WO2008127532A1 - Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules - Google Patents

Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules Download PDF

Info

Publication number
WO2008127532A1
WO2008127532A1 PCT/US2008/003422 US2008003422W WO2008127532A1 WO 2008127532 A1 WO2008127532 A1 WO 2008127532A1 US 2008003422 W US2008003422 W US 2008003422W WO 2008127532 A1 WO2008127532 A1 WO 2008127532A1
Authority
WO
WIPO (PCT)
Prior art keywords
particle
particles
protein
cells
polyketal
Prior art date
Application number
PCT/US2008/003422
Other languages
English (en)
Inventor
Niren Murthy
Michael Davis
Bali Pulendran
Stephen C. Yang
Original Assignee
Emory University
Georgia Tech Research Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Emory University, Georgia Tech Research Corporation filed Critical Emory University
Priority to AU2008239795A priority Critical patent/AU2008239795A1/en
Priority to EP08726853A priority patent/EP2146747A1/fr
Priority to JP2010503004A priority patent/JP2010523656A/ja
Priority to CA002684052A priority patent/CA2684052A1/fr
Publication of WO2008127532A1 publication Critical patent/WO2008127532A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L59/00—Compositions of polyacetals; Compositions of derivatives of polyacetals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/08—Bronchodilators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G4/00—Condensation polymers of aldehydes or ketones with polyalcohols; Addition polymers of heterocyclic oxygen compounds containing in the ring at least once the grouping —O—C—O—

Definitions

  • the invention relates to particle and micelle based strategies for delivering active agents, such as (i) vaccines; (ii) immune modulatory agents, (including TLR ligands or synthetic molecules, which modulate the function of innate immune cells such as dendritic cells, or synthetic molecules or siRNA that modulate signaling networks within cells (e.g., dendritic or other antigen presenting cells) and/or; (iii) drugs that target antigen- presenting cells so as to modulate innate and adaptive immunity, in a therapeutic or prophylactic setting.
  • active agents such as (i) vaccines; (ii) immune modulatory agents, (including TLR ligands or synthetic molecules, which modulate the function of innate immune cells such as dendritic cells, or synthetic molecules or siRNA that modulate signaling networks within cells (e.g., dendritic or other antigen presenting cells) and/or; (iii) drugs that target antigen- presenting cells so as to modulate innate and adaptive immunity, in a therapeutic or pro
  • Drug delivery vehicles based on polyesters and polyanhydrides have been widely used for the sustained release of therapeutics because of their excellent biocompatibility profiles and slow hydrolysis rates (Anderson, J.M. et al., Adv. Drug Delivery Rev., 1997, 28:5-24; Jain, R.A., Biomaterials, 2000, 21:2475-2490; Mathiowitz, E. et al., J. Appl. Polym. ScL, 1988, 35:755-774; Berkland, C. et al., J. Controlled Release, 2004, 94: 129- 141).
  • numerous medical applications such as targeting the acidic environment of lysosomes and tumors, require drug delivery systems that undergo rapid, pH-sensitive degradation (Stubbs, M.
  • Vaccines based on recombinant proteins, peptide antigens, or DNA vaccines encoding such vaccine antigens have tremendous therapeutic potential against infectious diseases and tumors, in which the antigenic epitopes have been defined.
  • Such vaccines have been capable of generating protective immunity against infectious diseases, in animal models, and numerous clinical trials with such vaccines are currently in progress (van Endert, PM, Biologicals, 2001, 29:285-8; Purcell, AW et al., Journal of Peptide Science, 2003, 9:255-81; Shirai, M. et al., Journal of Virology, 1994, 68:3334-42; Hunziker, IP et al., International Immunology, 2002, 14:615-26).
  • T-helper 1 or ThI
  • Th2 cytotoxic "killer” T cells
  • IgE immunoglobulin E
  • T regulatory responses can suppress over exuberant immune responses, and thus limit the immune pathology caused by allergies, autoimmunity, transplant rejection, or sepsis like symptoms.
  • DCs dendritic cells
  • TSTature's adjuvants express receptors which can recognize components of microbes and viruses.
  • receptors include the Toll-like receptors (TLRs), C-type lectins, and CATTERPILLAR proteins, which can "sense” microbial stimuli, and activate DCs and other immune cells (Pulendran, Immunol. Rev., 2004, 199:227-250; Pulendran, J. Immunol., 2005, 175:2457-2465; Pulendran & Ahmed, Cell, 2006, 124:849-863). It is now clear that DCs play essential roles in orchestrating the quality and quantity of the immune response.
  • TLRs There are currently some 13 TLRs described in mammals. Activating distinct TLRs on DCs induces qualitatively different types of immune responses (Pulendran, et al, 2001, supra; Dillon et al, 2004, supra; Agrawal et al, J. Immunol., 2003, 171 :4984-4989; Dillon et al, J. Clin. Immunol., 2006, 116:916-928).
  • activating most TLRs can induce ThI responses; activating TLR3, 7 or 9 can induce cytotoxic T cells that kill virally infected cells and tumors; and emerging evidence suggests that activating TLR2 induces Th2 responses, (which are associated with antibody responses that offer protection against viruses or extracellular bacteria or parasites), or even T regulatory or tolerogenic responses, (which suppress over exuberant immune responses, and thus offer protection against unbridled immunity in allergies, autoimmunity, sepsis, and transplantation).
  • Th2 responses which are associated with antibody responses that offer protection against viruses or extracellular bacteria or parasites
  • T regulatory or tolerogenic responses which suppress over exuberant immune responses, and thus offer protection against unbridled immunity in allergies, autoimmunity, sepsis, and transplantation.
  • DCs and TLRs and other recognition receptors represent attractive immune modulatory targets for vaccinologists and drug developers.
  • Acute myocardial infarction patients are traditionally treated at the hospital with blood thinning agents and/or angioplasty to try to clear the affected vessels.
  • Local cell death that occurs during this acute period leads to chronic heart failure, marked by increased ventricular size and reduced contractile function (Anversa, P., Myocyte death in the pathological heart Circ Res, 2000. 86(2): 121-4).
  • the only treatment for heart failure is transplantation surgery and it is estimated that less than 30% of transplant patients survive to receive their new heart (Rosamond et al., Heart Disease and Stroke Statistics—2007 Update. A Report From the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation, 2006).
  • a number of processes take place shortly following infarction have been the target of potential therapeutic approaches.
  • MAPK mitogen activated protein kinase cascades
  • the p38 MAPK cascade has been well characterized in stress and the inflammatory response (Lai et al., The role of MAP kinases in trauma and ischemia-reperfusion. J Invest Surg, 2004. 17(l):45-53; Lopez- Neblina and Toledo-Pereyra, Phosphoregulation of signal transduction pathways in ischemia and reperfusioa J Surg Res, 2006. 134(2):292-9).
  • p38 MAPK pathway has been implicated in the death of adult cardiac myocytes by use of transgenic mouse models as well as gene therapy studies (Engel et al., p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev, 2005. 19(10):l 175-87; Awad et al., Obese diabetic mouse environment differentially affects primitive and monocytic endothelial cell progenitors. Stem Cells, 2005. 23(4):575-83).
  • p38 is activated by dual phosphorylation (using ATP as a phosphate donor) through a variety of upstream mechanisms and its function is conserved across several cell types.
  • Activation of p38 in macrophages leads to superoxide production, as well as increased expression of pro-inflammatory cytokines.
  • cytokines pro-fibrotic cascades
  • p38 MAP kinases key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov, 2003. 2(9): 717-26).
  • Figure 29 shows a general schematic of p38 activation, including activation of upstream effectors and consequences of activation.
  • Phosphorylation of p38 leads to activation of 2 critical pathways in the disease process, including caspases and nuclear factor kappa B (NFKB) (Chen and Tu, Apoptosis and heart failure: mechanisms and therapeutic implications. Am J Cardiovasc Drugs, 2002. 2(l):43-57).
  • NFKB nuclear factor kappa B
  • phosphorylation of p38 induces activation of Bax, which eventually leads to mitochondrial permeability, resulting in cleavage of caspases and induction of apoptosis.
  • the inhibitory protein IKB is phosphorylated, releasing NFKB to the nucleus where activation of inflammatory cytokines can commence (Aggarwal et al., TNF blockade: an inflammatory issue. Ernst Schering Res Found Workshop, 2006(56): 161 -86). Both of these pathways have been targeted for a variety of inflammatory diseases including arthritis, ischemia/reperfusion injury and airway inflammation (Herlaar and Brown, p38 MAPK signalling cascades in inflammatory disease. MoI Med Today, 1999. 5(10):439- 47; Karin, M., Inflammation-activated protein kinases as targets for drug development Proc Am Thorac Soc, 2005.
  • the p38 inhibitor SB-282 When delivered to mice, the p38 inhibitor SB-282 improved cardiac function in a rat model of cardiac injury (Li et al., Selective inhibition of p38alpha MAPK improves cardiac function and reduces myocardial apoptosis in rat model of myocardial injury. Am J Physiol Heart Circ Physiol, 2006. 291(4):H 1972-7). Rats that were fed this compound for several days after L- NAME, salt and Angiotensin II treatment showed significant improvements in contractile function and other cardiac parameters. Additionally, in a mouse model of ischemia/reperfusion, SB239063 infused through the tail vein immediately prior to ischemia prevented p38 phosphorylation and reduced the infarct size after 24 hours.
  • polyketal particles are a new class of biomaterials that hydrolyze in a controllable manner at physiological pH values and degrade into neutral compounds.
  • the present invention provides biodegradable particles (e.g., three-dimensional particles) and micelles which can be used to encapsulate active agents for delivering to a subject.
  • the present invention further provides methods for producing and delivering such particles and micelles. Additionally, the invention provides vaccination strategies that encompass the use of the novel particles and micelles.
  • the present invention is directed to new type of hydrophobic polymers comprising ketal groups in the polymer backbone wherein the ketal groups are arranged in a way such that both oxygen atoms are located in the polymer backbone.
  • the ketal polymer can be formed via a ketal exchange reaction between a ketal and a diol.
  • one or more types of the ketal s and/or diols can be used for the formation of a homopolymer or copolymer.
  • polyketal polymers which are joined by other polymers (e.g. PEG, polyesters, polyamides, polysaccharides, polyethers, or polyanhydrides).
  • the resulting polymers can be alternating copolymers, random copolymers, block copolymers, or graft copolymers.
  • Polythioketal polymers, mixed polythio-amine ketals, polythio-hydroxyl ketals and polyhydroxyl-amine ketals are also in the scope of the present invention.
  • Polyketal polymers of the invention hydrolyze in aqueous solutions into low molecular weight, water soluble alcohols and ketones.
  • the advantage of a ketal linkage in the backbone is that it degrades under acidic conditions of phagosomes, within 1-2 days at pH 5.0.
  • Polyketals can therefore also be used for targeting the acidic environments of tumors, inflammation and phago-lysosomes. The degradation does not generate acidic degradation products.
  • the ketal polymers are suitable for biological use.
  • the present invention further provides novel biodegradable crosslinked micelles comprising multiple polymers, wherein the polymers are e.g., crosslinked by an external crosslinking agent (i.e., agents which are not introduced into the polymer chain).
  • an external crosslinking agent i.e., agents which are not introduced into the polymer chain.
  • the advantage of using of external agents is the faster crosslinking reaction compared to a reaction wherein only crosslinkable moieties within the polymer are used.
  • the external crosslinking agent also decreases the chances that the encapsulated is protein destroyed.
  • Figure 1 is a bar graph showing the fluorescence intensity of filtered and unfiltered PKNs with FITC-Ova (excitation 494 ran, emission 520 run). The data shown is the average of two samples. The FITC-Ova encapsulation efficiency is 60% (Example 3, infra).
  • Figure 2 is a diagrammatic representation showing the synthesis and degradation of Ketal-backbone polymer (polyketal).
  • A Ketal exchange reaction between 1,4- benzenedimethanol and 2,2-dimethoxypropane to produce the ketal intermediate 1.
  • B Stepwise polymerization of 1 to produce polyketal 2. Reaction steps A and B are driven forward by distilling off the methanol byproduct.
  • C Formation of drug-loaded particles by the solvent evaporation method. Particles exhibit pH-sensitive degradation into low molecular weight excretable compounds (Example 4, infra).
  • Figure 3(B) shows 1 H NMR spectrum of polyketal 2 (of Figure 2) in CDCl 3 (Varian Mercury Vx 400); repeating unit peaks at 7.3 ppm (4b), 4.5 ppm (4c), and 1.5 ppm (6a). Peaks at 2.5 and 1.0 are due to triethylamine added to prevent ketal hydrolysis. (Example 4, infra)
  • Figure 4 is a line graph showing the hydrolysis kinetics of polyketal 2 (of Fig. 2) (finely ground powder) at pH 1.0, 5.0, and 7.4. Exponential decay half-lives are 102 h (pH 7.4) and 35 h (pH 5.0). The pH 1.0 control batch was completely hydrolyzed before the first time point.
  • Figure 5 shows SEM images of particles made with polyketal 2 (of Fig. 2).
  • A,B Particles using 0.2:1 ratio of PVA to polyketal 2 (particle size: 0.5 - 30 ⁇ m).
  • C Dexamethasone-loaded particles made using 1:1 PVA:polyketal 2 (particle size: 200 - 500 nm). Scale bars are (A) 80 ⁇ m, (B) 3 ⁇ m, and (C) 4 ⁇ m.
  • FIG. 6 is a schematic representation showing particle formation.
  • A. Step 1 Dissolve polyketal and drug into chloroform; dissolve polyvinyl alcohol in water.
  • B. Add chloroform solution to water and sonicate, generate micron sized droplets.
  • Step 3 Let chloroform evaoporate, generates particles. (Example 6, infra)
  • Figure 7 is a photograph showing polyketal particles loaded with Fluorescein are taken up in the liver.
  • Murine liver tissue slice is shown releaing fluorescein from PKNs following intravenous injection. (Example 6, infra)
  • FIG 8 is a schematic diagram showing a peptide crosslinked micelle design and synthesis.
  • Step 1 ISS DNA and I are mixed to form micelles (uncrosslinked micelle).
  • Step 2 These micelles are then crosslinked with the antigenic peptide (II) to generate a delivery system that can encapsulate both immunostimulatory molecules and peptide antigens. After phagocytosis by APCs, the peptide-crosslinked micelles release their components. (Example 6, infra)
  • Figure 9 shows the synthesis and characterization of PEG-polylysine thiopyridal.
  • A. is a chemical diagram showing the synthesis of PEG-polylysine thiopyridal.
  • B is a IH-NMR spectrum of PEG-PLL-thiopyridal in D 2 O.
  • CfD are graphs depicting the dynamic light scattering analysis of PCMs uncross-linked (C) and peptide cross-linked (D).
  • E is a graph showing the crosslinking reaction of cysteines on peptide anigen II (Fig. 8).
  • F is a graph showing the UV analysis of crosslinking reaction between peptide anigen II (Fig. 8) and block copolymer micelles.
  • Figure 10 shows the effect of GSH on release of peptides and DNA
  • A is a graph showing GSH sensitive peptide release.
  • B is a gel elecrophoresis analysis showing GSH sensitive DNA release.
  • C is a gel electrophoresis analysis showing ISS-DNA is protected from serum nucleases in the PCMs. (Example 6, infra)
  • Figure 11 depicts block copolymer micelles.
  • A depicts the chemical structure of PEG- poly(lysine-thio-pyridyl).
  • B is a schematic diagram showing micelle formation.
  • C is a schematic diagram showing crosslinking of micelles.
  • D is a schematic diagram showing reducing of crosslinked micelles.
  • Figure 12 shows the immunology of micelle.
  • A Confocal microscopic analysis of the uptake of SIINFEKL-CFSE micelles by human monocyte derived DCs.
  • B FACS analysis of uptake of micelle encapsulated SIINFEKL peptide by human monocyte derived DCs.
  • C FACS analysis of uptake of micelle encapsulated SIINFEKL peptide by mouse DCs and Macrophages. (Example 6, infra)
  • Figure 13 is a bar graph showing micelle formulated SIINFEKL peptide induces potent T cell responses in-vitro (Example 6, infra).
  • Figure 14 shows the immunology of micelle.
  • A shows FACS analysis of efficient uptake of micelle encapsulated OVA protein by mouse DCs and Macrophages.
  • B is a graph showing that micelle encapsulated OVA/CpG activates DCs in-vitro. (Example 6, infra)
  • FIG 15 is a graph depicting the immunology of polyketal particles. Uptake of polyketal particle (PKN) encapsulated UO 126 by mouse DCs and Macrophages in-vitro. (Example 6, infra)
  • PNK polyketal particle
  • Figure 16 is a schematic diagram showing the experimental outline for T cell stimulation in-vitro using OVA-OT/ 1 transgenic model. (Example 6, infra)
  • Figure 17 shows the immunology of micelle.
  • A. Left panel is the flow cytometery analysis and the right panel is the summary showing that splenocytes pulsed with micelle formulated antigen induces potent antigen-specific CD8+ T cell responses in-vitro.
  • B. Left panel is the flow cytometery analysis and the right panel is the summary showing that DCs pulsed with micelle formulated antigens induce potent antigen-specific CD8+ T cell responses in vitro.
  • C Flow cytometery analysis showing that micelle formulated antigen activate DCs in-vivo. (Example 6, infra)
  • Figure 18 shows the immunology of micelle and polyketal particles.
  • A. Left panel is the flow cytometery analysis and the right panel is the summary showing that micelle formulated vaccines induce strong antigen-specific CD8+ IFN-gamma+ T cell responses in-vivo.
  • B. Left panel is the flow cytometery analysis and the right panel is the summary showing that micelle formulated vaccines induce strong antigen-specific CD8 + TNFa + T cell responses in-vivo.
  • C Line graphs showing the kinetics of specific CD8 + /IFN ⁇ + T cells after OVA/CpG vaccination.
  • D. Line graphs showing the kinetics of specific CD8 + /IFN ⁇ + T cells after OVA + UO 126 PKN vaccination.
  • Figure 19 shows polyketals from cyclohexane dimethanol (termed PCADK herein with an IUPAC designation poly(cyclohexane-l,4-diyl acetone dimethylene ketal)).
  • PCADK poly(cyclohexane-l,4-diyl acetone dimethylene ketal)
  • A Chemical representation showing polyketals from cyclohexane dimethanol.
  • B Line graph showing that PCADK degrades in an acid sensitive manner. (Example 6, infra)
  • Figure 20 shows SEM images depicting that particles from PCADK can encapsulate the hydrophobic compounds and drugs such as rhodhamine red and ebselen. (Example 6, infra)
  • Figure 21 shows line graphs showing that release of rhodhamine red from PCADK is pH sensitive. (Example 6, infra)
  • Figure 22 is a chemical representation showing that polyketals with almost any aliphatic diol can be made.
  • Figure 23 is a photograph showing that FITC labeled polyketals are phagocytosed by liver macrophages. (Example 6, infra)
  • Figure 24A is a schematic diagram showing double emulsion procedure used to encapsulate catalase and super oxide dismutase in polyketal particles.
  • B is a SEM image of catalase containing particles, and fluorescent microscope images of catalase containing particles.
  • C is a graph showing that catalase particles have enzymatic activity. (Example 6, infra)
  • Figure 25 is a chemical representation showing that polyketals made by acyclic diene metathesis (ADMET).
  • Figure 26 is a schematic diagram for the synthesis and acid degradation of drug loaded particles.
  • Figure 27 is a table showing the conditions used to make different rhodhamine containing PCADK particles.
  • Figure 28 is a graph showing that His-GFP stably bound to NTA-Ni for at least 15 hours (Example 9, infra).
  • Figure 29 is a schematic cartoon showing sources of p38 activation as well as downstream effects. (Example 7, infra)
  • FIG 30 is a schematic of the synthesis of PoIy(1, 4-cyclohexane-acetone dimethylene ketal) (PCADK).
  • PCADK is synthesized using the acetal exchange reaction with 1,4- cyclohexanedimethanol and 2,2 dimethoxypropane as reactants. PCADK hydrolyzes into 1 ,4-cyclohexanedimethanol and acetone (Example 7, infra).
  • Figure 32 is a photograph showing a representative SEM image of SB239063-loaded particles (PK-p38j). These images return an approximate particle size of 3-15 (Example 7, infra).
  • Figure 33 is a photograph showing that particle size can be easily modified.
  • Figure 34 is a photograph showing a representative SEM image of alternative porous particles. Addition of N-hexane to the initial dispersion resulted in formation of porous particles. Approximate size of these particles is 10-25 ⁇ m (Example 7, infra).
  • Figure 35 is a photograph showing SEM Images of SOD-PKNs.
  • A 6000X magnification.
  • B IOOOX maginification (Example 7, infra).
  • Figure 36 is a chart showing grouped data from Cultured macrophages pretreated with PK-SOD and stimulated with LPS. Pretreatment of macrophages with SOD-loaded polyketals resulted in a significant decrease in LPS-stimulated superoxide release as compared to empty PK and free SOD (Example 7, infra).
  • Figure 37 is a photograph showing representative fluorescent image of FITC-loaded polyketal incubated with cultured macrophages. Macrophages were incubated with PK- FITC for 2 hours prior to extensive washing and imaging. Arrows denote cells that have taken up FITC dye. Empty particles can also be seen bound to the surface of macrophages (Example 7, infra).
  • Figure 38 shows representative Western blot (top) and grouped densitometric data (bottom) from macrophages pretreated with polyketals for the indicated time (PK time) and stimulated with 10 ng/mL TNF- ⁇ for 20 minutes. Macrophages were incubated with empty PK or PK-p38, for 2-6 hours prior to extensive washing and TNF- ⁇ stimulation.
  • Figure 39 is a chart showing grouped data from macrophages pretreated with polyketals for 6 hours, stimulated with 10 ng/mL TNF- ⁇ for 20 minutesand dihydroethidium for 20 mins. HPLC data demonstrating a significant increase in extracellular superoxide release by TNF-a treatment. This increase was not blocked by empty PK but completely inhibited by PK-p38, pretreatment (*p ⁇ 0.05 ANOVA followed by Tukey-Kramer (Example 7, infra).
  • Figure 40 shows detection of extracellular superoxide production following infarction by DHE-HPLC.
  • Top panel is a representative trace from sham and infarct (MI) samples.
  • Bottom panel is normalized data showing an increase in extracellular superoxide production as detected by this method.
  • Animals were subjected to sham or coronary artery ligation surgery and left ventricular free walls were harvested 3 days following infarction.
  • Figure 41 is a photograph showing representative H&E Stain of leg muscle 3 days following injection.
  • PCADK injected form a defined injection area with little cellular staining outside the borders.
  • PLGA (right) in contrast shows evidence of fibrosis and inflammatory cell population (Example 7, infra).
  • Figure 42 is a photograph showing polyketal injection causes little inflammation.
  • the left panels show 2 representative immunofluoresence stains for CD45, a common inflammatory marker. There is little staining in the sections.
  • leg muscle injected with PLGA a commonly used polymer, generates a robust inflammatory response as measured by CD45 staining (Example 7, infra).
  • Figure 43 shows PK-p38, treatment inhibits p38 phosphorylation following infarction.
  • Figure 44 shows PK-p38i treatment inhibits IL-5 expression following infarction.
  • Grouped data demonstrates a significant increase in normalized IL- 5 levels in MI and MI+PK groups (* p ⁇ 0.05 vs. Sham and MI+PK-p38i).
  • Figure 45 is a chart showing polyketal-encapsulated SB239063 inhibits TNF- ⁇ production 3 days following infarction.
  • Grouped data demonstrating single injection of PK-p38, almost normalizes TNF- ⁇ levels in post-infarction rats (n 19 total rats). Data are represented as mean + SEM (Example 7, infra).
  • Figure 46 is a chart showing implanted cell death reversed by sustained growth factor treatment.
  • Neonatal cardiac myocytes were stained with a green membrane dye prior to injection, then stained for cleaved caspase-3 14 days following injection.
  • Data are mean + SEM from n>4 animals per group (**p ⁇ 0.01 vs. peptides alone or untethered IGF) (Example 7, infra).
  • Figure 47 shows tracking of cells Using GFP and HA adenoviruses.
  • Cells were incubated in suspension with 100 MOI of GFP or hemagglutanin adenoviruses for 2 hours immediately following isolation. Cells were plated for 24 hours and flow cytometry was performed (Example 7, infra).
  • Figure 48 is a chart showing grouped echocardiography data 3 days following sham or coronary artery ligation surgery. Fractional shortening measurements made from M- mode, short axis movies after 3 days of ischemia. There was a significant reduction in cardiac function as measured by fractional shortening in rats undergoing this procedure (*p ⁇ 0.05; t-test). These data demonstrate our ability to consistently perform this surgery in rats and induce cardiac dysfunction (Example 7, infra).
  • Figure 49 is a photograph showing cardiac stem cell isolation. Cells were isolated as described, fixed in 4% paraformaldehyde and stained with antibodies against the indicated maker followed by a fluorescent secondary antibody (Example 7, infra).
  • Figure 50 shows H-NMR analysis of PK-3.
  • the H-NMR spectrum was obtained using a Varian Mercury VX 400 MHz NMR spectrometer (Palo Alto, CA) using CDCl 3 as the solvent.
  • the molar ratio of 1,5-pentanediol to 1 ,4-cyclohexanedimethanol was obtained by obtaining the ratio of areas under the peaks a and b, respectively (Example 8, infra).
  • Figure 51 is a schematic showing synthesis of polyketal copolymers from 1,4- cyclohexanedimethanol, a second diol and 2,2-dimethoxypropane. Hydrolysis kinetics of polyketal copolymers can be controlled by copolymerization (Example 8, infra).
  • Figure 52 are graphs showing hydrolysis kinetics of polyketals can be tuned by copolymerization.
  • A Hydrolysis profiles of polyketal copolymers PKl to PK6 in pH 4.5 buffer
  • B hydrolysis profiles of PKl to PK6 in pH 7.4 buffer. Data are presented as mean ⁇ standard deviation. All experiments were conducted in triplicates at 37 0 C (Example 8, infra).
  • Figure 53 is a photograph showing SEM images of particles formulated with PK3. SEM image of empty particles formulated via double emulsion procedures (Example 8, infra).
  • Figure 54 is a schematic diagram of nitrilotriacetic acid (NTA) which, when loaded with nickel (Ni), binds histidine-tagged proteins (Example 9, infra).
  • NTA nitrilotriacetic acid
  • Figure 55 is a schematic diagram of a polyketal with a NTA linker forming microspheres where active agents are encapsulated by the microsphere and His-tagged proteins are bound to the surface of the microsphere.
  • B A diagram of DOGS-NTA (Example 9, infra).
  • Figure 56 is a photograph showing SEM micrographs of 10% NTA PCADK microparticles (Example 9, infra).
  • Figure 57 is a chart showing the spectrophotometric determination of Ni depletion in loading solution.
  • PCADK particle with varying concentrations of NTA-ligand (0%, 1%, 5%, 10%) were loaded in NiCl 2 solution. After incubating the particles overnight with agitation, solutions were centrifuged and the supernatants analyzed for Ni content spectrophotometrically.
  • the supernatant particles with no NTA-ligand had a nickel concentration of about 550 mM while 1%, 5%, and 10% NTA particles had approximately 500 mM Ni in their supernants. This data suggests that the surface of the particles is saturated with 1% NTA-ligand inclusion. Further quantative testing is currently underway using atomic absorption spectroscopy (Example 9, infra).
  • FIG 58 are photographs showing that His-tagged Green Fluorescent Protein (GFP) is Ni dependent. 10% NTA particles were charged with Ni, washed extensively and then incubated in a 100 nM solution of His-tagged GFP. Particles were washed extensively with PBS and imaged using fluorescent microscopy, (a) PCADK-NTA particles were loaded in PBS instead OfNiCl 2 . Very little fluorescence from GFP is seen, (b) PCADK- NTA particles loaded with NiCl 2 show extensive association with GFP (Example 9, infra).
  • Figure 59 is a graph of the Specific Binding curve for 1% NTA-PCADK particles (On- particle ELISA).
  • 1% NTA-PCADK particles were charged with Ni and loaded with varying concentrations of His-tagged GFP.
  • HRP horseradish peroxidase conjugated GFP antibody
  • ⁇ -GFP Ab horseradish peroxidase conjugated GFP antibody
  • Figure 60 is a graph of the Specific Binding curve for 10% NTA-PCADK particles (On- particle ELISA).
  • 10% NTA-PCADK particles were charged with Ni and loaded with varying concentrations of His-tagged GFP.
  • HRP horseradish peroxidase conjugated GFP antibody
  • ⁇ -GFP Ab horseradish peroxidase conjugated GFP antibody
  • FIG 61 is a graph showing that GFP Binding is reversible. Particles loaded with GFP were subjected to an spike of imidazole (200 mM final concentration), a competitive binding ligand to the NTA-Ni complex, and the fluorescence intensity of the supemantant measured as a function of time. Data suggests that GFP is maximally dissociated at 30 minutes in the presence of 200 mM imidazole (Example 9, infra).
  • Figure 62 is a chart showing that GFP binding increases with with amount of NTA-Ni (Example 9, infra).
  • micelle refers to a colloidal aggregate of polymer molecules having at least two different moieties which are linked to different properties in a liquid medium. The difference in the properties can occur due to different hydrophobicity/hydrophilicity, polarity, charge or charge distribution or other parameters which influence the solubility of a molecule. Micelles of the present invention are distinguished from and exclude liposomes which are composed of bilayers.
  • polymer refers to a covalently linked arrangement of monomeric molecules.
  • the arrangement can be realized in a linear chain or in a branched form.
  • the polymer can be a homopolymer which is composed of only one type of monomeric molecules, or it can be a copolymer wherein two or more different types of monomers are joined in the same polymer chain.
  • the polymer is called an alternating copolymer.
  • the two different monomers may be arranged in any order.
  • each type of monomer is grouped together.
  • a block copolymer can be thought of as two or more homopolymers joined together at the ends.
  • the term "particle” or “three-dimensional particle” refers to e.g., nanoparticles and/or microparticles. According to standard definitions, the term “nanoparticle” covers only particle having at least one dimension smaller than 100 nm. Larger particles which do not fulfill this requirement are termed "microparticles".
  • the present invention provides three-dimensional particles sized on the nanometer (nm) and micron ( ⁇ m) scale. The particles of the invention can range in size from about 50 nm to 1000 ⁇ m or from about 200 nm to 600 ⁇ m.
  • ketals and diols encompass ketals and diols comprising alkyl, cycloalkyl and aryl groups.
  • Alkyl group or “aliphatic group” as used herein, is linear or branched chain alkyl group. In one embodiment a linear alkyl group is preferred.
  • heteroalkyl groups wherein the heteroatom can be nitrogen, oxygen, phosphorus, sulfur and silicon.
  • cycloalkyl group or "cycloaliphatic group”, as used herein describes a ring- structured alkyl including at least three carbon atoms in the ring. In one embodiment, a cycloalkyl group having 5 or 6 carbons is preferred.
  • the cycloalkyl group also includes a heterocyclic ring, wherein the heteroatom can be nitrogen, oxygen, phosphorus, sulfur and silicon.
  • Aryl group or "aromatic group” as used herein, is an aromatic aryl ring such as phenyl, heterocyclic aromatic rings such as pyridine, furan, thiophene, pyrrole, indole and purine, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Included in the definition of alkyl, cycloalkyl and aryl groups are substituted alkyl, cycloalkyl and aryl groups. These groups can carry one or more substitutions. Suitable substitution groups include but are not limited to, halogens, amines, hydroxyl groups, carboxylic acids, nitro groups, carbonyl and other alkyl, cycloalkyl and aryl groups.
  • the present invention provides biodegradable hydrophobic polyketal polymers comprising multiple ketal groups, each ketal group having two oxygen atoms within the polymer backbone.
  • the biodegradable hydrophobic polyketal polymer of the invention is in the form of a solid molecule.
  • ketal groups include, but are not limited to, 2,2-dioxypropyl group, 2,2-dioxybutyl group, 1,1-dioxycyclohexyl group or dioxyacetophenyl group.
  • ketal polymers including aliphatic, cycloaliphatic or aromatic ketals containing one or more hetero-atom, such as nitrogen, sulfur, oxygen and halides.
  • the polymer further comprises a compound comprising alkyl, aryl, and cycloalkyl groups.
  • the compound may be directly attached to the ketal group.
  • suitable alkyl groups include, but are not limited to, methyl, ethyl and butyl groups.
  • suitable aryl groups include, but are not limited to, substituted or unsubstituted benzyl, phenyl or naphtyl groups, such as, for example, a 1,4- dimethylbenzene.
  • suitable cycloalkyl groups include, but are not limited to, substituted or unsubstituted cyclohexyl, cyclopropyl, cyclopentyl groups, such as, for example, 1 ,4-dimethylcyclohexyl group.
  • the polymer may be poly(l,4-phenylene-acetone dimethylene ketal). This polymer can be synthesized of 2,2-dimethoxypropane and 1,4-benzene dimethanol.
  • the polymer may also be a poly(l,4-cyclohexane-acetone dimethylene ketal), which can be synthesized of 2,2-dimethoxypropane and 1 ,4-cyclohexane dimethanol.
  • the polyketal polymer can be any one or more of PKl, PK2, PK3, PK4, PK5 and PK6 copolymers. These six polyketal copolymers exhibit varied hydrolysis kinetics at different pH levels.
  • PK4 is the fastest out of the six copolymers to hydrolyze with PK3 having the second faster hydrolysis rate.
  • PK3 has faster hydrolysis kinetics than PK2 or PK5, while PK2 and PK5 have faster hydrolysis kinetics than PKl or PK6.
  • pH 7.4 PK3 is hydrolyzed faster than PK4.
  • PKl has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e. percent component of the first monomer incorporated into the polymer) of about 98%
  • the second type of monomer is 1,5-pentanedioxy with a y value (i.e. percent component of the second monomer incorporated into the polymer) of about 2%.
  • the n value may be 3.
  • PKl is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1,5-pentanediol.
  • An IUPAC designation for PKl is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,5- pentane-acetone dimethylene ketal).
  • PK2 has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e. percent component of the first monomer incorporated into the polymer) of about 92%
  • the second type of monomer is 1,5-pentanedioxy with a y value (i.e. percent component of the second monomer incorporated into the polymer) of about 8%.
  • the n value may be 3.
  • PK2 is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1,5-pentanediol.
  • An IUPAC designation for PK2 is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,5- pentane-acetone dimethylene ketal).
  • PK3 has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e.
  • PK3 is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1,5-pentanediol.
  • An IUPAC designation for PK3 is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,5- pentane-acetone dimethylene ketal).
  • PK4 has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e. percent component of the first monomer incorporated into the polymer) of about 97%
  • the second type of monomer is 1 ,4-butanedioxy with a y value (i.e. percent component of the second monomer incorporated into the polymer) of about 3%.
  • the n value may be 2.
  • PK4 is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1 ,4-butanediol.
  • An IUPAC designation for PK4 is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,4- butane-acetone dimethylene ketal).
  • PK5 has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e. percent component of the first monomer incorporated into the polymer) of about 85%
  • the second type of monomer is 1 ,6-hexanedioxy with a y value (i.e. percent component of the second monomer incorporated into the polymer) of about 15%.
  • the n value may be 4.
  • PK5 is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1,6-hexanediol.
  • An IUPAC designation for PK5 is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,6- hexane-acetone dimethylene ketal).
  • PK6 has a structure as shown in Table 1 (Example 8) and is made up of at least 2 different monomers i.e., repeating units such as random repeating units.
  • one type of monomer is 1 ,4,-cyclohexanedimethoxy with an x value (i.e. percent component of the first monomer incorporated into the polymer) of about 87%
  • the second type of monomer is 1,8-octanedioxy with a y value (i.e. percent component of the second monomer incorporated into the polymer) of about 13%.
  • the n value may be 6.
  • PK6 is synthesized from two types of starting compounds, namely, 1 ,4-cyclohexanedimethanol and 1,8-octanediol.
  • An IUPAC designation for PK6 is poly(cyclohexane-l,4-diyl acetone dimethylene ketal-co-1,8- octane-acetone dimethylene ketal).
  • the PK1-PK6 copolymers may be synthesized using the acetal exchange reaction by copolymerizing 1 ,4-cyclohexanedimethanol with either butanediol, pentanediol, hexanediol or octanediol.
  • a PKl was synthesized with monomer A (1,4-cyclohexanedimethanol) plus monomer B (1,5- pentanediol); a PK2 was synthesized with monomer A (1,4-cyclohexanedimethanol) plus monomer B (1,5-pentanediol); a PK3 was synthesized with monomer A (1,4- cyclohexanedimethanol) plus monomer B (1,5-pentanediol); a PK4 was synthesized with monomer A (1,4-cyclohexanedimethanol) plus monomer B (1,4-butanediol); a PK5 was synthesized with monomer A (1,4-cyclohexanedimethanol) plus monomer B (1,6- hexanediol); a PK6 was synthesized with monomer A (1,4-cyclohex
  • the PKl, PK2, PK3, PK4, PK5 and PK6 copolymers of the invention can be intermingled to change or fine tune the release rate profile for attached active agents.
  • a polyketal with fast hydrolysis kinetics for example, a polyketal with fast hydrolysis kinetics
  • PK4 or PK3 can be mixed with a polyketal with slower hydrolysis kinetics (e.g., PKl or PK6) and co-administered to a subject.
  • the active agent joined to PK4 or PK3 will be released in a subject quickly to provide the subject with an immediate release (IR) dose of active agent, while the active agent joined to PKl or PK6 will be released at a slower rate allowing a gradual or extended release (ER) of the active agent in the subject.
  • IR immediate release
  • ER immediate release
  • the biodegradable hydrophobic polyketal polymers comprises (1) multiple ketal groups, each ketal group having two oxygen atoms within the polymer backbone and (2) a linker.
  • the ketal groups may comprise a 2,2-dioxypropyl group.
  • the invention further provides biodegradable particles comprising the polyketal polymers of the invention.
  • the sizes of the particles can vary.
  • biodegradable particles can be made at nanometer (ran) or micron ( ⁇ m) scale e.g., to form nanoparticles or microparticles.
  • the particles of the invention can range in size from about 50 nm to 1000 ⁇ m. In one embodiment, the particles range in size from about 200 nm to 600 ⁇ m. In another embodiment, the particles range in size from about 1 ⁇ m to 10 ⁇ m, 10 ⁇ m to 20 ⁇ m. 20 ⁇ m to 30 ⁇ m, 30 ⁇ m to 40 ⁇ m or 40 ⁇ m to 50 ⁇ m. In yet another embodiment, the particles range in size from about 1 ⁇ m to 50 ⁇ m.
  • a preferred particle size is between about 50 and 1000 nm, more preferred between about 200 and 600 nm. Another preferred particle size is about 30 ⁇ m.
  • the preferred size of a suitable polyketal polymer to form the biodegradable particle is between about 0.5 kDa and about 2 MDa, more preferred between 1 and about 150 kDa, most preferred between about 4 and about 6 kDa.
  • the number of the monomers in the polymer can range from about 2 to about 20,000, preferably about 10 to about 1,000, more preferably about 10 to about 50.
  • Polyketal polymers of the invention hydrolyze in aqueous solutions into low molecular weight, water soluble alcohols and ketones.
  • the degradation of poly(alkyl- acetone dimethylene ketal) is acid sensitive, with a half-life of 102.0 h at pH 7.4 and 35.0 h at pH 5.0.
  • the advantage of a ketal linkage in the backbone is that it degrades under acidic conditions of phagosomes, within 1-2 days at pH 5.0.
  • Polyketals can therefore also be used for targeting the acidic environments of tumors, inflammation and phago- lysosomes.
  • the degradation also does not generate acidic degradation products.
  • the ketal polymers are suitable for biological use.
  • the polyketal polymer particles can further comprise one or more active agents.
  • the biodegradable particle of the invention comprises: (a) a biodegradable hydrophobic polyketal polymers comprising multiple ketal groups, each ketal group having two oxygen atoms within the polymer backbone and (b) a linker.
  • Suitable polymers to form the particles of the invention include PCADK and PKl-PK6.
  • the polyketal polymer particle can further comprise one or more linkers which can bind an active agent.
  • Multiple linkers of the same or different types can be attached to a polymer particle.
  • the linkers are attached to the surfaces of the the particles i.e., the linkers are exposed to the solvent or aqueous solution surrounding the particles.
  • the biodegradable particle of the invention comprises (a) one or more of PKl, PK2, PK3, PK4, PK5 or PK6 (which can optionally comprise a linker), and (b) an active agent.
  • the biodegradable particle of the invention comprises (a) PCADK (which can optionally comprise a linker) and (b) an active agent.
  • the particles can comprise a single population of polyketals or a mixed population.
  • the term "active agent” refers to a protein, peptide, nucleic acid (DNA or RNA) or organic molecule, and other synthetic nucleic acid molecules, siRNA molecules, or antisense molecules.
  • the active agent can be a therapeutic, prophylactic or diagnostic agent.
  • the therapeutic agent can be an immunomodulatory agent such as specific ligands for RIG-I or TLRs, or C-type lectins (such as dectin-1 and DC-SIGN) or caterpillar proteins, or combinations of specific TLR ligands, or synthetic molecules or siRNAs which inhibit regulatory signaling networks with DCs and macrophages.
  • the active agent can further include proteins (e.g., recombinant proteins), peptides, carbohydrates, nucleic acids, small molecules (e.g., kinase inhibitors, phosphatase inhibitor, cytokine inhibitors, small molecule antioxidant mimetics, receptor blockers, cytoskeletal rearrangement inhibitors, small molecule receptor activators.), imaging agents, vaccine antigens, DNA vaccines, or vaccines themselves, such as the influenza vaccine.
  • proteins e.g., recombinant proteins
  • peptides e.g., recombinant proteins
  • carbohydrates e.g., nucleic acids, small molecules (e.g., kinase inhibitors, phosphatase inhibitor, cytokine inhibitors, small molecule antioxidant mimetics, receptor blockers, cytoskeletal rearrangement inhibitors, small molecule receptor activators.), imaging agents, vaccine antigens, DNA vaccines, or vaccines themselves, such as the influenza vaccine.
  • an active agent can comprise antibodies that target,
  • surface of these particles can be modified to contain targeting groups such as, for example, antibodies against subsets of dendritic cells, or proteins which stimulate subsets of dendritic cells, or macrophages such as CD40L, DEC-205, CDl Ic, langerin, MARCO, 33Dl etc.
  • targeting groups such as, for example, antibodies against subsets of dendritic cells, or proteins which stimulate subsets of dendritic cells, or macrophages such as CD40L, DEC-205, CDl Ic, langerin, MARCO, 33Dl etc.
  • Suitable active agents include, but are not limited to: (1) agonists and antagonists of TLRs (e.g., TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8, TLR-9, TLR- 10, and TLR-11), (2) agonists and antagonists of the receptor(s) activated by schistosome egg antigen (SEA), (3) molecules that stimulate or inhibit the expression or activity of a component of an intracellular signaling pathway that transduces the signal generated by activation of either of these types of receptors, (4) agents that stimulate or inhibit a transcription factor that is induced or stabilized by one or more of these signaling pathways, and (5) inhibitors of a regulatory pathway(s) within dendritic cells, macrophages or antigen-presenting cells.
  • TLRs e.g., TLR-2, TLR-3, TLR-4, TLR-5, TLR-7, TLR-8, TLR-9, TLR- 10, and TLR-11
  • SEA schistosome egg antigen
  • agonists include, but are not limited to, peptidoglycans (O. Takeuchi, et al., 1999 Immunity 11:443-451) or zymosans (Dillon et al, 2006 J. Clin. Invest. 116(4):916- 28). A. Ozinsky, et al., 2000 Proc. Natl. Acad. Sci. USA 97:13766-13771).
  • the agonists also include bacterial lipopeptides (e.g., diacylated and triacylated lipopeptides), lipoteichoic acid, lipoarabinomannan, phenol-soluble modulin, glycoinositolphospholipdis, glycolipids, porins, atypical LPS from Leptospira interrogns or Porphyromonas gingivalis, or HSP70 (for a review see K Takeda, et al., 2003 Annu. Rev. Immunol. 21 :335-376).
  • the agonists can be isolated and/or highly purified molecules.
  • the agonists include whole molecules or fragments thereof, or naturally-occurring or synthetic.
  • Examples include, but are not limited to, a non-toxic form of cholera toxin (Braun et al, J. Exp. Med. 189:541-552, 1999), certain forms of Candida albicans (d'Ostiani et al, J. Exp. Med. 191 :1661-1674, 2000), or P. gingivalis LPS (Pulendran et al., J. Immunol. 167:5067-5076, 2001).
  • bacterial lipopeptides include bacterial cell wall lipopeptides which differ in their fatty acid chain of the N-terminal cysteines, such as diacylated and triacylated lipopeptides.
  • diacylated lipopeptides include Macrophage Activating
  • Lipopeptide 2 kilo-Dalton from Mycoplasma fermentans or fragments thereof or synthetic analogues e.g., MALP2, Pam2CSK4, Pam2CGNNDESNISFKEK, and
  • the triacylated lipopeptides include Pam3cys ⁇ S- [2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH) ⁇ (Takeuchi, et al., 2001 International Immunology 13:933-940).
  • the agonist specifically effects TLR-2 or a receptor(s) bound by SEA (with respect to SEA, see MacDonald et al, J. Immunol 167:1982-1988, 2001).
  • the agonist can be, but is not limited to, a natural ligand, a biologically active fragment thereof, or a small or synthetic molecule.
  • Other useful agonists may include a non-toxic form of cholera toxin (Braun et al, J. Exp. Med. 189:541-552, 1999), certain forms of Candida albicans (d'Ostiani et al, J. Exp. Med.
  • the agonists can be agonists of TLR-4 (which bias the immune response toward the Th response, e.g. THl) include Taxol, fusion protein from Rous sarcomavirus, envelope proteins from MMTV, Hsp60 from Chlamydia pneumoniae or Hsp60 or Hsp70 from the host.
  • TLR-3 Other host factors that agonize TLR-3 include the type III repeat extra domain A of fibronectin, oligosaccharides of hyaluronic acid, polysaccharide fragments of heparan sulfate, and fibrinogen.
  • a number of synthetic compounds serve as agonists of TLR-7 (e.g., imidazoquinolin (imiquimod and R-848), loxoribine, bropirimine, and others that are structurally related to nucleic acids).
  • Suitable active agents include inhibitors of ERK, c-Fos, Foxp3, PD kinase, Akt, JNK, p38, NF-Kb, STAT 1, STAT2, IRF3, IRF7, IFN-alpha signaling or combinations thereof.
  • Suitable active agents can further include inhibitors of SOCS 1 -7 proteins.
  • Such inhibitors can be a small molecule, or a peptide, protein or nucleic acid (e.g., siRNA or antisense).
  • the agonist can be an exogenous or endogenous ligand, many of which are known in the art.
  • novel screening methods described below, particularly those that feature detecting TLR binding or activation, can be used to identify other ligands (whether naturally occurring molecules, fragments or derivatives thereof, antibodies, other peptides or protein-containing complexes, or synthetic ligands).
  • exogenous ligands of TLR-2 include LPS (lipopolysaccharide; a component of the outer membrane of Gram-negative bacteria), yeast-particle zymosan, bacterial peptidoglycans, lipoproteins from bacteria and mycoplasmas, and GPI anchor from Trypanosoma cruzi; endogenous ligands include heat shock (or "stress") proteins (e.g., an Hsp60 from, for example, a bacterial or mycobacterial pathogen) and surfactant protein-A.
  • LPS lipopolysaccharide
  • yeast-particle zymosan yeast-particle zymosan
  • bacterial peptidoglycans e.g., lipoproteins from bacteria and mycoplasmas
  • GPI anchor from Trypanosoma cruzi
  • endogenous ligands include heat shock (or "stress") proteins (e.g., an Hsp60 from, for example, a bacterial or mycobacterial pathogen) and
  • Exogenous ligands of TLR-3 include poly(I:C) (viral dsNRA); exogenous ligands of TLR-4 include LPS, and respiratory syncytial virus (endogenous ligands include stress proteins such as an Hsp60 or Hsp70, saturated fatty acids, unsaturated fatty acids, hyaluronic acid and fragments thereof, and surfactant protein-A).
  • Flagellin is an exogenous ligand of TLR-5.
  • CpG (cytosine-guanine repeat) DNA and dsDNA are exogenous and endogenous ligands, respectively, of TLR-9. See Zuany-Amorim et al, Nature Reviews 1:797-807, 2002, and Takeda et al., Ann. Rev. Immunol. 2J . :355-376, 2003.
  • suitable active agents include (a) an agent that inhibits the expression or activity of an AP-I transcription factor in a dendritic cell, (b) a dendritic cell treated in culture with an agent that inhibits the expression or activity of an AP-I transcription factor, or (c) syngeneic T cells stimulated in culture with dendritic cells treated as described in (b).
  • the transcription factor can include c-fos, fos-B, Foxp3, or c- jun, and the agent that inhibits expression (of the transcription factor or of any component of the pathways described herein (these components are known in the art)) can be an antisense oligonucleotide or an RNAi molecule that specifically inhibits c-fos, fos-B, Foxp3, or c-jun expression (or the expression of a kinase, phosphatase, or other component of the signaling pathways).
  • the inhibitory active agents discussed in the context of the present biodegradable particles can also be antibodies (or variants thereof (e.g., single-chain antibodies or humanized antibodies); preferably the antibodies are monoclonal antibodies).
  • the active agent is an antagonist (e.g., inhibitor or suppressor) of an intracellular pathway that impairs TLR2 signaling or activation.
  • the antagonists include gram negative LPS, Taxol, RSV fusion protein, MMTV envelope protein, HSP60, HSP70, Type III repeat extra domain A of fibronectin, oligosaccharides of hyaluronic acid, oligosaccharide fragments of heparan sulfate, fibrinogen and flagellin (for a review see K Takeda, et al., 2003 Annu. Rev. Immunol. 21 :335-376).
  • the active agent is an antagonist of an intracellular pathway that impairs SEA signaling or activation.
  • the molecule is an antagonist of a JNK 1/2 pathway.
  • the molecule is CpG DNA which activates p38 and ERK (A-K Yi, et al., 2002 The Journal of Immunolgy 168:4711-4720).
  • the active agent is an inhibitor of ERK 1 A which can inhibit maturation of dendritic cells and thus enhancing an IL 12 and ThI response.
  • the molecule include but are not limited to PD98059 and UO 126 (A. Puig-Kroger, et al., 2001 Blood 98:2175-2182).
  • the active agent inhibits c-fos signaling thus enhancing an IL 12 and ThI response.
  • Such molecules include a DEF domain mutant of c-fos or any polypeptide having a DEF domain mutation (L.O. Murphy, et al., 2002 Nature Cell Biology 4:556-564 and Supplementary information pages 1-3), including: rat Fra-1, and Fra-2; mouse FosB, JunD, c-Jun, c-Myc, and Egr-1; and human JunB, N-Myc, and mPerl.
  • Active agents can include nucleic acids such as DNA, RNA, anti-sense oligonucleotide or siRNA.
  • DNA include therapeutic genes.
  • therapeutic genes include suicide genes. These are genes sequences, the expression of which produces a protein or agent that inhibits tumor cell growth or tumor cell death.
  • Suicide genes include genes encoding enzymes, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or a gene encoding oncostatin. The purpose of the therapeutic gene is to inhibit the growth of or kill cancer cell or produce cytokines or other cytotoxic agents which directly or indirectly inhibit the growth of or kill the cancer cell.
  • Suitable oncogenes and tumor suppressor genes include neu, EGF, ras (including H, K, and N ras), p53, Retinoblastoma tumor suppressor gene (Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase (PTPase), and nm23.
  • Suitable toxins include Pseudomonas exotoxin A and S; diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-like toxins (SLT-I, -2), ricin, abrin, supporin, and gelonin.
  • Active agents can include enzymes. Suitable enzymes include thymidine kinase (TK), xanthine-guanine phosphoribosyltransferase (GPT) gene from E. coli or E. coli cytosine deaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT). Active agents can include cytokines. Suitable cytokines include interferons, GM-CSF, interleukins, tumor necrosis factor (TNF) (Wong G, et al., Science 1985; 228:810); WO9323034 (1993); Horisberger M.
  • TK thymidine kinase
  • GPT xanthine-guanine phosphoribosyltransferase
  • CD E. coli cytosine deaminase
  • HPRT hypoxanthine phosphoribosyl transferase
  • Active agents can include cytok
  • Active agents can include Growth factors.
  • Growth factors include Transforming Growth Factor-alpha. (TGF-alpha.) and beta (TGF-beta), cytokine colony stimulating factors (Shimane M., et al., Biochemical and Biophysical Research Communications, Feb. 28, 1994, 199(l):26-32; Kay A. B., et al., Journal of Experimental Medicine, Mar. 1, 1991, 173(3):775-8; de Wit H, et al., 1994 Feb., 86(2):259-64; Sprecher E., et al., Archives of Virology, 1992, 126(1 -4):253-69).
  • Active agents can further include the proteins catalase, superoxide dismutase, glutathione peroxidase, nitric oxide synthase.
  • the active agents of the invention can be can be naturally occurring, synthetic, or recombinantly produced, and includes, but are not limited to, any microbial or viral component or derivative thereof, including any component that is part of the structure of, or is produced by, the microbial cell or virus including, but not limited to, a cell wall, a coat protein, an extracellular protein, an intracellular protein, any toxic or non-toxic compound, a carbohydrate, a protein-carbohydrate complex, or any other component of a microbial cell or virus.
  • the microbial cell or virus can be pathological.
  • the polyketals of the invention may further comprise one or more linkers.
  • the linkers can be of the same type or can include a mixture of different types attached to the polyketal polymers.
  • the linkers can be attached to the surfaces of the the polyketal polymers e.g., the linkers can be exposed to the solvent or aqueous solution surrounding the particles.
  • the linker can range in size from 1-1000 nm or 100-200,000 Da. In one embodiment of the invention, the linker can range in size from 1-10 nm, 10-50 nm, 50-100nm, 100-200 nm, 200-300 nm, 300-400 nm, 400-500 nm, 500-600 nm, 600-700 nm, 700-800 nm, 800-
  • the linker can range in size from 100-10,000 Da, 10,000-50,000 Da, 50,000-100,000 Da, 100,000-150,000 Da and 150,000-200,000 Da. In yet another embodiment of the invention, the linker can be about 200-300 Da, 10,000 Da, 40,000 Da, 64,000 Da or 150,000 Da in size.
  • the linker can be a protein, polypeptide, nucleic acid or chemical compound.
  • protein or polypeptide linkers include, but are not limited to, an amino acid sequence such as arginine-aspartic acid-glycine (RDG), hemoglobin, glutathione-S-transferase (GST), streptavidin or an antibody.
  • nucleic acid linkers include, but are not limited to, RNA, DNA (e.g., ssDNA such as a 4 to 50 nucleic acid long tract of Poly A or Poly T sequence) or synthetic analogues.
  • linkers examples include, but are not limited to, polycarboxylic acid (e.g., polyacrylic acid) or chelating agents such as nitrilotriacetic acid (NTA) ligand, bipyradol and EDTA.
  • chelating agents bind to metal ions (e.g., nickel, zinc, copper, etc.) with the metal ions in turn facilitate binding of an active agent to the linker.
  • the linker tethers or joins (e.g., via a noncovalent association) an active agent to the polyketal of the invention.
  • the linker can bind to active agents including, but not limited to, a protein, peptide, nucleic acid and/or carbohydrate.
  • the protein, peptide and/or carbohydrate further comprises a histidine tag.
  • the histidine tag can comprise about 2-8 histidines.
  • the histidine tag comprises about 6 histidines.
  • the protein so bound to the linker can be a therapeutic, prophylactic or diagnostic protein.
  • the therapeutic protein can be an immunomodulatory protein.
  • the immunomodulatory protein can include, but is not limited to: (a) a ligand for any of TLR 2, 3, 4, 5, 7, 8, 9, 10 and 11 or combination thereof, (b) an inhibitor of a regulatory pathway within dendritic cells, macrophages or antigen-presenting cells; and (c) a ligand for RIG-I, any C-type lectins including dectin-1 and DC-SIGN, or Caterpillar proteins
  • the inhibitor of a regulatory pathway can include inhibitors of: (a) ERK, c-Fos, Foxp3, PI3 kinase, Akt, JNK, p38, NF-Kb, STAT 1, STAT2, IRF3, IRF7, IFN-alpha signaling; or (b) a SOCS 1, 2, 3, or other SOCS protein.
  • proteins to bind to the linker include growth factors, cytokines, antioxidant enzymes, antibodies, erythropoietin (EPO), receptor ligand.
  • EPO erythropoietin
  • a polyketal of the invention (e.g., PCADK or any of PKl to PK6) comprises a NTA linker.
  • the NTA linker can be loaded with nickel ions to produce a NTA-Ni linker that binds histidine tagged proteins.
  • the present invention further provides a biodegradable crosslinked micelle comprising multiple polymers of the invention.
  • the polymers can be crosslinked by an external crosslinking agent.
  • External crosslinking agents as used herein are agents which are not introduced into the polymer chain. The advantage of using of external agents is the faster crosslinking reaction compared to a reaction wherein only crosslinkable moieties within the polymer are used. The external crosslinking agent also reduces the probability that the encapsulated protein will be destroyed.
  • suitable crosslinking agents are compounds which comprise at least two thiol groups.
  • crosslinking agents include, but are not limited to, ethylene glycol dithiol, aliphatic dithiols, dithiols which are connected by ketal linkages, and diamine containing molecules.
  • thiol groups are the sensitivity to reducing conditions, thus enabling an easy degradation of the micelle.
  • crosslinking strategies include, but are not limited to, crosslinking by amines, esters, carbonates, thioesters, Schiff bases, vicinal diols, alkenes and alkynes, ketals, ketals orthoesters, thio-ketals, thio-orthoesters, sily-ketals, phenyl boronic acid-diol complexes, carbon-carbon bonds, sulfones, phosphate containing functional groups, azides, enzyme cleavable linkages, and urethanes, with Schiff bases, thiols and ketones being preferred in one embodiment of the present application (O'Reilly et al., 2005 Chem.
  • the external crosslinking agent comprises an antigen.
  • suitable antigens include, but are not limited to, proteins or peptides.
  • the antigens can be naturally occurring, chemically synthesized or recombinantly made.
  • suitable protein or peptide antigens include, but are not limited to, HIV antigens such as gpl20 protein (or fragment thereof), TAT protein (or fragment thereof), NEF protein (or fragment thereof), HCV protein (or fragment thereof), and env protein (or fragment thereof).
  • a preferred antigen includes a gpl20 peptide antigen chemically synthesized and modified to contain four additional cysteine residues to create therein additional disulfide bonds. Any antigen having crosslinkable groups can be used.
  • Antigens not having or having few crosslinkable groups can be modified (e.g., chemically modified) to comprise crosslinkable thiol groups, azides, alkynes, amines, maleimides, vinyl sulfones, ketones, hydrazines and thioesters.
  • Polymers to be used for micelle formation can be homopolymers or copolymers, such as block copolymers or graft polymers.
  • suitable polymers include, but are not limited to, PEG block copolymers, such as PEG-polyamino acids, for example, PEG- polylysine, PEG-polyglutamic acid, PEG-polyaspartic acid or PEG-polyarginine; PEG- polyesters, PEG-polyurethane, PEG-PPO, modified or unmodified, PEG-polyacrylate or PEG-polymethacrylate, synthesized by atom transfer polymerization, where the PEG acts as an initiator.
  • PEG block copolymers such as PEG-polyamino acids, for example, PEG- polylysine, PEG-polyglutamic acid, PEG-polyaspartic acid or PEG-polyarginine
  • PEG- polyesters PEG-polyurethane, PEG
  • the polymer can be modified to include chemical groups including, but not limited to, amines, esters, carbonates, thioesters, Schiff bases, vicinal diols, alkenes and alkynes, ketals, ketals orthoesters, thio-ketals, thio-orthoesters, sily-ketals, phenyl boronic acid-diol complexes, carbon-carbon bonds, sulfones, phosphate containing functional groups, azides, enzyme cleavable linkages, and urethanes, with Schiff bases, thiols and ketones being preferred in one embodiment of the present invention.
  • These groups can be introduced via chemical reactions known in the art, such as, among others, Michael addition or acylation (Example 1, Figure 9).
  • the modified polymer is PEG-polylysine thiopyridal ( Figure 9).
  • the polymethacrylate or polyacrylate block can contain modifications to allow for assembly with vaccine components and crosslinking.
  • a polyacrylate block can be a block copolymer consisting of polydimethylamino-acryalte-poly-glycidyl acrylate. Homopolymer of random copolymers composed of various acrylate or methacrylate monomers capable of forming micelles with vaccine components are also in the scope of the present invention.
  • the micelle can further comprise one or more active agents.
  • suitable active agents are found herein, supra.
  • the interaction between the micelle and the active agent can be electrostatic or hydrophobic or can occur due to hydrogen bond formation or molecular recognition depending on the type of polymer and agent.
  • the surface of this micelle can be modified to contain targeting groups such as, for example, antibodies against dendritic cells, or proteins which stimulate subsets of dendritic cells, or macrophages such as CD40L, DEC-205, CDl Ic, langerin, MARCO, 33Dl etc.
  • the micelle is designed to deliver peptide antigens and immunomodulatory molecules to antigen-presenting cells (APCs).
  • the micelle comprises immunomodulatory molecules, peptide antigens and a copolymer.
  • the peptide antigen acts as a crosslinker which allows the peptide antigen to be efficiently encapsulated into the peptide crosslinked micelles (PCMs) and also stabilizes them against degradation by serum components
  • the micelle can be used to encapsulate peptide or protein antigens together with immunomodulatory agents including multiple TLR ligands, or molecules such as synthetic compounds or siRNA that modulate signaling networks within cells (e.g., dendritic cells or other antigen presenting cells).
  • immunomodulatory agents including multiple TLR ligands, or molecules such as synthetic compounds or siRNA that modulate signaling networks within cells (e.g., dendritic cells or other antigen presenting cells).
  • the micelle targets dendritic cells and macrophages through their nanometer dimensions, as such cells robustly internalize nanometer sized materials, through phagocytosis.
  • the micelles are crosslinked by crosslinking agents comprising disulfide linkages, which should stabilize them against decomposition induced by serum proteins.
  • the micelle has a size of 5 to 50 microns.
  • the biodegradable particles and micelles of the invention will break down, and the encapsulated material such as peptide or antigens, and immune stimulatory agents [e.g: ISS DNA, TLR 7/8, TLR 3 ligands such as ss RNA, TLR 2 ligands, and inhibitors of regulatory pathways such as the ERK, c-Fos or Foxp3 pathway], will be released into the dendritic cell, or macrophage, and the immunomodulatory agent will induce the antigen-presenting cells, to secrete a variety of cytokines. This combination of signals will result in the optimal activation of T cells, and inhibition of regulatory T cells and dendritic cells.
  • immune stimulatory agents e.g: ISS DNA, TLR 7/8, TLR 3 ligands such as ss RNA, TLR 2 ligands, and inhibitors of regulatory pathways such as the ERK, c-Fos or Foxp3 pathway
  • the micelles of the invention can include active agents such as vaccines composed of antigens from the relevant pathogen, together with immune modulatory agents [e.g: ISS DNA, TLR 7/8 ligands such as ss RNA, TLR 2 ligands, and inhibitors of regulatory pathways such as inhibitors of ERK, c-Fos or Foxp3, PI3 kinase, Akt, SOCS 1-7 proteins, or siRNA molecules or antisense molecules that inhibit such regulatory pathways].
  • immune modulatory agents e.g: ISS DNA, TLR 7/8 ligands such as ss RNA, TLR 2 ligands, and inhibitors of regulatory pathways such as inhibitors of ERK, c-Fos or Foxp3, PI3 kinase, Akt, SOCS 1-7 proteins, or siRNA molecules or antisense molecules that inhibit such regulatory pathways.
  • the invention also provides methods for reversibly modifying proteins so that they have the appropriate charge to be encapsulated in the micelles. This strategy
  • the modified protein will then be encapsulated in the micelle, crosslinked and then the pH will be reduced in order to remove the inserted compound form the protein.
  • the compound is amine groups is cis-aconityl. This group adds to negative charges to each amine group and can be released at ph 4.0.
  • Examples for targeting strategies include synthesizing a heterobifunctional PEG that has a DNA binding domain at one end and another end that can attached to a protein. This PEG chain is then attached to a protein and then assembled into a preformed micelles that contains immunostimulatory DNA.
  • DNA binding domains include acridine or polyacridines.
  • Examples of targeting ligands include galactose, mannose phosphate, mannose, peptides, and antibodies.
  • the biodegradable particle or micelles can be further modified to incorporate antibodies or other molecules which target (1) specific receptors on particular subsets of DCs or macrophages or monocytes, including Langerhans cells, dermal DCs, myeloid DCs, plasmacytoid DCs or (2) specific receptors on antigen-presenting cells, such as DEC205, Langerin, DC-SIGN, dectin-1, 33Dl, MARCO.
  • specific receptors on particular subsets of DCs or macrophages or monocytes including Langerhans cells, dermal DCs, myeloid DCs, plasmacytoid DCs or (2) specific receptors on antigen-presenting cells, such as DEC205, Langerin, DC-SIGN, dectin-1, 33Dl, MARCO.
  • the present invention provides a pharmaceutical composition comprising the polymer
  • the present invention provides a pharmaceutical composition comprising the particle (which can optionally comprise a linker) of the invention.
  • the present invention provides a pharmaceutical composition comprising the micelle (which can optionally comprise a linker) of the invention.
  • the polymer of the invention whether in the form of a particle or micelle, can further comprise an active agent.
  • the present invention provides a pharmaceutical composition comprising PCADK, PKl, PK2, PK3, PK4, PK5 and/or PK6 particles with a linker bound to an active agent.
  • the invention provides methods for producing the particles of the invention.
  • the method comprises the steps of a) forming a hydrophobic polymer of a ketal and a diol or an unsaturated alcohol; b) forming a polymer particle of the polymer of a) in the presence of one or more active agents and thereby encapsulating the agent(s).
  • suitable chemistries for forming the hydrophobic polymer of a ketal and a diol or an unsaturated alcohol include acetal exchange reaction using single or double emulsions and acyclic diene metathesis (Heffernan MJ and Murthy N., 2005 Bioconjug. Chem. 16(6):1340-2; Jain RA., 2000 Biomaterials.
  • ketals for this method include, but are not limited to, 2,2- dimethoxypropane, 2,2-dimethoxybutane, 1 , 1 -dimethoxycyclohexane or dimethoxyacetophenole.
  • ketal polymers including aliphatic, cycloaliphatic or aromatic ketals containing one or more hetero-atom, such as nitrogen, sulfur, oxygen and halides.
  • the diol can be any of alkyl, aryl and cycloalkyl diols. Suitable examples of diols include, but are not limited to, 1 ,4-benzenedimethanol, 1 ,4- cyclohexanedimethanol, 1,5-pentane diol, 1,4-butane diol or 1,8-octane diol.
  • the invention provides a method for producing the particles of the invention comprising the steps of (a) forming PCADK polymer; and (b) forming a particle of PCADK in the presence of one or more active agents, thereby producing the particle of the invention.
  • the PCADK polymer can optionally comprise a linker.
  • the invention provides a method for producing the particles of the invention comprising the steps of (a) forming PKl, PK2, PK3, PK4, PK5 and/or PK6 polymer; and (b) forming a particle of one or more of PKl, PK2, PK3, PK4, PK5 and/or PK6 in the presence of one or more active agents, thereby producing the particle of the invention.
  • the PK1-PK6 polymers can optionally comprise a linker.
  • the micelles of the invention can be produced in a two step process.
  • the polymers of interest can be contacted with a liquid (polar or nonpolar liquid depending on the polymer to be used) under appropriate conditions so as to form a micelle.
  • the micelle can be crosslinked with an external crosslinking agent to produce the biodegradable micelle of the invention.
  • the invention further provides methods for delivering the active agents of the invention, via the particles of the invention or the micelles of the invention, to a subject in order to, for example, deliver active agents so as to treat the subject suffering from a disease (e.g. alleviate symptoms associated with the disease) or disorder.
  • a disease e.g. alleviate symptoms associated with the disease
  • the particles e.g., made of
  • PCADK, PKl, PK2, PK3, PK4, PK5 or PK6 polymer) or micelles may then be degraded or dissolved in the subject so as to release the active agent for delivery to the subject.
  • the particles or micelles of the invention have variable degradation rates at various pH ranges and can be designed to release an active agent according to a desired profile. For example, particles formed from PK3 polymers would degrade and release an active agent faster than particles formed from PK6 polymers.
  • the particles or micelles of the invention used for delivering active agents further comprise a linker.
  • a particle formed of the PCADK polymer further comprises a linker (e.g., NTA) bound to an active agent is used for delivery of the active agent to a subject.
  • the disease or disorder suffered by the subject can be any of HIV, malaria, TB, SARS, anthrax, Ebola, influenza, avian influenza and HCV.
  • Further disease or disorder can be any of an infectious disease, autoimmune disease, allergic disease, disorder or complications associated with transplantation, diabetes and cancer. Examples of autoimmune disease include lupus, rheumatoid arthritis, psoriasis, asthma and COPD.
  • the active agents are delivered through the particles of the invention by various administration means including, but not limited to, intravenous, subcutaneous, intramuscular, oral and inhalation means.
  • administration means including, but not limited to, intravenous, subcutaneous, intramuscular, oral and inhalation means.
  • the most effective mode of administration and dosage regimen for the compositions of the present invention depends upon the exact location of the disease or disorder being treated, the severity and course of the disease or disorder, the subject's health and response to treatment and the judgment of the treating physician. Accordingly, the dosages of the molecules should be titrated to the individual subject.
  • Adjustments in the dosage regimen maybe made to optimize the tumor cell growth inhibiting and killing response, e.g., doses may be divided and administered on a daily basis or the dose reduced proportionally depending upon the situation (e.g., several divided dose may be administered daily or proportionally reduced depending on the specific therapeutic situation). It would be clear that the dose of the compositions of the invention required to achieve treatment may be further reduced with schedule optimization.
  • the term "subject" may include a human, any animal such as equine, porcine, bovine, murine, canine, feline, and avian subject, a cell or a cell tissue.
  • the active agents can be delivered or administered to the subject (using the compositions of the invention) before, after, or during the onset of the disease or disorder.
  • the invention provides methods for regulating an immune response by administering active agents via the particles or micelles of the invention.
  • the particles or micelles used in the methods of the invention further comprise a linker.
  • an immune response can be biased towards a Th immune response in a TLR-dependent manner by delivering or administering to a subject a particle or micelle of the invention containing a desired active agent.
  • a TLR-expressing cell is contacted with an agent (delivered by the particle or micelle of the invention) that effects a bias towards a Th immune response (e.g., a ThO, Th2 or T regulatory cell immune response).
  • the agent e.g., a natural ligand, a biologically active fragment thereof, or a small or synthetic molecule that activatesTLR-2, ERK Vi, or c-fos.
  • the immune response can be regulated or modulated (e.g., increase biasing or decrease biasing toward a Th immune response) at a point in the signaling pathway downstream from receptor activation (e.g., downstream from TLR binding or downstream from TLR activation or recognition).
  • the patient can also be treated with an active agent or combination of active agents (delivered using the compositions of the invention) that bias the immune response by acting intracellularly on the elements of the downstream signaling pathway.
  • the present invention provides methods for biasing towards a Th2 immune response by inducing cell signaling (e.g., activation) of any of the MAP kinase pathways, including an ERK X A pathway using a desired active agent delivered using the compositions of the invention.
  • An induced MAP kinase pathway can be characterized by an increase in the amount and/or duration of phosphorylated components of the MAP kinase pathways, including ERK V 2 .
  • an active agent can be delivered, via the compositions of the invention, which modulates an ERK ⁇ ⁇ MAP kinase pathway so as to regulate a TH2 immune response.
  • an agonist of a TLR e.g., TLR-2
  • an active agent can be delivered via the compositions of the invention so as to modulate a c-FOS pathway in the cell thereby regulating a TH2 immune response.
  • an agonist of a c-fos pathway induces expression of c-fos and/or phosphorylation of c-fos so as to enhance a TH2 immune response.
  • an active agent can be delivered via the compositions of the invention, so as to modulate a Th2 immune response by affecting TLR2 or its downstream signaling pathway elements such as ERK Vi MAP kinase pathway and a c-FOS pathway.
  • the active agent, delivered via the compositions of the invention can be used to modulate production or activity of IL-IO (for example increase production or upregulate of IL- 10).
  • the methods for biasing towards a Th2 immune response includes decreasing or inhibiting signaling of p38 and/or JNK pathway(s) which mediate (e.g., inhibit) IL 12 production and thus biasing against a ThI response by administering an active agent delivered via the compositions of the invention.
  • the methods for biasing towards a Th2 immune response includes decreasing or inhibiting the amount of phosphorylated p38 and/or JNK, or decreasing or inhibiting the duration of phosphorylation of p38 and/or JNK which mediate (e.g., inhibit) IL12 production and thus biasing against a ThI response by administering micelles or particles of the invention containing a desired active agent (or combination thereof).
  • the present invention also provides methods for biasing towards a ThI immune response by inducing cell signaling (e.g., activation) of any of the MAP kinase pathways, including a p38 and/or JNK pathway by administering an active agent delivered via the compositions of the invention.
  • An induced p38 and/or JNK pathway can be characterized by an increase in the amount and/or duration of phosphorylated components of the MAP kinase pathways, including p38, and/or JNK.
  • the methods for biasing towards a ThI immune response includes decreasing or inhibiting signaling of ERK Vi and/or c-fos pathway(s) by administering an active agent delivered via the compositions of the invention.
  • the methods for biasing towards a ThI immune response includes decreasing or inhibiting the amount of phosphorylated ERK Vi and/or c-fos, or decreasing or inhibiting the duration of phosphorylation of ERK Vi and/or c-fos by administering an active agent delivered via the compositions of the invention.
  • the invention provides methods for regulating a TH2 immune response which comprises contacting a T cell (e.g., a naive T cell) with a TLR-positive cell (such as a DC) treated in culture with a TLR agonist (e.g., TLR-2 agonist), delivered via the compositions of the invention, which activates an ERK Vz pathway and/or which activates c-fos or c-fos pathway.
  • a T cell e.g., a naive T cell
  • a TLR-positive cell such as a DC
  • a TLR agonist e.g., TLR-2 agonist
  • the invention provides methods for regulating a THl immune response which comprises contacting a T cell (e.g., a na ⁇ ve T cell) with a TLR-positive cell treated in culture with a TLR agonist (e.g., TLR-4 agonist), delivered via the compositions of the invention, which activates a p38 pathway and/or a JNK pathway.
  • a T cell e.g., a na ⁇ ve T cell
  • TLR agonist e.g., TLR-4 agonist
  • the present invention provides methods for treating a subject having an immune-related condition or disease (e.g., allergies, autoimmune disease, and other immune-related conditions including cancer), comprising administering to the subject any of the agents of the invention, delivered via the compositions of the invention, in an amount effective to bias towards or against a ThI, Th2 or ThO immune response.
  • an immune-related condition or disease e.g., allergies, autoimmune disease, and other immune-related conditions including cancer
  • the subject can be bovine, porcine, murine, equine, canine, feline, simian, human, ovine, piscine or avian.
  • a subject having a condition or disease associated with an exhuberant Th2 response is treated with an agent of the invention, delivered via the compositions of the invention, that activates cell signaling in the subject so as to bias towards a ThI immune response.
  • Diseases characterized by exhuberant Th2 response include, but are not limited to allergy, asthma, and chronic obstructive pulmonary disease (COPD (e.g., emphysema or chronic bronchitis).
  • COPD chronic obstructive pulmonary disease
  • a subject having a condition or disease associated with an exhuberant Th2 response is treated with a molecule that inhibits biasing towards a Th2 immune response, delivered via the compositions of the invention.
  • a subject having a condition or disease associated with an exuberant ThI response is treated with agents of the invention, delivered via the compositions of the invention, that activate cell signaling in the subject so as to bias towards a Th2 immune response.
  • Disease characterized by exhuberant ThI response include, but are not limited to diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, and systemic lupus erythematosis.
  • a subject having a condition or disease associated with an exuberant ThI response is treated with an active agent, delivered via the compositions of the invention, that inhibits biasing towards a ThI immune response.
  • Polyketal particles of the invention comprise, or are capable of encapsulating, a wide range of active agents, from small inhibitors to large proteins, and can be used to deliver the active agents to a subject suffering from a disease or condition or as a preventative measure.
  • the active agents joined to or encapsulated by the polyketals can retain activity for several weeks in a subject. Additionally, the polyketals with the active agents can be freeze-dried, and are capable of being stored for months without degradation. The polyketals have a controllable degradation profile at various pH ranges, and if necessary can be fine tuned for precision release of active agents.
  • the polyketal polymer of the invention should overcome problems associated with administering active agents in areas around e.g., cardiac myocytes, namely, that the increased blood supply in areas surrounding the myocytes makes it highly likely that active agents will be immediately carried away following administration.
  • the larger, micron-scale polyketals of the invention should overcome this obstacle and allow the polyketals comprising or encapsulating active agents to remain in the local microenvironment.
  • the polyketals of the invention have several unique properties that make them ideal as a delivery vehicle of active agents for the treatment of a disorder or disease (e.g., myocardial treatment) or as a preventative measure: (1) they can degrade on a time scale that can be easily manipulated from 1-2 days to weeks at a variety of pH values; (2) they can hydrolyze into neutral excretable compounds, and should therefore cause less degradation and denaturation of proteins than polyester-based microparticles; (3) the polyketals can be solid particles and may be stable for extended periods after formulation; and (4) the properties of polyketals can be easily manipulated to alter particle size, shape and porosity.
  • a disorder or disease e.g., myocardial treatment
  • preventative measure (1) they can degrade on a time scale that can be easily manipulated from 1-2 days to weeks at a variety of pH values; (2) they can hydrolyze into neutral excretable compounds, and should therefore cause less degradation and denaturation of proteins than polyester-based microparticles; (3) the polyketals can be
  • altering the copolymer percentage of the polyketals can change or fine tune the hydrolysis kinetics of the polyketals in order to provide controllable release of active agents joined to or encapsulated by polyketals.
  • the monomer composition of PK4 provides for faster hydrolysis kinetics than PK3 at pH 4.5.
  • PK3 has faster hydrolysis kinetics than PK2 or PK5, while PK2 and PK5 have faster hydrolysis kinetics than PKl or PK6.
  • active agents joined to PK4 will be released faster than active agents joined to PK3; active agents joined to PK3 will be released faster than active agents joined to PK2 or PK5; and active agents joined to PK2 or PK5 will be released faster than active agents joined to PKl or PK6.
  • the polyketals of the invention can be mixed in order to change or fine tune the release rate for active agents joined to or encapsulated by polyketals.
  • a polyketal with fast hydrolysis kinetics e.g., PK4 or PK3
  • a polyketal with slower hydrolysis kinetics e.g., PKl or PK6
  • the active ingredient joined to PK4 or PK3 will be released in a subject quickly to provide the subject with an immediate release (IR) dose of active agent, while the active agent joined to PKl or PK6 will be released at a slower rate allowing a gradual or extended release (ER) of the active agent in the subject.
  • IR immediate release
  • ER immediate release
  • a linker can be attached to the polyketal polymer.
  • the polyketal polymers may then be used to form particles with linkers.
  • Particles with linkers allow dual delivery of one or more active agents to a subject, namely, via encapsulation of the active agents by the particle and attachment of the active agent to the particle to the linker.
  • this dual mode of delivery for active agents provides for dual release times for the active agents, with the linker bound active agents released faster than the encapsulated active agents.
  • Crosslinked micelles that contain the protein antigen Ovalbumin and immunostimulatory DNA were synthesized in a two step process.
  • micelles were formed between the cationic block copolymer, PEG-polylysine-thiopyridal and negatively charged FITC- Ovalbumin (FITC-OVA) and immunostimulatory DNA (ISS-DNA).
  • FITC-OVA FITC- Ovalbumin
  • ISS-DNA immunostimulatory DNA
  • the micelles were allowed to form for one hour and were then crosslinked with 0.4mg/ml of dithio- ethylene glycol.
  • the crosslinking reaction was monitored by U.V. activity (342nm), and indicated that the thiopyridal groups had been quantitatively reacted after 1 hour at room temperature.
  • the encapsulation efficiency of FITC-OVA in the micelles was determined by centrifuging the micelles through a 10OkD spin-filter (centricon) and analyzing the recovered solution for FITC fluorescence (excitation 494nm, emission 510nm). This indicated that over 95% of the FITC-OVA was encapsulated in the micelles.
  • Particles were synthesized with poly(l,4-phenylene-acetone dimethylene ketal) using an oil-in- water emulsion method. Briefly, 10 mg of 2, lmg of the ERK inhibitor UO 126 and 0.1 g of chloro-methyl fluorescein diacetate (CMFDA), were dissolved in 0.5 mL of CHCl 3 (with 0.1% triethylamine). This solution was then added to 5 mL of pH 9 buffer (10 mM NaHCO 3 ) containing 2mg/ml polyvinyl alcohol (PVA, 31-50 kDa, Aldrich).
  • CHCl 3 chloro-methyl fluorescein diacetate
  • Polyketal particles (PKNs) containing FITC-ovalbumin were fabricated using a double emulsion method.
  • 20 mg of poly(l,4-phenylene acetone dimethylene ketal) (PPADK) dissolved in 500 ⁇ L of chloroform was added to 100 ⁇ L of FITC-Ova solution (-0.7 mg). This mixture was sonicated at 40 watts for 1 minute to form the primary emulsion.
  • FITC-Ova solution -0.7 mg
  • This mixture was sonicated at 40 watts for 1 minute to form the primary emulsion.
  • 5 mL of 0.2% w/v polyvinyl alcohol (PVA, Aldrich) in 10 mM pH 9 sodium phosphate buffer was added, and this mixture was sonicated at 40 watts for at least 1 minute to form the secondary emulsion.
  • PVA poly(l,4-phenylene acetone dimethylene ketal)
  • the emulsion was mixed under nitrogen ventilation for 4 hours, after which the volume was made up to 5 mL with buffer. Two batches of PKNs containing FITC-Ova were prepared in this manner, as well as two batches of plain PKNs (without FITC-Ova). The PKN suspensions were stored at 4°C.
  • Particle sizing was determined by dynamic light scattering (DLS).
  • the two batches of FITC-Ovalbumin-loaded PKNs had effective diameters of 426 nm and 462 nm, and the empty PKN batches were 321 nm and 347 nm.
  • Chicken egg albumin (ovalbumin) was labeled with fluorescein as follows. Ovalbumin was dissolved at 10 mg/mL in 200 mM pH 9 NaHCO 3 buffer. Fluorescein isothiocyanate (FITC) was dissolved at 10 mg/mL in DMSO. Next, 2.5 mL of ovalbumin solution (25 mg, 0.56 mmol) was mixed with 50 ⁇ L of FITC/DMSO (0.5 mg, 1.28 mmol) for at least 1 hour at 30 0 C. The product was filtered in a Sephadex PD-10 column to remove the free dye; the column was loaded with 2.5 mL of product and was eluted with 2.5 mL water. The resulting ovalbumin concentration was approximately 7 mg/mL. The degree of labeling was calculated to be 1.09 by measuring the absorbance of FITC at 497 nm and using the estimated concentration of ovalbumin. Determination of FITC-Ova encapsulation in PKNs
  • the encapsulation efficiency was calculated using the fluorescence intensity of the filtered and unfiltered samples, as follows:
  • EncapsulationEfficiency 1 1 — Flltered
  • Figure 2 shows: (A) Ketal exchange reaction between 1 ,4-benzenedimethanol and 2,2- dimethoxypropane to produce the ketal intermediate 1. (B) Stepwise polymerization of 1 to produce polyketal 2. Reaction steps A and B are driven forward by distilling off the methanol byproduct. (C) Formation of drug-loaded particles by the solvent evaporation method. Particles exhibit pH-sensitive degradation into low molecular weight excretable compounds.
  • the Polyketals are synthesized via a new polymerization strategy based on the ketal exchange reaction (Lorette, N. B.; Howard, W. L. J. Org. Chem. 1960, 25, 521-525).
  • This reaction is generally used to introduce protecting groups onto low molecular weight alcohols and has not been used previously to synthesize polymers.
  • the ketal exchange reaction can be used to synthesize an acid sensitive polymer by simply reacting 2,2-dimethoxypropane (DMP) with a diol.
  • DMP 2,2-dimethoxypropane
  • the ketal exchange reaction is an equilibrium reaction involving protonation of DMP followed by nucleophilic attack by the alcohol. This equilibrium is shifted toward formation of the ketal intermediate 1 by distilling off the methanol byproduct. As the reaction proceeds, molecules of 1 combine in a stepwise manner to form polyketal 2.
  • a representative polymerization of DMP and 1 ,4-benzenedimethanol (BDM) gave the polyketal 2 with a 48% yield.
  • the polymerization was carried out in a 25 mL two- necked flask connected to a short-path distilling head.
  • BDM (1.0 g, 7.3 mmol, Aldrich) dissolved in 10 mL warm ethyl acetate was added to 10 mL distilled benzene kept at 100°C.
  • Re-crystallized />-toluene sulfonic acid 5.5 mg, 0.029 mmol, Aldrich
  • 550 ⁇ L ethyl acetate was then added.
  • distilled DMP (900 ⁇ L, 7.4 mmol, Aldrich) was added to initiate the reaction. Additional doses of DMP were added via a metering funnel, with each dose consisting of 2 mL benzene plus 300 to 500 ⁇ L DMP. Each dose was added over a 30 to 40 min period with a 30 min interval in between. The total duration of the reaction was 7 h. The reaction was stopped with the addition of 100 ⁇ L triethylamine and was precipitated in cold hexanes. The crude product was vacuum filtered, rinsed with ether and hexanes, and vacuum dried to yield 600 mg of white solid product (48% yield). The recovered polymer was analyzed by GPC and 1 H NMR.
  • the 1 H NMR spectrum ( Figure 3B) confirms that the repeating unit of 2 contains a dimethyl ketal group ('6a'). Together, the GPC and 1 H NMR data provide evidence for the successful synthesis of polyketal 2.
  • the hydrolysis kinetics of 2 were measured at pH values corresponding to lysosomes (pH 5.0) and the bloodstream (pH 7.4).
  • the hydrolysis rates were measured by grinding polyketal 2 into a fine powder and adding it to deuterated solutions at pH 7.4 (phosphate buffer), pH 5.0 (acetate buffer), and pH 1.0 (DCl).
  • the suspensions were stirred at 37°C and data points were taken at 3 h, 24 h, 48 h, and 72 h. Each suspension was centrifuged for 4 min at 1800 g, and the supernatant was analyzed by 1 H NMR.
  • the spectra contained peaks for BDM (7.24 and 4.47 ppm) and acetone (2.05 ppm).
  • the average of the two BDM peak integrals was used to determine the relative degree of hydrolysis.
  • the percent hydrolysis was calculated as the BDM peak average of the pH 7.4 or 5.0 sample divided by the BDM peak average of the pH 1.0 control batch.
  • Exponential decay half-lives were calculated to be 102 h at pH 7.4 and 35 h at pH 5.0, representing a 3-fold rate increase from pH 7.4 to 5.0 (Figure 4).
  • the pH sensitivity of 2 is significantly less than that reported by Kwon, et al. (Kwon, Y. J.; Standley, S. M.; Goodwin, A. P.; Gillies, E. R.; Frechet, J. M. J. MoI. Pharm. 2005, 2, 83-91) for a water- soluble ketal.
  • We hypothesize that the lower pH sensitivity of 2 is due to its water insolubility, which limits the diffusion of water and creates another rate limiting step that is insensitive to pH.
  • the diffusion kinetics of water into materials made of 2 will be dependent on the size of the particles and we would expect smaller particles to have greater pH sensitivity than ground particles.
  • Polyketal 2 (from Example 4, supra) was also used to synthesize micron sized particles.
  • An oil-in- water emulsion method (Panyam, J.; Williams, D.; Dash, A.; Leslie-Pelecky, D.; Labhasetwar, V. J. Pharm. Sci. 2004, 93, 1804-1814) was used to form the particles. Briefly, 50 mg of 2 dissolved in 1 mL CHCl 3 (with 0.1% triethylamine) was added to 5 mL of 10 mM NaHCO 3 pH 9 buffer containing various amounts of polyvinyl alcohol (PVA, 31-50 kDa, Aldrich) as the emulsifier.
  • PVA polyvinyl alcohol
  • the oil-water mixture was shaken briefly and then sonicated for 2 to 3 min at 40 watts (Branson Sonif ⁇ er 250) to form a fine oil/water emulsion.
  • the emulsion was stirred under N 2 flow for at least 3 h to evaporate the solvent and produce a particle suspension.
  • Particle sizes were analyzed by dynamic light scattering (DLS) and SEM.
  • DLS samples were prepared by diluting the particle suspension in 10 mL pH 9 buffer and allowing the larger particles to settle out. An aliquot from the liquid portion of each vial was then diluted for DLS particle sizing (Brookhaven 90Plus particle sizer).
  • An SEM sample was made with the 0.2:1 ratio of PVA:polyketal by centrifuging the particle suspension for 10 min (5000 g, 4°C), washing with distilled water, and lyophilizing the recovered pellet.
  • the particle size was sensitive to the ratio of PVA to polyketal.
  • the DLS particle diameters were 520 nm, 290 nm, and 280 nm for samples containing 0.2:1, 0.8:1, and 2:1 ratios of PVA:polyketal, respectively.
  • the SEM images of the 0.2:1 batch confirm that the polyketal does form micron sized particles, with particle size distribution ranging from 0.5 to 30 ⁇ m in diameter.
  • each particle batch was re-suspended in pH 9 buffer, and an aliquot was then further diluted. A portion was filtered through a 0.1 ⁇ m Supor membrane Acrodisc syringe filter (Pall Corp.), and the 242 nm absorbance of the filtrate was recorded with a Shimadzu UV- 1700 spectrophotometer.
  • the encapsulation efficiency was calculated as (Ao ex - A DexPo i y )/(A De ⁇ - Ap 0I y), where A is the absorbance at 242 run and the subscripts 'Poly', 'Dex', and 'DexPoly' refer to the 'Polyketal only', 'Dex only', and 'Dex + Polyketal' samples, respectively.
  • FIG 8 is a schematic representation showing peptide crosslinked micelle design and synthesis.
  • Step 1 ISS DNA and I are mixed to form micelles (uncrosslinked micelle).
  • Step 2 These micelles are then crosslinked with the antigenic peptide (II) to generate a delivery system that can encapsulate both immunostimulatory molecules and peptide antigens. After phagocytosis by APCs, the peptide-crosslinked micelles release their components.
  • An HIV peptide vaccine was synthesized using the PCM strategy with the peptide CGCRIQRGPGRAFVTIGKCGCG (II).
  • the peptide II comes from the GP-120 protein and contains the sequence RIQRGPGRAFVTIGK, which is both a class I and II antigen.
  • micelles were formed between I and ISS-DNA by mixing 0.5 mg of I with 0.1 mg of ISS DNA (5-TCCATGACGTTCCTGACGTT-S) (charge ratio was 1 to 15 (-/+)) in 0.5ml of 5OmM PBS. Dynamic light scattering of these micelles using the Cumulants method indicated that they had an average diameter of 57.0 nm.
  • micelles were then crosslinked by adding 0.1 mg of II to the micelles (equal molar ratio of cysteines on II to thiopyridal groups on I).
  • the peptide II was incorporated into the micelles through a disulfide exchange reaction.
  • Figure 9 shows synthesis and characterization of PEG-polylysine thiopyridal.
  • CfD Dynamic light scattering analysis of PCMs uncross-linked (C) and peptide cross-linked (D).
  • a 5OmM PBS buffer solution, at pH 7.4, containing 0.06 mg/ml of PEG-polylysine thiopyridal and 20 ⁇ g/ml of ISS DNA was made and filtered through a 200nm syringe filter.
  • This solution was then analyzed by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments), using the Cumulant method (C).
  • This solution was then crosslinked by adding 0.12 mg of peptide antigen (II), after 3 hours of reaction the solution was analyzed by DLS as described above, the size and the size distribution of the crosslinked micelles are shown in D).
  • Block copolymer micelles were formed between I and ISS DNA by mixing 0.5 mg of I with 0.1 mg of ISS DNA (representing a 15/1 amine to phosphate ratio), in 0.5 ml of 5OmM NaH 2 PO 4 buffer (pH 7.4), in an eppendeorf tube. After incubation for 2 hours at room temperature, 0.1 mg of II (representing a 1:1 cysteine to thiopyridal ratio) was added to the micelles.
  • the crosslinking reaction between the cysteines on II with the thiopyridal groups in the micelles was determined by UV analysis at 342nm (representing the released thiopyridone).
  • the percent of cysteine groups reacted was determined by the following formula:
  • Figure 10 shows the effects of GSH on release of peptides and DNA.
  • A. GSH sensitive peptide release The stimuli responsive release of peptides from the PCMs due to the presence of GSH was investigated to determine if the PCMs will release peptide antigens after phagocytosis.
  • the PCMs were incubated with different concentrations of GSH, for 24 hours in 5OmM pH 7.4 PBS buffer, and then analyzed by HPLC to determine the release of peptides.
  • This figure demonstrates that the release of peptides is triggered by the presence of GSH.
  • Incubation of the PCMs with 1OmM GSH (intracellular levels) induces the release of 71% of peptide, whereas incubation of the PCMs with just buffer causes the release of only 10% of peptides.
  • B. GSH sensitive DNA release The ability of the PCMs to protect encapsulated ISS-DNA from degradation by serum nucleases was investigated.
  • PCMs were synthesized (as described above) and incubated with 10% serum for 12 hours, these PCMs were then examined by gel electrophoresis to determine the stability of the encapsulated ISS-DNA.
  • ISS-DNA by itself was incubated with serum.
  • This figure demonstrates that ISS-DNA, by itself, is completely hydrolyzed in 10% serum (Lane 2), in contrast ISS-DNA encapsulated in the PCMs is protected from serum nucleases, presumably because of the effects of the crosslinking (Lane 3), which should prevent nucleases from entering the micelle.
  • C. ISS-DNA is protected from serum nucleases in the PCMs.
  • PCM strategy A key advantage of the PCM strategy is that it generates a crosslinked delivery system. This crosslinking should stabilize the PCMs in vivo.
  • the stability of the PCMs to decomposition was investigated by mixing the negatively charged polymer, poly(vinyl sulfate) (PVS) with the PCMs, this mixture was then analyzed by gel electrophoresis to determine the quantity of ISS-DNA displaced by the PVS.
  • PVS was also incubated with uncrosslinked micelles that were just composed of II and ISS-DNA.
  • Figure A, lane 4 demonstrates that PVS can disrupt uncrosslinked micelles that are just composed of ISS-DNA and II.
  • lane 5 demonstrates that PVS cannot displace ISS-DNA from the PCMs, presumably because the peptide crosslinking prevents the PVS from diffusing into the micelles and displacing the ISS-DNA.
  • the PCMs release encapsulated ISS-DNA, in the presence of PVS, demonstrating that the micelles should release their contents after phagocytosis, (lanes 2 and 3 in b).
  • Charge ratio of ISS-DNA to I is 1 to 15 (-/+); l ⁇ g of DNA was loaded in each lane; GSH (100/M) was added to lane 3 to induce release of encapsulated ISS-DNA. All samples were incubated with 10% serum at room temperature for 12 h.
  • Figure 11 depicts block copolymer micelles:
  • A Chemical structure of PEG-poly(Lysine- Thio-Pyridyl). PEG chain gives stability to the micelles, Polylysine segment is used for electrostatic interactions with proteins and DNA or RNA. Thiopyridal group is for subsequent crosslinking via a disulfide bond.
  • B Step I: Mix PEG-poly(Lysine-thio- pyridal) with DNA and protein, form micelles, with PEG on the outside.
  • Crosslink micelles with a di-thiol containing molecule, such as di-thioethylene glycol.
  • Crosslinked micelles are reduced by Glutathione, which has a much higher concentration in the cell than in the blood.
  • Figure 12 shows the immunology of micelles.
  • A Uptake of SIINFEKL-CFSE micelles by human monocyte derived DCs. Human PBMC derived DCs (day 6 of culture) were pulsed with SIINFEKL/CFSE micelles for 4h in 37°C at the concentration of lO ⁇ g/ml, 5x10*5 cell/well, 96-U wells, RPMI/10% FCS. Cells were washed, fixed on the figures and prepared for confocal microscope imaging.
  • B Efficient uptake of micelle encapsulated SIINFEKL peptide by human monocyte derived DCs.
  • Human PBMC derived DCs (day 6 of culture) were pulsed with SIINFEKL/CFSE plain (1) or micelle formulated (2,3) for 4h in 37°C at the concentration of lO ⁇ g/ml, 5x10*5 cell/well, 96-U wells, RPMI/10% FCS. Cells were washed, stained for CDl Ic and HLADR. Cells were gated for CDl Ic+, HL ADR+ cells and analysed for CFSE fluorescence. C. Efficient uptake of micelle encapsulated SIINFEKL peptide by mouse DCs and Macrophages.
  • Total mouse C57B1/6J FLT3-L splenocytes were pulsed with 1 or 10 ⁇ g/ml of CFSE labeled plain SIINFEKL (1) or micelle formulated peptide (2,3) for 4 hours (blue line), or remained untreated (red line) at 37°C at the concentration 1x10*6 cell/well, 96-U wells, RPMI/10% FCS. Cells were stained for CDl Ic and CDl Ib and analyzed for CFSE positive fluorescence.
  • Figure 13 is a bar graph showing that micelle formulated SIINFEKL peptide induces potent T cell responses in-vitro.
  • Purified CDl Ic + cells from a syngeneic C57B1/6J H-2 b FLT3-L treated mouse were pulsed for 4 hours with indicated concentrations and combinations of plain or micelle formulated SIINFEKL peptide.
  • Cells were washed and cocultured with total OT-I splenocytes for 2Oh (left panel) or 96h (right panel). Cells were stained for CD8 and intracellular IFN ⁇ and analyzed on a flow cytometer.
  • Graphs represent: micelle SIINFEKL 213.3 (black bar), micelle SIINFEKL 213.2 (grey bar), plain SIINFEKL (empty bar), medium (dotted bar) stimulated CDl lc+ DCs.
  • Figure 14 shows the immunology of micelles.
  • A Efficient uptake of micelle encapsulated OVA protein by mouse DCs and Macrophages.
  • Total mouse C57B1/6J FLT3-L splenocytes were pulsed with 1 or 10 ⁇ g/ml of FITC labeled plain ovalbumin (1) or micelle formulated protein (2) for 1 hour (black line), 5 hours (dotted line) or remained untreated (shaded). 37°C at the concentration 1x10*6 cell/well, 96-U wells, RPMI/10% FCS. Cells were stained for CDl Ic and CDl Ib and analyzed for FITC positive fluorescence.
  • B Micelle encapsulated OVA/CpG activates DCs in-vitro.
  • Total mouse C57B1/6J FLT3-L splenocytes were pulsed with 1 ⁇ g/ml of plain OVA (1) plain OVA + lug of CpG (2) or micelle formulated OVA (3) or micelle OVA + lug of CpG (4) for 24 hours.
  • CDl Ic+ cells were analysed for and CD80 or CD86 markers. Data represent isotype control (shaded), untreated (grey line) or stimuli treated (black line) cells.
  • Figure 15 is a graph depicting the immunology of polyketal particles.
  • PPN polyketal particle
  • FIG. 15 Shows the immunology of polyketal particles. Uptake of polyketal particle (PKN) encapsulated UO 126 by mouse DCs and Macrophages in-vitro.
  • Total mouse C57B1/6J FLT3-L splenocytes were pulsed with 1 or 10 ⁇ g/ml of CMFDA labeled Polyketal Particle carrying UO 126 ERK inhibitor for 5 hours at 37°C, 1x10*6 cell/well, 96-U wells, RPMI/10% FCS. Cells were stained for CDl Ic and CDl Ib and analyzed for CMFDA positive fluorescence. Data represent medium treated cells (shaded line) or 5 hours uptake (black line).
  • PPN polyketal particle
  • FIG 16 is a schematic diagram showing the experimental outline for T cell stimulation in-vitro using OVA-OT/ 1 transgenic model.
  • Figure 17 shows the immunology of micelles.
  • A. Micelle formulated antigen induces potent T cell responses in-vitro.
  • Purified CDl Ic + cells from a syngeneic C57B1/6J H-2 b mouse were pulsed for 4 hours with indicated concentrations and combinations of ovalbumin and CpG plain or micelle formulated. Cells were washed and 1x10*5 of CDl lc+ were cocultured with 1x10*6 of total OT-I (SIINFEKL specific) splenocytes for 5 days.
  • SIINFEKL specific total OT-I
  • Figure 18 shows the immunology of micelles and polyketal particles.
  • Micelle formulated vaccines induce strong T cell responses in-vivo - CD8 + IFNy + .
  • Cohorts of 4
  • C57B1/6J mice were vaccinated s.c. with 5 ⁇ g/mouse of OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA/CpG (4). Animals were boosted at day 36 (BOOST 1) and 84 (BOOST 2) using the same antigen formulations. Blood was harvested at days 6 post BOOST 2 and PBMCs were isolated using Histopaque gradient method, and restimulated with SIINFEKL peptide (lug/ml) and BFA (5ug/ml) for 6 hours and stained for CD8 and intracellular IFN ⁇ .
  • SIINFEKL peptide lug/ml
  • BFA 5ug/ml
  • mice Left panel represents the Flow Cytometry analysis and gating strategy for selected mice; right panel shows the summary of the data as a % of CD8 + IFNy + cells of total CD8 + T cells.
  • PBMCs from 4 mice were pooled and restimulated with SIESfFEKL peptide (lug/ml) and BFA (5ug/ml) for 6 hours and stained for CD8 and intracellular TNF ⁇ and IL-IO.
  • Left panel represents the Flow Cytometer analysis and gating strategy for selected mice; right panel shows the summary of the data as a % of CD8 + TNFa + cells of total CD8 + T cells.
  • C Kinetics of specific CD8 + /IFN ⁇ + T cells after OVA/CpG vaccination. Cohorts of 4 C57B1/6J mice were vaccinated s.c.
  • PBMCs from 4 mice were restimulated with SIINFEKL peptide (lug/ml) and BFA (5ug/ml) for 6 hours and stained for CD8 and intracellular IFN ⁇ .
  • Panels represent the summary of the data as % of CD8 + IFN ⁇ + cells of total CD8 + T cells including SEM error bars.
  • E. Micelle formulated vaccines induce strong antigen specific IgG antibody response in-vivo. Cohorts of 4 C57B1/6J mice were vaccinated s.c.
  • mice were vaccinated s.c. with 5 ⁇ g/mouse of OVA (1) or OVA + CpG (2), micelle OVA (3), micelle OVA/CpG (4) micelle OVA+ lOug of PKN UO 126 (5) or micelle OVA/CpG + lOug of PKN UO 126 (6). Animals were boosted at days 36 and 84 using the same antigen formulations.
  • Figure 19 shows polyketals from cyclohexane dimethanol
  • PCADK Polyketals from cyclohexane dimethanol
  • B PCADK degrades in an acid sensitive manner.
  • the ketal linkages in PCADK hydrolyze on the order of weeks under physiologic pH conditions.
  • the hydrolysis of the ketal linkages in PCADK were measured by H-NMR at the pHs of 4.5 and 7.4.
  • H-NMR the pHs of 4.5 and 7.4.
  • the ketal linkages of PCADK are approximately 30% hydrolyzed after 10 days. Based on this result, we anticipate that CAT-PKNs should be completely hydrolyzed, within 4-5 weeks after phagocytosis by macrophages.
  • Figure 20 shows SEM images depicting that particles from PCADK can encapsulate the hydrophobic compounds and drugs such as rhodhamine red and ebselen.
  • Figure 21 shows line graphs showing that release of rhodhamine red from PCADK is pH sensitive.
  • Step 6 is a schematic representation showing the steps of particle formation.
  • A. Step 1 Dissolve polyketal and drug into chloroform; dissolve polyvinyl alcohol in water.
  • B. Step 2: Add chloroform solution to water and sonicate, generate micron sized droplets.
  • Step 3 Let chloroform evaoporate, generates particles.
  • Figure 7 shows polyketal particles loaded with Fluorescein are taken up in the liver.
  • Murine liver tissue slice showing release of fluorescein from PKNs following intravenous injection.
  • Figure 22 is a chemical representation showing that polyketals with almost any aliphatic diol can be made. The hydrophobicity of the polyketal determines its hydrolysis kinetics.
  • FIG 23 is a photograph showing that FITC labeled polyketals are phagocytosed by liver macrophages. Phagocytosis of PKNs in vivo by Kupffer cells. Mice were injected with either FITC-PKNs or Empty PKNs. The livers of these mice were analyzed by histology. Left: FITC-PKNs are abundantly present in Kupffer cells, as evidenced by the punctate green fluorescence (10Ox magnification). Middle: empty PKNs generate very little background green fluorescence (10Ox magnification). Right: immunohistochemistry (IHC) for FITC (red) confirms uptake by Kupffer cells (40Ox magnification).
  • IHC immunohistochemistry
  • Figure 24 A. Double emulsion procedure used to encapsulate catalase and super oxide dismutase in polyketal particles.
  • a 50 ⁇ L aqueous solution of catalase (1 mg/ml) was dispersed into an organic phase, consisting of 75 mg of PCADK, dissolved in ImL of dichloromethane, using a homogenizer, generating a water in oil (w/o) emulsion.
  • This w/o emulsion was then dripped into 25mL of a 4% PVA solution, which was mechanically stirred with a homogenizer.
  • the resulting w/o/w emulsion was then poured into 225mL of a 4% PVA solution and mechanically stirred for several hours until the methylene chloride evaporated.
  • the resulting particles were isolated by centrifugation, freeze-dried and examined by SEM (B) The protein encapsulation efficiency was 35%.
  • the CAT-PKNs have an average diameter of approximately 8 microns.
  • C. Catalase particles have enzymatic activity, as evidenced by their ability to decrease the absorbance at 240nm of hydrogen peroxide.
  • EXAMPLE 7A Synthesis and Characterization of PK Particles for Treatment of Myocardial Infarction
  • PK polyketal particles
  • the PKs are designed to deliver therapeutics to the heart and treat acute myocardial infarction.
  • the PKs are formulated from a new class of polymers, the polyketals, which contain ketal linkages in their backbone ( Figure 30). Materials and Methods
  • PCADK was synthesized in a 50 mL two-necked flask, connected to a short-path distilling head. First, 5.5 mg of re-crystallized p-toluenesulfonic acid (0.029 mmol, Aldrich), dissolved in 6.82 mL of ethyl acetate, was added to a 30 mL benzene solution (kept at 100°C), which contained 1 ,4-cyclohexanedimethanol (12.98 g, 90.0 mmol, Aldrich).
  • the molecular weight of PCADK was determined by gel permeation chromatography (GPC) (Shimadzu) equipped with a UV detector as shown in Figure 31. THF was used as the mobile phase at a flow rate of 1 ml/min. Polystyrene standards from Polymer Laboratories (Amherst, MA) were used to establish a molecular weight calibration curve. This compound was used to generate the PCADK particles in all subsequent experiments.
  • Particles can be fabricated with SB239063 joined to PCADK
  • SB239063 is an inhibitor of p38.
  • Figure 29 shows a general schematic of p38 activation, including activation of upstream effectors and consequences of activation.
  • the resulting w/o emulsion was then poured into 25 mL of pH 7.4 buffer, and this mixture was stirred for several hours, evaporating the methylene chloride.
  • the resulting particles were isolated by centrifugation and freeze- dried, generating a white solid powder.
  • the protein encapsulation efficiency of the SB293063 micropaiticles was 25-50%, as determined by U.V. absorbance at 320 run using a standard curve for SB239063 alone.
  • An SEM image of the SB239063-PCADK particles, shown in Figure 32, demonstrates that they are 3 to 15 ⁇ m in diameter, which is suitable for both intracellular and extracellular delivery.
  • Porous PCADK microparticles can be synthesized
  • Porous microparticles 20-30 microns in size have recently attracted interest for lung drug delivery because their low density gives them the aerodynamic properties to be inhaled, via a spinhaler, yet their large size prevents them from being phagocytosed by macrophages.
  • Studies by Langer et al., with PLGA, have demonstrated that 20-30 micron sized particles are optimal for delivering therapeutics to the extracellular space of the alveolar region of the lung (Cheng et al., Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials, 2007. 28(5):869-76; Edwards et al., Large porous particles for pulmonary drug delivery. Science, 1997.
  • a water-in-oil emulsion was created by added 200 ⁇ l of aqueous FITC-labeled OVA solution (25 mg/ml) to the oil phase and dispersing the aqueous phase in the oil phase via homogenation at 21,500 rpm for 90 seconds.
  • the turbid w/o emulsion was then poured into 20 ml of 2% (w/v) aqueous polyvinyl alcohol solution and homogenized at 17,000 rpm for 60 seconds to generate the final water- in-oil- in- water emulsion.
  • the w/o/w emulsion was then decanted into 100 ml of 2% PVA solution and stirred at 500 rpm for 3 hours at 25 0 C in order to evaporate the methylene chloride in the oil phase causing the polymer to harden around the encapsulated OVA.
  • the hardened particles are then stirred at 500 rpm for 72 hours at 40 0 C to remove the hexane from the particles leaving pores in the interior and on the surface of the particles.
  • An SEM image of OVA-PCADK is shown in Figure 34 demonstrating that porous particles were synthesized.
  • Protein-loaded polyketals can be generated
  • the resulting w/o/w emulsion was then poured into 25 mL of pH 7.4 buffer, and this mixture 5 was stirred for several hours, evaporating the methylene chloride.
  • the resulting particles were isolated by centrifugation and freeze-dried, generating a white solid powder.
  • the protein encapsulation efficiency of the SOD-PCADK microparticles was 36.7%, as determined by U.V. absorbance at 280 nm.
  • An SEM image of the SOD-PCADK microparticles, shown in Figure 35, demonstrates that they are 3 to 15 ⁇ m in diameter
  • PCADK enhances the delivery of SOD to macrophages
  • SOD-PCADK microparticles The ability of SOD-PCADK microparticles to scavenge superoxide from macrophages was investigated in cell culture.
  • TIB-186 macrophages IxIO 5 cells/well, 96 well plate) were incubated with either 0.1 mg/mL SOD-PCADK microparticles, free SOD (1.14
  • SB239063 particles inhibit p38 phosphorylation in TNF- a-stimulated macrophages
  • RAW macrophages were plated at confluency in serum-free medium, and switched to serum-free medium containing empty PK or PK-p38i for 2, 4 or 6 hours, prior to 20 minutes TNF- ⁇ stimulation. Proteins were harvested in sample buffer and 20 ⁇ g run on a 12% SDS-polyacrylamide gel for Western analysis. Proteins were transferred to a PVDF membrane and were probed with an antibody to phospho-specific p38 (Cell Signaling). Blots were scanned and quantified using Quantity One software from Bio-Rad.
  • PK-p38 (0.1 mg/mL or 2 ⁇ M) prevented p38 phosphorylation by TNF- ⁇ in a time dependent manner. While there was no effect of empty PK at various time points, treatment of macrophages for 4 or 6 hours with PK-p38j completely prevented p38 phosphorylation by TNF- ⁇ . These data suggest that PK-p38j is capable of preventing cytokine-mediated p38 activation and superoxide production in macrophages.
  • SB239063 particles inhibit superoxide production in TNF- a-stimulated macrophages Increased superoxide production following myocardial infarction may play a role in post- infarction cardiac apoptosis and dysfunction. TNF- ⁇ stimulation can lead to extracellular superoxide production in macrophages and this response was shown to be p38 dependent.
  • Oxy-ethidium is stable and values can be quantified using the extinction coefficient and moles of superoxide are estimated to be the same as moles of oxy-ethidium as they react in a 1 :1 ratio. Macrophages were plated at 10 6 cells/well in serum-free medium the night before experiments. Cells were then
  • Polyketals are retained in muscle 3 days following injection 0
  • PLGA poly(lactic-co-glycolic acid)
  • leg muscles injected with PLGA particles showed intense fibrosis and possible inflammatory cell infiltration as examined in the subsequent figure.
  • the dark staining in the PCADK samples are most likely particles and not cells as confirmed by DAPI staining.
  • sections from the leg injection experiment above were stained with a fluorescent antibody against CD45, a common inflammatory cell marker. Specifically, after paraffin removal and rehydration, sections were incubated with proteinase K for antigen retrieval. Following retrieval, sections were blocked in 10% horse serum, then incubated with a fluorescently tagged anti-mouse CD45 antibody (eBioscience). After extensive washing, sections were incubated with DAPI (1 ⁇ g/mL) and coverslipped. The proper FITC-conjugated isotype was used as a control and no staining was evident. Images were taken with the exact same exposure times and merged using Matlab software.
  • SB239063 within polyketals inhibits p38 phosphorylation within the infarct zone
  • C57BL/6 mice underwent coronary artery ligation and immediately following infarction, were given either empty polyketals (PK) or polyketals containing SB239063 (PK-p38j 0.1 mg/mL) intramyocardially in a double-blinded manner.
  • Hearts were excised 3 days following infarction and the infarct zone of the left ventricular free wall was isolated and homogenized in a Triton-X buffer containing protease and phosphatase inhibitors.
  • PK-p38j When delivered to leg muscle, the particles were easily distinguishable from the surrounding tissue and were retained for several days following injection. In comparison to PLGA particles, which degrade in to acidic products, PCADK caused very little inflammation as measured by CD45 and CD 14 staining. When delivered intramyocardially, PK-p38j reduced left ventricular p38 phosphorylation in response to myocardial infarction. In addition, PK-p38j treatment reduced left ventricular levels of IL-5 and TNF- ⁇ , two pro-inflammatory cytokines. In contrast, there was no effect of empty PK, or even free SB239063.
  • this example shows that retention of compound SB239063, a p38 inhibitor, within the infarct area after a single injection will inhibit endogenous cardiac myocyte cell death, restore cardiac function following myocardial infarction and improve cell therapy in larger rodents.
  • PK micron-scale polyketal
  • Polyketals (PK) containing the p38 inhibitor SB239063 may inhibit p38 phosphorylation, pro-inflammatory cytokine production and apoptosis in cultured cells.
  • PK-encapsulated SB239063 may be used to inhibit TNF- ⁇ -induced p38 phosphorylation and apoptosis in rat neonatal cardiac myocytes in a dose-dependent manner over the course of several days.
  • PK-p38j will be generated as described in the methods section above. We will isolate rat neonatal cardiac myocytes as described in the methods and incubate them with 0, 0.1, 0.5 and 1 mg/mL of empty PK or PK-p38j for 6 hours, based on the time course shown in Figure 38. Following PK-p38j incubation, TNF- ⁇ (10 ng/mL) will be added to the cells for 30 minutes and proteins will be harvested in SDS- sample buffer. This dose of TNF- ⁇ should be sufficient for activation of p38 in most cell types. Samples will be run on an SDS-PAGE gel and probed with antibodies recognizing either the phosphorylated or total p38 protein.
  • rat neonatal cardiac myocytes will be incubated with 0, 0.1, 0.5 and 1 mg/mL of empty PK or PK-p38j for 6 hours. Following PK-p38i incubation, TNF- ⁇ (10 ng/mL) will be added to the cells for 18 hours and proteins will be harvested in SDS-sample buffer.
  • TNF- ⁇ (10 ng/mL) will be added to the cells for 18 hours and proteins will be harvested in SDS-sample buffer.
  • PK-encapsulated SB239063 may inhibit p38 phosphorylation and inflammatory cytokine production in both RAW and isolated primary macrophages.
  • PK-p38j will be generated as described in the methods section. We will perform these experiments using cultured RAW cells stimulated with TNF- ⁇ (10ng/mL) following incubation with 0, 0.1, 0.5 and 1 mg/mL of empty PK or PK-p38j for 6 hours. In one set of experiments, total proteins will be extracted following 30 minutes of stimulation and Western analysis will be performed for phosphorylated and total p38. Additionally, downstream effectors of p38 such as the transcription factor NFKB may be examined for activation. To measure cytokine production, we will stimulate cells with LPS and TNF- ⁇ following 6 hours of PK incubation.
  • cytokine production will be measured using the Bio-Plex cytokine assay from Bio-Rad.
  • Selected cytokines to be measured include, TNF- ⁇ , several members of the interleukin family, interferon- ⁇ , and several other well known inflammatory cytokines. Specifically, IL-I, IL-5, IL-6 and IL-8 have been implicated in cardiac dysfunction following myocardial infarction.
  • the above Bio-Plex assay may be followed up with Western analysis for confirmation.
  • mRNA from cultured and isolated cells will be isolated and we will perform real-time PCR for the same cytokines and using either 18S or a housekeeping gene ( ⁇ -tubulin) as a control.
  • PK-p38 will inhibit TNF- ⁇ -induced p38 phosphorylation in a dose- dependent manner. Additionally, we expect that this inhibition will be evident whether the PK-p38, is incubated for 6, 24, 48 or 72 hours before addition of TNF- ⁇ . In comparison, empty PK should have no effect on phosphorylation of p38, and free SB293063 should be a suitable positive control. It is possible that encapsulation may cause some of the inhibitor to be less active. If this is the case, we can either increase the starting amount of SB293063, or choose a replacement inhibitor. We also expect that PK-p38, treatment will reduce apoptosis induced by TNF- ⁇ .
  • JNK c-jun-N-terminal kinase
  • SAPK stress activated protein kinase
  • PK-p38 In addition to the effects on cardiac myocytes, we expect that PK-p38, treatment will dose-dependently inhibit phosphorylation of p38 and inflammatory cytokine production in cultured and isolated macrophages stimulated with TNF- ⁇ .
  • PKs containing the p38 inhibitor SB239063 may inhibit p38 phosphorylation, cardiac myocyte apoptosis, prevent inflammatory marker expression, and improve cardiac function following myocardial infarction.
  • PK-encapsulated SB239063 may inhibit p38 phosphorylation and apoptosis within the infarct zone for up to 7 days following myocardial infarction in rats, rescuing cardiac function.
  • PK-p38 will be generated as described in the methods section.
  • Adult male Sprague-Dawley rats will undergo sham surgery or coronary artery ligation as described in the methods and injected in a randomized and blinded manner with administration of empty PK, PK-p38, or free SB293063.
  • animals will be subjected to echocardiography and MRI by a trained technician under light anesthesia, and hearts will either be harvested for protein (n>5) or immunohistological studies (n>5) at the indicated time point.
  • the left ventricular free wall will be cut out and homogenized in a detergent buffer and proteins will be run on an SDS-PAGE gel and probed with antibodies recognizing both the phospho and total levels of p38.
  • This method has been used by our laboratory and others to reliably detect phospho-proteins in vivo following infarction (Hsieh et al., Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest, 2006. 116(l):237-48). Protein samples will also be examined for cleaved caspase-3 levels to determine levels of apoptosis.
  • blots will also be probed with antibodies against pro-inflammatory cytokines such as TNF- ⁇ and several members of the interleukin family, including IL-I, IL-5, IL-6, and IL-8 Densitometry will be performed using Quantity One software from Bio-Rad, and data compared statistically using ANOVA followed by the appropriate post-test. Our animal numbers are justified in the subsequent vertebrate animal section, but power analysis with our published variability has determined these numbers. Immunohistologically, sections will be probed with antibodies recognizing both phospho- p38 as well as a reliable cardiac myocyte marker (troponin I, ⁇ -sarcomeric actinin). We have published several papers using our standard protocol for immunohistochemistry.
  • tissue will be fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin for sectioning.
  • a standard microtome will be used to make 5 ⁇ m sections for all studies.
  • antigen retrieval will be performed with proteinase K (20 ⁇ g/mL). After washing, sections will be blocked with 10% serum prior to incubation with the indicated primary antibody and appropriate fluorescent-tagged secondary antibody. Finally, sections will be incubated with DAPI (1 ⁇ g/mL) for visualization of nuclei.
  • DAPI (1 ⁇ g/mL) for visualization of nuclei.
  • Both phospho-p38 staining intensity and double-positive cells will be quantified using ImagePro software. Intensity per unit area and percentage of double-positive cells will be compared statistically by ANOVA.
  • tissue sections will subjected to TUNEL staining per manufacturers protocol to determine a percentage of cardiac myocytes undergoing apoptosis. Data will be analyzed using ImagePro software and percentage of apoptotic cells will be counted and compared statistically with ANOVA followed by the appropriate post-test.
  • PK-encapsulated SB239063 may prevent long-term cardiac dysfunction and development of fibrosis.
  • PKs containing the p38 inhibitor SB239063 may improve cardiac stem cell survival and efficacy both in culture and in vivo.
  • PK-p38j may improve cultured cardiac stem cell survival and differentiation.
  • cells will be incubated for 18 hours with TNF- ⁇ and cells will be stained for common markers of apoptosis including cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Apoptosis will also be measured using TUNEL and Annexin-V staining. All images will be quantified using a Matlab program and expressed as percent-positive. Western blots will be scanned and quantified using Quantity One software from Bio-Rad. All experiments will be repeated at least 3 times before statistical comparison between groups with ANOVA followed the appropriate post-test.
  • Apoptosis including cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Apoptosis will also be measured using TUNEL and Annexin-V staining. All images will be quantified using a Matlab program and expressed as percent-positive. Western blots will be scanned and quantified using Quantity One software from Bio-
  • TNF-a (10 ng/mL)
  • TNF-a 10 ng/mL
  • cells will be fixed in 80% methanol for permeabilization and subsequent staining with cardiac-specific markers including the transcription factor Nkx2.5, cardiac myosin!, ⁇ -sarcomeric actinin, connexin-43 and troponin T.
  • cardiac-specific markers including the transcription factor Nkx2.5, cardiac myosin!, ⁇ -sarcomeric actinin, connexin-43 and troponin T.
  • PK-p38i will be generated as described in the methods.
  • Adult male Sprague-Dawley rats will be subjected to coronary artery ligation followed by immediate injection of empty PK, PK-p38j or SB239063 alone.
  • rats will be sacrificed and hearts fixed in 4% paraformaldehyde and embedded for immunohistological evaluation (n>5).
  • the presence, as well as the size, of c-Kit positive and Sca-1 positive cells in the infarct zone will be analyzed using immunofluorescence and imaging software and performed in a blinded and randomized fashion.
  • Double staining for the cardiac stem cell markers c-Kit or Sca-1 and the survival factor phospho-Akt will be examined as well using double immunofluorescence. Additionally, TUNEL and cleaved caspase-3 staining will also be co-localized with c-Kit and Sca-1 to determine if PK-p38j delivery improves survival of these endogenous stem cells. To measure maturation/differentiation, we will examine these progenitor cells for the expression of the cardiac markers ⁇ -sarcomeric actinin, troponin T, and for the presence of sarcomeres. Finally, to measure replication, we will examine BrdU incorporation and Ki67 staining of c-kit and sca-1 positive stem cells.
  • PK-p38i may improve implanted cardiac stem cell survival, growth and maturation.
  • Cardiac stem cells will be isolated and cultured as described in the methods section. Prior to use, cells will be infected with an adenovirus encoding hemagglutanin for tracking. In a randomized and double-blinded manner, immediately following coronary artery ligation in adult male Sprague-Dawley rats (n>5 per group), cells alone, cells with empty PK, cells with free SB239063, or cells with PK-p38j will be injected in to the infarct zone as inspected visually.
  • Sections will be co- stained for hemagglutanin, as well as cleaved caspase-3 and TUNEL for assessment of apoptosis.
  • cardiac maturation markers including cardiac myosin, ⁇ -sarcomeric actinin, and troponin T to determine differentiation/maturation.
  • sections will be stained with hemagglutanin and size will be measured with a program written in Matlab.
  • PCADK Polyketal synthesis- Poly(l,4-cyclohexane-acetone dimethylene ketal) (PCADK) will be synthesized, by reacting 1 ,4-cyclohexanedimethanol with 2,2-dimethoxypropane. PKs generated from PCADK should degrade in vivo to 1 ,4-cyclohexanedimethanol and acetone. PCADK will be synthesized in a 25 mL two-necked flask, connected to a short- path distilling head.
  • 1,4-cyclohexanedimethanol 1.0 g, Aldrich
  • 10 mL of warm ethyl acetate 10 mL of distilled benzene kept at 100°C.
  • Re- crystallized p-toluenesulfonic acid 5.5 mg, 0.029 mmol, Aldrich
  • ethyl acetate 550 ⁇ L of ethyl acetate will then be added to the benzene solution.
  • the ethyl acetate will be allowed to distill off, and distilled 2,2-dimethoxypropane (900 ⁇ L, 7.4 mmol, Aldrich) will be added to initiate the reaction.
  • PVA as the surfactant stabilizer.
  • a representative procedure is described below.
  • a 500 ⁇ L solution of SB239063 (1 mg/ml) will be dispersed into an organic phase, which consists of 75 mg of PCADK dissolved in ImL of dichloromethane. This solution will be homogenized in 4% PVA at the lowest setting for 1 minute, and should generate a water- in-oil (w/o) emulsion. The resulting w/o emulsion will then be poured into 30 mL of a
  • Myocardial injection/infarction- Adult male Sprague-Dawley rats weighing 250 grams will be subjected to myocardial infarction or injection surgeries as described. Briefly, the animals will be anesthetized (1-3% isoflurane) and, following tracheal intubation, the heart will exposed by separation of the ribs. Myocardial infarction will be performed by ligation of the left anterior descending coronary artery. For PK injection or cell therapy studies, immediately after coronary artery ligation, treatment (50-100 ⁇ L) will be injected into the infarct zone through a 30-gauge needle while the heart is beating. Following injection, the chests will be closed and animals will recover on a heating pad.
  • Echocardiograms will be performed when described in the experimental design. I have experience in this animal model and we have obtained reproducible infarcts as measured by echocardiography. We have performed several experiments in rats and Figure 48 shows a graph of grouped data from 10 animals undergoing sham or coronary artery ligation surgery. On the Y-axis is %fractional shortening after 3 days, a common measurement of cardiac function using echocardiography.
  • Neonatal cardiac myocyte isolation By following this protocol, we have been able to recover neonatal cardiac myocytes with 95-99% purity (Davis et al., Local myocardial insulin-like growth factor 1 (IGF-I) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci U S A, 2006. 103(21):8155-60; Davis et al., Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation, 2005.
  • IGF-I Local myocardial insulin-like growth factor 1
  • the resulting pellet will be resuspended in Medium 199 containing 20% fetal bovine serum and plated for 2 hours.
  • the nonadherent cells will be plated on fibronectin coated dishes for experiments or collected and analyzed by flow cytometry with the use of an antibody to ⁇ -sarcomeric actin to determine myocyte composition.
  • Cardiac stem cell isolation Hearts of adult Sprague-Dawley rats will be isolated, minced, and incubated with collagenase Type 2 (0.8 mg/ml) for 1 hour at 37°C. The supernatant will be isolated by centrifugation and then incubated with a c-Kit antibody conjugated to magnetic beads (Dynal). A second selection step will take place after cells reach confluency with an antibody directed against Sca-1. Cell phenotype will be determined with flow cytometry against c-Kit, Sca-1, and lineage markers. Lineage markers to be used are CD34, CD45, CD31, connexin43, cardiac myosin and von
  • Immunostaining- Hearts will be isolated at the indicated time point and fixed in 4% paraformaldehyde for 6 hours at room temperature. Following dehydration, hearts will be embedded in paraffin and 5 ⁇ m sections will be made for immunohistochemistry. Paraffin will be removed by immersion in Xylene and sections will be probed with antibodies as described in the methods. Antibodies to cardiac markers (troponin T, ⁇ - sarcomeric-actin, cardiac myosin) are commercially available and have been used in our laboratory.
  • Echocardiography/MRI- Echocardiography and MRI will be performed under light anesthesia by an experienced technician. All measurements will be taken and analyzed in a double-blinded manner. Left ventricular end systolic and diastolic measurements will be taken from M-mode short-axis images, and confirmed in long-axis views.
  • a strategy is presented for manipulating the hydrolysis kinetics of poly(cyclohexane-l,4-diyl acetone dimethylene ketal) (PCADK) by controlling its hydrophilicity through copolymerization with other diols, of varying hydrophilicity.
  • PCADK poly(cyclohexane-l,4-diyl acetone dimethylene ketal)
  • Polyketal copolymers were synthesized in a 25 mL two-necked flask, connected to a short-path distilling head.
  • the diols, 1 ,4-cyclohexanedimethanol (1.04 g, 7.25 mmol) and, either 1,4-butanediol, 1,5-pentanediol, 1 ,6-hexanediol, or 1,8-octanediol were dissolved in 20 mL of distilled benzene and kept at 100°C.
  • Re-crystallized p- toluenesulfonic acid (5.5 mg, 0.029 mmol), dissolved in 550 ⁇ L of ethyl acetate, was added to the benzene solution.
  • the ethyl acetate was distilled off, and distilled 2,2- dimethoxypropane (equal molar quantity as the two diols combined) was added to initiate the reaction.
  • Additional doses of 2,2-dimethoxypropane (500 ⁇ L) and benzene (2 mL) were subsequently added to the reaction, every hour for 6 hours, via a metering funnel, to compensate for 2,2-dimethoxypropane and benzene that had distilled off.
  • the hydrolysis of the polyketal copolymers was measured in buffered water at pH values of 1.0 (.1M HCL), 4.5 (100 mM AcOH) and 7.4 (100 mM) at 37 0 C. Briefly, 20mg of the polymer samples were placed in a 5 ml vial, 1 mL of buffer solution was added to each vial, and mixed with a magnetic stir bar. At specific time points, 1 mL of CDC13 was added to each vial and shaken vigorously, the CDCl 3 phase was isolated and analyzed by 1 H NMR, to determine the percent hydrolysis the of ketal linkages in polyketal copolymer.
  • Figure 51 shows a synthetic scheme of the acetal-exchange reaction used to make the polyketals and the degradation products generated from their hydrolysis.
  • the synthesis of all the polyketal copolymers was accomplished in one step, on a multi-gram scale, and with yields of 50 - 60%.
  • the introduction of diols other than CDM did not cause any complications in the synthesis, and procedures developed for the synthesis of PCADK were suitable for the synthesis of the copolymers.
  • all the copolyketals synthesized were crystalline, and therefore have the potential for formulation into microparticles.
  • PKl The hydrolysis kinetics of PKl to PK6 was measured at the pH values of 4.5 and 7.4 to determine their behavior in the acidic environment of phagosomes and in the blood.
  • Figure 52 demonstrates that all polyketal copolymers undergo acid-catalyzed hydrolysis and that their hydrolysis kinetics scale inversely with their hydrophobicity.
  • PKl, PK2, and PK3 were copolymers synthesized from 1 ,4-cyclohexanedimethanol and 1,5- pentanediol.
  • polyketal copolymers PK4, PK5, and PK6 were also synthesized from butanediol, hexanediol, and octanediol, which have differing hydrophobicity from pentanediol, and their hydrolysis kinetics were investigated.
  • Table 2 demonstrates that this set of copolymers also has an inverse relationship between hydrophobicity and hydrolysis kinetics.
  • PK4 a copolymer synthesized using 1 ,4- 10 cyclohexanedimethanol and 1,4-butanediol
  • PK6 synthesized using 1 ,4-cyclohexanedimethanol and a more hydrophobic monomer, 1,8-octanediol, had a pH 4.5 hydrolysis half-life of 18.6 days.
  • PK3 in Table 1 One of the polyketal copolymers synthesized (termed PK3 in Table 1), consisting of cyclohexane-dimethanol and pentane diol, had a hydrolysis half life of 2 days at pH 4.5 but several weeks at pH 7.4. This polyketal may be useful for delivering therapeutic drugs to a subject because it should hydrolyze rapidly after phagocytosis; however it is relatively stable at physiological pH. Additionally, PK3 may be suitable for microparticle drug delivery because of its rapid hydrolysis kinetics and biocompatible degradation products, which are 1,5-pentanediol, 1 ,4-cyclohexanedimethanol, and acetone.
  • microparticles were formulated from PK3 using a solvent evaporation procedure.
  • Figure 53 demonstrates that microparticles can be formed with PK3, and that the particles have a size between 1-5 microns, which suitable for phagocytosis by macrophages.
  • therapeutic drugs may be encapsulated into PK3 microparticles (for example by using a water/oil/water double emulsion procedure or other method) for adminstration into a subject.
  • Microparticles loaded with active agents will be formulated from the polyketal copolymers using a modified water/oil/water emulsion method (Ando et al., Journal of Pharmaceutical Sciences 1999, 88, 126-130).
  • PK3 (lOOmg) will be dissolved in 1 mL of dichloromethane, in a separate vial, 40 mg of the active agent will be dissolved in 400 ⁇ L of D.I. water.
  • the aqueous solution of the active agent will be mixed with the PK3 solution, and sonicated for 60 seconds (Misonix Incorporated, Farmigdale, NY).
  • the sonicated mixture will then immersed in liquid nitrogen for 15 sec, and 12 mL of a 5% w/w PVA solution (pH 7.45) will be added to it.
  • This mixture will be homogenized for 120 seconds with a Powergen 500 homogenizer (Fisher Scientific, Waltham, MA), and then transferred to a beaker containing 40 mL of 1 % w/w PVA (pH 7.45).
  • This solution will then be stirred for 3 hours with a magnetic stir bar to evaporate the organic solvent.
  • the particles will be isolated by centrifuging at 10,000 rmp for 15 min, washed twice with 15 mL of PBS buffer and freeze dried.
  • a polyketal molecule of the invention was modified with a linker that can bind an active agent.
  • a linker that can bind an active agent.
  • NTA nitrilotriacetic acid
  • the NTA linker when loaded with nickel (Ni) allowed the binding of histidine-tagged proteins (Figure 54).
  • Ni nickel
  • the modified PCADK then formed microspheres ideal for rapid delivery of active agents (e.g. growth factors) ( Figure 55).
  • PCADK was synthesized as previously described above (see also Lee S et al. Polyketal microparticles: a new delivery vehicle for superoxide dismutase. Bioconjug Chem. 2007 Jan-Feb;18(l):4-7 and Heffeman MJ and Murthy N. Polyketal nanoparticles: a new pH- sensitive biodegradable drug delivery vehicle. Bioconjug Chem. 2005 Nov-Dec; 16(6): 1340-2, herein incorporated by reference). After synthesis of the polyketal, 90 mg of PCADK was dissolved in 1 mL of a 10 mg/mL solution of DOGS-NTA (Avanti Polar Lipids) in dichloromethane (final concentration of 10% DOGS-NTA).
  • DOGS-NTA Advanti Polar Lipids
  • the mixture was sonicated for several seconds/minutes in a water bath sonicator.
  • the polymer solution was then poured into a vial containing 5 mL of PVA (2%) and homogenized for 60 seconds.
  • the resulting solution was stirred over 40 mL of 0.5% PVA for 4-6 hours.
  • the particles were then spun down and washed several times in deionized water and frozen in liquid nitrogen for lyophilization. The resulting freeze-dried particles were imaged using SEM and shown in Figure 56.
  • particles In order to get particles capable of binding to His-tagged proteins, particles needed to be loaded with nickel. Thus, particles were incubated with 50 mM NiCl 2 at 10 mg/mL and agitated for 2 hours at room temperature. To determine nickel uptake, we incubated the particles overnight with NiCl 2 , spun down the particles, and measured the unbound nickel in the supernatant spectrophotometrically. As the data in Figure 57 demonstrate, at varying NTA loading there was a decrease in free nickel in the solution, indicating the particles had been loaded with nickel.
  • NiNTA on particles was capable of binding His-tagged proteins
  • varying concentrations of His-GFP were incubated with PCADK-NiNTA (1% solution w/v) overnight at 4 degrees Celsius. Particles were centrifuged out and washed several times in PBS before resuspension for analysis.
  • Figure 58 is a representative fluorescent stain for GFP that was converted to grayscale for presentation. As the image shows, the particles retained GFP on their surface.
  • particles were loaded with increasing amounts of His-GFP, washed several times, and ELISA was run on the particles to determine levels of binding.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Pulmonology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Diabetes (AREA)
  • Nanotechnology (AREA)
  • Dermatology (AREA)
  • Polymers & Plastics (AREA)
  • Dispersion Chemistry (AREA)
  • Obesity (AREA)
  • AIDS & HIV (AREA)
  • Transplantation (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)

Abstract

La présente invention concerne des particules biodégradables (par exemple des particules tridimensionnelles) et des micelles qui peuvent être utilisées pour l'encapsulation d'agents actifs en vue de leur administration à un sujet. La présente invention concerne, en outre, des procédés de production et d'administration desdites particules et micelles. De plus, l'invention concerne des stratégies vaccinales impliquant l'utilisation de ces particules et micelles inédites.
PCT/US2008/003422 2007-04-12 2008-03-12 Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules WO2008127532A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2008239795A AU2008239795A1 (en) 2007-04-12 2008-03-12 Novel strategies for delivery of active agents using micelles and particles
EP08726853A EP2146747A1 (fr) 2007-04-12 2008-03-12 Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules
JP2010503004A JP2010523656A (ja) 2007-04-12 2008-03-12 ミセルおよび粒子を用いた、活性物質の送達のための新規の戦略
CA002684052A CA2684052A1 (fr) 2007-04-12 2008-03-12 Strategies inedites d'administration d'agents actifs au moyen de micelles et de particules

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92313607P 2007-04-12 2007-04-12
US60/923,136 2007-04-12

Publications (1)

Publication Number Publication Date
WO2008127532A1 true WO2008127532A1 (fr) 2008-10-23

Family

ID=39590347

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/003422 WO2008127532A1 (fr) 2007-04-12 2008-03-12 Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules

Country Status (5)

Country Link
EP (1) EP2146747A1 (fr)
JP (1) JP2010523656A (fr)
AU (1) AU2008239795A1 (fr)
CA (1) CA2684052A1 (fr)
WO (1) WO2008127532A1 (fr)

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010138193A2 (fr) 2009-05-27 2010-12-02 Selecta Biosciences, Inc. Nanosupports de synthèse ciblés se caractérisant par une libération sensible au ph d'agents immunomodulateurs
WO2011150240A1 (fr) 2010-05-26 2011-12-01 Selecta Biosciences, Inc. Compositions de nanovecteurs comportant un adjuvant découplé
WO2012149282A2 (fr) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogéniques pour la génération de lymphocytes t régulateurs cd8+
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
WO2013019669A2 (fr) 2011-07-29 2013-02-07 Selecta Biosciences, Inc. Nanosupports synthétiques qui génèrent des réponses immunitaires humorales et de lymphocytes t cytotoxiques (ltc)
WO2014197616A1 (fr) 2013-06-04 2014-12-11 Selecta Biosciences, Inc. Administration répétée d'agents immunothérapeutiques spécifiques de l'antigène non immunosuppresseurs
WO2015200728A1 (fr) 2014-06-25 2015-12-30 Selecta Biosciences, Inc. Méthodes et compositions de traitement par nanovecteurs de synthèse et inhibiteurs de point de contrôle immunitaire
WO2016037164A1 (fr) 2014-09-07 2016-03-10 Selecta Biosciences, Inc. Procédés et compositions pour atténuer des réponses immunitaires contre des vecteurs de transfert viraux modulant l'expression génique
WO2018064215A1 (fr) 2016-09-27 2018-04-05 Selecta Biosciences, Inc. Immunotoxines recombinantes destinées à être utilisées dans le traitement du cancer
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
WO2018129268A1 (fr) 2017-01-07 2018-07-12 Selecta Biosciences, Inc. Dosage systématique d'immunosuppresseurs couplés à des nanovecteurs synthétiques
WO2019075360A1 (fr) 2017-10-13 2019-04-18 Selecta Biosciences, Inc. Méthodes et compositions permettant d'atténuer les réponses en igm anti-vecteur de transfert viral
US20190365652A1 (en) * 2016-04-29 2019-12-05 Children's Medical Center Corporation Poly(ketals) and related compositions and methods
WO2020018583A1 (fr) 2018-07-16 2020-01-23 Selecta Biosciences, Inc. Procédés et compositions de constructions et de vecteurs otc
WO2020018587A1 (fr) 2018-07-16 2020-01-23 Selecta Biosciences, Inc. Procédés et compositions de constructions et de vecteurs mma
WO2020060908A1 (fr) * 2018-09-17 2020-03-26 The Trustees Of Columbia University In The City Of New York Cétals et polycétals en tant qu'agents de libération
WO2020223205A1 (fr) 2019-04-28 2020-11-05 Selecta Biosciences, Inc. Méthodes de traitement de sujets présentant une immunité préexistante à des vecteurs de transfert viraux
WO2020243261A1 (fr) 2019-05-28 2020-12-03 Selecta Biosciences, Inc. Procédés et compositions permettant d'atténuer les réponses immunitaires anti-virales aux vecteurs de transfert
EP3763359A1 (fr) 2013-05-03 2021-01-13 Selecta Biosciences, Inc. Procédé et compositions pour augmenter les cellules t régulatrices cd4+
WO2021081062A1 (fr) 2019-10-21 2021-04-29 Selecta Biosciences, Inc. Méthodes et compositions pour traiter des maladies et troubles hépatiques
WO2021174013A1 (fr) 2020-02-26 2021-09-02 Selecta Biosciences, Inc. Méthodes et compositions utilisant des nanovecteurs synthétiques comprenant un immunosuppresseur
WO2021211100A1 (fr) 2020-04-14 2021-10-21 Selecta Biosciences, Inc. Procédés et compositions pour induire une autophagie
US11331392B2 (en) 2014-12-04 2022-05-17 The Trustees Of Columbia University In The City Of New York Biodegradable thermo-responsive polymers and uses thereof
WO2022150335A1 (fr) 2021-01-05 2022-07-14 Selecta Biosciences, Inc. Protocoles de dosage de vecteurs viraux
WO2022217095A1 (fr) 2021-04-09 2022-10-13 Selecta Biosciences, Inc. Nanovecteurs synthétiques comprenant un immunosuppresseur en combinaison avec des agonistes du récepteur à l'il-2 à haute affinité destinés à améliorer la tolérance immunitaire
WO2023064350A1 (fr) 2021-10-12 2023-04-20 Selecta Biosciences, Inc. Protocoles de dosage de vecteurs viraux
WO2023064367A1 (fr) 2021-10-12 2023-04-20 Selecta Biosciences, Inc. Méthodes et compositions permettant d'atténuer les réponses anti-igm de vecteur de transfert viral
WO2023086615A1 (fr) 2021-11-14 2023-05-19 Selecta Biosciences, Inc. Dosage multiple avec vecteurs viraux
WO2023133319A1 (fr) 2022-01-10 2023-07-13 Selecta Biosciences, Inc. Agonistes du récepteur de l'il-2 à haute affinité et réduction de dose de nanotransporteur synthétique
WO2023172624A1 (fr) 2022-03-09 2023-09-14 Selecta Biosciences, Inc. Immunosuppresseurs en association avec des agents anti-igm et dosage associé
WO2023172628A1 (fr) 2022-03-09 2023-09-14 Selecta Biosciences, Inc. Immunosuppresseur en combinaison avec des agonistes du récepteur d'il-2 à haute affinité et dosage associé
WO2023183568A1 (fr) 2022-03-25 2023-09-28 Selecta Biosciences, Inc. Nanovecteurs synthétiques comprenant un immunosuppresseur en association avec des agonistes du récepteur il -2 à haute affinité et des agents anti-igm
WO2023196566A1 (fr) 2022-04-08 2023-10-12 Selecta Biosciences, Inc. Agonistes du récepteur de l'il-2 à haute affinité et immunosuppresseurs pour améliorer la tolérance immunitaire
WO2024107889A1 (fr) 2022-11-15 2024-05-23 Selecta Biosciences, Inc. Compositions et méthodes de traitement de la cholangite biliaire primitive

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023294A2 (fr) * 2003-09-05 2005-03-17 The General Hospital Corporation Systeme de liberation de medicament en deux phase
WO2006122223A2 (fr) * 2005-05-10 2006-11-16 Emory University Nouvelles strategies d'administration d'agents actifs utilisant des micelles et des particules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4586022B2 (ja) * 2003-09-29 2010-11-24 日東電工株式会社 インビボポリヌクレオチド送達用生分解性ポリアセタール

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023294A2 (fr) * 2003-09-05 2005-03-17 The General Hospital Corporation Systeme de liberation de medicament en deux phase
WO2006122223A2 (fr) * 2005-05-10 2006-11-16 Emory University Nouvelles strategies d'administration d'agents actifs utilisant des micelles et des particules

Cited By (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9884112B2 (en) 2009-05-27 2018-02-06 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US9006254B2 (en) 2009-05-27 2015-04-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
WO2010138193A2 (fr) 2009-05-27 2010-12-02 Selecta Biosciences, Inc. Nanosupports de synthèse ciblés se caractérisant par une libération sensible au ph d'agents immunomodulateurs
WO2010138194A2 (fr) 2009-05-27 2010-12-02 Selecta Biosciences, Inc. Composés agents immunomodulateurs-polymères
US8629151B2 (en) 2009-05-27 2014-01-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
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
WO2011150240A1 (fr) 2010-05-26 2011-12-01 Selecta Biosciences, Inc. Compositions de nanovecteurs comportant un adjuvant découplé
EP3388081A1 (fr) 2010-05-26 2018-10-17 Selecta Biosciences, Inc. Vaccins à nanovecteurs synthétiques multivalents
US9066978B2 (en) 2010-05-26 2015-06-30 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9764031B2 (en) 2010-05-26 2017-09-19 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
WO2012149393A2 (fr) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes destinés à une délétion, spécifique à un antigène, de cellules effectrices
EP3679933A1 (fr) 2011-04-29 2020-07-15 Selecta Biosciences, Inc. Nanosupports synthétiques tolérogènes afin de réduire les réponses immunitaires à des protéines thérapeutiques
EP3682878A1 (fr) 2011-04-29 2020-07-22 Selecta Biosciences, Inc. Libération contrôlée d'immunosuppresseurs à partir de nanosupports synthétiques
WO2012149259A1 (fr) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes destinés à réduire des réponses impliquant des anticorps
EP3848030A1 (fr) 2011-04-29 2021-07-14 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes destinés à réduire des réponses impliquant des lymphocytes t cytotoxiques
EP3682877A1 (fr) 2011-04-29 2020-07-22 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes
WO2012149268A1 (fr) 2011-04-29 2012-11-01 Selecta Biociences, Inc. Nanosupports synthétiques tolérogènes pour thérapie d'une allergie
EP3871691A1 (fr) 2011-04-29 2021-09-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes destinés à une délétion, spécifique à un antigène, de cellules effectrices t
WO2012149282A2 (fr) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogéniques pour la génération de lymphocytes t régulateurs cd8+
WO2012149301A2 (fr) 2011-04-29 2012-11-01 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogéniques pour l'induction de lymphocytes b régulateurs
EP3760201A1 (fr) 2011-04-29 2021-01-06 Selecta Biosciences, Inc. Nanovecteurs synthétiques tolérogènes destinés à réduire des réponses impliquant des anticorps
US10933129B2 (en) 2011-07-29 2021-03-02 Selecta Biosciences, Inc. Methods for administering synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte responses
WO2013019669A2 (fr) 2011-07-29 2013-02-07 Selecta Biosciences, Inc. Nanosupports synthétiques qui génèrent des réponses immunitaires humorales et de lymphocytes t cytotoxiques (ltc)
EP3763359A1 (fr) 2013-05-03 2021-01-13 Selecta Biosciences, Inc. Procédé et compositions pour augmenter les cellules t régulatrices cd4+
EP3858348A1 (fr) 2013-06-04 2021-08-04 Selecta Biosciences, Inc. Administration répétée d'agents immunothérapeutiques spécifiques de l'antigène non immunosuppresseurs
WO2014197616A1 (fr) 2013-06-04 2014-12-11 Selecta Biosciences, Inc. Administration répétée d'agents immunothérapeutiques spécifiques de l'antigène non immunosuppresseurs
WO2015200728A1 (fr) 2014-06-25 2015-12-30 Selecta Biosciences, Inc. Méthodes et compositions de traitement par nanovecteurs de synthèse et inhibiteurs de point de contrôle immunitaire
WO2016037164A1 (fr) 2014-09-07 2016-03-10 Selecta Biosciences, Inc. Procédés et compositions pour atténuer des réponses immunitaires contre des vecteurs de transfert viraux modulant l'expression génique
WO2016037162A1 (fr) 2014-09-07 2016-03-10 Selecta Biosciences, Inc. Procédés et compositions permettant d'atténuer les réponses immunitaires au vecteur de transfert anti-virales
US11331392B2 (en) 2014-12-04 2022-05-17 The Trustees Of Columbia University In The City Of New York Biodegradable thermo-responsive polymers and uses thereof
US20190365652A1 (en) * 2016-04-29 2019-12-05 Children's Medical Center Corporation Poly(ketals) and related compositions and methods
US11071714B2 (en) * 2016-04-29 2021-07-27 Children's Medical Center Corporation Poly(ketals) and related compositions and methods
WO2018064215A1 (fr) 2016-09-27 2018-04-05 Selecta Biosciences, Inc. Immunotoxines recombinantes destinées à être utilisées dans le traitement du cancer
WO2018129268A1 (fr) 2017-01-07 2018-07-12 Selecta Biosciences, Inc. Dosage systématique d'immunosuppresseurs couplés à des nanovecteurs synthétiques
WO2019075360A1 (fr) 2017-10-13 2019-04-18 Selecta Biosciences, Inc. Méthodes et compositions permettant d'atténuer les réponses en igm anti-vecteur de transfert viral
WO2020018587A1 (fr) 2018-07-16 2020-01-23 Selecta Biosciences, Inc. Procédés et compositions de constructions et de vecteurs mma
WO2020018583A1 (fr) 2018-07-16 2020-01-23 Selecta Biosciences, Inc. Procédés et compositions de constructions et de vecteurs otc
WO2020060908A1 (fr) * 2018-09-17 2020-03-26 The Trustees Of Columbia University In The City Of New York Cétals et polycétals en tant qu'agents de libération
WO2020223205A1 (fr) 2019-04-28 2020-11-05 Selecta Biosciences, Inc. Méthodes de traitement de sujets présentant une immunité préexistante à des vecteurs de transfert viraux
WO2020243261A1 (fr) 2019-05-28 2020-12-03 Selecta Biosciences, Inc. Procédés et compositions permettant d'atténuer les réponses immunitaires anti-virales aux vecteurs de transfert
WO2021081062A1 (fr) 2019-10-21 2021-04-29 Selecta Biosciences, Inc. Méthodes et compositions pour traiter des maladies et troubles hépatiques
WO2021174013A1 (fr) 2020-02-26 2021-09-02 Selecta Biosciences, Inc. Méthodes et compositions utilisant des nanovecteurs synthétiques comprenant un immunosuppresseur
WO2021211100A1 (fr) 2020-04-14 2021-10-21 Selecta Biosciences, Inc. Procédés et compositions pour induire une autophagie
WO2022150335A1 (fr) 2021-01-05 2022-07-14 Selecta Biosciences, Inc. Protocoles de dosage de vecteurs viraux
WO2022217095A1 (fr) 2021-04-09 2022-10-13 Selecta Biosciences, Inc. Nanovecteurs synthétiques comprenant un immunosuppresseur en combinaison avec des agonistes du récepteur à l'il-2 à haute affinité destinés à améliorer la tolérance immunitaire
WO2023064350A1 (fr) 2021-10-12 2023-04-20 Selecta Biosciences, Inc. Protocoles de dosage de vecteurs viraux
WO2023064367A1 (fr) 2021-10-12 2023-04-20 Selecta Biosciences, Inc. Méthodes et compositions permettant d'atténuer les réponses anti-igm de vecteur de transfert viral
WO2023086615A1 (fr) 2021-11-14 2023-05-19 Selecta Biosciences, Inc. Dosage multiple avec vecteurs viraux
WO2023133319A1 (fr) 2022-01-10 2023-07-13 Selecta Biosciences, Inc. Agonistes du récepteur de l'il-2 à haute affinité et réduction de dose de nanotransporteur synthétique
WO2023172624A1 (fr) 2022-03-09 2023-09-14 Selecta Biosciences, Inc. Immunosuppresseurs en association avec des agents anti-igm et dosage associé
WO2023172628A1 (fr) 2022-03-09 2023-09-14 Selecta Biosciences, Inc. Immunosuppresseur en combinaison avec des agonistes du récepteur d'il-2 à haute affinité et dosage associé
WO2023183568A1 (fr) 2022-03-25 2023-09-28 Selecta Biosciences, Inc. Nanovecteurs synthétiques comprenant un immunosuppresseur en association avec des agonistes du récepteur il -2 à haute affinité et des agents anti-igm
WO2023196566A1 (fr) 2022-04-08 2023-10-12 Selecta Biosciences, Inc. Agonistes du récepteur de l'il-2 à haute affinité et immunosuppresseurs pour améliorer la tolérance immunitaire
WO2024107889A1 (fr) 2022-11-15 2024-05-23 Selecta Biosciences, Inc. Compositions et méthodes de traitement de la cholangite biliaire primitive

Also Published As

Publication number Publication date
AU2008239795A1 (en) 2008-10-23
CA2684052A1 (fr) 2008-10-23
EP2146747A1 (fr) 2010-01-27
JP2010523656A (ja) 2010-07-15

Similar Documents

Publication Publication Date Title
WO2008127532A1 (fr) Stratégies inédites d'administration d'agents actifs au moyen de micelles et de particules
US8252846B2 (en) Strategies for delivery of active agents using micelles and particles
EP1893661B1 (fr) Nouvelles strategies d'administration d'agents actifs utilisant des micelles et des particules
Muhammad et al. Modulation of immune responses with nanoparticles and reduction of their immunotoxicity
Carrillo-Conde et al. Mannose-functionalized “pathogen-like” polyanhydride nanoparticles target C-type lectin receptors on dendritic cells
Yang et al. Estrone-modified pH-sensitive glycol chitosan nanoparticles for drug delivery in breast cancer
Ahmad et al. Gelatin-coated polycaprolactone nanoparticle-mediated naringenin delivery rescue human mesenchymal stem cells from oxygen glucose deprivation-induced inflammatory stress
Kwon et al. Enhanced antigen presentation and immunostimulation of dendritic cells using acid-degradable cationic nanoparticles
Yoon et al. A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice
Jain et al. PEG–PLA–PEG block copolymeric nanoparticles for oral immunization against hepatitis B
CA2735318C (fr) Nanoparticules pour immunotherapie
AU2005237270B2 (en) Pegylated nanoparticles
Park et al. Antioxidant and anti-inflammatory activities of hydroxybenzyl alcohol releasing biodegradable polyoxalate nanoparticles
Jain et al. Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B
EP2402032B1 (fr) Composition immunogène
Li et al. Surface-functionalized, pH-responsive poly (lactic-co-glycolic acid)-based microparticles for intranasal vaccine delivery: Effect of surface modification with chitosan and mannan
Abou-ElNour et al. Microparticles-in-thermoresponsive/bioadhesive hydrogels as a novel integrated platform for effective intra-articular delivery of triamcinolone acetonide
Song et al. Photoresponsive polypeptide-glycosylated dendron amphiphiles: UV-triggered polymersomes, OVA release, and in vitro enhanced uptake and immune response
Li et al. Guanidinylated cationic nanoparticles as robust protein antigen delivery systems and adjuvants for promoting antigen-specific immune responses in vivo
Gupta et al. Tolerogenic Modulation of the Immune Response by Oligoglycerol–and Polyglycerol–Peptide Conjugates
Anfray et al. Nanoparticles for immunotherapy
Tan et al. Epsilon-caprolactone modified polyethylenimine for highly efficient antigen delivery and chemical exchange saturation transfer functional MR imaging
Geisshüsler et al. Amphiphilic Cyclodextrin‐Based Nanoparticulate Vaccines Can Trigger T‐Cell Immune Responses
WO2022098467A1 (fr) Procédés, compositions et vaccin thérapeutique pour des maladies auto-immunes et le traitement des allergies
Coué et al. Development and in vitro Evaluation of Antigen‐Loaded Poly (amidoamine) Nanoparticles for Respiratory Epithelium Applications

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08726853

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2684052

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2010503004

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2008239795

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2008726853

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2008239795

Country of ref document: AU

Date of ref document: 20080312

Kind code of ref document: A