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WO 2017/019841 Al 2 February 2017 (02.02.2017) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/019841 Al 2 February 2017 (02.02.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 47/32 (2006.01) A61L 27/14 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 47/42 (2006.01) A61L 27/22 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US20 16/0444 11 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 28 July 2016 (28.07.2016) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 62/197,693 28 July 2015 (28.07.2015) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: TRUSTEES OF TUFTS COLLEGE TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, [US/US]; Ballou Hall, Medford, Massachusetts 02155 TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (US). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors: OMENETTO, Fiorenzo G.; 503 Concord Av SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, enue, Lexington, Massachusetts 02421 (US). KAPLAN, GW, KM, ML, MR, NE, SN, TD, TG). David L.; 46 Pond Street, Concord, Massachusetts 01742 (US). MARELLI, Benedetto; 42 Wachusett Drive, Lex Declarations under Rule 4.17 : ington, Massachusetts 02421 (US). LI, Chunmei; 109 Hill — as to applicant's entitlement to applyfor and be granted a St, Apt. 12, Stoneham, Massachusetts 02180 (US). patent (Rule 4.1 7(H)) (74) Agents: CAHILL, John J. et al; Choate, Hall & Stewart — as to the applicant's entitlement to claim the priority of the LLP, Two International Place, Boston, Massachusetts earlier application (Rule 4.1 7(in)) 021 10 (US). [Continued on nextpage] (54) Title: POLYPEPTIDE MONOLITHS 00 © (57) Abstract: The present application relates to amphiphilic polypeptide materials, methods for making and using the same. o Provided amphiphilic polypeptide materials are water-based and manufactured using all aqueous processing. Provided materials ex - hibit a capacity to encapsulate and store biologically active molecules or macromolecules. Such biologically active molecules or macromolecules retain their structure and the biological activity so that these biologically active molecules or macromolecules are not materially degraded, reduced, and/or inhibited by processing steps or exposure. Provided materials possess unique mechanical and structural properties, including size, density, moldability and machinability. w o 2017/019841 A llll II II 11III I II III II II I III IIII II I II Published: POLYPEPTIDE MONOLITHS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application claims priority to and the benefit of, United States provisional patent application serial number 62/197,693, filed on July 28, 2015, entitled "Polypeptide Monoliths", the contents of which are hereby incorporated by reference in their entirety herein GOVERNMENT SUPPORT [0002] This invention was made with government support under grant AR068048 awarded by the National Institutes of Health and grant FA9550-10-1-0172 awarded by the Air Force Office of Scientific Research. The government has certain rights in the invention. BACKGROUND [0003] Monoliths are three-dimensional blocks of material that are both solid and continuous. The term "monolith" is typically used to refer to materials characterized by properties (e.g., bulk density, hardness, resistance to fracture, etc.) that render them amenable to processing into hard structures. Thus monoliths of appropriate size may be machined into desirable shapes. Protein/polypeptide monoliths are of particular interest for a variety of reasons, including their potential biocompatibility and biodegradability. Additionally, appropriate monolith materials may tolerate inclusion or incorporation of additives that, for example, may provide some added functionality to the blocks. SUMMARY OF THE INVENTION [0004] Among other things, the present disclosure provides amphiphilic polypeptide materials. In some embodiments, provided amphiphilic polypeptide materials are characterized in that such materials are three-dimensional. In some embodiments, provided amphiphilic polypeptide materials are three dimensional "blocks" also referred to herein as "monoliths." Such amphiphilic polypeptide materials exhibit unique structural characteristics and properties when compared with known monoliths. In some embodiments, provided amphiphilic polypeptide materials are three dimensional structures that are surprising large relative to any known protein/polypeptide monolith materials. In some embodiments, provided amphiphilic polypeptide materials are characterized in that they are capable of being machined using machine tools. In some embodiments, amphiphilic polypeptide materials generated are replicate nano- scale structures of natural fibers. [0005] The present disclosure, in some embodiments, also provides methods of making and using amphiphilic polypeptide materials. [0006] Implementations of the present disclosure are useful for a wide range of applications, including but not limited to: anti-counterfeiting materials, biomaterials, biomedical devices, commercial products, controlled degradation applications, controlled release applications, drug delivery, drug release, electronics, extrusion injection molding, materials for tunable degradation, optics, orthopedic devices, photonics, preservation of biologically labile compounds, preservation of heat labile compounds, prosthetics, regenerative medicine, robotics, sensing, tissue engineering applications, tissue regeneration, tissue scaffolding, triggered release applications and/or wound clotting. [0007] In some embodiments, polypeptide materials are or comprise amphiphilic polypeptides and/or fragments thereof. [0008] In some embodiments, provided amphiphilic polypeptide materials comprise amphiphilic polypeptides and/or fragments thereof that have a specific molecular weight. In some embodiments, amphiphilic polypeptide and/or fragments thereof comprise amphiphilic polypeptides having varied molecular weights. In some embodiments, an average molecular weight of amphiphilic polypeptide is for example, between about 100 Da and about 400 kDa. [0009] In some embodiments, provided amphiphilic polypeptide materials are characterized by density. In some embodiments, provided amphiphilic polypeptide materials are characterized by a bulk density. In some embodiments, provided amphiphilic polypeptide materials have a bulk density that is comparable to the density of a native polypeptide when in its fibroin or globular structure form. In some embodiments, provided amphiphilic polypeptide materials are characterized by a bulk density that is uniform or continuous throughout the material. In some embodiments, provided amphiphilic polypeptide materials have a micro- density continuity. In some embodiments, provided amphiphilic polypeptide materials have a nano-density continuity. [0010] In some embodiments, provided amphiphilic polypeptide materials are characterized by a density near that of natural amphiphilic polypeptide materials. In some embodiments, provided amphiphilic polypeptide materials are characterized by a density of about 0.05 kg/dm3 and about 5.0 kg/dm3. In some embodiments, for example, when an amphiphilic polypeptide is silk, provided amphiphilic polypeptide materials are characterized by a density of about 1.4 kg/dm3. [0011] In some embodiments, provided amphiphilic polypeptide materials are water- based. In some embodiments, provided amphiphilic polypeptide materials contain residual water. In some embodiments, provided amphiphilic polypeptide materials have a low bound solvent content. In some embodiments, provided amphiphilic polypeptide materials are water- based and characterized by a low bound water content. For example, in some embodiments, provided amphiphilic polypeptide materials have a frozen and non-frozen bound water content of less than 50%. [0012] In some embodiments, provided amphiphilic polypeptide materials are free of or are substantially free of non-aqueous solvents. In some embodiments, provided amphiphilic polypeptide materials do not contain residual non-aqueous solvents. [0013] In some embodiments, provided amphiphilic polypeptide materials are characterized by amphiphilic polypeptides with structures self-assemble. In some embodiments, provided amphiphilic polypeptide materials self-assemble to form such three-dimensional structures. Amphiphilic polypeptides, in some embodiments, comprise a hydrophobic tail, an amino acid region, and a hydrophilic end. [0014] In some embodiments, a hydrophilic end of an amphiphilic polypeptide material is designed to allow solubility of the molecule in water. In some embodiments, a hydrophilic end permits or enhances biological function of an amphiphilic
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