Amphiphilic block copolymers, polymersomes, and methods of use

Abstract

Described herein are polymersomes (PSs) as a delivery platform that display enhanced macromolecular encapsulation efficiency with facile, streamlined processing. The formulations herein demonstrate rapid assembly of near-monodisperse PSs without organic solvents to circumvent purification issues. This is achieved through two design principles: 1) temperature responsive PS assembly and 2) affinity-driven payload encapsulation. The BCPs are solubilized in aqueous buffer when refrigerated (4 °C) but self-assemble at room temperature (20 °C) into homogeneous PSs. This is achieved by incorporating polymers segments with a lower critical solution temperature (LCST) below room temperature but above the freezing point of water. BCPs are dissolved alongside a hydrophilic payload, and their uniform self-assembly bypasses solvent and size-exclusion purifications.

Description

AMPHIPHILIC BLOCK COPOLYMERS, POLYMERSOMES, AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 63 / 616,005, filed December 29, 2023, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 29, 2024, is named “ARCDP0824WO.xml” and is 14,850 bytes in size.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under NIH 75N93019C00041 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTIONI. Field of the Invention

[0004] This invention relates to the field of molecular biology and methods of treating human disease.II. Background

[0005] Advancements in nanomedicine have allowed researchers to develop biomacromolecular therapies more efficiently and with greater precision, both considering small-interfering RNA (siRNA) therapy, messenger RNA (mRNA) therapies including vaccines, and protein therapies, including vaccines and inverse vaccines. However, effective payload protection and delivery are key requirements, as these macromolecules are highly sensitive to clearance and degradation when administered in vivo. To this extent, much promise has been demonstrated by the lipid nanoparticle (LNP) technology utilized by Pfizer / BioNTech® and Moderna® for their mRNA-based vaccines against SARS-CoV-2. Furthermore, LNPs designed by Alnylam® Pharmaceuticals have successfully demonstratedintravenous delivery of siRNA for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis in the liver using the RNA interference (RNAi) pathway. However, issues with storage stability and complex processing limit access to these novel therapies and necessitates improvements in formulation. Furthermore, LNPs are highly specialized for nucleic acid delivery, and therefore no formulations exist for protein payloads, e.g., use as subunit vaccines. Polymer nanoparticles have long been considered a key alternative to LNPs as the macromolecular nature of their substituents impart greater stability and tunability due to rapid advancements in the field. Furthermore, their synthetic versatility is particularly attractive for achieving delivery of a wide range of payloads such as proteins, small molecules and nucleic acids, namely mRNA and siRNA. By broadening the possible payloads that can be delivered in particulate formulations, a single encapsulation technology could be expanded to be applicable in a variety of treatments such as vaccination, tolerization, RNAi therapy and cancer treatment.

[0006] Polymersomes (PSs) are a class of vesicular polymer nanoparticles composed of self-assembled amphiphilic block copolymers (BCPs), first described by Discher and Eisenberg in the mid 1990s. These vesicles consist of a lyotropic membrane formed by macromolecular chain interactions between the hydrophobic blocks making them more stable than LNPs. The hydrophilic polymer block forms the PS corona and can prevent protein adsorption and unwanted immune recognition, thus prolonging circulation time. Moreover, their size (50-200 nm) and stability make PSs ideal for cellular uptake. Despite these benefits, formulation is complex as the organic solvents that are required to solubilize both domains must then be evaporated or diluted in an aqueous buffer. Self-assembly therefore occurs at an interface - either water-polymer or water-organic solvent - leading to inhomogeneous NPs that must be processed into uniform morphologies. Post-processing techniques require filter extrusions to ensure monodisperse populations and often lead to suboptimal payload encapsulation and material loss. Novel approaches address these limitations by rapidly mixing solvents with specialized microfluidic devices, but these require extensive operation time and greatly dilute the formulation. Furthermore, organic solvents can be toxic and denature biological payloads. Despite their synthetic tunability, PSs show modest loading efficiencies (-20% for proteins), and design complexity has prevented widespread clinical translation. Thus, there is a need in the art for improved drug delivery systems.SUMMARY OF THE INVENTION

[0007] The inventors have advanced polymersomes (PSs) as a delivery platform by enhancing macromolecular encapsulation efficiency with facile, streamlined processing. The formulations herein demonstrate rapid assembly of near-m onodi sperse PSs without organic solvents to circumvent purification issues. This is achieved through two design principles: 1) temperature-responsive PS assembly and 2) affinity-driven payload encapsulation. The BCPs are solubilized in aqueous buffer when refrigerated (4 °C) but self-assemble at room temperature (around 20 °C) into homogeneous PSs. This is achieved by incorporating polymers segments with a lower critical solution temperature (LCST) below room temperature but above the freezing point of water. BCPs are dissolved alongside a hydrophilic payload, and their uniform self-assembly bypasses solvent and size-exclusion purifications. Furthermore, cationic moieties are incorporated within the hydrophilic domains to attract negatively charged groups in protein and nucleic acid payloads. Electrostatics drive high loading efficiencies on the order of 75-99% and reduce the amount of synthetic material (50-200 pg) required for therapy. The inventors demonstrate the utility of such facile formulations in the context of protein subunit vaccination, tolerance induction, and RNA interference (RNAi) for cancer immunotherapy. Such a platform technology may prove useful in growing efforts to develop nonviral vectors for macromolecule delivery.

[0008] Described herein is an amphiphilic block copolymer (BCP) comprising a hydrophilic block comprising hydrophilic monomers and a hydrophobic membrane-forming block comprising hydrophobic monomers; wherein the lower critical solution temperature (LCST) of the copolymer is 0 °C - 22 °C. Also described is a polymersome comprising at least one amphiphilic BCP of the disclosure. In some aspects, the LCST is measured using water as a solvent. Also provided are compositions comprising at least one amphiphilic BCP of the disclosure and / or comprising a polymersome of the disclosure.

[0009] Methods include a method for making a block copolymer (BCP) comprising mixing hydrophilic monomers with hydrophobic monomers under conditions that allow for the polymerization of the hydrophilic and hydrophobic monomers. Methods include a method for making a block copolymer (BCP) comprising mixing at least one hydrophilic polymer block with at least one hydrophobic polymer block under conditions that allow for the polymerization of the hydrophilic and hydrophobic polymer blocks. Also provided is a method for making a polymersome comprising incubating a BCP or composition of the disclosure at a temperature greater than 15 °C or greater than the LCST to allow for the formation of polymersomes.Methods also include a method for inducing tolerance in a subject, the method comprising administering a polymersome or composition of the disclosure to the subject. Also provided is a method for vaccinating a subject, the method comprising administering a polymersome or composition of the disclosure to the subject. Further methods relate to a method for treating a disease in a subject, the method comprising administering a polymersome or composition of the disclosure to the subject. Also provided are polymersomes and BCPs made by the methods of the disclosure. Also described is a method comprising administering a polymersome or composition of the disclosure to a subject. In some aspects, a disulfide junction is included between a hydrophilic portion and a membrane-forming, LCST portion of the BCP.

[0010] The LCST of the BCP, of the hydrophilic monomers, of the hydrophobic monomers, of a hydrophilic block, and / or of a hydrophobic block may each be 0 °C - 22 °C. The LCST of the BCP, of the hydrophilic monomers, of the hydrophobic monomers, of a hydrophilic block, and / or of a hydrophobic block, may each be 0 °C - 17 °C. The LCST of the BCP, of the hydrophilic monomers, of the hydrophobic monomers, of a hydrophilic block, and / or of a hydrophobic block may each be, be at most, or be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 °C or any derivable range therein.

[0011] The BCP may be further defined as biocompatible. The hydrophilic weight fraction of the BCP may be about 10-25%. The hydrophilic weight fraction of the BCP may be, may be at least, or may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99%, or any derivable range therein.

[0012] The copolymer may comprise a membrane-forming block made of 120-175 membrane-forming block monomers. The copolymer may comprise a hydrophilic block made of 15-70 hydrophilic block monomers. The copolymer may comprise, comprise at least, or comprise at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or 400 monomers (or any derivable range therein) within a membrane-forming block or within a hydrophilic block.

[0013] The BCP may comprise an end modification. The end modification may comprise or exclude a mannose, mannose acrylate, a hydrophilic monomer, N-acetyl glucosamine, N- acetyl galactosamine, a targeting agent, or any combination thereof. The hydrophilic monomers may be uncharged. The hydrophilic monomers may be charged. The charge may be a positive charge. The charge may be a negative charge. In some aspects, a disulfide junction is included between an end modification and the BCP.

[0014] The hydrophobic monomers may comprise or exclude diethylene glycol ethyl ether acrylate (DEGEA). The hydrophilic monomers may comprise or exclude 2-hydroxyethyl acrylate (HEA). The hydrophilic monomers may comprise or exclude dimethyl aminoethyl acrylate (DMAEA), polyethylene glycol (PEG), poly(hydroxypropyl methacrylamide), poly(hydroxyethyl methacrylamide), poly(oxazoline). The hydrophilic monomers may comprise or exclude trimethylamino ethyl acrylate (TMAEA). The hydrophilic monomers may comprise or exclude mannose acrylate, N-acetyl glucosamine, and / or N-acetyl galactosamine. The hydrophilic monomers may comprise or exclude mannose acrylate and HEA. The hydrophilic block may comprise cationic moieties. The BCP may be lyophilized.

[0015] The BCP may comprise or exclude a polymer of formula (I):wherein Xi and Yi are independently an integer value ranging from 1-400 and Ri and R2 are each independently selected from an end modification or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0016] The BCP may comprise or exclude a polymer of formula (la):wherein Xi and Yi are independently an integer value ranging from 1-400 and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

[0017] The BCP may comprise or exclude a polymer of formula (II):wherein X2 and Y2 are independently an integer value ranging from 1-400 and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0018] The BCP may comprise or exclude a polymer of formula (Ila):wherein X2 and Y2 are independently an integer value ranging from 1-400 and R2 is selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

[0019] Ri may comprise or exclude H or mannose. The BCP may further comprise or exclude 2-(hexamethyleneimino)ethyl acrylate, 2,2-dimethylaminoethyl acrylate, and / or 2,2- diethylaminoethyl acrylate monomers. Other alkyl amine acrylate monomers, including but not limited to 2,2-dimethylaminoethyl acrylate, methylaminoethyl acrylate, ethylaminoethyl acrylate, and other alkylaminoethyl acrylate and dialkylaminoethyl acrylate monomers where the aminoalkyl group alkyl chain length ranges from 1 to 10 carbon atoms can be employed. Alkyl amine acrylate monomers can be employed where the ester alkyl chain can range in length from 1 to 10 carbon atoms, including but not limited to 2,2-dimethylaminomethyl acrylate, 2,2-diethylaminomethyl acrylate, 2-(hexamethyleneimino)methyl acrylate, methylaminomethyl acrylate, and ethylaminomethyl acrylate. The BCP may comprise or exclude a polymer block comprising 2-(hexamethyleneimino)ethyl acrylate, 2,2- dimethylaminoethyl acrylate, or 2,2-diethylaminoethyl acrylate monomers. The BCP may comprise or exclude a polymer of formula (III) or (IV):wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0020] The BCP may comprise or exclude a polymer of formula (Illa) or (IVa):wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and Ri and R2 are each independently selected from an end modification, ahydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0021] The BCP may comprise or exclude a polymer of formula (VI) or (VII):wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0022] The BCP may comprise or exclude a polymer of formula (Via) or (Vila):wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and R2 is selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

[0023] The BCP may comprise or exclude a polymer of formula (VIII) or (IX)wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

[0024] The BCP may comprise or exclude a polymer of formula (Villa) or (IXa):wherein W3, W4, Y3, and Y4 are each independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and R2 is selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

[0025] X3 and Z3 may be further defined as molar ratios. X4 and Z4 may be further defined as molar ratios. X3, Z3, X4, and Z4 may each independently be, be at least, or be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35,0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52,0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86,0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 or any derivable range therein. Furthermore, [X3 / (X3+Z3)] + [ZsAXi+Zs)] may equal 1, and [X4 / (X4+Z4)] + [Z4 / (X4+Z4)] may equal 1. X3 may be 0.5-0.9. X4 may be 0.5-0.9. Z3 may be 0.1-0.5. Z4 may be 0.1-0.5.

[0026] The polymersome may be 50-200 nm in diameter. The polymersome may be, be at least, or be at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480,485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575,580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955,960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm in diameter, or any derivable range therein. The size may be expressed as an average size of a composition comprising the polymersomes.

[0027] The polymersomes may include or exclude polymersomes that have undergone solvent purification. The polymersomes may include or exclude polymersomes that have undergone size exclusion purification. The polymersomes may include or exclude polymersomes that have been contacted with an organic solvent. The polymersomes may include those that have not been in contact with an organic solvent during the transition from BCP to polymersome.

[0028] The polymersome may comprise a payload. The payload may comprise or exclude a protein, an adjuvant, a nucleic acid, and / or a therapeutic molecule. The payload may comprise or exclude a protein antigen, a siRNA, mRNA, subunit vaccine, tolerogenic vaccine, and / or a siRNA-based cancer therapy. The polymersome may comprise at least two amphiphilic BCPs, wherein each amphiphilic BCP is independently selected from an amphiphilic BCP of the disclosure. The BCP:payload ratio may be 1 :5-1 :20. The BCP:payload ratio may be from, from at least, or from at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2,11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6,14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7,19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4,21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1,23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8,24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 to, to at least, or to at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6,11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3,13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7,16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4,18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1,20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8,21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5,23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2,25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 (or any derivable range therein).

[0029] The polymersome comprises 50-200 pg of payload. The polymersome may comprise, comprise at least, or comprise at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355,360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545,550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735,740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830,835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925,930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 pg of payload (or any derivable range therein).

[0030] The polymersome may be one that is unfiltered. The polymersome may be one that has not undergone filtration during and / or after the formation of the polymersome from theBCP composition. The RH (hydrodynamic radii) may be 75-200 nm. The RG (radius of gyration) may be 75-200 nm. The RH may be, be at least, or be at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425,430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615,620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710,715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805,810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900,905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm (or any derivable range therein). The RG may be, be at least, or be at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315,320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410,415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505,510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600,605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790,795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885,890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980,985, 990, 995, or 1000 nm (or any derivable range therein).

[0031] The form factor (RG / RH) may be 0.7-1.3. The form factor may be, be at least, or be at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5, or any range derivable therein. The polymersome may be at a temperature that is greater than the LCST. The polymersome may be or be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 °C above the LCST (or any derivable range therein). Thepolymersome may be at a temperature of more than 18 °C. The polymersome may be at a temperature that is, is at least, or is at most 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5,12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2,14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9,16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6,17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3,19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7,22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4,24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 °C (or any derivable range therein).

[0032] The composition may comprise at least a first and a second amphiphilic BCPs, wherein each of the first and second BCP is independently selected from an amphiphilic BCP of the disclosure. The composition may comprise a) a first amphiphilic BCP comprising a hydrophilic block and a membrane-forming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises TMAEA; and b) a second amphiphilic BCP comprising a hydrophilic block and a membraneforming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises HEA. The composition may further comprise a third amphiphilic BCP wherein the third BCP is independently selected from an amphiphilic BCP of the disclosure. The composition may comprise a fourth, fifth, sixth, or nth BCP, wherein each BCP is independently selected from a BCP of the disclosure and wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20) . The third BCP may comprise a hydrophilic block and a membrane-forming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises HEA and wherein the BCP is end modified with a HEA polymer monomer. The percentage of the first BCP may be 33% (of the total BCP). The percentage of the second BCP is 0, 10, or 33% (of the total BCP). The BCP, first, second, third, and / or nth BCP may be end modified. The end modification may comprise a mannose, mannose acrylate, a hydrophilic polymer subunit, N-acetyl glucosamine, N-acetyl galactosamine, or a targeting agent.

[0033] At least one of the first or second BCP (or nth BCP) may comprise a hydrophilic block, wherein the hydrophilic block comprises a charged monomer and wherein at least oneof the first or second BCP (or nth) comprises a hydrophilic block, wherein the hydrophilic block comprises an uncharged monomer. The composition may be at a temperature of greater than 16 °C. The composition may be at temperature of greater than 0 °C and less than 20 °C. The composition may be at a temperature of, of at least, or of at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3,11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13,13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7,14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4,16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1,18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8,19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5,21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2,23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 °C, or any derivable range therein. The BCP may be soluble in an aqueous solution at a temperature of less than 15 °C. The BCP may be soluble in an aqueous solution at a temperature of or of less than 13, 13.1, 13.2, 13.3,13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7,16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4,18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1,20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8,21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5,23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, or 25 °C, or any derivable range therein.

[0034] When referring to a composition comprising BCPs, the amount of BCP in the composition that it at a certain limitation may be, be at least, or be at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) of the total BCP in the composition. The composition may comprise an aqueous solution. The composition may comprise physiological salt concentrations, saline,and / or PBS (phosphate buffered saline). The total BCP in the composition may be 100 mg / mL or less. The total BCP in the composition may be, be at least, or be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 mg / mL, g / mL, pg / mL, ng / mL, g, ng, pg, or ng, or any derivable range therein.

[0035] The composition may comprise or exclude an immune shielding polymer (ISP) and wherein the ISP comprises a polymer that irreversibly binds to lysine residues. The ISP may have a molecular weight of 5-40 kDa. The ISP may have a molecular weight of, of at least, or of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, or 200 kDa, or any derivable range therein.

[0036] The ISP may comprise or exclude an amine-reactive chemical moiety. The aminereactive chemical moiety may comprise or exclude a N-hydroxysuccinimide (NHS) ester. The ISP may comprise or exclude one or more of (poly(ethylene glycol), poly(oligo(ethylene glycol) methyl ether methacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxyethyl methacrylamide), poly(oxazoline), dextran derivatives, and hyaluronic acid derivatives. The ISP may further comprise or may exclude conjugation to a sugar moiety. The sugar moiety may comprise or exclude mannose, glucose, galactose, N-acetyl glucosamine, and / or N-acetyl galactosamine. The ISP may be further defined as bioinert. The ISP may be further defined as pro-tolerogenic.

[0037] The ISP may comprise or exclude a compound of formula V or Va:The wavy line in the chemical formulas indicates an attachment to the molecule or, in the case of formula V, a continuation of the molecule. The ISP may be further defined as a random polymer.

[0038] A and B may be further defined as molar ratios. A and B may each independently be, be at least, or be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12,0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63,0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 or any derivable range therein. Furthermore, [X3 / (X3+Z3)] + [Z3 / (X3+Z3)] may equal 1, and [X4 / (X4+Z4)] + [Z4 / (X4+Z4)] may equal 1. A may be 0.5-0.9. B may be 0.1-0.5.

[0039] The composition may comprise a payload. The payload may comprise or exclude a protein, an adjuvant, a nucleic acid, and / or a therapeutic molecule. The payload may comprise or exclude a protein antigen, a siRNA, mRNA, subunit vaccine, tolerogenic vaccine, and / or a siRNA-based cancer therapy. The composition may be one in which at least 75% of the payload is encapsulated when the composition is at a temperature greater than the LCST. The composition may be one in which at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent (or any derivable range therein) of the payload is encapsulated when the composition is at a temperature greater than the LCST or when the composition is or is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 °C (or any derivable range therein) greater than the LCST. The molar ratio of ISP to payload may be 2: 1. The molar ratio of ISP to payload may be, be at least, or be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 (or any derivable range therein) to, to at least, or to at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 (or any derivable range therein).

[0040] The composition may comprise or exclude a quenching agent. The quenching agent may be non-toxic and biocompatible. The quenching agent may comprise or exclude a primary amine. The quenching agent may comprise or exclude lysine, glycine, or ethanolamine. The molar ratio of the quenching agent to the ISP may be 2: 1. The molar ratio of quenching agent to ISP may be, be at least, or be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 (or any derivable range therein) to, to at least, or to at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,9.8, 9.9, or 10 (or any derivable range therein). The composition may be one that has not been filtered. The composition may be one that has not been filtered after addition of the payload. The composition may be one that has not been filtered after transferring the composition to a temperature that is above the LCST of the BCP or polymer block comprised therein.

[0041] X, Y, Z, W, A, and B, as used in Formulas I, la, II, Ila, III, Illa, IV, IVa, V, and Va may be an integer from 1-400. X, Y, Z, W, A, and B may be, be at least, or be at most 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or 400, or any derivable range therein.

[0042] The ratio of X to Y, Z, W, A, or B may be, be at least, or be at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6,13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3,15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17,17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7,18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4,20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1,22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8,23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5,25.6, 25.7, 25.8, 25.9, or 30 to, to at least, or to at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6,10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3,12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14,14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4,17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1,19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8,20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5,22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2,24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 (or any derivable range therein).

[0043] The method may exclude contacting the BCP or the composition with an organic solvent. The method may comprise or exclude size exclusion purification. The method may comprise or exclude membrane filtration. The disease may comprise or exclude allergy or an allergic immune response. The payload may comprise an oncogene inhibitor(s). The oncogene may comprise Bcl-2 and / or VEGFA. The payload may comprise or exclude a Atf4, YTHDF2 inhibitor. The payload may comprise or exclude a siRNA, miRNA, or antisense oligonucleotide (ASO). The disease may comprise or exclude cancer. The cancer may comprise or exclude breast cancer. The subject may be one that a disease and / or has been diagnosed with a disease. The subject may be one that has not been diagnosed with a disease. The disease may comprise or exclude pre-cancer, dysplasia, and / or neoplasia.

[0044] The polymersome or composition may be administered intradermally, subcutaneously, intramuscularly, intratumorally, intralesional, intraocularly, orally, rectally, nasally, pulmonarily, or intravesicularly. Administration may exclude administration intradermally, subcutaneously, intramuscularly, intratumorally, intralesional, intraocularly, orally, rectally, nasally, pulmonarily, or intravesicularly.

[0045] The subject may be a human subject. The subject may be a laboratory test animal. The subject may be a mammal. The subject may be a rat, mouse, horse, cat, dog, pig, human, or non-human primate.

[0046] The copolymers described herein may be obtained using reversible additionfragmentation chain-transfer (“RAFT”) polymerization of an appropriate monomer with an initiator. The free terminus of the polymer may be one of a number of chemical groups or may be end modified to include a chemical group, including but not limited to hydroxyl, methoxy,benzyl, cyano, thiol, amine, maleimide, halogen, polymer chain transfer agents, protecting groups, drug, biomolecule, or tissue targeting moiety.

[0047] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0048] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in example 1 of this description and in the claims and disclosure of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.

[0049] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0050] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0051] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0052] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0053] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that maybe implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0054] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0056] FIG. 1A-1D. Thermoresponsive nanoparticles are composed of well-controlled block copolymers (BCPs) synthesized using RAFT polymerization. A. Schematic representation demonstrating the proposed mechanism of action for the BCPs described herein. Polymers are fully dispersed in aqueous solution at standard refrigeration temperatures and self-assemble homogeneously upon warming to room temperature. Red domains are thermoresponsive and blue are hydrophilic domains. B. BCPs used for blended formulations of polymersomes, with degrees of polymerization (subscripts) measured by NMR. End-groups for poly(diethylene glycol ethyl ether acrylate)-block-poly (hydroxyethyl acrylate) (D130-H70) are modified to incorporate either a single mannose moiety at the chain end or a bioinert hydroxyl moiety. All polymers are synthesized from the parent D130 homopolymer, and therefore the membrane-forming segment is the same length in all formulations. C. Gel permeation chromatography (GPC) for BCPs indicate monomodal distributions with low polydispersity. Note, D130-DM25 denotes p(DEGEA)-block-poly(dimethylaminoethyl acrylate) (pDEGEA-b-p(DMAEA)) prior to quatemization. D. Table of full chemical characterization of parent D130 and subsequent BCPs. Mn (NMR) was determined prior to end-group modification to retain the RAFT chain end protons, while Mn and poly dispersity (D) by GPC were measured post-modification.

[0057] FIG. 2A-2F. Polymersomes formed at room temperature are uniform in size and morphology, with high loading efficiency for protein and RNA payloads. A. Thermal response ofD 130-H70 in PBS as measured by temperature-varied dynamic light scattering (DLS) to probe total scattering intensity as well as particle size. Gradual increase followed by sudden drop in radius demonstrates distinct behavior associated with micelle-worm-vesicle transition. B. Quantification of BCP end-group using colorimetric assays to determine amounts of mannose (Phenol -H2SO4 assay) and residual free thiol (Ellman’s assay). C. Schematic representation for blended formulations consisting of three BCPs: D130-H70 (inert) , Di3o-H?o-Mann (mannosylated) and D130-TM25 (charged). For brevity, formulations are referred to only as weight percent of charge and mannosylated BCPs used (TXMY), with the remainder being the bioinert D130-H70. D. Size distributions of all nanoparticle formulations were measured by DLS and show favorable sizes (ca. 100 nm) with low dispersity (PDI<0.2) for blended formulations used in subsequent in-vitro and in-vivo work. E. Representative negative-stain TEM image of T0M0 polymersomes prepared at room temperature showing distinct vesicular morphology. Scale bar is 500 nm. F. Loading efficiency as determined by non-reducing, detergent-free gel electrophoresis for protein and RNA. Faintness in bands show effective loading relative to free payload control, demonstrating high loading efficiency for the BCP blend formulations indicated. Ovalbumin (OVA) was used as a model protein, and fluorescent (FAM)-siRNA and Luciferase (Luc)-mRNA used as model RNAs.

[0058] FIG. 3A-3G. Polymersomes enhance protein delivery in-vitro and stimulate antibodies in-vivo from a single dose subunit vaccine. A. Fluorescent OVA (FITC-OVA) uptake in bone-marrow derived dendritic cells (BMDCs) measured by flow cytometry with corresponding fluorescence histograms on right. Quantification of geometric mean fluorescence intensity (gMFI) of single, live population demonstrates significant uptake when the same amount of OVA is encapsulated in polymersomes. ****■. pO.OOOl. B. Antigen presentation probed using BMDC co-culture with CD8+ OVA-specific T-Cells (OTI) labelled with CFSE. Histogram on the left shows distribution of CFSE fluorescence signal, and populations left of the dotted line are new generations of OTI cells (“CFSEdiluted”), whose relative proportion is quantified by the bar graph on the right. ****■. p<0.0001. C. Vaccine timeline for mice (n=5) injected in all four hocks with single dose treatments. All non-saline treatments are adjuvanted with CpG ODN 1826, and blood was sampled periodically during a 90-day period. Statistics compare Day 84 antibody levels using oneway ANOVA with multiple comparisons corrected for with Tukey post-test. D. Antibody levels measured by ELISA and quantified as area under the curve (AUC) for 10-fold serial dilutions of sera. E. Antibody levelsmeasured 1 year post prime vaccination . F. In-vivo imaging (IVIS) for relative fluorescence in hock-draining lymph nodes of OVA647 injected either free or with polymersomes in all 4 hocks, where animals were sacrificed 4 hr after administration. Lymph nodes (LNs, n=2) were harvested from both sides of the animals from the following locations (top to bottom): axillary, brachial, inguinal, popliteal. G. Total radiant efficiency from brachial LN quantified. Data in A, B, and G are technical replicates plotted as mean ± s.d. and compared using ordinary oneway ANOVA with Tukey’s post-test for multiple comparisons. Data in c and d are biological replicates plotted as mean ± s.e.m. with p-values for Day 84 (in c) and 1 year antibody levels using one-way ANOVA with Tukey’s post-test for multiple comparisons.

[0059] FIG. 4A-4G. Polymersomes stimulate robust cellular and humoral immunity as a two-dose subunit vaccine, with marked enhancements in the CD8+ T-Cell response when using mannosylated formulations. A. Vaccine timeline showing prime-boost schedule with biweekly bleeds. All four hocks were injected and draining lymph nodes (dLNs) and spleen were harvested upon sacrifice. All non-saline treatments were adjuvanted with CpG ODN 1826. B. IFNy secreting CD8+ T-cells were measured in dLNs and spleen after 6 hr SIINFEKL (SEQ ID NOV) restimulation using intracellular staining. All percentages are reported by subtracting unstimulated from stimulated to remove non-specific responses. C. Double-positive TNFa+IFNy+ secreting CD8+ T-cells measured in dLNs and spleen after 6 hr SIINFEKL (SEQ ID NOV) restimulation D. Representative gating strategy for 6-hr restimulation for intracellular cytokines. Samples are first gated on single, live CD8+ T-Cells. E. Total OVA- specific antibody (IgG) levels measured by ELISA and reported as AUC from serum samples taken 2 wk post prime vaccination (Day 14) and 1 wk post boost (Day 27). F. OVA-specific CD8+ were probed using SIINFEKL (SEQ ID N0:9) / MHCI pentamer staining on antigen experienced, CD8+CD44+ T-cells 1 wk postboost. G. Lymphocytes from dLNs were stimulated for 3 d using the full OVA protein and IFNy, and TNFa levels measured in supernatants to determine a robust Type 1 immune responses for polymersome formulations. All values reported as amount from stimulated-unstimulated samples.

[0060] FIG. 5A-5H. Prophylactic intervention with antigen-loaded, mannosylated polymersomes ameliorates T cell-driven features of allergic airway inflammation. A. Schematic depicting a prophylactic treatment schedule in a mouse model of experimental allergic airway inflammation. Mice are prophylactically treated through intravenous injections of empty mannosylated polymersomes, or antigen-loaded mannosylated (or control) polymersomes prior to induction of allergic airway inflammation. Mice are sensitized through intraperitoneal injections of OVA (or saline) adsorbed to alum, prior to intratrachealinstillations with OVA. B. Representative flow cytometry plots depicting airway eosinophils (highlighted in box) isolated from bronchoalveolar lavage fluid. C. Airway eosinophil and CD4+ T cell quantification as determined by flow cytometry. D. Lung eosinophil and CD4 + T cell quantification as determined by flow cytometry. E. Lung CD4+ effector T cells (FoxP3- CD44+ CD62L-) and Type II lymphocytes (FoxP3- CD44+ ST2+ Gata3hl) as identified by flow cytometry. F. Systemic IgE levels as determined by ELISA, quantified through area under the curve of concentration vs. time as final amount determined on day 20 of study. G. Ex-vivo liver delivery comparing mannosylated to non-mannosylated polymersome delivery measured with in-vivo imaging (IVIS). H. Total radiance quantification from IVIS (n=2). Data are biological replicates plotted as box plots with max / min, median, and quartiles (C-F), line graphs (F, left) or bar graphs (H) with mean ± s.e.m and compared using ordinary one-way ANOVA with Sidak post-test for multiple comparisons.

[0061] FIG. 6A-6F. Nanoparticle-delivered siRNAs against VEGF-A and Bcl2 show enhanced gene knockdown in MCF-7 cancer cells in-vitro compared to commercial transfection agents, along with robust anti -turn oral efficacy in-vivo A. In-vitro gene, knockdown efficacy in MCF-7 cancer tumor lines using siVEGFa and siBcl-2 encapsulated either in polymersomes or Lipofectamine 2000 (Lipo), a commercial transfection agent. mRNA expression was measured by PCR relative to untreated groups and cells were treated under 2% serum conditions. B. Treatment timeline for intratumoral (i.t.) treatment in nude mice (n=5) inoculated with MCF-7 tumors behind the shoulder. Tumors were allowed to grow for 10 days, then mice were treated i.t. every alternate day for 10 days using either free or encapsulated siRNAs. Mice were sacrificed when tumor size reached 600 mm3C. Tumor growth curve obtained by measuring volume of tumors using calipers every other day from day 10 until day 35, with arrows indicated days of i.t. treatment. Nude mice lack the immune system necessary to fully reject tumors, though growth is halted during nanoparticle treatment. Tumor sizes were compared on day 20 after i.t. treatment halted (right) to measure significant difference between nanoparticle-treated and control groups. D. Survival curve for treated mice demonstrating enhanced survival for animals receiving nanoparticle-encapsulated siRNA. E. Tumor growth in nude mice inoculated with MCF-7 tumors and treated with either LNP-encapsulated or polymersome-encapsulated siRNAs. Arrows indicate days of i.t. treatment. F. Corresponding survival curves for head-to-head comparison of LNPs and polymersomes as the siRNA delivery material. Data in a are technical replicates plotted as mean ± s.d., compared with 2- way ANOVA with Sidak post-test for multiple comparisons. P-values in d and h determinedby Log-rank (Mantel-Cox) test against corresponding free siRNA and LNP-siRNA respectively.

[0062] FIG. 7A-7C. Synthetic Details. Synthetic scheme for pDEGEA used as a parent polymer for subsequent chain extensions and end-group modifications.

[0063] FIG. 8A-8B. Acrylate-modified mannose characterization. A) Synthetic scheme (top),JH NMR of mannose acrylate in a stock solution of DMF. NMR run in D2O B) Mass spectra of stock solution run on electrospray ionization mass spectrometry (ESI-MS) with acetonitrile as eluent.

[0064] FIG. 9A-9C. Synthetic Details. 1H NMR spectra for parent pDEGEA homopolymer as well as subsequent BCPs used for in-vitro and in-vivo work. All NMRs taken in de - DMSO, with 32 scans and 10s relaxation time. Peaks for D130-TM25 assigned using (Jesus-Tellez 2020) De Jesus-Tellez, M. A. et al. Macromolecular Chemistry and Physics 2020, 221 (9), 1900543.

[0065] FIG. 10A-10C. Endgroup modification. A) UV-Vis spectra for purified D130-H70 (left) and D130-TM25 at 5 mg / mL in DMF before and after endgroup modification. The peak at 375 in the latter is from residual lodomethane. Calibration Curve for B) Phenol-H2SO4 Assay using mannose monomer and C) Ellman’s Assay using 2-mercaptoethanol

[0066] FIG. 11A-11D. Di3o-(H3o-co-Mannio). A) DLS for particles formed at room temperature using the BCP alone without blending B) NMR spectra for BCP in de -DMSO. Peak assignments based on crude NMRs of Mannose Acrylate with pDEGEA prior to copolymerization with HEA (data not shown). C) GPC trace with pDEGEA superimposed. D) Full material characterization. Degrees of polymerization determined by NMR

[0067] FIG. 12A-12C. Multi-Angle Light Scattering (MALS) Data. A) Rayleigh-Gans approximation for scattering of spherical particles (Wyatt 2014). B) DLS of corresponding MALS samples denoting Rg / RH C) MALS scattering data for samples in B), note that M is only used as a fitting parameter, and cannot be used for Mwapproximation. Wyatt, P. J. Measurement of Special Nanoparticle Structures by Light Scattering. Anal. Chem. 2014, 86 (15), 7171-7183.

[0068] FIG. 13A-13B. Loading Efficiency using mannosylated polymersomes. Native gel electrophoresis (4-20% PAGE, TGX buffer) with quantification of loading efficiency for a variety of mannosylated nanoparticle formulations. 100 mg / mL polymer was loaded with 5 mg / mL OVA. Top standard (Lane 7) is 5 mg / mL OVA. Lanes 1,3 and 5 were also shown in FIG. 2F. B) Loading efficiency determined for therapeutic payloads such as Enbrel, anti-PDl and RBD-his peptide from Sars-Cov-2 spike protein. Odd-numbered lanes correspond to freepayload, and even-numbered lanes are the equivalent amounts with 33 wt% charged polymer. Quantification based on densitometry measurements of bands. Gels were run with 10% glycerol without loading dye to allow for short run times and similar migration distances.

[0069] FIG. 14A-14C. Toxicity, protein uptake and processing in antigen presenting cells. A) MTT toxicity assay in RAW 264.7 macrophages treated overnight with nanoparticle formulations consisting of varied weight percent of charged block copolymer. All formulations are treated at concentrations from 80-5000 ug / mL in serum-free media. B) Mannose receptor blocking overnight using blocking peptide (50 pg / mL) and treated with formulations from FIG. 3A. All data shown as technical replicates with mean ± s.d. Two-way ANOVA for technical replicates in B, with Sidak post-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. C) Fluorescence microscopy on RAW 264.7 macrophages treated overnight with T33M0 polymersomes co-encapsulating DQ-OVA and OVA647. Channels separated into columns and merged in right-most panel. Uptake probed with AF647 signal and endosomal processing and escape probed with DQ / FITC. Scale bars are 50 pm

[0070] FIG. 15A-15B S9. siRNA Knockdown controls. A) No off-target effects seen for housekeeping mRNA P-actin. B) polymer nanoparticles (33 wt% charged, T33M0), Lipo and siRNA have no inherent downregulation capacity.

[0071] FIG. 16A-16B. Gating Strategy (After gating lymphocytes, then single cells) for A) Cytokine-staining after 6 hour SIINFEKL (SEQ ID NO: 9) restimulation. Taken on LSRFortessa B) CD8+ T-cells after boost. Taken on NovoCyte Penteon. All gating on compensated data, using fluorescence-minus-one’s (FMOs).

[0072] FIG. 17. 3-day restimulation with OVA elicits enhanced IL-22 expression in dLNs and IL-6 in spleen

[0073] FIG. 18A-H Prophylactic intervention with antigen-loaded, mannosylated polymerosomes prevents B cell class-switching, germinal centre formation and differentiation into allergic IgE+ plasma cells. A - C. B cell inflammation as determined by B cells isolated from bronchoalveolar lavage (A), lung tissue (B) and lung-draining, mediastinal lymph node (C). D. Quantification on B cells that have undergone class-switch recombination and germinal centre formation. E. Accumulation of mediastinal lymph node germinal centre B cells. F. Ki- 67 expression within the germinal centre B cell compartment and quantification as assessed by flow cytometry. G. Plasma cell accumulation in the mediastinal lymph node. H. IgE containing plasma cells within the plasma cell compartment and quantification as assessed by flow cytometry.

[0074] FIG. 19A-19D. In-House LNP formulation (Onpattro mimics). A) formulation components and respective molar ratios used. B) DLS sizes of comparing

[0075] In-House LNP (after vortexed dispersion in PBS) to BCP blend polymersomes in this work. C) Loading efficiency determined by non-reducing gel electrophoresis (4-20% PAGE, TBE buffer). Nanoparticles each with 150 ng FAM-siRNA (also the control) D) Gene knockdown efficacy for novel siRNA Atf4 and YTHDF2 in RAW 264.7 macrophages compared to in-house Onpattro® mimics. Polymersome formulations consist of 33 wt% D130- TM25 (T33M0) while the mannosylated has 33 wt% mannosylated BCP (T33M33).

[0076] FIG. 20A-20B. Functionalized Polymers. A) DC-based polymers for enhanced mRNA delivery with i) DC-H and ii) DC-DM with “x” kept to roughly 0.9 along with B) Immune shielding polymer (ISP) for passivation of unencapsulated protein payloads.

[0077] FIG. 21A-21B. mRNA in-vitro delivery. A) Loading efficiency of Luciferase mRNA (mLuc) using varying charged weight percentages of DC-based polymers polymers (DC-DM is the charged polymer) B) DC-based polymers with 50 and 100 weight percent charged fraction (DM50 and DM100 respectively) were compared for delivery efficacy of mLuc. Successful delivery of mLuc was achieved with DM50 with enhanced efficacy over commercial Lipofectamie under 2% serum-containing conditions as measured by Luciferase Assay.

[0078] FIG. 22A-22D. Ionizable polymers synthesized using 2 different ionizable moieties. A) Synthetic scheme for sequential RAFT polymerization with the following conditions i) for pDC and pDE macroCTA synthesis, AIBN (0.05 molar equivalent relative to trithiocarbonate), DMSO (solvent), 80°C, trifluoroacetic acid (TFA, 1.5x molar equivalent relative to amine), ii) AIBN (0.05 equiv relative to trithiocarbonate), DMSO, 80°C iii) AIBN (0.05 equiv. relative to trithiocarbonate), DMSO, 80°C, TFA (1.5x relative to all amines). B) schematic overview of ionizable polymers utilizing both moieties. C) formulations synthesized with differing degrees of ionizability and different locations for secondary armine placement. D) DLS measurements for i) LOST and ii) total scattering intensity (counts) and size of particles as a function of pH at 0.1 mg / mL polymer (1 / 1, w / w, blend of Gen la).

[0079] FIG. 23A-23C. Polymers incorporating disulfide junctions and ionizable moieties for enhanced endosomal escape A) Synthesis of i) pryidyl disulfide (PDS) functionalized RAFT (top) and ATRP (bottom) agents and ii) functionalization of pHEA with disulfide containing RAFT agent (right) or ATRP initiator (left) for subsequent chain extension. B) Proposed structures for polymers synthesized using atom-transfer radical polymerization(ATRP) and C) RAFT polymerization using pHEA. A variety of ionizable and charged amines can be incorporated either as a block copolymer or statistical copolymer.

[0080] FIG. 24A-24G. Polymersome-based subunit vaccines elicit potent immune responses nearly 1 and a half years following single dose and two-dose injections. A) Immunization regimen for single dose vaccine (10 ug OVA, 20 ug CpG for all formulations besides saline) benchmarking T33M0 (PS-OVA, 100 ug) and T33M33 (MannPS-OVA, 100 ug). Mice were bled periodically to measure antibody levels and memory immune responses were stimulated with 5 ug OVA and 20 ug LPS 3 days prior to sacrifice and analysis of T-cell response. B) Antibody levels (Total OVA-specific IgG, area under the dilution curve (AUG)) measured over the 3 months following vaccination C) Antibody levels for i) Total IgG 1 year after vaccinaion ii) relative type 1 vs type 2 immunity measured by antibody subtypes (IgG2b / IgGl) and iii) Total IgG 2 days after LPS challenge. D) T-cell responses in the draining lymph nodes following restimulation measured by flow cytometry for IFNy+TNFa+ doublepositive i) CD8+ T-cells, stimulated with OVA peptide and ii) CD4+ T-cells stimulated with full OVA protein, along with iii) IFN / +CD4+ T-cells, iv) Full protein restimulation response also measuring IFNy amount in serum using Legendplex. A) Immunization regimen for two- dose vaccine (same dose as above) benchmarking T33M0 (PS-OVA, 100 ug) and T33M10 (MannPS-OVA, 100 ug). Mice were challenged with 2 million B16 mouse melanoma cells expressing OVA 16 months from the start of the experiment. F) survival and G) tumor growth3 was measured periodically, and mice were sacrificed when tumors reached 500 mm' .

[0081] FIG. 25A-25C. Polymersome-based siRNA therapies intended to modulate macrophage polarization promote antitumoral responses and enhance survival against a mouse colon carcinoma. A) Treatment timeline for mice inoculated with murine, MC38 colon cancer line, then treated periodically with siRNA (20 ug) with 200 ug vehicle in a variety of formulations. Polymer formulation is T33M10 and arrows indicate treatment, and B) tumor growth and C) survival were measured.

[0082] FIG. 26A-26C. Ionizable polymers show in-vitro efficacy in mRNA delivery and expression. A) Structures and nomenclatures used for polymers in in-vitro uptake and expression experiments. All polymers were used as a 1 : 1 (w: w) blend of charged and uncharged analogs. B) schematic overview of experiment C) i) uptake measured by gating on single, live cells and then gating Cy5+ population and iii) expression probed by measuring gMFI of FITC of single, live cells.

[0083] FIG. 27A-27H. Polymersomes stimulate robust cellular and humoral immunity as a two-dose subunit vaccine, with elevated CD8+ T-cell response and enhanced Type 1 immunity. A. Vaccination timeline with prime-boost schedule in C57BL / 6 mice (n=6). Four hocks were injected and draining lymph nodes (dLNs) and the spleen was harvested postsacrifice. All treatments were 10 pg OVA adjuvanted with 20 pg CpG ODN 1826, except saline and empty polymersomes (T33M10-empty). Encapsulated formulations consisted of 100 pg polymer. B. IFNy-secreting CD8+ T-cells were measured in dLNs and spleen after 6 hr SIINFEKL (SEQ ID NOV) restimulation using intracellular staining, with percentages reported by subtracting unstimulated from stimulated to remove non-specific responses. C. Double-positive TNFa+IFNy+-secreting CD8+ T-cells measured in dLNs and spleen after 6 hr SIINFEKL (SEQ ID NOV) restimulation. D. Representative gating strategy for 6-hr restimulation for intracellular cytokines. Samples are first gated on single, live CD8+ T cells. E. Gating strategy for SIINFEKL (SEQ ID N0:9) / MHCI tetramer staining on antigen experienced CD8+CD44+ T cells to measure antigen specificity. F. Quantification of antigenspecific CD8+ T cells. G. Splenocytes stimulated for 3 days using the full OVA protein, where IFNy, and H. TNFa levels measured in supernatants to indicate Type 1 immune response, i, Total OVA-specific IgG levels measured by ELISA and reported as area-under-the-curve (Log AUC) from serum samples taken 2 wk post-prime vaccination (Day 14) and 1 wk post boost (Day 27). Data are biological replicates plotted as box plots with max / min, median, and quartiles and compared using ordinary one-way ANOVA with Tukey’s post-test for multiple comparisons (for b, c) and Kruskal-Wallis test with Dunn’s post-test for multiple comparisons (for f). Welch’s ANOVA with Dunnet’s post-test was used for g, h and two-way ANOVA with Sidak post-test was used for i.

[0084] FIG. 28A-28C.JH NMR spectra (left column) for parent pDEGEA homopolymer as well as subsequent BCPs used for in-vitro and in- vivo work. All proton NMRs taken in d6- DMSO, with 32 scans and 10s relaxation time. Peaks for D130-TM25assigned using (Jesus-Tellez 132020). ' C NMR (right figure of each of A-C) spectra for parent pDEGEA homopolymer as well as subsequent BCPs used for in-vitro and in- vivo work. All carbon NMRs taken in d6-DMSO and peak splitting is due to chirality of polymer backbone. Endgroups not visible under the current conditions for carbon NMR. De Jesus-Tellez, M. A. et al. Macromolecular Chemistry and Physics 2020, 221 (9), 1900543.

[0085] FIG. 29A-29C. Polymersome size, zeta potential and morphology as a function of charge. A) Zeta potential measurements using elecotrophoretic light scattering (ELS) fornanoparticle formulations varying the amount of total charged polymer (D130-TM25) used. 10 Hz electric field was applied. B) Size and polydispersity measurements using DLS for varying amounts of charged material, in addition to those in Figure 2d in the main text. C) Multi-angle light scattering (MALS) data for charged polymer alone D130-TM25 with raw data (“RAW”) fit using both spherical and flexible coil models for extracting radius of gyration (RG) and form factor RG / RH.

[0086] FIG. 30A-30C. Loading and bioactivity of lyophilized and reconstituted formulations polymersomes. Lyophilized samples were reconstituted with deionized water on ice, then allowed to come to room temperature prior to gel electrophoresis. A) i) Image of gel for protein samples reconstituted to a concentration of 100 mg / mL polymer with 10 mg / mL OVA and run on a gradient, 4-20% polyacrylamide gel with Tris-Glycine (TGX) buffer under non-reducing, detergent free conditions, ii) Quantification of bands. B) siRNA samples were reconstituted to 0.1 mg / mL polymer and 0.01 mg / mL FAM-siRNA. Lane bands are shown in the following order from left to right (Lanes 1-4): T50M0, T33M0, T0M0, free payload. C) BMDC-OTI co-culture studies using lyophilized, reconstituted formulations of polymers and OVA, following previously described procedures and comparing to freshly prepared samples. ”OVA” is unencapsulated, while T33M0 and T33M10 refer to encapsulated formations of OVA. Values for CFSE-dilution should not be compared to study in Fig 3., due to biological variabilities in cell source.

[0087] FIG. 31A-31F | OVA and siRNA-loaded materials size, zeta potential and morphology. A) Size of nanoparticles when loaded with protein OVA or siRNA. Polymer: protein mass ratio is 10: 1, while polymersiRNA ratio is 5: 1, corresponding to conditions used for all in-vitro and in-vivo work. B) Zeta potential measurements of empty polymersomes (PSs), free protein and siRNA, loaded nanoparticles, and protein-loaded nanoparticles diluted in 0.2 mg / mL CpG. 10 Hz electric field was applied. Polymer: protein mass ratio is 10: 1, while polymer: siRNA ratio is 5: 1. C) Effect of polymer: siRNA weight ratio on zeta potential for siRNA. Polymers loaded with 50 pg / mL siRNA, with polymer concentration varied. D) Effect of polymer: OVA weight ratio on zeta potential for siRNA. Polymer at 100 mg / mL, OVA concentration varied. Samples diluted 1 : 100 for zeta potential measurement. E) Negative-stain TEM (uranyl acetate) of T33M0 loaded with siRNA. F) Negative-stain TEM of T33M0 loaded with OVA.

[0088] FIG. 32A-32H. In-vivo toxicity of empty polymer nanoparticles administered intravenously in naive female C57BL / 6 mice. A-G) Blood chemical analysis to measure the amounts of albumin, alanine transaminase (ALT), amylase (AMYL), blood urea nitrogen(BUN), total bilirubin (TBILI) and total protein (TP). Corresponding units shown in title. H) systemic cytokines in blood following injection of nanoparticles measured by Legendplex. Animals were injected intravenously via tail vein with 100 pg polymer in 100 pL saline. Animals were sacrificed 5 days following injection.

[0089] FIG. 33A-33B. Inherent immunostimulatory effect of T33M0 polymer and CpG DNA, along with loaded and admixed formulations. A) NF-kB activation meausured using RAW-Blue reporter macrophages treated with varying amounts of polymer, free CpG and particle-associated CpG. B) 1% agarose gel electrophoresis with TAE buffer and SYBR safe stain to measure association of admixed and encapsulated CpG with polymers. Admixed formulation has nearly 70% of CpG associated when compared to free CpG band density.

[0090] FIG. 34A-34B. Gating strategy for Fig 4, OVA-subunit prime-boost vaccine. A) Antigen-specific CD8 T-cells probed with SIINFEKL (SEQ ID N0:9) / MHCI tetramer on total CD8+and antigen- experienced (CD44 CD8 ) T-cells. All gating on compensated data, using fluorescence-minus-one’s (FMOs). B) Intracellular cytokine-staining after 6-hour restimulation with SIINFEKL (SEQ ID NO: 9) (in the case of CD8+response) or OVA (in the case of CD4+response). Data collected on BD LSRFortessa 4-15

[0091] FIG. 35A-35C. Additional, cellular and humoral readouts for Fig 4, OVA-subunit prime-boost vaccine A) Flow cytometry data probing i) percentage of antigen-specific, SIINFEKL (SEQ ID N0:9) / MHCI tetramer positive of total CD8+T-Cells ii) IL-2 and iii) IFNy- secreting CD4+T-cells after 6h restimulation with OVA. B) Legendplex data probing i) IL-6 secretion from splenocytes ii) IL-6 and iii) IFNy secretion from lymphocytes following 3 -day restimulation with OVA. iii) AUC of IgGl vs IgG2b from animal sera at endpoint to probe relative TH2 VS. TH1 responses respectively taken at experiment endpoint. Box and violin plots denote max / min, median and quartiles. All data are biological replicates compared with one-way (for A and Bi-ii) and two-way ANOVA (C).

[0092] FIG. 36A-36G. siRNA knockdown controls, dosing studies and in-vivo validation. A) Relative in-vitro mRNA degradation based on varying polymer weight ratio (uncharged: charged, D|30-H75:D|30-TM25) B) siRNA dose-dependent downregulation in-vitro C)No off-target effects seen for housekeeping mRNA P-actin in-vitro. D) polymer nanoparticles (33 wt% charged, T33M0), Lipo and siRNA have no inherent downregulation capacity in-vitro. E) In-vivo validation of VEGF-A mRNA downregulation F) Bcl2 mRNA downregulation and G) both proteins downregulation for MCF-7 tumors in nude mice treated twice (2-day intervals) withvarious formulations of siRNA. T33M0 polymer administered at 5: 1 (polymer: siRNA) weight ratio. All tumors treated starting day 11 and harvested at day 15 and flash frozen.

[0093] FIG. 37A-37B. Tumor sizes for MCF-7 in nude mice following siRNA intratumoral treatment as outlined in Fig 6. A) Initial efficacy study comparing T33M0-based delivery compared to free siRNA and B) follow-up efficacy study benchmarking to encapsulated siRNAs. Data are biological replicates compared with one-way ANOVA with Tukey’s post-test for multiple comparisons.

[0094] FIG. 38. Ex-vivo distribution of fluorescently labelled polymer injected intravenously and subcutaneously. A) Biodistribution of fluorescently tagged (AlexaFluor-647) polymers administered subcutaneously (s.c., rear flank) and intravenously (i.v., tail vein) in C57BL / 6 mice bearing MC38 tumors. Mice were sacrificed 24 hr post s.c. and 3 hr post i.v. injections, and IVIS imaging was conducted.DETAILED DESCRIPTION OF THE INVENTION

[0095] The current disclosure describes polymersomes that are formed by utilizing the thermoreversible behavior of the LCST polymer in the BCP. Specifically, the inventors are able to form stable, organized structures of predictable size ranges, and which is independent of any post-processing that is required of typical nanoparticles. The BCPs of the disclosure allow for rapid self-assembly into polymersomes upon dissolution in saline solution and roomtemperature warming, allowing for the generation of large batches (i.e. 10 mg of material makes 50-200 doses depending on the treatment) of polymersome treatments with no material loss due to purification.

[0096] By incorporating cationic moieties, the inventors achieve high loading efficiencies for both protein (75-90%) and nucleic acids (-99%), the former of which has not been previously demonstrated by polymersome systems. This polymer-based system provides key benefits over lipid nanoparticles in the context of protein encapsulation, as LNPs have demonstrated limitations in stability and efficiency when encapsulating and delivering proteins in animal models. The practical benefits of stability and high loading efficiency circumvent the need to purify unencapsulated payload since most is within the particles, and further concentrating of sample (common in the field of nanoparticle treatment) is not necessary to see a robust immune response.

[0097] The particles can also be decorated with mannose moieties to enhance immune effect both in the context of vaccination and tolerance induction. The benefit overmannosylated polymer conjugates is that the system is payload-agnostic, meaning that no chemical coupling is necessary to enhance the immunogenicity of the payload.

[0098] In the examples of the application, the inventors have demonstrated that this system can be used as a platform technology capable of delivering both protein and nucleic acid. The examples show delivery and treatment in three clinically-relevant settings using mouse models. These include: 1) Prophylactic vaccines using a model protein payload, 2) Prophylactic treatment for an allergy model using protein payload, and 3) Cancer therapy using smallinterfering (si)-RNA.I. Definitions

[0099] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0101] As used herein, the term “copolymer” refers to a polymer formed from two or more different monomers. Exemplary copolymers include alternating copolymers, random copolymers, block copolymers, etc.

[0102] As used herein, the term “block copolymer” refers to copolymers wherein the repeating subunits are polymeric blocks, i.e. a polymer of polymer blocks. In a copolymer of blocks A and B, A and B each represent polymeric entities themselves, obtained by the polymerization of monomers. Exemplary configurations of such block copolymers include branched, star, di-block, tri-block and so on. A copolymer block length refers to the number of monomers within a block.

[0103] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions. A “pharmaceutical composition” typically comprises at least one active agent (e.g., the copolymers described herein) and a pharmaceutically acceptable carrier.

[0104] As used herein, the term “biocompatible” refers to materials, compounds, or compositions means that do not cause or elicit significant adverse effects when administered to a subject. Examples of possible adverse effects that limit biocompatibility include, but are not limited to, excessive inflammation, excessive or adverse immune response, and toxicity.

[0105] The term “polymersome” refers to a vesicle that encloses a composition. The polymersome may be used for encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. The polymersome membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems.

[0106] The term “bioinert” refers to any material that does not elicit a response, such as an immune response, from the host.

[0107] The term “pro-tolerogenic” refers to a substance that produces immunological tolerance. Immune tolerance, or immunological tolerance, or immunotolerance, is a state of unresponsiveness of the immune system to substances or tissues that would otherwise have the capacity to elicit an immune response in a given organism. It can be induced by prior exposure to that specific antigen.

[0108] The term “lower critical solution temperature (LCST)” refers to the critical temperature below which the components of a mixture are miscible in all proportions.II. Payload

[0109] The compositions, polymersomes, and BCPs of the disclosure allows for the delivery of one or more therapeutic or other payload (including, for example, diagnostic agents)to an individual in need of the payload(s). The system, in particular cases, allows delivery of more than one payload, and such multiple payloads may be of the same type of agent (nucleic acid or drug, for example) or not. Thus, in a plurality of polymersomes, there may be a mixture of polymersomes with more than one agent but with each separate polymersome having only one agent; a therapeutically effective amount of the agent may be provided to the individual. Also provided is a mixture of polymersomes with more than one agent but with a particular polymersome having more than one agent. In any case, a therapeutically effective amount of the agent may be provided to the individual.

[0110] The payload may be one or more of a nucleic acid, small molecule, protein, peptide, or mixture thereof. The agent may be a drug. Particular nucleic acid examples include oligonucleotides, miRNA, shRNA, siRNA, DNA, RNA, mRNA, cDNA, double stranded nucleic acid, single stranded nucleic acid, and so forth. The size of the nucleic acid may be as large as a large plasmid or as small as a small oligonucleotide, as examples only. In some cases, the DNA is a vector comprising an expression construct for expression of one or more therapeutic polynucleotides or one or more polynucleotides that encodes a therapeutic gene product.[OHl] In some embodiments, the payload comprises a therapeutic gene product that is an entity that reduces at least in part if not in full the expression of an oncogene. Examples of the agent could be anti-sense RNA, miRNA oligo, or shRNA to silence a gene. The payload may be one that targets a tumor suppressor gene to increase the expression of the tumor suppressor gene.

[0112] The payload may be a small molecule, such as a drug that can be utilized to treat a medical condition. Hydrophilic drugs and / or hydrophobic small molecule drugs may be utilized.

[0113] The payload may be a protein or peptide. The protein or peptide may be therapeutic and / or diagnostic. The protein or peptide may be a vaccine. The protein may be a protein naturally endogenous to the individual to which the protein is delivered, but the endogenous level is deficient or naturally insufficient or would be beneficial to be present or abundant above a natural level.

[0114] Other therapeutic agents useful as payloads in the compositions and polymersomes of the disclosure include painkillers, diuretic, diabetic drugs, acid reflux drugs, high blood pressure drugs, thyroid hormone, high cholesterol drugs, and so forth. The medical condition may be heart disease, kidney disease, stroke, respiratory disease, septicemia, and so forth.

[0115] Other payload agents may be employed that are not therapeutic. In certain cases, one may employ the polymersomes to deliver one or more diagnostic agents as a payload. Such an agent may have a label, for example, that allows it to be tracked within an individual’s body. In specific embodiments, there are fluorescent microspheres and nanoparticles for imaging. Exemplary labels and colors include blue, green, orange, red and near-IR.

[0116] The agent may be employed systemically throughout the body or the agent may be delivered locally in a body. Besides human diseases, the polymersome delivery system can be also used in the treatment of animal diseases and in research in microorganisms, cell cultures and animal models, for example.

[0117] The payload in the polymersomes and compositions of the disclosure may comprise antigenic components including segments, fragments, or epitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipids and other molecules that provoke or induce an antigenic response, generally referred to as antigens. In one embodiment, the antigen is a peptide. In particular, antigens, or antigenic segments or fragments of such antigens, which lead to the destruction of a cell via an immune response, can be identified and used in the methods and compositions described herein.

[0118] Antigens associated with various diseases and infections are known in the art. It is contemplated that any antigen may be used in the methods and compositions described herein.

[0119] Non-limiting examples of viral antigens useful in the polymersomes and compositions of the disclosure include or exclude retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpl, gpll, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS1,NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components; picornavirus antigens, such as enteroviruses, rhinoviruses, hepamavirus, cardioviruses and aphthoviruses; viral antigens derived from a pestivirus, such as bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease (BDV); or antigens derived from a coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine transmissible gastroenteritis virus (TGEV). See, Vaccines, 6th Edition (Plotkin, Orenstein and Offit ed. 2012); Medical Microbiology 8th Edition (Murray, et al. ed. 2015) Fundamental Virology, 4th Edition, (Knipe, D. M., et al. eds. 2001) for additional examples of viral antigens.

[0120] Non-limiting examples of bacterial antigens useful in the polymersomes and compositions of the disclosure include or exclude bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diptheria bacterial antigens such as diptheria toxin or toxoid and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; Staphylococcal antigens, such as Staphylococcus aureus antigens, gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components, Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85 A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; haemophilus influenza bacterial antigens such as capsular polysaccharides and other haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens. Partial or whole pathogens may also be: haemophilus influenza; Plasmodium falciparum; neisseria meningitidis; streptococcus pneumoniae; neisseria gonorrhoeae; salmonella serotype typhi; shigella; vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis; Lyme disease; Yersinia pestis; west nile virus; yellow fever; Zika virus; tularemia; hepatitis (viral; bacterial); RS V (respiratory syncytial virus); HPIV 1 and HPIV 3; adenovirus; small pox; allergies and cancers.

[0121] Non-limiting examples of fungal antigens useful in the polymersomes and compositions of the disclosure include or exclude Candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.

[0122] Non-limiting examples of protozoal and other parasitic antigens can include or exclude plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, blood-stage antigen pf 155 / RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components; malaria antigens such as Plasmodium Glutamate dehydrogenase, histidine rich protein II, lactate dehydrogenase, and aldolase.

[0123] The antigen may also include or exclude one or more of viruses (inactivated, attenuated, and modified live), bacteria, parasites, nucleotides, polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including tumor cells), tissues, polysaccharides, carbohydrates, fatty acids, teichioc acid, peptidoglycans, lipids, or glycolipids, individually or in any combination thereof. The antigen may be wild-type or mutated.

[0124] The antigens used in methods and compositions described herein also include or exclude immunogenic fragments of nucleotides, polynucleotides, peptides, and polypeptides that can be isolated from the organisms referred to herein.

[0125] Live, modified-live, and attenuated viral strains that do not cause disease in a subject have been isolated in non-virulent form or have been attenuated using methods well known in the art, including serial passage in a suitable cell line or exposure to ultraviolet light or a chemical mutagen. Inactivated or killed viral strains are those which have been inactivated by methods known to those skilled in the art, including treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), sterilizing radiation, heat, or other such methods.

[0126] Two or more antigens can be combined to produce a polyvalent composition that can protect a subject against a wide variety of diseases caused by the pathogens. Currently, commercial manufacturers of vaccines, as well as end users, prefer polyvalent vaccine products. While conventional adjuvants are often limited in the variety of antigens with which they can be effectively used (either monovalently or polyvalently), the compositions and methods described herein can be used effectively with a wide range of antigens, both monovalently and polyvalently. Thus, the antigens described herein can be combined in a single composition comprising the conjugates described herein.

[0127] The cancer antigen can include or exclude any type of cancer antigen known in the art. The cancer antigen may be an epithelial cancer antigen, (e.g., breast, gastrointestinal, lung), a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen.

[0128] In one embodiment, the cancer antigen is a melanoma cancer antigen. Melanoma cancer antigens are useful for treating melanoma. Non-limiting exemplary melanoma cancer antigens include MART-1 (e.g., MART-1 26-35 peptide, MART-1 27-35 peptide); MART- 1 / Melan A; pMell7; pMell7 / gpl00; gplOO (e.g., gp 100 peptide 280-288, gp 100 peptide 154- 162, gp 100 peptide 457-467); TRP-1; TRP-2; NY-ESO-1; pl6; beta-catenin; mum-1; and the like.

[0129] In some embodiments, the antigen is a malaria antigen such as Plasmodium glutamate dehydrogenase, histidine rich protein II, P. falciparum lactate dehydrogenase, fructose-bisphosphate aldolase, or circumsporozite protein (CSP). In some embodiments, the antigen is an antigenic component found in Plasmodium sp. Such as P. falciparum.III. Delivery of polymersomes

[0130] The BCPs, polymersomes, and compositions of the disclosure may be delivered to an individual in need thereof in a variety of suitable ways. The BCPs, polymersomes, and compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art.

[0131] Vaccines including microorganisms and specific antigens can be delivered by the polymersomes. Examples of vaccines include those for cancer or infection, such as infection by a microbe. Examples of vaccines include live, attenuated vaccines; inactivated vaccines; subunit vaccines; toxoid vaccines; conjugate vaccines; DNA vaccines; and recombinant vector vaccines. Examples of vaccines include vaccines against hepatitis of any kind, rotavirus, DTaP, HIB, polio, MMR, and so forth.

[0132] The actual dosage amount of a composition of the present invention administered to an animal or patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0133] In certain instances, it will be desirable to have multiple administrations of the composition or polymersome, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0134] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0135] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0136] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0137] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0139] In certain aspects, the compositions or polymersomes for use in the methods are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration.Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0140] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0141] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In some aspects, the compositions are in an aqueous solution and / or a solution at physiological pH and physiological salt content. The compositions may be in a saline solution.

[0142] In certain aspects, the pharmaceutical compositions may include or exclude administration in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. The compositions may comprise a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like.

[0143] Examples of non-aqueous solvents that may be included or excluded in the compositions of the disclosure are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0144] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0145] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0146] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0147] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.IV. Methods of Use of the Compositions and Polymersomes

[0148] Methods may be employed with respect to individuals who have tested positive for such disease or who are deemed to be at risk for developing such a condition or related condition. The polymersomes comprising payload(s) may be useful for any kind of medical condition. In specific embodiments, the medical condition is cancer, such as brain, lung, breast, prostate, pancreatic, kidney, colorectal, blood, bone, stomach, spleen, gall bladder, testicular, ovarian, cervical, pituitary gland, thyroid gland, skin, and so forth. The medical condition maybe for treatment of an infection from a pathogen, including bacteria, virus, fungus, and so forth. The medical condition may be an injury or wound.

[0149] The disease may include or exclude cancer. The cancer may include or exclude any cancer known in the art or, for example, epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or a colorectal cancer. The cancer may include or exclude one or more of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing’s family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms’ tumor, and posttransplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs’ syndrome.

[0150] The disease may include or exclude an autoimmune condition. The autoimmune disease may include or exclude one or more of diabetes, graft rejection, GVHC, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still’s disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job’s syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn’s disease, autoimmune- mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratorydistress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen’s encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener’s granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and gianT cell (Takayasu’s) arteritis), medium-vesselvasculitis (including Kawasaki’s disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA- associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison’s disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer’s disease, Parkinson’s disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti -glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet’s disease / syndrome, Castleman’s syndrome, Goodpasture’s syndrome, Reynaud’s syndrome, Sjogren’s syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter’s disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto’s disease, chronic thyroiditis (Hashimoto’s thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave’s disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan’s syndrome, autoimmune hepatitis, chronic hepatitis, lupoidhepatitis, gianT cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger’s disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig’s disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan’s syndrome / nonsyphilitic interstitial keratitis, Bell’s palsy, Sweet’s disease / syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt’s syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressier’s syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas’ disease, rheumatic fever, recurrent abortion, farmer’s lung, erythema multiforme, postcard! otomy syndrome, Cushing’s syndrome, bird-fancier’s lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport’s syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampler’s syndrome, Caplan’s syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman’s syndrome, Felty’s syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch’s cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan’s syndrome, autoimmune gonadal failure, Sydenham’s chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspemiogenese, autoimmune hemolysis, Boeck’s disease, cryoglobulinemia, Dupuytren’s contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich’s disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain’s thyreoiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adultonset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loftier’ s syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmunedisease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton’s syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, graft versus host disease, contact hypersensitivity, asthmatic airway hyperreaction, and endometriosis.

[0151] Non-limiting examples of infectious diseases that can be prevented or treated by the methods described herein include or exclude anthrax, cervical cancer (human papillomavirus), diphtheria, hepatitis A, hepatitis B, haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza (Flu), Japanese encephalitis (JE), lyme disease, Zika, malaria, measles, meningococcal, monkeypox, mumps, pertussis, pneumococcal, polio, rabies, rotavirus, rubella, shingles (herpes zoster), smallpox, tetanus, typhoid, tuberculosis (TB), varicella (Chickenpox), yellow fever, HIV / AIDS, giardiasis, infectious mononucleosis, pneumonia, rocky mountain spotted fever, salmonella infections, severe acute respiratory syndrome, shingles, toxic shock syndrome, hepatitis C, West Nile virus, COVID, SARS-COV- 2, a coronavirus, sexually transmitted diseases, including gonorrhea, chlamydia, and syphilis, and the like.V. Kits

[0152] Any of the compositions, polymersomes, BCPs, and / or ISPs described herein may be comprised in a kit. In a non-limiting example, a BCP, polymersome, ISP, and / or one or more therapeutic or other payloads may be comprised in a kitThe kit will comprise its components in suitable container means. Such components may be suitably aliquoted. The components of the kits may be packaged either in aqueous media or in lyophilized form. Thecontainer means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a container. The kits of the present invention also will typically include a means for containing the component containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0153] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution may be an aqueous solution, with a sterile aqueous solution being particularly preferred. The compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and / or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and / or even applied to and / or mixed with the other components of the kit.

[0154] However, the components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0155] Irrespective of the number and / or type of containers, the kits of the invention may also comprise, and / or be packaged with, an instrument for assisting with the injection / administration and / or placement of the ultimate composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, and / or any such medically approved delivery vehicle.Examples

[0156] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: Room-Temperature Assembled Polymersomes for Protein and RNA Delivery

[0157] Although great advances in RNA delivery have been made in siRNA and mRNA, e.g. with lipid nanoparticles, there remains a pressing need for a versatile platform that can deliver both RNA and protein payloads. Here, the inventors demonstrate a highly efficient approach to forming vesicular polymer nanoparticles (polymersomes) that requires no organic solvents by utilizing thermally responsive block copolymers capable of self-assembling in aqueous media from unimers as the soltuion is raised to room temperature. This is achieved using polymers with a lower critical solution temperature (LCST) that are soluble in aqueous solvents under standard refrigeration (4-7°C) but have been designed to assemble upon warming to room temperature, resulting in large batches of nanoparticles with predictable size and morphology as dictated by polymer structure. Both siRNA and protein payloads could be incorporated during warming at high efficiencies. From a processing perspective, the inventors selected examples that did not require purification of unencapsulated payload, bypassing further purification and demonstrating a highly scalable approach for formulating nanoparticlebased treatments. Furthermore, the nanomaterials are designed with charged and biofunctional moieties to drive payload affinity as well as in-vivo targeting respectively. Finally, the inventors demonstrate that the system is capable of in-vivo delivery in the context of protein subunit vaccination, prophylactic immune tolerance induction, and siRNA interference therapy in cancer. The inventors believe this platform technology addresses many of the challenges preventing polymer nanoparticles from more broadly entering the clinic, such as scalability, loading efficiency, and quality control.

[0158] In this example, the inventors have advanced PSs as a vaccine platform by enhancing macromolecular encapsulation efficiency with facile, streamlined processing. The formulations herein demonstrate rapid assembly of near-m onodi sperse PSs without organic solvents to circumvent purification issues. This is achieved through two design principles: 1) temperature-responsive PS assembly and 2) affinity-driven payload encapsulation. The BCPs are solubilized in aqueous buffer when refrigerated (4 °C) but self-assemble at room temperature (20 °C) into homogeneous PSs. This is achieved by incorporating polymers segments with a lower critical solution temperature (LCST) below room temperature but above the freezing point of water. (16-18) BCPs are dissolved alongside a hydrophilic payload, and their uniform self-assembly bypasses solvent and size-exclusion purifications. Furthermore, cationic moieties are incorporated within the hydrophilic domains to attract negatively charged groups in protein and nucleic acid payloads. Electrostatics drive high loading efficiencies onthe order of 75-99% and reduce the amount of synthetic material (50-200 pg) required for therapy. The inventors demonstrate the utility of such facile formulations in the context of protein subunit vaccination, tolerance induction, and RNA interference (RNAi) for cancer immunotherapy. Such a platform technology may prove useful in growing efforts to develop nonviral vectors for macromolecule delivery. (1)A. Materials & Methods1. Reagents

[0159] Diethylene glycol ethyl ether acrylate (DEGEA), dimethyl aminoethyl acrylate (DMAEA) and 2-hydroxy ethyl acrylate (ELEA) were purchased from Sigma-Aldrich. DEGEA and DMAEA were distilled, passed through a basic alumina plug and stored at -80 °C prior to polymerization. HEA was purified following protocols from Matyjaszewski and coworkers then stored at -80 °C. (19) Azobisisobutyronitrile (AIBN) was purchased from Sigma-Aldrich, recrystallized from methanol and stored in -20 °C prior to use. Cyanomethyl dodecyl trithiocarbonate (CDT) was purchased from Sigma- Aldrich and used as received. Components for in-house lipid nanoparticles (LNPs) were ordered through Broadpharm. Ovalbumin (OVA) and endotoxin-free OVA were purchased through Invivogen. Fluorescent OVA was made by conjugating fluorescein isothiocyanate NHS ester (FITC-NHS ester) to OVA following standard literature procedures in carbonate / bicarbonate buffer. Fluorescent antibodies were ordered through BD and Biolegend. All other reagents and solvents were ordered through Sigma-Aldrich unless otherwise noted. Solvents used in polymerizations were dried overnight on molecular sieves prior to use. SiRNA sequences were designed and ordered through IDT, with dT sequences introduced to provide stability for the double stranded structures. The siRNA sequences (denoted as sense strands (SS) and antisense strands (AS)) used in the studies are as follows:“dT” refers to deoxythymidine2. Instrumentation & Data Analysis

[0160] Proton nuclear magnetic resonance (XH NMR) was conducted on a Bruker Avance 11+ 500 MHz spectrometer. For polymers, 32 scans were taken using a 10s relaxation time. Molecular weight distributions (Mn) and poly dispersity (D=MW / Mn) were determined by gel permeation chromatography (GPC) in DMF with 0.01 M LiBr on a Tosoh EcoSEC using PMMA standards. Small molecules were characterized via NMR and electrospray ionization mass-spectrometry (ESI-MS) on Agilent 6130 LCMS using methanol as the eluent. Dynamic light scattering (DLS) measurements were taken on a Wyatt Mobius™ at a fixed detection angle of 163.5° using 532 nm light. Data was analyzed using Dynamics® software and plotted using GraphPad Prism 9. Multi-angle light scattering (MALS) measurements were taken on a Wyatt DAWN HELEOS II MALS detector with a 658 nm light source using l x PBS as the eluent. UV-Visible (UV-Vis) spectroscopy was conducted on a Shimadzu UV-3600 Plus UV- VIS-NIR, scanning from 450 to 200 nm wavelength.

[0161] Flow cytometry measurements were taken on a Novoctye Penteon 5-30 or LSR Fortessa 4-15. Data was analyzed using BD FlowJo and plotted in GraphPad Prism 9. In-vitro and in-vivo statistics were based on one-way analysis of variance (ANOVA) comparisons or Kruskal-Wallis tests for parametric data when appropriate. All comparisons are denoted in the figures, and multiple comparisons are corrected for using Dunn’s post-test.3. Polymer synthesis

[0162] All BCPs are synthesized using reversible addition fragmentation chain-transfer (RAFT) polymerization, following standard literature protocols. CDT was employed as the initial chain transfer agent (CTA) for the parent polymer, which was then used for sequential chain extensions. Crude and purified materials are characterized by NMR throughout the synthetic steps and GPC is used to determine molecular weight of purified polymers. All degrees of polymerization are based on NMR measurements.4. Poly(diethylene glycol ethyl ether acrylate) (pDEGEA) macroCTA synthesis

[0163] In a 25mL, 3-neck flask, 3.33g DEGEA monomer (18 mmol, 2M in solution) and 26 mg CDT were dissolved in 8.6 mL DMF. AIBN was added as a stock solution of 1.3 mg in 50 uL DMF. The solution was degassed with four freeze-pump-thaw cycles and placed in a 70 °C oil bath for 1.8 hours under nitrogen flow to attain -60% conversion of monomer. This wasdone to maintain polymer chain end livingness. (20) The solution was quenched by immersion in liquid nitrogen and opening to air. DMF was removed via rotary evaporator and the crude mixture was reconstituted in minimal THF for precipitation into hexanes (*6). The resulting polymer was dried overnight and obtained as a yellow, viscous oil (yield: 99%). This was used as the “parent polymer” (RAFT macro-CTA) for all BCP constructs.5. p(DEGEA)-b-poly(hydroxyethyl acrylate) (pDEGEA-b-pHEA)

[0164] 1.4g pDEGEA macro-CTA and 770 mg HEA monomer (6.67 mmol, 0.6M in solution) were dissolved in 9 mL DMF. 1 mg of AIBN was added, and the solution was degassed and placed in a 70 °C oil bath for 3.75h to achieve -60% monomer conversion. The solution was diluted and dialyzed extensively against water for 48h with repeated buffer changes. pDEGEA-b-pHEA was lyophilized and obtained as a yellow, sticky solid (yield: 93%)6. p(DEGEA)-b-poly(trimethyl aminoethyl acrylate) (pDEGEA-b- pTMAEA)

[0165] 670 mg pDEGEA macro-CTA and 290 mg DMAEA monomer (2 mmol, 0.6M in solution) was dissolved in 2.4 mL 1,4 dioxane. 0.4 mg AIBN was added as a stock solution, and the solution was degassed and placed in a 70 °C oil bath for 1 ,5h. Due to the slower kinetics of the polymerization and relatively short target block length (-25 units) for DMAEA, monomer amount was adjusted to achieve the desired degree of polymerization at 20% conversion. Upon quenching, a small portion was precipitated in hexanes for GPC and NMR analysis of the resulting BCP, pDEGEA-pDMAEA. The remainder of the crude mixture was diluted with THF to a concentration of 2 wt% polymer and 500 uL of iodomethane (8 mmol) was added via positive pressure pipette. The reaction was run overnight at room temperature, dried via rotary evaporator, and the crude mixture was reconstituted in DMF for end-group modification.7. p(DEGEA)-b-poly(HEA-co-mannose acrylate) (pDEGEA-b- p(HEA-co-Mann))

[0166] pDEGEA-p(HEA-co-Mann) was synthesized by dissolving 300 mg of pDEGEA macro-CTA and 117 mg of HEA in 1.3 mL DMF. Then, 73 mg of mannose acrylate (monomer synthesis below) was added as a stock solution, followed by 0.2 mg AIBN. The solution wasdegassed, and reaction started by placing in 70 C oil bath for 2.75 h to achieve -60% of total monomer conversion. The reaction was quenched and precipitated twice in a 1 : 1 mixture of ether / hexanes (yield: 95%).8. Mannose Acrylate Synthesis

[0167] Acrylate-modified mannose was synthesized using a modified protocol for the acetal -exchange at the Cl position of mannose. (21) 2g mannose was dissolved in ImL water and added to 12g of HEA. 500 uL acetyl chloride was added to the reaction at 0 °C, which was then placed in a 70 °C oil bath for 4 hours. The reaction was monitored with thin-layer chromatography (TLC) using p-anisaldehyde and potassium permanganate stains. The crude mixture was adsorbed onto silica and purified by flash column chromatography using 20% MeOH in DCM as the eluent. The purified material was characterized by NMR (FIG. 8) and electrospray ionization mass spectrometry (ESI-MS). [M+Na]+theoreticai= m / z 301.1, [M+Na]+found= m / z 301.25, 10% yield. The acrylate was stored as a stock solution in DMF with 0.5 wt% MEHQ to prevent polymerization.9. Polymer Chain-End Modification

[0168] All polymers are modified via aminolysis of the trithiocarbonate chain-end followed by Thia-Michael addition of acrylate-functionalized moieties, using previously published techniques. (22, 23) Reactions are carried out by dissolving 50 mg / mL polymer in DMF, 20* molar excess acrylate and 5* excess TEA (relative to RAFT chain end). Reaction mixtures were degassed by four freeze-pump-thaw cycles, and 5* excess hexylamine was added under nitrogen as a stock solution in DMF after the first cycle. Reactions were run overnight, and UV-Vis was used to determine full cleavage of the polymers prior to workup (based on loss of RAFT-chain end absorbance at 308 nm). The resulting polymers are obtained by x precipitation in 1 : 1 mixture of ether / hexanes, followed by extensive dialysis against water. All purified polymers are then characterized with NMR, GPC and two colorimetric assays: Ellman’s assay is used for all formulations to ensure no residual free thiols and successful mannosylation is determined by the Phenol-EESCh assay. (24, 25)

[0169] For pDEGEA-b-pHEA, the chain end was cleaved and functionalized using either HEA monomer (final material referred to as “D130-H70”) or mannose acrylate (“D130-H70- Mann”). Both materials appear as a white, sticky solid after lyophilization. Yield: 93% (Mannose Modified), 95% (HEA modified).

[0170] For pDEGEA-b-pTMAEA, the chain end was cleaved and reacted with residual, unreacted TMAEA monomer remaining (~30x excess relative to chain end) from the crude mixture. The material is a white, fluffy solid after lyophilization and referred to hereafter as “D130-TM25”. Yield: 50%.

[0171] pDEGEA-b-(HEA-co-Mann) was cleaved and functionalized using HEA monomer (“Di30-(H-co-M)”) and appears as a white sticky solid following workup. Yield: 93%.10. p(DEGEA-co-(2-hexamethyleneiminoethyl acrylate)) (“pDC”) based pH-responsive polymers

[0172] 2-(hexamethyleneimino)ethyl acrylate, a seven-membered cyclic amine-bearing acrylate (referred to here as “C7”) was synthesized according to previous literature procedures. (26) the macroCTA p(DEGEA-co-C7) (pDC) was synthesized by dissolving 1.57g of DEGEA monomer with 175 mg of C7 monomer in 1.3mL dioxane (monomer concentration was 3M) with 1 mg AIBN and 11.3 mg CDT. Following four freeze-pump-thaw cycles, the reaction was allowed to proceed for 6.25h at 70 C to achieve 30% monomer conversion and the reaction was quenched in liquid nitrogen. The crude mixture was precipitated six times in cold hexanes, and the polymer was obtained as a viscous yellow oil (50% yield).

[0173] Subsequent chain extensions were conducted in a similar fashion, by dissolving 115 mg of pDC macroCTA in 1 mL dioxane. For chain extension with HEA monomer, 74 mg of HEA was added to the pDC solution along with 0.2 mg AIBN. The polymerization was run for 2.5h, quenched, and the crude mixture was dialyzed extensively against DI water followed by lyophilization. The polymer pDC-b-pHEA (DC-H) was obtained as a sticky yellow oil (50% yield). Chain extension with DMAEA monomer was done by adding 102 mg of DMAEA to the pDC solution with 0.2 mg AIBN. The polymerization was run for 2h at 70 C, quenched, and then precipitated five times in hexanes and dried overnight. The polymer pDC-b-pDMAEA (DC-DM) was obtained as a viscous yellow oil (80% yield). The resulting structures for DC-H and DC-DM are in FIG. 20A, with x (the mole fraction of DEGEA in the pDC block) being anywhere from 0.9-0.5.11. p(DEGEA-co-(2-hexamethyleneiminoethyl acrylate)) (“pDC”) and p(DEGEA-co-(2,2-diethylaminoethyl acrylate)) (“pDE”) based pH- responsive polymers

[0174] 2-(hexamethyleneimino)ethyl acrylate, a seven-membered cyclic amine-bearing acrylate (referred to here as “C7”) and 2,2-(diethylamino)ethyl acrylate (referred to here as “E”) were synthesized according to previous literature procedures. Generally, the macroCTAs p(DEGEA-co-C7) (pDC) and p(DEGEA-co-E) (pDE) were synthesized by dissolving 300 mg of DEGEA monomer with varying amounts of either C7 or E monomer (based on formulation, ranging from 0.1-1 molar equivalent relative to DEGEA monomer) in 1.3 mL DMSO to achieve a total monomer concentration of -1.5M. Molar amount of CDT is adjusted based on desired degree of polymerization, which typically ranges from 50-200 for the macroCTA once the polymerization reaches 70-80% conversion. AIBN is added at a 0.05 molar equivalent relative to CDT, while trifluoroacetic acid (TFA) is added at a 1.3 molar equivalent relative to the tertiary amine monomer. Following degassing for 45 minutes via bubbling nitrogen through the solution, the reaction was allowed to proceed for l-2h at 80°C to achieve -70-80% monomer conversion and the reaction was quenched in liquid nitrogen. To the crude mixture, 5-10x volume of 0.1 MNaOH in distilled water was added to deprotonate the amine. The crude mixture is dialyzed against 0.1 M NaOH for 2 hours with repeated solution changes, followed by dialysis against DI water for 36 hours with repeated changes. The polymer was obtained as a viscous yellow oil upon lyophilizing (50-70% yield).

[0175] Subsequent chain extensions were conducted in a similar fashion, by dissolving -100-200 mg of either pDC or pDE macroCTA in 1 mL DMSO (IM of monomer). For chain extension with HEA monomer, -100 mg of HEA monomer was added to the macroCTA solution along with typically 0.2 mg AIBN (0.1 molar equivalent relative to macroCTA. After bubbling nitrogen, the polymerizations were run for 2.5 hours at 80°C, quenched, and the crude mixtures were dialyzed extensively against DI water followed by lyophilization. The polymers pDC-b-pHEA (DC-H) and pDE-b-pHEA (DE-H) were obtained as sticky yellow solid (50% yield). Chain extension with DMAEA monomer was done by adding -100 mg of DMAEA to either the pDC or pDE solution above with AIBN (0.1 molar equiv. rel. macroCTA). TFA was added at a 1.5 x molar equivalent relative to all tertiary amines (DMAEA monomer and amines present in macroCTA). After bubbling nitrogen, the polymerizations were run for 2h at 80°C, quenched, added to 5 x volume of 0.1 M NaOH, and dialyzed similarly to the macroCTA above. The polymers pDC-b-pDMAEA (DC-DM) and pDE-b-pDMAEA (DE-DM) were obtained as viscous yellow oils or sticky solids (80% yield). The resulting structures for DC-H, DC-DM, DE-H and DE-DM are in FIG. 18 and and FIG. 22, with x (the mole fraction of DEGEA in the pDC or pDE block) being anywhere from 0.5-0.9. m and n correspond to the degrees ofpolymerization for HEA and DMAEA respectively in the two different BCPs, and are typically between 10-100.

[0176] Following similar protocols, but changing the order of monomer addition such that either “C” or “E” monomers are polymerized in the hydrophilic block of the polymer rather than the LCST-portion (“Gen 4a” in FIG. 22), polymers were also synthesized such that the corona, rather than membrane, of the nanoparticle is ionizable.12. pH-responsive polymers with disulfide junctions

[0177] To impart rapid biodegradability into the polymers, the inventors propose installing a disulfide junction between the hydrophilic portion and membrane-forming, LCST portion of the block copolymer. Previous literature suggests that such rapidly degradable motifs significantly improve mRNA release from lipid nanoparticles, and may be necessary for the more stable, polymer-based nanoparticles described herein.2,3Following a standard RAFT polymerization as outlined above, poly(hydroxy ethyl acrylate) (pHEA) will first be synthesized then the trithiocarbonate chain end cleaved via aminolysis, also described above, but in the absence of any electrophilic groups to expose a free thiol. The reaction mixture will be purified first with dialysis against tris(2-carboxyethyl)phosphine (TCEP) in DI water to reduce any disulfides formed during the reaction, then dialyzed briefly against water prior to lyophilizing to yield a free thiol-bearing pHEA (pHEA-SH). Separately, a pyridyl disulfide (PDS) functionalized RAFT chain transfer agent or ATRP initiator will be synthesized following previously established procedures (FIG. 23Ai).4,5 Briefly, a di-PDS molecule (Aldrithiol-2) will be reacted with mecraptoethanol in the MeOH with acetic acid as a catalyst to form the hydroxyl-functionalized PDS (hydroxyethyl pyridyl disulfide). The hydroxyl can then be coupled to carboxylic acid functionalized RAFT or ATRP agent following standard Fisher esterification protocols. The pHEA-SH polymer can then be reacted with the PDS- functionalized RAFT or ATRP agent to generate a disulfide-containing macroCTA - either pHEA-ss-RAFT or pHEA-ss-ATRP (FIG. 23 Aii). In both cases, the second, LCST-block of the polymer can be synthesized following RAFT polymerization protocols outlined above or standard ATRP conditions.6 The constructs will be generated with a variety of tertiary amines (either as block copolymers or statistical copolymers) to promote ionizability as well as mRNA loading (FIG. 23B and C).13. Polymersome Formation & Loading Efficiency for Proteins andNucleic Acids

[0178] BCP stock solutions are prepared by dissolving at 200 mg / mL in PBS overnight and stored at 4 °C. All stock solutions are used within 1 month to avoid excessive hydrolysis of the acrylate backbone. The thermal transition of materials was determined by preparing a 1 wt% solution of polymer in PBS, then placing in a quartz cuvette for DLS measurement. The instrument was cooled to 4 °C with a constant flow of nitrogen to prevent condensation. Once cooled, the solution was warmed to 37 °C with a slow heating rate of 0.1°C / min and light scattering measurements taken every 3 minutes. For determining particle size and morphology, the stock solutions were diluted 1 : 1 in cold PBS (-20-50 pL) and left at room temperature for 5 minutes to ensure warming and complete self-assembly. For DLS, particles are further diluted to 0.01 wt% in 0.22 pm filtered PBS and analyzed as the average of 3 sets of 5 scans, each with a 3 second relaxation time. Particle hydrodynamic radius (RH) was determined using the Stokes-Einstein Equation. MALS measurements were conducted by further diluting the same samples to 0.01 mg / mL in PBS. Scattering intensity (as Re / Kc) was plotted as a function of angle (sin2(9 / 2)), and analyzed using a Rayleigh-Gans approximation for spherical particles.27Radius of gyration (Rg) was obtained for polymer nanoparticles and compared against the RH from DLS to ascertain vesicular structure. For TEM, samples are diluted to 1 mg / mL, dropped on 200 mesh copper grids, and rinsed with deionized (DI) water. Grids are then stained with uranyl acetate and dried overnight prior to imaging.

[0179] Loading efficiencies of protein and nucleic acids were measured using gel electrophoresis with non-reducing, detergent-free buffers. For protein loading, a stock solution of OVA in cold PBS was mixed 1 : 1 with BCP stock to provide a solution of 10: 1 polymer: payload (wt / wt). The nanoparticles are formed at room temperature for 15 minutes, diluted, then run on a 4-20% gradient, stain-free polyacrylamide gel (BioRad) using Tris- Glycine (TGX) running buffer. The gels are removed, activated with UV, and analyzed using BioRad ImageLab. For nucleic acid loading, stock solutions in nuclease free water are diluted to 300 ng polymer with 30 ng FAM-siRNA (Thermo Fisher, Silencer Select). After room temperature assembly for 15 minutes, samples are diluted with loading dye and run on a 4-20% polyacrylamide gel using Tris-Boric Acid-EDTA (TBE) buffer to prevent RNA degradation. Gels are analyzed using the fluorescein channel on ImageLab. mRNA loading efficiency for Luciferase (Luc) mRNA (purchased through IDT) is quantified in a similar manner but using a 3% agarose gel and SYBR Safe dye for nucleic acid detection.14. Immune Shielding Polymer (ISP) design, synthesis and payload conjugation

[0180] The polymersome platform described here can be used to deliver a wide variety of payloads, including protein antigens for tolerogenic vaccination. In these cases, protein antigen is encapsulated at an efficiency of 75-90%. Some therapeutic applications of PS technology are tolerant to the presence of residual unencapsulated antigen, but others have skewed immunological responses to extra-particular antigen. This 10-25% of protein can theoretically be removed via filtration or other purification methods. However, to reduce clinical postprocessing requirements, the inventors propose a strategy to immunologically-passivate the unencapsulated antigen using bioinert or pro-tolerogenic polymers that irreversibly bind lysine residues, referred to as immune-shielding polymer (ISP).

[0181] The ISP could include (polyethylene glycol), poly(oligo(ethylene glycol) methyl ether methacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxyethyl methacrylamide), poly(oxazoline), dextran derivatives, hyaluronic acid derivatives, or others, and are terminated with an amine-reactive chemical moiety like N-hydroxysuccinimide (NHS) ester. The ISP could have a molecular weight of 5kDa-40kDa. The polymers may be decorated with sugar moieties like mannose, glucose, galactose, or others to enhance liver uptake at a monomer ratio <20% to prevent receptor crosslinking. An example ISP is shown in FIG. 20B, where x would be no greater than 0.2. The syntheses of such materials would be following previous literature procedures, but using an NHS-ester bearing RAFT agent for coupling to free amines on protein. (21)

[0182] Sequential steps were taken to chemically modify only the remaining antigen after polymersome formation. First, BCP and protein were dissolved and combined as described above. The solution was then allowed to warm to room temperature to allow for particle formation. To the turbid solution, the ISP was added at a 2: 1 molar ratio to original protein antigen, which equates to 8-20 polymers per unencapsulated antigen. Lower (1 : 1) and higher (5: 1) molar ratios were also explored to achieve maximal immune-shielding. The solution was mixed and allowed to react for 30 minutes. The unreacted NHS ester was then quenched with a non-toxic small molecule containing a primary amine, which could be lysine, glycine, ethanolamine, or others, at a 2: 1 molar ratio to ISP.15. OVA Cellular Uptake and Antigen Presentation in vitro

[0183] For protein uptake studies, FITC-OVA was encapsulated following the above procedures in nanoparticles at 20: 1 (polymer / payload, w / w) using 100 mg / mL of polymer. Nanoparticles are then diluted to 50-100 pg / mL in serum-free, RPML1640 media (Gibco) priorto treatment. Bone-marrow derived dendritic cells (BMDCs) are then plated at 100,000 cells / well in 96-well U-bottom plates and treated with nanoparticle formulations for 30 minutes. Cells were then washed with PBS, and incubated in complete Lutz media for 2 hours. (28) After, cells are stained for viability and fixed with 2% paraformaldehyde (PF A) prior to flow cytometry. For mannose-receptor blocking, 1 mg / mL of mannose / mannan blocking cocktail is prepared in serum-free media, and BMDCs are treated for 30 minutes prior to nanoparticle treatment to ensure binding. For antigen presentation studies, BMDCs were plated at 10,000 cells / well in 96-well U-bottom plates and treated in complete Lutz media containing lipopolysaccharide (LPS) with formulations for 6 hours. OVA-specific T-cells (OTs) were isolated from 6-10-week-old male, OTI transgenic mice, by collecting spleens and magnetically sorting for CD8+T-Cells. Then, OTI T-Cells were CFSE-labelled and co-cultured with BMDCs for 3 days in complete IMDM media (Gibco). Cells were then washed, stained for viability, and fixed with 2% PFA prior to flow cytometry. Proliferation is demonstrated by a decrease in fluorescence intensity, due to the CFSE stain becoming diluted as T-cells divide.16. In-house LNP Formulation for Nucleic Acids

[0184] To serve as a control lipid nanoparticle, the inventors formulated LNPs to mimic those of Onpattro®, following published structures and weight ratios of the relevant components. (5, 29) Briefly, stock solutions of DLin-MC3-DMA, DSPC, cholesterol and PEG2k-DMG were prepared in ethanol at concentrations of 157, 15.7, 30.4 and 4.72 mM respectively. When ready for use, stock solutions were mixed at a volumetric ratio of 5 : 10: 15 : 5, to give the prescribed molar ratio of 50:10:38.5: 1.5 (ionizable lipid:cationic lipid:cholesterol:PEG-lipid). Since the LNPs are only needed in small batches, simple vortexing was chosen as the method for encapsulating siRNA. (30) LNPs were formed by diluting the EtOH mixed stock solution 1 : 15 in nuclease-free water (either with or without RNA) under vortexing. For loading efficiency quantification, LNPs are diluted to identical mass concentration with FAM-siRNA as for polymers. Size, reproducibility and loading efficiency were then quantified with similar analytical techniques as above (FIG. 19).17. siRNA-mediated Gene Knockdown in vitro

[0185] MCF-7 cells, a human breast cancer line, were plated at 5xl04cells per well in a 48-well plate and incubated for 24 hours. Cell media was removed, and the cells were carefully washed using PBS. Various doses of siRNA (VEGF-A or Bcl-2 targeted siRNA, purchasedthrough IDT, sequences listed above) and polymer or Lipofectamine (Thermo Fisher Scientific) complex were suspended in RPMI media with 2% Fetal Bovine Serum (FBS) and cells were treated for 6 hours. Then, the cells were washed using PBS and incubated with complete media for 48 hours. After incubation, the cells were harvested for RNA isolation, and target mRNA expression was evaluated using standard PCR.

[0186] In a similar study, RAW 264.7 cells, a murine macrophage cell line, were plated at 7xl04cells per well in a 48-well plate and incubated for 24 hours. Cell media was removed and the cells were carefully washed using PBS. siRNA (Atf4 or YTHDF2 targeted siRNA, purchased through IDT, sequences listed above) and polymer or lipid nanoparticle complex (formulated as outlined above) were suspended in serum-free media and cells were treated for 2 hours. Then, the cells were washed using PBS and incubated in complete media for 24 hours. After the additional incubation, the cells were harvested for RNA isolation, and target mRNA expression was evaluated using standard PCR.18. mRNA transfection studies and Luciferase Assay

[0187] Luciferase mRNA (mLuc) was encapsulated into polymersomes pDC-based polymers in a similar manner as above. Briefly, 500 ng of mLuc was mixed with 5pg of pDC polymer or Lipofectamine (Lipo) in 8 uL total of Nuclease-Free Water at 4 °C. The mixture was then allowed to form polymersome-based complexes (in the case of polymers) and lipoplexes (in the case of Lipo) at room temperature for 15 minutes. Samples were then diluted in 200 pL of DMEM containing 2% FBS for in-vitro treatment. RAW 264.7 cells were cultured and plated at a density of 50,000 cells / well in a 48-well plate. Cell media was removed and the cells were washed, then 100 pL of diluted treatment was added to the wells and incubated for 6h. Cells were then washed and reconstituted in complete media overnight prior to Luciferase assay, which was conducted according to manufacturer protocols (Promega ®).

[0188] To evaluate both uptake and transcription, a Cy5-tagged mRNA encoding for GFP (Cy5mEGFP) was used, where Cy5 detection indicates uptake by RAW 264.7 cells and GFP expression indicates successful transcription. Only ionizable polymers were used for these studies, and all formulations were a 1 / 1 (w / w) mixture of pDC-H / pDC-DM or pDE-H / pDE- DM. The vehicle / payload ratio was reported as nitrogen / phosphate (N / P) ratio, which was varied from 12.5 to 100 by calculating the number of tertiary amines (from pDMAEA only) in the nanoparticle via NMR and the number of phosphates in the mRNA as per manufacturer documentation. Complexes were formed as described above, then diluted to 150 ng mRNA / 100uL in serum-free DMEM. 100,000 RAW 264.7 macrophages plated overnight in a 96-well U- bottom plate were then treated with 150 ng of mRNA / well for 2 hours. The mRNA-containing media was removed, cells were washed, then allowed to incubate in complete DMEM overnight. Cells were then washed, stained for viability, then analyzed with flow cytometry for live, Cy5+ populations and geometric mean fluorescence intensity (gMFI) of GFP.19. OVA Subunit Vaccination

[0189] 6-week-old, female C57BL / 6 (Charles River) were vaccinated intradermally in all four hocks with 100 pL total of nanoparticle formulations. Besides the saline treatment, all mice were treated with 10 pg OVA adjuvanted with 20 pg CpG ODN 1826 (Invivogen). 100 pg polymer was delivered for nanoparticle treatments. Blood was drawn weekly via submandibular bleeding, and sera was collected and stored at -20 °C. All mice were boosted on day 21 following prime vaccination and sacrificed at day 28. Draining lymph nodes (dLNs) and spleens were collected for analyzing the OVA-specific T-cell response using fluorescent probes and restimulation. After harvesting, dLNs were treated with collagenase IV and D for 30 minutes at 37°C and passed through a 70 pm filter with complete IMDM media. Spleens were passed through a 70 pm filter, treated with ACK lysis buffer (Thermo Fisher) for 5 min, then quenched with complete IMDM. Single-cell suspensions were counted, and all samples were brought to a concentration of 20 million cells / mL in complete IMDM.

[0190] For MHCI / SIINFEKL (SEQ ID NO:9) pentamer (Proimmune, PE-labelled) staining, isolated cells were plated at 1.5 million cells / well, stained following the manufacturer’s protocol and analyzed with flow cytometry (Novocyte Penteon). Pentamer+cells were determined on antigen experienced CD44+CD8+T-cells. Peptide restimulation experiments were conducted by plating 2 million cells / well, followed by the addition of 100 pL complete IMDM containing either saline (“unstimulated” condition), SIINFEKL (SEQ ID NO:9) peptide (Invivogen, 1 pg / mL, “stimulated” condition) or PMA / Ionomycin (positive control). After 6 hours, cells were isolated, stained for viability, surface markers (CD4 and CD8), and intracellular cytokines and markers (IFNy, TNFa, and CD3s) following standard protocols. Cells were then analyzed by flow cytometry (LSR Fortessa 4-15). For whole-protein restimulation studies, 500,000 cells / well were plated in 96-well U-bottom plates and stimulated with IMDM containing either saline (“unstimulated”) or 100 pg / mL OVA. Cells were centrifuged after 3 days, and media was collected for cytokine analysis using Legendplex (Murine Th 12-plex, Biolegend) and the manufacturer’s protocol (Novocyte Penteon). Allrestimulation studies are plotted as stimulated-unstimulated response. Serum antibodies (total IgG) were detected using direct enzyme-linked immunosorbent assay (ELISA) on Corning 9018 plates coated overnight with 10 pg / mL OVA in carbonate / bicarbonate buffer (pH 9.8). Sera were diluted in lx casein buffer as 10-fold serial dilutions (1 : 102- 107), incubated on coated plates and standard ELISA procedures were followed. Absorbance was read at 450 nm (characteristic for TMB buffer) and 570 nm (background) on a plate reader. Total IgG was reported as area under the curve (AUC) for the absorbance (A450-A570) vs. dilution curve of sera taken at 2 and 4 weeks.20. Long term, OVA Subunit Vaccination

[0191] 6-week-old, female C57BL / 6 (Charles River) were immunized as above either with a single dose of adjuvanted OVA or a two-dose (spaced 3 weeks apart) vaccine. Blood was taken periodically over ~1.5 years for analyzing total, OVA-specific IgG (as well as subtypes, IgG2b and IgGl). For the single-dose treated animals, mice were injected in the hocks with 5 ug OVA adjuvanted with 20 ug lipopolysaccharide (LPS) to recall immune response approximately 19 months following the initial vaccination. 2 days later, blood was drawn and mice were sacrificed the following day for analysis of T-cell responses in the lymph nodes as described above. For mice treated with the two-dose vaccine, a challenge model was implemented using a B16 murine melanoma cell line engineered to express OVA intracellularly. Approximately 16 months following injection, mice were injected intradermally with 2 million Bl 6-0 VA cells behind the shoulder. Tumor sizes were measured and mice were sacrificed when tumors reached 500 mm3.21. OVA Prophylactic Tolerogenic Vaccination

[0192] In a prophylactic allergy model, nanoparticles (either with or without protein antigen, OVA) were administered intravenously weekly for two weeks. 1 week post therapy, mice were sensitized using OVA mixed in a 1 : 1 (v / v) ratio with Imject Alum solution (Thermo Fisher Scientific). Mice were sensitized through two intraperitoneal injections set one week apart with 100 pg of alum / OVA. A week after the second sensitization, mice were challenged for four consecutive days with 25 pg of OVA grade V in PBS through intratracheal instillations. The submandibular vein was sampled through lancet puncture, and blood plasma was isolated through centrifugation at 20,000g in a tabletop centrifuge. Mice were euthanized three days after the last challenge to assess the extent of allergic airway inflammation. Bronchoalveolarlavage fluid (BALF) is isolated by cannulating the trachea and washing the lungs with PBS for a total recovery of 3 mL.

[0193] Perfused mouse lungs were mechanically dissociated and digested with Collagenase IV and Collagenase D in 3 mL Dulbecco’s modified Eagle’s medium for 1 hr. After RBC lysis, samples are then washed, filtered, and counted, prior to flow cytometry analysis. For immunophenotyping, 5 x 105cells are suspended in 50 pL buffer (PBS, 2% FBS), blocked using anti-CD16 / 32, and stained with surface antibodies. For intracellular staining, cells are fixed and permeabilized with the FoxP3 / Transcription Factor Staining Buffer Set following the manufacturer’s instructions. Samples were analyzed using an LSR Fortessa (BD Biosciences) or Cytek Aurora (Cytek Biosciences) and the data analyzed using FlowJo software.

[0194] To determine IgE-levels in the plasma, the inventors utilized an anti-IgE ELISA (Thermo Fisher Scientific) following the manufacturer instructions.22. siRNA Cancer Therapy

[0195] For in-vivo cancer studies probing RNA interference of VEGF-A and Bcl2, nude mice were inoculated with 2 million MCF-7 cancer cells (cultured in DMEM media) in a Matrigel extracellular matrix. Tumors were allowed to grow for 10 days to a size of 50-70 mm3before starting treatment. Then, mice were treated intratumorally on days 11,13,15,17 and 19 using nanoparticle-encapsulated or free siRNA (either si VEGF-A or siBcl2) formulations. 40 pg of siRNA and 200 pg of polymer or LNP was delivered. Tumor sizes were measured until day 35 and mice were sacrificed when tumors reached a size of 600 mm3.

[0196] Similar experiments were also conducted when probing RNA interference for Atf4 or YTHDF2 targeted siRNA. In this case, female, C57BL / 6 mice were inoculated with 500,000 MC38, murine colon carcinoma behind the left shoulder. Tumors were allowed to grow for 7 days to a size of 50-70 mm3before starting treatment. Then, mice were treated intratumorally on days 7,12,17 and 20 using nanoparticle-encapsulated siRNA (either siAtf4 or siYTHDF2) formulations and vehicle-only controls. 40 pg of siRNA and 200 pg of either polymer or LNP was delivered. Tumor sizes were measured until day 35 and mice were sacrificed when tumors reached a size of 600 mm3.B. Results1. Materials Design

[0197] In prior work, Lutz, Sumerlin, and Hoogenboom demonstrated the utility of oligo(ethylene glycol) acrylates as a more versatile LCST system compared to poly(N- isopropyl acrylamide)(NIPAAM), due to the variable number of pendant ethylene glycol units leading to a range of temperature responses from 0-100 °C. (17, 18, 31) Chilkoti further demonstrated a biological utility of such polymers as an alternative to the widely used poly(ethylene glycol) (PEG), as the short oligoethylene glycol side chains avoid the development of a humoral immune response, in contrast with the development of anti-PEG antibodies. (32) Poly(diethylene glycol ethyl ether acrylate) (pDEGEA) was chosen as the PS- membrane forming block due to its convenient LCST transition around 13-15°C and biocompatibility. Here the pDEGEA-containing BCPs have been designed with block-lengths chosen to allow for the self-assembly of vesicular structures characteristic of PSs (FIG. 1 A and FIG. 7). Based on previous literature, block lengths consisting of 120-175 monomers for the membrane-forming pDEGEA (denoted “D”) and block lengths of 15-70 monomers for the hydrophilic, corona forming blocks were targeted, (8) to maintain the hydrophilic weight fraction at 10-25% to guide vesicular morphology (FIG. IB). For all studies herein, D130 (subscript denotes repeat units) was used as the membrane-forming material, and either poly(hydroxyethyl acrylate) (pHEA) (“H70”) or poly(trimethylamino ethyl acrylate) (pTMAEA) or (“TM25”) was the hydrophilic segment for uncharged and charged polymers respectively (FIG. IB). Degrees of polymerization were determined by1H NMR (FIG. 9) and BCP end groups were modified to enhance particle stability and introduce bioactivity.

[0198] End-group functionalization of the D130-H70 and D130-TM25 polymers was achieved by aminolysis of the trithiocarb onate moiety, (23) followed by a thia-Michael reaction with a compound containing an acrylate derivative. For example, Di3o-H?o-Mann was modified with mannose-bearing an acrylate moeity. (21) Successful end group functionalization was determined by a combination of UV-visible spectroscopy (UV-Vis), to monitor the loss of the trithiocarbonate absorbance peak at 308 nm during aminolysis (FIG. 10), followed by two colorometric assays, Ellman’s and Phenol -H2SO4, to determine the efficiency of the thiaMichael reaction. (24, 25) The former was used to validate the absence of any remaining free thiols (which are generated as a part of the aminolysis reaction) while the latter was used to determine the quantity of mannose groups in the mannosylated formulations (FIG. 2B). The theoretical amount of mannose under the assay conditions (assuming one molecule per polymer chain) is 60 nmol and the slightly lower determined amount is attributed to variances in sample heating when following the literature procedure for the Phenol-ftSCh assay, along with some loss in BCP chain-end fidelity during purification. A final, mannosylated BCP, Di3o-(H3o-co-Manmo) was also synthesized as an alternative to Di3o-H?o-Mann by copolymerizing HEA and Mannose Acrylate into the hydrophilic domain of the polymer (FIG. 11). This material has shown initial promise in in-vitro uptake studies but was not explored in further experiments.

[0199] Final BCPs were characterized using Gel Permeation Chromatography (GPC) for molecular weight distribution. D130-H75 shows a monomodal molecular weight distribution with low dispersity (£) = 1.2), relative to PMMA standards (FIG. 1C,D). D130-TM25 cannot be run on the inventors’ GPC columns due to the highly charged, quaternary ammonium groups. (33, 34) However, the polymer prior to quaternization, D130-DM25 showed reasonably low dispersity (D = 1.3), with slight tailing due to the presence of tertiary amines. (35)

[0200] The LCST of the materials along with resulting nanoparticle size were determined using dynamic light scattering (DLS) in phosphate buffered saline (PBS, pH 7.4). A sharp increase in scattering intensity at 17°C suggests a morphology transition from unimers to polymer particles (FIG. 2A). Importantly, a sharp peak in hydrodynamic radius is observed upon initial transition, followed by a drastic decrease to a stable radius once the system reaches room temperature (20°C). Previous work from Armes and coworkers attributes this behavior to the micelle-worm -vesicle transition, as determined by rigorous microscopy studies. (36)

[0201] Polymer blends were made by mixing charged and uncharged BCPs at different weight percent to formulate nanoparticles with varying degrees of charge to enhance payload encapsulation efficiency. Nanoparticles are therefore reported here as the relative weight fractions of the charged BCP, D130-TM25 (“T”) and mannosylated BCP, Di3o-H?o-Mann (“M”). The remainder of the nanoparticle weight fraction consists of the inert BCP, D130-H70. The rest of this article refers to the blended particles as “TxMy”, with “x” and “y” being the relative fractions of charged and mannosylated BCPs (FIG. 2C).

[0202] Importantly, polymers are dissolved at high concentrations, but no more than 100 mg / mL to favor vesicular assembly as demonstrated by Battaglia and coworkers. (37) Following self-assembly, hydrodynamic radii (RH) and radius of gyration (RG) were determined by DLS (FIG. 2D) and MALS (FIG. 12) respectively. For nanoparticles used in- vitro and in-vivo, the inventors determined a form factor (RG / RH), which is theoretically equal to 1 for vesicle structures. (38) All structures used in these experiments have RG / RH between 0.93 and 1.18 and can be reasonably approximated as being vesicular (FIG. 12B). Morphologies are then visualized using negative-staining TEM, with sizes similar to those observed by light scattering, with size alterations attributed to drying upon sample preparation (FIG. 2E). As determined by both RG / RH and TEM imaging, all particles used in biological studies are indeed vesicular when assembled under physiological salt concentrations in salineor PBS. Importantly, it is possible to obtain reproducible particle sizes given that the selfassembly is dictated by polymer block lengths and relative weight fractions. It is hypothesized that narrow size distributions (PDI<0.1) are due to the complete solubility of polymers when cooled, thus avoiding inhomogeneities in dispersion. (39, 40) Taken together, this platform circumvents previous issues reported in polymersome processing by avoiding organic solvents for self-assembly along with narrow, predictable sizes to address issues of quality control.

[0203] For mRNA delivery, it is known that ionizability is a key parameter to drive endosomal scape and facilitate protein expression following nanoparticle uptake due to the high molecular weight and fragility of mRNA. For this reason, the inventors have also designed a class of materials incorporating ionizable amines with pKa’s that are just below physiological pH to allow protonation under mildly acidic conditions of the early endosome. Two amine moieties were chosen for these formulations, one is a 7-membered, cyclic amine-based acrylate (2 -hexamethyleneiminoethyl acrylate, denoted as “C”, pKa6.9) and the other is a diethylamino acrylate (2, 2-di ethylaminoethyl acrylate, denoted as “E”, pKa7.8) (FIG. 22 A). The more basic amine, “E”, was chosen because a slight decrease in apparent pKais expected when incorporating into a polymer, as the tertiary amine becomes more sterically hindered from protonation. The ionizable amine is positioned at the LCST-block of the polymer, and is intended to destabilize the particles (i.e., make the full polymer completely water soluble) upon reaching endosomal pH (FIG. 22B). Polymer molecular weight and mol percent of ionizable amine was tuned to generate a small library of polymers (Gen 1-4 in FIG. 22C). Each generation is then divided based on the specific tertiary amine used, (“a” for cyclic amine, “b” for diethylamine). All nanoparticle formulations used in mRNA delivery studies are a 1 / 1 (w / w) block copolymer blend of the p(DEGEA-co-ionizable amine)-b-pDMAEA and p(DEGEA-co- ionizable amine)-b-pHEA. It should be noted that for a given “generation”, the molecular weight of the p(DEGEA-co-ionizable amine) is the same for pDMAEA and pHEA-containing block copolymers to ensure homogenous self-assembly of the polymer blend. A Gen 4a material was also designed to contain the ionizable, “C” moiety in the hydrophilic, pHEA portion of the polymer to create an ionizable corona rather than nanoparticle membrane. All materials demonstrate a pH-dependent LCST, as expected since low pH (pH<pKaof either “C” or “E”) would increase solubility of the materials, therefore delaying or even ablating LCST transition (FIG. 22Di). This is also demonstrated by the lower scattering intensity at lower pH, indicating a lower number of self-assembled particles at the same concentration of polymer (FIG. 22Dii) Self-assembled nanostructures are still present at low pH, though much smaller than those assembled at physiological pH, indicating rearrangement (and possible payloadrelease) occurring upon acidification, which does not occur for the non-ionizable materials (data not shown).2. High Protein Loading Efficiency & Enhanced Protein Uptake in Antigen Presenting Cells

[0204] Encapsulating protein payloads has often proved challenging due to their weaker electrostatic interactions relative to nucleic acids, along with the generally dilute conditions under which nanoparticles are formed. (41,42) Due to the use of organic solvents, high concentrations are not achievable with typical formulations and the use of ionizable amines is insufficient for complexation with the sparse negative charges on proteins. To this extent, the temperature-sensitive PSs can be fully dissolved in aqueous medium in high concentrations (100 mg / mL), and the permanently charge ammonium moi eties were hypothesized to have enhanced electrostatic affinity for weakly charged residues on proteins.

[0205] The polymersomes encapsulate 75-90% of dissolved protein (OVA) in solution as determined by gel electrophoresis, with variances based on the weight percent of D130-TM25 used (FIG. 2D, top). RNAs such as mRNA and siRNA can be completely encapsulated (-100% efficiency) using polymer / payload ratios as low as a 1 :5 for formulations using T50M0 formulations (FIG. 2D, bottom). The loading efficiencies are consistent and reproducible and are similar when incorporating mannosylated Di3o-H?o-Mann BCPs (FIG. 13). Due to high loading efficiency and homogenous particle sizes upon warming, one can bypass tedious processing such as size exclusion and membrane filtration. (15) As a result, these materials can simply be diluted for biological application without needing further processing.

[0206] Indeed, uptake studies in murine bone-marrow derived dendritic cells (BMDCs) show enhanced uptake when delivering OVA formulations in this way. Viability studies were performed to determine dose and relative charge fraction based on the MTT assay (FIG. 14A). Due to the toxicity of highly charged moi eties, the inventors chose to use T33My based formulations to preserve cell viability. Subsequent screening studies also determined ideal ranges for mannosylation be within 10 and 33 wt% of the total particle formulation (T33M10 or T33M33) for optimal uptake. Previous literature also supports this claim that sparse ligand density is often favorable over full coverage of the particle with mannose. (43) For these formulations, nanoparticle encapsulated FITC-OVA achieves 3 to 6-fold greater uptake compared to unencapsulated OVA based on geometric mean fluorescence intensity (gMFI) onflow cytometry (FIG. 3 A, left). To mechanistically demonstrate the utility of the mannosylation, the inventors conducted the same study using a mannose-receptor blocking cocktail consisting of mannose-receptor blocking peptide. After blocking, mannosylated polymersomes had a statistically significant decrease in uptake compared to bioinert polymersomes and free protein (FIG. 14B). This suggests that incorporating targeting moieties favors uptake in a mannosereceptor dependent manner, which may prove beneficial for in-vivo nanoparticle delivery. After demonstrating enhanced uptake, the inventors sought to determine the material’s ability to allow for antigen presentation once the protein has been delivered intracellularly.3. Protein antigen presentation and in-vivo antibody response to protein subunit vaccination

[0207] Functional in-vitro assays were conducted to determine the material’s efficacy as a macromolecular delivery platform for prophylactic subunit vaccines. With the growing interest for eliciting strong CD8+T-cell responses, the inventors first probed the material’s ability to trigger proliferation in the OVA-specific transgenic T-cell line, OTI. (44) Upon co-culture with BMDCs treated with the formulations, CFSE-labelled OTIs underwent varying degrees of proliferation as measured by flow cytometry (FIG. 3B). The CD8+epitope SIINFEKL (SEQ ID NO: 9) was used as a positive control, as the peptide directly binds MHCI on BMDCs and is recognized by OTIs. A “CFSE-diluted” population was determined as all T-cells with diluted fluorescence compared to that of the original population (left of dotted line in FIG. 3B). Based on fluorescence histograms, the inventors observed enhanced OTI proliferation (%CFSE- diluted population) when OVA is delivered encapsulated versus in solution. The inventors believe that endosomal escape occurs due to the hydrolytic instability of the acrylate backbone, which is prone to cleavage by the slightly acidic environment and esterase activity within endosome. (45) However, further mechanistic studies utilizing microscopy and auto quenching fluorescent probes are required to prove this hypothesis. Based on these results, the inventors envisioned that the materials would lead to stronger CD8+T-cell responses upon protein subunit vaccination, motivating in-vivo work for these materials.

[0208] To highlight the material’s capacity to also stimulate robust humoral immunity, a pilot study was conducted as a single-dose vaccine adjuvanted with CpG ODN 1826 (FIG. 3C). Mice were vaccinated in all four hocks using either mannosylated (T33M33) or non- mannosylated (T33M0) encapsulating and compared to free, adjuvanted OVA formulations. Remarkably, the inventors observed potent antibody responses characterized by total OVA-specific IgG lasting over 3 months following initial vaccination (FIG. 3D). Though differences were not seen between the nanoparticle groups, it becomes apparent that in a single administration both formulations significantly enhance antibody levels over that of free protein.4. Protein Subunit Vaccination

[0209] In a model for protein subunit vaccines, C57BL / 6 mice were treated with formulations of the nanoparticles (T33M10 and T33M0) encapsulating OVA and adjuvanted with CpG ODN 1826 (FIG. 4A). Draining lymph nodes (dLNs) and spleens were harvested for analyzing T-cell responses, while blood samples were taken at weeks 2 and 4 to determine antibody levels 1 week before and after boost. Relative to protein delivered without encapsulation, both mannosylated and bioinert polymersomes demonstrated enhanced CD8+T-Cell response (FIG. 4B and D). As measured by pentamer staining, both formulations greatly improve CD44+CD8+T-cell-recognition for the vaccinated antigen relative to free protein (FIG. 4F). Importantly, mannosylated particles induced a greater percentage of IFNy+and IFNy+TNFa+double positive CD8+T-cells in the dLNs following peptide restimulation compared to the inert formulation (FIG. 4B and C). This is a promising result as it is known that CD8+immunity is particularly important in the clearing viral infections such as SARS- Cov-2 and other respiratory diseases. (46) Similarly, whole protein, 3-day stimulation show an enhanced Type-1 immune response, which has been shown using previous polymersome formulations from the inventors’ lab. (47) Briefly, restimulations of dLNs showed statistically significant enhancements in TNFa and IFNy, suggesting robust T-helper responses in addition to cytotoxic T-cells (FIG. 4G). Though slightly enhanced DC targeting was observed in-vitro, the inventors hypothesize that this response is magnified in-vivo due to the presence of interstitial flow and greater loss of bioinert polymersomes from the injection site. (48) However, given the enhanced uptake of nanoparticles in general, both delivery systems show improvements over free protein, also adjuvanted with CpG. Biodistribution studies are currently in progress to determine injection-site localization, using an in-vivo imaging system (IVIS). In summary, cellular immunity was enhanced using the nanoparticle formulations when delivering OVA antigen. The improvement is greater for mannosylated particles as determined by functional assays for stimulating CD8+T-cell response in the presence of its peptide epitope SIINFEKL (SEQ ID NO: 9) as well as the full protein.

[0210] The OVA-specific antibody response was also investigated by measuring total IgG are under the curve (AUC) (FIG. 4E). Not surprisingly, total IgG for encapsulated and freeOVA formulations show modest (albeit statistically significant) differences when using CpG ODN 1826, as the adjuvant is known for its robust B-cell activation. (49) Though the antibody response appears similar to that of adjuvanted free protein, the highlight of the nanoparticle system is enhanced cellular immunity, which is believed to be more robust against antigenic drift. (44) Taken together, the inventors demonstrate that these thermoresponsive nanoparticles present a facile delivery system for subunit vaccines intended for robust cellular and humoral immunity. The ease of formulation and lack of purification may prove highly beneficial in the context of vaccine clinics where large therapeutic doses can be made simply by diluting a stock of room-temperature solution.

[0211] For long term studies, mice were vaccinated with either one or two (spaced 3 weeks apart) doses of OVA adjuvanted with CpG, either encapsulated in polymersomes or delivered freely. For the single dose vaccine experiment, blood was measured periodically and greater antibody levels (compared to free, adjuvanted protein control) were detected in the nanoparticle formulations for up to 1 year following initial vaccination (FIG. 24B and C). Furthermore, the ratio of IgG2b to IgGl was also higher 1 year later, particularly in the mannosylated formulations, which indicates a strong type 1 immune response critical for antiviral immunity. This is further demonstrated by the stronger T-cell responses measured by restimulation, specifically showing a higher portion of IFNy TNFa double-positive CD8+and CD4+T-cells (FIG. 24D). Generally, a greater portion of IFNy secretion was also observed in the lymph nodes as a whole (FIG. 24D,iv). Such dose-sparing results demonstrate long-lasting immunity, which is critical for populations with limited resources or vaccine hesitancy that preclude multiple dosing.

[0212]

[0213] As it is well known that an enhanced CD8+T cell response contributes to antitumoral immunity, the inventors also conducted a challenge study to demonstrate functionality of the above-mentioned immune phenotype for mice vaccinated with two-dose formulations, approximately 1.5 years following initial injection. Mice were inoculated with an aggressive, murine melanoma (Bl 6) cell line engineering to express OVA antigen intracellularly, therefore preventing antibody-mediated tumor rejection (FIG. 24E). In this way, this model requires a strong CD8+response to suppress tumor growth and eventually eliminate the tumors. Indeed, the inventors see that the nanoparticle vaccination regime administered 1.5 years prior still retains appreciable antitumor response both characterized by enhanced survival and slowed tumor growth (FIG. 24F and G). Relative to the non- mannosylated polymersomes, the inventors note that mannosylation shows a higher proportionof complete responder (C.R.) mice fully able to eliminate tumors (2 / 4 versus 1 / 5), and both formulations outperform the free, adjuvanted OVA control and the untreated group.5. Prophylactic Tolerogenic Vaccination in Allergic Asthma

[0214] To determine the ability of the nanoparticle system at preventing an allergic immune response, a study was conducted by intravenously administering C57BL / 6J mice with either T33M10 without payload, or both T33M10 and T33M0 encapsulating OVA. Mice were then subjected to an experimental model of allergic airway inflammation during which, they are sensitized through intraperitoneal injections of OVA adsorbed to alum (FIG. 5 A). Upon challenge with intratracheal instillations of OVA, hallmarks of allergic airway disease in allergic asthma are observed, such as airway eosinophilia, systemic levels of IgE, and CD4+T- cell infiltration. (50) As a negative control, mice treated with T33M10 without payload were sensitized using alum without OVA, thus preventing any OVA-specific allergic responses upon challenge. The key benchmark in this study was to compare responses against empty T33M10 polymersomes without payload, sensitized using Alum / OVA. Compared to mice treated with empty polymersomes, mice that were prophylactically administered polymersomes encapsulating OVA show decreases in lung and airway eosinophilia (FIG. 5B, C, and D). Flow cytometry quantification of eosinophils (Siglec+CD1 lc’) in the bronchalveolar lavage fluid (BALF) as well as lungs showed significant decreases only for the mannosylated T33M10 delivering OVA, when compared to the positive control. Furthermore, a reduction in CD4+T- cells is indicative of a productive tolerogenic response, as allergies are known to be a type-2 mediated immune response. (51) In all cases, the mannosylated T33M10 platform delivered OVA in a systemic manner to reduce the total number of CD4+T-cells infiltrating the lungs and airways (FIG. 5D and E).

[0215] IgE mediates the allergic response by binding antigen and stimulating mast-cell degranulation, and the inventors examined systemic levels of this antibody post-sensitization (FIG. 5F). Not surprisingly, a sharp increase in systemic IgE was observed upon challenge of the empty T33M10 group sensitized with Alum / OVA. Though not a complete return to baseline, T33M10 delivering OVA is able to mitigate this response as seen by significantly reduced IgE levels following initial intratracheal challenge and upon conclusion of the study. Taken together, the T33M10 system provides a platform system for protein antigen delivery to effectively target immune cells in the context of vaccination and tolerance.6. siRNA Cancer Therapy

[0216] As a demonstration for siRNA therapy, the inventors turned to siRNA to downregulate cancer-promoting agents via the RNA interference (RNAi) pathway. (3) Vascular endothelial growth factor A (VEGF-A) is known to promote tumor cell survival and proliferation via angiogenesis. Bcl-2 is an anti-apoptotic protein that can be upregulated via the VEGFR pathway, providing a positive feedback loop for enhanced tumor cell survival. Therefore, the inventors chose VEGF-A and Bcl-2 as target molecules for RNAi in the human breast cancer line MCF-7, which is known to upregulate both. (52, 53) siRNA degrades rapidly in the presence of serum in cell-culture media, thus necessitating cationic delivery vehicles such as lipid nanoparticles for transfection. For this reason, the inventors compared their polymersomes to the commercially available transfection agent, Lipofectamine 2000 (Lipo). (54) In this head-to-head comparison, the polymersomes achieve greater gene downregulation when delivering siVEGF-A (-60% downregulation) and siBcl-2 (50% downregulation) and therefore were investigated further in in-vivo cancer studies (FIG. 6A). Control studies determined that nanoparticle formulations have no inherent capability to downregulate mRNA expression, and there is no off-target downregulation for housekeeping genes such as P-actin (FIG. 15). Since cancer cells are not known to express the mannose-receptor, only the inert polymersome formulations T33M0 were used for RNAi studies.

[0217] To establish the efficacy of the formulations for in-vivo siRNA delivery, mice inoculated with MCF-7 tumors were treated intratum orally (i.t.) with encapsulated and unencapsulated siVEGF-A and siBcl2 (FIG. 6B). Nude mice were chosen for this study to avoid immune rejection of the human tumors. As in previous in-vitro work, the target of this RNAi therapy are the tumor cells themselves, so mannosylated polymersomes were not used in this study.

[0218] During treatment, it becomes apparent that tumor growth is halted when siVEGF- A and siBcl-2 are delivered as encapsulated formulations (FIG. 6C). Statistically significant differences are observed when comparing tumor sizes taken at day 20, once i.t. treatment was stopped and neither siRNA nor polymer alone have antitumor efficacy in-vivo (FIG. 6C, left). Furthermore, a clear enhancement in survival is observed for mice treated with these polymersomes (FIG. 6D). These responses can be attributed to the downregulation of the tumor-promoting cytokines seen in the in-vitro work, though further studies are needed to quantify this phenomenon in-vivo. Tumors rapidly grow out once treatment is stopped due to the mice lacking an immune system, but this pilot study demonstrates the material’s ability to deliver siRNA therapeutics in-vivo. In a follow up study, the inventors also demonstrate the material’s ability to outperform clinically-used LNPs formulated following Alnylam’s molarratios in intratumoral siRNA delivery (FIG. 6, 19). This may be attributed to enhanced stability, and therefore payload retention, of polymer-based particles over lipid-based particles.

[0219] Recently, novel “undruggable” immune cell targets have also gained attention in the context of cancer immunotherapy. (55, 56) Instead of being expressed in cancer cells, these RNAi targets are present in tumor-associated macrophages (TAMs) and are known to promote an M2 -type tumor-promoting environment. It has been demonstrated, then, that targeting these specific pathways causes a shift towards Ml-phenotypes and antitumor immunity. For this reason, the inventors also investigated the ability of the particles to downregulate the genes Atf4 and YTHDF2 in RAW264.7 macrophages. Like results for siVEGF and siBcl-2, the particles can downregulate gene expression to a greater extent compared to Lipo as well as inhouse control LNP formulations (FIG. 19). Given that macrophages are known to express mannose receptors, targeting polymersomes to TAMs, as well as dendritic cells can be accentuated using the mannosylated formulations. The inventors also conducted an in-vivo study with mice inoculated with MC38, a murine colon cancer cell line, to test efficacy compared to clinically used LNPs (FIG. 25). In an intratumoral context, the inventors see enhanced efficacy in suppressing tumor growth as well as prolonging survival (FIG. 25B and 25C). Current studies are exploring this nanoparticle-mediated delivery in a more clinically- relevant context, by delivering these payloads subcutaneously, intravenously and with clinically used checkpoint inhibitors.7. mRNA transfection studies and Luciferase Assay

[0220] Similar to above, pDC-based polymers were mixed to form polymer blends by varying the portion of charged polymer (DC-DM, FIG. 20Ai). The polymer blends are refered to as DMx, where “x” represents the weight-percent of charged polymer used. pDC-based polymers successfully encapsulated Luciferase mRNA (mLuc) as measured by gel electrophoresis (FIG. 21 A), though a higher portion of charged polymer was required compared to the aforementioned TxMy block copolymer blend. This can be attributed to the lack of permanent charge on the tertiary amines present in the DC-DM polymer for mRNA complexation. For this reason, DM50 was chosen for subsequent in-vitro studies to probe mRNA delivery in RAW 264.7 macrophages. Indeed, the inventors observe enhanced expression of Luciferase when the inventors delivered mLuc to the macrophage cell line compared to Lipofectamine. As measured by Relative Luminsecence units from the Luciferase assay, it becomes apparent that a greater expression of protein is achieved by usingpolymersome system under serum-containing conditions. Similar to siRNA transfections studies, the inventors observed that simply using the charged polymer alone without blending in the uncharged analog (DM100), does not provide successful transfection. Furthermore, the presence of serum in the assay further validates the enhanced stability of the polymersome system over Lipofectamine, suggesting efficacy in subsequent in-vivo conditions.

[0221] Since the Luciferase assay does not provide details on specific uptake or cell viability, the inventors then conducted detailed in-vitro studies using fluorescently (Cy5) labeled mRNA. Importantly, the Cy5-mRNA also encodes for green fluorescent protein (Cy5- mEGFP), so expression can also be rapidly probed with flow cytometry (FIG. 26B). As mentioned above, for mRNA delivery all nanoparticles are a 1 / 1 (w / w) blend of the p(DEGEA- co-ionizable amine)-b-pDMAEA and p(DEGEA-co-ionizable amine)-b-pHEA polymers. Interestingly, they found that Gen la and 3a outperform all other formulations both in uptake and gene expression (FIG. 26B), which indicates that lower molecular weight polymers (~19kDa) are the more efficacious for mRNA delivery compared to higher molecular weight polymers (-40-50 kDa). Furthermore, the cylic amine-containing polymers outperformed the diethylamine containing polymers, though the specific mechanism behind both phenomena are a topic of current study.C. Conclusion

[0222] In this work, the inventors constructed thermally responsive polymersomes that address many of the challenges that currently limit the field’s viability in the clinic. Namely, they tackle issues in processing, loading efficiency and quality control by developing a scalable platform with predictable nanoparticle morphologies. This was done by designing a roomtemperature forming particle system decorated with cationic moieties to enhance payload affinity. Compared to current LNPs, the platform is highly stable due to its polymer components and is adaptable to a variety of payloads such as proteins and nucleic acids without further modification. Furthermore, enhancements in therapy localization can be achieved by introducing targeting moieties such as mannose. By simply warming a solution to room temperature one can form large batches of nanoparticle therapy within seconds, providing a practical solution to issues in scalability of previous polymer formulations. The particles are then ready to inject upon dilution, without requiring purification, since 75-99% of payload is encapsulated and the particles are of predictable size and poly dispersity, addressing the industry’s quality -by-design (QbD) principles. (57) The inventors have demonstrated thedevelopment of this technology to accommodate both protein and nucleic acid payloads, and they have shown applications in a subunit vaccine, a tolerogenic vaccine, and an siRNA-based cancer therapy.

[0223] The polymersomes are applicable for nucleotide and protein therapies targeting a wide variety of cell types in a wide variety of tissues, with payloads affecting pathways such as cancer progression, metabolism, effector immunity, tolerogenic immunity, and inflammation, among others. They are applicable for a wide variety of routes of administration, including injection intradermally, subcutaneously, intramuscularly, intratum orally, intralesional, intraocularly, orally, rectally, nasally, pulmonarily, and intravesicularly, among others.Example 2: Thermoreversibly assembled polymersomes for highly efficient loading, processing, and delivery of protein and siRNA biologies

[0224] This example may contain some of the same data as Example 1 and is not necessarily representative of additional replicates.

[0225] Although great advances in nucleic acid delivery have been made, versatile technologies that can deliver both RNA and protein payloads could streamline development, simplify manufacturing, and expand the capabilities of combination therapies. Here, the inventors demonstrate an efficient approach to forming ca. 100 nm polymer vesicles (polymersomes) capable of rapid self-assembly without organic solvents and thus the need for post-encapsulation purification. Block copolymers are designed with a lower critical solution temperature that renders them soluble in aqueous medium under standard refrigeration but spontaneously assemble at room temperature into large batches of nanoparticles with predictable size and morphology. The nanomaterials are designed with charged and biofunctional moieties to drive payload affinity and in-vivo targeting, while both siRNA and proteins can be encapsulated during warming at >75% loading efficiencies. Formulations can be stored in a dry state for greater hydrolytic stability under standard refrigeration and can be diluted directly from the vial, bypassing the need for purification for high scalability. The inventors utilize this system for in-vivo delivery in protein subunit vaccination, immune tolerance induction, and siRNA interference therapy in cancer. Overall, this versatile system address challenges in scalability, loading efficiency, quality control, and dry storage.A. Results

[0226] Advancements in nanomedicine have allowed researchers to develop biomacromolecular therapies efficiently and precisely, both considering small-interfering RNA (siRNA) therapy, messenger RNA (mRNA) therapies including vaccines, and protein therapies, including vaccines and tolerogenic, or inverse, vaccines. However, effective payload protection and delivery are key requirements for immunological efficacy, as these payloads are highly sensitive to clearance and degradation when administered in vivo. To this extent, much promise has been demonstrated by the lipid nanoparticle (LNP) technology utilized by Pfizer / BioNTech and Moderna® for their mRNA-based vaccines against SARS-CoV-2. Furthermore, LNPs designed by Alnylam® Pharmaceuticals have successfully demonstrated intravenous delivery of siRNA for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis in the liver using the RNA interference (RNAi) pathway. 4 However, issues with storage stability and complex processing limit broader access to such nanoparticulate therapies and motivate improvements in materials for formulation. Furthermore, LNPs are highly specialized for nucleic acid delivery, and therefore no such formulations exist for protein payloads, e.g., for use as subunit vaccines. Polymer nanoparticles have long been considered a key alternative to LNPs, as the high molecular weight (typically > 5 kDa) of their substituents imparts good stability, which is combined with high tunability on account of extensive advances in polymer chemistry. Furthermore, their synthetic versatility is particularly attractive for achieving delivery of a wide range of payloads such as proteins, small molecules and nucleic acids, including mRNA and siRNA. Here, the inventors’ interest was to develop a single encapsulation technology that would be applicable in a variety of treatments such as subunit and inverse vaccination with protein payloads along with nucleic acid adjuvants, and RNAi therapy.

[0227] Polymersomes (PSs) are a class of vesicular polymer nanoparticles composed of self-assembled amphiphilic block copolymers (BCPs), first described by Discher and Eisenberg in the mid 1990s. These vesicles consist of a lyotropic membrane formed by polymer chain interactions between hydrophobic domains, making them more stable than LNPs. The hydrophilic polymer block forms the PS corona and can reduce protein adsorption and unwanted immune recognition, thus prolonging circulation time. Moreover, their size (50-200 nm) and stability make PSs ideal for cellular uptake. Despite these benefits, formulation is complex, as the organic solvents that are required to solubilize both domains must then be evaporated or diluted in an aqueous medium. Self-assembly therefore occurs at an interface — either water-polymer or water-organic solvent — leading to kinetic trapping of structures and therefore inhomogeneous aggregates that must be processed into uniform morphologies. Post-processing techniques require filter extrusions to ensure monodisperse populations and often lead to suboptimal payload encapsulation and material loss. Novel approaches address these limitations by rapidly mixing solutions with specialized microfluidic devices, but these require extensive operation time and greatly dilute the formulation, which then requires further concentrating. Furthermore, organic solvents can be toxic and denature biological payloads, particularly detrimental to those that require their complex 3D architecture for retaining activity or immune recognition features, e.g., protein conformational antigens. Despite their synthetic tunability, PSs typically show modest loading efficiencies ( £ 20% for proteins), and the complexities described above have prevented widespread clinical translation.

[0228] In this work, the inventors have advanced PSs as a delivery vehicle for protein, nucleic acid adjuvant, and siRNA payloads by enhancing encapsulation efficiency through copolymer design, enabling facile, streamlined processing. The formulations herein demonstrate rapid assembly of near-monodisperse PSs without organic solvents to circumvent purification issues. This is achieved through two design principles: 1) thermoresponsive PS assembly and 2) affinity-driven payload encapsulation. The BCPs are solubilized in aqueous medium when refrigerated (4-7°C) and self-assemble at room temperature (above 20°C) into homogeneous PSs. This is achieved by incorporating polymer segments with a lower critical solution temperature (LCST) below room temperature but above the freezing point of water. The BCPs are dissolved alongside a hydrophilic payload, and their uniform self-assembly bypasses the need for solvent and size-exclusion purifications. Furthermore, cationic moieties are incorporated within the hydrophilic domains to attract negatively charged groups in protein and nucleic acid payloads. Electrostatics drives high loading efficiencies (75-99%) and reduces the amount of synthetic material (50-200 pg) required for treatment. The inventors’ ability to dissolve polymers at high solids content (~20 wt%) improves scalability and encapsulation of protein payloads. As is known in the LNP literature, low concentrations often prevent appreciable loading for protein payloads due to the sparse charge distribution, which requires modifications to the native protein. The system’s components can even be co-lyophilized for improved storage stability during distribution then reconstituted below the LCST, selfassembled at room temperature and still retain all material properties and performance. The inventors demonstrate the utility of such facile formulations in the contexts of protein subunit vaccination, protein inverse vaccination for tolerance induction, and RNA interference (RNAi) for cancer immunotherapy. Such a platform may prove useful in growing efforts to develop nonviral vectors for biomacromolecule delivery.1. Thermosensitive block copolymers for polymersome self-assembly at room temperature

[0229] In prior work, materials scientists demonstrated the utility of poly(oligo(ethylene glycol) acrylate)s as a versatile LCST polymer, whose LCST is tunable from 0-100°C by varying the number of ethylene glycol units in the side chain. Chilkoti further demonstrated a biological utility of such polymers for increasing hydrodynamic radius and reducing protein clearance as an alternative to the widely used poly(ethylene glycol) (PEG), as the short oligoethylene glycol side chains avoid the development of a humoral immune response, in contrast with the development of anti-PEG antibodies. Here, the inventors chose poly(diethylene glycol ethyl ether acrylate) (pDEGEA) as the PS membrane-forming block as its LCST transition is around 13-15°C, allowing for it be soluble at ca. 4°C and form selfassemblies at room temperature. The pDEGEA-containing BCPs were designed with blocklengths chosen to allow for the self-assembly of vesicular structures characteristic of PSs (FIG. 1A and FIG. 7). Based on previous literature, block lengths of 120-175 monomers for the membrane-forming pDEGEA block (denoted “D”) and block lengths of 15-70 monomers for the hydrophilic block, corona forming blocks were chosen to maintain the hydrophilic weight fraction at 10-25% and yield vesicular morphology (FIG. IB). For all studies herein, D130 (subscript denotes the number of monomers or repeat units) was used as the membrane-forming material, and either poly(hydroxy ethyl acrylate) (pHEA) (“H70”) or poly(trimethylamino ethyl acrylate) (pTMAEA) or (“TM25”) was the hydrophilic segment for uncharged and charged polymers respectively (FIG. 1B-D). Work from Eisenberg demonstrated that asymmetric vesicles comprised of a BCP blend can be formed by synthesizing the hydrophilic component in one BCP to be much shorter than the hydrophilic chain in the other, while maintaining the same length of membrane-forming segments in both. For this reason, the TM25 segment in the polymers was designed to be much shorter than H70 segment, while the same length of pDEGEA was used in both BCPs to ensure self-assembly of monomodally distributed particles. Degrees of polymerization were determined by1H NMR (FIG. 28) and BCP end groups were modified to enhance particle stability and introduce bioactivity (below).

[0230] End-group functionalization of the D130-H70 and D130-TM25 polymers was achieved by aminolysis of the trithiocarbonate terminal group, followed by a thia-Michael reaction with a compound containing an acrylate moiety. For example, Di3o-H?o-Mann was modified by reaction with a mannose-bearing acrylate (FIG. 8). Due to the high molecular weight of the polymers,1H NMR could not quantify chain ends, and successful end group functionalizationwas determined by a combination of UV-visible spectroscopy (UV-Vis), to monitor the loss of the trithiocarb onate absorbance peak at 308 nm during aminolysis (FIG. 10), followed by two colorimetric assays, Ellman’s and Phenol -H2SO4, to determine the efficiency of the thiaMichael reaction. The former was used to validate the absence of any remaining free thiols (which are generated as a part of the aminolysis reaction) while the latter was used to determine the quantity of mannose moieties in the mannosylated formulations (FIG. 2B). The theoretical amount of mannose under the assay conditions (assuming one molecule per polymer chain) was 60 nmol per 2 mg of material and the slightly lower determined amount (ca. 50 nmol mannose / 2 mg polymer) is attributed to variances in sample heating when following the literature procedure for the Phenol-FFSCh assay, along with some loss in BCP chain-end fidelity during purification. Final BCPs were characterized using gel permeation chromatography (GPC) for molecular weight distribution. D130-H75 shows a monomodal molecular weight distribution with low dispersity (D = 1.2), relative to PMMA standards (FIG. 1C,D). It was difficult to obtain good quality GPC data on D130-TM25 due to the quaternary ammonium groups. However, the polymer prior to quaternization, D130-DM25, showed relatively low dispersity (D = 1.3), with slight tailing due to the presence of tertiary amines.

[0231] The LCSTs of the materials along with resulting nanoparticle size were determined using dynamic light scattering (DLS) in phosphate buffered saline (PBS, pH 7.4). A sharp increase in scattering intensity at 17°C suggests a morphology transition from unimers to polymer nanoparticles (FIG. 2A). It is noteworthy that a sharp peak in hydrodynamic radius was observed upon initial transition, followed by a decrease to a stable radius once the system reached room temperature (20°C). Previous work from Armes and coworkers attributes this behavior to the micelle-worm-vesicle transition, as determined by rigorous microscopy studies. Polymer blends were made by mixing charged and uncharged BCPs at different weight ratios to formulate nanoparticles with varying degrees of charge to enhance payload encapsulation efficiency. Nanoparticles are therefore reported here as the relative weight fractions of the charged BCP, D130-TM25 (“T”) and mannosylated BCP, Di3o-H?o-Mann (“M”). The remainder of the nanoparticle weight fraction consists of the inert BCP, D130-H70. The blended particles are referred to as “TxMy”, with “x” and “y” being the relative weight percentages of charged and mannosylated BCPs (FIG. 2C).

[0232] The polymers were highly soluble in aqueous media at low temperatures and could be dissolved at high concentrations, although the concentrations were kept less than 100 mg / mL in order to favor vesicular assembly as demonstrated by Battaglia and coworkers. Following self-assembly by warming to room temperature, size and morphology weredetermined by DLS (FIG. 2D), transmission electron microscopy (TEM) (FIG. 2E) and multiangle light scattering (MALS) for determining radius of gyration (RG) (FIG. 12C). By DLS, the inventors noted hydrodynamic radii (RH) of favorable size (-100 nm) and distribution (PDI < 0.1) with a single peak in distribution (FIG. 2D), indicating uniform assembly for the BCP blended system. The zeta potential for the charged BCP-only polymersome (T100M0) is highly positive (+ 14m V), but incorporating the uncharged BCPs lowers this charge of these mixed polymersomes as expected to within biologically nontoxic values (FIG. 29). To quantitatively assess vesicular structure for nanoparticles used in vitro and in vivo, the inventors determined a form factor, RG / RH, which should be equal to 1 for hollow sphere (i.e., vesicles). This is because RG quantifies the mass distribution of a particle, which is theoretically distributed along its radius rather than its core for a polymersome. All structures used in these experiments have RG / RH between 0.93 and 1.18 and can be reasonably approximated as being spherical vesicles (FIG. 12C). Morphologies are also qualitatively confirmed with negative-staining TEM, showing sizes similar to those observed by light scattering, with slight deviations attributed to drying upon sample preparation (FIG. 2E). As determined by both RG / RH and TEM imaging, all particles used in biological studies were indeed vesicular when assembled under physiological salt concentrations in saline or PBS. Importantly, it was possible to obtain reproducible particle sizes given that the self-assembly is dictated by polymer block lengths and relative weight fractions, rather than assembly kinetics, thus leading to structures near thermodynamic equilibrium. It is hypothesized that narrow size distributions (PDI<0.1) are due to the complete solubility of the polymers when cooled, thus avoiding inhomogeneities in dispersion. Taken together, this material circumvents previous issues reported in polymersome processing by avoiding organic solvents for self-assembly along with yielding narrow, predictable sizes to address issues of quality control.2. High macromolecule loading within PSs and enhanced uptake in antigen-presenting cells

[0233] Encapsulating protein payloads has often proved challenging due to their weaker electrostatic interactions relative to nucleic acids, along with the generally dilute conditions under which nanoparticles are formed. Though some reports do show LNPs encapsulating protein, these instances often required modification of the payload and still require solubilizing the amphiphilic components of the delivery vehicle in an organic solvent such as ethanol. On account of the use of organic solvents, high concentrations are not achievable with typicalformulations, and the use of ionizable amines is insufficient for complexation with the sparse negative charges on proteins. By contrast, the temperature-sensitive PSs can be fully suspended in aqueous medium at high concentrations (100 mg / mL), and the permanently charged ammonium moieties were hypothesized to have enhanced electrostatic affinity for weakly charged residues on proteins. Such high solids content is critical for maximizing encapsulation efficiency while still maintaining vesicular morphology. Previous attempts to achieve this rely on complex synthetic or processing approaches, such as polymerization-induced self-assembly (PISA), microfluidic devices, or flash nanoprecipitation (FNP), which require specialized reactors for scale-up and complex manipulations that present challenges for translation to the clinic.

[0234] The PSs consisting of BCP blends without mannosylated polymer, TxMO, encapsulate 75-90% of dissolved protein (here, the model protein ovalbumin, or OVA) in solution as determined by gel electrophoresis, with increased efficiency using higher weight percent of charged polymer (“x” from 33 to 100). (FIG. 2F). As to nucleotide payload, siRNA can be completely encapsulated (-100% efficiency) using polymer / payload weight ratios as low as a 1 :5 for formulations using the T50M0 BCP blend (FIG. 2F). The loading efficiencies are consistent and reproducible, and they are similar when incorporating mannosylated D130- H?o-Mann BCPs (FIG. 13 A). Since OVA has a net-negative charge (isoelectric point -4.7) at physiologic pH (7.4), other therapeutically relevant biologies such as Enbrel®, anti-program cell death protein 1 (PD-1) antibody and SARS-CoV-2 RBD peptide were encapsulated (FIG. 13B). All of these payloads have a slight net-positive charge at physiologic pH, but could still be incorporated in the particles due to the presence of negatively charged amino acids. However, the inventors noticed that this efficiency is decreased for low molecular weight peptides compared to larger macromolecules such as antibodies. To demonstrate successful loading from dry formulations, the inventors also dissolved the BCPs alongside the same protein and siRNA payloads, lyophilized the fully soluble mixture, and then reconstituted in deionized water on ice. Samples were then allowed to warm to room temperature, and the inventors detected similar, high loading efficiencies for both payloads (FIG. 30A,B). The reconstituted polymers are then able to deliver OVA to in-vitro cell lines and demonstrate retained bioactivity in antigen presentation assays (see below, and FIG. 30C). Such an approach to loading may be particularly beneficial for PS storage and distribution at large scales, with sensitive payloads prone to hydrolysis. The materials were also characterized post-loading and demonstrate similar sizes and spherical morphology with both protein and siRNA payloads (FIG. 31). Zeta potential was payload dependent, with PS-siRNA having more negative zetapotential (-0.2 to -2 mV) and PS-OVA being positive (0.6 to 9 mV). Admixing with the nucleic acid adjuvant CpG 1826 post-encapsulation also lowered the zeta potential for protein-based formulations to being slightly negative. Due to high loading efficiency and homogenous particle sizes upon warming, the inventors can bypass tedious processing such as size exclusion and membrane filtration. Furthermore, the high solids content remains a highly attractive feature of the system, as current polymer and lipid nanoparticle technologies rely on further processing for concentration of suspensions in order to achieve a robust biological response. As a result, the materials can simply be diluted for administration without needing further processing.

[0235] Indeed, uptake studies in murine bone-marrow derived dendritic cells (BMDCs) show enhanced uptake when delivering OVA formulations in this way. Viability studies were performed to determine dose and relative charge fraction based on the MTT assay (FIG. 14A). On account of the toxicity of highly charged moi eties, the inventors chose to use T33My based formulations to preserve cell viability. Subsequent screening studies also determined ideal ranges for mannosylation be between 10 and 33 wt% of the total particle formulation (T33M10 or T33M33) for optimal uptake. Previous literature also supports this observation that sparse ligand density is often favorable over full coverage of the particle with mannose. For these formulations, nanoparticle-encapsulated FITC-OVA achieved 3 to 6-fold greater uptake by BMDCs compared to unencapsulated OVA based on geometric mean fluorescence intensity (gMFI) in flow cytometry (FIG. 3A). To mechanistically demonstrate the utility of mannosylation for BMDC uptake, the inventors conducted the same study using a mannosereceptor blocking cocktail consisting of mannose-receptor blocking peptide. After blocking, mannosylated polymersomes showed a statistically significant decrease in uptake compared to bioinert polymersomes and free protein (FIG. 14B). This suggests that incorporating targeting moieties favors uptake in a mannose-receptor dependent manner, which may prove beneficial for in vivo nanoparticle delivery in the context of antigen delivery.3. PSs enhance protein antigen presentation in-vitro and antibody responses in-vivo

[0236] After demonstrating enhanced uptake, the inventors sought to determine the material’s ability to allow for antigen presentation once the protein has been delivered to the antigen-presenting cell (APC). Thus, functional in-vitro assays were conducted to determine the material’s efficacy as an antigen delivery platform for prophylactic subunit vaccines. Withthe growing interest for eliciting strong CD8+T cell responses, the inventors first probed the material’s ability to trigger proliferation in the OVA-specific transgenic CD8+T cell line, OTI. Upon co-culture with BMDCs treated with the formulations, CFSE-labelled OTIs underwent varying degrees of proliferation as measured by flow cytometry (FIG. 3B). A “CFSE-diluted” population (i.e., proliferated to various generations) was determined as all T cells with diluted fluorescence compared to that of the original population (FIG. 3B). Based on fluorescence histograms, the inventors observed enhanced OTI proliferation (%CFSE-diluted population) when OVA was delivered encapsulated in the PSs versus free in solution. The inventors hypothesize that endosomal escape occurs due to the hydrolytic instability of the acrylate backbone, which is prone to cleavage by the slightly acidic environment and esterase activity within the endosome. The inventors then probed this phenomenon using fluorescence microscopy studies codelivering a fluorescently-quenched DQ-OVA and fluorescently-active OVA647 in RAW 264.7 macrophages to probe endosomal processing and uptake respectively (FIG. 14C) The inventors observed that the nanoparticle constructs not only enhance uptake based on increased Alexa Fluor 647 signal, but promote endosomal processing and release into cytosol as observed by the diffuse fluorescence of DQ-OVA compared to more punctate fluorescence seen when delivering free protein alone. Based on these results, the inventors envisioned that the materials would lead to stronger CD8+T-cell responses upon protein subunit vaccination compared to free antigen under equivalent adjuvant stimulation. To establish the materials’ capabilities for robust immunity, the inventors also probed the humoral response to demonstrate that the PSs can also elicit antigen-specific antibodies, which necessitated in-vivo studies. Polymer alone was first delivered intravenously to ensure no systemic toxicity as identified by blood chemistry and inflammatory cytokine secretion (FIG.32).

[0237] Considering humoral responses, a pilot experiment was conducted as a single-dose vaccine using OVA admixed with the Toll-like receptor 9 (TLR9) agonist CpG ODN 1826 (FIG. 3D). CpG was the benchmark due to its current use as a clinical adjuvant for a proteinbased subunit vaccines, including HEPLISAV-B® and PreHevbrio®, and it is particularly known to stimulate strong antibody responses. CpG is also attractive due to its solubility in aqueous buffer, broad safety profile in elderly and immune-suppressed patients, and well- understood mechanism of action. Mice were vaccinated in all four hocks using either mannosylated (T33M33) or non-mannosylated (T33M0) PS-encapsulated OVA admixed with CpG and compared to free, adjuvanted OVA formulations. The inventors observed potent antibody responses characterized by total OVA-specific IgG measured periodically over 3months following initial vaccination, with PS formulations significantly outperforming free protein with CpG (FIG. 3D). Though differences were not seen between the mannosylated and non-mannosylated PS groups, it became apparent that even a single administration of either formulation significantly enhanced antibody levels over that of free adjuvanted protein at equal doses. Remarkably, the PS-encapsulated OVA maintained antibody levels even one year after single vaccination, while antibodies from unencapsulated OVA-CpG declined to levels similar to those of untreated animals (FIG. 3E). In addition to antigen delivery, the surface charges on the PSs cause association with CpG, which may further contribute to immune stimulation in- vivo, though no such effect was seen in-vitro (FIG. 33).

[0238] Initial biodistribution studies suggest that this response may be due to the enhanced retention of PS-encapsulated OVA in injection site-draining lymph nodes following vaccination (FIG. 3F,G). Interestingly, Alexa-Fluor 647 tagged OVA showed greater fluorescence in the brachial and popliteal lymph nodes when delivered in a PS formulation, whereas free OVA showed low measurable fluorescence 4 hr after administration. The differences in lymph-node distribution could be associated with the varying tissue architecture at each of the injection sites along with the time point chosen for measurement (4 hr). These results suggest greater consistency of lymph-node trafficking when protein was delivered in encapsulated formulations, with mannosylation enhancing delivery to the brachial lymph nodes. This observation further motivated probing the lymphocyte response upon vaccination, as the inventors hypothesized that the enhanced delivery from this platform may lead to greater immunogenicity of encapsulated OVA in both cellular and humoral responses.4. PSs enhance cellular immunity upon subunit vaccination in-vivo

[0239] In a model for protein subunit vaccines, C57BL / 6 mice were treated with formulations of the PSs (T33M10 and T33M0) encapsulating OVA and adjuvanted with CpG ODN 1826 in a prime-boost regimen. The benchmark for this study was free OVA admixed with equivalent CpG, akin to clinical subunit vaccine formulations (FIG. 27A). Draining lymph nodes (dLNs) and spleens were harvested for analyzing T cell responses, while blood samples were taken at weeks 2 and 4 to determine antibody levels 1 week before and after boost. Relative to protein delivered without encapsulation, both mannosylated and non- mannosylated polymersomes demonstrated enhanced CD8+T cell responses (FIG. 27B-F). As measured by peptide-MHCI tetramer staining of lymphocytes, both formulations greatly increased populations in both CD8+T cells (FIG. 34A) and antigen-experienced, CD44+CD8+T cells that were tetramer , i.e. that recognize the vaccinal antigen, relative to free protein with the same adjuvant (FIG. 27E,F). To explore the functionality of OVA-specific T cells, lymphocytes and splenocytes were subjected to 6-hr restimulation studies to probe the CD8+and CD4+T cell responses as measured by intracellular cytokine staining. Importantly, responses from mannosylated particles trended towards a greater percentage of IFNy+and IFNy+TNFa+double-positive CD8+T cells in the dLNs following peptide restimulation compared to the non-mannosylated PS formulation (FIG. 27B-D and FIG. 34B). This strong response in the CD8+T cell compartment is promising, as it is known that CD8+T cell immunity is particularly important in clearing viral infections such as SARS-CoV-2 and other respiratory infections, as well as in cancer immunotherapy. Furthermore, CD4+T cells also demonstrated a greater percentage of proinflammatory cytokine-secreting populations, measured by IL-2 and IFNy (FIG. 35A). Similarly, whole protein, 3 -day restimulation of splenocytes, indicative of a systemic immune response, showed strong Type-1 immunity, which has also been shown using a different PS formulation from the inventors’ lab. Briefly, restimulations of splenocytes led to secretions of significant levels of IFNy and TNFa, suggesting robust T-helper responses in addition to cytotoxic T-cells (FIG. 27G,H). Splenocytes also demonstrated enhanced secretion of IL-6 in response to the PS-encapsulated formulations, though significance was only observed in the mannosylated formulation (FIG. 35Bi), and similar trends were observed in the draining lymph nodes for IFNy and IL-6 (FIG. 35Bii-iii). The inventors hypothesize that the enhanced immunogenicity of PS-encapsulated OVA is a result of enhanced uptake and cross presentation, as seen in-vitro (FIG. 3A,B and FIG. 14C), and a greater loss of free protein from the injection site. However, given the enhanced uptake of nanoparticles in general, both delivery systems show improvements over free protein, also adjuvanted with CpG. In summary, cellular and Type 1 immunity was enhanced using the PS formulations when delivering OVA antigen. Slight improvements were seen when using the mannosylated particles as determined by functional assays for stimulating CD8+T cell response in the presence of OVAs peptide epitope SIINFEKL (SEQ ID NOV) and systemic immune response observed by whole OVA protein stimulation.

[0240] The OVA-specific antibody response was also investigated in this prime-boost model by measuring antigen-specific IgG area under the curve (AUC) (FIG. 27i). Not surprisingly, total IgG for encapsulated and free OVA formulations showed modest (albeit statistically significant) differences when using CpG ODN 1826, as the adjuvant is known for its robust B cell activation. Though the antigen-specific IgG response appears similar to that of adjuvanted free protein, the highlight of the nanoparticle system is enhanced cellular immunity,which is believed to be more robust against antigenic drift. Furthermore, upon looking at specific subtypes of IgG, the inventors see enhancements in IgG2b relative to vaccination with free protein, with similar amounts of IgGl (FIG. 35C). This is a vital result, as higher levels of IgG2b are associated with enhanced Type 1 immunity, critical in controlling anti-viral responses. Taken together, the inventors demonstrate that these therm oresponsive PSs present a facile delivery system for subunit vaccines intended for robust cellular and humoral immunity. The enhancement in Type-1 and cytotoxic T-cell immunity over that of free protein admixed with adjuvant demonstrates the benefit of the PS platform as a subunit vaccine, which commonly have not elicited strong cellular immunity in the clinic using CpG as an adjuvant. Due to the enhanced effects of the formulation, it may also be possible to lower the overall dose of antigen and adjuvant for more vulnerable populations. The ease of formulation and lack of purification may prove beneficial in the context of vaccine clinics where large therapeutic dose batches can be made simply by diluting a stock of room-temperature solution.5. PS-encapsulated protein antigens as an inverse vaccine in a model for allergic asthma

[0241] In addition to subunit vaccination, the use of the material was also expanded towards applications in tolerance induction towards a protein antigen, for which the inventors also used OVA as a model protein antigen. To determine the ability of the nanoparticle system at preventing an allergic immune response, a study was conducted by intravenously administering C57BL / 6J mice with either T33M10 without payload, or both T33M10 and T33M0 encapsulating OVA. Mice were then subjected to an experimental model of allergic airway inflammation during which mice are sensitized through intraperitoneal injections of OVA adsorbed to alum (FIG. 5A). Upon challenge with intratracheal instillations of OVA, hallmarks of allergic airway disease in allergic asthma are measured, including airway eosinophilia, systemic levels of IgE, and CD4+T cell inflammation. As a negative control, mice were treated with T33M10 without payload and sensitized using alum without OVA, thus preventing any OVA-specific allergic responses upon challenge. The key benchmark and positive control in this study was empty T33M10 PS without payload, sensitized using alum / OVA. Compared to mice treated with empty PSs, mice that were prophylactically administered polymersomes encapsulating OVA showed decreases in lung and airway eosinophilia (FIG. 5B-D). Flow cytometric quantification of eosinophils (SiglecUCDl lc ) in the bronchoalveolar lavage fluid (BALF) as well as lungs showed significant decreases onlyfor the mannosylated T33M10 delivering OVA, when compared to the positive control. Furthermore, a reduction in CD4+T cells is indicative of a productive tolerogenic response, as allergies are known to be a Type-2 mediated immune response. In all cases, our mannosylated T33M10 platform delivered OVA in a systemic manner to reduce the total number of CD4+T- cells infiltrating the lungs and airways upon pulmonary challenge (FIG. 5D,E).

[0242] In addition to T cells and eosinophils, B cells are known to mediate the allergic response through class-switching and antibody secretion. Specifically, IgE binds antigen and stimulates mast cell degranulation. The inventors therefore examined systemic levels of IgE post-sensitization and during the challenge phase of the model (FIG. 5F). Not surprisingly, a sharp increase in systemic IgE was observed upon challenge of the empty T33M10 PS treated group sensitized with alum / OVA. Though not a complete return to baseline, T33M10 encapsulating OVA was able to mitigate this response as seen by significantly reduced IgE levels following initial intratracheal challenge and at endpoint. Furthermore, allergic B cell responses were also measured by flow cytometry, with reductions seen in class-switching (IgM‘ IgD ), germinal central formation (GL-7+CD95+) and differentiation into IgE+plasma cells (CD138+) (FIG. 18). Taken together, the T33M10 nanoparticles provide a platform system for protein antigen delivery to effectively target immune cells in the context of tolerance induction (inverse vaccination) in the absence of an adjuvant (FIG. 5), as well as vaccination in the presence of an adjuvant (FIG. 27). Though both PS systems show trends toward reducing the allergic response, mannosylation plays a key role in further suppression, consistent with previous results from the lab using a soluble polymer-antigen conjugate platform. This may be due to the mannosylated PSs promoting delivery to APCs in the liver, which is known to present antigen in a tolerogenic manner (FIG. 5G,H).6. In-vitro and in-vivo delivery of PS-encapsulated siRNA for RNA interference in cancer

[0243] As a demonstration of the utility of the PS platform in siRNA therapy, the inventors turned to siRNA to downregulate cancer-promoting pathways via RNA interference (RNAi), employing validated pathways to lead to clear interpretation. Vascular endothelial growth factor A (VEGF-A) is known to promote tumor cell survival and proliferation via induction of angiogenesis. Bcl-2 is an anti-apoptotic protein that can be upregulated via the VEGFR pathway, providing a positive feedback loop for enhanced tumor cell survival. Therefore, the inventors chose VEGF-A and Bcl-2 as target molecules for RNAi in the human breast cancerline MCF-7, which is known to upregulate both. Without chemical modification, siRNA degrades rapidly in the presence of serum in cell-culture medium, thus necessitating cationic delivery vehicles such as lipid nanoparticles for transfection. For this reason, the inventors first compared the PSs to the commercially available transfection agent, Lipofectamine 2000 (Lipo). In this head-to-head comparison, the PSs achieved greater gene downregulation when delivering siVEGF-A (-60% downregulation) and siBcl-2 (50% downregulation) and therefore were investigated further in in-vivo cancer studies (FIG. 6A). Control studies determined the optimal dosing for PS formulations, that vehicle alone does not downregulate mRNA expression, and that there is no off-target downregulation for housekeeping genes such as P- actin (FIG. 36A-D). Since the MCF-7 cancer line does not express the mannose-receptor, only the inert PS formulations T33M0 were used for RNAi studies.

[0244] To establish the capability of the formulations for in-vivo siRNA delivery, mice inoculated with MCF-7 tumors were treated intratum orally (i.t.) with encapsulated and unencapsulated siVEGF-A and siBcl2 (FIG. 6B). Nude mice were chosen for this study to avoid immune rejection of the human tumors. As in the in-vitro work, the cellular target of this RNAi therapy is the tumor cells themselves, so mannosylated polymersomes were not used in this study. To ensure downregulation of siRNA targets in vivo, tumor-bearing mice were treated days 11 and 13 post-inoculation and sacrificed on day 14 to ensure tumor sizes in control- treated groups did not grow to sacrifice criteria. Both mRNA and protein content were measured to confirm downregulation of both VEGFA and Bcl2 (FIG. 36E-G). The inventors then proceeded with an efficacy study to determine if this downregulation provides therapeutic benefit. During treatment, tumor growth was halted when either siVEGF-A and siBcl-2 were delivered intratumorally (i.t.) as encapsulated formulations (FIG. 6C,D). Statistically significant differences were observed when comparing tumor sizes taken at day 20 (FIG. 37A), at which point i.t. treatment was stopped, and neither unformulated siRNA nor empty PSs alone had antitumor efficacy in vivo (FIG. 6C). Furthermore, a clear enhancement in survival was observed for mice treated with the siRNA-loaded PSs (FIG. 6D). Tumors rapidly grew out once treatment was stopped, due to the lack of an adaptive immune response in the host mice, but this pilot study demonstrates the material’s ability to deliver siRNA therapeutics in vivo via halting tumor growth progression. Finally, to benchmark our materials against a clinically- relevant siRNA delivery technology, the inventors formulated lipid nanoparticles (LNPs) using lipids at molar ratios similar to those of Alnylam’s (FIG. 19). Following a treatment schedule similar to that above, the inventors demonstrate that the PS-siRNA formulations indeed enhance survival and suppress tumor growth over that of LNPs when delivered intratumorally(FIG. 6E,F). Alnylam’s formulations have been optimized for intravenous, liver targeting, but the inventors believe that this approach demonstrates the versatility of the PS platform in its ability to deliver multiple payloads with a variety of administration routes. Furthermore, the intratumoral efficacy of the technology could be attributed to the greater stability of PSs due to their macromolecular chain entanglements and may present an approach for delivering biomacromolecules without require payload-specific optimization.

[0245] Though the study was conducted with intratumoral injections, biodistribution studies administering a fluorescent nanoparticle show trafficking to tumors upon intravenous and subcutaneous injections (FIG. 38). Kidney accumulation is also observed suggesting clearance particularly upon intravenous delivery. Future work will optimize nanoparticle size, surface charge and additional targeting moieties to allow for intravenous or subcutaneous delivery as it pertains to cancer therapy. As a result, the inventors envision that this platform may allow for nanoparticle trafficking to the tumor site when parentally administered other than i.t., a topic for future study.B. Conclusions

[0246] In this work, the inventors have designed and developed thermosresponsive polymersomes that address many of the challenges that currently limit the polymer nanoparticle field’s broad progression to clinic. Namely, the inventors tackle issues in processing, loading efficiency, and quality control by developing a scalable platform with predictable nanoparticle morphologies. This was done by designing a room-temperature forming particle system decorated with cationic moieties to enhance payload affinity and thus encapsulation efficiency. Compared to current LNPs, the polymersome platform is highly stable due to its polymer components and is adaptable to a variety of payloads, here considering proteins and nucleic acids, without further modification. Furthermore, enhancements in therapy localization can be achieved by introducing targeting moieties such as mannose for vaccine or inverse vaccine applications. By simply warming a solution to room temperature, one can rapidly form large batches of nanoparticle therapies, providing a practical solution to issues in scalability of previous polymer formulations. The polymersomes are then ready to inject upon dilution, without requiring purification, since 75-99% of payload is encapsulated and the nanoparticles are of predictable size and poly dispersity, addressing the industry’s quality -by-design (QbD) principles. Furthermore, the inventors envision these materials being used as dry formulations alongside sensitive biomacromolecular payloads, with reconstitution in cold aqueous mediumprior to room-temperature warming. In this way, the inventors retain maximal stability potentially without (or at least less stringent) need for cold-chain logistics and storage, depending on the inherent thermal stability of the payload in the lyophilized state. As a result, these stable, easy-to-use materials may provide an avenue for improving distribution and accessibility of next-generation vaccines, particularly in areas with limited resources for coldchain logistics, storage, and manufacturing.C. Outlook

[0247] The inventors have demonstrated the development of a polymersome (PS) that accommodates both protein and nucleic acid payloads, and they have shown applications in a subunit (protein) vaccine, an inverse (protein) vaccine, and an siRNA-based cancer therapy. The key benefit of this payload-agnostic vehicle is that it allows for rapid formulation of multiple types of biomacromolecules for in-vitro and in-vivo delivery without any payload modification. This not only streamlines development, but may allow for subunit vaccines with greater immunogenicity, inverse vaccines inducing multi-antigen tolerance and nucleic acid delivery vehicles that can be stored at room temperature. As to subunit vaccines, the inventors’ laboratory has previously described glycopolymer-antigen conjugate vaccines, where the polymer is mannosylated and comprises TLR7 / 8 agonist moieties, thus producing strong cellular and humoral responses. By comparison, the PS system is much simpler and thus more economical to produce, requiring mere formulation in the vial, and can be used to develop more potent subunit vaccines consisting of multiple antigens. Furthermore, the benchmarking against formulations akin to HEPLISAV-B® demonstrate superior, long-lasting antibodies after a single immunization, which may be critical for communities with limited vaccine supply or patient hesitancy. As to tolerogenic, or inverse, vaccines, the inventors’ laboratory has also developed glycopolymer-antigen conjugate inverse vaccines used in autoimmune therapy, food allergy prevention and therapy, and anti-drug antibody prevention cases. The need for multiantigen delivery is emphasized in cases of hypersensitivity, such as in allergic asthma where a single patient may respond to multiple allergens. Once again, delivering proteins using the antigen-agnostic, non-immunogenic platform facilitates implementing therapies quickly and broadly. Finally, for nucleotide delivery - specifically siRNA therapy in cancer - the inventors outperform clinically used LNPs. In addition to efficacy, the PS platform also offers distinct advantages over LNPs and other siRNA delivery methods, such as 1) mild storage conditions as a lyophilized, room-temperature stable powder without additional preservative 2) rapidformulation without tedious processing (e.g., concentrating, dialysis, etc.) and 3) payloadagnostic biofunctionalization through targeting moieties as demonstrated in this work. In multiple use cases, the thermoresponsive PSs demonstrate efficacy, but broadly speaking, this material may be amenable to a variety of immunological applications with features that simplify production and ultimately increase formulation potency.D. Methods1. Reagents

[0248] Di(ethylene glycol) ethyl ether acrylate (DEGEA), 2-(dimethylamino)ethyl acrylate (DMAEA) and 2-hydroxy ethyl acrylate (HEA) were purchased from Sigma-Aldrich. DEGEA and DMAEA were distilled, passed through a basic alumina plug and stored at -80°C prior to polymerization. HEA was purified following protocols from Matyjaszewski and coworkers then stored at -80°C. Azobisisobutyronitrile (AIBN) was purchased from Sigma-Aldrich, recrystallized from methanol and stored in -20°C prior to use. Cyanomethyl dodecyl trithiocarbonate (CDT) was purchased from Sigma-Aldrich and used as received. Ovalbumin (OVA) and endotoxin-free OVA were purchased from Invivogen. Fluorescent OVA conjugates (FITC-OVA and OVA-647) were made by reacting either fluorescein isothiocyanate N- hydroxysuccinimide ester (FITC-NHS ester) or Alexa-Fluor 647 NHS ester (AF647 NHS ester) to OVA following standard literature procedures in carbonate / bicarbonate buffer. DQ-OVA was purchased from Fisher Scientific. All lipids used for formulating lipid nanoparticles were ordered from Broadpharm. Fluorescent antibodies were ordered through BD and Biolegend (Supplementary Tables 1 and 2). Western blot antibodies were ordered through Cell Signaling Technologies. All other reagents and solvents were ordered through Sigma-Aldrich unless otherwise noted. Solvents used in polymerizations were dried overnight on molecular sieves prior to use. siRNA sequences were designed and ordered through IDT, with dT sequences introduced to provide stability for the double stranded structures. The siRNA sequences (denoted as sense strands (SS) and antisense strands (AS)) used in the studies are as follows: Human VEGF-A (SS): 5’-AAAUGUGAAUGCAGACCAAAGdTdT-3' (SEQ ID NO: 1); Human VEGF-A (AS): 5’-CUUUGGUCUGCAUUCACAUUUdTdT-3' (SEQ ID NO:2); Human Bcl-2 (SS): 5’-AGUCAUCCACAGGGCGAUGUUdTdT-3' (SEQ ID NO:3); and Human Bcl-2 (AS): 5’-AACAUCGCCCUGUGGAUGACUdTdT-3' (SEQ ID NO:4).2. Instrumentation

[0249] Proton nuclear magnetic resonance (XH NMR) was conducted on a Bruker Avance 11+ 500 MHz spectrometer. For polymers, 32 scans were taken using a 10s relaxation time. Molecular weight distributions (Mn) and poly dispersity (D=MW / Mn) were determined by gel permeation chromatography (GPC) in DMF with 0.01 M LiBr on a Tosoh EcoSEC using PMMA standards. Small molecules were characterized via NMR and electrospray ionization mass-spectrometry (ESI-MS) on Agilent 6130 LCMS using methanol as the eluent. Dynamic light scattering (DLS) measurements were taken on a Wyatt Mobius™ at a fixed detection angle of 163.5° using 532 nm light. Data was analyzed using Dynamics® software and plotted using GraphPad Prism 9. Multi-angle light scattering (MALS) measurements were taken on a Wyatt DAWN HELEOS II MALS detector with a 658 nm light source using lx PBS as the eluent. UV-Visible (UV-Vis) spectroscopy was conducted on a Shimadzu UV-3600 Plus UV- VIS-NIR, scanning from 450 to 200 nm wavelength. Flow cytometry measurements were taken on a Novoctye Penteon 5-30 or BD LSRFortessa 4-15. Data was analyzed using BD FlowJo and plotted in GraphPad Prism 10. Fluorescence microscopy was conducted on a Leica Microsystems microscope equipped with 96-well plate holder.3. Polymer synthesis

[0250] All BCPs were synthesized using reversible addition fragmentation chain-transfer (RAFT) polymerization, following standard literature protocols. CDT was employed as the initial chain transfer agent (CTA) for the parent polymer, which was then used for sequential chain extensions. Crude and purified materials were characterized by NMR throughout the synthetic steps and GPC was used to determine molecular weight of purified polymers. All degrees of polymerization are based on NMR measurements.4. Poly(diethylene glycol ethyl ether acrylate) (pDEGEA) macroCTA synthesis

[0251] In a 25 mL, 3 -neck flask, 3.33 g DEGEA monomer (18 mmol, 2M in solution) and 26 mg CDT were dissolved in 8.6 mL DMF. AIBN was added as a stock solution of 1.3 mg in 50 pL DMF. The solution was degassed with four freeze-pump-thaw cycles and placed in a 70°C oil bath for 1.8 hr under nitrogen flow to attain -60% conversion of monomer. This was done to maintain polymer chain end livingness. The solution was quenched by immersion in liquid nitrogen and opening to air. DMF was removed via rotary evaporator, and the crude mixture was reconstituted in minimal THF and precipitated into hexanes (x6). The resultingpolymer was dried overnight and obtained as a yellow, viscous oil (yield: 99%). This was used as the “parent polymer” (RAFT macro-CTA) for all BCP constructs.5. p(DEGEA)-b-poly(hydroxyethyl acrylate) (pDEGEA-b-pHEA)

[0252] 1.4 g pDEGEA macro-CTA and 770 mg HEA monomer (6.67 mmol, 0.6M in solution) were dissolved in 9 mL DMF. 1 mg of AIBN was added, and the solution was degassed and placed in a 70°C oil bath for 3.75 hr to achieve -60% monomer conversion. The solution was diluted and dialyzed extensively against water for 48 hr with repeated buffer changes. pDEGEA-b-pHEA was lyophilized and obtained as a yellow, sticky solid (yield: 93%)6. p(DEGEA)-b-poly(trimethyl aminoethyl acrylate) (pDEGEA-b- pTMAEA)

[0253] 670 mg pDEGEA macro-CTA and 290 mg DMAEA monomer (2 mmol, 0.6M in solution) was dissolved in 2.4 mL 1,4 dioxane. 0.4 mg AIBN was added as a stock solution, and the solution was degassed and placed in a 70°C oil bath for 1.5h. Due to the slower kinetics of the polymerization and relatively short target block length (-25 monomers) for DMAEA, the monomer amount was adjusted to achieve the desired degree of polymerization at 20% conversion. Upon quenching, a small portion was precipitated in hexanes for GPC and NMR analysis of the resulting BCP, pDEGEA-pDMAEA. The remainder of the crude mixture was diluted with THF to a concentration of 2 wt.% polymer, and 500 pL of iodomethane (8 mmol) was added via positive pressure pipette. The reaction was run overnight at room temperature, dried via rotary evaporator, and the crude mixture was reconstituted in DMF for end-group modification.7. Mannose acrylate synthesis

[0254] Acrylate-modified mannose was synthesized using a modified protocol for the acetal -exchange at the Cl position of mannose. 2 g mannose was dissolved in 1 mL water and added to 12 g of HEA. 500 pL acetyl chloride was added to the reaction at 0°C, which was then placed in a 70°C oil bath for 4 hr. The reaction was monitored with thin-layer chromatography (TLC) using p-anisaldehyde and potassium permanganate stains. The crude mixture was adsorbed onto silica and purified by flash column chromatography using 20% MeOH in DCM as the eluent. The purified material was characterized by 'H NMR andelectrospray ionization mass spectrometry (ESLMS). [M+Na]+theoreticai= m / z 301.1, [M+Na]+found= m / z 301.25, 10% yield. The acrylate was stored as a stock solution in DMF with 0.5 wt% MEHQ to prevent polymerization.8. Polymer chain-end modification

[0255] All polymers were modified via aminolysis of the trithiocarb onate chain-end followed by Thia-Michael addition of acrylate-functionalized moieties, using previously published techniques. Reactions were carried out by dissolving 50 mg / mL polymer in DMF, 20x molar excess acrylate and 5x molar excess TEA (relative to RAFT chain end). Reaction mixtures were degassed by four freeze-pump-thaw cycles, and 5x molar excess hexylamine was added under nitrogen as a stock solution in DMF after the first cycle. Reactions were run overnight, and UV-Vis was used to determine full cleavage of the polymers prior to workup (based on loss of RAFT-chain end absorbance at 308 nm). The resulting polymers were obtained by 2x precipitation in 1 : 1 mixture of ether / hexanes, followed by extensive dialysis against water. All purified polymers were then characterized with NMR, GPC and two colorimetric assays: Ellman’s assay was used for all formulations to ensure no residual free thiols and successful mannosylation is determined by the Phenol-EESCh assay. For pDEGEA- b-pHEA, the chain end was cleaved and functionalized using either HEA monomer (final material referred to as “D130-H70”) or mannose acrylate (“Diso-Ebo-Mann”). Both materials appear as a white, sticky solid after lyophilization. Yield: 93% (Mannose Modified), 95% (HEA modified). For pDEGEA-b-pTMAEA, the chain end was cleaved and reacted with residual, unreacted TMAEA monomer remaining (~30x excess relative to chain end) from the crude mixture. The material is a white, fluffy solid after lyophilization and referred to hereafter as “D130-TM25”. Yield: 50%.9. Polymersome formation and loading efficiency for proteins and nucleic acids

[0256] BCP stock solutions were prepared by dissolving at 200 mg / mL in PBS overnight and stored at 4 °C. All stock solutions are used within 1 month to avoid appreciable hydrolysis of the acrylate backbone. The thermal transition of materials was determined by preparing a 1 wt% solution of polymer in PBS, then placing in a quartz cuvette for DLS measurement. The instrument was cooled to 4°C with a constant flow of nitrogen to prevent condensation. Once cooled, the solution was warmed to 37°C with a slow heating rate of 0.1°C / min and lightscattering measurements taken every 3 min. For determining particle size and morphology, the stock solutions were diluted 1 : 1 in cold PBS (-20-50 pL) and left at room temperature for 5 min to ensure warming and complete self-assembly. For DLS, particles were further diluted to 0.01 wt% in 0.22 pm filtered PBS and analyzed as the average of 3 sets of 5 scans, each with a 3 sec relaxation time. Particle hydrodynamic radius (RH) was determined using the Stokes- Einstein equation. MALS measurements were conducted by further diluting the same samples to 0.01 mg / mL in PBS. Scattering intensity (as Re / Kc) was plotted as a function of angle (sin2(9 / 2)) and analyzed using a Rayleigh-Gans approximation for spherical particles. Radius of gyration (Rg) was obtained for polymer nanoparticles and compared against the RH from DLS to ascertain vesicular structure. For TEM, samples were diluted to 1 mg / mL, dropped on 200 (lines / inch) mesh copper grids, and rinsed with deionized (DI) water. Grids were then stained with uranyl acetate and dried overnight prior to imaging. Loading efficiencies of protein and nucleic acids were measured using gel electrophoresis with non-reducing, detergent-free buffers. For protein loading, a stock solution of OVA in cold PBS was mixed 1 : 1 with BCP stock to provide a solution of 10: 1 polymerpayload (wt / wt). The nanoparticles were formed at room temperature for 15 min, diluted, then run on a 4-20% gradient, stain-free polyacrylamide gel (BioRad) using Tris-Glycine (TGX) running buffer. The gels were removed, activated with UV, and analyzed using BioRad ImageLab. For nucleic acid loading, stock solutions in nuclease free water are diluted to 300 ng polymer with 30 ng FAM-siRNA (Thermo Fisher, Silencer Select). After room temperature assembly for 15 min, samples were diluted with loading dye and run on a 15% polyacrylamide gel using Tris-Boric Acid-EDTA (TBE) buffer to prevent RNA degradation. Gels were analyzed using the fluorescein channel as well as staining with SYBR Safe nucleic acid dye on ImageLab. To determine loading efficiency for materials reconstituted from dry formulations, BCPs were mixed with either OVA or FAM- siRNA as above, then lyophilized prior to gel electrophoresis. Once fully dried, polymer / payload powder was resuspended with cold, nuclease-free water to similar concentrations as prior to drying. Solutions were then allowed to warm to room temperature, followed by similar conditions for gel electrophoresis as above.10. OVA cellular uptake, antigen processing and presentation in vitro

[0257] For protein uptake studies, FITC-OVA was encapsulated following the above procedures in nanoparticles at 20: 1 (polymer / payload, w / w) using 100 mg / mL of polymer. Nanoparticles were then diluted to 50-100 pg / mL in serum-free, RPMI-1640 media (Gibco)prior to treatment. Bone-marrow derived dendritic cells (BMDCs) were then plated at 100,000 cells / well in 96-well U-bottom plates and treated with nanoparticle formulations for 30 min. Cells were then washed with PBS and incubated in complete Lutz media for 2 hr. After, cells were stained for viability and fixed with 2% paraformaldehyde (PF A) prior to flow cytometry. For mannose-receptor blocking, 50 pg / mL of mannose blocking peptide (LSBio Cat#: LS- E4304-500) is prepared in serum-free media, and BMDCs are treated for 30 min prior to nanoparticle treatment to ensure binding. For antigen presentation studies, BMDCs were plated at 10,000 cells / well in 96-well U-bottom plates and treated in complete Lutz media containing lipopolysaccharide (LPS) with formulations for 6 hr. OVA-specific T-cells (OTs) were isolated from 6-10-week-old male, OTI transgenic mice, by collecting spleens and magnetically sorting for CD8+T cells. Then, OTI T cells were CFSE-labelled and co-cultured with BMDCs for 3 days in complete IMDM media (Gibco). Cells were then washed, stained for viability, and fixed with 2% PFA prior to flow cytometry. Proliferation was demonstrated by a decrease in fluorescence intensity, due to the CFSE stain becoming diluted as T cells divide. To probe antigen processing, RAW 264.7 macrophages were treated with 100 ug / mL polymer and 10 ug / mL fluorescent OVA. A high concentration of polymer, 100 mg / mL, encapsulated a mixture of DQ-OVA and OVA647 (1 : 1 mass ratio) at a total concentration of 10 mg / mL. The concentrated solution was diluted in complete DMEM and RAW macrophages plated at a concentration of 10k cells / well in a 96-well plate were treated overnight with 100 uL media. Media was removed the next day, cells were washed with PBS, then stained with DAPI (diluted 1 :5000) for 5 minutes. The cells were washed again, PBS was added, then 96-well plates were imaged on a Leica microscope using the DAPI channel for cell nuclei, FITC channel for DQ- OVA and APC channel for OVA647 visualization.11. In-vitro immune stimulation using NF-kB reporter line

[0258] RAW -Blue macrophages were plated at a concentration of 100k cells / well in 96- well plates for overnight incubation with CpG ODN 1826 and polymers. CpG was either delivered free, encapsulated, or admixed with polymer formulations. For encapsulated formulations, polymers (10 mg / mL) were fully dissolved alongside CpG (500 pg / mL) in cold PBS, then the solution was warmed to room temperature. Admixed formulations had polymers assembled at 100 mg / mL, then diluted lOOx in 200 pg / mL CpG. All formulations were then diluted to equivalent concentrations of CpG in RAW-Blue media, and cells were treated with 100 pL of this suspension. NF-kB activation was measured the following day by collecting 20pL supernatant, mixing with 180 pL QUANTLBlue solution (Invivogen) and ODeso was measured with a plate reader.12. Biodistribution upon hock and intravenous injection using in-vivo imaging system (IVIS)

[0259] 5 pg of fluorescent OVA-647 was encapsulated in PSs (100 pg) as above or delivered unencapsulated in 100 pL saline into all 4 hocks (intradermal) of healthy C57BL / 6 (Charles River, 6 weeks old, n=2) male mice. For tail vein (intravenous, i.v.) injections, encapsulated OVA-647 was similarly administered (n=2). For lymph node collection, animals were sacrificed after 4 hr for harvesting all 4 pairs of hock-draining lymph nodes: axillary, brachial, inguinal, and popliteal. Lymph nodes were immediately stored in PBS, then arranged onto a black background for imaging on an IVIS Spectrum (Perkin Elmer) for OVA fluorescence. For i.v. administration, livers were harvested after 3 hours and OVA fluorescence quantified using IVIS Spectrum. For tumor distribution, subcutaneous (rear flank, same side as tumor) and tail vein injections were similarly conducted in MCF-7 tumor-bearing nude mice (Jackson Laboratory, n=2 or 1 if untreated), using fluorescently labelled (AF-647) polymer. Liver, spleen, heart, lungs, thymus, kidney, pancreas and tumor was harvested 3 hours post i.v. and 24 hours post s.c. injection and polymer fluorescence visualized on IVIS Spectrum.13. OVA subunit vaccination

[0260] 6-week-old, female, C57BL / 6 (Charles River) mice were vaccinated intradermally in all four hocks with 100 pL total of nanoparticle formulations. Besides the saline treatment, all mice were treated with 10 pg OVA adjuvanted with 20 pg CpG ODN 1826, which was admixed post-encapsulation (Invivogen). OVA was first encapsulated into the polymersomes at high concentration (100 mg / mL polymer, 10 mg / mL protein) in PBS, then diluted lOOx into sterile saline containing 200 ug / mL CpG. 100 pg polymer was delivered for nanoparticle treatments. Blood was drawn weekly via submandibular bleeding, and sera was collected and stored at -20°C. All mice were boosted on day 21 following prime vaccination and sacrificed at day 28. Draining lymph nodes (dLNs) and spleens were collected for analyzing the OVA- specific T cell response using fluorescent probes and restimulation. After harvesting, dLNs were treated with collagenase IV and D for 30 min at 37°C and passed through a 70 pm filter with complete IMDM media. Spleens were passed through a 70 pm filter, treated with ACK lysis buffer (Thermo Fisher) for 5 min, and then quenched with complete IMDM. Single-cellsuspensions were counted, and all samples were brought to a concentration of 20 million cells / mL in complete IMDM. For MHCI / SIINFEKL (SEQ ID NO:9) tetramer (NIH, APC- labelled) staining, isolated cells were plated at 1 million cells / well, stained following literature protocols and analyzed with flow cytometry (LSR Fortessa). Briefly, lymphocytes were plated on 96-well U-bottom plates, washed with PBS, and stained for viability. To prevent membrane turnover of the T cell receptor, cells were then treated with 50 nM of dasatinib for 30 min at 37°C, washed, then stained with tetramer for 15 min at room temperature. All other surface stains were done after, along with cell fixation following standard literature procedures. Tetramer+CD8+T-cells were identified on bulk (all live, TCRb+CD8+cells) and antigen experienced CD44+CD8+T-cells. 6-hour restimulation experiments were conducted by plating 2 million cells / well, followed by the addition of 100 pL complete IMDM containing either saline (“unstimulated” condition), SIINFEKL (SEQ ID NO:9) peptide (Invivogen, 1 pg / mL, “CD8-stimulated” condition) or a high concentration of endotoxin-free OVA (1 mg / mL, “CD4- stimulated” condition). Pooled samples stimulated with PMA / Ionomycin were used as a positive control. After 6 hr, cells were isolated, stained for viability, surface markers (CD4 and CD8), and intracellular cytokines and markers (IFNy, TNFa, IL-2 and CD3s) following standard protocols. Cells were then analyzed by flow cytometry (LSR Fortessa). 6-hour restimulation studies are plotted as stimulated minus unstimulated response. For whole-protein restimulation studies, 500,000 cells / well were plated in 96-well U-bottom plates and stimulated with IMDM containing 100 pg / mL OVA. Cells were centrifuged after 3 days, and media was collected for cytokine analysis using Legendplex (Murine Th 12-plex, Biolegend) following the manufacturer’s protocol (Novocyte Penteon flow cytometer). Serum antibodies (total IgG) were detected using direct enzyme-linked immunosorbent assay (ELISA) on Coming 9018 plates coated overnight with 10 pg / mL OVA in carbonate / bicarbonate buffer (pH 9.8). Sera were diluted in lx casein buffer as 10-fold serial dilutions (1 :102-107), incubated on coated plates and standard ELISA procedures were followed. Absorbance was read at 450 nm (characteristic for TMB buffer) and 570 nm (background) on a plate reader. Total IgG was reported as area under the curve (AUC) for the absorbance (A450-A570) vs. dilution curve of sera taken at 2 and 4 weeks. It should be noted that panels in the figures represent separate ELISAs, so AUC values can only be compared within one panel (i.e., between treatment groups), and not between panels (such as Fig. 3., d and e are not comparable to each other).14. OVA prophylactic inverse vaccination

[0261] In a murine, prophylactic allergy model, nanoparticles (either with or without protein antigen, OVA) were administered intravenously weekly for two weeks. 1 week post therapy, mice were sensitized using OVA mixed in a 1 : 1 (v / v) ratio with Imject Alum solution (Thermo Fisher Scientific). Mice were sensitized through two intraperitoneal injections set one week apart with 100 pg of alum / OVA. A week after the second sensitization, mice were challenged for four consecutive days with 25 pg of OVA grade V in PBS through intratracheal instillations. The submandibular vein was sampled through lancet puncture, and blood plasma was isolated through centrifugation at 20,000g in a tabletop centrifuge. Mice were euthanized three days after the last challenge to assess the extent of allergic airway inflammation. Bronchoalveolar lavage fluid (BALF) was isolated by cannulating the trachea and washing the lungs with PBS for a total recovery of 3 mL. Perfused mouse lungs were mechanically dissociated and digested with Collagenase IV and Collagenase D in 3 mL DMEM for 1 hr. After red blood cell lysis, samples were then washed, filtered, and counted prior to flow cytometry analysis. For immunophenotyping, 5 x 105cells were suspended in 50 pL buffer (PBS, 2% FBS), blocked using anti-CD 16 / 32, and stained with surface antibodies. For intracellular staining, cells were fixed and permeabilized with the FoxP3 / Transcription Factor Staining Buffer Set (Invitrogen) following the manufacturer’s instructions. Samples were analyzed using an LSR Fortessa (BD Biosciences) or Cytek Aurora (Cytek Biosciences) and the data analyzed using FlowJo software. To determine total IgE-levels in the plasma, the inventors utilized an anti-IgE ELISA (Thermo Fisher Scientific) following the manufacturer instructions.15. siRNA-mediated gene knockdown in vitro and in-vivo

[0262] MCF-7 cells, a human breast cancer line, were plated at 5xl04cells per well in a 48-well plate and incubated for 24 hr. Cell media was removed, and the cells were washed using PBS. Various doses of siRNA (VEGF-A or Bcl-2 targeted siRNA, purchased through IDT, sequences listed above) and polymer or Lipofectamine (Thermo Fisher Scientific) complex were suspended in RPMI media with 2% fetal bovine serum (FBS) and cells were treated for 6 hr. Then, the cells were washed using PBS and incubated with complete media for 48 hr. After incubation, the cells were harvested for RNA isolation (RNEasy Mini Kit, Qiagen) following manufacturer’ s protocols, and target mRNA expression was evaluated using standard RT-PCR techniques with commercially available primers for human VEGF-A and Bcl-2 (IDT, sequences above). cDNA (Superscript III, Invivogen) was synthesized using manufacturerprotocols and PCR was conducted using an annealing temperature 5°C below the melting temperature for primers (usually ~55°C) and run for 26 amplification cycles to prevent signal saturation.

[0263] For in-vivo measurements, nude mice were inoculated with 2 million MCF-7 cancer cells in a Matrigel extracellular matrix. Tumors were allowed to grow for 10 days to a size of 50-70 mm3before starting intratumoral treatment of siVEGFA and siBcl2 on days 11 and 13. Mice were sacrificed at day 14, and tumors were harvest and stored at -80°C. For measuring mRNA knockdown, RT-PCR protocols were used as outlined above, after tumors were sectioned - roughly 20% of tumor was excised, while the rest remained frozen. For measuring protein, similarly sized tumor sections were taken from siRNA treated animals, and protein was extracted using RIPA buffer containing protease inhibitors (Pierce, Invivogen) with light sonication for Western blot. Total protein concentration was measured using a BCA assay, and equivalent amounts of protein (50 pg) were loaded onto a 4-20% PAGE gel under reducing, detergent-containing conditions (TGS buffer, 6x reducing loading dye). Separated protein was electrophoretically transferred onto a PVDF membrane and then blocked with 5% skim milk in PBS with 0.1% Tween20 (PBS-T) for Ih at room temperature. Subsequent staining was done with rabbit antibodies in 2% milk (in PBS-T) against human VEGFA (E9X8Q, dilute 1 : 1000), Bcl2 (D17C4, diluted 1 : 1000) and GAPDH (D16H11, diluted 1 : 10,000, loading control) overnight at 4°C, washed with PBS-T, and then stained with secondary antibody -HRP conjugate for Ih at room temperature. Blots were washed and imaged using a Bio Rad blot chemiluminescence imager following treatment with Western blot substrate (BioRad).16. siRNA cancer therapy

[0264] For in-vivo cancer studies probing RNA interference of VEGF-A and Bcl2, nude mice were inoculated subcutaneously behind the shoulder with 2 million MCF-7 cancer cells (cultured in DMEM media) in a Matrigel extracellular matrix. Tumors were allowed to grow for 10 days to a size of 50-70 mm3before starting treatment. Then, mice were treated intratumorally on days 11,13,15,17 and 19 using nanoparticle-encapsulated or free siRNA (either si VEGF-A or siBcl2) formulations. 40 pg of siRNA and 200 pg of polymer were administered. Tumor sizes were measured until day 35, and mice were sacrificed when tumors reached a size of 600 mm3. For LNP benchmarking studies, 20 ug of siRNA was used with 100 ug of either polymer or LNP formulation. Mice were treated every 5 days starting at day 11, and sacrificed when tumors grew to 600 mm3.17. Lipid nanoparticle (LNP) formulation for in-vivo cancer therapy

[0265] To serve as an in-vivo control for siRNA delivery, the inventors formulated LNPs to mimic those of Onpattro® (Alnylam® Pharmaceuticals), following published structures and weight ratios of the relevant components. Stock solutions of DLin-MC3-DMA, DSPC, cholesterol and PEG2k-DMG (Broadpharm) were prepared in ethanol at concentrations of 157, 15.7, 30.4 and 4.72 mM respectively. When ready for use, stock solutions were mixed at a volumetric ratio of 5: 10:15:5, to give the prescribed molar ratio of 50: 10:38.5: 1.5 (ionizable lipid: cationic lipid: cholesterol: PEG-lipid). Since the LNPs are only needed in small batches, dilution and rapid vortexing was chosen as the method for encapsulating RNA. LNPs were formed by diluting the EtOH mixed stock solution 1 : 15 in nuclease-free water (either with or without RNA) under vortexing. For loading efficiency quantification, LNPs are diluted to identical mass concentration with FAM-siRNA as for polymers. Size, reproducibility and loading efficiency was then quantified with similar analytical techniques as above (FIG. 19).18. Statistical Analyses

[0266] All statistical analyses were conducted using GraphPad Prism 10. In-vitro and in- vivo statistics were based on one-way or two-way (when appropriate) analysis of variance (ANOVA) comparisons or Kruskal-Wallis tests for nonparametric data. Welch’ s ANOVA was used when assumptions could not be made regarding standard deviations of samples. All comparisons are shown in the figures, and multiple comparisons are corrected for using posttests as described in the figures. Exact p-values are denoted in the figures and may also appear as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant, unless otherwise noted. Survival analyses in Fig. 6 was determined using a log-rank Mantel-Cox test separately conducted for the denoted comparisons.* * *

[0267] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Claims

WHAT IS CLAIMED IS:

1. An amphiphilic block copolymer (BCP) comprising a hydrophilic block comprising hydrophilic monomers and a hydrophobic membrane-forming block comprising hydrophobic monomers; wherein the lower critical solution temperature (LCST) of the copolymer is 0 °C - 22 °C.

2. The BCP of claim 1, wherein the LCST of the copolymer is 0 °C - 17 °C3. The BCP of claim 1, wherein the BCP is biocompatible.

4. The BCP of claim 1 or 3, wherein the hydrophilic weight fraction is 10-25%.

5. The BCP of any one of claims 1-4, wherein the copolymer comprises a hydrophobic membrane-forming block length of 120-175 monomers and a hydrophilic block length of 15- 70 monomers.

6. The BCP of any one of claims 1-5, wherein the BCP comprises an end modification.

7. The BCP of claim 6, wherein the end modification comprises mannose, mannose acrylate, a hydrophilic monomer, N-acetyl glucosamine, N-acetyl galactosamine, or a targeting agent.

8. The BCP of claim 7, wherein the end modification comprises mannose.

9. The BCP of claim 7 or 8, wherein the end modification comprises N-acetyl glucosamine.

10. The BCP of any one of claims 7-9, wherein the end modification comprises a N-acetyl galactosamine.

11. The BCP of any one of claims 1-10, wherein the hydrophilic monomers are uncharged.

12. The BCP of any one of claims 1-10, wherein the hydrophilic monomers are charged.

13. The BCP of any one of claims 1-12, wherein the hydrophobic monomers comprise diethylene glycol ethyl ether acrylate (DEGEA).

14. The BCP of any one of claims 1-13, wherein the hydrophilic monomers comprise 2- hydroxyethyl acrylate (ELEA).

15. The BCP of any one of claims 1-14 wherein the hydrophilic monomers comprise dimethyl aminoethyl acrylate (DMAEA), polyethylene glycol (PEG), poly(hydroxypropyl methacrylamide), poly(hydroxyethyl methacrylamide), and / or poly(oxazoline).

16. The BCP of any one of claims 1-15, wherein the hydrophilic monomers comprise trimethylamino ethyl acrylate (TMAEA).

17. The BCP of any one of claims 1-16, wherein the hydrophilic monomers comprise mannose acrylate, N-acetyl glucosamine, or N-acetyl galactosamine.

18. The BCP of any one of claims 1-17, wherein the hydrophilic monomers comprise mannose acrylate and HEA.

19. The BCP of any one of claims 1-18 wherein the hydrophilic block comprises cationic moieties.

20. The BCP of any one of claims 1-19, wherein the BCP is lyophilized.

21. The BCP of any one of claims 1-20, wherein the BCP comprises a polymer of formula (I):wherein Xi and Yi are independently an integer value ranging from 1-200 and Ri and R2 are each independently selected from an end modification or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

22. The BCP of claim 21, wherein the BCP comprises a polymer of formula (la):wherein Xi and Yi are independently an integer value ranging from 1-200 and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

23. The BCP of any one of claims 1-20, wherein the BCP comprises a polymer of formula (II):wherein X2 and Y2 are independently an integer value ranging from 1-200 and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

24. The BCP of claim 23, wherein the BCP comprises a polymer of formula (Ila):wherein X2 and Y2 are independently an integer value ranging from 1-200 and R2 is selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

25. The BCP of claim 21 or 23, wherein Ri comprises H or mannose.

26. The BCP of any one of claims 1-25, wherein the BCP further comprises 2- (hexamethyleneimino)ethyl acrylate monomers.

27. The BCP of any one of claims 1-25, wherein the BCP further comprises 2,2- (dimethylamino)ethyl acrylate monomers.

28. The BCP of any one of claims 1-25, wherein the BCP further comprises 2,2- (diethylamino)ethyl acrylate monomers.

29. The BCP of claim 26, wherein the BCP comprises a polymer block comprising 2- (hexamethyleneimino)ethyl acrylate monomers.

30. The BCP of claim 26 or 29, wherein the BCP comprises a polymer of formula (III) or (IV):wherein W3, W4, Y3, and Y4 are independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is 1- X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

31. The BCP of claim 30, wherein the BCP comprises a polymer of formula (Illa) or (IVa):wherein W3, W4, Y3, and Y4 are independently selected from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and R2 is an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

32. The BCP of claim 26 or 29, wherein the BCP comprises a polymer of formula (VI) or(VII):wherein W3, W4, Y3, and Y4 are independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is 1- X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, amannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

33. The BCP of claim 30, wherein the BCP comprises a polymer of formula (Via) or (Vila):wherein W3, W4, Y3, and Y4 are independently selected from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and R2 is an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

34. The BCP of claim 26 or 29, wherein the BCP comprises a polymer of formula (VIII) or(IX):wherein W3, W4, Y3, and Y4 are independently an integer value ranging from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is 1- X4; and Ri and R2 are each independently selected from an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between Ri and the BCP and / or R2 and the BCP.

35. The BCP of claim 30, wherein the BCP comprises a polymer of formula (Villa) or(IXa):wherein W3, W4, Y3, and Y4 are independently selected from 1-200; X3 and X4 are each independently selected from a decimal number between 0 and 1; Z3 is I-X3, Z4 is I-X4; and R2is an end modification, a hydrogen, a mannose, or a nitrile, with a disulfide junction optionally included between R2 and the BCP.

36. A polymersome comprising at least one amphiphilic BCP of any one of claims 1-31.

37. The polymersome of claim 36, wherein the polymersome is 50-200 nm in diameter.

38. The polymersome of claim 36 or 37, wherein the polymersome has not undergone solvent or size exclusion purification.

39. The polymersome of any one of claims 36-38, wherein the polymersome comprises a payload.

40. The polymersome of claim 39, wherein the payload comprises a protein, an adjuvant, a nucleic acid, and / or a therapeutic molecule.

41. The polymersome of claim 40, wherein the payload comprises a protein antigen, a siRNA, mRNA, subunit vaccine, tolerogenic vaccine, and / or a siRNA-based cancer therapy.

42. The polymersome of any one of claims 36-41, wherein the polymersome comprises at least two amphiphilic BCPs, wherein each amphiphilic BCP is independently selected from an amphiphilic BCP of any one of claims 1-30.

43. The polymersome of any one of claims 36-42, wherein a BCP:payload ratio is 1 :5-l :20.

44. The polymersomes of any one of claims 36-43, wherein the polymersome comprises 50-200 pg of payload.

45. The polymersome of any one of claims 36-43, wherein the polymersome is unfiltered.

46. The polymersome of any one of claims 36-45, wherein the RH (hydrodynamic radii) is 75-200 nm.

47. The polymersome of any one of claims 36-46, wherein the RG (radius of gyration) is 75-200 nm.

48. The polymersome of any one of claims 36-47, wherein the form factor (RG / RH) is 0.7- 1.3.

49. The polymersome of any one of claims 36-45, wherein the polymersome is at a temperature of more than 18 °C.

50. A composition comprising at least one amphiphilic BCP of any one of claims 1-31 or the polymersome of any one of claims 36-49.

51. The composition of claim 50, wherein the composition comprises at least a first and a second amphiphilic BCP, wherein each of the first and second BCP is independently selected from an amphiphilic BCP of any one of claims 1-31.

52. The composition of claim 51, wherein the composition comprises:a) a first amphiphilic BCP comprising a hydrophilic block and a membrane-forming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises TMAEA; and b) a second amphiphilic BCP comprising a hydrophilic block and a membrane-forming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises HEA.

53. The composition of claim 51 or 52, wherein the composition further comprises a third amphiphilic BCP wherein the third BCP is independently selected from an amphiphilic BCP of any one of claims 1-31.

54. The composition of claim 53, wherein the third BCP comprises a hydrophilic block and a membrane-forming block; wherein the lower critical solution temperature of the copolymer is 0 °C - 22 °C, and wherein the hydrophilic block comprises HEA and wherein the BCP is end modified with an HEA monomer.

55. The composition of claim 52, wherein a weight percentage of the first BCP is 33%, based on a total weight of the composition or a combined weight of the first amphiphilic BCP and the second amphiphilic BCP.

56. The composition of any one of claims 51-55, wherein a weight percentage of the second BCP is 0, 10, or 33%, based on a total weight of the composition or a combined weight of the first amphiphilic BCP and the second amphiphilic BCP.

57. The composition of any one of claims 51-56, wherein each of the first, second, and / or third BCP are end modified.

58. The composition of claim 57, wherein the end modification comprises a mannose, mannose acrylate, a hydrophilic monomer, N-acetyl glucosamine, N-acetyl galactosamine, or a targeting agent.

59. The composition of claim 58, wherein the end modification comprises mannose.

60. The composition of claim 58 or 59, wherein the end modification comprises N-acetyl glucosamine.

61. The composition of any one of claims 58-60, wherein the end modification comprises a N-acetyl galactosamine.

62. The composition of any one of claims 51-61, wherein at least one of the first and second BCPs comprises a hydrophilic block, wherein the hydrophilic block comprises a charged monomer and wherein at least one of the first and second BCPs comprises a hydrophilic block, wherein the hydrophilic block comprises an uncharged monomer.

63. The composition of any one of claims 50-62, wherein the composition is at temperature of greater than 16 °C.

64. The composition of any one of claims 50-63, wherein the composition is at temperature of greater than 0 °C and less than 20 °C.

65. The composition of any one of claims 50-64, wherein the BCP is soluble in an aqueous solution at a temperature of less than 15 °C.

66. The composition of any one of claims 50-65, wherein the composition comprises an aqueous solution.

67. The composition of any one of claims 50-66, wherein the composition comprises physiological salt concentrations, saline, and / or PBS (phosphate buffered saline).

68. The composition of any one of claims 50-67, wherein a concentration of the BCP in the composition is 100 mg / mL or less.

69. The composition of any one of claims 50-68, wherein the composition comprises an immune shielding polymer (ISP) and wherein the ISP comprises a polymer that irreversibly binds to lysine residues.

70. The composition of claim 69, wherein the ISP has a molecular weight of 5-40 kDa.

71. The composition of claim 69 or 70, wherein the ISP comprises an amine-reactive chemical moiety.

72. The composition of claim 71, wherein the amine-reactive chemical moiety comprises a N-hydroxysuccinimide (NHS) ester.

73. The composition of any one of claims 69-72, wherein the ISP comprises one or more of (polyethylene glycol), poly(oligo(ethylene glycol) methyl ether methacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxyethyl methacrylamide), poly(oxazoline), dextran derivatives, and hyaluronic acid derivatives.

74. The composition of any one of claims 69-73, wherein the ISP further comprises a conjugated sugar moiety.

75. The composition of claim 74, wherein the sugar moiety comprises mannose, glucose, galactose, N-acetyl glucosamine, and / or N-acetyl galactosamine.

76. The composition of any one of claims 69-75, wherein the ISP is bioinert or pro- tolerogenic.

77. The composition of any one of claims 69-75, wherein the ISP comprises a compound of formula V or Va:wherein A is selected from a decimal number between 0 and 1; B is 1-A; and R3 is selected from hydrogen, mannose, N-acetyl glucosamine, or N-acetyl galactosamine.

78. The composition of any one of claims 69-75, wherein the ISP comprises a random copolymer.

79. The composition of any one of claims 50-78, wherein the composition comprises a payload.

80. The composition of claim 79, wherein the payload comprises a protein, an adjuvant, a nucleic acid, and / or a therapeutic molecule.

81. The composition of claim 80, wherein the payload comprises a protein antigen, a siRNA, mRNA, subunit vaccine, tolerogenic vaccine, and / or a siRNA-based cancer therapy.

82. The composition of any one of claims 50-81, wherein at least 75% of the payload is encapsulated when the composition is at a temperature greater than the LCST.

83. The composition of any one of claims 69-82, wherein a molar ratio of ISP to payload is 2: 1.

84. The composition of any one of claims 50-83, wherein the composition comprises a quenching agent.

85. The composition of claim 84, wherein the quenching agent is non-toxic and biocompatible.

86. The composition of claim 84 or 85, wherein the quenching agent comprises a primary amine.

87. The composition of claim 86, wherein the quenching agent comprises lysine, glycine, or ethanolamine.

88. The composition of any one of claims 69-87, wherein a molar ratio of the quenching agent to the ISP is 2: 1.

89. The composition of any one of claims 50-88, wherein the composition is unfiltered.

90. A method for making a block copolymer (BCP) comprising mixing hydrophilic monomers with hydrophobic monomers under conditions that allow for the polymerization of the hydrophilic and hydrophobic monomers.

91. A method for making a block copolymer (BCP) comprising mixing hydrophilic polymer blocks with hydrophobic polymer blocks under conditions that allow for the polymerization of the hydrophilic and hydrophobic polymer blocks.

92. A method for making a polymersome comprising incubating the BCP of any one of claims 1-31 or the composition of any one of claims 50-89 at a temperature greater than 15 °C or greater than the LCST to allow for the formation of polymersomes.

93. The method of claim 93, wherein the method excludes contacting the BCP or the composition with an organic solvent.

94. The method of claim 92 or 93, wherein the method excludes size exclusion purification and / or membrane filtration.

95. A polymersome made by the method of any one of claims 92-94.

96. A method for treating a disease in a subject, the method comprising administering the polymersome of any one of claims 36-49 or 95 or the composition of any one of claims 50-89.

97. The method of claim 96, wherein the disease comprises an allergy or an allergic immune response.

98. A method comprising administering the polymersome of any one of claims 36-49 or 95 or the composition of any one of claims 50-89 to a subject.

99. The method of any one of claims 96-98, wherein the payload comprises an oncogene inhibitor(s).

100. The method of claim 99, wherein the oncogene comprises Bcl-2 and / or VEGFA.

101. The method of any one of claims 96-100, wherein the payload comprises a Atf4, YTHDF2 inhibitor.

102. The method of any one of claims 96-101, wherein the payload comprises a siRNA, miRNA, or antisense oligonucleotide (ASO).

103. The method of any one of claims 96-102, wherein the disease comprises cancer.

104. The method of claim 103, wherein the cancer comprises breast cancer.

105. A method for inducing tolerance in a subject, the method comprising administering the polymersome of any one of claims 36-49 or 95 or the composition of any one of claims 50-89.

106. A method for vaccinating a subject, the method comprising administering the polymersome of any one of claims 36-49 or 95 or the composition of any one of claims 50-89.

107. The method of any one of claims 96-106, wherein the subject has a disease and / or has been diagnosed with a disease.

108. The method of any one of claims 96-106, wherein the subject has not been diagnosed with a disease.

109. The method of any one of claims 96-108, wherein the polymersome or composition is administered intradermally, subcutaneously, intramuscularly, intratumorally, intralesional, intraocularly, orally, rectally, nasally, pulmonarily, or intravesicularly.

110. The method of any one of claims 96-109, wherein the subject is a human subject.

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