Block copolymer nanoparticles for sustained drug delivery

Abstract

Disclosed herein are POEGMA-based block copolymers that have phase transition and self-assembly properties. The disclosed block copolymers can take advantage of these properties to form particles that can effectively encapsulate and deliver drugs. An example block copolymer includes a first block that includes POEGMA with ethylene glycol side chains of 2 monomers, 3 monomers, or combinations of both; and a second block that includes POEGMA with ethylene glycol side chains of 1 monomer, 2 monomers, or combinations of both. Also disclosed herein are compositions that include the block copolymers, methods of treating a disease or disorder, and methods of delivering a drug.

Description

Attorney Docket No.028193-0045-WO01 BLOCK COPOLYMER NANOPARTICLES FOR SUSTAINED DRUG DELIVERY AND METHODS OF MAKING AND USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 543,143 filed on October 9, 2023, and U.S. Provisional Patent Application No.63 / 678,955 filed on August 2, 2024, both of which are incorporated fully herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Federal Grant no. R01 DK124276-04 awarded by the National Institutes of Diabetes and Digestive and Kidney Disease / National Institutes of Health (NIH / NIDDK). The Federal Government has certain rights to this invention. TECHNICAL FIELD

[0003] This disclosure relates to block copolymers that can self-assemble into nanoparticles and can effectively deliver drugs for biomedical applications, such as drug delivery. INTRODUCTION

[0004] Polyethylene glycol (PEG) is the most common hydrophilic block in self-assembled block copolymer nanoparticles (NPs), as the PEG corona provides “stealth” behavior that imparts a long plasma residence time to the NPs. However, the prevalence of pre-existing anti- PEG antibodies in more than 60% of the human population raises several concerns, including opsonization of the PEG NPs, leading to their premature clearance through the reticuloendothelial system and the incidence of rare—but potentially fatal—anaphylactoid reactions. Furthermore, the core-forming block in these NPs can be composed of a more hydrophobic or relatively less hydrophilic polymer such as polypropylene oxide (PPO), polystyrene oxide (PSO), polylactic acid (PAA), etc., some of which are poorly degradable, or are potentially toxic. Thus, biomaterials including amphiphilic block copolymers that can overcome these foregoing issues would be beneficial to the field of drug delivery.Attorney Docket No.028193-0045-WO01 SUMMARY

[0005] In one aspect, disclosed are block copolymers comprising a first block (A), wherein the first block comprises recurring units of formula (I)wherein x is 2 or 3; and a second block (B), wherein the second block comprises recurring units of formula (II)wherein y is 1 or 2.

[0006] In another aspect, disclosed are compositions comprising a plurality of block copolymers as disclosed herein self-assembled into a particle; and a drug encapsulated within the particle.

[0007] In another aspect, disclosed are methods of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition as disclosed herein.

[0008] In another aspect, disclosed are methods of delivering a drug to a subject in need thereof, the method comprising systemically or locally administering to the subject a composition as disclosed herein, wherein the drug is released from the particle following administration.

[0009] In another aspect, disclosed are methods of delivering a scaffold to a subject in need thereof, the method comprising administering a block copolymer as disclosed herein to aAttorney Docket No.028193-0045-WO01 location of the subject, whereupon administration to the location, the block copolymer exhibits phase transition behavior defined by a Tt, the block copolymer forming a porous network above the Tt. BRIEF DESCRIPTION OF THE DRAWING

[0010] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] FIG.1 is a schematic of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) copolymers as disclosed herein.

[0012] FIG.2A is a schematic design of POEGMA diblock copolymers including EG2 and EG3 side chains in the respective blocks that can self-assemble into nano-scale particles in aqueous media.

[0013] FIG.2B is a plot showing size exclusion chromatography (SEC) chromatograms of example diblocks to determine Mn, Mwand polydispersity index (PDI).

[0014] FIG.2C is a table showing a summary for Mn, Mwand PDI of example copolymers determined by SEC-MALS. The ID of the diblock POEGMA represents degree of polymerization (DP) of individual block determined by Mndivided by molecular weights of the respective monomer. Each polymer is given a random alphabet letter as a label for simplicity.

[0015] FIG.3A is a plot of optical density for polymer D: EG3167-EG285at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines. Sharp increase in absorbance at 42 °C indicates the cloud point / transition temperature (Tt) for this polymer.

[0016] FIG.3B is a plot showing change in size distribution of polymer D from 25 °C (below CMT) to 37°C (above CMT but below Tt) at low (12.5 μM) and high concentrations (200 μM).

[0017] FIG.3C is a bar chart of hydrodynamic diameter of polymer D for a wide range of relevant temperatures.

[0018] FIG.3D is a plot of small-angle X-ray scattering (SAXS) of polymer D at 5, 2, and 1 mg / ml concentrations at 37 °C: Zero slope of Intensity (I) vs scattering vector (q) at low q showing globular nanoparticles.

[0019] FIG.3E is a Guinier plot (linear region of ln(I) vs. q2) to calculate Rgof the self- assembled NPs using the formula Rg= sqrt(-3*slope), resulting in Rg= 16.8 nm for polymer D atAttorney Docket No.028193-0045-WO01

[0020] FIG.3F is a Kratky plot for polymer D at 37 °C where the sharp peak is validating the globular self-assembly.

[0021] FIG.3G is a cryo-electron microscopy (EM) image of polymer D at 37 °C. Electron contrast for POEGMA is known to be extremely low due to its highly hydrated structure but circles outline nanoparticles (NPs).

[0022] FIG.3H is a plot showing boundary conditions for self-assembly of example block- POEGMAs: Contribution from relative length of each block should be at least 25% to impart enough amphiphilicity to the diblock for its self-assembly in aqueous media.

[0023] FIG.3I is a plot showing the effect of concentration on Ttfor simple diblock coPOEGMAs: The concentration dependence on Ttof polymers that do not exhibit self- assembly is more prominent than the ones that self-assemble.

[0024] FIG.4A is a schematic of a complex diblock where hydrophobicity of the hydrophobic block is increased by doping in a more hydrophobic monomer (EG1), polymer F: EG3119- EG2120 / EG167.

[0025] FIG.4B is a plot of optical density for polymer F at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines. Sharp increase in absorbance at 44.5 °C indicates the Ttfor polymer F.

[0026] FIG.4C is a plot of size distribution at room temperature (RT) and body temperature for polymer F at two different concentrations.

[0027] FIG.4D is a bar chart of hydrodynamic diameter of polymer F for a wide range of relevant temperatures.

[0028] FIG.4E is a plot of SAXS of F at 5, 2, and 1 mg / ml concentrations at 25 °C: Zero slope of Intensity (I) vs scattering vector (q) at low q showing globular nanoparticles.

[0029] FIG.4F is a Guinier plot (linear region of ln(I) vs. q2) to calculate Rgof the self- assembled NPs using the formula Rg= sqrt(-3*slope), resulting in Rg= 12.6 nm for polymer F at 25 °C.

[0030] FIG.4G is a Kratky plot for polymer F at 25 °C where the sharp peak is validating the globular self-assembly.

[0031] FIG.4H is a micrograph image of polymer F in the presence of ThT dye showing its phase transition behavior above Tt.

[0032] FIG.4I is a cryo-EM image of polymer F at RT. Electron contrast for POEGMA is known to be extremely low due to its highly hydrated structure but circles outline NPs.Attorney Docket No.028193-0045-WO01

[0033] FIG.4J is an image of an agarose gel showing free doxorubicin (Dox) (left) and Dox encapsulated in polymer F (right).

[0034] FIG.4K is a plot showing in vitro potency of free Dox vs encapsulated Dox on MC38 cell line after 48 h incubation with the drug.

[0035] FIG.5A is a schematic of a complex diblock where hydrophilicity of the hydrophilic block is decreased by doping in a more hydrophobic monomer (EG2), polymer rB-2: EG311 / EG264- EG28 / EG145.

[0036] FIG.5B is a plot of optical density for polymer rB-2 at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines.

[0037] FIG.5C is a plot of size distribution at RT for polymer rB-2 at two different concentrations.

[0038] FIG.5D is a bar chart of hydrodynamic diameter for polymer rB-2 below Ttat low (12.5 μM) and high concentrations (200 μM).

[0039] FIG.5E is a plot of SAXS of rB-2 at 5, 2, and 1 mg / ml concentrations at 25 °C showing cylindrical self-assembly.

[0040] FIG.5F is a Guinier plot (linear region of ln(I) vs. q2) to calculate Rgof the self- assembled NPs using the formula Rg= sqrt(-3*slope), resulting in Rg= 17.4 nm for polymer rB-2 at 25 °C.

[0041] FIG.5G is a confocal micrograph image of rB-2 labelled with a green fluorophore imaged above Tt of the polymer.

[0042] FIG.5H is a plot of fluorescence recovery after photobleaching (FRAP) analysis of AF488 conjugated complex diblock polymer rB-2 vs simple diblock polymer A.

[0043] FIG.5I is a plot of FRAP analysis of AF488 conjugated complex diblock polymer rB-2 vs simple diblock polymer A above their Tt.

[0044] FIG.6A is a plot of optical density for triblock POEGMA 1 (T1) at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines.

[0045] FIG.6B is a bar chart of hydrodynamic radii for triblock T1 at 50 μM at 25 °C and 32 °C.

[0046] FIG.6C is a plot of optical density for triblock POEGMA 2 (T2) at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines.Attorney Docket No.028193-0045-WO01

[0047] FIG.6D is a plot of optical density for triblock POEGMA 3 (T3) at various concentrations with change in temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines. DETAILED DESCRIPTION

[0048] Disclosed herein are POEGMA block copolymers designed to form non-immunogenic nanoparticles (NPs) that can be composed of only POEGMA. AB-type block polymers with varied monomer— OEGMA with EG1-3as sidechains— ratio and degree of polymerization (DP) were designed and synthesized. Hypothesizing that the EG3 monomer is slightly more hydrophilic than EG2 monomer, the diblock EG3-EG2 library was screened for self-assembling block constructs and characterized for their unique properties. Additionally, complexity was introduced into the diblock EG3-EG2 library by either making the hydrophobic block more hydrophobic or decreasing the hydrophilicity of the hydrophilic block. Overall, it is shown herein that the disclosed diblock POEGMA system is highly tunable in the range of polymeric materials and NPs that can be produced for a variety of drug delivery applications. 1. Definitions

[0049] 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. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0050] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0051] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should alsoAttorney Docket No.028193-0045-WO01 be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0052] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0053] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0054] The term “antigen” refers to a molecule capable of being bound by an antibody or a T cell receptor. The term “antigen” also encompasses T-cell epitopes. An antigen is additionally capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B-lymphocytes and / or T-lymphocytes. In some embodiments, the antigen contains or is linked to a Th cell epitope. An antigen can have one or more epitopes (B-epitopes and T-epitopes). Antigens may include polypeptides, polynucleotides, carbohydrates, lipids, small molecules, polymers, polymer conjugates, and combinations thereof. Antigens may also be mixtures of several individual antigens.

[0055] The term “antigenicity” refers to the ability of an antigen to specifically bind to a T cell receptor or antibody and includes the reactivity of an antigen toward pre-existing antibodies in a subject.Attorney Docket No.028193-0045-WO01

[0056] The term “critical micelle temperature” or “CMT” refers to the minimum temperature required for self-assembly, or micelle formation of a thermo-responsive material (e.g., disclosed block copolymers).

[0057] The term “drug” refers to a substance that can act on a cell, virus, tissue, organ, organism, or the like, to create a change in the functioning of the cell, virus, tissue, organ, or organism. Examples of drugs include, but are not limited to, peptide-based drugs and chemotherapeutics. A drug is capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Drugs of the present disclosure also include prodrug forms of the agent.

[0058] The term “effective dosage” or “therapeutic dosage” or “therapeutically effective amount” or “effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results, to modulate a biological process, and / or treat a disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. A therapeutically effective amount is also one in which any toxic or detrimental effects of substance are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In reference to treating cancer, an effective or therapeutically effective amount can include an amount sufficient to, among other things, decrease or slow cancer cell growth.

[0059] The term “immunogenicity” refers to the ability of any antigen to induce an immune response and includes the intrinsic ability of an antigen to generate antibodies in a subject. As used herein, the terms “antigenicity” and “immunogenicity” refer to different aspects of the immune system and are not interchangeable.

[0060] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described block copolymers or compositions thereof, or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgous monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be ofAttorney Docket No.028193-0045-WO01 any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment.

[0061] The term “transition temperature” or “Tt” refers to the temperature at which the block copolymer changes from one state to another, for example, soluble to insoluble. For example, below the Tt, the conjugate may be highly soluble. Upon heating above the transition temperature, for example, the conjugate may aggregate, forming a separate phase. The Ttcan also be defined as the inflection point of temperature versus the optical density curve and calculated as the maximum of the first derivative using, e.g., GraphPad Prism 8.0 software.

[0062] The terms “treatment” or “treating” refer to the medical management of a patient with the intent to heal, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0063] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. 2. Block Copolymers

[0064] Provided herein are POEGMA-based block copolymers having structures that can take advantage of phase transition and self-assembly properties to effectively deliver drugs. POEGMA has a poly(methacrylate) backbone and a plurality of side chains covalently attached to the backbone. The side chains are oligomers of ethylene glycol (EG). The oligoethylene glycol side chains may include a first end and a second end. The first end may be attached to the backbone and the second end may include a capping moiety. The capping moiety may be hydroxyl or C1-C3alkyl. In some embodiments, the capping moiety is a C1-C3alkyl. The length ofAttorney Docket No.028193-0045-WO01 each side chain is dependent on the monomers used to synthesize the block copolymer. The block copolymer disclosed herein takes advantage of differing the length of these side chains between the different blocks to achieve a threshold difference in hydrophilicity / hydrophobicity between the blocks that can allow the block copolymer to self-assemble into particulate structures.

[0065] For example, the block copolymer can include two different polymer blocks – the first block can be referred to as an A block and the second block can be referred to as a B block. Each block includes POEGMA but with a different EG side chain length. The A block can include POEGMA having side chains of either 2 or 3 monomers of EG. In contrast, the B block can include POEGMA having side chains of either 1 or 2 monomers of EG. Surprisingly, it was found that a mere difference of 1 EG monomer between the different blocks can afford the block copolymer self-assembly properties.

[0066] The self-assembly ability of the block copolymer can be described as the block copolymer having a critical micelle temperature (CMT). The block copolymer can have a CMT of about 15 °C to about 45 °C, such as about 18 °C to about 42 °C, about 20 °C to about 40 °C, about 22 °C to about 37 °C, about 15 °C to about 35 °C, about 20 °C to about 30 °C, about 25 °C to about 45 °C, or about 35 °C to about 45 °C. In some embodiments, the block copolymer has a CMT of greater than 15 °C, greater than 20 °C, greater than 25 °C, greater than 30 °C, or greater than 35 °C. In some embodiments, the block copolymer has a CMT of less than 45 °C, less than 40 °C, less than 35 °C, less than 30 °C, or less than 25 °C. In some embodiments, the block copolymer has a CMT of less than body temperature (e.g., about 37 °C). In some embodiments, the block copolymer has a CMT of less than room temperature (e.g., about 20 °C to about 25 °C). CMT can be measured by a combination of UV-Vis and dynamic light scattering as described in the Examples.

[0067] The block copolymer may undergo self-assembly at varying concentrations. For example, the block copolymer can self-assemble at a concentration of about 1 μM to about 200 μM, such as about 10 μM to about 175 μM, about 15 μM to about 150 μM, about 1 μM to about 100 μM, or about 100 μM to about 200 μM. In some embodiments, the block copolymer self- assembles at a concentration of greater than 1 μM, greater than 10 μM, greater than 50 μM, or greater than 100 μM. In some embodiments, the block copolymer self-assembles at a concentration of less than 200 μM, less than 175 μM, less than 150 μM, or less than 100 μM. The foregoing concentrations can be applied to the CMT at which the block copolymer can self- assemble. For example, the block copolymer can have a CMT of about 15 °C to about 45 °C at a concentration of about 1 μM to about 200 μM.Attorney Docket No.028193-0045-WO01

[0068] In addition to self-assembly, the block copolymer can have phase transition behavior. Phase transition refers to the aggregation of the block copolymer(s), which may occur sharply, and in some instances, reversibly at or above a Ttof the block copolymer. Below the Tt, for example, the block copolymer may be highly soluble. Upon heating above the transition temperature, for example, the block copolymer may hydrophobically collapse and aggregate, forming a separate phase.

[0069] The phase of the block copolymer may be described as, for example, soluble or an aggregate. When below its Tt, the phase of the block copolymer may be described as, for example, soluble. When above its Tt, rather than soluble, the phase of the block copolymer can be described as, for example, particulate form or an aggregate. The aggregate may be a variety of forms. The form and size of the aggregate may depend on the temperature, the composition of the block copolymer, or a combination thereof. The aggregate may be, for example, nanoscale aggregates, micron-sized aggregates, or macroscale aggregates. In some embodiments, at a temperature above the Ttand above the CMT, the aggregate has a diameter or length of about 1 ^m to about 1 cm. In some embodiments, the aggregate is a coacervate.

[0070] The block copolymer may have a varying Ttdepending on its application. The block copolymer may have a Ttof about 20 °C to about 50 °C, such as about 22 °C to about 46 °C, about 25 °C to about 45 °C, about 30 °C to about 46 °C, or about 20 °C to about 30 °C. In some embodiments, the block copolymer has a Ttof room temperature (e.g., about 20 °C to 25°C) to body temperature (e.g., about 37 °C). In some embodiments, the block copolymer has a Ttof greater than 37 °C. In some embodiments, the block copolymer has a Tt of less than 37 °C. In some embodiments, the block copolymer has its Ttbelow a body temperature at a concentration at which the block copolymer or composition thereof is administered to a subject. The Ttof the block copolymer can depend on the molecular weight of the block copolymer and blocks thereof, monomer composition, the block copolymer’s concentration, or a combination thereof. Accordingly, the Ttcan be adjusted by varying the aforementioned parameters and properties. In addition, the Ttof the block copolymer can be measured by optical density via a UV-vis spectrophotometer as described in the Examples.

[0071] The block copolymer may undergo phase transition at varying concentrations. For example, the block copolymer can phase transition at a concentration of about 10 μM to about 200 μM, such as about 15 μM to about 175 μM, about 20 μM to about 150 μM, about 10 μM to about 100 μM, or about 100 μM to about 200 μM. In some embodiments, the block copolymer phase transitions at a concentration of greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM. In some embodiments, the block copolymer phase transitions at aAttorney Docket No.028193-0045-WO01 concentration of less than 200 μM, less than 175 μM, less than 150 μM, or less than 100 μM. The foregoing concentrations can be applied to the Ttat which the block copolymer can phase transition. For example, the block copolymer can have a Ttof about 22 °C to about 46 °C at a concentration of about 10 μM to about 200 μM. In some embodiments, the block copolymer has a Ttof greater than 37 °C at a concentration below 200 μM. In some embodiments, the block copolymer has a Ttof less than 37 °C at a concentration above 10 μM.

[0072] The block copolymer can also have different combinations of Ttand CMT as described herein. For example, the block copolymer can have a Ttof greater than 37 °C and a CMT of less than 37 °C at a concentration of about 1 μM to about 200 μM. In some embodiments, the block copolymer has a Ttof less than 37 °C and a CMT of less than 25 °C at a concentration of about 1 μM to about 200 μM. The foregoing are example temperature combinations and any combination of Ttand CMT, as well as the concentrations, as disclosed herein can be used for the block copolymers.

[0073] The block copolymer can have a varying molecular weight. For example, the block copolymer can have a number average molecular weight of about 5,000 Daltons (Da) to about 100,000 Da, such as about 10,000 Da to about 90,000 Da, about 15,000 Da to about 80,000 Da, about 20,000 Da to about 70,000 Da, about 5,000 Da to about 70,000 Da, about 25,000 Da to about 100,000 Da, about 25,000 Da to about 70,000 Da, about 25,000 Da to about 65,000 Da, or about 25,000 Da to about 60,000 Da. In some embodiments, the block copolymer has a number average molecular weight of greater than 5,000 Da, greater than 10,000 Da, greater than 15,000 Da, greater than 20,000 Da, greater than 25,000 Da, greater than 30,000 Da, or greater than 35,000 Da. In some embodiments, the block copolymer has a number average molecular weight of less than 100,000 Da, less than 90,000 Da, less than 80,000 Da, less than 70,000 Da, less than 60,000 Da, less than 50,000 Da, or less than 40,000 Da.

[0074] The term “molecular weight” in relation to the polymer refers to number average molecular weight (Mn) unless noted otherwise. Molecular weight of the block copolymer and the individual blocks can be measured by techniques used within the art, such as size exclusion chromatography (SEC), SEC combined with multi-angle light scattering (SEC-MALS), gel permeation chromatography, intrinsic viscosity, nuclear magnetic resonance (NMR), and the like. In some embodiments, the molecular weight is measured by SEC-MALS.

[0075] The block copolymer can be synthesized in a variety of different structures. For example, the block copolymer can be an A-B diblock copolymer or an A-B-A triblock copolymer. In some embodiments, the block copolymer is an A-B diblock copolymer or an A-B-A triblock copolymer. Different copolymer structures can facilitate the formation of different particulateAttorney Docket No.028193-0045-WO01 structures. The block copolymer can have a varying structure associated with a varying degree of polymerization. For example, the block copolymer can have a structure of A10-300-B10-300, A10-250-B10-200-A10-250, A10-200-B10-250, or A20-200-B20-200-A20-200. The foregoing are example structures and any combination of degree of polymerization and block alignment as disclosed herein can be used in the block copolymers.

[0076] The block copolymer can also have advantageous immune response properties. It has been found that by using POEGMA, rather than polyethylene glycol (PEG), the immune response to POEGMA and block copolymers thereof can be reduced or eliminated compared to PEG. The reduced or eliminated immune response can include a reduced or eliminated antigenicity, a reduced or eliminated immunogenicity, or both of the block copolymer. Accordingly, the disclosed block copolymer can have beneficial interactions with a subject’s immune system.

[0077] The beneficial immune interactions of the block copolymer can also be seen in that the block copolymer may not induce an anti-POEGMA antibody response. An anti-POEGMA antibody response can include inducing IgG class antibodies, inducing IgM class antibodies, inducing a IgM response that lasts longer than 10 days, or a combination thereof. Accordingly, in some embodiments, the block copolymer does not induce anti-POEGMA IgG class antibodies, induce anti-POEGMA IgM class antibodies, and / or induce an anti-POEGMA IgM response that lasts longer than 10 days. In addition, in some embodiments, the block copolymer is not reactive with pre-existing anti-PEG antibodies in a subject.

[0078] With respect to PEG as a control, this can be a PEG polymer, PEG copolymer, and / or PEG block copolymer having a similar molecular weight as the block copolymer. These PEG polymers can be considered a control as to what the disclosed block copolymer is compared to when assessing reducing or eliminating antigenicity, immunogenicity, or both. The control can also be branched or linear, as long as it has more than the disclosed number of consecutive ethylene glycol monomers in tandem. For example, a suitable control PEG can include linear or branched PEG having more than 3 consecutive ethylene glycol monomers in tandem.

[0079] Further discussion on POEGMA and its application can be found in U.S. Patent No. 8,497,356, U.S. Patent No.10,364,451, and U.S. Patent Application No.18 / 553,135, each of which are fully incorporated herein by reference in their entirety. A. First Block (A)

[0080] The first block of the block copolymer can be a hydrophilic block, e.g., more hydrophilic than the second block. Accordingly, the first block can include a polymer(s) that is more hydrophilic than the polymer(s) of the second block. The first block may act as aAttorney Docket No.028193-0045-WO01 hydrophilic corona block (e.g., in particulate form). The first block may also instill stealth properties (e.g., evading a subject’s immune system and / or clearance from the blood stream) to particles formed by the block copolymer. The first block can include POEGMA having side chains of either 2 or 3 monomers of EG. For example, the first block can include recurring units of formula (I)(I), wherein x is 2 or 3. In some embodiments, x is 3. In some embodiments, x is 2. In some embodiments, the first block includes at least one unit of x is 2 and at least one unit of x is 3. In embodiments that include both units of x is 2 and x is 3, said units can be included in the first block as a random copolymer.

[0081] The block copolymer can include varying amounts (e.g., degree of polymerization) of the recurring unit of formula (I). For example, the block copolymer can include the recurring unit of formula (I) at a degree of polymerization of 10 to 300, such as 12 to 280, 15 to 250, 10 to 250, 20 to 230, 10 to 200, 10 to 100, 50 to 300, 100 to 300, or 150 to 300. In some embodiments, the block copolymer includes the recurring unit of formula (I) at a degree of polymerization of greater than 10, greater than 12, greater than 15, greater than 18, greater than 20, greater than 50, or greater than 100. In some embodiments, the block copolymer includes the recurring unit of formula (I) at a degree of polymerization of less than 300, less than 280, less than 275, less than 260, less than 250, less than 200, less than 150, or less than 100.

[0082] Formula (I) of the first block can include units of x is 2 and units of x is 3 in varying amounts. For example, the recurring unit of formula (I) can include a unit of x is 2 at a degree of polymerization of 0 to 100, such as 0 to 75, 0 to 70, 1 to 100, 2 to 100, 5 to 80, 5 to 70, 1 to 90, 1 to 80, 1 to 50, 1 to 35, 25 to 100, 50 to 100, or 60 to 100. In some embodiments, the recurring unit of formula (I) includes a unit of x is 2 at a degree of polymerization of greater than 0, greater than 1, greater than 2, greater than 5, greater than 10, greater than 20, or greater than 30. In some embodiments, the recurring unit of formula (I) includes a unit of x is 2 at a degree of polymerization of less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, or less than 40.Attorney Docket No.028193-0045-WO01

[0083] The recurring unit of formula (I) can include a unit of x is 3 at a degree of polymerization of 10 to 250, such as 20 to 250, 20 to 225, 20 to 200, 10 to 200, 20 to 150, 10 to 150, 10 to 150, 10 to 100, 50 to 250, 100 to 250, or 20 to 80. In some embodiments, the recurring unit of formula (I) includes a unit of x is 3 at a degree of polymerization of greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 40, or greater than 50. In some embodiments, the recurring unit of formula (I) includes a unit of x is 3 at a degree of polymerization of less than 250, less than 225, less than 200, less than 175, less than 150, less than 100, or less than 75.

[0084] The recurring unit of formula (I) can include any foregoing combination of x is 2 and x is 3 as described herein. In addition, in triblock copolymer embodiments, each of the A blocks of the triblock copolymer can include any of the foregoing description of the first block. B. Second Block (B)

[0085] The second block can be more hydrophobic than the first block. Accordingly, the second block can include a polymer(s) that is more hydrophobic than the polymer(s) of the first block. The second block may act as a hydrophobic core block (e.g., in particulate form). The second block can include POEGMA having side chains of either 1 or 2 monomers of EG. For example, the second block can include recurring units of formula (II)(II), wherein y is 1 or 2. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, the second block includes at least one unit of y is 1 and at least one unit of y is 2. In embodiments that include both units of y is 1 and y is 2, said units can be included in the second block as a random copolymer.

[0086] The block copolymer can include varying amounts of the recurring unit of formula (II). For example, the block copolymer can include the recurring unit of formula (II) at a degree of polymerization of 10 to 300, such as 15 to 250, 20 to 250, 30 to 230, 30 to 225, 10 to 250, 20 to 230, 10 to 200, 10 to 100, 50 to 300, 100 to 300, 150 to 300, or 50 to 200. In some embodiments, the block copolymer includes the recurring unit of formula (II) at a degree of polymerization of greater than 10, greater than 15, greater than 20, greater than 25, greaterAttorney Docket No.028193-0045-WO01 than 30, greater than 40, greater than 50, or greater than 100. In some embodiments, the block copolymer includes the recurring unit of formula (II) at a degree of polymerization of less than 300, less than 280, less than 275, less than 260, less than 250, less than 235, less than 230, less than 220, or less than 100.

[0087] Formula (II) of the second block can include units of y is 1 and units of y is 2 in varying amounts. For example, the recurring unit of formula (II) can include a unit of y is 1 at a degree of polymerization of 0 to 100, such as 0 to 75, 0 to 70, 1 to 100, 2 to 100, 5 to 80, 5 to 70, 1 to 90, 1 to 80, 1 to 50, 1 to 35, 15 to 85, 20 to 85, 25 to 100, 50 to 100, or 60 to 100. In some embodiments, the recurring unit of formula (II) includes a unit of y is 1 at a degree of polymerization of greater than 0, greater than 1, greater than 2, greater than 5, greater than 10, greater than 20, or greater than 30. In some embodiments, the recurring unit of formula (II) includes a unit of y is 1 at a degree of polymerization of less than 100, less than 90, less than 85, less than 80, less than 70, less than 60, or less than 50.

[0088] The recurring unit of formula (II) can include a unit of y is 2 at a degree of polymerization of 0 to 250, such as 10 to 250, 20 to 250, 20 to 225, 20 to 200, 10 to 200, 20 to 235, 30 to 230, 20 to 150, 10 to 150, 10 to 150, 10 to 100, 50 to 250, 100 to 250, 20 to 80, or 0 to 80. In some embodiments, the recurring unit of formula (II) includes a unit of y is 2 at a degree of polymerization of greater than 0, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 40, or greater than 50. In some embodiments, the recurring unit of formula (II) includes a unit of y is 2 at a degree of polymerization of less than 250, less than 235, less than 230, less than 200, less than 175, less than 150, less than 100, or less than 75.

[0089] The recurring unit of formula (II) can include any foregoing combination of y is 1 and y is 2 as described herein. C. Synthesis of Block Copolymers

[0090] Example block copolymer synthetic details can be found in the Examples herein. Generally, the block copolymers can be synthesized by employing a two-step sequence of reversible addition-fragmentation chain-transfer (RAFT) polymerization with 4-Cyano-4- (thiobenzoylthio) pentanoic acid as the chain transfer agent (CTA) and AIBN as the radical initiator. The first block can be polymerized by mixing the monomers of the hydrophilic block, CTA, AIBN in approximately 4x : 4 : 1 ratio, respectively, where x = 1.2 times the total desired degree of polymerization of the hydrophilic block. The polymerization reaction can be continued under an inert environment at approximately 70 °C for a few hours depending on the target molecular weight and the initial monomer concentration. The reaction can be quenched byAttorney Docket No.028193-0045-WO01 submersion of the round bottom flask in liquid nitrogen and the resulted polymer can be purified by hexane precipitation (e.g., three rounds thereof). To complete the synthesis of the diblock, the second block can be polymerized by utilizing the first-POEGMA block as a macromolecular CTA. Thus, the first polymer block can be resuspended in toluene, and the hydrophobic monomers of the second block along with AIBN can be added to the reaction mix. The reaction conditions and purification procedure for the second block can be identical to those implemented for the first block. D. Particles

[0091] The composition and structure of the block copolymer can afford the block copolymers the ability to self-assemble into particulate structures. For example, the block copolymer can have a CMT and at a temperature above the CMT, the block copolymers can self-assemble, e.g., with other block copolymers into a particulate structure. Accordingly, also disclosed herein are particles that can include a plurality of self-assembled block copolymers. Example structures include micelles, vesicles, inverted micelles, spherical polymersomes, tubular polymersomes, and nanofibers. Micelles can be a variety of shapes with varied aspect ratios. For example, the particle can be a filomicelle, a rodlike micelle, a cylindrical micelle, and / or a wormlike micelle.

[0092] The particle can have a varying diameter. For example, the particle can have a hydrodynamic diameter of about 20 nm to about 120 nm, such as about 25 nm to about 100 nm, about 20 nm to about 100 nm, about 20 nm to about 80 nm, about 25 nm to about 75 nm, about 25 nm to about 55 nm, about 20 nm to about 50 nm, or about 50 nm to about 120 nm. In some embodiments, the particle has a hydrodynamic diameter of greater than 20 nm, greater than 25 nm, greater than 30 nm, greater than 35 nm, greater than 40 nm, or greater than 50 nm. In some embodiments, the particle has a hydrodynamic diameter of less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, or less than 60 nm. Particle size can be measured by techniques known within the art, such as dynamic light scattering and electron microscopy (e.g., TEM). E. Example Block Copolymers

[0093] In some embodiments, the first block and the second block each individually comprise a POEGMA polymer or a POEGMA copolymer. In some embodiments, the first block and the second block each individually consist of a POEGMA polymer or a POEGMA copolymer.

[0094] In some embodiments, the block copolymer includes the recurring unit of formula (I) with a degree of polymerization of 10 to 300 and the recurring unit of formula (II) with a degree of polymerization of 10 to 300.Attorney Docket No.028193-0045-WO01

[0095] In some embodiments, the recurring unit of formula (I) includes a unit of x is 2 at a degree of polymerization of 0 to 100 and a unit of x is 3 at a degree of polymerization of 20 to 250; and the recurring unit of formula (II) includes a unit of y is 1 at a degree of polymerization of 0 to 100 and a unit of y is 2 at a degree of polymerization of 20 to 250.

[0096] In some embodiments, the recurring unit of formula (I) includes a unit of x is 2 at a degree of polymerization of 0 to 80 and a unit of x is 3 at a degree of polymerization of 20 to 200; and the recurring unit of formula (II) includes a unit of y is 1 at a degree of polymerization of 0 to 85 and a unit of y is 2 at a degree of polymerization of 25 to 230.

[0097] In some embodiments, the block copolymer includes the recurring unit of formula (I) where x is 3, and the recurring unit of formula (II) where y is 2. In embodiments where x is 3 and y is 2, the block copolymer can include the first and second block individually at greater than 25% of the total polymer length in terms of degree of polymerization. 3. Compositions including the Block Copolymers

[0098] Also disclosed herein are compositions that include a plurality of block copolymers self-assembled into a particle as described above. Accordingly, the description of the block copolymers, first block, second block, and particles above can be applied to the disclosed compositions. The composition can also include a drug encapsulated within the particle or a drug decorating the surface of the particle.

[0099] Any suitable drug can be used in the disclosed compositions. Example drugs include, but are not limited to, a peptide-based drug, a chemotherapeutic, and a combination thereof. In some embodiments, the drug is a hydrophobic chemotherapeutic. Example hydrophobic chemotherapeutics include, but are not limited to, doxorubicin (DOX), paclitaxel (PTX), tamoxifen (TAM), methotrexate, camptothecin (CPT), maytansine, auristatin, and their derivatives, or a combination thereof. In some embodiments, the hydrophobic chemotherapeutic is DOX. Example peptide-based drugs include, but are not limited to, GLP1 agonist, GIP1 agonist or antagonist, or an ova-based peptide, or a combination thereof.

[0100] The composition can also include one or more pharmaceutically acceptable excipients, where such compositions can also be referred to as a pharmaceutical composition. The term “pharmaceutically acceptable excipient,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to,Attorney Docket No.028193-0045-WO01 sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, citrate buffers, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The route by which the composition is administered, and the form of the composition can dictate the type of excipient to be used.

[0101] In some embodiments, the pharmaceutically acceptable excipient includes buffering agents (e.g., phosphate buffered saline), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or a combination thereof. The compositions and pharmaceutical compositions can be used in the methods disclosed herein.

[0102] General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated by reference herein in its entirety. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a block copolymer. An excipient or accessory ingredient may be incompatible with a component of a block copolymer if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect. 4. Uses of the Block Copolymers and Compositions Thereof A. Administration

[0103] The block copolymer and composition thereof may be suitable for administration to a subject (such as a patient, which may be a human or non-human) well known to those skilled in the pharmaceutical art. The block copolymer and composition thereof may be prepared for administration to a subject. Such block copolymers and compositions thereof can be administered in dosages and by techniques well known to those skilled in the medical artsAttorney Docket No.028193-0045-WO01 taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.

[0104] The block copolymer and composition thereof can be administered prophylactically or therapeutically. In prophylactic administration, the block copolymer and composition thereof can be administered in an amount sufficient to induce a response. In therapeutic applications, the block copolymer and composition thereof can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the conjugate regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0105] The block copolymer and composition thereof can be administered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, the block copolymer or composition thereof is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally. In some embodiments, the block copolymer or composition thereof is administered intravenously or subcutaneously.

[0106] The block copolymer and composition thereof may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0107] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular drugs employed, and the specific use for which these drugs are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.

[0108] Dosage amount and interval may be adjusted individually to provide plasma levels of the biologically active agent which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each agent but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, assays well known to those in the art canAttorney Docket No.028193-0045-WO01 be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions can be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, such as between 30-90% or between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0109] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine. B. Methods of Treating a Disease or a Disorder

[0110] Disclosed herein are methods of treating a disease or disorder in a subject (e.g., in need thereof). The method can include administering to the subject a therapeutically effective amount of the disclosed block copolymer or composition thereof.

[0111] The disease or disorder can be any that would benefit from effective delivery by the disclosed block copolymers. Example diseases or disorders include, but are not limited to, cancers, autoimmune diseases, or metabolic disorders. In some embodiments, the disease or disorder is a cancer. In some embodiments, the subject is human.

[0112] The disclosed methods can take advantage of the block copolymers’ phase transition and self-assembling behavior. For example, the block copolymer can have a Ttgreater than a body temperature of the subject and a CMT below the body temperature of the subject. Practically speaking, this can allow the block copolymer or composition thereof to be administered in liquid / suspension form as particles through, e.g., a syringe to a subject, and then following injection, the block copolymer or composition thereof can maintain its particle structure. An example administration route for this embodiment is intravenous or systemic administration.

[0113] In another embodiment, the block copolymer or composition thereof can be administered at a temperature below the block copolymer’s Ttand above the block copolymer’s CMT to an area of the subject that has a temperature above the block copolymer’s Tt. Practically speaking, this can allow the block copolymer or composition thereof to be administered in liquid / suspension form as particles through, e.g., a syringe to a subject, andAttorney Docket No.028193-0045-WO01 then following injection, the block copolymer or composition thereof can phase transition to an aggregate (e.g., depot) at the site of administration. The aggregate / depot can restrict the release of the drug and thus sustain its release over a longer period of time. An example administration route for this embodiment is subcutaneous or local administration.

[0114] As discussed elsewhere, the block copolymers and compositions thereof can have advantageous immune properties. For example, following administration, the block copolymer or composition thereof can have a reduced immune response relative to a composition including PEG; may not induce an anti-PEOGMA antibody response; may not react with pre-existing anti- PEG antibodies in the subject; or a combination thereof. C. Methods of Delivering a Drug

[0115] Further disclosed herein are methods of delivering a drug to a subject (e.g., in need thereof). The method can include administering, either systemically or locally, to the subject the block copolymer or composition thereof. The drug is then released from the particle following administration. Depending on how the block copolymer is designed, the release mechanism, location, and / or release kinetics can be modulated for a tailored application.

[0116] As the methods of treating a disease or disorder may include delivering a drug, the description of methods of treating a disease or disorder may also be applied to methods of delivering a drug. Likewise, the description of methods of delivering a drug can be applied (when applicable) to the methods of treating a disease or disorder. Similarly, where applicable, the methods of treating a disease or disorder and delivering a drug can be applied to the methods of delivering a scaffold. D. Methods of Delivering a Scaffold

[0117] Also disclosed herein are methods of delivering a scaffold to a subject (e.g., in need thereof). The method can include administering a block copolymer to a location of the subject. The block copolymer can exhibit phase transition behavior that can be defined by a transition temperature of the block copolymer. The temperature-dependent phase transition behavior can allow the block copolymer to form a porous network above the transition temperature. Practically speaking, this can allow the block copolymer to be administered through, e.g., a syringe to a subject, and then following injection, the block copolymer can phase transition into, e.g., a network-like structure or scaffold. This scaffold can be used for cell recruitment and wound healing applications.

[0118] The description of the block copolymers, first block, second block, particles, compositions, and drug can also be applied to the uses and methods disclosed herein. The disclosed technology has multiple aspects, illustrated by the following non-limiting examples.Attorney Docket No.028193-0045-WO01 5. Examples Materials and Methods for Example 1

[0119] Materials: All reagents for synthesis of the block coPOEGMA were purchased from Sigma Aldrich. The monomers—triethylene glycol methyl ether methacrylate (EG3, CAS No. 24493-59-2), diethylene glycol methyl ether methacrylate (EG2, CAS No.45103-58-0), ethylene glycol methyl ether methacrylate (EG1, CAS No.6976-93-8)—were passed through a column of activated basic aluminum oxide (CAS No.1344-28-1, Sigma # 199443) to remove inhibitors before polymerization reactions.4-Cyano-4-(thiobenzoylthio) pentanoic acid (CAS 201611-92-9, Sigma # 722995,) and 2,2’-Azobis (2-methyl propionitrile) (AIBN) (CAS 78-67-1, Sigma # 441090,) and solvents were used as received.

[0120] POEGMA di-block copolymers were synthesized in a two-step sequence of reversible addition-fragmentation chain-transfer (RAFT) polymerizations employing 4-Cyano-4- (thiobenzoylthio) pentanoic acid as the chain transfer agent (CTA) and AIBN as the radical initiator. The first block (EG3 POEGMA) was polymerized by mixing the EG3 monomer, CTA, AIBN in 4x : 4 : 1 ratio, respectively, in toluene as a solvent, where x = 1.2 times the desired degree of polymerization. The reaction mixture was prepared in a round bottom (RB) flask on ice bath and purged with nitrogen gas for 40 minutes to create an inert environment before initiating the polymerization at 70 °C for varied time with continuous nitrogen purging. Reaction time ranged from one to five hours depending on the target molecular weight and the initial monomer concentration. The reaction was quenched by submersion of the RB flask in liquid nitrogen and the resulted EG3 polymer was purified with three rounds of hexane precipitation. The purified polymer was freeze dried and stored in -20 °C until further use. To complete the synthesis of the diblock, the second block (EG2 POEGMA) was polymerized by utilizing the EG3-POEGMA block as a macromolecular CTA. Thus, the first polymer block was resuspended in toluene, and EG2 monomer along with AIBN were added into RB flask on ice bath. The reaction conditions and purification procedure were identical to those implemented for the EG3 polymerization. For the second set of the diblock polymers the individual blocks were synthesized and purified in a similar manner except a combination of two monomers (EG3 / EG2; EG2 / EG1) were randomly copolymerized in one or both blocks.

[0121] CTA end group removal: The active part— thiobenzoylthio —of the chain transfer agent present as an end group on RAFT synthesized polymers was cleaved off from theAttorney Docket No.028193-0045-WO01 polymer chain by treatment with excess radical generators. Briefly, the pink colored polymer was treated with 20x excess AIBN at 80 °C for 3 hours in an inert environment resulting in a colorless solution. The CTA-free polymer was purified out by three rounds of hexane precipitation.

[0122] Chemical characterization of POEGMA: All polymerization reactions were monitored with proton nuclear magnetic resonance (1H NMR) spectroscopy on 500MHz Varian spectrophotometer to determine percent monomer to polymer conversion and to predict molecular weight. After quenching the polymerization reaction, a small amount of the mixture was dissolved in deuterated chloroform (CDCl3) containing 1%(v / v) TMS as an internal reference to record1H NMR. The data was analyzed using MNOVA software.

[0123] All purified polymers were additionally characterized for molecular weights (Mn, Mw) and polydispersity (PDI) by size exclusion chromatography in line with multi angle light scattering (SEC-MALS) using an Agilent 1260 Infinity HPLC equipped with DAWN HELEOS II MALS detector and an Optilab T-rEX refractive index detector (Wyatt Technology). The polymers were resuspended in HPLC grade tetrahydrofuran (THF) at concentration of 2 - 5 mg / ml and filtered through 0.2 μm syringe filters (Whatman).50 ul of the sample was injected to dual Agilent gel columns (7.5 x 300 mm, 179911GP-503 (103 Å) and a 179911GP-504 (104 Å) and run at room temperature using THF (containing 100 ppm butylated hydroxytoluene (BHT) as a stabilizer) mobile phase at a flow rate of 1 mL / min. The light scattering data was analyzed for Mnand Mwusing ASTRA software (Wyatt) by inputting the refracting index increment (dn / dc) = 0.07 for POEGMA in THF. The dn / dc value for POEGMA was calculated using the refractive index detector with an online method for a known concentration of the polymer sample.

[0124] Thermo-responsive behavior and hydrodynamic size characterization: The polymers were resuspended in 1x DPBS at 200 μM concentration at 4 °C, and a series dilution in DPBS was performed resulting in 200, 100, 50, 25, 12.5, 6.25 μM as the concentrations to be tested. The optical density (OD) of all polymers was monitored at 600 nm as a function of temperature on a temperature-controlled UV-vis spectrophotometer (Cary 300 Bio, Varian instruments). Starting at 20 °C, the temperature of the samples in cuvette containing chamber was increased at a rate of 0.3 °C / min until ~ 40 °C and was cooled back to 20 °C at a rate of 0.3 °C / min. The absorbance was recorded at each 0.6 °C interval. A sharp increase in the OD with temperature is indicative of phase transition and the temperature at the inflection point of the optical density is defined as the Tt. The OD vs temperature was plotted and analyzed for Ttby finding the maximum of the first derivative using GraphPad Prism software. The size distribution of the diblock POEGMA samples was measured using a Malvern NanoZS Zetasizer equipped with aAttorney Docket No.028193-0045-WO01 173° backward scatter detector. The DLS measurements were performed in triplicates at various temperatures for various concentrations in a low-volume quartz cuvette (Malvern #ZEN2112). Data were analyzed by regularization fit for Raleigh spheres using Malvern sizing software. Combination of the UV-Vis spectrophotometry and the DLS data was used to determine critical aggregate (micelle) temperatures (CMT).

[0125] Small Angle X-ray Scattering and cryo-EM: diblock POEGMA samples were resuspended in 1x DPBS at concentrations of 1, 2, 5 mg / ml for NP structure analysis below Ttof the polymer.

[0126] Evaluation of critical aggregate concentration (CAC): A pyrene fluorescence assay was utilized to determine CAC. Briefly, a 12mM pyrene stock in methanol was diluted to 720 nM in PBS to get pyrene working reagent. The diblock POEGMA samples were resuspended in the pyrene solution at a polymer concentration of 50 μM and ten serial dilutions of the polymer were prepared in pyrene solution to achieve 0.05 μM as the lowest concentration tested. Each polymer sample of varying concentrations was maintained at constant pyrene concentration of 720 nM and the fluorescence emission (355 to 450 nm) spectra of pyrene was recorded at an excitation wavelength of 334 nm below Ttof the polymer on Spectrofluorometer Shimadzu RF- 5301PC. The ratio between the first (~372 nm) and third (~383 nm) peak intensities (I1 / I3) was plotted against polymer concentration and data was analyzed with Excel by fitting two lines— one for the flat region and one for the negatively sloped region. The concentration at which the I1 / I3 values begins to decrease is CAC.

[0127] Light microscopy: The polymers were resuspended in PBS at various concentrations (2 – 30 mg / ml) for envisioning them under a light microscope. A 50x Thioflavin T (ThT, 2mM) dye stock solution in PBS was added to polymer samples in a 1:50 v / v ratio to visualize the partition of the dye into polymer condensates. For imaging, 3ul of sample was placed on a glass slide with double-sided tape lining the sample area to allow sufficient space for condensates to form between the slide and coverslip. The samples were imaged on a benchtop Zeiss microscope (Axio Imager.D2m) with a custom heating insert, below and above Ttof the polymer. Brightfield and green fluorescence images were taken under 20x magnification and overlayed in ImageJ.

[0128] Confocal microscopy and FRAP: The polymers were labelled with AlexaFluor 488 amine (Lumniprobe # 118C0) using EDC-NHS chemistry and available terminal -COOH group on the POEGMA; unreacted free dye was removed by extensive dialysis against 1xDPBS. Alexafluor 488-labelled polymer was mixed with unlabeled polymer in PBS for a final polymer concentration of 200 μM and labelling percentage of 1-2%. Confocal imaging was performed onAttorney Docket No.028193-0045-WO01 a Zeiss 710 inverted confocal microscope heated to slightly above the Ttof tested polymer with a custom incubator box.30 ul of each sample was added to a black 384-well glass-bottom Greiner plate and topped with 50ul mineral oil immediately to prevent evaporation during imaging. FRAP was performed following the methods and principles outlined in Ozer at el.,^ Polyethylene GlycolǦLike Brush Polymer Conjugate of a Protein Drug Does Not Induce an Antipolymer Immune Response and Has Enhanced Pharmacokinetics than Its Polyethylene Glycol Counterpart, Adv. Sci., 2022, Apr; 9(11), which is incorporated by reference herein in its entirety. Briefly, a small rectangular portion of a droplet or gel globule was photobleached using the Argon / 2488nm laser line at 100% intensity under 63x magnification, then the region of interest was imaged at 5 fps for 40 seconds to observe recovery of fluorescence at the bleached region. Spots were analyzed by correcting for photofading using an unbleached region, then normalizing pre-bleach intensity to 100% and post-bleach intensity to 0%. Three spots per sample were averaged to generate recovery curves.

[0129] Doxorubicin (Dox) encapsulation: A 500 μM stock Dox solution in distilled water was used to resuspend the polymer at Dox:polymer of 10:1 at 4 °C. After ensuring proper solubilization of the polymer, the solutions were incubated at a convenient temperature (above CMT but below Tt) for aggregation and hence drug encapsulation, for e.g., polymer F was incubated at room temperature for 30 minutes. The solution was then centrifuged at 12,000 rcf for 5 minutes at the same temperature to remove any precipitate but unencapsulated Dox. The supernatant containing polymer and Dox mixture was passed through several rounds of Amicon (10k Mw cutoff) tube filters in PBS to remove free Dox and isolate Dox encapsulated in polymeric NPs. With each round, the absorbance of flowthrough was recorded before discarding it, and the sample was diluted with PBS. This process was repeated until the the absorbance of flowthrough was zero was Dox. The Dox encapsulating NPs were tested for purity by running the positively charged Dox on agarose gel towards negative terminal at 130V for 30 minutes. The fluorescence of the Dox was used to image presence and location of the Dox in the gel. Any significant amount of free Dox in the sample was measured by its migration in the gel while the encapsulated Dox stayed in the well, along with the polymer, at the loading site. Additionally, a standard Dox curve was generated by plotting absorbance of free Dox at 480 nm against its known concentrations to obtain a linear equation which was used to determine Dox concentrations in unknown samples. The concentration of encapsulated Dox was calculated by breaking the NPs in 70 % acetonitrile and measuring the absorbance of the Dox at 480 nm.

[0130] In vitro potency of Dox loaded NP: The efficacy of free Dox vs encapsulated Dox was investigated in MC38 murine colon adenocarcinoma cell line purchased from Sigma Aldrich.Attorney Docket No.028193-0045-WO01 The cell line was maintained in culture as per manufacturer’s instructions and 5 x 103cells / well, 90ul were plated in a 96-well format a day before the assay. Cells were treated with dilutions of the drug (free vs encapsulated), 30 ul / well (n = 3) and were incubated for 48 hours. Blank wells and wells treated with PBS were defined as 0 and 100% viability, respectively.20 ul of CellTiter 96 AQueous (Promega) reagent was added to each well and incubated for 1h before absorbance measurement at 490nm. The data was analyzed by plotting dox concentration vs Absorbance and a variable slope (four parameter) model was fitted in GraphPad Prism to calculate IC50 of the drug. Example 1 Diblock POEGMA copolymers

[0131] Design and synthesis of simple EG3-EG2 diblock POEGMA library: It was hypothesized that a block polymer of EG3MA and EG2MA (FIG.1) can impart enough amphiphilicity for the polymer to self-assemble into non-immunogenic NPs with EG3 forming the corona and EG2 forming core of the NPs (FIG.2A). To test this hypothesis, a library of EG3- EG2 diblock copolymers were synthesized with varied ratios of EG2 monomer— as the hydrophobic block— and EG3 monomer —as the hydrophilic block. The synthesis was accomplished in two steps of RAFT polymerizations and purification:Scheme 1: Two-step chemical synthesis scheme of the diblock POEGMA via RAFT polymerization reaction.

[0132] The first RAFT reaction was quenched at 80-90 % monomer conversion to ensure an intact CTA-end group on resulted EG3 POEGMA that can be further employed as a macro-CTA for synthesis of EG2 block using RAFT. The resulted EG3 block and the final EG3-EG2 diblock were characterized for monomer conversion and apparent molecular weight by NMR. Additionally, all the purified polymers including the first EG3 block and final diblock were characterized for number average molecular weight (Mn), weight average molecular weight (Mw)Attorney Docket No.028193-0045-WO01 and polydispersity index (PDI = Mw / Mn) using size exclusion chromatography in line with multi angle light scattering (SEC-MALS) (FIG.2B and FIG.2C). Only diblock polymers with PDI ^ 1.2 were studied further to ensure quality control. The DP of individual block was determined by its Mndivided by molecular weights of the respective monomer. Each diblock polymer in the library was given a unique ID representing relative DPs of the individual blocks and a random short alphabetical label for easy identification of the diblock.

[0133] Physical characterization of the diblock POEGMA library: Each diblock POEGMA was characterized for signs of self-assembly in detail. For simplicity the physical characterization data is shown for only one polymer, polymer D as a representative of the simple diblock library in the figures and the data for the other polymers is summarized in Table 1.

[0134] First, thermal phase behavior of all the diblock polymers were studied by recordingtheir optical density with temperature at various concentrations, and their cloud points (Tt) weredetermined by sharp and significant increase in the absorbance on the heating curve. To test the reversibility of the LCST phase behavior of the diblock POEGMA, the polymers were also cooled at the same rate as heating while recording the absorbance. All the polymers returned to their absorbance baseline after cooling, with slight variations in their hysteresis behavior, showing completely reversible phase transition behavior. Based on the optical density data, a Tt= 42.5 °C was determined for polymer D, corresponding to the sharp increase in absorbance before plateauing at high absorbance (FIG.3A). Second, dynamic light scattering (DLS) was performed for all the diblock polymers at various concentrations below their Ttto study self- assembly of polymers. For the diblock polymer D, hydrodynamic diameter is 7 ± 2 nm at room temperature (RT = 25 °C), corresponding to the monomeric form of the POEGMA, but increases to 40 ± 10 nm upon heating to 30 °C indicating self-assembly of the polymer. This temperature also corresponds to and explains the small hump at 30 °C in the optical density curve for high concentrations. The minimum temperature required for self-assembly, or micelle formation of a thermo-responsive material is defined as critical micelle temperature (CMT). The size distribution for polymer D at RT and body temperature shows a single peak with normal distribution, but differences in sizes, respectively, at both a low and high concentration (FIG. 3B). The size of self-assembly is stable for a wide range of temperatures for polymer D above its CMT until the phase transition occurs at Tt(FIG.3C). To further study the morphology of the self-assembled polymer D, a small angle X-ray scattering (SAXS) experiment at 37 °C was performed at 1, 2 and 5 mg / ml in PBS. The zero slope of the intensity (I) curves at small scattering vectors (q) (FIG.3D) is a ‘fingerprint’ of globular nanoparticles, which was also validated by the sharp peak in Kratky plot (FIG.3F) indicating compact assembly. To calculateAttorney Docket No.028193-0045-WO01 the radius of gyration (Rg), the Guinier approximation was employed that at low q, the scattering profile can be approximated as I(q) § ^(0) ^(-q2Rg2 / 3), where ^(0) is the intensity at zero scattering angle. A linear fit to a plot of ln(I) vs. q2(Guinier plot) in the linear fitting region gives the formula Rg= sqrt(-3*a), where ‘a’ is the slope of the Guinier fit. The Rgof self-assembled polymeric NPs of polymer D was determined to be 16.8 ± 1.45 nm using the Guinier linear fit (FIG.3E). To visualize the diblock POEGMA NPs in their true form, they were imaged with cryo- transmission electron microscopy (cryo-TEM) at a temperature between CMT and Tt. The electron contrast of these polymeric NPs is thought to be low compared to the solvent due to their highly hydrated corona, and only the core structure of the NPs can be visualized. As a representation, (FIG.3G) shows a cryo-TEM image of polymer D at 37 °C, where circles outline a few NPs that are visible in the micrograph.

[0135] Overall, based on the simple EG3-EG2 diblock library, it was found that even the difference of a single EG side chain in each block imparts sufficient amphiphilicity to a subset of these diblock copolymers to drive their self-assembly into NPs. Polymers B, C and D, show strong self-assembly whereas polymer A and E stay in their monomeric form as evident by their hydrodynamic size, Table 1. To determine the boundary conditions for self-assembly of simple EG3-EG2 diblock, relative EG2 block lengths were plotted in terms of DP against relative EG3 block length (FIG.3H). The area outside the vertical bar represents compositions that would stay monomeric whereas the area in green box in the middle represent diblock compositions that are likely to self-assemble. It was determined that the relative DP of an individual block in a simple EG3-EG2 block should be at least 25% of the total DP for it to impart enough amphiphilicity to the block for its self-assembly. Another interesting observation made is that the dependence of concentration on Ttof a simple EG3-EG2 diblock is much less prominent for polymers that self-assemble into NPs compared to the ones that do not (FIG.3I). This can be attributed to differences in the intrinsic particle concentration for phase transition to occur from monomeric polymer particles to coacervation as compared to transition from self-assembled polymer NPs to coacervation. Table 1: Simple library of EG3-EG2 diblock POEGMAAttorney Docket No.028193-0045-WO01Determined byaSEC-MALS,bDLS,cSAXS; n / m: not measured. The Tt and CMT are reported at 100 μM.

[0136] The simple diblock library in Table 1 shows that self-assembly of EG3-EG2 diblock POEGMA can be achieved by varying the relative block length to more than 25% of total DP, but all these polymers required temperature higher than RT for self-assembly to occur. To achieve a more tunable and a wide range of CMT, a slightly more complex library of diblock POEGMA was designed and synthesized with improved amphiphilicity. This complex diblock POEGMA library is designed to have some random copolymerization of EGn, n ^ 3, in either of the individual block or in both the blocks.

[0137] Increasing the hydrophobicity of the hydrophobic block: To achieve self-assembly of diblock POEGMA at RT and hence get a wide range of working temperatures with NPs, the amphiphilicity of the diblock POEGMA was modulated further by doping in a more hydrophobic monomer EG1 into the hydrophobic block, see, e.g., polymer F, G, H, I in Table 2. For simplicity, the physical characterization data is shown for only one polymer for this subset of the complex diblock library, polymer F (FIG.4A) as a representative in the figures and the data for the other polymers is summarized in Table 2.

[0138] First, thermal phase behavior of polymer F at various concentrations is shown in FIG. 4B, showing Tt = 44.5 °C. The polymer F returned to its start absorbance baseline after cooling, showing completely reversible phase transition behavior, but has more hysteresis compared to the polymer D, attributed to its more hydrophobicity. Interestingly, despite having variable amounts of hydrophobic EG1 monomer, the Ttfor polymers F, G, H, I was high and constant around 44 °C irrespective of EG1 doping levels. This can be attributed to the fact that any differences in the hydrophobic core are masked by the similar EG3 corona in all these polymers, hence, a constant thermal energy is required for the self-assembled polymer NPs to coacervate during the phase transition. Second, DLS data for these polymers revealed stable self-assembly for various concentrations and a wide range of temperatures below their Ttwith a convenient CMT below RT. The size distribution for polymer F at RT and body temperature shows a normally distributed single peak at both a low and high concentration with size ~ 44 ± 10 nm in hydrodynamic diameter (FIG.4C and FIG.4D). To further study the morphology of the self- assembled polymer F at 25 °C, SAXS experiment was performed at 1, 2 and 5 mg / ml in PBS.Attorney Docket No.028193-0045-WO01 The zero slope of the intensity curves at small q (FIG.4E) is a ‘fingerprint’ of globular nanoparticles, which was also validated by the sharp peak in Kratky plot (FIG.4G) indicating a compact assembly. The Rgof self-assembled polymeric NPs of polymer F was determined to be 12.6 ± 1.37 nm using the Guinier linear fit (FIG.4F). To study the phase transition of these polymers, polymer F was also visualized under light microscope above its Ttin presence of Thioflavin T (ThT) dye (FIG.4H). ThT is a fluorescent dye that is generally used in investigation of amyloid formations due to its enhanced fluorescence when bound to protein aggregates. Here ThT was used for its non-specific fluorescence when present in a hydrophobic environment, to visualize the phase transition of polymer F with more contrast. The appearance in ThT’s green fluorescence in presence of polymer F, above its Tt—at 45 °C, is indicative of increase in the local hydrophobicity on phase transition of the polymer. Self-assembly of polymer F was further visualized by cryo-TEM at RT where the circles outline a few NPs on the micrograph (FIG.4I). Only the tightly packed core of these NP was able to be visualized and not the whole NP due to its highly hydrated EG3 corona.

[0139] To showcase the utility of these easy-to-handle and stable NPs, a therapeutically relevant hydrophobic molecule, Doxorubicin (Dox), was encapsulated with polymer F by simple co-incubation. The free Dox was separated from the encapsulated Dox using amicon filtration and the purity of Dox encapsulated inside polymer F was confirmed by electrophoresis (FIG. 4J). The positively charged free Dox migrated in the agarose gel whereas the charge of Dox is masked when encapsulated in the polymer and hence encapsulated Dox stayed in the well. The migration of Dox in the agarose gel was visualized by its fluorescence. The loading efficiency of the purified Dox encapsulated in polymer F was calculated using absorbance of Dox at 480 nm. The cytotoxicity of free Dox vs encapsulated Dox was evaluated in vitro for MC38 murine colon adenocarcinoma cell line (FIG.4K). The free Dox was found to be more potent as compared to the Dox encapsulated inside polymer F.

[0140] Overall, the first set of four complex diblocks, polymer F, G, H, I, shows that diblock POEGMA composition can be easily manipulated to create thermo-responsive stealth polymers that self-assemble into stable NPs for a wide range if temperature including RT and body temperature. These polymeric nanoparticles can be injected systemically as a colloidal suspension and circulate as NPs in the blood stream, with drugs that are either physically encapsulated or covalently conjugated in their core. Next, diblock POEGMA were designed and synthesized that form NPs for a wide range of temperatures with CMT below RT but their Ttcan be tuned to slightly under body temperature such that upon subcutaneous or intramuscularAttorney Docket No.028193-0045-WO01 injection as a colloidal suspension, the NPs undergo LCST phase separation and form a depot that enables sustained release of the NPs with their drug payload into systemic circulation. Table 2: Complex library of EG3 / EG2 - EG2 / EG1 diblock POEGMA with some random copolymerization in either of the individual block or in both the blocks.Determined byaSEC-MALS,bDLS,cSAXS; n / m: not measured. The Tt and CMT are reported at 100 μM. Decreasing the hydrophilicity of the hydrophilic block

[0141] To get highly tunable and unique diblock POEGMA compositions that have CMT below RT and Ttslightly under body temperature for sustained release of NPs from a depot, the hydrophilicity of the hydrophilic block was decreased while still maintaining the amphiphilicity of the diblock. The second set, represented by rB series, in complex diblocks, has the hydrophilic block as a random copolymer of EG3 and EG2, and the hydrophobic block as either just EG1 or a random copolymer of EG2 and EG1. For simplicity, the physical characterization data is shown for only rB-2 diblock (FIG.5A) and the data for other polymers is summarized in Table 2. Additionally, the CTA-end group was removed from the rB-2 diblock to show that this small hydrophobic end-group has no significant impact on the self-assembly of the diblock and that the self-assembly is driven by the hydrophilicity differences between the two blocks.

[0142] First, the thermal phase behavior of polymer rB-2 after CTA-end group removal at various concentrations is shown in FIG.5B, exhibiting Tt= 26 °C and completely reversible phase transition behavior with some hysteresis. The Ttvalues for the rB series before their CTA removal are summarized in Table 2 proving that the Ttcan indeed be tuned by decreasing the hydrophilicity of the hydrophilic block, exhibiting high tunability of the system. Second, DLS dataAttorney Docket No.028193-0045-WO01 for rB-2 polymer, both before and after CTA-end group removal, revealed stable self-assembly for various concentrations below their Ttwith a convenient CMT below RT (FIG.5C and FIG. 5D). The normal size distribution with a small hydrodynamic diameter of 25 ± 8 at RT demonstrates tight self-assembly of polymer rB-2. The self-assembly was additionally studied by SAXS (FIG.5E and FIG.5F).

[0143] To characterize the structurally complex rB-series further, the phase behavior of polymer rB-2 was examined above its Tt. The polymer was covalently labelled with a green fluorophore and visualized at 30 °C with a confocal microscope (FIG.5G). The confocal microscopy revealed an arrested network-like structure for rB-2 above its Ttas compared to the classic liquid droplets for polymers exhibiting LCST phase behavior. Not shown here but a control diblock polymer that exhibit classic LCST phase behavior shows spherical droplets that fuse with time to minimize the surface tension. The unique phase behavior of rB-2 polymer was further evaluated by fluorescence recovery after photobleaching (FRAP) technique as compared to a control polymer. The diblock polymer A, from the simple diblock library, that shows classic LCST phase behavior and has a similar Ttwas selected as an appropriate control for the FRAP experiment. For both the labelled polymers, a few spots were locally bleached using the laser and the fluorescence recovery afterwards was monitored. The fluorescence intensity of the control polymer was quickly recovered, FIG.5H and FIG.5I, whereas the fluorescence intensity of polymer rB-2 stayed the same as after bleaching revealing that the dynamic molecular exchange in rB-2 is arrested and the coacervate is more solid-like.

[0144] The first system described includes POEGMA diblock copolymers that are micelles at body temperature, so that they can be injected systemically as a colloidal suspension and circulate as NPs in the blood stream, with drugs that are either physically encapsulated or covalently conjugated in their core. The second system described includes NPs that have a micelle to bulk coacervate Ttthat is between room and body temperature, so that upon injection under the skin or intramuscularly as a colloidal suspension, the micelles undergo LCST phase separation and form a depot that enables sustained release of the NPs with their drug payload into systemic circulation.

[0145] The library is categorized into three subsets depending on the complexity of the individual blocks in the diblock copolymer. First, it was established that even the difference of a single EG unit in each block imparts sufficient amphiphilicity to a subset of these block copolymers to drive their self-assembly into NPs. It was found that the Ttof non-self-assembling block POEGMA or random coPOEGMA remain concentration dependent, whereas the Ttof block POEGMA becomes concentration independent when polymer self-assembles intoAttorney Docket No.028193-0045-WO01 micelles— highlighting a shift in thermodynamic parameters. Additionally, the Ttof non-self- assembling block POEGMA or random coPOEGMA is positively correlated with the total hydrophilic monomer weight fraction whereas the Ttof micelle forming block POEGMA tend to lose that positive correlation. Example 2 Triblock POEGMA Copolymers

[0146] Synthesis of triblock POEGMA copolymers: ABA-type POEGMA tri-block copolymers were also synthesized using RAFT polymerization, but a di-functionalized chain transfer agent (diCTA) was first synthesized to extend the polymer chain from CTA in two directions and hence avoiding the need to do three sequential reactions and reducing the overall poly-dispersity index (PDI) of the polymer. For diCTA synthesis, CTA and ethylene glycol (Sigma Aldrich 324558, CAS 107-21-1) were suspended in dimethylformamide (DMF) (CAS 68-12-2). EDC hydrochloride (Sigma Aldrich 341006, CAS 25952-53-8), and 4-dimethylamino pyridine (DMAP) (Sigma Aldrich 8.51055, CAS 1122-58-3) were added on ice bath, and reaction was purged with N2 for 30 minutes. To initiate the reaction, reaction vessel was moved to room temperature (Scheme 2) and the reaction was continued overnight. Progress of the reaction was monitored by thin layer chromatography (TLC) and the crude product was concentrated and purified on silica column with eluent slowly changed from dichloromethane (DCM) to ethyl acetate / DCM (v / v= 1 / 10). Small fractions were collected while monitoring the presence of diCTA in each fraction by TLC and fractions with no contamination of CTA were combined. Purified diCTA were dried under high vacuum yielding red solid that was characterized by NMR.Scheme 2: Synthesis scheme of di-functional chain transfer agent – diCTA.Attorney Docket No.028193-0045-WO01

[0147] ABA-type tri-block POEGMA copolymers were synthesized using a combination of EG3, EG2, and ethylene glycol methyl ether methacrylate (EG1) (Sigma Aldrich 415332, CAS 6976-93-8). First, inhibitor-free monomer for block “B”, di-CTA, and AIBN were suspended in toluene over cold bath, purged with N2for 30 minutes, and initiated RAFT polymerization reaction at 70°C. The reaction time (2 - 4 h) was decided based on the desired block length. This first reaction resulted in polymer B with cta-groups still present on both ends of the polymer (cta-(B)n-cta), which was purified by three rounds of hexane precipitation. The purified polymer was vacuum dried for storage or to be used as a macromolecular di-functional CTA in further synthesis of the triblock. Monomer for blocks “A”, macro diCTA (polymer cta-(B)n-cta) and AIBN were resuspended in toluene and RAFT polymerization was again initiated at 70°C. Similar hexane precipitation was employed to obtain pure ABA-type triblock polymer (cta-(A)m-(B)n- (A)m-cta) after this second reaction (Scheme 3).

[0148] At last, to ensure that the hydrophobic cta-end groups on polymer do not interfere with the self-assembly of block copolymers, these cta groups were removed by treatment of polymers with excess AIBN and lauroyl peroxide (LPO) (Sigma Aldrich 290785, CAS 105-74-8) at 80°C for 2 – 4 h or until the pink color completely disappears (Scheme 3, step 3).Scheme 3: Synthesis scheme of ABA-type triblock POEGMA and cta-end group removal.

[0149] Triblock POEGMA polymers were characterized as described above in Example 1 regarding molecular weight, phase transition behavior, and nanoparticle assembly (FIG.6A, FIG.6B, FIG.6C, and FIG.6D).Attorney Docket No.028193-0045-WO01

[0150] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosed technology, may be made without departing from the spirit and scope thereof.

[0151] For reasons of completeness, the following Embodiments are provided.

[0152] Clause 1. A block copolymer comprising: a first block (A), wherein the first block comprises recurring units of formula (I)(I), wherein x is 2 or 3; and a second block (B), wherein the second block comprises recurring units of formula (II)(II), wherein y is 1 or 2.

[0153] Clause 2. The block copolymer of clause 1, wherein the recurring unit of formula (I) has a degree of polymerization of 10 to 300 and the recurring unit of formula (II) has a degree of polymerization of 10 to 300.

[0154] Clause 3. The block copolymer of clause 1 or 2, wherein: the recurring units of formula (I) comprise a unit of x is 2 at a degree of polymerization of 0 to 100 and a unit of x is 3 at a degree of polymerization of 20 to 250; and the recurring units of formula (II) comprise a unitAttorney Docket No.028193-0045-WO01 of y is 1 at a degree of polymerization of 0 to 100 and a unit of y is 2 at a degree of polymerization of 20 to 250.

[0155] Clause 4. The block copolymer of any one of clauses 1-3, wherein x is 3 and y is 2.

[0156] Clause 5. The block copolymer of clause 4, comprising the first block and the second block individually at greater than 25% of the total polymer length in terms of degree of polymerization.

[0157] Clause 6. The block copolymer of any one of clauses 1-3, wherein x is 3 and the second block comprises a random copolymer including recurring units of formula (II).

[0158] Clause 7. The block copolymer of any one of clauses 1-6, having a transition temperature (Tt) of greater than 37 °C at a concentration below 200 μM.

[0159] Clause 8. The block copolymer of any one of clauses 1-6, having a Ttof less than 37 °C at a concentration above 10 μM.

[0160] Clause 9. The block copolymer of any one of clauses 1-6, having a Ttof about 22 °C to about 46 °C at a concentration of about 10 μM to about 200 μM.

[0161] Clause 10. The block copolymer of any one of clauses 1-9, having a critical micelle temperature (CMT) of about 15 °C to about 45 °C at a concentration of about 1 μM to about 200 μM.

[0162] Clause 11. The block copolymer of any one of clauses 1-10, having a Ttof greater than 37 °C and a CMT of less than 37 °C at a concentration of about 1 μM to about 200 μM.

[0163] Clause 12. The block copolymer of any one of clauses 1-10, having a Ttof less than 37 °C and a CMT of less than 25 °C at a concentration of about 1 μM to about 200 μM.

[0164] Clause 13. The block copolymer of any one of clauses 1-12, having a number average molecular weight of about 5,000 Daltons (Da) to about 100,000 Da as measured by SEC-MALS.

[0165] Clause 14. The block copolymer of any one of clauses 1-13, wherein the block copolymer is an A-B diblock copolymer or an A-B-A triblock copolymer.

[0166] Clause 15. The block copolymer of any one of clauses 1-14, wherein the block copolymer is A10-300-B10-300or A10-250-B10-200-A10-250.

[0167] Clause 16. The block copolymer of any one of clauses 1-15, wherein the block copolymer has a reduced immune response relative to polyethylene glycol (PEG).

[0168] Clause 17. The block copolymer of any one of clauses 1-16, wherein the block copolymer does not induce an anti-POEGMA antibody response.

[0169] Clause 18. The block copolymer of any one of clauses 1-17, wherein the block copolymer is not reactive with pre-existing anti-PEG antibodies in a subject.Attorney Docket No.028193-0045-WO01

[0170] Clause 19. A composition comprising: a plurality of block copolymers according to any one of clauses 1-18 self-assembled into a particle; and a drug encapsulated within the particle.

[0171] Clause 20. The composition of clause 19, wherein the drug comprises a peptide- based drug, a hydrophobic chemotherapeutic, or a combination thereof.

[0172] Clause 21. The composition of clause 19 or 20, wherein the drug comprises doxorubicin (DOX), paclitaxel (PTX), tamoxifen (TAM), methotrexate, camptothecin (CPT), maytansine, auristatin, a derivative thereof, or a combination thereof.

[0173] Clause 22. The composition of any one of clauses 19-21, further comprising a pharmaceutically acceptable excipient.

[0174] Clause 23. The composition of any one of clauses 19-22, wherein the particle has a hydrodynamic diameter of about 20 nm to about 120 nm.

[0175] Clause 24. The composition of any one of clauses 19-23, wherein the particle is a micelle.

[0176] Clause 25. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of clauses 19-24.

[0177] Clause 26. The method of clause 25, wherein the disease or disorder is a cancer, an autoimmune disease, or a metabolic disorder.

[0178] Clause 27. The method of clause 25 or 26, wherein the composition is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally.

[0179] Clause 28. The method of any one of clauses 25-27, wherein the block copolymer has a Ttof greater than a body temperature of the subject and a CMT below the body temperature of the subject.

[0180] Clause 29. The method of any one of clauses 25-27, wherein the composition is administered at a temperature below the block copolymer’s Ttand above the block copolymer’s CMT to an area of the subject that has a temperature above the block copolymer’s Tt.

[0181] Clause 30. A method of delivering a drug to a subject in need thereof, the method comprising systemically or locally administering to the subject the composition of any one of clauses 19-24, wherein the drug is released from the particle following administration.

[0182] Clause 31. A method of delivering a scaffold to a subject in need thereof, the method comprising administering the block copolymer of any one of clauses 1-18 to a location of the subject, whereupon administration to the location, the block copolymer exhibits phase transition behavior defined by a Tt, the block copolymer forming a porous network above the Tt.

Claims

Attorney Docket No.028193-0045-WO01 CLAIMS What is claimed is:

1. A block copolymer comprising: a first block (A), wherein the first block comprises recurring units of formula (I)wherein x is 2 or 3; and a second block (B), wherein the second block comprises recurring units of formula (II)wherein y is 1 or 2.

2. The block copolymer of claim 1, wherein the recurring unit of formula (I) has a degree of polymerization of 10 to 300 and the recurring unit of formula (II) has a degree of polymerization of 10 to 300.

3. The block copolymer of claim 1, wherein: the recurring units of formula (I) comprise a unit of x is 2 at a degree of polymerization of 0 to 100 and a unit of x is 3 at a degree of polymerization of 20 to 250; and the recurring units of formula (II) comprise a unit of y is 1 at a degree of polymerization of 0 to 100 and a unit of y is 2 at a degree of polymerization of 20 to 250.Attorney Docket No.028193-0045-WO01 4. The block copolymer of claim 1, wherein x is 3 and y is 2.

5. The block copolymer of claim 4, comprising the first block and the second block individually at greater than 25% of the total polymer length in terms of degree of polymerization.

6. The block copolymer of claim 1, wherein x is 3 and the second block comprises a random copolymer including recurring units of formula (II).

7. The block copolymer of claim 1, having a transition temperature (Tt) of greater than 37 °C at a concentration below 200 μM.

8. The block copolymer of claim 1, having a Ttof less than 37 °C at a concentration above 10 μM.

9. The block copolymer of claim 1, having a Ttof about 22 °C to about 46 °C at a concentration of about 10 μM to about 200 μM.

10. The block copolymer of claim 1, having a critical micelle temperature (CMT) of about 15 °C to about 45 °C at a concentration of about 1 μM to about 200 μM.

11. The block copolymer of claim 1, having a Ttof greater than 37 °C and a CMT of less than 37 °C at a concentration of about 1 μM to about 200 μM.

12. The block copolymer of claim 1, having a Ttof less than 37 °C and a CMT of less than 25 °C at a concentration of about 1 μM to about 200 μM.

13. The block copolymer of claim 1, having a number average molecular weight of about 5,000 Daltons (Da) to about 100,000 Da as measured by SEC-MALS.

14. The block copolymer of claim 1, wherein the block copolymer is an A-B diblock copolymer or an A-B-A triblock copolymer.Attorney Docket No.028193-0045-WO01 15. The block copolymer of claim 1, wherein the block copolymer is A10-300-B10-300or A10-250- B10-200-A10-250.

16. The block copolymer of claim 1, wherein the block copolymer has a reduced immune response relative to polyethylene glycol (PEG).

17. The block copolymer of claim 1, wherein the block copolymer does not induce an anti- POEGMA antibody response.

18. The block copolymer of claim 1, wherein the block copolymer is not reactive with pre- existing anti-PEG antibodies in a subject.

19. A composition comprising: a plurality of block copolymers according to claim 1 self-assembled into a particle; and a drug encapsulated within the particle.

20. The composition of claim 19, wherein the drug comprises a peptide-based drug, a hydrophobic chemotherapeutic, or a combination thereof.

21. The composition of claim 19, wherein the drug comprises doxorubicin (DOX), paclitaxel (PTX), tamoxifen (TAM), methotrexate, camptothecin (CPT), maytansine, auristatin, a derivative thereof, or a combination thereof.

22. The composition of claim 19, further comprising a pharmaceutically acceptable excipient.

23. The composition of claim 19, wherein the particle has a hydrodynamic diameter of about 20 nm to about 120 nm.

24. The composition of claim 19, wherein the particle is a micelle.

25. A method of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition according to claim 19.Attorney Docket No.028193-0045-WO01 26. The method of claim 25, wherein the disease or disorder is a cancer, an autoimmune disease, or a metabolic disorder.

27. The method of claim 25, wherein the composition is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally.

28. The method of claim 25, wherein the block copolymer has a Tt of greater than a body temperature of the subject and a CMT below the body temperature of the subject.

29. The method of claim 25, wherein the composition is administered at a temperature below the block copolymer’s Ttand above the block copolymer’s CMT to an area of the subject that has a temperature above the block copolymer’s Tt.

30. A method of delivering a drug to a subject in need thereof, the method comprising systemically or locally administering to the subject the composition according to claim 19, wherein the drug is released from the particle following administration.

31. A method of delivering a scaffold to a subject in need thereof, the method comprising administering the block copolymer of claim 1 to a location of the subject, whereupon administration to the location, the block copolymer exhibits phase transition behavior defined by a Tt, the block copolymer forming a porous network above the Tt.

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