WO2016025747A1 - Copolymères non linéaires en tant que véhicules d'administration intracellulaire et procédés associés - Google Patents

Copolymères non linéaires en tant que véhicules d'administration intracellulaire et procédés associés Download PDF

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WO2016025747A1
WO2016025747A1 PCT/US2015/045114 US2015045114W WO2016025747A1 WO 2016025747 A1 WO2016025747 A1 WO 2016025747A1 US 2015045114 W US2015045114 W US 2015045114W WO 2016025747 A1 WO2016025747 A1 WO 2016025747A1
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copolymer
nonlinear
block
copolymers
polymer
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Patrick S. Stayton
Anthony J. Convertine
John Wilson
John Chiefari
Almar Postma
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol

Definitions

  • CD8+ cytotoxic T cell (CTL) responses Activation of CD8+ cytotoxic T cell (CTL) responses is widely considered to be an essential component for effective vaccination against many intracellular pathogens and cancers.
  • CTL cytotoxic T cell
  • the vast majority of currently approved vaccine formulations primarily elicit humoral immune responses with minimal activation of cellular immunity.
  • Recombinant viral vectors have been engineered that elicit strong CTL responses, but anti-vector immunity can dramatically compromise efficacy and safety concerns remain.
  • recombinant protein-based vaccines provide antigen specificity and generally favorable safety profiles, but at the expense of immunogenicity and cellular immunity.
  • nanoparticles that efficiently promote antigen presentation on class I major histocompatibility complex (MHC) by dendritic cells.
  • MHC major histocompatibility complex
  • nanoparticles in the viral size range (about 20-200 nm) that enhance class I cross-presentation of exogenous protein antigens.
  • these include liposomes, immune stimulating complexes (ISCOMs), inorganic nanoparticles, and polymer-based nanoparticles such as polymerosomes, dendrimers, and micelles.
  • ISCOMs immune stimulating complexes
  • RAFT-synthesized block copolymers have been developed as carriers that alter intracellular trafficking pathways and enhance cytosolic delivery of proteins. These micellar carriers have been shown to enhance class I antigen presentation in vitro and elicit CD8+ T cell responses in vivo.
  • RAFT reversible-addition fragmentation chain transfer
  • the invention provides nonlinear copolymers.
  • the nonlinear copolymer comprises a pH-responsive, endosomal releasing block covalently coupled to a hydrophilic block, wherein at least one of the pH-responsive, endosomal releasing block and the hydrophilic block is a nonlinear block.
  • the pH-responsive, endosomal releasing block is nonlinear. In certain embodiments, the hydrophilic block is nonlinear. In certain embodiments, the pH-responsive, endosomal releasing block is nonlinear and the hydrophilic block is nonlinear. In certain embodiments, the pH-responsive, endosomal releasing block is nonlinear and the hydrophilic block is linear.
  • the copolymer is a branched copolymer. In other embodiments, the copolymer is a crosslinked copolymer. In further embodiments, the copolymer is a star copolymer.
  • the copolymer comprises one or more degradable groups.
  • Representative nonlinear copolymers of the invention include copolymers comprising constitutional units prepared from one or more monomers selected from the group consisting of acrylates, alkacrylates, acrylamides, alkacrylamides, vinylidenes, and styrenes.
  • the pH-responsive, endosomal releasing block comprises constitutional units prepared from one or more monomers selected from the group consisting of an alkyl acrylate, an aminoalkyl acrylate, and an acrylic acid.
  • the hydrophilic block comprises constitutional units prepared from one or more monomers selected from the group consisting of uncharged monomers, cationic monomers, anionic monomers, and zwitterionic monomers.
  • the nonlinear copolymers of the invention advantageously further include a biological moiety covalently coupled to the copolymer.
  • the biological moiety covalently coupled to the copolymer by a cleavable linkage.
  • the biological moiety covalently coupled to the copolymer by a reducible linkage.
  • Representative biological moieties are selected from a nucleic acid, a protein, a lipid, a carbohydrate, and a small molecule.
  • the biological moiety is an antigen, such as an immunogen.
  • compositions comprising a nonlinear copolymer and an acceptable carrier.
  • Representative compositions include pharmaceutical compositions such as immunogenic compositions.
  • the invention provides particles that include the nonlinear copolymers.
  • the particle comprises one or more nonlinear copolymers.
  • the particle comprises a single nonlinear copolymer.
  • the particle is a micelle.
  • the particle is a core-shell nanoparticle.
  • the invention provides a method for intracellular delivery of a biological molecule.
  • a cell is contacted with a nonlinear copolymer of the invention, whereby the nonlinear copolymer is incorporated into an endosomal membrane within the cell through endocytosis, the endosomal membrane is destabilized by the presence of the nonlinear copolymer, and the biological molecule is released from the nonlinear copolymer and delivered to the cytosol of the cell.
  • the invention provides a method for preventing or treating a disease or condition. In the method, an effective amount of a nonlinear copolymer of the invention is administered to a subject in need thereof.
  • FIGURES 1A-1C depict pH-responsive nanoparticles assembled using polymer chains of different architecture that were utilized as carriers for delivery of a biological moiety into the MHC-I antigen processing pathway.
  • pH-Responsive diblock copolymers were synthesized with linear (L), crosslinked (CL), or hyperbranched (HB) architectures (1A).
  • Representative nanoparticle having pH-responsive, endosomal releasing component that is a copolymer of butyl methacrylate (BMA) and diethylaminoethyl methacrylate (DEAMEA) that was chain extended with a copolymer of dimethylacrylamide (DMA) doped with a pyridyl-disulfide functionalized monomer (PDSMA) for antigen conjugation (1B).
  • BMA butyl methacrylate
  • DEAMEA diethylaminoethyl methacrylate
  • DMA dimethylacrylamide
  • PDSMA pyridyl-disulfide functionalized monomer
  • Nanocarriers composed of architecturally distinct polymer chains were evaluated for their ability to enhance MHC-I antigen presentation by dendritic cells.
  • FIGURES 1D-1H are schematic illustrations of nonlinear copolymers of the invention depicting branch points and branch point density.
  • each region is a block copolymer region (e.g., a pH-responsive, endosomal releasing block, or a hydrophilic block).
  • the dots in these figures represent branch points.
  • the distribution of branch points in each region is shown for various representative nonlinear polymers of the invention and these figures schematically illustrate branch point density for these polymers.
  • FIGURE 1D is a schematic illustration of a representative nonlinear polymer of the invention (110) in which the first block region (112) includes branch points and the second block region (114) does not.
  • FIGURE 1E is a schematic illustration of a representative nonlinear polymer of the invention (120) in which the first block region (122) and the second block region (124) include branch points. In this embodiment, the density of branch points in the first and second block regions is substantially the same.
  • FIGURE 1F is a schematic illustration of a representative nonlinear polymer of the invention (130) in which the first block region (132) and the second block region (134) include branch points. In this embodiment, the density of branch points in the first block region is greater than the density of branch points of the second block region.
  • FIGURE 1G is a schematic illustration of a representative nonlinear polymer of the invention (140) in which the first block region (142) and the second block region (144) include branch points.
  • FIGURE 1H is a schematic illustration of a representative nonlinear polymer of the invention (150) in which the second block region (154) includes branch points and the first block region (152) does not.
  • F IGURE 2 depicts an evolution plot of molar mass progression (M GPC
  • ⁇ molar mass dispersity with DMA conversion percent for DMA/PDSMA copolymerisation block extension of 4-arm star macroRAFT agent (marked on plot at x-axis origin) showing DMAc-GPC measured M n of star and M n of the tri n-butyl phosphine (TBP) reduced star.
  • FIGURE 3 compares number average diameter (nm) determined by dynamic light scattering for linear (L1:1), hyperbranched (HB1:1), crosslinked (DV1:1), and star (star) copolymers. All polymer systems show similar micelle size (about 25 nm) in HEPES buffered glucose at pH 7.4. DLS at 0.7 mg/mL polymer.
  • FIGURE 4 illustrates the pH-dependent transition from micelle to unimer transition for linear (L1:1), hyperbranched (HB1:1), crosslinked (DV1:1), and star (star) copolymers (number average diameter (nm) determined by dynamic light scattering as a function of pH).
  • FIGURES 5A and 5B illustrate antigen conjugation to pH-responsive polymer nanoparticles via disulfide exchange reaction. Size of linear (L1:1), hyperbranched (HB1:1), crosslinked (DV1:1), and star (star) copolymer nanoparticles (number average diameter by dynamic light scattering) after reaction with thiolated ovalbumin (ova-SH) at 0, 0.35, 0.175, and 0.0875 mg ova/mg polymer (5A).
  • L1:1 hyperbranched
  • DV1:1 crosslinked
  • star star copolymer nanoparticles (number average diameter by dynamic light scattering) after reaction with thiolated ovalbumin (ova-SH) at 0, 0.35, 0.175, and 0.0875 mg ova/mg polymer (5A).
  • FIGURES 6A and 6B compare reducible antigen conjugation.
  • TCEP tris(2-carboxyethyl)phosphine
  • FIGURES 7A and 7B compare antigen conjugation of a physical mixture (m) of copolymers and non-thiolated ovalbumin and copolymer conjugates (c).
  • endogenous thiols in ovalbumin are largely buried, but can undergo some polymer conjugation. Some minimal amount of antigen conjugation is observed upon mixture of polymer and non-thiolated ovalbumin.
  • FIGURES 8A-8D compare erythrocyte lysis assays demonstrating pH-dependent membrane destabilizing activity of copolymers (% hemolysis as a function of pH).
  • Total polymer concentration fixed at 1.25 ⁇ g/mL for linear (L1:1), hyperbranched (HB1:1), crosslinked (DV1:1), and 4-arm star (star) copolymers (8A) and for linear (L1:2), hyperbranched (HB1:2), and crosslinked (DV1:2) copolymers (8B).
  • FIGURE 9 illustrates the effect of polymer architecture on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • free antigen ova
  • B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • T cell response A 570
  • ovalbumin concentration ⁇ g/mL
  • L1:1 linear
  • HB1:1 hyperbranched
  • DV1:1 crosslinked
  • star star
  • FIGURE 10 illustrates the effect of 1:2 block ratio on polymer architecture on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • ova free antigen
  • B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • T cell response A 570
  • ovalbumin concentration ⁇ g/mL
  • L1:2 linear
  • HB1:2 hyperbranched
  • DV1:2 crosslinked copolymer conjugates.
  • Data is from a single representative experiment conducted in quadruplicate (mean ⁇ standard deviation).
  • FIGURE 11 illustrates the effect of physical mixture of polymer architectures on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • free antigen ova
  • B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • T cell response A 570
  • ovalbumin concentration ⁇ g/mL
  • FIGURE 12 compares fluorescence measurements of antigen uptake by DC2.4 cells at 0.35 g ova/g polymer, 245 ⁇ g/mL ova, incubated for 5 hours, for linear (L1:1 and L1:2), hyperbranched (HB1:1 and HB1:2), crosslinked (DV1:1 and DV1:2), and star (star) copolymer conjugates.
  • the present invention provides nonlinear copolymers useful for intracellular delivery of therapeutic agents, pharmaceutical compositions that include the nonlinear polymers, immunological compositions that include the nonlinear copolymers, nanoparticles that include the nonlinear polymers, and methods for making and using the nonlinear polymers.
  • the nonlinear copolymers of the invention have been found to be effective in the intracellular delivery of therapeutic agents, such as therapeutic biological molecules. In certain embodiments, the nonlinear copolymers have been found to be more effective in intracellular delivery relative to their linear counterparts.
  • FIGURE 1A compares the architectures of linear copolymers and nonlinear copolymers (e.g., crosslinked and hyperbranched) of the invention.
  • FIGURE 1B is a schematic illustration of a representative nonlinear copolymer of the invention that includes a branched copolymer bearing a therapeutic agent attached to the polymer by a reducible (i.e., disulfide) linkage.
  • FIGURE 1C schematically illustrates the interaction of the branched copolymer of FIGURE 1B with a dendritic cell.
  • the invention provides nonlinear copolymers.
  • the nonlinear copolymer comprises two blocks: (1) a pH-responsive, endosomal releasing block covalently coupled to (2) a hydrophilic block, wherein at least one of the pH-responsive, endosomal releasing block and the hydrophilic block is a nonlinear block.
  • the terms "nonlinear polymer,” “nonlinear copolymer,” and nonlinear block” refers to a polymer, copolymer, or polymer block, respectively, in which the respective polymer backbone is nonlinear (i.e., includes a nonlinear chain of atoms).
  • a “nonlinear chain” refers to a chain of atoms that includes branch points along the polymer backbone intermediate the polymer boundary units (i.e., the end-groups or other branch points). At the branch point, the nonlinear polymer has a branch from the polymer backbone. The branch is covalently coupled to the polymer backbone through the branch point. The branch is polymeric or oligomeric.
  • Branching points of the nonlinear copolymers and copolymer blocks can be characterized by the following degree of branching formula (see Simon, P. and Müller, A. Macromol. Theory Simul. 2000, 9, 621–627):
  • Range of DB can be based on expected branching vinyl consumptions. However, due to overlap of monomer peaks and polymer peaks, it is not always possible to determine DB directly via 1 H NMR analysis. In such cases, indirect methods, such as viscosity measurements (GPC viscometry) and light-scattering (GPC–LS detection), can be used to determine the branched architecture. These techniques also use assumptions based on "ideal" polymer structures (e.g., HDPE, high density polyethylene).
  • DB degree of branching
  • Block extension will further reduce DB.
  • the nonlinear block of the copolymers of the invention have a degree of branching less than about 0.03 (low DB, e.g., from about 0.01 to about 0.03).
  • the nonlinear block of the copolymers of the invention have a degree of branching of about 0.04 (mid-range DB, e.g., from about 0.03 to about 0.05).
  • the nonlinear block of the copolymers of the invention have a degree of branching greater than about 0.2 (high DB, e.g., from about 0.1 to about 0.3).
  • FIGURES 1D-1H are schematic illustrations of nonlinear copolymers of the invention depicting branch points and branch point density.
  • the nonlinear copolymer is schematically illustrated as a macromolecule having two regions.
  • each region is a block copolymer region (e.g., a pH-responsive, endosomal releasing block, or a hydrophilic block).
  • the two block regions result from the method for preparing representative nonlinear copolymers of the invention: the first region results from preparation of a first block and the second region results from extending those first blocks with second blocks (chain extension polymerization).
  • the dots in these figures represent branch points.
  • the distribution of branch points in each region is shown for various representative nonlinear polymers of the invention and these figures schematically illustrate branch point density for these polymers.
  • FIGURE 1D is a schematic illustration of a representative nonlinear polymer of the invention (110) in which the first block region (112) includes branch points and the second block region (114) does not.
  • the second block region is branch free and is formed by chain extension from polymers of the first block.
  • FIGURE 1E is a schematic illustration of a representative nonlinear polymer of the invention (120) in which the first block region (122) and the second block region (124) include branch points.
  • the density of branch points in the first and second block regions is substantially the same.
  • FIGURE 1F is a schematic illustration of a representative nonlinear polymer of the invention (130) in which the first block region (132) and the second block region (134) include branch points.
  • the density of branch points in the first block region is greater than the density of branch points of the second block region.
  • FIGURE 1G is a schematic illustration of a representative nonlinear polymer of the invention (140) in which the first block region (142) and the second block region (144) include branch points.
  • the density of branch points in the first block region is greater than the density of branch points of the second block region.
  • FIGURE 1H is a schematic illustration of a representative nonlinear polymer of the invention (150) in which the second block region (154) includes branch points and the first block region (152) does not.
  • FIGURES 1D-1H schematically illustrate representative nonlinear polymers of the invention having two block regions and have the appearance of a first block copolymer core surrounded by a second block copolymer shell. It will be appreciated that these illustrations are schematic representations depicting only the nature of branch point distribution and density for representative embodiments, and are not presented for the purpose of necessarily defining a core-shell structure for these polymers. The description of the nonlinear polymers and methods for their production set forth herein makes clear that the structure of the nonlinear copolymers can be varied greatly depending on the number and nature of branches and as such, FIGURES 1D-1H are schematic illustrations of representative nonlinear polymers of the invention. In certain embodiments, the nonlinear polymers of the invention form nanoparticles having a core and a shell.
  • FIGURES 1D-1H schematically illustrate representative nonlinear polymers of the invention having two block regions. It will be appreciated that the nonlinear copolymers of the invention can include more than two block regions. As described herein, the nonlinear copolymers of the invention are formed by chain extension polymerization methods and as such the nature of the copolymers of the invention can be varied depending on the number and nature of chain extensions (i.e., additional generations). The nonlinear copolymers of the invention can include three, four, five, or more block regions (i.e., additional generations, second, third, fourth, fifth, or more generations).
  • the nonlinear polymers of the invention are polymeric networks or systems and are macromolecules.
  • macromolecule refers to a nonlinear copolymer of the invention.
  • T he molecular weights (number-average (M n )) and dispersities ( ⁇ ) of the nonlinear copolymers of the invention may vary.
  • the nonlinear copolymers have a number-average (M n ) from about 10 kDa to about 1000 kDa. In other embodiments, the nonlinear copolymers have a number-average (M n ) from about 10 kDa to about 1000 kDa.
  • Dispersities ( ⁇ ) for the copolymers may vary from about 1.0 to about 4.0 (or greater); for example, whereas the linear copolymers have ⁇ from about 1.05 to greater than about 1.50, the branched and crosslinked nonlinear copolymers have ⁇ from about 1.10 to about 4.0 or greater, and the star copolymer (e.g., 4-arm) have ⁇ from about 1.05 to about 1.5 or greater.
  • the nonlinear copolymers of the invention include one or more pH-responsive, endosomal releasing blocks.
  • pH-responsive, endosomal releasing polymer or “pH-responsive, endosomal releasing block” refers to a polymer or polymer block, respectively, that, at about physiologic pH (7.4), undergoes a transition at the lower pH environment of the endosome and becomes endosomal membrane destabilizing thereby releasing cargo (e.g., therapeutic agent) transported by the polymer to the surrounding cytosol.
  • cargo e.g., therapeutic agent
  • the pH-responsive endosomal, releasing block has constitutional units derived from one or more of an alkyl acrylate (e.g., a C1-C6 alkyl methacrylate such as BMA), an aminoalkyl acrylate (e.g., a di- C1-C6 alkylamino acrylate such as DEAEMA), and an acrylic acid (e.g., propylacrylic acid).
  • an alkyl acrylate e.g., a C1-C6 alkyl methacrylate such as BMA
  • an aminoalkyl acrylate e.g., a di- C1-C6 alkylamino acrylate such as DEAEMA
  • an acrylic acid e.g., propylacrylic acid
  • Representative C1-C6 alkyl acrylates include methyl acrylates such as methyl acrylate, methyl methacrylate, and methyl ethacrylate, and ethyl acrylates such as ethyl acrylate, ethyl methacrylate, and ethyl ethacrylate; and representative C1-C6 alkyl acrylic acids include methacrylic acid, ethacrylic acid, propylacrylic acid, and butylacrylic acid.
  • the pH-responsive, endosomal releasing block comprises constitutional units derived from dimethylaminoethyl methacrylate (DMAEMA), diethylaminoethyl methacrylate (DEAEMA), butylmethacrylate (BMA), propylacrylic acid (PAA), and lauryl methacrylate.
  • DMAEMA dimethylaminoethyl methacrylate
  • DEAEMA diethylaminoethyl methacrylate
  • BMA butylmethacrylate
  • PAA propylacrylic acid
  • lauryl methacrylate lauryl methacrylate
  • the pH-responsive, endosomal releasing block is branched. In certain embodiments, the pH-responsive polymer, endosomal releasing block is crosslinked. In certain embodiments, the pH-responsive, endosomal releasing block comprises or consists of star polymers.
  • T he molecular weights (number-average (M n )) the pH-responsive, endosomal releasing block of the nonlinear copolymers of the invention may vary.
  • the pH-responsive, endosomal releasing block has a number-average (M n ) from about 10 kDa to about 100 kDa. In other embodiments, the pH-responsive, endosomal releasing block has a number-average (M n ) of about 10 kDa.
  • the nonlinear copolymers of the invention include one or more hydrophilic blocks. As used herein, the terms "hydrophilic block" refers to a polymer block that includes constitutional units derived from hydrophilic monomers.
  • Suitable hydrophilic monomers include monomers that include polar groups (e.g., hydroxy, amino, ether, polyether, polyamino), anionic groups or groups that become anionic at physiological pH (e.g., carboxylate, carboxylic acid), and cationic groups or groups that become cationic at physiological pH (e.g., amino and amine groups).
  • polar groups e.g., hydroxy, amino, ether, polyether, polyamino
  • anionic groups or groups that become anionic at physiological pH e.g., carboxylate, carboxylic acid
  • cationic groups or groups that become cationic at physiological pH e.g., amino and amine groups
  • the hydrophilic block of the copolymer of the present invention may be used to contribute a range of properties or functions to the copolymer.
  • the number and composition of the constituent units of the hydrophilic block may be selected to impart the desired degree of water solubility/dispersability to the copolymer.
  • the number and composition of the constituent units of the hydrophilic block may be selected to provide micelles having a desired size, critical micelle concentration or other property. Independent of micelle formation, the number and composition of the constituent units of the hydrophilic block may be selected to target the polymer to a cellular or other biological target.
  • the hydrophilic block may be copolymer block comprising charged repeat units (i.e., cationic repeat units, anionic repeat units, zwitterionic repeat units or a combination thereof), non-charged repeat units, or a combination thereof.
  • the hydrophilic block may contain anionic repeat units, cationic repeat units, zwitterionic repeat units, a combination of two or more charged repeat units (e.g., anionic and cationic repeat units, anionic and zwitterionic repeat units, cationic and zwitterionic repeat units, or anionic, cationic and zwitterionic repeat units), substantially non-charged repeat units, or a combination thereof, provided that its overall character is hydrophilic.
  • the constitutional units may include a residue selected from the group consisting of residues which are hydrophilic at physiologic pH and are substantially non-charged at physiologic pH (e.g., hydroxy, polyoxylated alkyl, polyethylene glycol, polypropylene glycol, thiol, or the like).
  • the hydrophilic block comprises repeat units derived from cationic monomers having a pKa ranging anywhere between about 6.0 and about 10.0, typically between about 6.2 and about 9.5, and in some embodiments between about 6.5 and about 8.5.
  • the pKa of the residue tends to decrease relative to the unpolymerized monomer; in general, therefore, the pKa of the incorporated repeat units will be between about 6.0 and 10.0, typically between about 6.2 and 9.0, and in some embodiments, between about 6.5 and 8.0.
  • the hydrophilic block comprises a monomeric species comprising an acyclic amine (e.g., an amine, an alkyl amine, a dialkyl amine, or the like), an acyclic imine (e.g., an imine, an alkyl imine, or the like), a cyclic amine (e.g., piperidine), a nitrogen containing heterocycle (e.g., pyridine or quinoline), or the like.
  • an acyclic amine e.g., an amine, an alkyl amine, a dialkyl amine, or the like
  • an acyclic imine e.g., an imine, an alkyl imine, or the like
  • a cyclic amine e.g., piperidine
  • a nitrogen containing heterocycle e.g., pyridine or quinoline
  • a cationic species utilized herein includes a protonated acyclic amine (e.g., an amine, an alkyl amine, a dialkyl amine, or the like), an acyclic imine (e.g., an imine, an alkyl imine, or the like), a cyclic amine (e.g., piperidine), a nitrogen containing heterocycle (e.g., pyridine or quinoline), or the like.
  • a protonated acyclic amine e.g., an amine, an alkyl amine, a dialkyl amine, or the like
  • an acyclic imine e.g., an imine, an alkyl imine, or the like
  • a cyclic amine e.g., piperidine
  • a nitrogen containing heterocycle e.g., pyridine or quinoline
  • Non-limiting examples of acyclic amines include methylamine, dimethylamine, ethylamine, diethylamine, propylamine, isopropylamine, diisopropylamine, diisopropylethylamine, n-butylamine, sec-butylamine, tert-butylamine, pentylamine, neo-pentylamine, iso-pentylamine, hexanamine or the like.
  • Non-limiting examples of acyclic imines include methylimine, ethylimine, propylimine, isopropylimine, n-butylimine, sec-butylimine, pentylimine, neo-pentylimine, iso-pentylimine, hexylimine or the like.
  • Non-limiting examples of cyclic amines include cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, cycloheptylamine, piperidine, pyrazine, pyrolidine, homopiperidine, azabicylcoheptane, diazabicycloundecane, or the like.
  • Non-limiting examples of cyclic imines include cyclopropylimine, cyclobutylimine, cyclopentylimine, cyclohexylimine, cycloheptylimine, or the like.
  • Non-limiting examples of nitrogen containing heteroaryls include imidazolyl, pyrrolyl, pyridyl, indolyl, or the like.
  • the hydrophilic block comprises a plurality of monomeric residues of optionally substituted, amino(C 1 -C 6 )alkyl-ethacrylate, amino(C 1 -C 6 )alkyl- methacrylate, amino(C 1 -C 6 )alkyl-acrylate, (N-(C 1 -C 6 )alkyl-amino(C 1 -C 6 )alkyl- ethacrylate, N-(C 1 -C 6 )alkyl-amino(C 1 -C 6 )alkyl-methacrylate, N-(C 1 -C 6 )alkyl-amino(C 1 - C 6 )alkyl-acrylate, (N,N-di(C 1 -C 6 )alkyl-amino(C 1 -C 6 )alkyl-ethacrylate, N,N-di(C 1 - C 6 )alkyl-amino(C 1 -C 6
  • the hydrophilic block comprises a plurality of anionic monomeric residues.
  • the anionic monomeric residues can have a species charged or chargeable to an anion, including a protonatable anionic species.
  • the chargeable species can preferably be anionic at serum physiological pH and substantially neutral or non-charged at the pH.
  • the carrier block comprises a plurality of anionic hydrophobic monomeric residues, monomeric residues comprising both hydrophobic species (e.g., a C 2 -C 8 alkyl substituent) and species charged or chargeable to an anion.
  • the hydrophilic block comprises one or more monomeric residues comprising a conjugatable or conjugated side chain (e.g., a pendant group of a monomeric residue).
  • a conjugated side chain as used herein is meant to include monomeric residues that when initially polymerized possessed a conjugatable side chain, which was later conjugated, e.g., to a biological molecule.
  • a conjugatable side chain is a group bearing one or more reactive groups that can be used for post-polymerization introduction of additional functionalities via known in the art chemistries, for example, "click” chemistry (for example of "click” reactions, see Wu, P.; Fokin, V. V.
  • conjugatable or functionizable side chains provided herein comprise one or more of any suitable electrophilic or nucleophilic functional group, such as but not limited to N-hydrosuccinimide (NHS)ester, HOBt (1-hydroxybenzotriazole) ester, p-nitrophenyl ester, tetrafluorophenyl ester, pentafluorophenyl ester, pyridyl disulfide group, maleimide, aldehyde, ketone, anhydride, thiol, amine, hydroxyl, alkyl halide, or the like.
  • NHS N-hydrosuccinimide
  • HOBt 1-hydroxybenzotriazole
  • p-nitrophenyl ester tetrafluorophenyl ester
  • pentafluorophenyl ester pentafluorophenyl ester
  • pyridyl disulfide group maleimide, aldehyde, ketone,
  • the hydrophilic block comprises one or more of such functionizable side chains that is bioconjugated with a biomolecule, e.g., a polynucleotide or peptide.
  • a biomolecule e.g., a polynucleotide or peptide.
  • the conjugatable group is a pyridyl disulfide group, which allows the biological molecule to be cleaved from the copolymer under reducing conditions (i.e., a reducible group)
  • the hydrophilic block comprises constitutional units derived from N,N-dimethylacrylamide (DMA), diethylacrylamide (DEA), 2-hydroxypropyl methacrylamide (HPMA), hydroxyethylacrylamide, hydroxyethylmethacrylate, polyethylene glycol acrylate, propylene glycol methacrylamide (PEGMA), and hydroxy- or methoxy-terminated diethylene glycol methacrylate (950– 10 kDa).
  • DMA N,N-dimethylacrylamide
  • DEA diethylacrylamide
  • HPMA 2-hydroxypropyl methacrylamide
  • HPMA 2-hydroxyethylacrylamide
  • hydroxyethylmethacrylate polyethylene glycol acrylate
  • PEGMA propylene glycol methacrylamide
  • hydroxy- or methoxy-terminated diethylene glycol methacrylate 950– 10 kDa
  • the hydrophilic block can further include constitutional units derived from pyridine disulfide methacrylamide (PDSMA).
  • PDSMA pyridine disulfide methacrylamide
  • T he molecular weights (number-average (M n )) the hydrophilic block of the nonlinear copolymers of the invention may vary.
  • the hydrophilic block has a number-average (M n ) from about 10 kDa to about 100 kDa. In other embodiments, the hydrophilic block has a number-average (M n ) from about 10 kDa to about 20 kDa.
  • Monomers are selected for the preparation of the nonlinear copolymers of the invention depending on the desired properties of the individual blocks (i.e., pH-responsive, endosomal releasing block, and hydrophilic block) and overall copolymer.
  • ethylenically unsaturated monomers are used to prepare the nonlinear copolymers.
  • ethylenically unsaturated monomers is defined as a compound having at least one carbon double bound or triple bond.
  • Suitable ethylenically unsaturated monomers include alkyl (alkyl)acrylates, methacrylates, acrylates, alkylacrylamides, methacrylamides, and acrylamides.
  • Non-limiting examples of the ethylenically unsaturated monomers are: an alkyl (alkyl)acrylate, a methacrylate, an acrylate, an alkylacrylamide, a methacrylamide, an acrylamide, a styrene, an allylamine, an allylammonium, a diallylamine, a diallylammonium, an N-vinyl formamide, a vinyl ether, a vinyl sulfonate, an acrylic acid, a sulfobetaine, a carboxybetaine, a phosphobetaine, or maleic anhydride.
  • monomers suitable for use in the preparation of the polymers provided herein include, by way of non-limiting example, one or more of the following monomers: methyl methacrylate, ethyl acrylate, propyl methacrylate (all isomers), butyl methacrylate (all isomers), 2-ethylhexyl methacrylate, isobornyl methacrylate, methacrylic acid, benzyl methacrylate, phenyl methacrylate, methacrylonitrile, alpha-methylstyrene, methyl acrylate, ethyl acrylate, propyl acrylate (all isomers), butyl acrylate (all isomers), 2-ethylhexyl acrylate, isobornyl acrylate, acrylic acid, benzyl acrylate, phenyl acrylate, acrylonitrile, styrene, acrylates and styrenes selected from glycid
  • a functionalized monomer is a monomer comprising a masked or non-masked functional group, e.g. a group to which other moieties can be attached following the polymerization.
  • a masked or non-masked functional group e.g. a group to which other moieties can be attached following the polymerization.
  • Non-limiting examples of such groups are primary amino groups, carboxyls, thiols, hydroxyls, azides, and cyano groups.
  • suitable masking groups are available (see, e.g., T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis (2nd edition) J. Wiley & Sons, 1991. P. J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994).
  • monomers suitable for use in the preparation of the polymers include, for example, one or more of the following monomers: methyl methacrylate, ethyl acrylate, propyl methacrylate (all isomers), butyl methacrylate (all isomers), 2-ethylhexyl methacrylate, methacrylic acid, benzyl methacrylate, phenyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate (all isomers), butyl acrylate (all isomers), 2-ethylhexyl acrylate, acrylic acid, benzyl acrylate, phenyl acrylate, acrylates selected from glycidyl methacrylate, 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate (all isomers), hydroxybutyl methacrylate (all isomers), or combinations thereof.
  • the nonlinear copolymers include additional degradable groups that can result in the degradation of the copolymer architectures into linear copolymer chains or into block segments or a combination of these. This structural transformation of the nonlinear copolymers may provide for enhanced availability of the biological moiety and also facilitate metabolism and clearance of the polymers (and/or its residues).
  • degradable groups may be one or more functional groups selected from ester, anhydride, carbonate, peroxide, peroxyester, phosphate, thioester urea, thiourethane, ether, disulfide, carbamate (urethane) and boronate ester.
  • Degradation of a degradable group may be facilitated in the presence of an acid, a base, an enzyme and/or another endogenous biological compound that can catalyze or at least assist in the bond cleavage process.
  • an ester may be hydrolytically cleaved to produce a carboxylic acid group and an alcohol group
  • an amide may be hydrolytically cleaved to produce a carboxylic acid group and an amine group
  • a disulfide may be reductively cleaved to produce thiol groups.
  • the degradable group is a disulfide.
  • the degradable groups can be introduced into the nonlinear copolymer be selection of the appropriate monomer or comonomers. See, for example, Examples 6 and 7.
  • FIGURE 2 depicts an evolution plot of molar mass progression (M GPC
  • the nonlinear copolymers of the invention include branched, crosslinked, and star copolymers.
  • Each of the branched, crosslinked, and star copolymers of the invention includes one or more branch points from which branches extend from the polymer backbone.
  • FIGURE 1A compares the architectures of linear and nonlinear copolymers (e.g., crosslinked and hyperbranched).
  • Scheme 1 illustrates the synthesis of a linear copolymer and the syntheses of representative branched and crosslinked copolymers of the invention (poly(BMA-co- DEAEMA)-b-(DMA-co-PDSMA)).
  • Refe rring to Scheme 1 B illustrates t he RAFT s ynthesis o f a branche d copolym er from m onomers BMA and DEAEMA to provide (2), which was chain extended by polymerization with DMA and PDSMA as shown in D to provide a representative branched copolymer of the invention.
  • C illustrates the RAFT synthesis of a crosslinked copolymer from monomers BMA, DEAEMA, and EGDMA to provide (3), which was chain extended by polymerization with DMA and PDSMA as shown in D to provide a representative crosslinked copolymer of the invention.
  • FIGURES 1D-1H schematically illustrate branch point distribution and density for representative nonlinear copolymers of the invention.
  • branched polymer As used herein, the terms “branched polymer,” “branched copolymer,” and “branched block” refer to a polymer, copolymer, or polymer block, respectively, that include a polymeric branch covalently attached to the polymer backbone at the branch point.
  • branched and “hyperbranched” are used interchangeably.
  • the term “branched chain” refers to a chain with at least one branch point intermediate between the boundary units (i.e., the end-groups or other branch points).
  • branch point refers to a point on a chain at which a branch is attached.
  • branch refers to an oligomeric or polymeric offshoot or extension from a chain (e.g., backbone).
  • the branch can be a result of the CPA used in the polymerization, the crosslink of the crosslinked polymers, or the core of the star copolymers.
  • the branched copolymer of the invention includes a pH-responsive, endosomal releasing block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates), one or more aminoalkyl acrylates (e.g., mono- and di- C1-C6 alkylamino acrylates), one or more alkyl acrylamides (e.g., C1-C6 alkyl acrylamides), one or more aminoalkyl acrylamides (e.g., mono- and di- C1-C6 alkyl acrylamides), and/or one or more acrylic acids (e.g., C1-C6 alkyl acrylic acids), and a hydrophilic block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates) and/or one or more alkyl acrylamides (e.g., C1-C6 alkyl acrylamides (
  • the branched copolymer of the invention includes a pH-responsive endosomal, releasing block having constitutional units derived from an alkyl acrylate (e.g., a C1-C6 alkyl methacrylate such as BMA), an aminoalkyl acrylate (e.g., a di- C1-C6 alkylamino acrylates such as DEAEMA), and optionally an acrylic acid (e.g., propylacrylic acid), and a hydrophilic block having constitutional units derived from an alkyl acrylamide (e.g., a C1-C6 alkyl acrylamide such as DMA or PDSMA), where the pH-responsive, endosomal releasing block is chain extended by polymerization with the monomers used to make the hydrophilic block.
  • an alkyl acrylate e.g., a C1-C6 alkyl methacrylate such as BMA
  • an aminoalkyl acrylate e.g., a di- C
  • Example 2 The preparation and characterization of a representative branched nonlinear copolymer of the invention is described in Example 2, and the biological activity of a representative branched nonlinear copolymer of the invention is described in Example 8.
  • the non-linear copolymer of the invention is a crosslinked copolymer.
  • a "crosslink” is a constitutional unit connecting two parts of a macromolecule.
  • the crosslink comprises or consists of ethylene glycol.
  • the crosslinked copolymer of the invention includes a pH-responsive, endosomal releasing block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates), one or more aminoalkyl acrylates (e.g., mono- and di- C1-C6 alkylamino acrylates), one or more alkyl acrylamides (e.g., C1-C6 alkyl acrylamides), one or more aminoalkyl acrylamides (e.g., mono- and di- C1-C6 alkyl acrylamides), and/or one or more acrylic acids (e.g., C1-C6 alkyl acrylic acids), and a hydrophilic block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates) and/or one or more alkyl acrylamides (e.g., C1-C6 alkyl acryl acrylate),
  • the crosslinked copolymer of the invention includes a pH-responsive endosomal, releasing block having constitutional units derived from an alkyl acrylate (e.g., a C1-C6 alkyl methacrylate such as BMA), an aminoalkyl acrylate (e.g., a di- C1-C6 alkylamino acrylates such as DEAEMA), and optionally an acrylic acid (e.g., propylacrylic acid), and a hydrophilic block having constitutional units derived from an alkyl acrylamide (e.g., a C1-C6 alkyl acrylamide such as DMA or PDSMA), where the pH-responsive, endosomal releasing block is crosslinked with a diacrylate crosslinking agent, and where the pH-responsive, endosomal releasing block is chain extended by polymerization with the monomers used to make the hydrophilic block.
  • an alkyl acrylate e.g., a C1-C6 alkyl meth
  • Example 3 The preparation and characterization of a representative crosslinked nonlinear copolymer of the invention is described in Example 3, and the biological activity of a representative crosslinked nonlinear copolymer of the invention is described in Example 8.
  • the non-linear copolymer of the invention is a star copolymer.
  • a star copolymer or "star macromolecule” is a macromolecule containing a constitutional unit from which more than two chains or arms emanate.
  • a star macromolecule with n linear chains (arms) attached to the central unit is termed an n-star, e.g., five-star.
  • the star polymer is a three-star, four-star, five-star, six-star, or more polymer.
  • the star copolymer of the invention includes a pH-responsive, endosomal releasing block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates), one or more aminoalkyl acrylates (e.g., mono- and di- C1-C6 alkylamino acrylates), one or more alkyl acrylamides (e.g., C1-C6 alkyl acrylamides), one or more aminoalkyl acrylamides (e.g., mono- and di- C1-C6 alkyl acrylamides), and/or one or more acrylic acids (e.g., C1-C6 alkyl acrylic acids), and a hydrophilic block having constitutional units derived from one or more alkyl acrylates (e.g., C1-C6 alkyl acrylates) and/or one or more alkyl acrylamides (e.g., C1-C6 alkyl acrylamide
  • the star copolymer of the invention includes a pH-responsive endosomal, releasing block having constitutional units derived from an alkyl acrylate (e.g., a C1-C6 alkyl methacrylate such as BMA), an aminoalkyl acrylate (e.g., a di- C1-C6 alkylamino acrylate such as DEAEMA), and optionally an acrylic acid (e.g., propylacrylic acid), and a hydrophilic block having constitutional units derived from an alkyl acrylamide (e.g., a C1-C6 alkyl acrylamide such as DMA or PDSMA), where the pH-responsive, endosomal releasing block is chain extended by polymerization with the monomers used to make the hydrophilic block.
  • an alkyl acrylate e.g., a C1-C6 alkyl methacrylate such as BMA
  • an aminoalkyl acrylate e.g., a di- C1-
  • Example 4 The preparation and characterization of a representative star copolymer of the invention is described in Example 4, and the biological activity of a representative star copolymer of the invention is described in Example 8.
  • representative C1-C6 alkyl acrylates include methyl acrylates such as methyl acrylate, methyl methacrylate, and methyl ethacrylate, and ethyl acrylates such as ethyl acrylate, ethyl methacrylate, and ethyl ethacrylate;
  • representative mono- and di- C1-C6 alkylamino acrylates include monomethyl methylamino acrylate and dimethyl ethylamino acrylate;
  • representative C1-C6 alkyl acrylamides include (e.g., C1-C6 alkyl C1-C6 alkylacrylamides) include methyl acrylamides such as methyl acrylamide, methyl methacrylamide, and methyl ethacrylamide, and ethyl acrylamides such as ethyl acrylamide, eth
  • the nonlinear copolymers of the invention were shown to possess advantageous properties for MHC class I presentation in an in vitro co-culture model.
  • Example 8 describes the effect of polymer architecture on MHC class I presentation in an in vitro co-culture model.
  • ovalbumin was used as the model antigen (i.e., biological moiety).
  • Murine dendritic cells (DC2.4) were incubated with free antigen (ova) or conjugates prepared using polymers of different architecture and subsequently co-cultured with B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • constitutional unit of a polymer refers to an atom or group of atoms in a polymer, comprising a part of the chain together with its pendant atoms or groups of atoms, if any.
  • the constitutional unit can refer to a repeat unit.
  • the constitutional unit can also refer to an end group on a polymer chain.
  • the constitutional unit of polyethylene glycol can be–CH 2 CH 2 O- corresponding to a repeat unit, or–CH 2 CH 2 OH corresponding to an end group.
  • the term “repeat unit” corresponds to the smallest constitutional unit, the repetition of which constitutes a regular macromolecule (or oligomer molecule or block).
  • the term “end group” refers to a constitutional unit with only one attachment to a polymer chain, located at the end of a polymer.
  • the end group can be derived from a monomer unit at the end of the polymer, once the monomer unit has been polymerized.
  • the end group can be a part of a chain transfer agent or initiating agent that was used to synthesize the polymer.
  • the term "monomer” is a polymerizable compound that, on polymerization, contributes one or more constitutional units in the structure of the polymer.
  • polymer refers to the product that is the result of polymerization of a single monomer.
  • copolymer refers to a polymer that is the result of polymerization of two or more different monomers.
  • the number and the nature of each constitutional unit can be separately controlled in a copolymer.
  • the constitutional units can be disposed in a purely random, an alternating random, a regular alternating, a regular block, or a random block configuration unless expressly stated to be otherwise.
  • a purely random configuration can, for example, be: x-x-y-z-x-y-y-z-y-z-z... or y-z-x-y-z-y-z-x-x....
  • An alternating random configuration can be: x-y-x-z-y-x-y-z-y-x-z..., and a regular alternating configuration can be: x-y-z-x-y-z-x-y-z....
  • block copolymer refers to a polymer formed of two or more covalently joined segments of polymers.
  • a regular block configuration has the following general configuration: ...x-x-x-y-y-y-z-z-z-x-x...
  • a random block configuration has the general configuration: ...x-x-x-z-z-x-x-y-y-y-y-z-z-x-x-z-z-z-....
  • the nonlinear of the invention may be prepared by a variety of suitable means.
  • the nonlinear block copolymers can be made by polymerizing ethylenically unsaturated monomers. Polymerization of the ethylenically unsaturated monomers is preferably conducted using a living polymerization technique.
  • Non–linear copolymer prepared by living polymerization can advantageously exhibit a well-defined molecular architecture, a predetermined molecular weight and narrow molecular weight distribution or low polydispersity.
  • living polymerization examples include ionic polymerization and controlled radical polymerization (CRP) (also known as reversible-deactivation radical polymerization, RDRP).
  • CRP controlled radical polymerization
  • examples of CRP include, but are not limited to, iniferter polymerization, stable free radical mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), and reversible addition fragmentation chain transfer (RAFT) polymerization.
  • CRP controlled radical polymerization
  • SFRP stable free radical mediated polymerization
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition fragmentation chain transfer
  • RAFT polymerization and RAFT agents are described in numerous publications (see, for example, WO 98/01478, Moad G.; Rizzardo, E; Thang S, H. Polymer 2008, 49, 1079-1131; Aust. J. Chem., 2005, 58, 379-410; Aust. J. Chem., 2006, 59, 669-692; and Aust. J.
  • Suitable RAFT agents for use in preparing the nonlinear copolymers include xanthate, dithioester, dithiocarbamate and trithiocarbonate compounds.
  • a source of initiating radicals can be provided by any suitable means of generating free radicals, such as the thermally induced homolytic scission of suitable compound(s) (thermal initiators such as peroxides, peroxyesters, or azo compounds), the spontaneous generation from monomers (e.g. styrene), redox initiating systems, photochemical initiating systems or high energy radiation such as electron beam, X- or gamma-radiation.
  • the nonlinear copolymers of the invention form nanoparticles that can be advantageously used to administer embodiments of the copolymers that include biological moieties (e.g., therapeutic agents, immunological agents).
  • biological moieties e.g., therapeutic agents, immunological agents.
  • the nature of the nanoparticle will vary depending on the composition and size of the nonlinear polymer as well as the environment of the vehicle for administration and the mode of administration.
  • the nanoparticle is a micellar assembly.
  • the nanoparticle is a core-shell nanoparticle.
  • core-shell nanoparticle refers to a particle comprising a nonlinear copolymer of the invention and in which one block forms the nanoparticle core and the second block forms the nanoparticle shell.
  • the core-shell nanoparticle has a pH-responsive, endosomal releasing core and a hydrophilic shell.
  • the core-shell nanoparticle has a hydrophilic core and a pH-responsive, endosomal releasing shell.
  • the nanoparticles of the invention have diameters from about 10 to about 30 nm.
  • micellar assembly is a composition comprising one or more polymers that form a micelle in solution.
  • the micellar assembly comprises nonlinear block copolymers.
  • the micellar assembly comprises hydrophilic blocks and pH-responsive, endosomal releasing blocks.
  • the mass ratio of the pH-responsive, endosomal releasing blocks is between about 45, 50, 55, 60, 65, and 70%.
  • the micellar assembly forms a micelle in particular pH ranges and not in others. In certain preferred embodiments, the micellar assembly does not form a micelle at a pH less than about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or 7.3.
  • the micellar assemblies are between 10 and 100 nm in diameter. In certain further embodiments, the micellar assemblies are between 15 and 80 nm. In certain preferred embodiments, the micellar assemblies are about 15 nm, 20 nm, 25 nm, 30 nm, or 35 nm in diameter.
  • one or more biological molecules can be covalently coupled to the nonlinear copolymers of the invention to provide nonlinear copolymer conjugates that include biological moieties. Coupling to the nonlinear copolymers is achieved by conjugating the one or more biological molecules to the hydrophilic block. The conjugation can be achieved using a cleavable linkage.
  • a "cleavable linkage” is a chemical linkage or bond that can be broken under certain conditions. In certain embodiments, the cleavable linkage can be broken at a particular pH.
  • the cleavable linkage is reduced, and thereby broken, at a pH of between about 5.5 and 7.0 (i.e., the cleavable linkage is a reducible linkage).
  • the cleavable linkage comprises a disulfide bond.
  • the cleavable linkage comprises or consists of a pyridyl-disulfide bond.
  • the cleavable linkage is a disulfide bond.
  • the nonlinear copolymer that includes one or more biological moieties deliver their cargo (i.e., biological molecules), for example, intracellularly via endocytosis followed by endosomal release.
  • Suitable biological molecules include biopolymers (e.g., proteins, peptides, oligonucleotides, and polysaccharides), lipids, and small molecules (i.e., an organic, inorganic, or organometallic compound having a molecular weight of less than about 800 g/mole).
  • biopolymers e.g., proteins, peptides, oligonucleotides, and polysaccharides
  • lipids lipids
  • small molecules i.e., an organic, inorganic, or organometallic compound having a molecular weight of less than about 800 g/mole.
  • Exemplary biological molecules that may be used in accordance with the present invention include proteins (including multimeric proteins, protein complexes, peptides, antigens, and antigen fragments), nucleic acids, lipids, carbohydrates, and small molecules.
  • the biological molecule is a protein or peptide.
  • the terms "protein,” “polypeptide,” and “peptide” can be used interchangeably.
  • peptides include from about 5 to about 5000, 5 to about 1000, about 5 to about 750, about 5 to about 500, about 5 to about 250, about 5 to about 100, about 5 to about 75, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, or about 5 to about 10 amino acid residues.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation. In some embodiments, polypeptides may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof, as described herein.
  • the biological molecules is a hormone, erythropoietin, insulin, cytokine, antigen for vaccination, or growth factor.
  • the biological molecule is an antibody and/or functional fragment (characteristic portion) thereof.
  • the antibody is a polyclonal, monoclonal, chimeric (i.e., "humanized"), single chain (recombinant), or bispecific antibody.
  • antibodies may have reduced effector functions and/or bispecific molecules.
  • antibodies may include Fab fragments and/or fragments produced by a Fab expression library (e.g., Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments).
  • the biological molecule is a nucleic acid (e.g., DNA, RNA, derivatives thereof).
  • the nucleic acid agent is a functional RNA.
  • a "functional RNA" is an RNA that does not code for a protein but instead belongs to a class of RNA molecules whose members characteristically possess one or more different functions or activities within a cell. It will be appreciated that the relative activities of functional RNA molecules having different sequences may differ and may depend at least in part on the particular cell type in which the RNA is present.
  • RNAi-inducing entities e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and microRNAs
  • ribozymes e.g., tRNAs, rRNAs, RNAs useful for triple helix formation.
  • the biological molecule is a carbohydrate.
  • the carbohydrate is a carbohydrate that is associated with a protein (e.g., glycoprotein, proteogycan).
  • a carbohydrate may be natural or synthetic.
  • a carbohydrate may also be a derivatized natural carbohydrate.
  • a carbohydrate may be a simple or complex sugar.
  • a carbohydrate is a monosaccharide, including but not limited to glucose, fructose, galactose, and ribose.
  • a carbohydrate is a disaccharide, including but not limited to lactose, sucrose, maltose, trehalose, and cellobiose.
  • a carbohydrate is a polysaccharide, including but not limited to cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, and pullulan.
  • a carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol.
  • the biological molecules is a lipid.
  • the lipid is a lipid that is associated with a protein (e.g., lipoprotein).
  • Exemplary lipids that may be used in accordance with the present invention include, but are not limited to, oils, fatty acids, saturated fatty acid, unsaturated fatty acids, essential fatty acids, cis fatty acids, trans fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, and lipoproteins.
  • the lipid may comprise one or more fatty acid groups or salts thereof.
  • the fatty acid group may comprise digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons.
  • the fatty acid group is one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid.
  • the fatty acid group is one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
  • the invention provides compositions that include a nonlinear copolymer of the invention that includes a therapeutic agent and a pharmaceutically acceptable carrier or diluent.
  • Suitable carriers and diluents include those known in the art, such as saline and dextrose.
  • Representative useful therapeutic agents administered by the nonlinear copolymer compositions are described above (e.g., biological molecules).
  • the invention provides immunological compositions.
  • the nonlinear copolymer includes a biological moiety that stimulates an immunological response (e.g., antigen for immunogenic response).
  • the immunological compositions of the invention advantageously enhance antigen presentation and elicit T cell responses.(e.g., CD8+ response).
  • the method comprises contacting a cell with a nonlinear copolymer of the invention that includes a biological molecule covalently coupled thereto, the nonlinear polymer incorporated into an endosomal membrane within the cell through endocytosis, the endosomal membrane is destabilized by the nonlinear copolymer, and the biological molecule is released from the nonlinear copolymer and delivered to the cytosol of the cell.
  • the present invention provides methods of preventing or treating a disease or condition.
  • the methods comprise administering a nonlinear copolymer of the invention that includes a biological molecule (e.g., therapeutic agent) coupled to the copolymer to a subject, wherein the biological molecule is released from the copolymer (e.g., as a result of a pH change), thereby treating, preventing, or ameliorating the disease or condition.
  • a biological molecule e.g., therapeutic agent
  • each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component.
  • the transitional terms “comprise” or “comprises” or “comprising” allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified.
  • the transitional phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment.
  • BMA butyl methacrylate
  • DEAEMA N,N- diethylaminoethyl methacrylate
  • DMA N,N-dimethylacrylamide
  • ELDMA ethyleneglycol dimethacrylate
  • Bond- Breaker TCEP solution Traut's reagent (2-iminothiolane-HCl), and Ellman's reagent (5,5'-dithio-bis-[2-nitrobenzoic acid]; DTNB) were obtained from Thermo Scientific.
  • N,N-Dimethylacetamide (DMAc) (containing 2.1 g L -1 lithium chloride (LiCl)) was used as an eluent with a flow rate of 1 mL/min at 80 °C.
  • Number (M n ) and weight- average (M w ) molar masses were evaluated using Shimadzu LC Solution software.
  • the GPC columns were calibrated with low dispersity polystyrene (PSt) standards (Polymer Laboratories) ranging from 3100 to 650,000 g mol -1 and molar masses are reported as PSt equivalents.
  • PSt dispersity polystyrene
  • Dynamic light scattering measurements were conducted using a Malvern Zetasizer Nano ZS (Worcestershire, UK) at a constant scattering angle of 173°. Briefly, particle sizes of copolymer micelles and protein-polymer conjugates were determined by DLS at room temperature (RT) in 4-(2 -hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered glucose HBG (pH 7.4) at 1 mg/mL polymer.
  • RT room temperature
  • HEPES 4-(2 -hydroxyethyl)-1-piperazineethanesulfonic acid
  • DLS measurements were performed with copolymers (0.5 mg/mL) incubated with 10 mM sodium phosphate buffer (supplemented with 150 mM NaCl) in the pH range of the endosomal processing pathway (7.4, 7.0, 6.6, 6.2, and 5.8). Mean diameters are reported as the number average ⁇ standard deviation from three or more measurements.
  • ECT ethylcyanovaleric acid trithiocarbonate
  • RAFT copolymerization of DEAEMA and BMA was conducted with ECT and 1,1′-azobis(cyclohexanecarbonitrile) (ABCC) as the RAFT chain transfer agent and i nitiator, respectively, in dioxane at 90°C.
  • the initial monomer ([M] o ) to CTA ([CTA] o ) to initiator ([I] o ) ratio was 100 : 1 : 0.05.
  • Individual polymerization solutions from the stock solution were degassed and then preheated in an oil bath at 90°C and allowed to polymerize for the prescribed time period. Following polymerization the solutions were quenched by immersion in liquid nitrogen, opened and evaluated via 1 H NMR in CD 3 OD 30 and GPC (DMAc eluent).
  • Stock solution was DEAEMA (3.89 g, 21 mmol), BMA (1.99 g, 14 mmol), ECT (92 mg, 0.35 mmol), dioxane (5.88 g), and ABCC (4.3 mg, 0.017 mmol).
  • Individual polymerization solutions from the stock solution were degassed and pre-heated in an oil bath at 90 °C and allowed to polymerize for the prescribed time period. Following polymerization the solutions were quenched by immersion in liquid nitrogen. The polymers were precipitated from the polymerization mixture into pentane, cooled to -10 °C and decanted; the polymer oil was re-suspended in fresh pentane.
  • the polymer was isolated via rotary evaporation followed by drying under high vacuum.
  • Monomer conversions were determined via 1 H NMR spectroscopy by diluting the polymerization solutions in CD 3 OD and comparing the DEAEMA vinyl resonances at 6.08 ppm to the BMA vinyl resonances at 6.05 ppm relative to an anisole spike (6.88 ppm).
  • Copolymer composition was determined by comparison of the DEAEMA aliphatic amine resonances at 2.5– 3.0 ppm to the combined ester resonances between 3.9 - 4.3 ppm.
  • Linear Copolymer Poly(DEAEMA-co-BMA)-b-(DMA-co- PDSMA)
  • the polymerization solution was then transferred to glass ampoules, freeze-evacuate-thaw degassed to constant high vacuum (8-9 ⁇ 10 -3 mbar) and flame sealed. After this time the polymerization ampoules were transferred to a preheated oil bath at 60 °C and allowed to polymerize for 9 hours. The polymer was precipitated from the polymerisation mixture into pentane, filtered, redissolved and re-precipitated into pentane, repeated 3 times. After this time the polymer was isolated via filtration, air-drying, followed by drying under high vacuum.
  • the final M n of the diblock copolymer 30 500 g/mol (M n 19 800 g/mol 2 nd block) with a ⁇ of 1.22 and a 2 nd block composition of 92 mol % DMA and 8 mol % PDSMA (92:8 feed).
  • Monomer conversions were determined via 1 H NMR spectroscopy by diluting the polymerization solutions in CD 3 OD and comparing the DMA vinyl resonances at 6.17 ppm to the PDSMA vinyl resonances at 5.37 ppm relative to an anisole spike (6.88 ppm).
  • the theoretical copolymer composition was determined via comonomer conversion.
  • [M]:[RAFT] 100:1 ([M]:[RAFT] is the molar ratio of monomers to RAFT agent)
  • ECT 1.0 g, 3.8 mmol
  • HEMA hydroxyethyl methacrylate
  • DCM dry dichloromethane
  • DMAP dimethylaminopyridine
  • DIC diisopropylcarbodiimide
  • Branched poly(DEAEMA-co-BMA) macroCTA was prepared as described for the linear macroCTA under the control and copolymerization of the inimer RAFT agent
  • the initial monomer ([M] o ) to CTA ([CTA] o ) to initiator ([I] o ) ratio was 100: 1 : 0.05.
  • Inimer macroRAFT block extension via copolymerization of N,N- dimethylacrylamide (DMA) and pyridyl disulphide methacrylamide (PDSMA) with a representative poly(DEAEMA-co-BMA) macroCTA provided representative branched copolymers [poly(DEAEMA-co-BMA)-b-(DMA-co-PDSMA)].
  • Polymerization provided a final molecular weight of the diblock copolymer 43 000 g/mol (21 400 g/mol 2 nd block) with a ⁇ of 1.39 and a 2 nd block composition of 93 mol % DMA and 7 mol % PDSMA (93:7 feed).
  • a representative poly(DEAEMA-co-BMA-co-EGDMA) macroCTA was synthesized in the same manner as for the linear macroCTA described above, with the addition of a divinyl crosslinker, ethyleneglycol dimethacrylate (EGDMA).
  • the initial monomer ([M] o ) to CTA ([CTA] o ) to initiator ([I] o ) ratio was 100: 1 : 0.05.
  • the ratio of ECT RAFT agent to EGDMA was 1 : 0.7.
  • the polymer oil was re-suspended in fresh pentane, repeated 3 times. After this time the polymer was isolated via rotary evaporation followed by drying under high vacuum.
  • M n , ⁇ , and composition of the poly(DEAEMA-co-BMA-co-EGDMA) macroCTA was 13 000 g/mol, 1.33, and 60:40 DEAEMA:BMA (60:40 feed), respectively.
  • MacroRAFT block extension via copolymerization of N,N-dimethylacrylamide (DMA) and pyridyl disulphide methacrylamide (PDSMA) with a representative poly(DEAEMA-co-BMA-co-EGDMA) macroCTA provided a representative crosslinked copolymer [poly(DEAEMA-co-BMA-co-EGDMA)-b-(DMA-co-PDSMA)].
  • the polymer was precipitated from the polymerization mixture as previously.
  • the polymer was isolated via filtration, air-drying, followed by drying under high vacuum.
  • the final molecular weight of the diblock copolymer 30 500 g/mol (Mn 19 800 g/mol 2 nd block) with a ⁇ of 1.22 and a 2 nd block composition of 92 mol % DMA and 8 mol % PDSMA (92:8 feed).
  • Monomer conversions were determined via 1 H NMR spectroscopy.
  • the 4-arm RAFT agent has the structure:
  • Step 1 ECT (1.04 g, 3.97 mmol), 2-(pyridine-2-yldisulfanyl)ethanol (0.81 g, 4.32 mmol), diisopropylcarbodiimide (DIC) 0.55 g, 4.36 mmol) in dichloromethane (50 mL) and DMAP (N,N-dimethylaminopyridine), 0.05g, 0.41 mmol ) were allowed to stir at room temperature for three hours.
  • DIC diisopropylcarbodiimide
  • Step 2 (S)-2-(Pyridin-2-yldisulfanyl)ethyl 4-cyano-4-(((ethylthio)carbonothioyl) thio) pentanoate (1.53 g, 3.53 mmol) prepared as described in Step 1 above was allowed to react with pentaerythritol tetrakis(3-mercaptopropionate) (0.422 g, 0.86 mmol) in dichloromethane solvent (20 mL) with 6 drops of glacial acetic acid. The reaction was stirred at room temperature for 48 h.
  • the crude reaction mixture was purified by column chromatography on a silica column (Merck 60, 70-230 mesh) first using ethyl acetate : pentane 1:1 (v/v) as the eluent to remove unreacted pyridinyl RAFT agent, then using ethyl acetate : DCM 1:19 (v/v) solvent to isolate the desired title product, four-arm star RAFT agent (0.21 g, 14 % yield) as a yellow oil.
  • a 4-arm poly(DEAEMA-co-BMA) macroCTA was synthesized in a manner analogous to the linear copolymer as described in Example 1, under the control and co- 30 polymerization of the 4-arm RAFT agent.
  • the initial monomer ([M] o ) to CTA ([CTA] o ) to initiator ([I] o ) ratio was 400 : 1 : 0.05.
  • MacroRAFT block extension via copolymerization of N,N-dimethylacrylamide (DMA) and pyridyl disulphide methacrylamide (PDSMA) with a representative poly(DEAEMA-co-BMA) macroCTA provided representative star copolymers [poly(DEAEMA-co-BMA)-b-(DMA-co-PDSMA) 4 ].
  • Polymerization provided a final molecular weight of the diblock copolymer 40 950 g/mol (Mn 40 950 g/mol 2 nd block) with a ⁇ of 1.16 and a 2 nd block composition of 95 mol % DMA and 5 mol % PDSMA (93:7 feed).
  • Table 9 Summary of final monomer conversions, theoretical molar masses (based on monomer conversion), DMAc–GPC molar masses and dispersities of 4-arm star EB polymerization at 90 °C.
  • Table 11 Summary of final monomer conversions, theoretical molar masses (based on monomer conversion), DMAc–GPC molar masses, and dispersities of 4-arm EB-b-DMA-co-PDSMA polymerisation at 60 °C.
  • the reducible inimer RAFT agent has the structure:
  • EDC ⁇ HCl (0.90 g, 4.68 mmol, 1.2 eq.) was dissolved in 20 mL and slowly added to the solution at room temperature. The solution was left stirring for 16h. The crude was washed with 150 mL of water & 150 mL of brine. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude was purified on silica eluting from pentane to 7 pentane : 3 ethyl acetate affording the product as a yellow-brown oil (0.41g, 23%).
  • reducible branched copolymers of the invention can be prepared by the polymerization procedures described herein.
  • the reducible crosslinker has the structure:
  • poly(DEAEMA-co-BMA-co-DSDMA) macroCTA can be prepared and then used to provide reducible crosslinked copolymers of the invention by the polymerization procedures described herein.
  • backbone reducible nonlinear copolymers of the invention can be prepared by the polymerization procedures described herein.
  • Aqueous polymer solutions were prepared by first dissolving dry copolymer into ethanol at 50 mg/ml followed by rapid dilution into HEPES buffered glucose (HBG; 20 mM HEPES, 5% glucose, pH 7.4) to a final concentration of 10 mg/ml.
  • HEPES buffered glucose HEPES buffered glucose
  • Ovalbumin ova; EndoGrade®, Hyglos GmbH
  • Thiol groups were incorporated onto ova using a 22 molar excess of 2-iminothiolane (Traut's reagent).
  • Non-reacted 2-iminothiolane was removed using a desalting column equilibrated with HBG.
  • the average number of thiol groups per ova was determined using Ellman's reagent (Thermo Scientific). For all studies 4-5 thiols per ova were introduced. In some instances, ova was labeled with AlexaFluor488®-TFP (Invitrogen) prior to thiolation with about 1 dye/protein. Thiolated ova was subsequently reacted with polymer micelles at a polymer concentration of 7.5 mg/ml. The extent of conjugation was determined via non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of conjugates prepared with fluorescently-labeled ova. To demonstrate the reducibility of the disulfide bond between polymer and protein, conjugates were incubated with 20 mM tris(2-carboxyethyl)phosphine for 1 h at room temperature.
  • HBG - HEPES buffered glucose 20 mM HEPES, 5% glucose, pH 7.4 solvent was thus tried and all polymers are completely soluble. No turbidity or precipitation in HBG or DC2.4 cell culture media. Micelle-sized particles (20-30 nm) were detected via DLS, see FIGURE 3.
  • Dynamic light scattering (DLS) measurements were conducted using a Malvern Zetasizer Nano ZS (Worcestershire, UK) at a constant scattering angle of 173°. Particle sizes of copolymer micelles and protein-polymer conjugates were determined by DLS at RT in HBG (pH 7.4) at 1 mg/mL polymer. In some cases, DLS measurements were performed with copolymers (0.5 mg/mL) incubated with 10 mM sodium phosphate buffer (supplemented with 150 mM NaCl) in the pH range of the endosomal processing pathway (7.4, 7.0, 6.6, 6.2, and 5.8). Mean diameters are reported as the number average ⁇ standard deviation from three or more measurements.
  • FIGURE 3 shows the number average diameter of the polymer systems tested under HEPES buffered glucose at pH 7.4, DLS at 0.7 mg/mL polymer.
  • FIGURE 4 shows the micelle to unimer (linear polymer) transition under a range of different pH.
  • the data show similar trends between architectures, though HB1:1 appears to transition more slowly than L1:1 and DV1:1. At pH 6.6 different particle sizes are evident.
  • FIGURE 5A shows the size of nanoparticles (number average diameter) by dynamic light scattering after reaction with thiolated ovalbumin (ova-SH) at 0, 0.35, 0.175, and 0.0875 mg ova/mg polymer.
  • FIGURE 5B shows SDS-PAGE of fluorescently labeled ovalbumin (ova) and ova-nanoparticle conjugates (0.35 mg ova/mg polymer) prepared using linear (L), divinyl crosslinked (DV), hyperbranched (HB), or 4-arm star polymers.
  • L linear
  • DV divinyl crosslinked
  • HB hyperbranched
  • 4-arm star polymers At lower ova loading, instability/aggregation of 1:1 branched materials (HB, DV, star) occurs, likely as a result of polymer crosslinking. Note dramatic increase in particle size via DLS.
  • FIGURES 6A and 6B show SDS-PAGE of fluorescently labeled ovalbumin (ova) and ova-nanoparticle conjugates (0.35 mg ova/mg polymer) prepared using linear (L), divinyl crosslinked (DV) hyperbranched (HB) or 4-arm star polymers (star): 1:1 diblock ratio conjugates (6A), and 1:2 diblock ratio conjugates (6B). Incubation of conjugates with TCEP liberates ova from the carrier.
  • FIGURES 7A and 7B show SDS-PAGE of a physical mixture of polymer systems of the present disclosure and non-thiolated, fluorescently-labelled ovalbumin: 1:1 diblock ratio conjugates (7A), and 1:2 diblock ratio conjugates (7B). Some minimal amount of antigen conjugation is observed upon mixture of polymer and non-thiolated ova.
  • Cells from the mouse dendritic cell line DC2.4 were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine, 100 ⁇ /mL penicillin/100 ⁇ g/mL streptomycin (Gibco), 55 ⁇ M 2-mercaptoethanol (Gibco), 1X nonessential amino acids (Cellgro), and 10 mM HEPES (Invitrogen). Cells were passaged at about 60-70% confluency using 0.25% trypsin- EDTA (Gibco).
  • B3Z T cells a lacZ-inducible T-cell hybridoma specific for the SIINFEKL-H-2Kb complex, were obtained from Nilabh Shastri (UC Berkeley) and cultured in RPMI 1640 (Gibco) supplemented with 10% FBS, 100 U/mL penicillin/100 ⁇ g/mL streptomycin (Cellgro), 50 ⁇ M 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Gibco). Both cell types were grown in a humidified atmosphere with 5% carbon dioxide at 37 °C.
  • Intracellular uptake of ovalbumin was evaluated by flow cytometry using AlexaFluor488®-labeled ovalbumin.
  • DC2.4 cells were plated at 75k cells/well in 24-well plates and allowed to adhere overnight. Cells were subsequently incubated with formulations containing fluorescently-labeled ova for 5 hrs, rinsed 2x with DPBS, trypsinized (0.25%, 5 min), pelleted by centrifugation, and re-suspended in DPBS containing 2% FBS.
  • Flow cytometry was performed on a FACSCantoII (BD) and analyzed using FlowJo software (Tree Star Inc.).
  • polymeric nanoparticles to enhance MHC class I antigen presentation was assessed by an in vitro antigen presentation assay using a DC2.4 cells as the antigen presenting cell.
  • This assay utilizes a specialized LacZ B3Z T cell hybridoma that produces ⁇ -galactosidase upon recognition of the immunodominant ovalbumin class I epitope SIINFEKL presented on MHC class I H-2Kb on DC2.4 cells.
  • DC2.4 cells were plated at 5x10 4 cells/well in U-bottom 96-well cell culture plates and grown overnight.
  • ova containing formulations were added to the final indicated concentration (7.7-245 ⁇ g/mL) and incubated with DC2.4 cells for 5 hours at 37 °C in a 5% carbon dioxide incubator.
  • Cells were then carefully rinsed 3x with DPBS and 1x10 5 B3Z T cells were added to each well and co-cultured for 20 h in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 55 ⁇ M beta-mercaptoethanol, 1 mM pyruvate, and 100 U/mL penicillin/100 ⁇ g/mL streptomycin.
  • FIGURE 9 illustrates the effect of polymer architecture on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • free antigen ova
  • conjugates prepared using polymers of different architecture ova
  • B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • Data is from a single representative experiment conducted in quadruplicate (mean ⁇ standard deviation).
  • HB1:1 significantly enhances antigen cross presentation
  • DV1:1 and Star show some enhancement
  • L1:1 has no activity.
  • Trends were largely reproducible between three independent studies.
  • FIGURE 10 illustrates the effect of 1:2 block ratio on polymer architecture on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • ova free antigen
  • B3Z T-cells B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • Data is from a single representative experiment conducted in quadruplicate (mean ⁇ standard deviation).
  • FIGURE 11 illustrates the effect of physical mixture of polymer architectures on MHC class I presentation in an in vitro co-culture model.
  • Murine dendritic cells DC2.4
  • free antigen ova
  • B3Z T-cells which produce ⁇ -galactosidase in response antigen presentation on MHC-I.
  • FIGURE 12 compares fluorescence measurements of antigen uptake by DC2.4 cells 0.35 g ova/g polymer, 245 ⁇ g/mL ova, 5 hr incubation. The differences in uptake between architectures are not substantial. Increasing the 2nd block length decreases antigen uptake.

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Abstract

La présente invention concerne des copolymères non linéaires possédant un bloc de libération endosomique sensible au pH et un bloc hydrophile, au moins l'un des blocs étant non linéaire. L'invention porte également sur des compositions qui comprennent des copolymères non linéaires, et sur des procédés de fabrication et d'utilisation de ces copolymères.
PCT/US2015/045114 2014-08-13 2015-08-13 Copolymères non linéaires en tant que véhicules d'administration intracellulaire et procédés associés Ceased WO2016025747A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018156600A1 (fr) * 2017-02-21 2018-08-30 University Of Florida Research Foundation, Incorporated Nouveaux agents anticancéreux à petites molécules
CN108659241A (zh) * 2018-04-27 2018-10-16 同济大学 具有温度响应性、pH响应性、二氧化碳响应性的星型聚合物、自愈合水凝胶及制备方法
WO2018195067A1 (fr) * 2017-04-17 2018-10-25 The University Of Chicago Matières polymères pour l'administration d'acides gras à chaîne courte à l'intestin pour des applications de santé humaine et de traitement de maladie
CN110183363A (zh) * 2019-06-18 2019-08-30 天津大学 一种甲基丙烯酸-3,4-二硫代羟己酯的衍生物及其制备方法与应用
WO2020084471A1 (fr) * 2018-10-22 2020-04-30 Takeda Pharmaceutical Company Limited Encapsulation de nanoparticules pour cibler des récepteurs couplés à la protéine g dans des endosomes
WO2022231676A1 (fr) * 2021-04-30 2022-11-03 Arkema Inc. Monomères de trithiocarbonate-acrylate et leurs procédés de fabrication
JP2023553010A (ja) * 2020-12-03 2023-12-20 バテル・メモリアル・インスティテュート 非ウイルス性送達のためのポリマーナノ粒子およびｄｎａナノ構造組成物ならびに方法
US12031128B2 (en) 2021-04-07 2024-07-09 Battelle Memorial Institute Rapid design, build, test, and learn technologies for identifying and using non-viral carriers
US12441996B2 (en) 2023-12-08 2025-10-14 Battelle Memorial Institute Use of DNA origami nanostructures for molecular information based data storage systems
US12454590B2 (en) 2019-12-02 2025-10-28 Ilumina Cambridge Limited Hydrogel
US12458606B2 (en) 2023-09-29 2025-11-04 Battelle Memorial Institute Polymer nanoparticle compositions for in vivo expression of polypeptides

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100150952A1 (en) * 2008-11-07 2010-06-17 University Of Washington pH-RESPONSIVE POLYMER CARRIER COMPOSITIONS FOR CYTOSOLIC PROTEIN DELIVERY
US20110281354A1 (en) * 2008-11-06 2011-11-17 Phaserx, Inc. Bispecific intracellular delivery vehicles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110281354A1 (en) * 2008-11-06 2011-11-17 Phaserx, Inc. Bispecific intracellular delivery vehicles
US20100150952A1 (en) * 2008-11-07 2010-06-17 University Of Washington pH-RESPONSIVE POLYMER CARRIER COMPOSITIONS FOR CYTOSOLIC PROTEIN DELIVERY

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COVERTINE, AJ ET AL.: "Development of a novel endosomolytic diblock copolymer for siRNA delivery.", JOURNAL OF CONTROLLED RELEASE., vol. 133, no. 3, 10 February 2009 (2009-02-10), pages 221 - 229 *
TAI, H ET AL.: "pH-responsive hyperbranched copolymers from one-pot raft copolymerization.", MACROMOLECULAR MATERIALS AND ENGINEERING., vol. 297, 2012, pages 1175 - 1183 *
WILSON, JT ET AL.: "pH-responsive nanoparticle vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides.", vol. 7, no. 5, 2013 *

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US11633486B2 (en) 2017-04-17 2023-04-25 The University Of Chicago Polymer materials for delivery of short-chain fatty acids to the intestine for applications in human health and treatment of disease
WO2018195067A1 (fr) * 2017-04-17 2018-10-25 The University Of Chicago Matières polymères pour l'administration d'acides gras à chaîne courte à l'intestin pour des applications de santé humaine et de traitement de maladie
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