Thermo-responsive polymer for use in biological applications and method of making and using the same

Abstract

Thermo-responsive polymers that can be used to provide extracellular matrices for cell / organoid growth and / or for using in isolating / purifying biomolecules. Also disclosed are methods for making and using the polymers. The thermo-responsive polymers exhibit the ability to transition between different phases with changes in temperature and thus provide the ability to grow cells / organoids and then be easily separated from the cells / organoids without destroying them. The thermo-responsive polymer facilitates isolating biomolecules from contaminants that might be produced in biological assays.

Description

Docket No. TP385946WO1THERMO-RESPONSIVE POLYMER FOR USE IN BIOLOGICAL APPLICATIONS AND METHOD OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 696,551, filed September 19, 2024, the disclosure of which is considered part of, and incorporated in its entirety by reference in the disclosure of this application.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on September 16, 2025, is named TP385946WO1 , and is 29,919 bytes in size.FIELD

[0003] The present disclosure is directed to a thermo-responsive polymer, compositions comprising the thermo-responsive polymer, and methods of making and using the same in biological applications, such as cell culture, organoid synthesis, and biomolecule isolation / purification.BACKGROUND

[0004] Commonly used, commercially available extracellular matrices (ECM) typically are animal-derived formulations comprising numerous, poorly-defined biological components. ECM manufacturing consistency is complicated by the complexity of these formulations, which results in experimental inconsistencies due to lot-to-lot variation. The poorly-defined, animal-derived ECM compositions limit the applications of cells and organoids that are produced using such ECMs. Additionally, the application and removal of reagents associated with these poorly- defined, animal-derived ECM compositions is cumbersome. Upon application to cells, conventional ECM reagents crosslink uncontrollably to form hydrogels of varying consistencies and stiffness. The removal of cell models from such ECMs requires tedious and potentially destructive processes, such as mechanical trituration. As such, there exists a need for new materials that can be used for ECMs that allow for facile cell purification.Docket No. TP385946W01

[0005] Recent advances in messenger RNA (m NA) therapeutics are expected to provide opportunities for new treatments for disease prevention. These advances opened the way for the first approved mRNA-based vaccines against COVID-19. mRNA therapies are attractive because they can be easily modified and rapidly developed with high efficacy; however, many challenges remain in their large-scale purification. T raditional chromatography techniques that implement porous resins are limited by low binding capacity. RNA molecules typically have a hydrodynamic size that is 10-100 times greater than traditional biomolecules and thus cannot access the pores of many types of resins used in standard column chromatography purifications. Because typical mRNA therapeutics have an even larger hydrodynamic size (e.g., about 3-5 kb), new emerging modalities such as self-amplifying mRNA (>10 kb) will pose even greater challenges for resin-based purifications. As such, there is a need for materials that can improve RNA molecule (and other biomolecule) purification, particularly in resin-based purification methods.SUMMARY

[0006] Disclosed herein is a thermo-responsive polymer, having a structure according to Formula I as disclosed herein wherein, for Formula I, Z represents a coupling component; TG is a terminating group; each X and each Y, for each occurrence of X and Y, independently is selected from an acryloyl-derived monomer having a structure according to Formula A, or an N- isopropylacrylamide-derived monomer having a structure according to Formula B, provided that (i) at least one X is the acryloyl-derived monomer and at least one Y is the N- isopropylacrylamide-derived monomer; or (ii) at least one X is the N-isopropylacrylamide- derived monomer and at least one Y is the acryloyl-derived monomer; each n, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + mtotai = p; each m, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + mtotai = p; and p is an integer selected from 2 to 1600; wherein Formula A is as described herein and wherein, for Formula A, each X1independently is (i) NH or O if q' is 1 , or (ii) is NH2if q' is 0; each R" independently is H, aliphatic, or OH; each q independently is an integer selected from 0 to 20; and q' is 0 or 1 ; and Formula B is as described herein.

[0007] In some aspects, a composition is disclosed wherein the composition comprises one or more cells; and the thermo-responsive polymer according to aspects of the present disclosure, wherein the thermo-responsive polymer is coupled to a cell-binding peptide.

[0008] In some other aspects, disclosed herein is a composition, comprising: one or moreDocket No. TP385946W01 biological components; and the thermo-responsive polymer according to aspects of the present disclosure wherein the thermo-responsive polymer is coupled to the biomolecule-coupling component; or a thermo-responsive polymer according to other aspects of the disclosure.

[0009] Also disclosed is a method, comprising: exposing a cell to a thermo-responsive polymer according to the present disclosure at a first temperature to provide a seeding composition comprising the cell dispersed in the thermo-responsive polymer, wherein the first temperature is a temperature below the critical temperature point of the thermo-responsive polymer; applying the seeding composition to a culture apparatus comprising a growth medium to provide a seeding layer on the growth medium; warming the culture apparatus by exposing it to a second temperature, wherein the second temperature is a temperature that is above the critical temperature point of the thermo-responsive polymer, wherein warming the culture apparatus facilitates cell growth from the seeding layer; and removing the thermo-responsive polymer from the culture apparatus by exposing the culture apparatus to the first temperature; wherein the thermo-responsive polymer is coupled to a cell-binding peptide.

[0010] Disclosed herein is a method for making the thermo-responsive polymer according to aspects of the present disclosure, the method comprising: combining a multi-armed polymer with N-isopropylacrylamide, an acryloyl monomer, and a radical initiator to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer.

[0011] In some aspects, the method for making the thermo-responsive polymer according to aspects of the present disclosure comprises: combining a chain transfer agent, a radical initiator, an acryloyl monomer, and N-isopropylacrylamide to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer; wherein (i) the chain transfer agent is a compound comprising the Z group of Formula I and a thiourea group; (ii) the acryloyl monomer and the N- isopropylacrylamide are provided at a ratio to provide a value for p ranging from 2 to 1600; and (iii) the concentration ratio of the acryloyl monomer and / or the N-isopropylacrylamide to the chain transfer agent ranges from greater than 0 to 20 molo / o.

[0012] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.Docket No. TP385946WO1BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a schematic illustration of a method for using a thermo-responsive polymer according to aspects of the present disclosure, wherein the method comprising coupling cellbinding peptides to the thermo-responsive polymer to attract peptides that become conjugated to the thermo-responsive polymer.

[0014] FIG. 2 is a schematic illustration of a method for using a thermo-responsive polymer according to aspects of the present disclosure, wherein a thermo-responsive polymer is coupled to a nucleic acid-coupling component and the resulting product is used to attract and associate with target nucleic acids that can be purified from any contaminants using the thermo- responsive polymer.

[0015] FIG. 3 is a schematic illustration of a method for coupling a thermo-responsive polymer to a nucleic acid-coupling component for use in the method illustrated in FIG. 2.

[0016] FIG. 4 is a schematic illustration of a representative method for using a thermo- responsive polymer according to aspects of the disclosure to isolate and purify a biomolecule (e g., a nucleic acid) from contaminants in a biological sample.

[0017] FIG. 5 is a schematic illustration of a method for making a thermo-responsive polymer according to aspects of the present disclosure, wherein the thermo-responsive polymer comprises a multi-armed polymer.

[0018] FIG. 6 is a schematic illustration of a method for making a thermo-responsive polymer according to aspects of the present disclosure, wherein protecting groups on amine functional groups of the multi-armed polymer are removed.

[0019] FIG. 7 is a schematic illustration of a method for making a thermo-responsive polymer according to aspects of the present disclosure, wherein the thermo-responsive polymer comprises a functional group for use in adding a coupling component to the polymer.Docket No. TP385946W01

[0020] FIG. 8 is a chromatogram obtained from using gel permeation chromatography (GPC) to analyze a four-armed acrylamide polymer as described herein, wherein different degrees of polymerization were utilized.

[0021] FIG. 9 is a chromatogram obtained from using light scattering detection to analyze a four-armed thermo-responsive polymer as described herein, wherein different degrees of polymerization were utilized.

[0022] FIG. 10 shows spectra obtained from using1H nuclear magnetic resonance (NMR) to analyze the thermo-responsive polymer illustrated in the figure in protected and deprotected forms.

[0023] FIG. 11 is a graph of transition temperature as a function of molo / o of butyl acrylate showing the different transition temperatures that can be obtained by modifying the mole o / o of butyl acrylate used to make a thermo-responsive polymer.

[0024] FIG. 12 is a chromatogram showing the change in elution time for thermo-responsive polymers having different molecule weights.

[0025] FIGS. 13A and 13B are1H-NMR spectra of a thermo-responsive polymer according to an aspect of the present disclosure wherein a functional group of the thermo-responsive polymer is coupled to a maleimide group; FIG. 13A shows the spectrum of the polymer before maleimide coupling and FIG. 13B shows the spectrum of the polymer after maleimide coupling.

[0026] FIGS. 14A and 14B are1H-NMR spectra of a thermo-responsive polymer according to an aspect of the present disclosure wherein a maleimide group of the polymer becomes coupled to a cell-binding peptide; FIG. 13A shows the spectrum of the polymer before the cell-binding peptide is bound and FIG. 13B shows the spectrum of the polymer after the cell-binding peptide is bound.

[0027] FIGS. 15A and 15B are microscopic images of an adult liver organoid (ALO) generated using a Geltrex™-derived ECM (FIG. 15A) and an ALO generated using a thermo-responsive polymer-derived ECM according to aspects of the present disclosure; as can be seen byDocket No. TP385946W01 comparing the images, the thermo-responsive polymer-derived ECM provides smaller sized ALOs.

[0028] FIGS. 16A-16C show results obtained from comparing organoid size and number compared with a Geltrex™ ECM, wherein FIGS. 16A and 16B show size distribution results from analyzing the size distributions of ALOs generated using ECMs comprising thermo- responsive polymers comprising a multi-armed polymer as compared with the Geltrex™ ECM (wto / o values listed in FIGS. 16A and 16B correspond to total wto / o of the thermo-responsive polymer in solution); and FIG. 16C shows the results from measuring the average number of organoids.

[0029] FIGS. 17A-17E are microscopic images of ECMs from FIG. 16 showing that the ECMs made using the thermo-responsive polymers according to aspects of the disclosure (FIGS. 17A- 17C) can generate organoids having a similar average size, whereas organoids in the Geltrex™ ECM (FIG. 17D) and the NEO ECM (FIG. 17E) have statistically different sizes.

[0030] FIGS. 18A and 18B are microscopic images showing growth of adult liver organoids and their release from thermo-responsive polymer according to aspects of the disclosure, wherein organoid release can be achieved within 1 minute caused by temperature-mediated phase changes and this rapid release allows for isolation of organoids grown with ECMs comprising the polymers.

[0031] FIGS. 19A and 19B are bar graphs obtained from using ECMs comprising the thermo- responsive polymer according to aspects of the present disclosure for PCR gene expression for the stem cell markers, LGR5 and SOX9, and which show that the same cell model is being grown in vitro between EHS-BME and the thermo-responsive polymer.

[0032] FIGS. 20A-20C are bar graphs obtained from using ECMs comprising the thermo- responsive polymer according to aspects of the present disclosure for PCR gene expression for the cell specific markers, HNFa (a hepatocyte marker), KRT-19 (a cholangiocyte marker), and albumin (a hepatocyte marker), establishing that the same cell model is being grown in vitro between EHS-BME and the thermo-responsive polymer.Docket No. TP385946WO1

[0033] FIGS. 21A-21C are microscopic images taken before and after organoid recovery, wherein FIG. 21 A shows the morphology of ALO organoids at day 6, embedded in TRP, FIG. 21 B shows the almost immediate (less than 1 minute) polymerization of the TRP, releasing organoids form the TRP and starting into cell medium suspension, and FIG. 21 C shows that, within one minute after the addition of cold cell media or cold DPBS, the gel was completely unpolymerized and all organoids are completely suspended in cell medium.

[0034] FIGS. 22A-22C are UV-vis spectra taken at different reaction times (FIG. 22A = hour zero; FIGS. 22B and 22C = hour 20) that confirm a successful click chemistry reaction has occurred between azide functional group of a thermo-responsive polymer (with varying degrees of polymerization) according to aspects of the present disclosure and a DBCO moiety of a nucleic acid-coupling component.

[0035] FIGS. 23A and 23B are UV-vis spectra showing results after subjecting a thermo- responsive polymer-nucleic acid-coupling component conjugate to a temperature change that facilitates precipitation of the conjugate, wherein FIG. 22B shows the spectrum without baseline subtraction for the supernatant.DETAILED DESCRIPTION

[0036] Overview of Terms

[0037] The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

[0038] Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, steps described sequentially may in some cases beDocket No. TP385946W01 rearranged or performed concurrently. Additionally, the description sometimes uses terms like "produce" or "provide" to describe the disclosed methods. These terms are high-level abstractions of the actual steps that are performed. The actual steps that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

[0039] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[0040] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term "about." Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and / or limits of detection under standard test conditions / methods and in some aspects encompasses a range up to ± 15o / o of that numerical value, unless the context clearly dictates otherwise. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word "about" is recited. Furthermore, not all alternatives recited herein are equivalents.

[0041] Certain functional group terms used herein include a symbol which is used to show how the defined functional group attaches to, or within, the compound to which it is bound. Also, a dashed bond (i.e. , ") as used in certain formulas described herein indicates an "optional" bond to a substituent or atom of the formula other than hydrogen in the sense that the bond (and in some embodiments, the substituent) may or may not be present. In any formulas comprising a dashed bond, if the optional bond and / or any corresponding substituent is not present, then the valency requirements of any atom(s) bound thereto is completed by a bond to a hydrogen atom.Docket No. TP385946W01

[0042] The symbol " " is used to indicate a bond disconnection in abbreviated structures / formulas provided herein. A person of ordinary skill in the art recognizes that the definitions provided below and the compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever a functional group or other atom is not illustrated. For example, a phenyl ring that is drawn as pF comprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the "a" carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0043] T o facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided. o

[0044] Acryloyl: I , wherein the dashed line represents a methyl group that may or may not be present.

[0045] Activated Ester: A functional group that is susceptible to nucleophilic attack. In some aspects of the disclosure, activation can be imparted by modifying an acyl or alkoxy portion of a normal ester, such as by adding electronegative substituents. In exemplary aspects of the disclosure, an activated ester can comprise a carboxyl group attached to a succinimide via the oxygen bound via a single bond to the carbonyl carbon.

[0046] Aldehyde: -C(O)H.

[0047] Aliphatic: A hydrocarbon group having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1.25), or one to ten carbon atoms (C1-10), and which includes alkanes (or alkyl), alkenes (or alkenyl), alkynes (or alkynyl), including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo andDocket No. TP385946W01 position isomers as well. Aliphatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0048] Alkoxy: -O-aliphatic, such as -O-alkyl, -O-alkenyl, -O-alkynyl; with exemplary embodiments including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy (wherein any of the aliphatic components of such groups can comprise no double or triple bonds, or can comprise one or more double and / or triple bonds). Alkoxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0049] Amide: -C(O)NRbRcor -NRbC(O)R0wherein each of Rband R° independently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0050] Amino: -NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group, and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0051] Aromatic: A cyclic, conjugated group or moiety of, unless specified otherwise, from 5 to15 ring atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized TT-electron system.Typically, the number of out of plane TT-electrons corresponds to the Huckel rule (4n + 2). The point of attachment to the parent structure typically is through an aromatic portion of the condensed ring system. For example,However, in certain examples, context or express disclosure may indicate that the point of attachment is through a non-aromatic portionDocket No. TP385946W01 of the condensed ring system. For example,. An aromatic group or moiety may comprise only carbon atoms in the ring, such as in an aryl group or moiety, or it may comprise one or more ring carbon atoms and one or more ring heteroatoms comprising a lone pair of electrons (e.g. S, O, N, P, or Si), such as in a heteroaryl group or moiety. Aromatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0052] Aroxy: -O-aromatic. Aroxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0053] Azo: -N=NRawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Azo groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0054] Biological Component: A biomolecule (e.g., nucleic acid, protein, peptide, antibody, or the like) or a cellular structure (e.g., a cell or an organoid).

[0055] Click Chemistry: Chemical synthetic methods for forming covalent bonds using compounds that can be joined together using efficient reagent conditions and that can be performed in benign solvents or solvents that can be removed or extracted using facile methods, such as evaporation, extraction, or distillation.

[0056] Clickable Functional Group: A functional group that can be used in click chemistry to form covalent bonds between a thermo-responsive polymer and a biological component.

[0057] Carbamate: -OC(O)NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbamate groups can be substituted with one or more groups other thanDocket No. TP385946WO1 hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0058] Carbonate: -OC(O)ORa, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbonate groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. In independent embodiments, Racan be hydrogen.

[0059] Carboxyl: -C(O)OH.

[0060] Carboxylate: -C(0)O or salts thereof, wherein the negative charge of the carboxylate group may be balanced with an M+counterion, wherein M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]o.5, or [Ba2+]05.

[0061] Coupling Component: A functional group or molecule that facilitates binding or associating the thermo-responsive polymer to / with a biological component. In aspects of the disclosure where the coupling component facilitates associating the thermo-responsive polymer with a biological component, the biological component can be a cell or an organoid and the association can involve a physical association between the thermo-responsive polymer and the cell or the organoid.

[0062] Cyano: -CN.

[0063] Degree of Polymerization: The number of monomer units in a polymer. In the context of the present disclosure, when discussing block polymers comprising repeat units of different monomers, the degree of polymerization is represented by the formula ntotai + mtotai = P (wherein n is the number of monomer X units and m is the number of monomer Y units, wherein X and Y are as described herein for Formula I) is intended to correspond to the number of monomer units and not to the number of repeat units of monomer X + monomer Y units.Docket No. TP385946W01

[0064] Disulfide: -SSRa, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Disulfide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0065] Dithiocarboxylic: -C(S)SRawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Dithiocarboxylic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0066] Ester: -C(O)ORaor -OC(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0067] Ether: -aliphatic-O-aliphatic, -aliphatic-O-aromatic, -aromatic-O-aliphatic, or -aromatic- O-aromatic, including any polymers thereof having repeats of any such groups (e.g., polyalkene oxide compounds). Ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0068] Halo (or halide or halogen): Fluoro, chloro, bromo, or iodo. In some embodiments, halo can also include astatine.

[0069] Haloaliphatic: An aliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. Haloaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0070] Haloheteroaliphatic: A heteroaliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such asDocket No. TP385946WO1 fluoro, bromo, chloro, or iodo. Haloheteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0071] Heteroatom: An atom other than carbon or hydrogen, such as (but not limited to) oxygen, nitrogen, sulfur, silicon, boron, selenium, or phosphorous. In particular disclosed embodiments, such as when valency constraints do not permit, a heteroatom does not include a halogen atom.

[0072] Heteroaliphatic: An aliphatic group comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized forms thereof within the group. Alkoxy, ether, amino, disulfide, peroxy, and thioether groups are exemplary (but non-limiting) examples of heteroaliphatic. Heteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0073] Ketone: -C(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0074] Maleimide: A chemical group having a core structure, wherein the core structure can comprise substituents bound to the ring carbon atoms.

[0075] Organic Functional Group: A functional group that may be provided by any combination of aliphatic, heteroaliphatic, aromatic, haloaliphatic, and / or haloheteroaliphatic groups, or that may be selected from, but not limited to, aldehyde; aroxy; acyl halide; halogen; nitro; cyano; azide; carboxyl (or carboxylate); amide; ketone; carbonate; imine; azo; carbamate; hydroxyl; thiol; sulfonyl (or sulfonate); oxime; ester; thiocyanate; thioketone; thiocarboxylic acid; thioester; dithiocarboxylic; phosphonate; phosphate; silyl ether; sulfinyl; sulfonamide; thial; or combinations thereof. Organic functional groups can be substituted with one or more groupsDocket No. TP385946W01 other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0076] Oxime: -CRa=NOH, wherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Oxime groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0077] Peroxy: -O-ORawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Peroxy groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0078] Phosphate: -O-P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]o.s, [Mg2+]o.s, or [Ba2+]o.s. The Ragroups of the phosphate can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0079] Phosphonate: -P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5. The Ragroups of the phosphonate group can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.Docket No. TP385946W01

[0080] Silyl Ether: -OSiRaRbRc, wherein each of Ra, Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Silyl ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0081] Sulfinyl: -S(O)Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfinyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0082] Sulfonyl: -SC>2Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0083] Sulfonamide: -SC>2NRbRcor-N(Rb)SC>2Rc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonamide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0084] Sulfonate: -SO 3, wherein the negative charge of the sulfonate group may be balanced with an M+counter ion, wherein M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]os, [Mg2+]os, or [Ba2+]o5-

[0085] Thial: -C(S)H.

[0086] Thiocarboxylic acid: -C(O)SH, or -C(S)OH.

[0087] Thiocyanate: -S-CN or -N=C=S.Docket No. TP385946WO1

[0088] Thioester: -C(O)SRaor -C(S)ORawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0089] Thioether: -S-aliphatic or -S-aromatic, such as -S-alkyl, -S-alkenyl, -S-alkynyl, -S-aryl, or -S-heteroaryl; or -aliphatic-S-aliphatic, -aliphatic-S-aromatic, -aromatic-S-aliphatic, or - aromatic-S-aromatic. Thioether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0090] Thioketone: -C(S)Rawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0091] Terminating Group: A functional group that is used to terminate polymerization of monomeric components into a thermo-responsive polymer and that becomes covalently bound to a monomer component positioned at an end of the polymer chain. In some aspects of the disclosure, the terminating group can be provided by a radical initiator used in the polymerization step.

[0092] Thermo-Responsive Polymer: A polymer having a structure according to Formula I described herein and that exhibits the ability to change phase with particular changes in temperature.

[0093] Introduction

[0094] Current methods for cell culture typically rely on using extra cellular matrices (or ECMs) that are prepared from animal-derived formulations, which can limit the applications of cell / organoids grown in such ECMs. In addition to being derived from animals, these conventional formulations often include undefined biological components / contaminates that canDocket No. TP385946W01 have undesired effects on experimental results and also complicate isolating pure cells / organoids. Additionally, upon exposure to cells and / or organoids, the conventional ECM materials can exhibit levels of crosslinking that cannot be controlled, which can negatively impact the physical properties of the ECM (e.g., producing stiff hydrogels). Isolation of cells or organoids from these conventional ECM materials also can require extended incubation on ice and mechanical trituration, techniques which can disrupt or damage the cells / organoids.

[0095] Difficulties with isolating and separating large biomolecules, such as nucleic acids (particularly mRNA), also present an opportunity for developing new materials for such applications. For example, large biomolecules, such as mRNA and other nucleic acids, are often not compatible with traditional affinity chromatography methods used to isolate and / or purify such biomolecules because the resins used in such methods comprise smaller pore sizes that cannot provide adequate binding capacity for larger biomolecules targets. As such, arriving at a material that can be used for separating such targets from other materials (e.g., separating mRNA from transcription-related contaminates, such as DNA, enzymes, salt, and the like) without having to utilize conventional chromatography methods (e.g., pore / size exclusion-based techniques) presents an opportunity to address the need in the art for methods of preparing large biomolecule constructs needed for use in various fields, such as next-generation vaccines where self-amplifying mRNA is needed (which has a predicted particle size ten times greater than a typical 1.2 kb ssRNA molecule).

[0096] Disclosed herein are thermo-responsive polymers that can address the above- mentioned needs in the biological arts. In some aspects of the disclosure, the thermo- responsive polymers can be used in cell culturing to provide ECMs that facilitate facile isolation of cells and / or organoids. In such aspects, the thermo-responsive polymers can be used to form highly-defined and phase-reversible synthetic ECMs that comprise handles for chemical modification (e.g., adding peptides and / or other components to facilitate cell adhesion and / or growth). By using the thermo-responsive polymer to provide the ECM scaffold, cells grown on the ECM can be easily isolated by manipulating temperature to promote phase changes in the thermo-responsive polymer. In particular aspects, the thermo-responsive polymer exists in liquid form below a lower critical solution temperature (LCST) point and in solid form above the LCST. By controlling the phase of the thermo-responsive polymer using temperature, cellsDocket No. TP385946W01 grown in ECMs made from the thermo-responsive polymers can be easily isolated without having to use conventional isolation techniques (e.g., mechanical trituration).

[0097] In yet other aspects of the disclosure, the thermo-responsive polymer can be used to isolate and / or purify biomolecules. In particular disclosed aspects, the thermo-responsive polymer is used to isolate large biomolecules, such as nucleic acids (e.g., mRNA) from a mixture. Given its ability to under reversible phase transitions upon application of temperature changes, the thermo-responsive polymer according to aspects of the present disclosure can be used to bind a biomolecule and isolate it from other undesired components that might be present in a biological sample. In particular aspects, the thermo-responsive polymer is designed to comprise functional groups that facilitate binding the thermo-responsive polymer to groups that can bind to the biomolecule of interest. In representative aspects of the disclosure, the thermo-responsive polymer can be used for affinity-based purification and provide large scale production of mRNA therapeutics with higher yields and with lower production times than other resin-based purification approaches currently used in the field. As such, in some aspects, the thermo-responsive polymer disclosed herein can be used in various types of RNA enrichment approaches for the sequence specific capture and precipitation of RNA in place of existing biotinylation procedures, such as Pierce™ RNA 3'End Biotinylation Kit (Thermo Fisher Scientific). Methods disclosed herein also can provide an alternative to "pull-down" type approaches (e.g., beads that bind a target molecule and then can be spun out of solution or pulled out by magnet), such as Magnetic RNA-Protein Pull-Down Kit and Ultralink™ acrylamide- based support resins (available from Thermo Fisher Scientific). While mRNA purification is presented herein as one exemplary method for using the disclosed thermo-responsive polymer, the present disclosure is not so limited and can be applied for other types of biomolecules (e.g., DNA, proteins, peptides, antibodies, or the like).

[0098] Thermo-Responsive Polymers and Compositions Thereof

[0099] Disclosed herein is a thermo-responsive polymer component that can be used in biological applications, such as cell culturing and / or organoid development, as well as for biomolecule purification methods. In some aspects of the disclosure, the thermo-responsive polymer has a structure according to Formula I, shown below.Docket No. TP385946W01Formula I

[0100] With reference to Formula I, Z represents a coupling component, which can be a functional group or molecule that facilitates binding or associating the thermo-responsive polymer to / with a biological component. In particular aspects, Z is a functional group capable of coupling the thermo-responsive polymer to a biomolecule-coupling component or a multi-armed polymer.

[0101] The TG of Formula I is a terminating group. In particular aspects, the terminating group can be -C(Me)2CN.

[0102] With reference to Formula I, each X and each Y, for each occurrence of X and Y, independently is selected from an acryloyl-derived monomer having a structure according to Formula A, or an N-isopropylacrylamide-derived monomer having a structure according to Formula B, provided that (i) at least one X is the acryloyl-derived monomer and at least one Y is the N-isopropylacrylamide-derived monomer; or (ii) at least one X is the N-isopropylacrylamide- derived monomer and at least one Y is the acryloyl-derived monomer.

[0103] With reference to Formula I, each n, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + mtotai = p; and each m, for each occurrence, independently is an integer selected to satisfy a formula of ntotai+ mtotai = p. In these aspects, p is an integer selected from 2 to 1600. In such aspects, p represents the degree of polymerization and in representative aspects can be selected from an integer ranging from 100 to 1500, or 100 to 1400, or 100 to 1300 or 100 to 1200 or 100 to 1100, or 100 to 1000. In some particular aspects of the disclosure, each n is an integer ranging from 1 to 500, such as from 1 to 400, or 1 to 300 or 1 to 200, or 1 to 100, or 1 to 50. In some particular aspects of the disclosure, each m is an integer ranging from 1 to 500, such as from 1 to 400, or 1 to 300 or 1 to 200, or 1 to 100, or 1 to 50. In some particular aspects of the disclosure, the thermo-responsive polymer can have a molecular weight ranging from 10 kDa to 100 kDa or higher.

[0104] In the above-described aspects, Formula A isDocket No. TP385946W01Formula A wherein each X1independently is (i) NH or O if q' is 1 , or (ii) is NH2if q' is 0; each R" independently is H, aliphatic (such as alkyl, alkenyl, or alkynyl), or OH; each q independently is an integer selected from 0 to 20; and q' is 0 or 1. In particular aspects of the disclosure, q' is 1 , each X1is O, each R" is H and each q is an integer selected from 1 to 4. In particular aspects of the disclosure, q' is 0 and X1is NH2. In particular aspects of the disclosure, the compound according to Formula A is butyl acrylate, N-2-hydroxypropyl methacrylamide, methyl acrylate, ethyl acrylate, 2-hydroxyethyl methacrylate, N-propylacrylamide, N, N-(2- hydroxyethyl)acrylamide, methyl methacrylate, methyl methacrylamide, or N-butylacrylamide.

[0105] In the above-described aspects, Formula B isFormula B.

[0106] In particular aspects of the disclosure, the thermo-responsive polymer has a structure according to Formula IAFG ■■WYk J-pTGFormula IA wherein FG represents a coupling component that is a functional group capable of coupling the thermo-responsive polymer to a biomolecule-coupling component. In particular aspects of the disclosure, the FG is a clickable functional group, a reactive group, a heterobifunctional linker, or a combination thereof. In such aspects, the clickable functional group can be selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cycloocteneDocket No. TP385946W01 group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group. In representative aspects of the disclosure, the clickable functional group is an azide. In yet other aspects of the disclosure, the FG is a heterobifunctional linker that comprises an alkylene or alkylene oxide spacer and a reactive end comprising, for example, an NHS ester, an acid, a sulfo-NHS ester, a biotin group, a maleimide group, a TFP ester, an amine, an STP ester, a haloacetamido group, and the like. Exemplary FG groups that can be used include those contained in various DBCO- containing reagents sold by Vector Laboratories. In yet additional aspects of the disclosure, the FG is a reactive group, such as a maleimide, a protected amine, an activated ester, or the like.

[0107] In particular aspects of the disclosure, the FG is further bound to a biomoleculecoupling component. In particular disclosed aspects, the biomolecule-coupling component is a nucleic acid-coupling component (e.g., an RNA-coupling component) or other component capable of coupling with a protein, peptide, antibody, or the like. In particular aspects of the disclosure, the biomolecule-coupling component is a nucleic acid-coupling component and can be selected from an oligonucleotide ligand (e.g., an oligo(deoxythymidine) ligand or an oligo(deoxyadenosine) ligand). Other representative biomolecule-coupling components can include a member of a specific binding pair (e.g., biotin, streptavidin, or avidin), a group that facilitates a protein-protein interaction (e.g., a functional group, domain, or atom of a protein that promotes hydrophobic binding, van der Waals interactions, and / or salt bridges between proteins), or a member of a guest-host pair (e.g., beta-cyclodextrin derivatives, crown ethers, streptavidin, biotin, BSA, and / or combinations thereof). In representative aspects, the nucleic acid-coupling component is an oligonucleotide ligand and is an oligo(deoxythymidine) (OdT) ligand further comprising a 5'dibenzocyclooctyne (DBCO) group. In such aspects of the disclosure, the OdT is bound to the DBCO group through a linker group, such as a carbonylcontaining linker group (e.g., -C(0)[CH2]I-IOC(0)-, or amide linkages). The DBCO group of such an exemplary nucleic acid-coupling component can undergo a "click" reaction (e.g., a strain promoted alkyne azide cycloaddition) with a terminal azide group of the thermo-responsive polymer to thereby provide the polymer bearing the nucleic acid-coupling component.

[0108] In some aspects of the disclosure, the thermo-responsive polymer can have a structure according to any of the structures provided below.Docket No. TP385946WO1Docket No. TP385946WO1Docket No. TP385946WO1Docket No. TP385946W01

[0109] In yet other aspects of the disclosure, the thermo-responsive polymer can have a structure according to Formula IBFormula IB wherein MAP represents a multi-armed polymer. The MAP can comprise a multi-armed PEG- containing group or a multi-armed acrylamide-containing group. In some such aspects, a terminal X group of Formula B is attached directly or indirectly to at least one arm of the multiarmed PEG-containing group. In other aspects of the disclosure, a terminal X group of Formula IB is attached directly or indirectly to at least one arm of the multi-armed acrylamide-containing group. In some aspects of the disclosure, the multi-armed PEG-containing group is an eightarmed PEG-containing group, such as an eight-armed PEG polymer comprising a hexaglycerol core and a terminal functional group capable of binding to an X group of the thermo-responsive polymer. In other aspects of the disclosure, the multi-armed acrylamide-containing group is a four-armed acrylamide-containing group comprising four arms formed by repeating units of (i) a monomer having a structure according to Formula C and (ii) a monomer having a structure according to Formula D, wherein Formula C and Formula D are shown below.Docket No. TP385946W01Formula D

[0110] With reference to Formula D, each R' independently is an amine protecting group, a linker group, a linker group bound to a cell-binding peptide, or hydrogen; and each r' independently is an integer selected from 1 to 3. In some aspects of the disclosure, R' is hydrogen and the nitrogen bound to R' can further comprise a third hydrogen atom bound thereto to provide a quaternary amine ion. Representative compounds that can act as amine protecting groups can include, but are not limited to, a 9-fluorenylmethyl carbamate (or "Fmoc"), t-butyl carbamate (or "Boc"), benzyl carbamate (or "Cbz"), acetamide (or "Ac"), trifluoroacetamide, phthalimide, benzylamine (or "Bn"), triphenylmethylamine (or "trityl"), benzylideneamine, or p-toluenesulfonamide (or "Ts"). Representative linker groups can include, but are not limited to, heteroaliphatic linker groups, including carbonyl-containing linker groups, maleimide-containing linker groups, or combinations thereof. In some aspects of the disclosure comprising a linker group, the linker group can further be coupled to a peptide (e.g., a cellbinding peptide). The peptide (including any cell-binding peptide) can be a natural peptide or it can be a synthetic peptide. In particular aspects of the disclosure, a natural peptide can be used. In particular aspects of the disclosure, a synthetic peptide can be used. In particular aspects of the disclosure, the peptide can be selected from those listed in Table 1 below, and the like.Docket No. TP385946W01

[0111] The cell-binding peptide can be bound to the linker group through a sulfur atom present in a cysteine residue of the cell-binding peptide. Other suitable groups can be used to bind the peptide to the linker, such as amine groups and / or carboxylic acid groups.

[0112] In some aspects of the disclosure utilizing multi-armed PEG-containing and / or acrylamide groups, the thermo-responsive polymer comprises one or more additional groups having a formula -[(X)n-(Y)m]p-TG, wherein each of X, Y, TG, n, m, and p are as recited for Formula I, and wherein the one or more additional groups independently are attached to other arms of the multi-armed PEG-containing group or the multi-armed acrylamide-containing group. In some aspects of the disclosure, a single arm of a multi-armed acrylamide-containing group is independently coupled to a number of peptides through one or more arms comprising a -[(X)n- (Y)m]P-TG group as defined above, wherein the number of peptides can range from 1 to 20, such as 1 to 15, or 1 to 10, or 1 to 5 (e.g„ 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0113] In representative aspects of the disclosure, thermo-responsive polymers according to Formula IB can have a structure according to Formula III, wherein each R group is as illustrated within the outermost brackets shown in Formula III.Docket No. TP385946W01Formula III

[0114] With reference to Formula III, each Q and each T, for each occurrence of Q and T, independently is selected from an acrylamide-derived monomer having a structure according to Formula C, or an amino-aliphatic methacrylamide-derived monomer having a structure according to Formula D, provided that (i) at least one Q is the acrylamide-derived monomer and at least one T is the amino-aliphatic methacrylamide-derived monomer; or (ii) at least one Q is the amino-aliphatic methacrylamide-derived monomer and at least one T is the acrylamidederived monomer; and each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 100 to 500. With reference to such aspects of the disclosure, Formula C and Formula D are as described above.

[0115] In some aspects of the disclosure, the thermo-responsive polymer according to Formulas IB and III has a structure as shown belowwherein each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 200 to 300.Docket No. TP385946W01

[0116] Representative examples of thermo-responsive polymers according to Formulas IB and III are illustrated below.Docket No. TP385946WO1

[0117] With reference to the above formulas, wherein r, s, n, and m are selected to provide a ratio of (r + s) : (n + m) that ranges from 0.25 to 3, such as 0.25 to 2.5 or 0.25 to 2, or 0.25 to 1.5 or 0.25 to 1 , or 0.25 to 0.5.

[0118] In particular aspects of the disclosure, the thermo-responsive polymer exists as a liquid at temperatures up to a lower critical solution temperature (LCST) point of the thermo-Docket No. TP385946WO1 responsive polymer and as a hydrogel at temperatures above the LCST of the thermo- responsive polymer. In some aspects, the thermo-responsive polymer can have an LCST ranging from 25 °C to 60 °C, such as 25 °C to 55 °C, or 25 °C to 50 °C, or 25 °C to 45 °C, or 25 °C to 40 °C, or 25 °C to 35 °C, or 25 °C to 30 °C. In particular aspects wherein the thermo- responsive polymer is used for cell-based methods, the thermo-responsive polymer can have an LCST ranging from 25 °C to 40 °C, such as 25 °C to 35 °C, or 25 °C to 34 °C, or 25 °C to 32 °C. In particular such aspects of the disclosure, the thermo-responsive polymer has an LCST of 32 °C. In particular aspects wherein the thermo-responsive polymer is used for methods for separating and / or purifying biomolecules, the thermo-responsive polymer can have an LCST ranging from 25 °C to 60 °C, such as 25 °C to 50 °C, or 25 °C to 40 °C, or 25 °C to 35 °C. In representative aspects of the disclosure, the thermo-responsive polymer can be prepared so as to exhibit different transition temperatures between phases of the thermo-responsive polymer. In some aspects of the disclosure, NIPAM can be copolymerized with a hydrophobic monomer, such as an acryloyl monomer to lower transition temperature. In another aspect, NIPAM can be copolymerized with acrylamide and / or N-2-hydroxyl propyl methacrylamide to raise the transition temperature.

[0119] Also disclosed herein are compositions comprising the thermo-responsive polymer. In such aspects of the disclosure, the composition can comprise the thermo-responsive polymer in combination with one or more biological components (e.g., cells, organoids, or one or more biomolecules, such as nucleic acid molecules, proteins, peptides, antibodies, associated adenoviruses, etc.). In particular aspects of the disclosure, the composition comprises cells in combination with the thermo-responsive polymer, which typically comprises a coupling component that is a multi-armed polymer that can facilitate binding the polymer to one or more cell-binding peptides. The cells can be primary cells, stem cells, induced pluripotent stem cells, or a combination thereof. In some aspects, the cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, epidermal cells, or other cells. In exemplary aspects of the disclosure, the composition can comprise cells in the form of one or more organoids. In some aspects of the disclosure, the thermo-responsive polymer is in the form of a liquid when part ofDocket No. TP385946W01 the composition. In some other aspects of the disclosure, the thermo-responsive polymer is in the form of a hydrogel when part of the composition.

[0120] In yet other aspects of the disclosure, the composition comprises a biomolecule in combination with the thermo-responsive polymer, which typically is coupled to a biomoleculecoupling component. The biomolecule can include a nucleic acid molecule (e.g., oligonucleotide or polynucleotide, such as RNA, DNA, mRNA, tRNA, or any other type of natural or synthetic nucleic acid, including fragments thereof), a protein, a peptide, an antibody, an associated adenovirus, or the like. In particular aspects of the disclosure, the biomolecule is a nucleic acid, such as RNA (including mRNA). The biomolecule-coupling component that is attached to the thermo-responsive polymer can be selected from compounds disclosed herein and can be selected based on the type of biomolecule that is to be isolated using the thermo- responsive polymer. In some aspects of the disclosure, the thermo-responsive polymer is in the form of a liquid when part of the composition. In some other aspects of the disclosure, the thermo-responsive polymer is in the form of a hydrogel when part of the composition. In yet additional aspects of the disclosure, the thermo-responsive polymer can be conjugated to the biomolecule or it can be separated from (i.e., not conjugated to) the biomolecule.

[0121] Method of Use

[0122] Disclosed herein are methods for using the thermo-responsive polymers described herein.

[0123] In some aspects of the disclosure, the thermo-responsive polymer is used for cell culturing and / or organoid development. In some such aspects, the method comprises exposing a cell to a thermo-responsive polymer disclosed herein at a first temperature to provide a seeding composition comprising the cell dispersed in the thermo-responsive polymer. In such aspects, the thermo-responsive polymer typically is bound to a coupling component that is a multi-armed polymer as described herein. The multi-armed polymer can facilitate coupling the thermo-responsive polymer to one or more cell-binding peptides, which can facilitate cell binding and / or dispersion within the seeding composition. A representative illustration describing using the thermo-responsive polymer to bind one or more cell-binding peptides is provided in FIG. 1 .Docket No. TP385946WO1As illustrated in FIG. 1, free amine groups 100 of thermo-responsive polymer 102 can bind to cell-binding peptide 104 to provide cell-binding peptide-containing conjugate 106. As further illustrated in FIG. 1, cell-binding peptide-containing conjugate 106 can be used to attract and bind peptides 108. The peptide-bearing construct 110 can then be used in the cell culturing method.

[0124] In some aspects of the disclosure, the first temperature is a temperature that is below or equal to an LOST of the thermo-responsive polymer. In particular aspects, the first temperature causes the thermo-responsive polymer to be in a liquid state and thus the cell (or plurality of cells) can be dispersed in the thermo-responsive polymer and easily transferred. In particular aspects of the disclosure, the first temperature can range from 1 °C to 35 °C, such as 1 °C to 34 °C, or 1 °C to 32 °C, or 1 °C to 30 °C, or 1 °C to 28 °C, or 1 °C to 26 °C. In particular aspects of the disclosure, the thermo-responsive polymer has an LCST of 32 °C and the first temperature ranges from 1 °C to 32 °C. In some aspects of the disclosure, the first temperature can be achieved by cooling the composition comprising the thermo-responsive polymer and the cell on an ice bath or adding ice / cold water to the composition.

[0125] The method can further comprise applying the seeding composition to a culture apparatus comprising a growth medium to provide a seeding layer on the growth medium. Because the thermo-responsive polymer exists as a liquid at this point in the method, the cells can be positioned on the growth medium by spreading the thermo-responsive polymer solution over the growth medium (e.g., as sheets or droplets). Seeding can take place using any technique known to those in the art with the benefit of the present disclosure.

[0126] The culture apparatus can then be warmed at a second temperature, which facilitates cell growth on the growth medium. In some aspects of the present disclosure, warming the culture apparatus at the second temperature comprises warming the culture apparatus to a temperature sufficient to promote cell growth. Not only does warming the culture apparatus promote cell growth, but it also facilitates converting the thermo-responsive polymer from a liquid to a hydrogel as the second temperature can be selected to be above the LCST of the thermo-responsive polymer. In such aspects of the disclosure, having the thermo-responsive polymer in the form of a hydrogel gives it the ability to act as an ECM for cell growth. InDocket No. TP385946W01 particular aspects of the disclosure, the temperature can range from 32 °C to 40 °C, such as 32 °C to 39 °C, or 32 °C to 38 °C, or 32 °C to 37 °C, or 32 °C to 36 °C, or 32 °C to 35 °C, or 32 °C to 34 °C.

[0127] After a desired level of cell growth has occurred, the thermo-responsive polymer can be separated or isolated from the seeding layer by cooling the culture apparatus at the first temperature used in the method. By cooling the culture apparatus at the first temperature, the thermo-responsive polymer can be converted to a liquid from its hydrogel form, which facilitates separating it from the solid cells and / or organoids grown on the growth medium. In such aspects of the disclosure, the culture apparatus can be cooled at the first temperature by placing the culture apparatus on ice and / or by adding ice water to the culture apparatus. In particular aspects of the disclosure temperature, the first temperature used for the isolation step need not be exactly the same as the first temperature used to form the seeding composition but can typically be within ± 1 to 5 degrees of the first temperature, so long as it does not exceed the LCST of the thermo-responsive polymer.

[0128] In some aspects of the disclosure, the thermo-responsive polymer is used for cell culturing wherein the thermo-responsive polymer acts as an ECM that facilitates growing cells selected from primary cells, stem cells, induced pluripotent stem cells, or a combination thereof. In particular aspects of the disclosure, the cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, epidermal cells, or combinations thereof. In some aspects of the disclosure, the thermo-responsive polymer can serve as an ECM that facilitates growing organoids, such as liver organoids (e.g., adult liver ductile organoids) or other organoids, such as cerebral organoids, gastrointestinal organoids, lingual organoids, lung organoids, kidney organoids, and others. The ability to change the phase of the thermo-responsive polymer after cells have been added to the ECM formed by the thermo-responsive polymer facilitates the ability to easily isolate the cells and / or organoids as the thermo-responsive polymer can be converted to a liquid phase that is easily separated from the solid cells / organoids.

[0129] In yet other aspects of the disclosure, the thermo-responsive polymer can be used to isolate and / or purify a biomolecule (e.g., nucleic acid, protein, antibody, peptide or the like) fromDocket No. TP385946W01 one or more other components. In such aspects of the disclosure, the method comprises adding a thermo-responsive polymer described herein to a biomolecule-containing composition to provide a solution wherein the thermo-responsive polymer is coupled to biomolecules present in the biomolecule-containing composition to form a biomolecule-polymer conjugate. In particular aspects of the disclosure, the thermo-responsive polymer used for the method comprises a biomolecule-coupling component. The biomolecule-coupling component facilitates binding the thermo-responsive polymer to any biomolecules present in the biomoleculecontaining composition. A representative method of coupling a thermo-responsive polymer comprising a biomolecule-coupling component to bind the thermo-responsive polymer to a biomolecule is shown in FIG. 2. With reference to FIG. 2, thermo-responsive polymer 200 can be combined with a biomolecule-coupling component 204 (e.g., an OdT ligand) to provide conjugated product 206 which can then be reacted with biomolecule 208 (e.g., mRNA comprising a PolyA tail) to provide thermo-responsive polymer-biomolecule conjugate 210, which can undergo different phase transitions using temperature control as described below. FIG. 3 provides a schematic illustration of a representative method for making conjugated product 206. As shown in FIG. 3, azide-term inated thermo-responsive polymer 300 can be combined with a DBCO-functionalized oligo dT tag 302 to provide conjugated product 206.

[0130] The method further comprises exposing the solution to a heat source at a first temperature that is above an LOST of the thermo-responsive polymer to convert the biomolecule-polymer conjugate to a non-liquid state (e.g., a hydrogel or other solid). In some aspects of the disclosure, the first temperature ranges from greater than 25 °C to a temperature of 60 °C, such as 28 °C to 60 °C, or 30 °C to 60 °C, or 32 °C to 60 °C or 35 °C to 60 °C, or 40 °C to 60 °C, or 45 °C to 60 °C, or 50 °C to 60 °C, or 55 °C to 60 °C. After the thermo- responsive polymer is converted to its non-liquid stated, it will precipitate in the solution, thereby causing the biomolecule-polymer conjugate to precipitate, which facilitates isolation from contaminants in the solution. Any undesired contaminants can be removed from the precipitated biomolecule-polymer conjugate by filtration, rinsing, centrifugation, or similar techniques. In such aspects of the disclosure, the precipitated biomolecule-polymer conjugate is separated from contaminants that might be solubilized in the solution. After the contaminants have been removed, the precipitate can be redispersed in a solution (e.g., water or otherDocket No. TP385946WO1 suitable solvent). The solution can then be cooled to a second temperature ranging from 1 °C to a temperature below 32 °C to convert the biomolecule-polymer conjugate to a liquid state. The biomolecule-polymer conjugate can then be exposed to an additive to de-couple the biomolecule and the thermo-responsive polymer. Suitable additives include compounds or a stimulus that disrupts interactions between the biomolecule and the thermo-responsive polymer. Exemplary additives can include, but are not limited, to buffers and / or salts (or other reagents capable of modifying pH), water miscible solvents (e.g., acetonitrile, methanol, and the like), a reagent capable of cleaving a cleavable linker and / or capable of promoting competitive dissociation (e.g., DTT, TCEP, or the like), excess ODT to facilitate release of mRNA, or any combination thereof. In some aspects, the biomolecule can then be isolated from the thermo- responsive polymer. In particular aspects of the disclosure, one or more of the above-described method steps can be performed using a column or other suitable separation (or purification) apparatus. In some aspects of the disclosure, the method can further comprise performing a washing step and / or a centrifugation step to remove any contaminants from the solution and / or the biomolecule-polymer conjugate. In yet additional aspects of the disclosure, the method can further comprise one or more filtering steps to isolate the biomolecule-polymer conjugate. In some such aspects, tangential flow filtration can be used.

[0131] The disclosed method for purifying biomolecules using the disclosed thermo-responsive polymer overcomes drawbacks associated with conventional resins typically used to purify biomolecules (e.g., pore size-based techniques and / or other affinity precipitation methods). For example, the present disclosed method facilitates achieving separations and / or purifications of biomolecules with larger-sized biomolecule species that could not otherwise be purified using conventional pore size-based techniques given the capacity limitations associated with such techniques (e.g., RNA molecules often have a hydrodynamic size that far surpasses other biomolecules and thus they cannot be purified using pore size-based techniques). The thermo- responsive polymers described herein can be useful in various types of RNA enrichment approaches for the sequence specific capture and precipitation of RNA in place of existing biotinylation procedures, such as Pierce™ RNA 3'End Biotinylation Kit (Thermo Fisher Scientific). The method described above also can provide an alternative to "pull-down" type approaches (e.g., beads that bind a target molecule and then can be spun out of solution orDocket No. TP385946W01 pulled out by magnet), such as Magnetic RNA-Protein Pull-Down Kit and Ultralink™ acrylamide- based support resins (available from Thermo Fisher Scientific).

[0132] In one representative aspect of the disclosure, the thermo-responsive polymer is used to purify mRNA from an in vitro transcription (or IVT) reaction mixture. In such aspects, the thermo-responsive polymer of the present disclosure overcomes binding capacity limitations of conventional OdT resins and provides a high yield of purified mRNA molecules from the IVT reaction. This representative example is illustrated schematically in FIG. 4. With reference to FIG. 4, target mRNA can be purified from an IVT reaction using a method and thermo- responsive polymer as described herein. Solution 400 which contains RNA products 402 and contaminants 404 (such as polymerase, NTPs, and plasmid). A thermo-responsive polymer 406 can be added to solution 400 and, in solution 408, will form a conjugate 410 with RNA products 402 by associating the nucleic acid-coupling component of the thermo-responsive polymer (e.g., an OdT ligand) with a polyA portion of the mRNA molecule. Solution 408 is then exposed to a first temperature above an LOST of the thermo-responsive polymer (e.g., a temperature greater than 32 °C to a temperature of 60 °C) to promote precipitation of the conjugates 410 from contaminants 404 in solution 412. While this temperature converts the mRNA-polymer conjugate from a liquid to a solid, other components from the IVT reaction and other impurities remain in solution. Contaminants 404 can be removed using a suitable method (e.g., tangential flow filtration followed by washing) and conjugates 410 can be isolated by centrifugation or filtration. The temperature is then lowered to a second temperature to facilitate converting the mRNA-polymer conjugate back to a liquid to provide solution 414. In some aspects of the disclosure, the target mRNA compound can be separated from the thermo- responsive polymer using an additive that disrupts the hydrogen bonding of the polyA tail to the polyT ligand. The recovery of the mRNA can be evaluated by the absorbance 260 (A260) and the residual PolyT-polymer can be quantified by, for example, a CAD instrument or another UV / Fluorescence technique.

[0133] Method of Making

[0134] Also disclosed are methods for making the thermo-responsive polymers described herein, including any conjugated structures comprising such polymers.Docket No. TP385946W01

[0135] Thermo-responsive polymers disclosed herein for use in cell growth method described herein can be made according to a method wherein a combining a multi-armed polymer with N- isopropylacrylamide, an acryloyl monomer, a radical initiator, and an optional chain transfer agent, to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer. In some aspects, the method can further comprise exposing the thermo-responsive polymer to a deprotecting reagent and a linker group. The method can also further comprise coupling the thermo-responsive polymer to a cell-binding peptide.

[0136] In some aspects, the method can further comprise making the multi-armed polymer. In such aspects, the multi-armed polymer can be made by combining acrylamide, an aminoaliphatic methacrylamide monomer, a multi-armed starting reagent, and a radical initiator to provide a preliminary reaction mixture and heating the preliminary reaction mixture. The resulting polymerization can be terminated by exposing the reaction mixture to air. In some aspects of the disclosure, the amino-aliphatic methacrylamide monomer has a structure according to Formula D"Formula D' wherein R' is an amine protecting group; and r' is an integer selected from 1 to 4. Multi-armed starting reagents can include a 4-arm start DDMAT RAFT agent, such as pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate.

[0137] A representative method for making thermo-responsive polymers comprising a multiarmed polymer is illustrated schematically in FIG. 5. With reference to FIG. 5, a four-armed acrylamide-containing group 500 is prepared by polymerizing dimethylacrylamide monomers 502 with Boc-protected aminopropyl methacrylamide monomers 504 in the presence of chain transfer agent 506. The four-armed acrylamide-containing group is then combined with n-Docket No. TP385946W01 isopropylacrylamide monomers 508 and butyl acrylate monomers 510 to provide thermo- responsive polymer product 512 comprising chain transfer agent end groups. These end groups can be removed to provide thermo-responsive polymer 514. In some additional exemplary aspects of the disclosure, protecting groups on amines of thermo-responsive polymer 514 (e.g., Boc protecting groups) can be removed to provide a thermo-responsive polymer product with free amine groups, such as illustrated in FIG. 6, wherein thermo- responsive polymer 514 is converted to free amine-containing thermo-responsive polymer 600.

[0138] Also disclosed herein is a method for making the thermo-responsive polymer comprising a biomolecule-coupling component that can be used in the biomolecule purification methods described herein. In particular aspects of the disclosure, the method comprises combining a chain transfer agent, a radical initiator, an acryloyl monomer, and N-isopropylacrylamide to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer. In some such aspects, an initiator can also be used to facilitate polymerization. Suitable initiators can include radical initiators, such as azobisisobutyronitrile (or Al BN). A representative schematic summarizing the synthesis is provided in FIG. 7. As illustrated in FIG. 7, aliphatic and n-isopropylarylamide monomers 700 and 702, respectively, are polymerized into polymer 704 using chain transfer agent 706, which is terminated by the two structural groups of the chain transfer agent, 708 and 710, which include the Z group of Formula I and a thiourea group, respectively. In exemplary aspects of the disclosure, the Z group is an azide and the thiourea group comprises an aliphatic group bound to a sulfur atom of the thiourea group. In some additional exemplary aspects of the disclosure, thiourea end group 708 can be removed to provide thermo-responsive polymer 712.

[0139] In some aspects of the disclosure, the acryloyl monomer and the N-isopropylacrylamide are provided at a ratio to provide a value for p ranging from 2 to 1600; and the concentration ratio of the acryloyl monomer and / or the N-isopropylacrylamide to the chain transfer agent ranges from greater than 0 to 20 molo / o. In particular aspects of the disclosure, the acryloyl monomer has a structure according to Formula A'Docket No. TP385946W01Formula A' wherein X1is NH or O if q' is 1 or is NH2 if q' is 0; R" is H, aliphatic, or OH; q is an integer selected from 0 to 20; and q' is 0 or 1.

[0140] The chain transfer agent can have a structure according to Formula FS R2'S A^R11Formula F wherein R1is an ether or thioether group; and R2is an aliphatic or heteroaliphatic group comprising the Z group of Formula I.

[0141] In particular aspects, the chain transfer agent has a structure selected fromwherein Z is selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group. In some exemplary aspects of the disclosure, the chain transfer agent is 3-azidopropyl 2- (((dodecylthio)carbonothioyl)thio)-2-methylpropanoate.

[0142] In particular aspects of the disclosure, the chain transfer agent reacts with the acryloyl monomer or the N-isopropylacrylamide monomer such that R2of the chain transfer agent becomes bound to a first terminus of the thermo-responsive polymer and a sulfur atom of theDocket No. TP385946WO1 thiourea becomes bound to a second terminus of the thermo-responsive polymer. In some aspects of the disclosure, the method further comprises cleaving the thiourea from the second terminus of the thermo-responsive polymer and capping the second terminus with a terminating group. A representative scheme of this additional step of the method is provided by Scheme 1 below. As shown in Scheme 1 , the thiourea group is cleaved from the thermo-responsive polymer using excess Al BN, which provides a terminal group as illustrated.Scheme 1

[0143] Processing methods described above can be modified as needed to arrive at other thermo-responsive polymers contemplated by the present disclosure using techniques recognized by those in the art with the benefit of the present disclosure. For example, different monomers as described herein can be used and can be integrated into the methods described above. Additionally, other chain transfer agents can be used, as well as other initiators known to those in the art with the benefit of the present disclosure. Specific molecular weights of the thermo-responsive polymer can be targeted by varying the concentration ratio of each monomer to the chain transfer agent. Molecular weight and the transition temperatures associated with any particular thermo-responsive polymer can be determined using light scattering methods known in the art with the benefit of the present disclosure (e.g., dynamic light scattering (DLS) and / or UV-vis spectroscopy).Docket No. TP385946W01

[0144] Overview of Several Embodiments

[0145] Disclosed herein is a thermo-responsive polymer, having a structure according toFormula IFormula I wherein Z represents a coupling component; TG is a terminating group; each X and each Y, for each occurrence of X and Y, independently is selected from an acryloyl-derived monomer having a structure according to Formula A, or an N-isopropylacrylamide-derived monomer having a structure according to Formula B, provided that (i) at least one X is the acryloyl-derived monomer and at least one Y is the N-isopropylacrylamide-derived monomer; or (ii) at least one X is the N-isopropylacrylamide-derived monomer and at least one Y is the acryloyl-derived monomer; each n, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + mtotai = p; each m, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + mtotai = p; and p is an integer selected from 2 to 1600; wherein Formula A isFormula A wherein each X1independently is (i) NH or O if q' is 1 , or (ii) is NH2 if q' is 0; each R" independently is H, aliphatic, or OH; each q independently is an integer selected from 0 to 20; and q' is 0 or 1 ; and Formula B isFormula B.Docket No. TP385946W01

[0146] In any or all aspects, the coupling component is a functional group or molecule that facilitates binding the thermo-responsive polymer to a biological component or associating the thermo-responsive polymer with a biological component.

[0147] In any or all aspects, the coupling component is (i) a functional group capable of coupling the thermo-responsive polymer to a biomolecule-coupling component or (ii) a multiarmed polymer.

[0148] In any or all aspects, the thermo-responsive polymer has a structure according to Formula IAFormula IA wherein FG represents the functional group capable of coupling the thermo-responsive polymer to the biomolecule-coupling component.

[0149] In any or all aspects, the FG is a clickable functional group or a heterobifunctional linker.

[0150] In any or all aspects, the clickable functional group is selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group.

[0151] In any or all aspects, the FG is an azide group.

[0152] In any or all aspects, the FG is further bound to the biomolecule-coupling component.

[0153] In any or all aspects, the biomolecule-coupling component is an oligonucleotide ligand, a member of a specific binding pair, a group that facilitates a protein-protein interaction, or a member of a guest-host pair.

[0154] In any or all aspects, the oligonucleotide ligand is an oligo deoxythymidine (OdT) ligand further comprising a 5'dibenzocyclooctyne group.Docket No. TP385946W01

[0155] In any or all aspects, the thermo-responsive polymer has a structure according to a formula selected from

[0156] In any or all aspects, the thermo-responsive polymer has a structure according to a formula selected fromDocket No. TP385946W01

[0157] In any or all aspects, the thermo-responsive polymer has a structure according toFormula IBFormula IB wherein MAP represents the multi-armed polymer.

[0158] In any or all aspects, the MAP is a multi-armed PEG-containing group or a multi-armed acrylamide-containing group and wherein (i) X is attached directly or indirectly to at least one arm of the multi-armed PEG-containing group or (ii) X is attached directly or indirectly to at least one arm of the multi-armed acrylamide-containing group.

[0159] In any or all aspects, the multi-armed PEG-containing group is an eight-armed PEG- containing group; or wherein the multi-armed acrylamide-containing group is a four-armed acrylamide-containing group.

[0160] In any or all aspects, the four-armed acrylamide-containing group comprises four arms formed by repeating units of(i) a monomer having a structure according to Formula CFormula C; and(ii) a monomer having a structure according to Formula DFormula DDocket No. TP385946W01 wherein each R' independently is an amine protecting group, a linker group, a linker group bound to a cell-binding peptide, or hydrogen; and each r' independently is an integer selected from 1 to 3.

[0161] In any or all aspects, the thermo-responsive polymer comprises one or more additional groups having a formula -[(X)n-(Y)m]P-TG, wherein each of X, Y, TG, n, m, and p are as recited for Formula I, and wherein the one or more additional groups independently are attached to other arms of the multi-armed PEG-containing group or the multi-armed acrylamide-containing group.

[0162] In any or all aspects, a single arm of the multi-armed acrylamide-containing group is independently coupled to a number of peptides ranging from 1 to 20.

[0163] In any or all aspects, the thermo-responsive polymer has a structure according to a Formula IIIFormula III wherein each Q and each T, for each occurrence of Q and T, independently is selected from an acrylamide-derived monomer having a structure according to Formula C, or an amino-aliphatic methacrylamide-derived monomer having a structure according to Formula D, provided that (i) at least one Q is the acrylamide-derived monomer and at least one T is the amino-aliphatic methacrylamide-derived monomer; or (ii) at least one Q is the amino-aliphatic methacrylamidederived monomer and at least one T is the acrylamide-derived monomer; and each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 100 to 500; wherein Formula C isDocket No. TP385946W01Formula C and Formula D isFormula D wherein each R' independently is an amine protecting group, a linker group, a linker group bound to a cell-binding peptide, or hydrogen; and each r' independently is an integer selected from 1 to 3.

[0164] In any or all aspects, the thermo-responsive polymer has a structure according to a formulawherein each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 200 to 300.

[0165] In any or all aspects, the thermo-responsive polymer has a structure according to a formula selected fromDocket No. TP385946WO1Docket No. TP385946W01Cell-binding Peptidewherein each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer selected from 200 to 300.

[0166] In any or all aspects, wherein r, s, n, and m are selected to provide a ratio of (r + s) : (n + m) that ranges from 0.25 to 3.

[0167] In any or all aspects, the thermo-responsive polymer exists either (i) as a liquid at temperatures up to a critical temperature point of the thermo-responsive polymer or (ii) as a hydrogel at temperatures above the critical temperature point of the thermo-responsive polymer.

[0168] Also disclosed is a composition, comprising: one or more cells; and the thermo- responsive polymer according to any or all of the above aspects, wherein the thermo-responsive polymer is coupled to a cell-binding peptide.

[0169] In any or all aspects, at a temperature below 32 °C, the thermo-responsive polymer is in the form of a liquid.

[0170] In any or all aspects, at a temperature above 32 °C, the thermo-responsive polymer is in the form of a hydrogel.Docket No. TP385946W01

[0171] In any or all aspects, the one or more cells are primary cells, stem cells, induced pluripotent stem cells, or a combination thereof.

[0172] In any or all aspects, the one or more cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, epidermal cells, or any combination thereof.

[0173] Also disclosed is a composition, comprising: one or more biological components; and the thermo-responsive polymer according to any or all of the above aspects, wherein the thermo-responsive polymer is coupled to the biomolecule-coupling component; or the thermo- responsive polymer according to any or all of the above aspects and without being coupled to the biomolecule-coupling component.

[0174] Also disclosed is a method, comprising: exposing a cell to a thermo-responsive polymer according to any or all of the above aspects at a first temperature to provide a seeding composition comprising the cell dispersed in the thermo-responsive polymer, wherein the first temperature is a temperature below the critical temperature point of the thermo-responsive polymer; applying the seeding composition to a culture apparatus comprising a growth medium to provide a seeding layer on the growth medium; warming the culture apparatus by exposing it to a second temperature, wherein the second temperature is a temperature that is above the critical temperature point of the thermo-responsive polymer, wherein warming the culture apparatus facilitates cell growth from the seeding layer; and removing the thermo-responsive polymer from the culture apparatus by exposing the culture apparatus to the first temperature; wherein the thermo-responsive polymer is coupled to a cell-binding peptide.

[0175] In any or all aspects, the first temperature causes the thermo-responsive polymer to be in a liquid state.

[0176] In any or all aspects, the second temperature causes the thermo-responsive polymer to transition from a liquid to a hydrogel.

[0177] In any or all aspects, the one or more cells are primary cells, stem cells, induced pluripotent stem cells, or a combination thereof.Docket No. TP385946W01

[0178] In any or all aspects, the one or more cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, or epidermal cells.

[0179] Also disclosed is a a method, comprising: adding the thermo-responsive polymer according to any or all of the above aspects to a biomolecule-containing composition to provide a solution wherein the thermo-responsive polymer is coupled to biomolecules present in the biomolecule-containing composition to form a biomolecule-polymer conjugate; exposing the solution to a heat source at a first temperature that is a temperature above the critical temperature point of the thermo-responsive polymer to convert the biomolecule-polymer conjugate to a non-liquid state; isolating the biomolecule-polymer conjugate from the solution; cooling the biomolecule-polymer conjugate by exposing the biomolecule-polymer conjugate to a second temperature that is a temperature below the critical temperature point of the thermo- responsive polymer to convert the biomolecule-polymer conjugate to a liquid state; exposing the biomolecule-polymer conjugate to an additive to de-couple the biomolecule and the thermo- responsive polymer; and isolating the biomolecule from the thermo-responsive polymer.

[0180] In any or all aspects, the biomolecule-coupling component of the thermo-responsive polymer is an oligonucleotide ligand and is coupled to the molecule via non-covalent interactions.

[0181] In any or all aspects, the biomolecule-polymer conjugate is isolated by filtering the solution to separate the biomolecule-polymer conjugate from liquid in the solution.

[0182] In any or all aspects, filtering the mixture comprises exposing the mixture to tangential flow filtration.

[0183] In any or all aspects, the method further comprises performing a washing step and / or a centrifugation step to remove any contaminants from the solution and / or the biomolecule- polymer conjugate.

[0184] In any or all aspects, the additive is a compound or stimulus that disrupts interactions between the biomolecule and the thermo-responsive polymer.Docket No. TP385946W01

[0185] In any or all aspects, the additive is selected from a buffer, a salt, a water miscible solvent, a reagent capable of cleaving a cleavable linker and / or capable of promoting competitive dissociation, excess ODT to facilitate release of mRNA, or any combination thereof.

[0186] Also disclosed is a method for making the thermo-responsive polymer according to any or all of the above aspects, the method comprising: combining a multi-armed polymer with N- isopropylacrylamide, an acryloyl monomer, and a radical initiator to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer.

[0187] In any or all aspects, the method further comprises exposing the thermo-responsive polymer to a deprotecting reagent and a linker group.

[0188] In any or all aspects, the method further comprises coupling the thermo-responsive polymer to a cell-binding peptide.

[0189] In any or all aspects, the method further comprises making the multi-armed polymer by combining acrylamide, an amino-aliphatic methacrylamide monomer, a multi-armed starting reagent, and a radical initiator to provide a preliminary reaction mixture; and heating the preliminary reaction mixture.

[0190] In any or all aspects, the amino-aliphatic methacrylamide monomer has a structure according to Formula D"Formula D1wherein R' is an amine protecting group; and r' is an integer selected from 1 to 4.

[0191] Disclosed herein is a method for making the thermo-responsive polymer according to any or all of the above aspects, comprising: combining a chain transfer agent, a radical initiator, an acryloyl monomer, and N-isopropylacrylamide to provide a reaction mixture; and heating theDocket No. TP385946W01 reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo- responsive polymer; wherein (i) the chain transfer agent is a compound comprising the Z group of Formula I and a thiourea group; (ii) the acryloyl monomer and the N-isopropylacrylamide are provided at a ratio to provide a value for p ranging from 2 to 1600; and (iii) the concentration ratio of the acryloyl monomer and / or the N-isopropylacrylamide to the chain transfer agent ranges from greater than 0 to 20 molo / o.

[0192] In any or all aspects, the acryloyl monomer has a structure according to Formula A'Formula A' wherein X1is NH or O if q' is 1 or is NH2 if q' is 0; R" is H, aliphatic, or OH; q is an integer selected from 0 to 20; and q' is 0 or 1.

[0193] In any or all aspects, the chain transfer agent has a structure according to Formula F SR2'S^R 11Formula F wherein R1is an ether or thioether group; and R2is an aliphatic or heteroaliphatic group comprising the Z group of Formula I.

[0194] In any or all aspects, the chain transfer agent has a structure selected fromwherein Z is selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group.Docket No. TP385946W01

[0195] In any or all aspects, the chain transfer agent reacts with the acryloyl monomer or the N-isopropylacrylamide monomer such that R2of the chain transfer agent becomes bound to a first terminus of the thermo-responsive polymer and a sulfur atom of the thiourea becomes bound to a second terminus of the thermo-responsive polymer.

[0196] In any or all aspects, the method further comprises cleaving the thiourea from the second terminus of the thermo-responsive polymer and capping the second terminus with a terminating group.

[0197] Examples

[0198] The examples provided herein utilize the following general methods unless indicated otherwise. Molecular weights of 4-armed poly(dimethylacrylamide-co-N-(3-BOC-aminopropyl methacrylamide), 4-armed poly(dimethylacrylamide-co-N-(3-BOC-aminopropyl methacrylamide)-block-(A / -isopropylacrylamide), 4-Armed poly(dimethylacrylamide-co-N-(3- BOC-aminopropyl methacrylamide)-block-(A / -isopropylacrylamide-co-butyl acrylate), linear poly( / V-isopropylacrylamide-co-butyl acrylate), linear poly(A / -isopropylacrylamide) were measured by standard gel permeation chromatography (GPC) methods using 5 mM Li Br methanol / dimethylformamide (50 / 50 v / vo / o) as an eluent, TSkgel® Hnrorganic size exclusion columns (TOSOH Bioscience LLC, King of Prussia, PA) with Wyatt Technology Corporation's miniDAWN® TREOS® multi-angle light scattering (MALS) and Optilab® T-rEX differential refractometer detectors (Santa Barbara, CA). Copolymer compositions were determined from1HNMR using a 400 MHz Bruker Instrument. Rheological properties of the temperature responsive hydrogel materials were monitored using a HAAKE™ MARS™ 60 Rheometer (Thermo Fisher Scientific; Waltham, MA) at 37°C.EXAMPLE 1Polymerization of 4-Armed Poly(dimethylacrylamide-co-N-(3-BOC-aminopropyl methacrylamide)) Copolymer

[0199] In this example, 4-armed copolymers with BOC protected amine reactive side chains were prepared using controlled free radical polymerization methods. Copolymers with 5 molo / oDocket No. TP385946W01N-(3-BOC-aminopropyl methacrylamide (bocAPMA; Polysciences, Warrington, PA) and 95molo / o A / ,A / -dimethylacrylamide (DMA, TCI America, Portland, OR) were prepared with targeted molecular weights ranging from 42,500 g / mol to 170,000 g / mol (e.g. Degree of polymerization per arm = 100 to 400). For example, 1.88g (19mmol) of DMA, 0.242g (1 mmol) of bocAPMA and 38mg (25 p.mol) of pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2- methylpropionate] (Sigma-Aldrich, St. Louis, MO), were added to a 25mL round bottomed flask equipped with a stir bar and dissolved in 10mL of 1 ,4-dioxane. Once dissolved, 1.6 mg (9.8|imol) of azobisisobutyronitrile (AIBN; Sigma-Aldrich, St. Louis, MO) was added and the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C for 3.5 hours. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. These 4-armed copolymers were isolated by removing dioxane by rotary evaporation, redissolving in 5mL tetra hydrofuran (THF) and precipitating into 100mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and1H NMR, respectively. Results are shown in FIG. 8.EXAMPLE 2Polymerization of 4-Armed Poly(dimethylacrylamide-co-N-(3-BOC-aminopropyl methacrylamide)-block-(N-isopropylacrylamide) Copolymer

[0200] In this example, 4-armed block copolymers with N-isopropylacrylamide (NIPAM) were prepared using controlled free radical polymerization methods. Block copolymers with NIPAM were prepared using 4-armed copolymers described in Example 1 . Total targeted molecular weights ranged from 80,000 g / mol to 240,000 g / mol with NIPAM block lengths approximately equal to 40,000 to 120,000 g / mol (e.g. degree of NIPAM polymerization per arm = 100 to 300). For example, 0.638g (5.6 mmol) of NIPAM and 650mg (7.5 jimol) of 4-armed Poly(DMA-co- bocAPMA) (Mn = 86,700 g / mol) were added to a 25mL round bottomed flask equipped with a stir bar and dissolved in 6mL of 1,4-dioxane. Once dissolved, 0.49 mg (3 pmol) of AIBN from a 20mg / mL stock solution was added and the flask was septa sealed and sparged with nitrogen for 45 minutes and subsequently placed in an oil bath at 70 °C for 3.5 hours. The polymerizationDocket No. TP385946WO1 was terminated by exposing the reaction to air and placing in a -20°C freezer. These 4-armed copolymers were isolated by removing dioxane by rotary evaporation, redissolving in 5mL THF and precipitating into 100mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and1H NMR, respectively.EXAMPLE 3Polymerization of 4-Armed Poly(dimethylacrylamide-co-N-(3-BOC-aminopropyl methacrylamide)-block-(N-isopropylacrylamide-co-butyl acrylate) Copolymer

[0201] In this example, 4-armed block copolymers with N-isopropylacrylamide (NIPAM) were prepared using controlled free radical polymerization methods. Block copolymers with NIPAM and butyl acrylate were prepared using 4-armed copolymers described in Example 1 . Total targeted molecular weights ranged from 80,000 g / mol to 240,000 g / mol with NIPAM block lengths approximately equal to 40,000 to 120,000 g / mol (e.g. degree of NIPAM polymerization per arm = 100 to 300). For example, 0.638g (5.6 mmol) of NIPAM, 46mg (0.36mmol) of butyl acrylate (BA) and 650mg (7.5 pmol) of 4-armed Poly(DMA-co-bocAPMA) (Mn = 86,700 g / mol) were added to a 25mL round bottomed flask equipped with a stir bar and dissolved in 6mL of 1 ,4-dioxane. Once dissolved, 0.49 mg (3 pmol) of AIBN from a 20mg / mL stock solution was added and the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C for 3.5h. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. These 4-armed copolymers were isolated by removing dioxane by rotary evaporation, redissolving in 5mL THF and precipitating into 100mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and1H NMR, respectively. Results are shown in FIGS. 9 and 10.

[0202] The Boc protecting group in the 4-armed block copolymers was removed using trifluoroacetic acid (TFA). For example, 260mg (Mn= 162,000 g / mol, 1.6 .mols polymers, ~Docket No. TP385946WO168|a.mols BOC groups) of capped 4-Armed Poly(DMA-co-bocAPMA)-block-(N I PAM-co-BA) was weighed into a round bottomed flask equipped with a stir bar and dissolved into 1 ,33ml_ of TFA and 2.67mL of dioxane. The reaction was heated to 50°C in an oil bath and allowed to react overnight The deprotection was monitored using1H NMR and tracking the disappearance of the t-butyl methyl protons near 1.45ppm (MeOD used as solvent). Once the removal of the BOC group was confirmed by NMR the deprotected polymer was isolated by removing TFA and dioxane by rotary evaporation, redissolving in THF and precipitating into cold diethyl ether. This step was repeated to ensure no residual TFA was present. Following precipitation, the precipitate was collected by filtration, washed twice with 50ml_ of cold diethyl ether and dried in vacuo overnight at room temperature. In addition, approximation of amine functionality present was also determined by performing the colorimetric assay with pH 9.3 100mM borate buffer and 2,4,6-trinitrobenzene sulfonic acid (TNBS) and monitoring the absorbance at 420nm using a UV-vis spectrometer.EXAMPLE 4 co-end group removal from 4-Armed Copolymers

[0203] In this example, co-end groups (thiocarbonylthio) on synthesized 4-armed block copolymers were removed using common procedures familiar to those in the art, particularly with the benefit of the present disclosure. For example, 610mg (Mn= 162,000 g / mol, 15pmols of thiocarbonylthio) of 4-Armed Poly(DMA-co-bocAPMA)-block-(N I PAM-co-BA), 49mg (300 jimols) of AIBN, and 9.7mg (30umol) of benzoyl peroxide (BPO; Sigma-Aldrich, St. Louis, MO) were weighed into a round bottomed flask equipped with a stir bar and dissolved into 6mL of dioxane. Once dissolved, the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C overnight. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. The 4-armed copolymer with removed end-group was isolated by removing dioxane by rotary evaporation, redissolving in 10mL THF and precipitating into 100mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Greater than 90o / o removal of the thiocarbonylthio end-Docket No. TP385946W01 group was confirmed by monitoring the UV absorbance of the thiocarbonylthio group at 310nm during GPC analysis.EXAMPLE 5Synthesis of Maleimide Functional 4-Armed Poly(dimethylacrylamide-co-N-(3-aminopropyl methacrylamide) -block-(N-isopropylacrylamide-co-butyl acrylate) Copolymer

[0204] Following deprotection of the 4-Armed Poly(DMA-co-bocAPMA)-block-(NIPAM-co-BA), the primary amine of the APMA repeat unit was converted to a maleimide group using the heterobifunctional cross-linker Succinimidyl-4-(A / -maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), as summarized in Scheme 2 below. Briefly, 4-Armed Poly(DMA-co-APMA)-block- (Nl PAM-co-BA) and 5-fold molar excess SMCC were weighed and added to a round bottomed flask equipped with a stir bar and dissolved in DMSO at an APMA concentration of - 5mM. After complete dissolution, triethylamine (TEA) was pipetted into the reaction in 2-fold molar excess relative to APMA concentration. This reaction was allowed to stir overnight at room temperature. The next day water was added to the reaction mixture to be 50 / 50 v / vo / o with deionized water and placed in 12-14kDa molecular weight cut-off dialysis tubing. The product was dialyzed for a minimum of 72 hours with 3 DI water changes per day and isolated by lyophilization. After freeze drying, the presence of maleimide group on the 4-armed copolymer was confirmed by1H NMR in MeOD an integrating the protons at 6.83ppm.Scheme 2Docket No. TP385946WO1

[0205] The coupling to the maleimide group was confirmed using1H-NMR spectroscopy. FIGS. 13A and 13B show the1H-NMR spectrum for the free amine starting material (FIG. 13A) and for the maleimide-coupled product (FIG. 13B).EXAMPLE 6Conjugation of GFOGER (SEQ ID NO:29) orRGD peptides to Maleimide Functional 4-Armed Poly(dimethylacrylamide-co-N-(3-aminopropyl methacrylamide)-block-(N-isopropylacrylamide- co-butyl acrylate) Copolymer

[0206] Following synthesis and isolation of maleimide functional 4-armed Poly(DMA-co-APMA)- block-(NIPAM-co-BA), peptides GFOGER (SEQ ID NO:29) and / or RGD with either C- or N- terminus cysteine groups (SciLight Biotechnology, LLC; Beijing, China) were conjugated to the maleimide groups of the 4-armed block copolymer. An exemplary synthesis is summarized in Scheme 3, below.The molar amount of GFOGER (SEQ ID NO:29) and / or RGD per arm was varied between 2 to 4 peptides per arm of block copolymer. Briefly, maleimide functional block copolymer, GFOGER (SEQ ID NO:29) and or RGD were weighed into a round bottomed flask equipped with a stir bar and dissolved into degassed dimethylformamide (DMF) to yield a maleimide concentration of 5mM. DMF was degassed for 20 to 30minutes with nitrogen prior to using to prevent disulfide formation between peptides. If the polymer / peptide solution was not fully dissolved, then degassed DI water was added until solution turned clear (~10vo / o relative to total volume). TEA was then added to the solution in 3- fold molar excess relative to total peptide concentration. Following TEA addition, the round bottomed flask was septa sealed and the headspace was purged with nitrogen for 5 minutes.The conjugation was allowed to proceed for 24 to 48h at room temperature. Following conjugation, any remaining maleimide groups were quenched with 5-fold molar excess2- (■- mercaptoethanol (BME) relative to starting maleimide groups. Quenching step was allowed to react for 2h at room temperature. DMF was then removed via rotary evaporation and the conjugate was dissolved in deionized water and placed in 5mL 50kDA MWCO SpectraPor® Float-A-Lyzers (Repligen, Waltham, MA). The conjugate was dialyzed against DI water for 3 days, with 3 water changes a day. The conjugate was then isolated via lyophilization, and the presence of peptide conjugated to the 4-armed block copolymer was confirmed by1H NMR. The appearance of Tyrosine protons in MeOD was present in the 6.7 to 7.1 ppm range.Docket No. TP385946W01Scheme 3

[0207] The coupling to the maleimide group was confirmed using1H-NMR spectroscopy. FIGS. 14A and 14B show the1H-NMR spectrum for the maleimide starting material (FIG. 14A) and for the peptide-coupled product (FIG. 14B).EXAMPLE 7Polymerization of linear Poly(N-isopropylacrylamide-co-butyl acrylate)

[0208] Random linear copolymers with NIPAM and BA were prepared using controlled free radical polymerization methods. Total targeted molecular weights ranged from 10,000 to 25,000g / mol (i.e. degree of polymerization = 150 to 300). BA molo / o was varied in the monomer feed ratio from 0 to 20molo / o in the reactions. For example, 2.13g (18.8 mmol) of NIPAM, 0.154g (1.2 mmol) of BA and 53.8mg (133 jamol) of 4-Cyano-4- [(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDPA; Sigma-Aldrich, St. Louis, MO), were weighed and added to a 50mL round bottomed flask equipped with a stir bar. All monomers and CDPA were dissolved in 20 mL of 1,4-dioxane. Once dissolved, 2.74 mg (16.7 .mol) of Al BN was added and the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C for 5h. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. All copolymers were isolated through removing dioxane by rotary evaporation, redissolving in 5mL THF and precipitating into 100mL cold diethyl ether. The precipitate was then redissolved in THF and precipitated again into cold diethyl ether and collected by centrifugation. The precipitate was then washed twice with 50mLDocket No. TP385946W01 of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and1H NMR, respectively.EXAMPLE 8Conjugation of8-arm PEG-vinyl sulfone with Poly(N-isopropylacrylamide-co-butyl acrylate)

[0209] Poly(NI PAM-co-BA) copolymers prepared as described in Example 8 were conjugated to 8-arm PEG-vinyl sulfone (PEG-VS) through a thiol-vinyl sulfone coupling reaction. For example, 36 mg (14p.mol of VS) of 20,000 g / mol 8-arm PEG-VS (JenKem Technology; Plano, TX), 200mg (14pmol) of Poly(NI PAM-co-BA) (Mn = 14,000 g / mol, 6molo / o BA) were added to a round bottomed flask equipped with a stir bar and dissolved in degassed DMF (2.8mL) to give a total VS and copolymer concentration of 5mM. The flask was sealed with a septum and sparged for 15min with nitrogen. 2.8 iL (28pmol) of Butyl amine and 4 iL (28pmol) of TEA were added to 200pL of DMF. This butyl amine, TEA and DMF solution was then added to the polymer and PEG-VS solution using a syringe and needle. The reaction was allowed to proceed at room temperature overnight. DMF was then removed via rotary evaporation, redissolved in 2mL THF and precipitated into 40mL of cold diethyl ether. The precipitate was then redissolved in THF and precipitated again into cold diethyl ether and collected by centrifugation. The precipitate was then washed twice with 40mL of cold diethyl ether and dried in vacuo overnight at room temperature. The success of the conjugation of Poly(N I PAM-co-BA) to the vinyl sulfone groups of the 8-arm PEG-VS was determined by1H NMR and integrating the vinyl sulfone protons (6.1 to 6.4 ppm) before and after conjugation. From1H NMR analysis it was determined that 40 to 50o / o of the vinyl sulfone groups were reacted.EXAMPLE 9Polymerization of azide functional linear Poly(N-isopropylacrylamide-co-butyl acrylate)

[0210] a-terminated azide random linear copolymers with NIPAM and BA were prepared using controlled free radical polymerization methods. Total targeted molecular weights ranged from 10,000 to 25,000g / mol (i.e. degree of polymerization = 150 to 300). BA molo / o was varied in the monomer feed ratio from 0 to 20molo / o in the reactions. For example, 1 ,04g (9.2 mmol) ofDocket No. TP385946WO1NIPAM, 0.103g (0.8 mmol) of BA and 29.9mg (67 jarnol) of 2-(dodecylthiocarbonothioylthio)-2- methylpropionic acid 3-azido-1 -propanol ester (Azido-CTA; Sigma-Aldrich, St. Louis, MO), were weighed and added to a 25mL round bottomed flask equipped with a stir bar. All monomers and Azido CTA were dissolved in 10 mL of 1 ,4-dioxane. Once dissolved, 1 .37 mg (8.3 pmol) of Al BN was added and the flask was septa sealed and sparged with nitrogen for45min and subsequently placed in an oil bath at 70 °C for 5h. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. All copolymers were isolated through removing dioxane by rotary evaporation, redissolving in 2.5 mL THF and precipitating into 40mL cold diethyl ether. The precipitate was then redissolved in 2.5 mL THF and precipitated again into cold diethyl ether and collected by centrifugation. The precipitate was then washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Copolymer molecular weight and compositions were characterized by GPC and1H NMR, respectively.EXAMPLE 10 co-end group removal from azide functional linear Poly(N-isopropylacrylamide-co-butyl acrylate)

[0211] <z>-end groups (thiocarbonylthio) on synthesized a-terminated azide Poly(NI PAM-co-BA) copolymers 4 were removed using common procedures familiar to those skilled in the art. For example, 400mg (Mn= 14,000 g / mol, 29pmols of thiocarbonylthio) of azide terminatedPoly(N I PAM-co-BA), 94mg (570 pimols) of Al BN, and 18.5mg (57 jarnols) of BPO were weighed into a round bottomed flask equipped with a stir bar and dissolved into 6mL of dioxane. Once dissolved, the flask was septa sealed and sparged with nitrogen for 45min and subsequently placed in an oil bath at 70 °C overnight. The polymerization was terminated by exposing the reaction to air and placing in a -20°C freezer. The copolymer with removed end-group was isolated by removing dioxane by rotary evaporation, redissolving in 5 mL THF and precipitating into 50 mL cold diethyl ether. Following precipitation, the precipitate was collected by filtration, washed twice with 50mL of cold diethyl ether and dried in vacuo overnight at room temperature. Greater than 90o / o removal of the thiocarbonylthio end-group was confirmed by monitoring the UV absorbance of the thiocarbonylthio group at 310nm during GPC analysis.Docket No. TP385946W01EXAMPLE 11Conjugation of cysteine modified peptides to 8-arm PEG-vinyl sulfone

[0212] Cysteine modified peptides GFOGER (SEQ ID NO:29), RGD and or MMP) with either C- or N-terminus cysteine groups were conjugated to either 20kDa or 40kDa 8-arm PEG-VS through thiol-vinyl sulfone coupling. MMP peptide with an N-terminus dibenzocyclooctyne (DBCO) functionality and C-terminus cysteine group (SciLight Biotechnology, LLC; Beijing, China) was used so that the azide terminate Poly(NIPAM-co-BA) polymers prepared in Example 11 could be conjugated to the peptide modified 8-arm PEG-VS. For example, 25 mg (10 jimol VS group) 8-arm PEG-VS (Mn= 20,000 g / mol), 17 mg (4.15 .mol) GFOGER (SEQ ID NO:29) and 13.3 mg (6.9 ol) MMP were weighed into a 4mL glass scintillation vial equipped with a stir bar and dissolved into 1mL of nitrogen degassed 1x HEPES / 1xPBS buffer pH 8.47. This reaction was allowed to react for 5h at room temperature and stored at 4°C for 48h. The reacted conjugate was then placed in 5mL 10kDa MWCO SpectraPor® Float-A-Lyzer and dialyzed against DI water for 3 days, with 3 water changes per day. The conjugate was then isolated via lyophilization and analyzed by1H NMR in D2O. Successful conjugation was confirmed by the disappearance of the vinyl sulfone protons between 6.3 and 6.5 ppm, and the appearance of the valine methyl protons from the MMP peptide at 1 ppm and appearance of the tyrosine protons from the GFOGER (SEQ ID NO:29) peptide at 6.8 ppm.EXAMPLE 12Conjugation of azide Poly(N I PAM-co-BA) copolymers to 8-arm PEG-peptide-DBCO conjugates

[0213] The DBCO groups of the 8-arm PEG-peptide-DBCO conjugates, similar to those highlighted in Example 12, were then conjugated to azide poly(N I PAM-co-BA) copolymers (synthesized in Example 11) through copper-free click chemistry. Briefly, 40 mg of a 40kDa 8- arm PEG-peptide-DBCO conjugate and 70 mg of azide poly(NI PAM-co-BA) copolymer (17.5kDa) were weighed into a 4mL glass scintillation vial with a stir bar. Both materials were then dissolved in 1mL of DMF to give a DBCO concentration of 3.33mM and an azide concentration of 4mM. 75uL of deionized water was added to facilitate with complete dissolution of the 8-arm PEG-peptide-DBCO conjugates. The vial was placed in a water bath at 40°C and allowed to react for 48h. The reacted conjugate was then diluted to 5mL in deionizedDocket No. TP385946W01 water and placed in 5mL 50kDa MWCO SpectraPor® Float-A-Lyzer and dialyzed against DI water for 3 days, with 3 water changes per day. The conjugate was then isolated via lyophilization and analyzed by1H NMR in MeOD. Successful conjugation was confirmed by 1 H NMR by the appearance of the NIPAM protons near 4ppm and the retention of the PEG protons near 3.6 ppm and tyrosine protons 7.7 ppm. Further evidence of successful conjugation was seen by monitoring the disappearance of the DBCO absorbance at 309nm us UV-vis spectroscopy. The ratio of poly(NIPAM) to PEG was determined through integration of 1 H NMR spectra.EXAMPLE 13Polymerization of azide functional linear Poly(N-isopropylacrylamide) and w-end group removal

[0214] Linear homopolymers of only NIPAM were polymerized and the w-end groups were removed identically to the procedures outlined in Example 10 and 11 without the presence of butyl acrylate. Molecular weights varying from 30kDa up to 100 kDa.EXAMPLE 14Conjugation of DBCO modified oligonucleotide to azide Poly(N-isopropylacrylamide)

[0215] 5' DBCO modified oligonucleotide (25mer thymidine; Integrated DNA Technologies; Coralville, IA) was conjugated to azide poly( / \ / -isopropylacrylamide) using copper free click chemistry. For example, 48 mg of azide poly(NIPAM) (Mn = 62.4kDa; 0.8 pmol) and 4.0 mg (0.5 jimol) of oligonucleotide were weighed into a 2mL microcentrifuge tube. 1mL of 1x phosphate buffered saline (PBS) was added to dissolve both materials. This vial was then placed at 25 °C for 20h. Reaction of the DBCO group with azide was monitored using UV-vis spectroscopy and recording the absorbance of the DBCO group at 309nm. Once the absorbance at 309nm decreased by ~90o / o, the material was diluted with deionized water and dialyzed in a 5mL 50kDa MWCO SpectraPor® Float-A-Lyzer to remove any unreacted oligonucleotide. Dialysis against DI water proceeded for 3 days, with 3 water changes per day. The conjugate was then isolated via lyophilization and characterized by UV-vis spectroscopy. The amount of remaining oligonucleotide was estimated by the absorbance at 260nm and using the provide extinction coefficient for the oligonucleotide of 203,100 M'1cm'1. Any absorbance contribution at 260nmDocket No. TP385946W01 from the polymer would be minimal compared to the high extinction coefficient of the oligonucleotide.EXAMPLE 15Formulating temperature responsive reversible hydrogel solutions in cell culture media or phosphate buffered saline

[0216] 4-armed copolymers prepared as described in Example 5 and 7 or 8-armed copolymers prepared as described in Example 9 and 13 were dissolved in either 1x PBS or GIBCO DMEM (Thermo Fisher Scientific, Waltham, MA) cell culture media at copolymer concentrations between 5 and 10wto / o. For example, copolymers were weighed into microcentrifuge tubes and the final weight of copolymer + 1x PBS or cell culture media was 0.2g. The material was allowed to dissolve into solution on ice with intermittent mixing and sonication until completely dissolved. For copolymers dissolved in cell culture media, following complete copolymer dissolution the material was sterile filtered using centrifugal filtration micro centrifuge tubes outfitted with 0.2 micron pore size filters. These centrifugal filter devices were placed in a microcentrifuge at 4°C and spun at 10,000g for 15 minutes. Collected filtrate was then used for all cell studies.Copolymer solutions prepared in 1x PBS were used for rheometry measurements following complete dissolution without filtration.EXAMPLE 16Transition temperature determination by dynamic light scattering

[0217] Synthesized poly(N I PAM-co-BA) copolymers with varying BA molar content, as described in Example 11 , were dissolved in 1x PBS at a concentration of 5wto / o solids on ice. Once completely dissolved, a temperature sweep between 8°C and 24 °C was run using a Malvern-Zetasizer Nano Series DLS detector with a laser operating at A = 632.8 nm, an avalanche photodiode detector with high quantum efficiency and an ALV / LSE-5003 multiple r digital correlator electronics system. Using the included software the aggregation point determination was used to assign transition temperature. This was also apparent from the rapid rise in count rate as temperature increased above the transition point. Results are shown in FIG. 11.Docket No. TP385946W01EXAMPLE 17Rheometry measurements on 4-armed copolymer solutions

[0218] 4-armed copolymers dissolved in 1x PBS, as prepared in Example 16, were used to measure storage modulus at 37°C and to determine transition temperature as the material went from liquid to hydrogel. To determine transition temperatures an oscillation temperature ramp experiment was performed. Briefly, 200|iL was pipetted onto the rheometer equipped with a cone and plate geometry (diameter 35mm with a 1 ° angle for the cone, gap height = .053 mm). The starting temperature was set to 10°C and increased at 2°C per minute up to 37°C. A strain of 0.02 and frequency of 1Hz was used throughout the entire experiment.

[0219] For direct storage modulus measurements an oscillation time sweep experiment, 200 .L was pipetted onto the rheometer equipped with a cone and plate geometry (diameter 35mm with a 1 ° angle for the cone, gap height = 0.053 mm). The initial temperature was 10°C so that the liquid would not gel immediately. The plate was then heated to 37°C and the experiment was run with a strain of 0.02 and frequency of 1 Hz for 600s.EXAMPLE 18Poly(N-isopropylacrylamide) oligonucleotide conjugate precipitation

[0220] To test the efficiency of precipitating poly( / V-isopropylacrylamide) (PNIPAM) oligonucleotide conjugates prepared in Example 15, the following procedure was performed. First, unconjugated PNIPAM (prepared in Example 8; Mn= 80kDa) was dissolved in TRIS buffer (10mM TRIS, 140 mM NaCI, 1 mm EDTA at pH 8.0) at a concentration of 3wto / o. Next PNIPAM- oligonucleotide conjugate (Mn = 80.3 kDa) was dissolved in TRIS buffer at a concentration of 1.4wto / o (250 jiM oligonucleotide concentration as measured by UV-vis spectroscopy). The PNIPAM-oligonucleotide conjugate was then diluted into the 3wto / o PNIPAM solution to give a oligonucleotide concentration of 25|4.M with a final volume of 500 .L (total PNIPAM concentration = 2.4wto / o). The unconjugated PNIPAM solution was added to serve as a bulking agent to encourage flocculation of the PNIPAM-oligonucleotide conjugate. The solution was then mixed and incubated at 45°C for 15 minutes. The solution was then centrifuged at 13,000g for 1 minute and the supernatant was collected for UV-vis analysis. The remaining PNIPAM-Docket No. TP385946W01 oligonucleotide conjugate concentration in the supernatant was determined to be 21o / o by absorption at 260nm, indicating that ~80o / o of the conjugate was successfully removed through the precipitation procedure.EXAMPLE 19

[0221] In this example, adult liver organoids were prepared. As a general procedure, ductal liver cells were isolated by enzymatic digestion from human adult liver donors. Then, the digestion was stopped, and the cells were collected. Isolated cells were resuspended in EHS- BME or thermo-responsive polymer. The material was deposited in wells by adding a droplet of cells embedded in the material of choice. Droplets with cells were incubated for 30mins at 37°C (cell incubator) to solidify the EHS-BME (Engelbreth-Holm-Swarm-Basement Membrane Extracts) or the thermo-responsive polymer. Cell media was then added to wells containing droplets. Cells were grown under standard tissue culture conditions (37°C, 5o / o CO2). Medium was changed every 3-4 days. Organoids were visible within 7 days and ready for passaging before day 14 of culture.

[0222] In specific examples, biopsies were minced and resuspended in Wash Medium ((DMEM high glucose + Glutamax+ Pyruvate (Gibco) supplemented with 1o / o v / v Fetal Bovine Serum (Gibco)). Supernatant was removed to remove red blood cells and fat. Liver cells were isolated by collagenase digestion as follows: minced liver biopsies (0.5-1cm3) were incubated with the digestion solution (2.5mg / ml Collagenase IV (Gibco) + 0.1 mg / ml DNAse I (Thermo Fisher Scientific) in EBSS (Gibco)), for 30 minutes at 37°C. The digestion was stopped by adding cold DMEM / F12 (Gibco) and the suspension was then filtered through a 70pm nylon cell strainer (Falcon) and spun for 5mins at 300g. The pellet was washed twice with Wash Medium and spun for 5 mins at 300g. The pellet was then washed once with Basal Medium (Advanced DMEM / F12 (Gibco) supplemented with 10,000 U / ml Penicillin-Streptomycin (Gibco) + 1o / o v / v GlutaMax (Gibco) + 10mM HEPES (Gibco)) and spun for at 200g for 5mins at 8°C. The cell pellet was mixed and re-suspended at a cell density of 20,000 cells / 50ul with Geltrex (Gibco). A 50 ul droplet per well was seeded in non-coated 24-well plates (NUNC). Plate was incubated for 30 minutes at 37°C. After matrix had solidified, cell media was added. Cell media was changed every 3 days and culture was sub-passaged every 7-10 days.Docket No. TP385946W01

[0223] Next, clump passaging is used to establish and expand the human liver organoid culture. Briefly, the basement membrane extract, Geltrex droplet, is disrupted by scarping and pipetting droplet up and down using a P1 ,000 pipette. Cold basal media is then added and pipetted up and down using a P200 pipette to break Geltrex droplets and cell model into smaller pieces. Centrifugation at 400g for 5mins at 4°C was used. The supernatant is aspirated, leaving minimal volume in the tube without aspirating pellet. The pellet is re-suspended in Wash medium and pipette up and down using P200. The sample is centrifuged at 400g for 5mins at 4 °C. The supernatant is aspirated, leaving minimal volume in the tube without aspirating pellet. Cell aggregates are suspended in Geltrex and seeded as droplets by adding a 50ul droplet at the center of each well. They are incubated at 37°C for 20 minutes to solidify Geltrex and then overlayed with cell media. The medium is changed every 3 days and passage every 7-10 days at a split ratio of 1 :4 - 1 :8. As can be seen in FIGS. 17A-17E, at day 6 after cell seeding, organoid morphology in all configurations of the thermo-responsive polymer (FIGS. 17A-17C) was similar to the gold standard EHS-BME, known in the market as Geltrex / Matrigel (FIG. 17D).

[0224] Organoid cultures were dissociated into single cells using TrypLE express (GIBCO). A Geltrex droplet was disrupted by scarping and pipetting droplet up and down using a P1 ,000 pipette. It is centrifuged at 400g for 5mins at 4 °C. The supernatant is aspirated, leaving minimal volume in the tube without aspirating pellet. The sample is washed with T rypLE express, 1 ml per dissociated Geltrex droplet, and centrifuged at 400g for 5mins at 4 °C. The supernatant is aspirated, leaving minimal volume in the tube without aspirating pellet, and T rypLE express was added, 1 ml per dissociated Geltrex droplet and incubated at 37°C for 10 minutes. After 10 minutes, the sample was centrifuged at 400g for 5mins at 4 °C. The supernatant was aspirated, leaving minimal volume in the tube without aspirating pellet and rinsed Ixwith cell media. Cells were counted by trypan blue exclusion. Cells were then suspended in Geltrex or thermo-responsive polymer at a concentration of 250 cells / ul and seeded as droplets by adding a 50ul droplet at the center of each well. Samples were incubated at 37°C for 20mins to solidify Geltrex and then overlayed with cell media. The cell medium was changed every 3 days. Phase contrast pictures were acquired using EVOS FL Auto (Thermo Fisher Scientific) microscope at days 7-10.Docket No. TP385946W01

[0225] Quantification of organoid size and number: Phase contrast images of organoids suspended in Geltrex or thermo-responsive polymer were acquired using EVOS FL Auto (Thermo Fisher Scientific) microscope with a 1 .25X objective at day 7-10. A single image was taken per droplet and was processed using an open-sourced software for quantifying organoid metrics (Orgaquant, MIT). Organoid metrics such as number of organoids per image and size was processed using the statistical software JMP (JMP). Two-way ANOVA and Tukey"s post hoc test were used to assess the difference of organoid size in the different treatments. In general, the sample size consisted of at least 4 technical replicates each. Statistical significance was considered at a value of a<0.05. FIGS. 15A and 15B show images of an adult liver organoid (ALO) generated using a GeltrexTM-derived ECM (FIG. 15A) and an ALO generated using a thermo-responsive polymer-derived ECM according to aspects of the present disclosure. See FIGS. 16A, 16B, and 16C for additional size and / or number analysis results. At day 6, after cell seeding in an embedded format (droplet deposition), all configurations of the Thermo-Responsive polymer had a comparable size to the gold standard matrix (EHS-BME) known in the art as Geltrex / Matrigel. The results establish that TRP can support the organoid growth to a size that is similar to the gold standard EHS-BME matrix. Also, at day 6, after cell seeding in an embedded format (droplet deposition), organoid number was similar between the EHS-BME and the Thermo-Responsive Polymer (TRP). The results establish that TRP can support the organoid formation at a similar level compared to the gold standard EHS-BME matrix.

[0226] After culturing the ALO for 6 days in the respective matrix, gene expression was assessed and compared. The expression of adult liver and ductal progenitor markers such as LGR5 and SOX9 were comparable between EHS-BME and the Thermo-Responsive Polymer (FIG. 19A and 19B). The expression of hepatocyte related markers, such as HNF4A and albumin, was comparable between EHS-BME and the Thermo-Responsive Polymer (FIGS. 20A and 20C). Similar expression of a cholangiocyte marker KRT-19 was observed between EHS- BME and the Thermo-Responsive Polymer (FIG. 20B). Overall results showed that the same cell model is being grown in vitro between EHS-BME and the Thermo- Responsive Polymer.Docket No. TP385946WO1

[0227] Real-time PCR (qPCR): Human Liver Organoids were grown as described previously in in vitro growth. At day 7-10 RNA was extracted from organoid cultures using TRIzol Plus RNA Purification Kit and Phasemaker Tubes Complete System (Thermo Fisher Scientific). RNA concentration was quantified using Agilent TapeStation System (Agilent). RNA from each sample was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). For each quantitative PCR reaction, generated cDNA was combined with primers for genes of interest (see Table 2) and Taqman reagents (Thermo Fisher Scientific) in a real-time PCR system QuantStudio 12K Flex (Thermo Fisher Scientific). Changes in relative expression for the genes of interest were calculated using GAPDH as reference gene.Docket No TP385946WO1

[0228] Organoid recovery from Thermo-Responsive Polymer: At day 7-10, cell media was removed and substituted with cold DMEM or 1x DPBS. Phase contrast videos of organoids being released from the thermo-responsive polymer were acquired using EVOS FL Auto (Thermo Fisher Scientific) microscope with a 1.25X objective. See FIGS. 21A-21C.EXAMPLE 20

[0229] General information regarding preparing thermo-responsive polymers conjugated with nucleic acid-coupling components. In some examples, the thermo-responsive polymer is conjugated to a nucleic-acid coupling component (e.g., polyT oligonucleotide ligand) using a functional group positioned at the end of a thermo-responsive polymer. In some additional examples, a heterobifunctional cross-linker is used to facilitate reaction between polyT amine and a functional group of the thermo-responsive polymer. Conjugates are purified using dialysis and are characterized by NMR. Transition temperatures of the conjugate are remeasured postconjugation using DLS to identify if adding the PolyT ligand impacts the transition temperature of the thermo-responsive polymer.

[0230] Preliminary pull-down assays are used to test the ability of the PolyT-polymer conjugates to bind test mRNA comprising 25-mer PolyA tags. The efficacy of the thermo- responsive PolyT-polymer conjugates to bind and precipitate the test mRNA is determined by monitoring the supernatant absorbance at 260 nm using UV-Vis spectroscopy. This can be done before and after inducing precipitation of the mRNA / PolyT-polymer complex.

[0231] The polymer conjugated to the OdT ligand can be reused. The reusability of the polyT- polymer is evaluated by reusing the polyT-polymer after the purification in another round of mRNA capture and release. Binding and release conditions are tuned by changing the buffer, salt, EDTA, and the like.EXAMPLE 21

[0232] Preparation of Azide-Terminated Thermo-Responsive Polymers. In this example, azide-terminated PNIPAM thermo-responsive polymers are synthesized. 6.72 mg (15 pmol) of 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester (DMP-N3) wasDocket No TP385946WO1 weighed out into a 25mL round bottomed flask equipped with a stir bar followed by 1.70g (15 mmol) of NIPAM monomer. These materials were then dissolved into 15mL of dioxane. Once dissolved, a 20mg / ml_ stock solution of the radical initiator azobisisobutyronitrile (Al BN) in dioxane was prepared. 25pL (3 jimol) of this stock was added to the round bottomed flask. This gives a monomer:DMP-N3:AIBN ratio of 1000:1.0:0.2. The flask was sealed with a rubber septa and copper wire and sparged under nitrogen for 45 minutes. After sparging, the flask was immersed in an oil bath at 70 °C for 4 hours. After 4 hours, the reaction was quenched by cooling to room temperature and exposing the polymerization to the atmosphere. The polymer was isolated by first removing dioxane via rotary evaporation, redissolving in ~ml_ THF and precipitating into ice cold diethyl ether. The precipitated polymer was then washed with 50mL ether. The material was then redissolved in THF and reprecipitated before drying under vacuum overnight at room temperature. The PNIPAM was characterized by1H NMR and size exclusion chromatography (see FIG. 12). PNIPAM polymers of varying molecular weight were synthesized by varying the ratio of NIPAM monomer to DMP-N3from 300 up to 1600.EXAMPLE 22

[0233] Removing Dodecylthiocarbonothioylthio Functionality From Azide Terminated Thermo- Responsive Polymer. In this example, a dodecylthiocarbonothioylthio functionality present on a representative thermo-responsive polymer was removed from the p-terminal of the synthesized azide-terminated PNIPAM polymer described in Example 2 using a method as summarized in Scheme 1. Briefly, 1.0 gram of azide-terminated PNIPAM was weighed into a round bottomed flask equipped with a stir bar and dissolved in dioxane to give a polymer concentration of 2 to 5mM. Once dissolved, 2-fold molar excess of benzoyl peroxide (BPO) and 20-fold molar excess Al BN were weighed and added to the flask. The flask was sealed with a rubber septa and copper wire and sparged under nitrogen for 35 min. After sparging the flask was immersed in an oil bath at 70°C overnight (~18h). The polymer was then isolated by first removing dioxane via rotary evaporation, redissolving in ~mL THF and precipitating into ice cold diethyl ether. The precipitated polymer was then washed with 50mL ether. The material was then redissolved in THF and reprecipitated before drying under vacuum overnight at room temperature. The PNIPAM was characterized size exclusion chromatography and monitoring the absorbance at 310nm to indicate removal of the dodecylthiocarbonothioylthio functionality.Docket No TP385946WO1EXAMPLE 23

[0234] Preparation of Oligo dT-Polymer Conjugates. In this example, an oligo dT-polymer conjugate was made according to the method summarized by FIG. 3. The azide-terminated thermo-responsive polymer (in this example, azide-terminated PNIPAM without dodecylthiocarbonothioylthio functionalities) was conjugated to a DBCO-modified oligo dT tag (in this example, 5'dibenzocyclooctyne (DBCO) modified oligo deoxythymidine (OdT; 25mer) purchased form Integrated DNA Technologies (IDT; Coralville, IA)). Briefly, 47.8 mg of azide- terminated PNIPAM (0.995 .mol; Mn= 48,000 g / mol) and 4 mg of DBCO-OdT (0.497pmol; MW = 8,038 g / mol) were dissolved in 1x phosphate buffer saline to give a polymer concentration of 1mM. This reaction was then allowed to proceed overnight at 21°C and conversion of the DBCO to a triazole-conjugated oligo dT-polymer conjugate as shown upon reaction with the azide group was monitored by UV-vis absorption at 309nm. See FIGS. 22A-22C. The conjugate was then isolated by dialyzing against deionize water using 50kDA molecular weight cut-off Float-A-Lyzers (Repligen). Additional results are provided in Table 3, below.

[0235] FIG. 23A shows the precipitation of the conjugate and polymer using UV-vis measurement and FIG. 23B shows the measurement without the polymer baseline subtraction for the supernatant.EXAMPLE 24

[0236] Preparation of Azide-Terminated poly(NIPAM-co-Butylacrylate) Polymers. In this example, an azide-terminated poly(NIPAM-co-butylacrylate) polymer is made using a method asDocket No TP385946WO1 summarized in FIG. 7. Briefly, 22.4 mg (50 jimol) (2-(Dodecylthiocarbonothioylthio)-2- methylpropionic acid 3-azido-1 -propanol ester (DMP-N3) was weighed out into a 25mL round bottomed flask equipped with a stir bar followed by 1.56g (13.8 mmol) of NIPAM monomer and 0.15g (1.2 mmol) of butyl acrylate monomer. These materials were then dissolved into 15ml_ of dioxane. Once dissolved, a 20mg / mL stock solution of the radical initiator azobisisobutyronitrile (Al BN) in dioxane was prepared. 51 .L (6 |amol) of this stock was added to the round bottomed flask. This gives a total monomer: DM P-N3: Al BN ratio of 300:1.0:0.125. The flask was sealed with a rubber septa and copper wire and sparged under nitrogen for 45 min. After sparging, the flask was immersed in an oil bath at 70°C for 4 hours. After 4 hours, the reaction was quenched by cooling to room temperature and exposing the polymerization to the atmosphere. The polymer was isolated by first removing dioxane via rotary evaporation, redissolving in ~mL THF and precipitating into ice cold diethyl ether. The precipitated polymer was then washed with 50ml_ ether. The material was then redissolved in THF and reprecipitated before drying under vacuum overnight at room temperature. The poly(NIPAM-co-butylacrylate) polymer was characterized by1H NMR and size exclusion chromatography. In additional examples, poly(NIPAM-co-butylacrylate) copolymers of varying molo / o of butyl acrylate were synthesized by varying the feed ratio of NIPMA to butyl acrylate from 6 to 20 molo / o.EXAMPLE 25

[0237] Synthesis of 5'dibenzocyclooctyne (DBCO) modified oligo deoxythymidine (OdT). In this example, a 5'dibenzocyclooctyne (DBCO) modified oligo deoxythymidine (OdT) is prepared as an alternative to purchasing an already DBCO-modified oligo deoxythymidine 25mer. A DBCO-linker-N-hydroxysuccinimide (NHS) ester crosslinker, such as dibenzocyclooctyne-N- hydroxysuccinimidyl ester, is provided and reacted with a 5'Amino- (NH2) modified oligo deoxythymidine using amine-activated ester chemistry and purification conditions known to those in the art with the benefit of the present disclosure.

[0238] In view of the many possible embodiments to which the principles of the present disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of theDocket No TP385946W01 present disclosure. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. Docket No TP385946WO1What is claimed is:

1. A thermo-responsive polymer, having a structure according to Formula I z-fxHYJpTGFormula I whereinZ represents a coupling component;TG is a terminating group; each X and each Y, for each occurrence of X and Y, independently is selected from an acryloyl-derived monomer having a structure according to Formula A, or an N- isopropylacrylamide-derived monomer having a structure according to Formula B, provided that (i) at least one X is the acryloyl-derived monomer and at least one Y is the N- isopropylacrylamide-derived monomer; or (ii) at least one X is the N-isopropylacrylamide- derived monomer and at least one Y is the acryloyl-derived monomer; each n, for each occurrence, independently is an integer selected to satisfy a formula of ritotal "*■ nritotal—p, each m, for each occurrence, independently is an integer selected to satisfy a formula of ntotai + rritotai = p; and p is an integer selected from 2 to 1600; wherein Formula A isFormula A wherein each X1independently is (i) NH or O if q' is 1 , or (ii) is NH2if q' is 0; each R" independently is H, aliphatic, or OH; each q independently is an integer selected from 0 to 20; and q' is 0 or 1 ; and Formula B isDocket No TP385946W01Formula B.

2. The thermo-responsive polymer of claim 1 , wherein the coupling component is a functional group or molecule that facilitates binding the thermo-responsive polymer to a biological component or associating the thermo-responsive polymer with a biological component.

3. The thermo-responsive polymer of claim 1 or claim 2, wherein the coupling component is (i) a functional group capable of coupling the thermo-responsive polymer to a biomolecule-coupling component or (ii) a multi-armed polymer.

4. The thermo-responsive polymer of any one of claims 1-3, having a structure according to Formula IAFormula IA wherein FG represents the functional group capable of coupling the thermo-responsive polymer to the biomolecule-coupling component.

5. The thermo-responsive polymer of claim 4, wherein the FG is a clickable functional group or a heterobifunctional linker.

6. The thermo-responsive polymer of claim 4 or claim 5, wherein the clickable functional group is selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group.Docket No TP385946WO17. The thermo-responsive polymer of any one of claims 4-6, wherein the FG is an azide group.

8. The thermo-responsive polymer of any one of claims 1-7, wherein the FG is further bound to the biomolecule-coupling component.

9. The thermo-responsive polymer of claim 8, wherein the biomolecule-coupling component is an oligonucleotide ligand, a member of a specific binding pair, a group that facilitates a protein-protein interaction, or a member of a guest-host pair.

10. The thermo-responsive polymer of claim 8, wherein the oligonucleotide ligand is an oligo deoxythymidine (OdT) ligand further comprising a 5'dibenzocyclooctyne group.11 . The thermo-responsive polymer of any one of claims 1-7 having a structure according to a formula selected from12. The thermo-responsive polymer of any one of claims 1-7 or 11 having a structure according to a formula selected fromDocket No TP385946WO113. The thermo-responsive polymer of claim 3, having a structure according toFormula IBFormula IB wherein MAP represents the multi-armed polymer.

14. The thermo-responsive polymer of claim 13, wherein the MAP is a multi-armed PEG-containing group or a multi-armed acrylamide-containing group and wherein (i) X is attached directly or indirectly to at least one arm of the multi-armed PEG-containing group or (ii) X is attached directly or indirectly to at least one arm of the multi-armed acrylamide-containing group.

15. The thermo-responsive polymer of claim 14, wherein the multi-armed PEG- containing group is an eight-armed PEG-containing group; or wherein the multi-armed acrylamide-containing group is a four-armed acrylamide-containing group.

16. The thermo-responsive polymer of 15, wherein the four-armed acrylamide- containing group comprises four arms formed by repeating units ofDocket No TP385946WO1(i) a monomer having a structure according to Formula CFormula C; and(ii) a monomer having a structure according to Formula DFormula D wherein each R' independently is an amine protecting group, a linker group, a linker group bound to a cell-binding peptide, or hydrogen; and each r' independently is an integer selected from 1 to 3.

17. The thermo-responsive polymer of any one of claims 14-16, wherein the thermo- responsive polymer comprises one or more additional groups having a formula -[(X)n-(Y)m]P-TG, wherein each of X, Y, TG, n, m, and p are as recited for Formula I, and wherein the one or more additional groups independently are attached to other arms of the multi-armed PEG-containing group or the multi-armed acrylamide-containing group.

18. The thermo-responsive polymer according to any one of claims 14-17, wherein a single arm of the multi-armed acrylamide-containing group is independently coupled to a number of peptides ranging from 1 to 20.

19. The thermo-responsive polymer of any one of claims 13-17 having a structure according to a Formula IIIDocket No TP385946W01Formula III wherein each Q and each T, for each occurrence of Q and T, independently is selected from an acrylamide-derived monomer having a structure according to Formula C, or an amino-aliphatic methacrylamide-derived monomer having a structure according to Formula D, provided that (i) at least one Q is the acrylamide-derived monomer and at least one T is the amino-aliphatic methacrylamide-derived monomer; or (ii) at least one Q is the amino-aliphatic methacrylamidederived monomer and at least one T is the acrylamide-derived monomer; and each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 100 to 500; wherein Formula C isFormula C and Formula D isFormula D wherein each R1independently is an amine protecting group, a linker group, a linker group bound to a cell-binding peptide, or hydrogen; and each r1independently is an integer selected from 1 to 3.Docket No TP385946W0120. The thermo-responsive polymer of any one of claims 13-17 or 19 having a structure according to a formulawherein each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer ranging from 200 to 300.

21. The thermo-responsive polymer of any one of claims 13-17, 19, or 20 having a structure according to a formula selected fromDocket No TP385946WO1Docket No TP385946WO1Cell-binding Peptidewherein each of r and s independently is an integer selected to satisfy a formula r + s = t, wherein t is an integer selected from 200 to 300.

22. The thermo-responsive polymer according to any one of claims 18-23, wherein r, s, n, and m are selected to provide a ratio of (r + s) : (n + m) that ranges from 0.25 to 3.

23. The thermo-responsive polymer according to any one of claims 1 -23, wherein the thermo-responsive polymer exists either (i) as a liquid at temperatures up to a critical temperature point of the thermo-responsive polymer or (ii) as a hydrogel at temperatures above the critical temperature point of the thermo-responsive polymer.

24. A composition, comprising: one or more cells; and the thermo-responsive polymer according to any one of claims 1 or 13-23, wherein the thermo-responsive polymer is coupled to a cell-binding peptide.Docket No TP385946WO125. The composition according to claim 24, wherein, at a temperature below 32 °C, the thermo-responsive polymer is in the form of a liquid.

26. The composition according to claim 24 or claim 25, wherein, at a temperature above 32 °C, the thermo-responsive polymer is in the form of a hydrogel.

27. The composition according to any one of claims 24-26, wherein the one or more cells are primary cells, stem cells, induced pluripotent stem cells, or a combination thereof.

28. The composition according to any one of claims 24-27 wherein the one or more cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, epidermal cells, or any combination thereof.

29. A composition, comprising: one or more biological components; and the thermo-responsive polymer according to any one of claims 1-7 or 11 , wherein the thermo-responsive polymer is coupled to the biomolecule-coupling component; or the thermo- responsive polymer according to any one of claims 8-10.

30. A method, comprising: exposing a cell to a thermo-responsive polymer according to any one of claims 1 or 13- 23 at a first temperature to provide a seeding composition comprising the cell dispersed in the thermo-responsive polymer, wherein the first temperature is a temperature below the critical temperature point of the thermo-responsive polymer; applying the seeding composition to a culture apparatus comprising a growth medium to provide a seeding layer on the growth medium; warming the culture apparatus by exposing it to a second temperature, wherein the second temperature is a temperature that is above the critical temperature point of the thermo- responsive polymer, wherein warming the culture apparatus facilitates cell growth from the seeding layer; andDocket No TP385946WO1 removing the thermo-responsive polymer from the culture apparatus by exposing the culture apparatus to the first temperature; wherein the thermo-responsive polymer is coupled to a cell-binding peptide.

31. The method of claim 30, wherein the first temperature causes the thermo- responsive polymer to be in a liquid state.

32. The method of claim 30 or claim 31 , wherein the second temperature causes the thermo-responsive polymer to transition from a liquid to a hydrogel.

33. The method according to any one of claims 30-32, wherein the one or more cells are primary cells, stem cells, induced pluripotent stem cells, or a combination thereof.

34. The method according to any one of claims 30-33 wherein the one or more cells are selected from hepatic cells, colonic cells, gastric cells, pancreatic cells, prostate cells, lung cells, kidney cells, intestinal cells, rectal cells, mammary cells, corneal cells, or epidermal cells.

35. A method, comprising: adding the thermo-responsive polymer according to any one of claims 1-10 or 21 to a biomolecule-containing composition to provide a solution wherein the thermo-responsive polymer is coupled to biomolecules present in the biomolecule-containing composition to form a biomolecule-polymer conjugate; exposing the solution to a heat source at a first temperature that is a temperature above the critical temperature point of the thermo-responsive polymer to convert the biomolecule- polymer conjugate to a non-liquid state; isolating the biomolecule-polymer conjugate from the solution; cooling the biomolecule-polymer conjugate by exposing the biomolecule-polymer conjugate to a second temperature that is a temperature below the critical temperature point of the thermo-responsive polymer to convert the biomolecule-polymer conjugate to a liquid state; exposing the biomolecule-polymer conjugate to an additive to de-couple the biomolecule and the thermo-responsive polymer; andDocket No TP385946W01 isolating the biomolecule from the thermo-responsive polymer.

36. The method of claim 35, wherein the biomolecule-coupling component of the thermo-responsive polymer is an oligonucleotide ligand and is coupled to the molecule via non- covalent interactions.

37. The method of claim 35 or claim 36, wherein the biomolecule-polymer conjugate is isolated by filtering the solution to separate the biomolecule-polymer conjugate from liquid in the solution.

38. The method of claim 37, wherein filtering the mixture comprises exposing the mixture to tangential flow filtration.

39. The method of any one of claims 35-38, further comprising performing a washing step and / or a centrifugation step to remove any contaminants from the solution and / or the biomolecule-polymer conjugate.

40. The method of any one of claims 35-38, wherein the additive is a compound or stimulus that disrupts interactions between the biomolecule and the thermo-responsive polymer.

41. The method of any one of claims 35-40, wherein the additive is selected from a buffer, a salt, a water miscible solvent, a reagent capable of cleaving a cleavable linker and / or capable of promoting competitive dissociation, excess ODT to facilitate release of mRNA, or any combination thereof.

42. A method for making the thermo-responsive polymer according to any one of claim 1 or 13-23, comprising: combining a multi-armed polymer with N-isopropylacrylamide, an acryloyl monomer, and a radical initiator to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer.Docket No TP385946WO143. The method of claim 42, further comprising exposing the thermo-responsive polymer to a deprotecting reagent and a linker group.

44. The method of claim 43, further comprising coupling the thermo-responsive polymer to a cell-binding peptide.

45. The method of any one of claims 42-44, further comprising making the multiarmed polymer by combining acrylamide, an amino-aliphatic methacrylamide monomer, a multiarmed starting reagent, and a radical initiator to provide a preliminary reaction mixture; and heating the preliminary reaction mixture.

46. The method of claim 45, wherein the amino-aliphatic methacrylamide monomer has a structure according to Formula D"Formula D' wherein R' is an amine protecting group; and r' is an integer selected from 1 to 4.

47. A method for making the thermo-responsive polymer according to any one of claims 1-12 or 23, comprising: combining a chain transfer agent, a radical initiator, an acryloyl monomer, and N- isopropylacrylamide to provide a reaction mixture; and heating the reaction mixture at a temperature ranging from 25 °C to 100 °C to provide the thermo-responsive polymer; wherein(i) the chain transfer agent is a compound comprising the Z group of Formula I and a thiourea group;Docket No TP385946WO1(ii) the acryloyl monomer and the N-isopropylacrylamide are provided at a ratio to provide a value for p ranging from 2 to 1600; and(iii) the concentration ratio of the acryloyl monomer and / or the N-isopropylacrylamide to the chain transfer agent ranges from greater than 0 to 20 molo / o.

48. The method of claim 47, wherein the acryloyl monomer has a structure according to Formula A'Formula A' wherein X1is NH or O if q' is 1 or is NH2if q' is 0; R" is H, aliphatic, or OH; q is an integer selected from 0 to 20; and q' is 0 or 1.

50. The method of claim 47 or claim 48, wherein the chain transfer agent has a structure according to Formula FSR2'S-^R i1Formula F wherein R1is an ether or thioether group; and R2is an aliphatic or heteroaliphatic group comprising the Z group of Formula I.

51. The method of any one of claims 47-49, wherein the chain transfer agent has a structure selected fromDocket No TP385946WO1 wherein Z is selected from an azide group, a terminal or di-substituted alkyne group, a tetrazine group, a trans-cyclooctene group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group.

52. The method of any one of claims 47-50, wherein the chain transfer agent reacts with the acryloyl monomer or the N-isopropylacrylamide monomer such that R2of the chain transfer agent becomes bound to a first terminus of the thermo-responsive polymer and a sulfur atom of the thiourea becomes bound to a second terminus of the thermo-responsive polymer.

53. The method of any one of claims 47-51 , further comprising cleaving the thiourea from the second terminus of the thermo-responsive polymer and capping the second terminus with a terminating group.

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