resulting emulsion was poured into a second PVA solution (50 mL, 0.3% w/w in PBS) and stirred for 4 h allowing the organic solvent to evaporate.
  • the single emulsion particles were isolated in the same manner as described for the double emulsion particles above.
  • Single emulsion particles encapsulating imiquimod were prepared according to the same procedure used to encapsulate pyrene, but CHCI3 was used in place Of CH 2 Cl 2 and imiquimod was used in place of pyrene.
  • the single emulsion particles were isolated in the same manner as described for the double emulsion particles above.
  • Single emulsion particles encapsulating doxorubicin were prepared according to a procedure adapted from Tewes et al. (Tewes, F.; Munnier, E.; Antoon, B.; Ngaboni Okassa, L.; Cohen- Jonathan, S.; Marchais, H.; Douziech-Eyrolles, L.; Souce, M.; Dubois, P.; Chourpa, L, Eur J Pharm Biopharm 2007, 66, 488-92). Briefly, Dox (1 mg) was dissolved in a sodium borate buffer (2ml, 50 mM, pH 8.8) and mixed extensively overnight with CH 2 Cl 2 .
  • the CH 2 Cl 2 was then isolated and evaporated to a final volume of 1 mL.
  • a solution of Ac-Dex (100 mg) in CH 2 Cl 2 (1 mL) was added to the Dox.
  • the resulting solution was added to a PVA solution (4 mL, 3% w/w in PBS) and emulsified by sonicating for 60 cycles on ice using a probe sonicator (Branson Sonifier 450) with an output setting of 4 and a duty cycle of 10%.
  • the resulting emulsion was poured into a second PVA solution (10 mL, 0.3% w/w in PBS) and stirred for 4 h allowing the organic solvent to evaporate.
  • the resulting particles were isolated in the same manner as described for the double emulsion particles above.
  • Ac-DEX particles can encapsulate organic molecules using noprecipitation. Camptothecin (2.4 mg) was dissolved in hot DMF (400 ⁇ L) then diluted with acetone (600 ⁇ L) containing Ac-DEX (100 mg). This solution was added dropwise to H 2 O (10 mL, pH 8). Particles were isolated by concentration using Centricon spin filters. Dry state storage could be obtained by lyophilization in the presence of 10 % sucrose as a cryoprotectant.
  • the toxicity of bioactive material loaded degradable particles can be measured with the yellow tetrazolium salt, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), assay using RAW 309.CR1 macrophage cells (ATCC No. TIB-69, American Type Culture Collection, Manassas, VA).
  • the cells are incubated with the degradable particles in DMEM media with 10% F. B. S.
  • the degradable particles are aspirated from the cells and then washed several times with PBS and allowed to grow for 24- 48 hours.
  • the cell viability is determined by measuring the absorbance of the reduced MTT reagent using the protocol described in Freshney et al.
  • MTT yellow
  • the measurement of the ability of cells to reduce the MTT reagent metabolically is a measurement of the health of the cell population.
  • the cell viability is determined by measuring the absorbance of the reduced MTT reagent.
  • the MTT assay is performed using 0.5, 1, 2.5 and 5 mg particles/mL serum in each well with a particle loading of 10 micrograms protein/mg particle. After 24 hours, number of viable cells remaining is observed. It is preferred that at least 50%, more preferably 80-90% viable cells remain. Thus, it can be found whether the degradable particles of the invention are not toxic to mammalian cells if more than 50% of the cells remain viable.
  • degradation products of empty Ac-DEX particles were tested using RAW macrophages ( Figure 20b). Additionally, empty Ac-DEX particles and empty PLGA particles were incubated with RAW macrophages ( Figure 20a).
  • the degradation products were obtained by incubating Ac-DEX particles in a 0.3 M acetate buffer (pH 5.0) at 37 0 C under gentle agitation for 3 d.
  • the resulting solution was desalted using a Microcon 3 centrifugation filter (Millipore, USA) and lyophilized. During the desalting and lyophilization steps the methanol and acetone released during degradation was removed. Before use in the viability experiment, the lyophilized degradation products were dissolved in medium, and methanol and acetone were added corresponding to the maximum amount of these byproducts released, as found in the 1 H-NMR degradation study described above.
  • Degradable particles are made according to Example 4 with the degradable polymers in Example 1 , encapsulating fluorescently labeled dextran and fed to macrophage cells and compared to non-degradable control particles.
  • a nondegradable particle When a nondegradable particle is used, the fluorescence is more localized showing that when nondegradable particles have been taken up by the cells, they remain sequestered in the lysosome without a mechanism of release.
  • the acid degradable particles are used, the fluorescence is more diffuse within the cytoplasm of cells, which is indicative of cytoplasmic release of the degradable particle contents.
  • EG7 is a derivative of the thymoma EL4, which was transfected with the ovalbumin gene, making it a target cell for CTLs activated against ovalbumin.
  • the modified polyhydroxylated polymer chosen for in vivo study should demonstrate good dispersibility which may be an important consideration for the study if the particles must be suspended in saline and injected into animals. Certain modified polyhydroxylated polymers may be somewhat more difficult to suspend, most likely due to a degree of particle aggregation as a result of higher ovalbumin content.
  • the modified polyhydroxylated particles are injected into the foot pad of CD4 or CD8 transgenic mice to show that the particles can activate cytotoxic T lymphocytes in vivo. More preferably, delivery is by injection of 50 ⁇ l of resuspended particle using a 25 gauge syringe in the flanks of these transgenic mice. At least 50 ⁇ g of O V A/mouse should suffice per injection with at least 3 mice per group injected. Also 150 ⁇ g of particles with OVA and a similar amount of particles used for control are injected. The lymph nodes are isolated 7 days after the injection and analyzed for antigen specific T cell priming.
  • mice are performed with female C57BL/6 mice and all immunizations are administered by subcutaneous injections using 26 gauge needles. There are three groups (15 mice per group): control mice injected with saline (200 ⁇ L); mice injected with free ovalbumin in saline (50 ⁇ g in 200 ⁇ L); and mice injected with ovalbumin encapsulated in particles dispersed in saline (1.13 mg particles, corresponds to 50 ⁇ g ovalbumin, in 200 ⁇ L). A second identical immunization is delivered 2 weeks after the first.
  • tumors are established by administering an injection of 1 x 10 6 EG7-OVA tumor cells in 100 ⁇ L saline into the shaved left flank of each mouse.
  • the EG7 cells are stained with the anti- SIINFEKL/K b monoclonal antibody 25.D1-16 and the secondary goat anti-mouse antibody labeled with R-phycoethrin (PE).
  • PE R-phycoethrin
  • highly ovalbumin-expressing cells are collected using flourescence-activated cell sorting (FACS) and proliferated.
  • FACS flourescence-activated cell sorting
  • Ac-DEX particles will encapsulate OVA similar to (Example 9 with B3Z assay) and be used in a tumor prophylactic and treatment vaccination against cancer.
  • MO5 is a B16 tumor cell line that expresses OVA (FaIo et al. 1995, Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity. Nat Med 1 :649-53).
  • Mice are immunized subcutaneously with 2 mg of Ac-DEX particles with a 2.5 wt% loading of OVA (50 ⁇ g of OVA) in the left flank on day 0.
  • mice which are injected with either OVA, saline, or Ac-DEX particles encapsulating OVA and an immunostimulatory CpG stimulant.
  • CpG can be either co-injected with the particles, or it can be encapsulated inside the particles.
  • mice injected with PBS and nonencapsulated OVA have the fastest tumor onset and tumor growth.
  • Mice injected with OVA encapsulated in Ac-DEX particles have a delay in cancer onset and increase in survival time.
  • Mice receiving encapsulated OVA and coencapsulated or coinjected CpG have the slowest tumor onset and the longest survival time.
  • treatment experiments are performed. Mice are implanted with 200,000 MO5 tumor cells on day 0. After day 0, mice are immunized on day 6 with either saline, 50 micrograms of OVA, OVA encapsulated in Ac- DEX particles, or OVA encapsulated in Ac-DEX particles with coencapsulated or coinjected CpG. After injection mice are monitored for tumor growth. Tumors shrink and disappear after a coinjection of OVA and CpG in Ac-DEX particles.
  • Ac-DEX particles will encapsulate natural antigens inherent in the tumors against which are vaccinated.
  • the mouse B16 tumor possess the antigen tyrosinase-related protein (TRP2), which is also present in many human melanoma tumors (Jerome et al. 2006, Cytotoxic T lymphocytes responding to low dose TRP2 antigen are induced against B16 melanoma by liposome-encapsulated TRP2 peptide and CpG DNA adjuvant. J Immunother 29:294-305).
  • TRP2 antigen tyrosinase-related protein
  • the recombinant form of TRP2 will be encapsulated in Ac-DEX particles similar to the experiments done with OVA.
  • mice are immunized in the left flank with 2 mg of particles that are 2.5 wt% (50 ⁇ g of TRP2).
  • other groups include immunization with saline, free TRP2, or TRP2 particles with coencapsulated or coinjected CpG.
  • 200,000 B16 tumor cells are implanted in the right flank and mice are monitored for tumor growth. Mice receiving encapsulated OVA and coencapsulated or coinjected CpG have the slowest tumor onset and the longest survival time.
  • mice are implanted with 200,000 MO5 tumor cells on day 0. After day 0, mice are immunized on day 6 with either saline, 50 micrograms of TRP2, TRP2 encapsulated in Ac- DEX particles, or TRP2 encapsulated in Ac-DEX particles and CpG. After injection mice are monitored for tumor growth. Tumors shrink and disappear after a coinjection of OVA and CpG in Ac-DEX particles.
  • Ac-DEX particles will encapsulate chemotherapeutic agents such as doxorubicin (DOX) inside the particles by using the method described above.
  • chemotherapeutic agents such as doxorubicin (DOX)
  • particles can be coated with polyethylene glycol (PEG). It is well known to those in the art that particles coated with PEG have increased blood circulation in vivo (Shenoy et al. 2005, Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 2. In vivo distribution and tumor localization studies. Pharm Res 22:2107-14).
  • mice After 3 days mice are injected with PBS, free DOX, Ac-DEX particles encapsulating DOX, pegylated-Ac-DEX particles, or Doxcil (a commercially available doxorubicin/liposome conjugate). Mice are monitored for tumor growth. Once the tumor reaches an average diameter of 1.5 cm, the mice are sacrificed. Mice injected with PBS have the fastest tumor growth followed by, free Dox, Ac-DEX particles, Doxcil and finally PEGylated Ac-DEX particles.
  • PBS free DOX
  • Ac-DEX particles encapsulating DOX
  • pegylated-Ac-DEX particles pegylated-Ac-DEX particles
  • Doxcil a commercially available doxorubicin/liposome conjugate
  • Ac-DEX polymer is used as a bioengineering scaffold.
  • tissue engineering scaffolds out of biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA).
  • PLGA poly(lactic-co-glycolic acid)
  • PLGA has been used to make sutures, macroscopic implantable disks, and tissue scaffolds. Since Ac-DEX particles are soluble in most of the organic solvents PLGA is soluble in, Ac-DEX can be used to replace PLGA in these applications that are known by those in the art.
  • Alternatives for PLGA are needed because in certain in vivo applications, PLGA has been known to lower the local pH in and around the polymer (Shenderova et al.
  • the scaffolds will then be placed under high pressure CO2 gas and allowed to equilibrate.
  • the pressure will be rapidly returned to ambient conditions leading to a thermodynamic instability and causing the polymer to foam and create an interconnected structure around the NaCl.
  • Both types of particles foam and fuse together to create the scaffold, and no distinct particles or microspheres are present in the scaffold after this processing.
  • the NaCl is leached in a CaC12 solution to create a macroporous structure.
  • Bioengineering scaffolds can be used to encapsulate growth factors for bones, blood vessles, cardiovascular tissue, nerve cells and other tissue systems.
  • Growth factors such as vascular endothelial growth factor (VEGF) can be incorporated in the Ac-DEX particles. Incorporation of VEGF inside the scaffold can increase blood vascular growth and an increase in vascularization (Sun et al. 2005, Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb. Pharm Res 22:1110-6).
  • VEGF vascular endothelial growth factor
  • Another application where Ac-DEX may be an advantageous replacement for PLGA is absorbable sutures. Sutures are prepared using methods known to those familiar with the art.
  • Ac-DEX sutures are found to have tensile strength that is comparable to that of PLGA closure and sufficient flexibility for use. Rate of absorption is adjusted according to the needs of the wound closure. Generally, Ac-DEX sutures are appropriate for situations where absorption must occur faster than occurs with PLGA sutures or in situations where it is desirable to avoid local acidification from PLGA degradation.
  • An embodiment for the use of Ac-DEX polymer is the encapsulation of DNA inside Ac-DEX particles. Plasmid DNA was encapsulated into Ac-DEX using a double emulsion technique (Gwak and Kim, 2008, Poly(lactic-co-glycolic acid) nanosphere as a vehicle for gene delivery to human cord blood-derived mesenchymal stem cells: comparison with polyethylenimine. Biotechnol Lett).
  • luciferase plasmid DNA pCMV- Luc, 2 mg/ml
  • Tris/EDTA buffer 5% w/v in methylene chloride
  • the water-in-oil emulsion was further emulsified in 25 ml of a 2% (w/v) PBS buffered aqueous solution of polyvinyl alcohol using a homogenizer for an additional minute.
  • the emulsion was stirred for 8 h at room temperature to remove the methylene chloride.
  • the nanospheres were recovered by centrifugation at 12,00Og for 15 min at 4°C.
  • Particles were compared to Polyethylenimine (PEI), a polycationic polymer used commonly in transfecting mammalian cells with DNA.
  • PEI Polyethylenimine
  • the first experiment was to compare the toxicity of Ac-DEX particles to PEI polymers. HeLa cells were plated at 40000 cells/well in 96 well plates. Varying concentrations of PEI or Ac-DEX particles are cultured with cells for 4, 8, or 24 hours.
  • CPP Cell penetrating peptides
  • Particles encapsulating plasmid and functionalized with CPP were prepared in the same manner, except the reaction was only allowed to proceed for 1 d. This reaction leads to the formation of oxime linkages between the aminoxy group and the latent aldehydes at the reducing ends of the modified polysaccharide.
  • the use of hydrazides or hydrazines can similarly lead to the formation of acid-labile hydrazone linkages.
  • reaction with amines followed by reduction with NaBH 3 CN or similar reducing agents can lead to amine linkages through reductive amination. .
  • the toxicity of Ac-DEX particles with surface CPPs was tested. HeLa cells were seeded at 40 000 cells per 96 well plate.
  • Restenosis typically occurs after angioplasty is done on blood vessles. Restenosis in part is due to an inflammatory response around the site where the angioplasty occurred. Restenosis can be prevented by the treatment with rapamycin and dexamethasone (Zweers et al. (2006) Release of anti-restenosis drugs from poly(ethylene oxide)-poly(DL-lactic-co- glycolic acid) nanoparticles. J Control Release 114:317-24). Ac-DEX particles can encapsulate both rapamycin and dexamethasone using single emulsion techniques or salting methods. Particles can be pegylated as earlier stated.
  • a HPLC Cl 8 column is used to study the drug loading of rapamycin inside the Ac-DEX particles. Particles are dissolved with acetonitrile, and then injected into the HPLC. Based on a standard curve, the rapamycin content is calculated. While the dexamethasone content is calculated by dissolving approximately 5 mg of Ac-DEX particles in deuterated DMSO and comparing the integral of the dexamethasone peak to the anomeric peak on dextran.
  • SMCs smooth muscle cells
  • restenosis an exaggerated neointimal proliferative response
  • coronary stents as a treatment for obstructive cardiovascular disease.
  • drug-eluting stents have been used (i.e., Randade et al. (2004) Physical characterization of controlled release of paclitaxel from the TAXUSTM Express 2 TM drug-eluting stent.
  • Restenosis is a result of a number of processes occurring both acutely (i.e., the inflammatory response caused by mechanical injury to the arterial wall secondary to balloon dilation and stent deployment) as well as in the intermediate/long-term (proliferation of SMCs). Due to the multiple processes involved in restenosis, which occur after various periods of time, it is beneficial to control the release rate of drugs from drug-eluting stents (see Venkatraman et al. (2007) Release profiles in drug- eluting stents: Issues and uncertainties. Journal of Controlled Release 120:149-160).
  • Ac-DEX which have easily controlled degradation rates should be promising materials for stent coatings or for the production of fully biodegradable stents.
  • Ac-DEX or other acetal- modified polyhydroxylated polymers can be used to coat metal stents with formulations containing various concentrations of paclitaxel, rapamycin (sirolimus), dexamethasone or any mixture of these drugs.
  • these drugs can be encapsulated in microparticles using techniques such as emulsion/solvent evaporation or nanoprecipitation and the microparticles can be used to coat stents.
  • fully biodegradable stents can be made from Ac-DEX using methods known to those familiar in the art. Based on the degree and type of acetal modification, the release rates of drugs from the stents described above can be varied.
  • the drug release profiles from the stents described above are determined by incubating individual stents in medium at 37 0 C and pH 7.4. The medium is removed after various time points and analyzed using high performance liquid chromatography (HPLC) to determine the amount of each drug released.
  • HPLC high performance liquid chromatography
  • the drug-eluting stents prepared above using Ac-DEX or other acetal-modified polyhydroxylated polymers can be tested in vitro for their ability to reduce proliferation of SMCs using the antiproliferation assay described in Example 18. Following in vitro tests, the stents will be evaluated for their in vivo efficacy using methods known to those familiar in the art.
  • HeLa-/wc cells were seeded (-15,000 cells/well) into each well of a 96-well clear tissue culture plate and allowed to attach overnight in growth medium.
  • Growth medium was composed of DMEM (with phenol red), 10% FBS, and 1% glutamine.
  • Particle samples encapsulating siRNA were prepared at 1000 ⁇ g/mL in medium (without antibiotics) by alternately vortexing and sonicating in a Branson 2510 water bath for 20 s to generate homogeneous suspensions. The samples were then serially diluted in medium to give the indicated particle concentrations.
  • Existing medium was replaced with 100 ⁇ l of each particle dilution (or medium only) in triplicate wells of each 96-well plate.
  • the cells were allowed to grow for an additional 48 h before being analyzed for gene expression.
  • Lipofectamine 2000 was used as a positive control for siRNA delivery and was prepared according to the manufacturer's instructions.
  • complexes of DOTAP and siRNA were prepared by mixing DOTAP and siRNA solutions and incubating for 30 min prior to adding to the cells.
  • the cells of one of the plates were washed with PBS (containing Mg2+ and Ca2+, 3 x 100 ⁇ L), GLO LYSIS Buffer (120 ⁇ L, Promega, USA) was added to each well and the plate was incubated at rt. After 20 min, 100 ⁇ L from each well was transferred to the wells of a white 96-well tissue culture plate.
  • STEADY-GLO luciferase assay reagent (Promega) was reconstituted according to the manufacturer's instructions and added to each well (100 ⁇ L) using an automatic injector. The plate was read using a GLOMAX 96 microplate luminometer (Promega) with a 2 s integration rate.

Abstract

L'invention permet de faireen sorte que des matériaux à base d'un polymère polyhydroxylé sensibles aux acides tels que des microparticules, des sutures, des structures, des endoprothèses, des matériaux de revêtement, et autres matériaux en vrac implantables, se dégradent d'une manière dépendante du pH. Des charges à la fois hydrophobes et hydrophiles ont été introduites avec succès dans des particules à base des présents polymères par des techniques de simple et double émulsion, respectivement. Dans un modèle d'application vaccinale, des particules chargées avec la protéine ovalbumine (OVA) ont augmenté de 16 fois la présentation de peptides dérivés d'OVA à des cellules T CD8+ par rapport à l'OVA seule. Dans une autre application, un siARN encapsulé a significativement stimulé l'inactivation de l'expression protéique. Grâce à leur facilité de préparation, aptitude au traitement, sensibilité au pH, et biocompatibilité, les polymères polyhydroxylés modifiés selon la présente invention devraient être utiles dans de nombreuses applications d'administration de médicaments.
PCT/US2009/049415 2006-03-24 2009-07-01 Matériaux polyhydroxylés modifiés dégradables par des acides et par bio-érosion WO2010005847A1 (fr)

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US8349308B2 (en) 2010-03-26 2013-01-08 Mersana Therapeutics, Inc. Modified polymers for delivery of polynucleotides, method of manufacture, and methods of use thereof
US10207001B2 (en) 2013-10-23 2019-02-19 Lawrence Livermore National Security, Llc Techniques for release of material into an environment
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CN113350526A (zh) * 2021-06-28 2021-09-07 西南大学 基于主客作用的多糖超分子聚合物药物载体及其制备方法
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