Methods of inhibiting cancer growth

Abstract

The present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting a cell surface of the subject with an effective amount of a recombinant collagen fragment comprising a fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Also provided are therapeutic biomaterials for use in the methods described herein.

Description

[0001] Reference to a sequence listing submitted electronically

[0002] The contents of the electronically submitted sequence listing XML (named 4431_088PC01_SequenceListing_ST26.xml; size 1,312,028 bytes; and creation date May 30, 2023) submitted with this application are incorporated herein by reference in their entirety. Background Art

[0003] Collagen is one of the most important proteins in the human body and is found in connective tissues such as cartilage, bone, tendons, ligaments and skin. It is the major protein in the extracellular matrix of human cells. "Recombinant collagen" refers to a family of at least 28 different naturally occurring collagen types that are prepared using recombinant technology.

[0004] Despite recent advances in cancer therapy, cancer remains the leading cause of death worldwide, causing nearly 10 million deaths in 2020. A defining characteristic of cancer is the rapid generation of abnormal cells that invade and seed distant organs away from the primary tumor or neoplasm. These metastatic cells can remain dormant for many years before forming clinically detectable metastatic tumors. Extensive metastasis is the leading cause of cancer mortality. While cancer treatment modalities such as immunotherapy are an active area of ​​clinical research, there remains a need in the field of oncology for creative approaches and novel therapeutic agents for treating primary tumors and preventing metastatic seeding cells from becoming incurable metastatic tumors. Summary of the invention

[0005] The present disclosure provides novel inventive methods and compositions for inhibiting cancer growth in a subject in need thereof. In some embodiments, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0006] In some embodiments, the collagen fragment has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the collagen fragment having the amino acid sequence shown in SEQ ID NO: 1 is not hydroxylated. In some embodiments, the collagen fragment having the amino acid sequence shown in SEQ ID NO: 1 is hydroxylated.

[0007] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting the surface of a cell of the subject with an effective amount of a recombinant collagen fragment having an amino acid sequence as shown in any one of SEQ ID NOs: 2-972.

[0008] In some embodiments, contacting the cell surface with the fragment induces cell quiescence.

[0009] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the foregoing cancers.

[0010] In some embodiments, the subject has not undergone resection of a tumor. In some embodiments, the subject has undergone resection of a primary tumor. In some embodiments, the cell surface of the subject includes residual cancer cells after resection of a primary tumor.

[0011] In some embodiments, contacting the cell surface with the fragment maintains the cell in quiescence.

[0012] In some embodiments, the present disclosure provides a method of inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0013] In some embodiments of the methods of inhibiting the proliferation of cancer cells disclosed herein, the collagen fragment has an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0014] In some embodiments, the present disclosure provides a method of inhibiting proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having an amino acid sequence shown in any one of SEQ ID NOs: 2-972.

[0015] In some embodiments of the methods disclosed herein, the collagen fragment is formulated in a pharmaceutically acceptable composition. In some embodiments, the fragment is formulated in a therapeutic biomaterial.

[0016] In some embodiments, the polydispersity (weight average molecular weight of the collagen fragments divided by the number average molecular weight of the collagen fragments) of the collagen fragments provided herein may be about 1. In some embodiments, the polydispersity of the collagen fragments may be in the range of about 1 to about 2. In some embodiments, the polydispersity may be in the range of about 1 to about 3 or about 1 to about 5.

[0017] In some embodiments, the therapeutic biomaterial can be a protein polyurethane alloy. In some embodiments, the protein is dissolved in the polyurethane. In some embodiments, when the alloy is exposed to water, the protein migrates out of the alloy to contact the cell surface. In some embodiments, when the alloy is exposed to a buffer solution, the protein migrates out of the alloy to contact the cell surface. In some embodiments, the alloy may include about 10% to about 50% by weight of the fragment and about 50% to about 90% by weight of the polyurethane. In some embodiments, the alloy may include about 20% to about 35% by weight of the protein and about 65% to about 80% by weight of the polyurethane. In some embodiments, the alloy may not contain or may be substantially free of particles of recombinant collagen fragments having an average diameter greater than 1 micron.

[0018] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment within the pharmaceutically acceptable scaffold may be unhydroxylated. In some embodiments, the recombinant collagen fragment within the pharmaceutically acceptable scaffold may be hydroxylated.

[0019] In some embodiments, the recombinant collagen fragments in the pharmaceutically acceptable scaffold have the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragments in the pharmaceutically acceptable scaffold may be non-hydroxylated. In some embodiments, the recombinant collagen fragments in the pharmaceutically acceptable scaffold may be hydroxylated.

[0020] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence according to any one of SEQ ID NOs: 2-972 and a pharmaceutically acceptable scaffold.

[0021] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the therapeutic biomaterial is used in the treatment of cancer in a subject in need thereof. In some embodiments, the cancer can be characterized by the presence of a solid tumor. In some embodiments, the cancer can be skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers.

[0022] In some embodiments, the pharmaceutically acceptable stent can be a protein polyurethane alloy, wherein the protein is dissolved in the polyurethane. Protein polyurethane alloys are described in, for example, US2021-0355326, the entire contents of which are incorporated herein by reference. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein migrates out of the alloy to contact the cell surface. In some embodiments, when the alloy is exposed to the cell surface, the protein migrates out of the alloy.

[0023] In some embodiments, the alloy comprises about 10% to about 50% by weight of the fragment and about 50% to about 90% by weight of the polyurethane. In some embodiments, the alloy comprises about 20% to about 35% by weight of the protein and about 65% to about 80% by weight of the polyurethane. In some embodiments, the alloy contains no or substantially no particles of recombinant collagen fragments having an average diameter greater than 1 micron.

[0024] In some embodiments, the pharmaceutically acceptable stent comprises a protein hyaluronic acid alloy. In some embodiments, hyaluronic acid and protein are cross-linked with themselves, cross-linked with each other, or both. In some embodiments, the protein is dissolved in a cross-linked hyaluronic acid matrix. In some embodiments, when the alloy is exposed to water, a buffer solution, and / or a cell surface, the protein migrates out of the alloy.

[0025] In some embodiments, the pharmaceutically acceptable scaffold comprises a protein dissolved in a gelatin matrix. In some embodiments, the gelatin is cross-linked. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein migrates out of the alloy to contact the cell surface.

[0026] In some embodiments, the biomaterial is attached directly to the cancer cell.

[0027] In some embodiments, the present disclosure provides for the use of a biomaterial described herein for treating a wound resulting from the resection of a primary tumor.

[0028] In some embodiments, the present disclosure provides for the use of the biomaterials described herein for maintaining cell dormancy.

[0029] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0030] In some embodiments, the collagen fragment has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0031] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has a sequence comprising an amino acid sequence shown in any one of SEQ ID NOs: 2-972 or an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0032] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the therapeutic biomaterial is used in the treatment of cancer in a subject in need thereof. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers.

[0033] In some embodiments, the present disclosure provides a biomaterial comprising a pharmaceutically acceptable scaffold. In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of hyaluronic acid and recombinant collagen fragments. In some embodiments, the recombinant collagen fragments are mixed with hyaluronic acid. In some embodiments, the hyaluronic acid scaffold is cross-linked. In some embodiments, the hyaluronic acid is a biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA.

[0034] In some embodiments, the present disclosure provides a biomaterial comprising a pharmaceutically acceptable scaffold, wherein the pharmaceutically acceptable scaffold comprises a mixture of polyvinyl pyrrolidone and recombinant collagen fragments.

[0035] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of polyacrylamide and recombinant collagen fragments.

[0036] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of poly(ethylene oxide) and recombinant collagen fragments.

[0037] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of poly(2-oxazoline) and recombinant collagen fragments.

[0038] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of polyethyleneimine and recombinant collagen fragments.

[0039] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of carboxymethyl cellulose and recombinant collagen fragments.

[0040] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of chondroitin sulfate and recombinant collagen fragments.

[0041] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of acetylated hyaluronate and recombinant collagen fragments.

[0042] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of zinc hyaluronate and recombinant collagen fragments.

[0043] In some embodiments, the biomaterial is attached directly to the cancer cell.

[0044] In some embodiments, the present disclosure provides for the use of the disclosed biomaterials for treating wounds resulting from the resection of a primary tumor.

[0045] In some embodiments, the present disclosure provides uses for maintaining cell dormancy.

[0046] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting a cell surface of the subject with a therapeutic biomaterial comprising an effective amount of a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0047] In some embodiments, the present disclosure provides a method of inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a therapeutic biomaterial comprising a recombinant collagen fragment having an amino acid sequence as shown in SEQ ID NO: 973. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0048] In some embodiments, the present disclosure provides a method of inducing cell dormancy in a subject in need thereof, the method comprising contacting the cell surface of the subject with a therapeutic biomaterial comprising a recombinant collagen having an amino acid sequence as shown in SEQ ID NO: 973. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0049] In some embodiments of the methods of treating cancer provided herein, the cancer is characterized by the presence of solid tumors. In some embodiments, the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers.

[0050] In some embodiments of the methods provided herein, the therapeutic biomaterial is a protein polyurethane alloy.

[0051] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyurethane, hyaluronic acid, or gelatin.

[0052] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyvinylpyrrolidone.

[0053] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyacrylamide.

[0054] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises poly(ethylene oxide).

[0055] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises poly(2-oxazoline).

[0056] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyethyleneimine.

[0057] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises carboxymethylcellulose.

[0058] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises chondroitin sulfate.

[0059] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises acetylated hyaluronate.

[0060] The method of any one of claims 60-66, wherein the therapeutic biomaterial comprises acetylated hyaluronate.

[0061] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises zinc hyaluronate.

[0062] In some embodiments of the methods provided herein, the therapeutic biomaterial is directly attached to the cancer cell.

[0063] In some embodiments of the methods provided herein, the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5. In some embodiments, the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1A is a graph showing the mean tumor growth of each group over time (days).

[0065] Figure 1B is a graph showing the median tumor growth of each group over time (days).

[0066] Figures 2A-2J is a graph showing the effect of test collagen (different types and concentrations as described herein) compared to untreated controls over time (days).

[0067] Figure 2A is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL bovine collagen compared to untreated controls.

[0068] Figure 2B is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL bovine collagen and 10 mg / mL compared to untreated controls.

[0069] Figure 2C is a graph showing the mean estimated tumor volume up to day 23 for 200 mg / mL of SEQ ID NO 1 compared to untreated controls.

[0070] Figure 2D is a graph showing the mean estimated tumor volume up to day 23 for 10 mg / mL of SEQ ID NO 1 compared to untreated controls.

[0071] Figure 2E is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL of SEQ ID NO 1 compared to untreated controls.

[0072] Figure 2F is a graph showing the mean estimated tumor volume up to day 23 for all three concentrations of SEQ ID NO 1 compared to untreated controls.

[0073] Figure 2G is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL bovine collagen and 10 mg / mL hydroxylated SEQ ID NO 1 compared to untreated controls.

[0074] Figure 2H is a graph showing the mean estimated tumor volume up to day 23 for 10 mg / mL SEQ ID NO 1 and 10 mg / mL hydroxylated SEQ ID NO 1 compared to untreated controls.

[0075] Fig.2I is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL bovine collagen and 1.5 mg / mL bovine collagen in 0.1% ethanol compared to untreated controls.

[0076] Figure 2J is a graph showing the mean estimated tumor volume up to day 23 for 1.5 mg / mL bovine collagen and 1.5 mg / mL bovine collagen in 0.1% ethanol.

[0077] Figure 3 is a graph showing the progression-free survival rate of the test group.

[0078] Figure 4 A-4D is a series of graphs showing individual tumor growth curves for each group.

[0079] Figures 5A-5E is a series of photographs showing tumors after autopsy.

[0080] Figures 6A-6J is a series of representative photomicrographs of subcutaneous tumors from untreated and treated BALB / c mice.

[0081] Figure 7 Shown is the workflow for extracting ECM from tumor samples.

[0082] Figure 8 Enrichment of type III collagen in tumor samples is shown.

[0083] Figures 9A-9F The results of an in vitro proliferation assay using bovine collagen.

[0084] Fig. 9A Results in MCF-7 LUC cells are shown.

[0085] Fig. 9BResults in HCT-116 cells are shown.

[0086] Fig. 9C Results in HepG2 cells are shown.

[0087] Fig.9D Results in 4T1 cells are shown.

[0088] Fig.9E Cell image results in 4T1 cells are shown.

[0089] Fig.9F 4T1 cell proliferation assay readout at time 0 is shown.

[0090] Figures 10A-10E is the result of in vitro proliferation assay of SEQ ID NO 1.

[0091] Fig. 10A Results in MCF-7 LUC cells are shown.

[0092] Fig. 10B Results in HCT-116 cells are shown.

[0093] Fig. 10C Results in HepG2 cells are shown.

[0094] Fig. 10D Results in 4T1 cells are shown.

[0095] Fig. 10E Cell image results in 4T1 cells are shown.

[0096] Fig.11A is a standard curve used to convert UV absorbance to protein concentration by showing the relationship between absorbance (Y-axis) and protein concentration (X-axis).

[0097] Fig. 11B Shown is the concentration of protein released from a solution of protein in PBS (Y-axis) over time (days) (X-axis) compared to a formulation of HA and protein in PBS. Fig.11A The measurements in -B were taken outside a 300 kDa molecular weight cutoff (MWCO) dialysis membrane.

[0098] Fig. 12A is a standard curve used to convert absorbance to protein concentration by showing the relationship between absorbance (Y-axis) and protein concentration (X-axis).

[0099] Fig. 12B Shown is the concentration of protein released from PBS (Y-axis) compared to protein released from HA formulations over time (days) (X-axis). Figures 12A-12BThe measurements were taken outside the 300 kDa MWCO dialysis membrane, as Figures 11A-11B shown.

[0100] Fig.13A is a standard curve used to convert absorbance to protein concentration by showing the relationship between absorbance (Y-axis) and protein concentration (X-axis).

[0101] Fig. 13B Protein concentrations are shown plotted against time points to give a trend for the amount of protein remaining inside the PBS solution and HA formulations after dialysis. In this experiment, the data were acquired from inside the starting solution, in contrast to Figures 12-13 which were acquired from outside the 300 kDa MWCO dialysis membrane.

[0102] Fig.14A is a standard curve used to convert absorbance to protein concentration by showing the relationship between absorbance (Y-axis) and protein concentration (X-axis).

[0103] Fig. 14B Shown is the concentration of protein released from PBS solution (Y-axis) compared to protein released from HA formulations over time (days) (X-axis). DETAILED DESCRIPTION

[0104] definition

[0105] The indefinite articles "a" and "an" describing an element or component mean that there is one or at least one of these elements or components. Although these articles are usually used to indicate that the modified noun is a singular noun, unless otherwise specified in a specific context, as used herein, the articles "a" and "an" also include plural forms. Similarly, unless otherwise specified in a specific context, as used herein, the definite article "the" also indicates that the modified noun can be singular or plural.

[0106] As used herein, unless expressly stated otherwise, the term "about" used with a numerical value means "within 10% of the stated value." For example, "about 5 weight percent" means 4.5 weight percent to 5.5 weight percent. Nevertheless, "about" should not be understood as modifying a value to be greater than or equal to 100%. For example, a composition containing "about 95" weight percent of a given element can have 85.5 weight percent to 99.999 weight percent of that component in the composition.

[0107] The term "collagen" refers to any known type of collagen, whether natural, synthetic, semisynthetic or recombinant. The term collagen includes collagen, collagen fragments, collagen-like proteins, triple helical collagen, alpha chains, monomers, gelatin, trimers and combinations thereof. Collagen includes all collagens, modified collagens and collagen-like proteins described herein. The term also encompasses procollagen and collagen-like proteins or collagenous proteins comprising the motif (Gly-XY)n (wherein n is an integer). The term encompasses molecules of collagen and collagen-like proteins, trimers of collagen molecules, collagen fibrils, and fibers of collagen fibrils. The term also refers to collagen or collagen-like molecules that can be fibrillated through chemical, enzymatic or recombinant modification, and fragments of collagen, collagen-like molecules and collagenous molecules that can be assembled into nanofibers. Natural or engineered recombinant collagen molecules will typically include repeated -(Gly-XY)n-sequences.

[0108] As used herein, collagen is a generic term for a family of at least 28 different collagen types. Various collagen types have been identified in a range of species, including cattle, sheep, pigs, chickens, marine, plant and human collagen. Animal skin is usually type I collagen. The term "collagen" encompasses unprocessed (e.g., procollagen), as well as collagen with a triple helical structure that is post-translationally modified and proteolytically hydrolyzed. Type I collagen is the main fibrous collagen of bones and skin, accounting for approximately 80%-90% of the total collagen of an organism. Type I collagen is the main structural macromolecule present in the extracellular matrix of multicellular organisms, and accounts for approximately 20% of the total protein mass. Type I collagen is a heterotrimeric molecule comprising two α1 (I) chains and one α2 (I) chain, encoded by COL1A1 and COL1A2 genes, respectively. The in vivo assembly of type I collagen fibrils, fibers and fiber bundles occurs during development, and provides mechanical support to tissues while allowing cell movement and nutrient transport. The other collagen types are less abundant than type I collagen and exhibit different distribution patterns. Type III collagen is the major fibrillar collagen found in skin and vascular tissue. Type III collagen is a homotrimeric collagen containing three identical α1(III) chains encoded by the COL3A1 gene.

[0109] When applied to the collagen fragments disclosed herein, the term "modified" refers to a collagen fragment comprising an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical or similar to the amino acid sequence of a biologically active molecule. In some embodiments, the modified collagen fragment comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of a native sequence or a previously engineered sequence. The modified sequence may comprise additions, deletions, substitutions or combinations thereof to the amino acid sequence of a native molecule or a previously engineered molecule. For example, compared to the native collagen sequence, the modified collagen fragment may be combined with or lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. Such selection can be performed to change the looseness or compactness of the recombinant collagen. The degree of hydroxylation of collagen is related to the looseness or compactness of the collagen triple helix. The modified collagen fragments may also include chemical modifications to the polypeptide, such as cross-linking between cysteine ​​residues, or hydroxylated or glycosylated residues, or hydrolysis to lower molecular weight by any mechanism.

[0110] The term "pharmaceutically acceptable" when applied to carriers, excipients, or stabilizers that can be used in the compositions described herein refers to carriers, excipients, or stabilizers that are non-toxic to recipients at the dosages and concentrations employed.

[0111] As used herein, the term "protein" refers to any collagen fragment described herein, including: 1) "50kDarCol"; 2) a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:1; 3) a recombinant collagen fragment having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity or similarity with SEQ ID NO:1; and 4) a combination of any of the foregoing.

[0112] As used herein, the terms "hyaluronic acid" and "HA" refer to acidic polysaccharides with different molecular weights composed of residues of D-glucuronic acid and N-acetyl-D-glucosamine acid, and their corresponding salts, including but not limited to sodium hyaluronate. As used herein, unless otherwise indicated, a composition, scaffold, therapeutic biomaterial or any intended embodiment comprising HA should be understood as comprising a combination of hyaluronic acid and sodium hyaluronate.

[0113] As used herein, the terms "sodium carboxymethylcellulose", "carboxymethylcellulose" and "CMC" are used interchangeably and refer to polymers of varying molecular weight ranges that are derivatives of cellulose in which carboxymethyl groups are bonded to some of the monomers that make up the cellulose backbone. Unless otherwise indicated, compositions described herein as comprising carboxymethylcellulose should be understood to comprise a combination of carboxymethylcellulose and sodium carboxymethylcellulose.

[0114] As used herein, the term "miscible" refers to a substance that is soluble in another substance so that a molecularly uniform mixture is formed when the two components are mixed together in water or in a buffer system. In solution, collagen and hyaluronic acid are completely miscible and refer to a solution of a uniformly mixed or blended combination that forms a transparent single phase under suitable processing conditions, wherein no visible collagen particles are present in the solution. Solid films prepared from completely miscible solutions may appear transparent, turbid, and / or may exhibit phase separation. Transparency can be easily determined by one of ordinary skill in the art and can be measured, for example, using a haze meter.

[0115] In the context of protein polyurethane alloys, a protein that is miscible with polyurethane means that the protein is miscible with only one of the multiple phases of polyurethane or with multiple polyurethanes blended therewith.

[0116] A. Methods of treating cancer

[0117] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting a cell surface of the subject with an effective amount of a recombinant collagen fragment.

[0118] As used herein, the term "recombinant collagen" refers to a family of at least 28 different naturally occurring collagen types prepared using recombinant technology, including but not limited to type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX and XX collagen. The term collagen includes collagen, collagen fragments, collagen-like proteins, triple helical collagen, α chains, monomers, gelatin, trimers and combinations thereof. The recombinant expression of collagen and collagen-like proteins is known in the art (see, e.g., Bell, EP 1232182B1, Bovine collagen and method for producing recombinant gelatin; Olsen et al., U.S. Pat. No. 6,428,978 and Van Heerde et al., U.S. Pat. No. 8,188,230, which are incorporated herein by reference in their entirety). Collagen is characterized by repeating triplets of amino acids, -(Gly-XY)n-, such that approximately one-third of the amino acid residues in collagen are glycine. X is typically proline, and Y is typically hydroxyproline. The structure of collagen may consist of three interwoven peptide chains of varying lengths.

[0119] In some embodiments, the recombinant collagen described herein is a recombinant collagen fragment. The recombinant collagen fragment can be a fragment of the complete amino acid sequence of a native collagen molecule capable of forming tropocollagen (trimeric collagen), or the fragment can be a modified collagen molecule or a truncated collagen molecule having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity or similarity with the native collagen amino acid sequence (or with its fibril-forming region or with a segment substantially comprising [Gly-XY]n).

[0120] Exemplary collagen sequences from which fragments can be derived include the amino acid sequences of Col1A1, Col1A2, and Col3A1, such as those described by the following accession numbers: P02461.4 (SEQ ID NO: 1003; human Col3A1) (www.ncbi.nlm.nih.gov / protein / 124056490), P02452 (SEQ ID NO: 982; human Col1A1), P08123 (SEQ ID NO: 983; human Col1A2), NP_001029211.1 (SEQ ID NO: 1004; bovine Col1A1) (www.ncbi.nlm.nih.gov / protein / 77404252), NP_776945.1 (SEQ ID NO: 983; human Col1A2), NO: 1005; bovine Col1A2) (www.ncbi.nlm.nih.gov / protein / 27806257) and NP_001070299.1 (SEQ ID NO: 1006; bovine Col3A1) (www.ncbi.nlm.nih.gov / protein / 116003881), which are incorporated herein by reference.

[0121] In some embodiments, the collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the collagen fragment has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the collagen fragment has the amino acid sequence shown in any one of SEQ ID NOs: 2-972.

[0122] In some embodiments, the collagen fragment may be a collagen fragment sequence variant having an amino acid sequence according to any one of SEQ ID NOs: 975-1002.

[0123] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 has a molecular weight of about 50 kDa. In some embodiments, the recombinant collagen fragment may have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1.

[0124] The amino acid sequence of SEQ ID NO:1 is:

[0125] DVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENG APGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGA GEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPG PSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPAIAGIGGEKAGGFAPYYG

[0126] In some embodiments, the lysine, proline or lysine residues and proline residues present in the recombinant collagen fragment are not hydroxylated. In other embodiments, the recombinant collagen fragments described herein may be hydroxylated. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (or any intermediate value or sub-range) of lysine, proline or lysine residues and proline residues in the recombinant collagen fragment may be hydroxylated. Hydroxylated collagen can establish rheology and improve the thermal stability of collagen molecules or fragments. Hydroxylated collagen and hydroxylated collagen fragments are also resistant to high concentration pepsin digestion, such as 1:25 to 1:1 pepsin: high concentration pepsin digestion of total protein ratio. In some embodiments, the recombinant collagen fragment with the amino acid sequence shown in SEQ ID NO: 1 is not hydroxylated. In some embodiments, the recombinant collagen fragment having the amino acid sequence shown in SEQ ID NO: 1 is hydroxylated.

[0127] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having an amino acid sequence as shown in any one of SEQ ID NOs: 2-972. In some embodiments, the recombinant collagen may include a hydrolyzate of the collagen fragment, wherein the hydrolyzate may have a sequence as a portion of SEQ ID NO: 1. In some embodiments, the hydrolyzate may have a sequence according to one of SEQ ID NOs: 2-972.

[0128] In some embodiments, the methods provided herein for treating cancer in a subject in need thereof do not comprise contacting the cell surface of the subject with an effective amount of recombinant collagen having the amino acid sequence shown in SEQ ID NO: 973 in the absence of a pharmaceutically acceptable scaffold.

[0129] In some embodiments, the recombinant collagen fragments described herein may have an amino acid sequence as shown in Table 1 below.

[0130] Table 1.

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] As noted above, it is well known in the art that cancer cells spread and seed in distant organs, and they can remain dormant for many years before forming clinically detectable metastases. In addition, disseminated tumor cells can sense and reshape the extracellular matrix (ECM) to remain dormant. Dormant cancer cells assemble an ECM niche enriched with type III collagen. Without being bound by any particular theory, it is believed that enrichment of type III collagen in the ECM can limit or prevent metastasis by inducing dormancy in disseminated tumor cells.

[0163] In some embodiments of the methods of treating cancer disclosed herein, contacting the cell surface of a subject with cancer with a recombinant collagen fragment induces cell dormancy. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer can be skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers.

[0164] In some embodiments, the subject has not undergone resection of a tumor. In some embodiments, the subject has undergone resection of a primary tumor. In some embodiments, the cell surface of the subject includes residual cancer cells after resection of a primary tumor.

[0165] In some embodiments of the methods of treating cancer disclosed herein, contacting the surface of a cell of a subject having cancer with the fragment maintains the cell in quiescence.

[0166] In some embodiments, the present disclosure provides a method of inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and / or 973. When the present specification uses the phrase "1 and / or 973", it should be understood that any percentage identity preceding the phrase means that the contemplated collagen sequence can have the indicated percentage identity with a single sequence or two sequences (i.e., as in a mixture). In some embodiments, the collagen fragment can have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity or similarity with SEQ ID NO: 1 and / or 973. In some embodiments, the present disclosure provides a method of inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and / or 973. In some embodiments, the collagen fragment may have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity or similarity to SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence shown in any one of SEQ ID NO: 974-1002.

[0167] In some embodiments, the recombinant collagen fragment may be non-hydroxylated. In some embodiments, the recombinant collagen fragment may be hydroxylated.

[0168] In some embodiments, the present disclosure provides a method of inhibiting proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having an amino acid sequence shown in any one of SEQ ID NOs: 2-972.

[0169] In some embodiments, the methods of inhibiting the proliferation of cancer cells provided herein do not comprise contacting the cancer cells with the recombinant collagen having the amino acid sequence shown in SEQ ID NO: 973 in the absence of a pharmaceutically acceptable scaffold.

[0170] In some embodiments, the fragment is formulated in a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition comprises at least one pharmaceutically acceptable excipient. In a specific embodiment, the composition may comprise a recombinant collagen fragment according to SEQ ID NO: 1 and / or 973 and at least one pharmaceutically acceptable excipient. In still other embodiments, the composition may comprise a recombinant collagen fragment having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 973 and at least one pharmaceutically acceptable excipient, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NO: 974-1002. In some embodiments, the composition may include a recombinant collagen fragment having an amino acid sequence of any one of SEQ ID NOs: 2-972 and at least one pharmaceutically acceptable excipient.

[0171] In another embodiment, the composition may comprise a recombinant collagen fragment according to SEQ ID NO: 1 and / or 973 and a plurality of hydrolysates having such sequences according to any one of SEQ ID NO: 2-972, which may be the same or different, and at least one pharmaceutically acceptable excipient. In certain embodiments, the number of hydrolysates present in the plurality of hydrolysates in the composition may increase with time, temperature, pH, or due to other conditions that would normally cause the recombinant collagen fragment (such as the recombinant collagen fragment according to SEQ ID NO: 1 or 973) to hydrolyze or otherwise decompose. In other embodiments, the composition may be stabilized with one or more stabilizers such that the concentration of the recombinant collagen fragment according to SEQ ID NO: 1 and / or 973 and the concentration of each of the plurality of fragments remain substantially constant (i.e., vary by no more than ±5% as measured by HPLC over a given period of time) or remain constant. In certain embodiments, the recombinant collagen fragment, such as the recombinant collagen fragment according to SEQ ID NO: 1, can be hydrolyzed such that less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the unhydrolyzed recombinant fragment remains in the composition as measured by HPLC. In other embodiments, the composition may comprise a mixture of recombinant collagen fragments (e.g., recombinant collagen fragments according to SEQ ID NO: 1) and multiple hydrolysates of the recombinant collagen fragments (e.g., multiple collagen fragments according to any one of SEQ ID NOs: 2-972), such that the weight of the hydrolysate in the composition is less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the weight of the collagen-related protein in the composition.

[0172] Pharmaceutically acceptable compositions for use in the methods disclosed herein have the desired purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0173] In some embodiments, a topical mixture comprising a recombinant collagen fragment can be prepared for topical application. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, lavage, spray, bandage, skin patch, or any other formulation suitable for topical application. The topical mixtures described herein can be formulated for topical application to the skin and mucous membranes in the form of gels, creams, and lotions for transdermal delivery.

[0174] Transdermal patches (including iontophoresis and electrophoresis devices) are well known to those skilled in the art and can be used to administer recombinant collagen fragments. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0175] B. Therapeutic biomaterials

[0176] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0177] As used herein, pharmaceutically acceptable stents may include synthetic, polymeric, non-immunogenic, three-dimensional stents. Suitable stents may be biocompatible and biodegradable support structures, and may be solid, liquid or gel stents, such as injectable gel stents. Suitable stents may also include those made of nanomaterials such as nanopolymers and nanofibers. After the cell surface of a subject (e.g., a human subject) is damaged or operated on, such stents may be placed on the cell surface. Suitable stents include any conventionally used and / or available stents for surgery, wound healing and / or tissue repair and / or engineering approaches. For example, some intelligent thermoresponsive polymers such as chitosan, polyvinyl pyrrolidone, alginate and poly (ε-caprolactone) may be used to produce biocompatible and biodegradable stents. These processed thermoresponsive biomaterials have a 3D architecture similar to that of a human structure, and may be used as an active agent delivery system.

[0178] Additional polymers suitable for use in the pharmaceutically acceptable scaffolds described herein include, but are not limited to, biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA, polyvinylpyrrolidone, polyacrylamide, poly(ethylene oxide), poly(2-oxazoline) and protein, polyethyleneimine and protein, carboxymethylcellulose, chondroitin sulfate, acetylated hyaluronate, and zinc hyaluronate.

[0179] Therefore, in some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and a biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0180] Therefore, in some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and polyvinyl pyrrolidone. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and polyvinyl pyrrolidone. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and polyvinyl pyrrolidone, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0181] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a polyacrylamide. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and a polyacrylamide. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a polyacrylamide, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0182] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 and poly(ethylene oxide). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs:2-972 and poly(ethylene oxide). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 and poly(ethylene oxide), wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs:974-1002.

[0183] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and poly(2-oxazoline). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs:2-972 and poly(2-oxazoline). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and poly(2-oxazoline), wherein the recombinant collagen fragment has the amino acid sequence shown in any one of SEQ ID NOs:974-1002.

[0184] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and polyethyleneimine. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and polyethyleneimine. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and polyethyleneimine, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0185] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and carboxymethyl cellulose. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and carboxymethyl cellulose. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and carboxymethyl cellulose, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0186] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and chondroitin sulfate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and chondroitin sulfate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and chondroitin sulfate, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0187] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and acetylated hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NO: 2-972 and acetylated hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and acetylated hyaluronate, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NO: 974-1002.

[0188] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and zinc hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 and zinc hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and zinc hyaluronate, wherein the recombinant collagen fragment has an amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

[0189] In some embodiments, the polydispersity of the collagen fragments disclosed herein may be about 1. In some embodiments, the polydispersity of the collagen fragments disclosed herein may be in the range of about 1 to about 2. In some embodiments, the polydispersity of the collagen fragments disclosed herein may be in the range of about 1 to about 3 or about 1 to about 5.

[0190] In some embodiments, the pharmaceutically acceptable scaffold can be used to treat cancer in a subject in need thereof. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer can be skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers. In some embodiments, the biomaterial can be directly attached to the cancer cell.

[0191] In some embodiments of the methods described herein, the biomaterial may comprise recombinant collagen fragments that may be non-hydroxylated. In some embodiments of the methods described herein, the biomaterial may comprise recombinant collagen fragments that may be hydroxylated.

[0192] In some embodiments, the biomaterial can be used to treat wounds caused by resection of a primary tumor. In some embodiments, the biomaterial can be directly attached to the wound. In some embodiments, the biomaterial can be used to maintain cell dormancy after resection of a primary tumor. In some embodiments, the biomaterial can be used to maintain cell dormancy in a subject who has not undergone resection of a primary tumor.

[0193] In some embodiments, the biomaterial can be attached directly to cells. Without being bound by a particular theory, placing therapeutic biomaterials comprising recombinant collagen fragments directly on wounds or cell surfaces is believed to enrich type III collagen in the ECM and limit or prevent metastasis by inducing dormancy in disseminated tumor cells.

[0194] In some embodiments, the therapeutic biomaterial can be a protein polyurethane alloy. In some embodiments, the protein can be any collagen fragment (or combination thereof) described herein, and when the alloy is exposed to water, a buffer solution, and / or a cell surface, the fragment migrates out of the alloy to contact the cell surface.

[0195] In some embodiments, the alloy may include collagen fragments of about 10% to about 50% by weight and polyurethane of about 50% to about 90% by weight. In some embodiments, the alloy may include proteins of about 20% to about 35% by weight and polyurethane of about 65% to about 80% by weight. In some embodiments, the alloy may not contain or may be substantially free of particles of recombinant collagen fragments with an average diameter greater than 1 micron. In some embodiments, the protein polyurethane alloy may be mechanically attached, such as with sutures or glue, or may be otherwise directly attached to the cell surface of a subject suffering from cancer. In some embodiments, the protein polyurethane alloy may be used as a dry film or powder application, and the dry film or powder may be naturally hydrated by the subject or hydrated in place with suitable water or buffer after application. In the embodiment in which the protein polyurethane alloy or hyaluronic acid protein alloy is applied to the cell surface as a dry film or powder, it is not bound by a specific theory, and it is believed that after application, the film or powder will be hydrated by the cell surface. Alternatively, the film or powder can be hydrated by applying a suitable buffer or water. In some embodiments, films may be prepared by drying any alloy described herein using any standard drying method known in the art, such as in a continuous oven. Powder forms may be prepared by spray drying or freeze drying a solution, grinding a previously dried alloy, or by any other preparation method known in the art. Again, and without being bound by a particular theory, placing any alloy described herein containing recombinant collagen fragments directly onto the cell surface is thought to enrich type III collagen in the ECM, thereby limiting or preventing metastasis by inducing dormancy in disseminated tumor cells.

[0196] In some embodiments, pharmaceutically acceptable stent may include protein hyaluronic acid alloy. In some embodiments, alloy may include about 10 wt % to about 50 wt % fragment and about 50 wt % to about 90 wt % hyaluronic acid. In some embodiments, alloy may include about 20 wt % to about 35 wt % protein and about 65 wt % to about 80 wt % hyaluronic acid. In some embodiments, hyaluronic acid or protein or both and protein are cross-linked with themselves, cross-linked with each other or both situations are all there. In some embodiments, protein is soluble, suspended or otherwise present in a cross-linked hyaluronic acid matrix. In some embodiments, when alloy is exposed to water or buffer solution, protein migrates out of alloy to contact cell surface.

[0197] In some embodiments, the pharmaceutically acceptable scaffold may include proteins dissolved in a gelatin matrix. In some embodiments, the alloy may include about 10% to about 50% by weight of the fragment and about 50% to about 90% by weight of gelatin. In some embodiments, the alloy may include about 20% to about 35% by weight of the protein and about 65% to about 80% by weight of the gelatin. In some embodiments, the gelatin may be cross-linked. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein migrates out of the alloy to contact the cell surface.

[0198] In some embodiments, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising contacting the cell surface of the subject with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and an effective amount of recombinant collagen having the amino acid sequence shown in SEQ ID NO:973.

[0199] The amino acid sequence of SEQ ID NO:973 is:

[0200]

[0201] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and a recombinant collagen fragment having the amino acid sequence shown in SEQ ID NO:973.

[0202] In some embodiments, the present disclosure provides a method of inducing cell dormancy in a subject in need thereof, the method comprising contacting the cell surface of the subject with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and recombinant collagen having the amino acid sequence shown in SEQ ID NO:973.

[0203] In some embodiments, cancer can be characterized by the presence of solid tumors. In some embodiments, the cancer can be skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the above cancers. In some embodiments, the recombinant collagen can be non-hydroxylated. In some embodiments, the recombinant collagen can be hydroxylated.

[0204] In some embodiments of the methods of using the recombinant collagen having the amino acid sequence shown in SEQ ID NO: 973 provided herein, the biomaterial can be a protein polyurethane alloy. In some embodiments of the methods of using the recombinant collagen having the amino acid sequence shown in SEQ ID NO: 973 provided herein, the biomaterial can include a pharmaceutically acceptable scaffold. In some embodiments, the biomaterial can be directly attached to cancer cells.

[0205] The protein polyurethane alloys described herein comprise proteins dissolved in one or more polyurethanes, such as any fragment disclosed herein, including, for example, SEQ ID NO. 1-1002. In some embodiments, the protein polyurethane alloys described herein comprise collagen fragments having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and / or 973. In some embodiments, the protein polyurethane alloys described herein comprise recombinant collagen fragments having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity or similarity with SEQ ID NO: 1 and / or 973. In some embodiments, the protein polyurethane alloys described herein comprise collagen fragments having the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the protein polyurethane alloys described herein comprise collagen fragments having an amino acid sequence as shown in any one of SEQ ID NOs: 2-972. In some embodiments, the protein polyurethane alloys described herein comprise recombinant collagen having an amino acid sequence as shown in SEQ ID NO: 973 dissolved in one or more polyurethanes. In some embodiments, the protein polyurethane alloys described herein may comprise recombinant collagen having an amino acid sequence as shown in any one of SEQ ID NOs: 974-1002 dissolved in one or more polyurethanes.

[0206] In a specific embodiment, the protein polyurethane alloy described herein may include a protein that is miscible with only one of the multiple phases of the polyurethane or with a variety of polyurethanes blended therewith. For example, in some embodiments, the protein polyurethane alloy may include a protein that is miscible with only the hard phase of the polyurethane or with the multiple polyurethanes having both hard and soft phases. The protein polyurethane alloy described herein may be free of or substantially free of protein in the form of particles dispersed in the polyurethane. For example, in some embodiments, the protein polyurethane alloy may be free of or substantially free of protein particles having an average diameter greater than 1 micron (μm). In some embodiments, the protein polyurethane alloy may be free of or substantially free of particles of recombinant collagen fragments having an average diameter greater than 1 micron (μm). In some embodiments, the polyurethane may also include a urea bond. In such embodiments, the polyurethane is a polyurethane urea.

[0207] In some embodiments, the protein used in the therapeutic biomaterial comprising the protein polyurethane alloy can be a succinylated protein. Succinylated protein is a protein modified by adding a succinyl group to the side chain of an amino acid in a protein. Most commonly, a succinyl group is added to a lysine side chain. The method of adding a succinyl group to the side chain of an amino acid in a protein is commonly referred to as protein succinylation. Adding a succinyl group to a protein can change the functional and structural properties of the protein. In some cases, adding a relatively large modification (such as a succinyl moiety) is expected to change the tertiary structure of the protein. In addition, by adding a succinyl moiety, the lysine side chain can be changed from a primary amine to an acid, making it more hydrophilic, and its charge can be changed from a positive charge to a negative charge at physiological pH. Succinylation can be accomplished using techniques and chemical methods well known in the art.

[0208] Suitable polyurethanes for use in therapeutic biomaterials containing protein polyurethane alloys described herein include those containing at least two phases, including a "soft phase" and a "hard phase". The soft phase is formed by polyol segments within the polyurethane that are separated from the urethane-containing phase due to polarity differences. The urethane-containing phase is referred to as the hard phase. This phase separation is known in the art and is the basis of many properties of polyurethanes.

[0209] The soft phase is typically elastomeric at room temperature and typically has a softening point or glass transition temperature (Tg) below room temperature. Tg can be measured by dynamic mechanical analysis (DMA) and quantified by the peak value of tan (δ) or the onset of the drop in storage modulus. Alternatively, Tg can be measured by differential scanning calorimetry (DSC). In some cases, there may be crystallinity in the soft phase, which can be considered as a melting point, typically between 0°C and about 60°C.

[0210] The hard phase typically has a Tg or melting point above room temperature, more typically above about 80° C. Softening of the hard phase can be measured by measuring the onset of the drop in storage modulus (sometimes referred to as stiffness) as measured by DMA.

[0211] The "soft phase" of polyurethane or protein polyurethane alloys comprising polyurethane comprises the polyol component of polyurethane. Its function is to become soft and flexible at a temperature higher than its Tg, to give polyurethane toughness, elongation and flexibility. Typical soft segments may include polyether polyols, polyester polyols, polycarbonate polyols and mixtures thereof. The molecular weight of the soft segment is generally in the range of about 250D to greater than about 5kD. The "hard phase" for polyurethane or protein polyurethane alloys comprising polyurethane comprises the carbamate segments of polymers given by isocyanates for connecting polyols and short-chain glycols such as butanediol, propylene glycol, etc. In some embodiments, chain extenders may include amines such as ethylenediamine, hexamethylenediamine, etc. to introduce urea bonds. Urea bonds may also be introduced by adding water, and water reacts with isocyanates to produce amines and carbon dioxide that can serve as a blowing agent. Therefore, the term polyurethane is understood in some embodiments to include urea bonds in the hard phase. Typical isocyanates that can be used for the polyurethane of the present invention include, but are not limited to, hexamethylene diisocyanate, isophorone diisocyanate, methylene diisocyanate, toluene diisocyanate, phenyl diisocyanate, and the like. These molecules are more polar and harder than the polyols used to prepare the soft segments. Therefore, the hard segments are harder and have a higher softening point than the soft segments. The function of the hard phase is to provide properties such as strength, temperature resistance, and wear resistance to the polyurethane.

[0212] In some embodiments described herein, the protein may be miscible only with the hard phase, leaving the soft phase transition substantially unchanged. Without wishing to be bound by a particular theory, it is believed that when the protein is dissolved in the hard phase, it significantly increases the temperature at which the hard phase begins to soften, thereby increasing the temperature resistance of the alloy.

[0213] In a protein polyurethane alloy comprising one or more miscible proteins and polyurethanes, the one or more proteins may be dissolved in the hard phase of the one or more polyurethanes. The protein polyurethane alloy may include at least one protein that is miscible with the hard phase of one or more polyurethanes in the alloy. In some embodiments, the protein polyurethane alloy may include multiple proteins and / or multiple polyurethane hard phases that are miscible with each other. In all of these embodiments, and without wishing to be bound by a particular theory, it is believed that the protein or multiple proteins are dissolved in the hard phase of the polyurethane or multiple polyurethanes.

[0214] When blended, one or more proteins dissolved in the hard phase of one or more polyurethanes can form a homogenized mixture. In some embodiments, the protein polyurethane alloy can include multiple proteins dissolved in one or more polyurethanes, so that the protein and polyurethane form a homogenized mixture when blended and dried. Typically, the protein polyurethane alloy comprising a homogenized mixture of protein and polyurethane does not include a large amount of protein that is not dissolved in the polyurethane. That is, and in some embodiments, the protein polyurethane alloy can include a portion of the protein dispersed in the polyurethane.

[0215] Suitable polyurethanes according to the embodiments described herein include, but are not limited to, aliphatic polyurethanes, aromatic polyurethanes, bio-based polyurethanes, or acrylic modified polyurethanes. Suitable polyurethanes are commercially available from manufacturers including Covestro, Lubrizol, Hauthaway, Stahl, etc. In some embodiments, the polyurethane used for protein polyurethane alloys may be bio-polyurethanes. In some embodiments, the polyurethane is a water-dispersible polyurethane. In some embodiments, the polyurethane may be a polyester polyurethane. In some embodiments, the polyurethane may be a polyether polyurethane. In some embodiments, the polyurethane may be a polycarbonate-based polyurethane. In some embodiments, the polyurethane may be an aliphatic polyester polyurethane. In some embodiments, the polyurethane may be an aliphatic polyether polyurethane. In some embodiments, the polyurethane may be an aliphatic polycarbonate polyurethane. In some embodiments, the polyurethane may be an aromatic polyester polyurethane. In some embodiments, the polyurethane may be an aromatic polyether polyurethane. In some embodiments, the polyurethane may be an aromatic polycarbonate polyurethane.

[0216] In some embodiments, polyurethane may have a soft segment selected from the group consisting of: polyether polyols, polyester polyols, polycarbonate polyols and mixtures thereof. In some embodiments, polyurethane may have a hard segment comprising a diisocyanate and an optional short-chain diol or diamine. Suitable diisocyanates may be selected from the group consisting of: aliphatic dicyanates, such as hexamethylene diisocyanate, isophorone diisocyanate; aromatic diisocyanates, such as 4,4' diphenylmethylene diisocyanate, toluene diisocyanate, phenyl diisocyanate and mixtures thereof. Suitable short-chain diols include ethylene glycol, propylene glycol, butanediol, 2,2 methyl 1,3 propanediol, pentanediol, hexanediol and mixtures thereof. Suitable short-chain diamines include ethylenediamine, hexamethylenediamine and mixtures thereof. In some embodiments, crosslinking agents such as polyfunctional alcohols, for example trimethylolpropane triol; or diamines, such as ethylenediamine or 4,4' diaminodiphenyl diamine.

[0217] Exemplary commercial polyurethanes include, but are not limited to, L3360 and Hauthane HD-2001 available from CL Hauthaway & Sons Corporation, SANCURE polyurethane available from Lubrizol Corporation, BONDTHANE available from Bond Polymers International, and TM Polyurethanes, such as UD-108, UD-250 and UD-303, are available from BASF ECO 3702 and P100ECO, and Permutex Evo EX-RC-2214 (RC-2214) available from Stahl. L3360 is an aqueous aliphatic polyester polyurethane polymer dispersion having a solid content of 35%, a viscosity of 50 cps (centipoise) to 500 cps, and a density of about 8.5 lb / gal (pounds per gallon). HD-2001 is an aqueous aliphatic polyester polyurethane polymer dispersion having a solid content of 40%, a viscosity of 50 cps to 500 cps, and a density of about 8.9 lb / gal. BONDTHANE TM UD-108 is an aqueous dispersion of an aliphatic polyether polyurethane polymer having a solids content of 33%, a viscosity of 300 cps, and a density of 8.7 lb / gal. TM UD-250 is an aqueous dispersion of an aliphatic polyester polyurethane polymer having a solids content of 35%, a viscosity of 200 cps, and a density of 8.8 lb / gal. BONDTHANE TM UD-303 is an aqueous dispersion of an aliphatic polyether polyurethane polymer having a solids content of 35%, a viscosity of less than 500 cps, and a density of 8.7 lb / gal. P100ECO is a polyester polyurethane elastomer aqueous dispersion having approximately 40% solids and a viscosity of about 40 mPas. RC-2214 is an aliphatic polyether polyurethane polymer aqueous dispersion having a solids content of 58% to 60%, a viscosity of 4,000 cps to 15,000 cps, and a density of about 8.9 lb / gal.

[0218] Exemplary bio-based polyurethanes include, but are not limited to, L3360 available from CL Hauthaway & Sons Corporation, Eco DLS, Eco DL 519, Eco DLP-R and DL-5249. Eco DLS is an anionic aliphatic polyester polyurethane polymer aqueous dispersion having a solids content of approximately 50%, a viscosity of less than 1,200 MPa·s, and a density of about 1.1 g / cc. Eco DL 519 is an anionic, aliphatic polyester polyurethane polymer aqueous dispersion. Eco DLP-R is an anionic, aliphatic polyester polyurethane polymer aqueous dispersion. DL 5249 is an anionic, aliphatic polyester polyurethane polymer aqueous dispersion.

[0219] In some embodiments, the polyurethane may include reactive groups that can crosslink with proteins. Exemplary reactive groups include, but are not limited to, sulfonates, aldehydes, carboxylic acids or esters, blocked isocyanates, and the like, and combinations thereof. In such embodiments, the polyurethane can be crosslinked with proteins in a protein-polyurethane alloy by reacting reactive groups on the protein with reactive groups present in the polyurethane.

[0220] In some embodiments, the amount of protein (a recombinant collagen fragment having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973, or a collagen fragment having an amino acid sequence as shown in any one of SEQ ID NO: 2-972, or a recombinant collagen having an amino acid sequence as shown in SEQ ID NO: 973, or a recombinant collagen having an amino acid sequence as shown in any one of SEQ ID NO: 974-1002) in the protein polyurethane alloy can be in the range of about 5 wt % to about 50 wt % protein, including subranges. For example, in some embodiments, the amount of protein in the polyurethane alloy can be in the range of about 5 wt % to about 50 wt %, about 10 wt % to about 50 wt %, about 15 wt % to about 50 wt %, about 20 wt % to about 50 wt %, about 25 wt % to about 50 wt %, about 30 wt % to about 50 wt %, about 35 wt % to about 50 wt %, about 40 wt % to about 50 wt %, about 45 wt % to about 50 wt %, about 5 wt % to about 45 wt %, about 5 wt % to about 40 wt %, about 5 wt % to about 35 wt %, about 5 wt % to about 30 wt %, about 5 wt % to about 25 wt %, about 5 wt % to about 20, about 5 wt % to about 15 wt %, or about 5 wt % to about 10 wt %, or in the range having any two of these values ​​as endpoints, including the endpoints. In some embodiments, the amount of protein in the protein polyurethane alloy can be in the range of about 20 wt % to about 35 wt %.

[0221] In some embodiments, the amount of polyurethane in the protein polyurethane alloy may be in the range of about 50 wt % to about 95 wt %, inclusive of subranges. For example, in some embodiments, the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50% to about 95% by weight, about 55% to about 95% by weight, about 60% to about 95% by weight, about 65% to about 95% by weight, about 70% to about 95% by weight, about 75% to about 95% by weight, about 80% to about 95% by weight, about 85% to about 95% by weight, about 90% to about 95% by weight, about 50% to about 90% by weight, about 50% to about 85% by weight, about 50% to about 80% by weight, about 50% to about 75% by weight, about 50% to about 70% by weight, about 50% to about 65% by weight, about 50% to about 60% by weight, or about 50% to about 55% by weight, or in the range having any two of these values ​​as endpoints, including the endpoints. In some embodiments, the amount of polyurethane in the protein polyurethane alloy can be in the range of about 65% to about 80% by weight.

[0222] Any of the above-listed ranges of the weight percentages of protein and polyurethane in the protein polyurethane alloy can be combined. For example, in some embodiments, the weight percentages of protein and polyurethane in the protein polyurethane alloy can be any of the following weight percentages. The amount of protein in the polyurethane alloy can be in the range of about 5 weight % to about 50 weight %, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50 weight % to about 95 weight %. The amount of protein in the polyurethane alloy can be in the range of about 15 weight % to about 50 weight %, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50 weight % to about 85 weight %. The amount of protein in the polyurethane alloy can be in the range of about 20 weight % to about 50 weight %, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50 weight % to about 80 weight %. The amount of protein in the polyurethane alloy can be in the range of about 25 weight % to about 50 weight %, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50 weight % to about 75 weight %. The amount of protein in the polyurethane alloy can be in the range of about 30% by weight to about 50% by weight, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 50% by weight to about 70% by weight. The amount of protein in the polyurethane alloy can be in the range of about 10% by weight to about 40% by weight, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 60% by weight to about 90% by weight. The amount of protein in the polyurethane alloy can be in the range of about 15% by weight to about 40% by weight, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 60% by weight to about 85% by weight. The amount of protein in the polyurethane alloy can be in the range of about 20% by weight to about 40% by weight, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 60% by weight to about 80% by weight. The amount of protein in the polyurethane alloy can be in the range of about 20% by weight to about 35% by weight, and the amount of polyurethane in the protein polyurethane alloy can be in the range of about 65% by weight to about 80% by weight. In some embodiments, the weight percentage values ​​and ranges listed above can be based on the gross weight of the protein polyurethane alloy. In some embodiments, the weight percentage values ​​and ranges listed above may be based on the total weight of only protein and polyurethane in the protein polyurethane alloy. Unless otherwise specified, the weight percentage values ​​or ranges of polyurethane and protein are based on the total weight of only protein and polyurethane in the protein polyurethane alloy.

[0223] In some embodiments, the total amount of protein (a recombinant collagen fragment having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973, or a collagen fragment having the amino acid sequence shown in any one of SEQ ID NO: 2-972, or a recombinant collagen having the amino acid sequence shown in SEQ ID NO: 973) plus the amount of polyurethane in the protein-polyurethane alloy may be about 80 weight % or more. For example, in some embodiments, the sum of the amount of protein plus the amount of polyurethane in the protein-polyurethane alloy may be in the range of about 80 wt % to 100 wt %, about 82 wt % to 100 wt %, about 84 wt % to 100 wt %, about 86 wt % to 100 wt %, about 88 wt % to 100 wt %, about 90 wt % to 100 wt %, about 92 wt % to 100 wt %, about 94 wt % to 100 wt %, about 96 wt % to 100 wt %, or about 98 wt % to 100 wt %.

[0224] In some embodiments, protein polyurethane alloys may include water that is a part of the total weight percentage of the material. In some embodiments, the amount of water in the protein polyurethane alloy may be in the range of about 1 wt % to about 10 wt %, including sub-ranges. For example, in some embodiments, the amount of water in the protein polyurethane alloy may be in the range of about 1 wt % to about 10 wt %, about 2 wt % to about 10 wt %, about 3 wt % to about 10 wt %, about 4 wt % to about 10 wt %, about 5 wt % to about 10 wt %, about 6 wt % to about 10 wt %, about 7 wt % to about 10 wt %, about 8 wt % to about 10 wt %, about 1 wt % to about 9 wt %, about 1 wt % to about 8 wt %, about 1 wt % to about 7 wt %, about 1 wt % to about 6 wt %, about 1 wt % to about 5 wt %, about 1 wt % to about 4 wt % or about 1 wt % to about 3 %, or in the range of having any two of these values ​​as endpoints, including endpoints.

[0225] Example

[0226] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art based thereon, and are to be included within the spirit and scope of the present application.

[0227] Example 1: Effective coagulation of recombinant collagen in polyurethane

[0228] To determine whether recombinant collagen can be effectively coagulated with commercially available polyurethane dispersions designed for medical applications, 50 kDa recombinant collagen comprising the amino acid sequence of SEQ ID NO: 1 ("50 KDa rCol") was formulated in various polyurethane compositions, as described below.

[0229] 50KDa rCol: AD111

[0230] The sample was prepared by dissolving 0.86 g of 50 KDa rCol in 4.29 mL of deionized water and stirring at 600 rpm for 30 min at 20 °C with a magnetic stir bar. AD111 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. The polyurethane and 50 KDa rCol solution were then transferred to a 10 cm diameter The dish was dried in an oven at 45°C overnight. After drying, the dried solution was conditioned in a standard reference atmosphere (23°C, 50% humidity) for 24 hours to produce a 50 KDa rCol-AD111 solidified film.

[0231] The film was tested using DMA-850 from TA Instruments. A 1cm×2.5cm strip was cut from each film using a metal die. The cut film sample was loaded into a film and fiber tension fixture for testing. During the test, a preload of 0.01N was applied to the cut film sample. The instrument was cooled to -80°C, held for 1 minute, and then the temperature was raised to 200°C at 4°C / min, or until the sample was too weak to maintain tension. During the temperature ramp, the sample oscillated 0.1% strain at a frequency of 1Hz. For both films, storage modulus, loss modulus, and tan (δ) were plotted with temperature. The resulting second storage modulus transition (as the starting point of the last drop in the measured storage modulus, i.e., the second DMA modulus transition starting temperature) of the 50KDa rCol-AD111 film was 125.0°C. The control sample of AD111 was too soft for DMA testing.

[0232] In addition, four tensile specimens were cut from the dried and conditioned sample film using a metal mold (according to the standard test method for tensile properties of plastics "ASTM D638"). The cut film samples were loaded onto 5960 (Instron, Norwood, MA) series machine, and stretched at a tension of 100 mm / min until it broke. The average Young's modulus, average tensile strength (maximum tensile stress), and average elongation at break were recorded. For the 50KDa rCol-AD111 film, the Young's modulus was 131 MPa, the maximum tensile stress was 4.8 MPa, and the elongation at break was 209%.

[0233] 50KDarCol:BaymedixCD102

[0234] The sample was prepared by dissolving 0.75 g of 50 KDa rCol in 3.75 mL of deionized water and stirring at 600 rpm for 30 min at 20 °C with a magnetic stir bar. CD102 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. The polyurethane and 50 KDa rCol solution were then transferred to a 10 cm diameter The dish was dried in an oven at 45°C overnight. After drying, the dried solution was conditioned in a standard reference atmosphere (23°C, 50% humidity) for 24 hours to produce a 50 KDa rCol polyurethane coagulated film.

[0235] Dynamic mechanical analysis (DMA) testing was performed as outlined above. The resulting second storage modulus transition (as the starting point of the last drop in measured storage modulus, i.e., the second DMA modulus transition onset temperature) of the 50 KDa rCol-CD102 film was 155.9°C, an increase of 27.3°C over the CD102 control sample (128.6°C).

[0236] Tensile testing was performed as outlined above. The 50 KDa rCol-CD102 film had an average Young's modulus of 48 MPa, measured an average tensile stress of 13 MPa, and an average elongation at break of 754%.

[0237] 50KDa rCol: CD104

[0238] The sample was prepared by dissolving 1.25 g of 50 KDa rCol in 6.25 mL of deionized water and stirring at 600 rpm for 30 min at 20 °C with a magnetic stir bar. CD104 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. The polyurethane and 50 KDa rCol solution were then transferred to a 10 cm diameter The dish was dried in an oven at 45°C overnight. After drying, the dried solution was conditioned in a standard reference atmosphere (23°C, 50% humidity) for 24 hours to produce a 50 KDa rCol polyurethane coagulated film.

[0239] DMA testing was performed as outlined above.The resulting second storage modulus transition (as the starting point of the last drop in measured storage modulus, ie, second DMA modulus transition onset temperature) of the 50 KDa rCol-CD104 film was 109.7°C, an increase of 37.8°C over the CD104 control sample (71.9°C).

[0240] Tensile testing was performed as outlined above. The 50 KDa rCol-CD104 film had an average Young's modulus of 150 MPa, measured an average tensile stress of 19 MPa, and an average elongation at break of 598%.

[0241] 50KDa rCol: FD103

[0242] The sample was prepared by dissolving 0.94 g of 50 KDa rCol in 4.72 mL of deionized water and stirring at 600 rpm for 30 min at 20 °C with a magnetic stir bar. FD103 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. The polyurethane and 50 KDa rCol solution were then pipetted into a 10 cm diameter The dish was dried in an oven at 45°C overnight. After drying, the dried solution was conditioned in a standard reference atmosphere (23°C, 50% humidity) for 24 hours to produce a 50 KDa rCol polyurethane coagulated film.

[0243] DMA testing was performed as outlined above.The resulting second storage modulus transition (as the starting point of the last drop in measured storage modulus, ie, second DMA modulus transition onset temperature) of the 50 KDa rCol-FD103 film was 178.1°C, an increase of 14.2°C over the FD103 control sample (163.9°C).

[0244] Tensile testing was performed as outlined above. The 50 KDa rCol-FD103 film had an average Young's modulus of 45 MPa, measured an average tensile stress of 19 MPa, and an average elongation at break of 414%.

[0245] FD103 Foam

[0246] pass The samples were prepared by mechanical foaming of FD103. After foaming, the foam was dried at an elevated temperature (e.g., 120°C) for a sufficient time (e.g., 10-30 min) depending on the foam thickness. Afterwards, the dried foam was soaked with a 50 kDa rCol deionized water solution at a certain concentration (e.g., 200 mg / ml).

[0247] FD103:AD111 foam

[0248] pass The samples were prepared by mechanical foaming of a mixture of FD103:AD111. After foaming, the foam was dried at an elevated temperature (e.g., 120°C) for a sufficient time (e.g., 10-30 min) depending on the foam thickness. Afterwards, the dried foam was soaked with a 50 kDa rCol deionized water solution at a certain concentration (e.g., 200 mg / ml).

[0249] 50KDa rCol: FD103 Foam

[0250] By 50kDa rCol deionized water and The samples were prepared by mechanical foaming of a mixture of 1,2-dimethoxy-1,4 ...

[0251] 50KDa rCol: FD103:AD111 foam

[0252] Prepare a 16.7% (w / w) stock solution of 50 kDa rCol in water. Baymedix FD103 and AD111 is each an aqueous polyurethane dispersion. Baymedix FD103:AD111 ( FD103 and FD103:AD111) stock solution. Prepare 10% (w / w) stock solution of Rheolate208 (Elementis, London, UK) in water. 9.16g recombinant collagen stock solution and 11.50g Baymedix FD103:AD111 stock solution are mixed with an overhead mixer in a 50mL beaker. Afterwards, 1.55g Rheolate 208 stock solution is added and the solution is further mixed. Then 0.72g ChemTex 2216 (Cumberland, RI), 0.65g ChemTex 2317 and 0.03g ChemTex 2243 are added and the resulting mixture is further mixed. The resulting mixture is mechanically foamed by violent mixing with an overhead mixer. After bubbling to introduce bubbles, the foamed mixture is coated on a glass plate on a TQC film (TQC automatic film applicator, Gardco) with a gap of 600 μm. The coating was dried at 75° C. for 30 minutes, and a porous foam sheet was obtained after drying.

[0253] This example demonstrates that a 50 kDa recombinant collagen comprising the amino acid sequence of SEQ ID NO: 1 can be effectively coagulated with a polyurethane dispersion designed for medical applications.

[0254] 50kDa rCol:Hauthaway L3360 polyurethane dispersion

[0255] Samples were prepared by dissolving 1g of 50KDa rCol in 5mL deionized water and stirring with a magnetic stirring bar at 1000rpm for 1 hour at 20°C. After stirring for 1 hour, 6.7g of L3360 (Hauthaway L3360 polyurethane dispersion) was added to the solution and stirred at 1000rpm for 30 minutes. The polyurethane and 50KDa rCol solution were then transferred to a Teflon evaporating dish with a diameter of 10cm. The dish was dried overnight in an oven at 45°C. After drying, the dried sample was conditioned for 24 hours to produce a 50KDa rCol polyurethane alloy film in a standard reference atmosphere (23°C, 50% humidity).

[0256] The film was tested using a DMA-850 from TA Instruments. The resulting second storage modulus transition (the starting point of the last drop in the measured storage modulus, i.e., the second DMA modulus transition starting temperature) of the 50KDa rCol polyurethane alloy was 177.8°C, an increase of 62.9°C over the control sample. The storage modulus measured for the control sample was 114.9°C.

[0257] By loading into Tensile testing was performed in a 5960 series machine at a tension of 100 mm / min until break. The average Young's modulus of the 50KDa rCol polyurethane alloy was 161 MPa, the average tensile stress was measured to be 17 MPa, and the average elongation at break was 173%.

[0258] Example 2: In vivo evaluation of collagen-supplemented extracellular matrix

[0259] The anticancer activity of the extracellular matrix (ECM) was evaluated by treatment with commercial bovine collagen III ("bovine collagen III") (Uniprot sequence Q08E14; SEQ ID NO: 1009) and 50 kDa recombinant collagen (SEQ ID NO: 1). Treatment solutions of bovine collagen III or SEQ ID NO: 1 were mixed with 4T1-Luc-1A4 murine breast cancer cells and then implanted subcutaneously into female Balb / c mice. Tumors were collected at the end of life, photographed, and then bisected. The experimental details and results are as follows.

[0260] 4T1-Luc2-1A4 cells

[0261] Mouse breast cancer cells (4T1-Luc2-1A4) were grown in Park Memorial Institute (Park Memorial Institute, RPMI) 1640 medium (ThermoFisher Scientific), 1mM sodium pyruvate, 10mM HEPES buffer, 2.8mL45% glucose (1.25g), 10% NHI fetal bovine serum (FBS), 1% PSG. Cells were dissociated with 0.25% trypsin / 2.21mM EDTA in Hanks' balanced salt solution (HBSS), and 5.00E+05 trypan-excluding cells were resuspended in 200μL serum-free RPMI 1640 medium. Collagen was added to the cells during the preparation of the inoculum. Each inoculum was supplemented with 1.2mL collagen. Test samples of different concentrations were prepared as shown below. Bovine collagen III was obtained from Nippi-inc (Japan; catalog number PSC-3-100-20).

[0262] Eight groups of animals (1 control group and 7 experimental groups; N=8) were included in the study and treatments were administered as shown in Table 2. The inoculum dose per animal was 5.00E+05 cells.

[0263] Table 2.

[0264] Group deal with Concentration (mg / mL) 1 Comparison 1.5 2 Hydroxylated bovine collagen III 1.5 3 Hydroxylated bovine collagen III + 0.1% EtOH 1.5 4 Hydroxylated bovine collagen III + 0.01% EtOH 1.5 5 200mg / ml SEQ ID NO:1 200 6 10mg / ml SEQ ID NO:1 10 7 1.5mg / ml SEQ ID NO:1 1.5 8 10 mg / ml hydroxylated SEQ ID NO: 1 10

[0265] Each mouse was injected subcutaneously with 200 μL of inoculum (right axilla (high)). All procedures performed in this experiment were performed in compliance with applicable laws, regulations and guidelines of the National Institutes of Health (NIH). Cell viability was measured before and after injection, as shown in Table 3.

[0266] Table 3.

[0267]

[0268]

[0269] On the 23rd day, tissue was excised, photographed, and the tumor was divided into two. Half of the tumor was placed in 10% neutral buffered formalin (NBF) (Sigma) for 24 hours and transferred to 70% EtOH to be processed into FFPE blocks. The 23rd day was selected for evaluation because this was the last day that all animals remained in the study. These blocks were stored at ambient temperature. The other half of the tumor was quickly frozen in liquid nitrogen. The tissue was stored at -80 ° C. A summary of the tolerance of each group to treatment and the response to treatment is provided in Table 4.

[0270] Table 4.

[0271]

[0272] *Median ΔT / ΔC (%) - ΔC and ΔT - individual animal endpoints, calculated for each animal as follows: ΔT = Tt-T0 and ΔC = Ct-C0, where Tt and T0 are the tumor burden of the treated animal at time t or the start of dosing, respectively. ΔC reflects a similar calculation for control animals.

[0273] result

[0274] All treatments were well tolerated and did not result in weight loss during the study duration (Table 3). One mouse in Group 3 was found dead on Day 28, and one mouse in Group 6 was euthanized on Day 28 due to severe impairment of movement. No other treated animals were euthanized for any reason except reaching the maximum tumor volume or study endpoint specified by the Institutional Animal Care and Use Committee (IACUC). Although the deaths observed in Groups 3 and 6 were outside the traditional treatment window of 14 days after treatment, both groups of animals were considered dead in the treatment window due to the nature of the treatment administration and its uncertain effect on disease progression.

[0275] The control group (Group 1) inoculated with 5.00E+05 cells per animal resulted in a median tumor doubling time of 5.0 days and a median time to progression of 25 days. All tumor growth assessments were compared to Group 1 (untreated control) (Figure 1, Figure 2A-2I , Figure 3 and Figure 4 ).

[0276] Addition of bovine collagen (Group 2) resulted in a median tumor doubling time of 5.8 days, a median ΔT / ΔC value at day 23 of 60%, and an increase in time to progression (ITP) of 16.0%. There were no tumor-free survivors in this group.

[0277] Addition of bovine collagen and 0.1% EtOH (Group 3) or bovine collagen and 0.01% EtOH (Group 4) resulted in median tumor doubling times of 6.2 and 6.6 days, median ΔT / ΔC values ​​at day 23 of 70% and 58%, and ITP values ​​of 8.0% and ≥16.0%, respectively. There were no tumor-free survivors in these groups.

[0278] Surprisingly, treatment of tumors with hydroxylated collagen did not show statistically significant inhibition of tumor growth compared to untreated controls (data not shown). Without being bound by a particular theory, it is believed that residual ethanol in the hydroxylated collagen samples interfered with the experimental results. See, e.g., Alcohol Res. 2015; 37(2): 311–322 (2015), which is incorporated herein by reference, explaining that ethanol can induce tumor growth. To study the effects of ethanol, 0.1% and 0.01% ethanol were added to Groups 3 and 4 (bovine collagen controls) to determine the effect of ethanol on tumor size compared to Groups 5-8. In all cases, the addition of ethanol induced tumor growth and inhibited the effects of bovine collagen, as observed in Group 2 (bovine collagen alone).

[0279] Addition of 200 mg / mL, 10 mg / mL or 1.5 mg / mL of SEQ ID NO: 1 (Groups 5, 6 and 7, respectively) resulted in median tumor doubling times ranging from 5.5 to 6.2 days, median ΔT / ΔC values ​​at day 23 ranging from 51% to 64%, and ITP values ​​ranging from 12.0% to ≥16.0%. There were no tumor-free survivors in these groups. SEQ ID NO: 1 was most effective at the 200 mg / mL dose (Group 5).

[0280] Addition of SEQ ID NO: 1 at 10 mg / mL (Group 8) resulted in a median tumor doubling time of 5.1 days, a median ΔTΔC value of 75% at day 23, and an ITP value of 4.0%. There were no tumor-free survivors in this group.

[0281] This example demonstrates that treatment with SEQ ID NO: 1 produces anticancer activity as effective as treatment with bovine collagen.

[0282] Effects of treatment on estimated tumor volume Figures 2A-2J The tumor volume is labeled as "estimated" because it is an indirect measure of tumor volume. The true tumor volume ( Figure 1A , Figure 1B and Figure 4 ).

[0283] Anatomical pathology analysis

[0284] To understand the effect of SEQ ID NO: 1 on cancer dormancy, H&E and Masson's Trichrome stained 4T1-Luc-1A4 murine breast cancer tumor masses from female BALB / c mice in the above examples were analyzed for the relative amounts of live tumor cells, necrosis, and collagen in live tumor areas compared to untreated samples. Tumor masses were obtained from mice administered 5E+05 4T1-Luc1A4 cells / animal combination control (Group 1), 1.5 mg / ml bovine collagen III (Group 2), or 200 mg / ml, 10 mg / ml, or 1.5 mg / ml SEQ ID NO: 1 (Groups 5, 6, and 7, respectively) by subcutaneous injection (200 μL) (above experiment). H&E and Masson's Trichrome stained slides were digitally scanned from three separate areas of each tumor mass (beginning, middle, and end), and the scanned images were examined and recorded (Table 4). Fourteen tumor blocks from the following animals were stained with H&E, Masson's trichrome, and immunohistochemistry (IHC).

[0285] All slides (H&E, Masson's trichrome, and immunohistochemistry (IHC)) were digitally scanned at 40x and evaluated. Approximate values ​​for the percentage of relative areas of live tumor cells, necrosis, and collagen within the live tumor cell area were generated solely by evaluation of the scanned images and are shown in Table 5.

[0286] Table 5.

[0287]

[0288] In all groups, regardless of the area of ​​the tumor mass, the area of ​​necrosis was located in the center of the tumor and occupied approximately 40% to 60% of the total tumor area on average for each group ( Figures 5A-5E). Areas of viable tumor cells were located mostly along the tumor periphery and collectively comprised approximately 40% to 60% of the total tumor area, regardless of group or tumor area within the block. Groups administered 200 mg / ml SEQ ID NO:1, 10 mg / ml SEQ ID NO:1, and 1.5 mg / ml SEQ ID NO:1 or bovine collagen had a greater overall mean percentage of collagen in the viable tumor area compared to controls. Additionally, all three groups administered SEQ ID NO:1 had a greater mean percentage of collagen in the viable tumor area compared to Group 2 animals administered 1.5 mg / mL bovine collagen. Representative photomicrographs of subcutaneous tumors from untreated and treated BALB / c mice are shown in Figures 6A-6J Shown in.

[0289] Among the test article treated groups, Group 2 animals administered 1.5 mg / mL bovine collagen had the lowest mean collagen percentage in the viable tumor area (mean 8.33 [± 2.50], range 5 to 10). However, the mean collagen percentage in the viable tumor area was still greater than that of the untreated control animals.

[0290] Group 5 animals (200 mg / mL SEQ ID NO: 1) had the greatest relative mean collagen percentage in the viable tumor area (mean 10.56 [±1.67%], range 10% to 15%) compared to the control group and all other groups.

[0291] Group 6 animals (10 mg / mL SEQ ID NO: 1) had greater mean relative collagen percentage in viable tumor percentage area (mean 10.00 [±2.50%], range 5 to 15) compared to control animals, Group 2 animals administered 1.5 mg / mL bovine collagen, and Group 7 animals administered 1.5 mg / mL SEQ ID NO: 1.

[0292] Group 7 animals administered 1.5 mg / mL of SEQ ID NO: 1 had a greater mean percentage of collagen in viable tumors (mean 9.44 [±3.91%], range 5 to 15) compared to control animals and Group 4 animals administered 1.5 mg / mL of commercial collagen III.

[0293] Mass spectrometry analysis of tumor tissue

[0294] The extracted tumor tissue was evaluated by mass spectrometry and the results were Figure 7 and Figure 8 The method is provided below.

[0295] The samples were loaded onto an LC-MS (Agilent 6545XT AdvanceBio LC / Q-TOF) for analysis. LC separation was achieved using a 140-minute gradient LC method (2.1×150 mm, 2.7 μm, catalog number: 653750-902). The gradient parameters were as follows (solvent A: 0.1% formic acid, solvent B: 80% acetonitrile, 0.1% formic acid): 0-3 min: 2-10% B, 3-107 min: 10-30% B, 107-121 min: 30-40% B, 121-126 min: 40-60% B, 126-127: 60-100% B, 127-137 min: 100% B, 137-138 min: 100-0% B, 138-140 min: 0% B.

[0296] Each sample was analyzed on LC-MS using a conventional automated MS / MS method (data dependent mode). The MS was run at a resolution of approximately 30,000 over the m / z range of 100-3000, with an acquisition rate of 6 spectra / sec. MS / MS analysis was performed on the first 8 precursor ions over the m / z range of 50-3000 using normalized (based on size and charge) collision energies in each cycle, with an acquisition rate of 3 spectra / sec, dynamic exclusion of 2 spectra, and release after 0.1 min.

[0297] Byologic software (Protein Metrics Inc., San Carlos, CA) was used to identify and quantify peptides. All original Agilent*.d data files were searched against the Mus musculus (mouse) proteome protein database (Uniprot, proteome ID: UP000000589) linked to SEQ ID NO: 1. Peptide search parameters were set to completely specific trypsin digestion, allowing up to two missed cuts. Precursor mass tolerance was set to 15ppm, fragment mass tolerance was set to 20ppm, with QTOF / HCD fragmentation type.

[0298] Carbamidomethylation (Cys; +57.021464 Da) was set as a fixed modification. The following were set as rare (1 occurrence per peptide) variable modifications: Gln->pyro-Glu (N-terminal Gln; -17.026549 Da), Glu->pyro-Glu (N-terminal Glu; -18.010565), carbamylation (peptide N-terminus; +43.005814), phosphorylation (Ser, Thr, Tyr; +79.966331), and oxidation (Met; +15.994915). Deamidation (Asn; +0.984016) was set as a common variable modification. Hydroxyproline (up to three per peptide) and hydroxylysine were specified as variable modifications only on "collagen family" proteins to improve peptide coverage.

[0299] After peptide identification and sequence matching, all scans with a score < 100 were filtered out from the data set. The relative protein abundance of each sample was calculated by summing the XIC areas of all peptide matches for a specific protein and dividing by the total peptide XIC area for that sample. The relative protein abundance of the target protein was then compared between the experimental conditions and the negative (Group 1) / positive (Group 2) control groups.

[0300] like Figure 8 As shown in Table 5, collagen enrichment in each experimental group was more than 100% compared to the negative control.

[0301] The collagen composition of tumor samples determined by mass spectrometry is shown in Table 6.

[0302] Table 6.

[0303]

[0304]

[0305] This example demonstrates that treatment with bovine collagen and treatment with SEQ ID NO: 1 increases the amount of collagen in tumor ECM. Treatment with SEQ ID NO: 1 is as effective as treatment with commercially available bovine collagen III.

[0306] In vitro cancer cell line viability assay

[0307] In vitro cell viability assays were performed to evaluate whether SEQ ID NO: 1 (50 kDa rCol protein) induces dormancy in various cancers.

[0308] Fourteen different conditions were compared (negative control (basal medium + 1.5% FBS), positive control (puromycin (500 μg / mL), 6 different concentrations of SEQ ID NO: 1, and 6 different concentrations of bovine type III collagen. Cell lines 4T1, HCT 116, Hep G2, and MCF7 were treated with SEQ ID NO: 1 and bovine collagen. Criteria for selecting cell lines included (1) cell doubling time ≤ 30 hours, (2) cells from human origin, and (3) cells that can grow on plastic without Matrigel ECM matrix. Proliferation levels between different treatments were determined as described herein, and the results are presented in Table 1. Figures 9A-9F (cow) and Figures 10A-10C Fourteen conditions were selected to evaluate each cell line.

[0309] 1. Negative control - untreated (minimum n = 6)

[0310] 2. Positive control - Puromycin (a potent inhibitor of translation that causes rapid cell death) (minimum n = 6)

[0311] 3.2% w / w SEQ ID NO: 1 (minimum n=6) [final solution concentration]

[0312] 4.1% w / w SEQ ID NO: 1 (minimum n=6) [final solution concentration]

[0313] 5. 0.5% w / w SEQ ID NO: 1 (minimum n = 6) [final solution concentration]

[0314] 6. 0.25% w / w SEQ ID NO: 1 (minimum n = 6) [final solution concentration]

[0315] 7. 0.125% w / w SEQ ID NO: 1 (minimum n=6) [final solution concentration]

[0316] 8. 0.0625% w / w SEQ ID NO: 1 (minimum n=6) [final solution concentration]

[0317] 9.2.7 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0318] 10.2 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0319] 11.1.5 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0320] 12.1 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0321] 13.0.5 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0322] 14.0.1 mg / mL bovine type III collagen (minimum n=6) [final solution concentration]

[0323] Results of bovine collagen Figures 9A-9F Surprisingly, higher bovine collagen concentrations were associated with higher absorbance, suggesting greater cell proliferation. Despite this data, lower bovine collagen concentrations (e.g., 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL) had reduced absorbance relative to higher concentrations, suggesting reduced cell proliferation. The apparent differences caused by bovine collagen concentrations suggest that bovine collagen interferes with the assay, and at 2.7 mg / mL, 2 mg / mL, and 1.5 mg / mL, bovine collagen is likely to inhibit cell growth, although Figures 9A-9E This is supported by the fact that the data for higher concentrations are given in Fig.9F This is supported by the data in which the effects of different concentrations of bovine collagen on 4T1 cells were measured by a proliferation assay readout at time 0. In addition, Fig.9E The microscopy images in show that bovine collagen inhibited cell growth at all concentrations, including a high concentration of 2.7 mg / mL.

[0324] The results of SEQ ID NO:1 are Figures 10A-10E Based on the in vivo data presented above, it is expected that SEQ ID NO: 1 will inhibit cell proliferation in vitro. Despite these expectations, Figures 10A-10E This indicates that SEQ ID NO: 1 does not inhibit cell growth in vitro. Without being bound by a particular theory, it is believed that the conditions used to plate different cell types in this example (different from Figures 9A-9F The conditions given in the data for bovine collagen) affect the ability of SEQ ID NO:1 to inhibit cell proliferation.

[0325] Example 3: Application of therapeutic biomaterials with recombinant collagen fragments

[0326] T-HEp3 cells expressing CDK2 biosensors were injected orthotopically into mice. At the time of tumor resection, therapeutic biomaterials loaded with recombinant collagen fragments (or controls loaded with DPBS) were applied to the wound area. Mice were monitored for tumor cell recurrence after primary tumor resection and compared with controls. Cancer cells were viewed using in vivo imaging. Cells that were arrested in G0 were identified by nuclear localization of the CDK2 sensor.

[0327] Example 4: Application of therapeutic biomaterials with recombinant collagen fragments

[0328] T-HEp3 cells expressing CDK2 biosensor were injected orthotopically into mice. Tumors were excised and sutured. After the skin healed, the sutures were removed and the scar was visible. Therapeutic biomaterials loaded with recombinant collagen fragments (or controls loaded with DPBS) were applied to the scar. Mice were monitored for tumor cell recurrence at the scar site compared to the control group. Cancer cells were viewed using in vivo imaging. Cells that were arrested in G0 were identified by nuclear localization of the CDK2 sensor.

[0329] Example 5: Application of therapeutic biomaterials with recombinant collagen fragments

[0330] T-HEp3 cells expressing CDK2 biosensors were injected orthotopically into mice. Tumors were excised and sutured. The therapeutic biomaterial loaded with recombinant collagen fragments (or DPBS-loaded controls) was applied as wound dressings immediately after surgery. Mice were monitored for tumor cell recurrence after removal of the wound dressings compared to the control group. Cancer cells were visualized using in vivo imaging. Cells that were arrested in G0 were identified by nuclear localization of the CDK2 sensor.

[0331] Example 6: Application of therapeutic biomaterials with recombinant collagen fragments

[0332] T-HEp3 cells expressing CDK2 biosensors were injected in situ into mice. The tumor was excised and the therapeutic biomaterial loaded with recombinant collagen fragments (or a control loaded with DPBS) was applied internally to the excision site and retained there while applying sutures. The mice were monitored for tumor cell recurrence after suture removal compared to the control group. Cancer cells were viewed using in vivo imaging. Cells that were arrested in G0 were determined by nuclear localization of the CDK2 sensor.

[0333] Example 7-68 - Research on therapeutic biomaterials

[0334] As described herein, mixtures of HA and collagen, as well as mixtures of various other additional polymers and collagen, have been investigated for use as therapeutic biomaterials.

[0335] Example 7

[0336] Polyethylene oxide (PEO) with a molecular weight of 400 kDa was purchased from Sigma (Sigma-Aldrich, St. Louis, MO) ("Polymer 1"). A recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared in yeast as described in PCT / US2022 / 027016, which is incorporated herein by reference in its entirety ("Polymer 2"). A 5 wt % polyethylene oxide (PEO) solution was prepared by dissolving 2.5 g PEO in 47.5 ml deionized (DI) water and mixing at 400 RPM on a stirring plate for 16 hours. A 5% (w / v) solution of a recombinant collagen fragment containing an amino acid sequence of SEQ ID NO: 1 was prepared by dissolving 5 grams of 50 kDa protein in 100 ml of water. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 10 RPM for 16 hours. The conical tube was placed vertically on a rack overnight before evaluating the stability of the solution. The final solution was turbid and slowly phase separated with white or slightly yellow aggregates settling to the bottom of the tube.

[0337] Example 8

[0338] A mixture comprising polyethylene oxide (PEO) and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared and dried and visually inspected. PEO having a molecular weight of 100 kDa was purchased from Sigma ("Polymer 1"). The remainder of the method was the same as described in Example 7. The final solution was transparent. The final solution phase separated, with a turbid solution at the top and a turbid and light yellow solution at the bottom.

[0339] Example 9

[0340] A first polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and dried and visually inspected. Example 9 was prepared using the same method described in Example 7, except that polyvinylpyrrolidone (Sigma; molecular weight of 10 kDa) was used instead of polymer 1. The final solution was transparent. Approximately 13 grams (g) of the solution was cast into a 100 ml polytetrafluoroethylene (PTFE) evaporating dish and dried overnight at 45°C. The dried film was visually transparent.

[0341] This example demonstrates that solutions comprising 10 kDa PVP and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible and can be prepared into films or articles.

[0342] Example 10

[0343] A second polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared, dried, and visually inspected. Example 10 was prepared using the method described in Example 7, except that polyvinylpyrrolidone (Sigma; molecular weight of 40 kDa) was used instead of polymer 1. The final solution was clear. The membrane was prepared using the method described in Example 9. The dried membrane was visually clear.

[0344] This example demonstrates that solutions comprising 40 kDa PVP and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible and can be prepared into films or articles.

[0345] Embodiment 11

[0346] A third polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and dried and visually inspected. Example 11 was prepared using the method described in Example 7, except that polyvinylpyrrolidone (Sigma; molecular weight of 360 kDa) was used instead of polymer 1. The final solution was transparent. The membrane was prepared using the same method as in Example 9. The dried membrane was visually transparent.

[0347] This example demonstrates that solutions containing 360 kDa PVP and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible and can be prepared as a membrane.

[0348] Example 12

[0349] Prepare a first chitosan and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture and visually inspect. Example 12 was prepared using the method described in Example 7, except that chitosan (molecular weight range of 50 kDa to 190 kD; Sigma) was used instead of polymer 1. The pH of the chitosan solution was adjusted to pH 4 using 0.287 ml of acetic acid from Sigma to completely dissolve the solution during mixing. The final solution phase separated into two layers. The viscosity was visually inspected by inspecting the difference between the two layers. The top layer had a lower viscosity and was more transparent, while the bottom layer had a higher viscosity and was more turbid. The viscosity was visually inspected by inspecting the difference between the two layers.

[0350] Embodiment 13

[0351] A mixture of polyethylene glycol and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and dried and visually inspected. Example 14 was prepared using the method described in Example 7, except that polyethylene glycol (molecular weight of 35 kDa; Sigma) was used instead of polymer 1. The final solution phase separated with a clear and transparent solution on the top and a clear and light yellow solution on the bottom.

[0352] Embodiment 14

[0353] Prepare the first alginate and collagen (recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1) mixture and dry, and visually inspect.Use the method described in Example 7 to prepare Example 14, except that polymer 1 is replaced with a medium viscosity sodium alginate salt (Sigma) from brown algae. At 25°C, the viscosity of 2% sodium alginate in water is equal to or greater than 2,000cps. Prepare 2.5 wt% alginate solution by dissolving 2.5g sodium alginate in 97.5ml deionized ("DI") water and mixing on a stirring plate at 400RPM for 24 hours (due to the high viscosity of the solution). Visually inspect the difference between the two layers for viscosity. The two solutions are mixed in a 50mL conical tube at a ratio of 1:1 (v / v) and mixed at 10RPM for 16 hours. Before evaluating the solution stability, the conical tube is placed vertically on a shelf overnight. The final solution is transparent. Use the method described in Example 9 to prepare the film. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0354] Embodiment 15

[0355] Prepare the second alginate and collagen (recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1) mixture and dry, and visually inspect.Use the method described in Example 7 to prepare Example 15, except that polymer 1 is replaced with a low viscosity sodium alginate salt (Sigma) from brown algae.At 25°C, the viscosity of 1% sodium alginate in water is about 4-12cps.Alginate sodium salt and collagen stock solution are both 5 weight %.Two solutions are mixed in a 50mL conical tube with a 1:1 (v / v) ratio and mixed at 10RPM for 16 hours.Finally, the solution is transparent.Use the method described in Example 9 to prepare the film.The dried film is visually turbid and has a uniformly distributed turbidity throughout the film.

[0356] Example 16

[0357] A mixture of polyacrylamide and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and dried and visually inspected. Example 16 was prepared using the method described in Example 7, except that polyacrylamide (molecular weight range of 5,000 kDa to 6,000 kDa; Sigma) was used instead of polymer 1. The rest of the method was the same as described in Example 7. The final solution was clear. The membrane was prepared using the same method as described in Example 9. The dried membrane was visually clear.

[0358] This example demonstrates that solutions comprising 5,000 kDa to 6,000 kDa polyacrylamide and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are miscible and can be prepared into films or articles.

[0359] Embodiment 17

[0360] A mixture of polyacrylamide and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and dried and visually inspected. Example 17 was prepared using the method described in Example 7, except that polyacrylamide (molecular weight of 40 kDa; Sigma) was used instead of polymer 1. The rest of the method was the same as described in Example 7. The final solution was clear. The membrane was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried membrane was visually clear.

[0361] This example demonstrates that solutions comprising 40 kDa polyacrylamide and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are miscible and can be prepared into films or preparations.

[0362] Embodiment 18

[0363] A first polyvinyl alcohol (PVA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and visually inspected. Example 18 was prepared using the method described in Example 7, except that PVA (molecular weight range of 89 kDa to 98 kDa; Sigma) was used instead of polymer 1. A 4 wt % PVA solution was prepared by dissolving 2 g PVA in 48 ml DI water and mixing on a stirring plate at 400 RPM for 16 hours. The two solutions were mixed in a 50 mL conical tube at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution phase separated into two layers. The top layer was turbid and a large amount of white powdery aggregates settled at the bottom.

[0364] Embodiment 19

[0365] A second PVA and collagen (recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and visually inspected. Example 19 was prepared using the method described in Example 18, except that 4% PVA (w / v) solution (molecular weight unknown; Fisher Scientific) was used instead of polymer 1. The rest of the method was the same as described in Example 18. The final solution phase separated into two layers. The viscosity was visually inspected by inspecting the difference between the two layers. The top layer had a lower viscosity and was more turbid and had small suspended particles. The bottom layer had a higher viscosity and was transparent.

[0366] Embodiment 20

[0367] A first hyaluronic acid (HA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and dried and visually inspected. Example 20 was prepared using the method described in Example 7, except that hyaluronic acid (HA) (molecular weight of 1,000 kDa; purchased from Pure Health Botanicals (Saint Charles, IL)) was used instead of polymer 1. A 2.5 wt% HA solution was prepared by dissolving 5 g HA in 195 ml DI water and mixing at 400 RPM for 40 hours (due to the high viscosity of the solution) with a high shear impeller. A 5% (w / v) collagen solution containing a recombinant collagen fragment of SEQ ID NO: 1 was prepared as described in Example 7. The HA solution and the collagen solution were mixed in a 50 ml conical tube at a ratio of 2: 1 (w / w) and mixed at 10 RPM for 16 hours to maintain a dry mass ratio of 1: 1. The final solution was transparent. The membrane was prepared using the method described in Example 9. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0368] Embodiment 21

[0369] A second hyaluronic acid (HA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and dried and visually inspected. Example 21 was prepared using the method in Example 7, except that non-animal-based HA (molecular weight of 50 kDa; Pure Health Botanicals (Saint Charles, IL) and DSM (Kaiseraugst, Switzerland)) was used instead of polymer 1. A 5 wt% HA solution was prepared by dissolving 5 g HA in 95 ml DI water and mixing at 400 RPM on a stirring plate for 16 hours. A 5% (w / v) collagen solution containing a recombinant collagen fragment of SEQ ID NO: 1 was prepared as described in Example 7. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 10 RPM for 16 hours. The final solution was transparent. The film was prepared using the method described in Example 9. The dried film was visually turbid and had a uniformly distributed turbidity throughout the film. Additionally, an aliquot of the HA and collagen solution mixture was placed in a 4°C refrigerator overnight and the solution remained transparent.

[0370] Embodiment 22

[0371] Prepare the first gelatin and HA mixture and visually inspect. Example 22 was prepared using the method in Example 21, except that polymer 2 was replaced with a low molecular weight bovine gelatin dietary supplement (Great Lakes Wellness, Grayslake, IL). A 2.2 wt% gelatin solution was prepared by dissolving 1 g of gelatin in 45 ml of 0.01 N HCl and mixing at 400 RPM for 16 hours (due to the high viscosity of the solution) with a high shear impeller. A 5 wt% 50 kDa HA solution was prepared using the method described in Example 21. The HA solution and the gelatin solution were mixed in a 50 ml conical tube at a ratio of 1:2.4 (w / w) and mixed at 10 RPM for 16 hours to maintain a dry mass ratio of 1:1. The final solution phase separated into two layers. The viscosity was visually inspected by examining the difference between the two layers. The top layer had a lower viscosity and was more transparent. The bottom layer had a higher viscosity and was more turbid.

[0372] Embodiment 23

[0373] A second gelatin and HA mixture was prepared and visually inspected. Example 23 was prepared using the method described in Example 22, except that polymer 2 was replaced with gelatin from pig skin (molecular weight 300 kDa; Sigma). A 1.1 wt% gelatin solution was prepared by dissolving 0.5 g of gelatin in 45 ml of 0.01 N HCl and mixing with a high shear impeller at 400 RPM for 16 hours (due to the high viscosity of the solution). A 5 wt% 50 kDa HA solution was prepared using the method described in Example 21. The HA solution and the gelatin solution were mixed in a 50 ml conical tube at a ratio of 1:4.5 (w / w) and mixed at 10 RPM for 16 hours to maintain a dry mass ratio of 1:1. The final solution phase separated, with a large amount of white aggregates settling at the bottom and dispersed white aggregates suspended or floating on the top.

[0374] Embodiment 24

[0375] HA and type I collagen mixtures were prepared and visually inspected. Example 24 was prepared using the method described in Example 21, except that type I collagen (molecular weight 414kDa; Sigma) was used instead of polymer 2. A 0.7 wt% type I collagen solution was prepared by dissolving 0.3g type I collagen in 45ml 0.01N HCl and mixing at 400RPM for 16 hours (due to the high viscosity of the solution) with a high shear impeller. A 5 wt% 50kDa HA solution was prepared using the method described in Example 21. The HA solution and type I collagen solution were mixed together in a 50ml Falcon conical tube at a ratio of 1:7 (w / w) and mixed at 10RPM for 16 hours with a Hula mixer to maintain a dry mass ratio of 1:1. The final solution phase separated, with a large amount of white fibrils settling at the bottom and dispersed white fibrils suspended or floating at the top.

[0376] Embodiment 25

[0377] Prepare the cellulase and HA mixture and visually inspect. Example 25 was prepared using the method described in Example 21, except that cellulase (Sunson Industry Group Co., Ltd.) was used instead of polymer 2. A 5% (w / v) cellulase solution was prepared by dissolving 2.5g of cellulase in 47.5ml of DI water and mixing on a stirring plate at 400RPM for 16 hours. A 5 wt% 50kDa HA solution was prepared using the same method described in Example 21. The two solutions were mixed together in a 1:1 (w / w) ratio in a 50mL conical tube and mixed at 10RPM for 16 hours. The final solution was turbid with a yellow powder settled at the bottom.

[0378] Embodiment 26

[0379] A mixture of HA, chitosan and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and visually inspected. Example 26 was prepared using the method described in Example 21, except that the chitosan used in Example 12 was added as polymer 3. A 5 wt% 50 kDa HA solution was prepared by dissolving 5 g of HA in 95 ml of DI water and mixing at 400 RPM on a stirring plate for 16 hours. A 5% (w / v) collagen solution containing a recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 5 grams of 50 kDa protein in 100 ml of water, as described in Example 7. A 5 wt% chitosan solution was prepared by dissolving 2.5 g of chitosan in 47.5 ml of DI water and mixing at 400 RPM on a stirring plate for 16 hours. The pH of the chitosan solution was adjusted to pH 4 using 0.287 ml of acetic acid from Sigma to completely dissolve the solution during mixing. The three solutions were mixed in a 50 mL conical tube at a 1:1:1 (w / v) ratio and mixed at 10 RPM for 16 hours. The final solution phase separated, with large yellow aggregates suspended or floating in the low viscosity medium.

[0380] Embodiment 27

[0381] A mixture of hyaluronic acid and a recombinant collagen fragment (hydroxylated to 22%) with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 27 was prepared using the method in Example 21, except that polymer 2 was replaced with 22% hydroxylated collagen with a molecular weight of 50 kDa. The solid content of the 22% hydroxylated collagen solution was 1.3% and the pH was about 6. A 5% by weight 50 kDa HA solution (polymer 1) was prepared by dissolving 5 g HA in 95 ml DI water and mixing at 400 RPM on a stirring plate for 16 hours. The two solutions were mixed in a 15 mL Falcon tube at a ratio of 1: 1 (w / w) and mixed at 10 RPM for 16 hours. The final solution was turbid, indicating that the two polymers were not miscible in solution at this level of hydroxylation.

[0382] Embodiment 28

[0383] A mixture of hyaluronic acid and a recombinant collagen fragment (hydroxylated to 44%) with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 28 was prepared using the same method described in Example 27, except that 44% hydroxylated collagen with a molecular weight of 50 kDa was used as polymer 2. The solid content of the 44% hydroxylated collagen solution was 1.3% and the pH was about 6. A 5% by weight 50 kDa HA solution (polymer 1) was prepared by dissolving 5 g HA in 95 ml DI water and mixing at 400 RPM on a stirring plate for 16 hours. The two solutions were mixed in a 15 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 10 RPM for 16 hours. The final solution was turbid and slowly phase separated with white aggregates settling to the bottom of the tube.

[0384] Embodiment 29

[0385] A mixture of biphasic cross-linked hyaluronic acid (comprising cross-linked HA and uncross-linked HA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and visually inspected. Example 29 was prepared using the method described in Example 21, except that biphasic cross-linked HA (particle size of 1.25-2 mm; Beijing Meng Bo Run Biotechnology Co., Ltd.) was used instead of polymer 1. A 2.4% (w / v) HA solution was used. A 20% by weight solution of the recombinant collagen fragment of SEQ ID NO: 1 (polymer 2) was prepared by dissolving 2g of 50kDa recombinant collagen in 18ml DI water and mixing at 10RPM for 4 hours. The two solutions were mixed together in a 50mL conical tube at a ratio of 1: 1 (w / w) and mixed at 10RPM for 16 hours. The mixed solution was visually transparent.

[0386] This example demonstrates that solutions comprising biphasic cross-linked HA and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible.

[0387] Embodiment 30

[0388] A single-phase cross-linked hyaluronic acid and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and visually inspected. Example 30 was prepared using the same method as Example 21, except that polymer 1 was replaced with a single-phase cross-linked HA (Huaxi Biotechnology Co., Ltd. (Jinan, Shandong, China)). A 2% (w / v) single-phase cross-linked HA solution was used. A 20 wt% collagen solution containing a recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 2g 50kDa recombinant collagen in 18ml DI water and mixing at 10RPM for 4 hours. The two solutions were mixed in a 50mL conical tube at a 1:1 (w / w) ratio and mixed at 10RPM for 16 hours. The solution was visually transparent.

[0389] This example demonstrates that a solution comprising single-phase cross-linked HA and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 is completely miscible.

[0390] Embodiment 31

[0391] A mixture of biphasic cross-linked hyaluronic acid (comprising cross-linked HA and uncross-linked HA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and visually inspected. Example 31 was prepared using the method described in Example 30, except that polymer 1 was replaced with biphasic cross-linked HA (Restylane). A 2.0% (w / v) biphasic cross-linked HA solution was used. A solution of a recombinant collagen fragment of 40% by weight of SEQ ID NO: 1 was prepared by dissolving 4g of 50 kDa recombinant collagen in 16ml DI water and mixing at 10RPM for 4 hours. The two solutions were mixed with a pipette in a 1ml Eppendorf tube at HA: collagen ratios of 1: 3, 1: 1, 10: 3 and 100: 3 (w / w), and mixed at 10RPM for 16 hours. The solution was visually transparent.

[0392] This example demonstrates that solutions comprising biphasic cross-linked hyaluronic acid and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible.

[0393] Embodiment 32

[0394] Prepare a 5% hyaluronic acid and collagen (a recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO: 1) mixture and visually inspect. Example 32 was prepared using the method described in Example 21, except that the pH of the 5 wt% 50kDa HA solution was adjusted to pH 5.2-7.2 with sodium hydroxide before adding the collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1. The two solutions were mixed together in a 50mL tube at a ratio of 1: 1 (w / w) and mixed at 10RPM for 16 hours. The solution was visually transparent. The film was prepared using the method of Example 9. The dried film was visually turbid and had a uniformly distributed turbidity throughout the film.

[0395] This example demonstrates that solutions containing 5% hyaluronic acid (pH 5.2-7.2) and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible.

[0396] Embodiment 33

[0397] 5% hyaluronic acid and collagen (recombinant collagen fragment with a molecular weight of 50 kDa of SEQ ID NO: 1) mixtures were prepared at different pH and visually inspected. Example 33 was prepared using the method described in Example 21, except that the pH of the 5 wt% 50 kDa HA solution was adjusted to pH 9, 10, 11 and 12 with sodium hydroxide before adding the recombinant collagen fragment of SEQ ID NO: 1. The two solutions were mixed in a 20 ml glass bottle at a ratio of 1: 1 (w / w) and mixed at 400 RPM for 30 minutes using a stirring rod (due to the lower viscosity of the alkaline solution). The solution was then readjusted to pH 7 ± 1 with concentrated hydrochloric acid while mixing at 400 RPM with a stirring rod. Before and after the pH was adjusted to 7 ± 1, the solution was visually transparent.

[0398] This example demonstrates that solutions containing 5% hyaluronic acid (pH 9, 10, 11, 12) and a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 are completely miscible.

[0399] Embodiment 34

[0400] Hyaluronic acid and collagen mixtures were prepared and visually inspected. Example 34 was prepared using the method of Example 21, except that 2% Solution (Geltor) was used instead of polymer 1. A 2 wt% 50 kDa HA solution was prepared using the method described in Example 21. The two solutions were mixed in a 1:1 (w / w) ratio in a 2 ml glass bottle and mixed on a stir plate at 400 RPM for 1 hour. The final solution was visually clear.

[0401] This example demonstrates that the inclusion of 2% The solution and the solution of the recombinant collagen fragment having a molecular weight of 50 kDa having SEQ ID NO: 1 are completely miscible.

[0402] Embodiment 35

[0403] Hyaluronic acid and full-length recombinant collagen and 50kDa recombinant collagen fragment are blended, mixed with HA, and visually inspected. Full-length recombinant collagen and 50kDa recombinant collagen fragment are blended and mixed with HA. Solution is visually inspected. The blend of 50kDa recombinant collagen fragment and full-length recombinant collagen and / or hydrolyzed recombinant collagen (or any one combination of these three) is mixed with HA, and visually inspected. Then, the blend of full-length recombinant collagen and 50kDa recombinant collagen fragment and the blend of full-length recombinant collagen and hydrolyzed full-length recombinant collagen are mixed with HA. Solution is visually inspected.

[0404] Embodiment 36

[0405] A solution of poly(2-ethyl-2-oxazoline) (PEOx) was mixed with a solution containing a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1. Example 36 was prepared using the same method described in Example 7, except that PEOx (molecular weight 50 kDa; Sigma) 1 was used instead of polymer 1. The remainder of the method was the same as described in Example 7. The final solution was visually transparent, indicating that the polymers were miscible in the solution. The membranes were prepared using the same method described in Example 9, except that they were performed in aluminum weighing pans. The dried membranes visually appeared to phase separate during the drying process.

[0406] Embodiment 37

[0407] A polyethyleneimine (PEI) solution was mixed with a solution containing a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1. Example 37 was prepared using the method described in Example 7, except that PEI (molecular weight of 100 kDa; Polysciences (Warrington, Pennsylvania)) was used instead of polymer 1. A 5 wt% polyethyleneimine solution was prepared by dissolving 0.5 g PEI in 9.5 g Milli-Q water at 80°C and mixing at 450 RPM using a stirring bar for 3 hours or until the solution was completely transparent. The solution was then cooled to room temperature and the pH was adjusted to 7 using 12N HCl. The remainder of the method was the same as described in Example 7. The solution was transparent, indicating that the polymer was miscible in the solution. The membrane was prepared using the method described in Example 3. The dried membrane was visually opaque.

[0408] Embodiment 38

[0409] Prepare the first sodium carboxymethylcellulose (NaCMC) and collagen (recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1) mixture and dry, and visually inspect. Use the method described in Example 7 to prepare Example 38, except that sodium carboxymethylcellulose (molecular weight is about 250kDa and degree of substitution is 1.22; Sigma) replaces polymer 1. Prepare 5% by weight sodium carboxymethylcellulose solution by dissolving 0.5g sodium carboxymethylcellulose in 9.5g DI water and mixing at 5RPM in a conical tube for 16 hours. The two solutions are mixed in a 50mL conical tube at a ratio of 1:1 (v / v) and mixed at 10RPM for 16 hours. The final solution is transparent and light yellow, indicating that the solution is miscible. Use the method described in Example 9 to prepare the film, except that an aluminum weighing pan is used. The dried film is visually turbid and has uniformly distributed turbidity throughout the film.

[0410] Embodiment 39

[0411] Prepare a second sodium carboxymethylcellulose and collagen (a recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1) mixture and dry it, and visually inspect it. Use the same method described in Example 7 to prepare Example 39, except that sodium carboxymethylcellulose (molecular weight of about 250kDa and degree of substitution of 0.79; Sigma) is used to replace polymer 1. A 2.5 wt% sodium carboxymethylcellulose solution is prepared by dissolving 0.25g sodium carboxymethylcellulose in 9.75g DI water and mixing it in a conical tube at 5RPM for 16 hours. A 2.5 wt% collagen solution containing a recombinant collagen fragment with an amino acid sequence of SEQ ID NO:1 is prepared by dissolving 0.25g 50kDa recombinant collagen in 9.75g DI water. The two solutions are mixed in a 50mL conical tube at a ratio of 1:1 (v / v) and mixed at 10RPM for 16 hours. The final solution is transparent, indicating that the polymer is miscible in the solution. The film is prepared using the method described in Example 9, except that an aluminum weighing pan is used. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0412] Embodiment 40

[0413] A third sodium carboxymethylcellulose and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) mixture was prepared and dried and visually inspected. Example 41 was prepared using the method described in Example 7, except that sodium carboxymethylcellulose (a 1% aqueous solution with a high viscosity of 2,700 cps and a degree of substitution of 0.87; Sigma) was used instead of polymer 1. A 2.5 wt% sodium carboxymethylcellulose solution was prepared by dissolving 0.25 g of sodium carboxymethylcellulose in 9.75 g of DI water and mixing at 5 RPM in a conical tube for 16 hours. A 2.5 wt% solution of a recombinant collagen fragment containing an amino acid sequence of SEQ ID NO: 1 was prepared by dissolving 0.25 g of recombinant collagen fragment in 9.75 g of water. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (v / v) and mixed at 10 RPM for 16 hours. The final solution was transparent, indicating that the polymer was miscible in the solution. The membrane was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0414] Example 41 (HA / SEQ ID NO: 1007)

[0415] A recombinant collagen fragment having an amino acid sequence of SEQ ID NO: 1007 and a HA mixture were prepared, dried and visually inspected. SEQ ID NO: 1007 has 98% identity to SEQ ID NO: 1. Example 41 was prepared using the method described in Example 21, except that polymer 2 was replaced with a collagen polymer having an amino acid sequence of SEQ ID NO: 1007. A 5 wt % solution of a recombinant collagen fragment having an amino acid sequence of SEQ ID NO: 1007 was prepared by dissolving 0.25 g of the recombinant collagen fragment in 4.75 g of water. A 5 wt % 50 kDa HA solution was prepared by dissolving 0.25 g of 50 kDa HA (polymer 1) in 4.75 g of water. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 10 RPM for 16 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The final solution was transparent. The membrane was prepared using the method described in Example 9, except that an aluminum pan was used. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0416] Example 42 (HA / SEQ ID NO: 1008)

[0417] A recombinant collagen fragment having an amino acid sequence of SEQ ID NO: 1008 and HA mixture was prepared, dried and visually inspected. SEQ ID NO: 1008 has 50% identity to SEQ ID NO: 1. Example 42 was prepared using the method described in Example 21, except that a collagen polymer having an amino acid sequence of SEQ ID NO: 1008 was used instead of polymer 2. A 5 wt % collagen solution containing a collagen polymer having an amino acid sequence of SEQ ID NO: 1008 was prepared by dissolving 0.25 g of recombinant collagen fragment in 4.75 g of Milli-Q water (water purified using a Millipore Milli-Q laboratory water system; "MQ water") and purified by centrifugation at 3214 relative centrifugal forces (RCF) for 1 hour. A 5 wt % 50 kDa HA (polymer 1) solution was prepared using the same method described in Example 41. The supernatant of the collagen solution and the HA solution were mixed in a 50 ml Falcon tube at a 1: 1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was clear, indicating that the two polymers were miscible in solution. The dried film was visually turbid.

[0418] Example 43 (HA / SEQ ID NO: 973)

[0419] A full-length collagen and HA mixture was prepared, dried, and visually inspected. Example 43 was prepared using the method described in Example 41, except that polymer 2 was replaced with a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 973. A 5 wt % collagen solution containing a recombinant collagen fragment of SEQ ID NO: 973 was prepared by dissolving 0.25 g of recombinant collagen in 4.75 g of MQ water. A 5 wt % 50 kDa HA solution was prepared using the method described in Example 42. The remainder of the method was the same as described in Example 42. The solution was transparent, indicating that the polymers were miscible in the solution. The dried film was visually turbid and had a uniformly distributed turbidity throughout the film.

[0420] Example 44 (Chondroitin Sulfate / Collagen)

[0421] A mixture of chondroitin sulfate and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared, dried, and visually inspected. Example 44 was prepared using the method described in Example 7, except that chondroitin sulfate from bovine trachea (molecular weight of 50 kDa; EMD Millipore (Burlington, MA)) was used instead of polymer 1. A 5 wt % chondroitin sulfate solution was prepared by dissolving 0.5 g of chondroitin sulfate in 9.5 g of MQ water. The remainder of the method was the same as described in Example 7. The final solution was transparent, indicating that the polymer was miscible in the solution. The membrane was prepared using the same method described in Example 9, except that an aluminum weighing pan was used. The dried membrane was visually turbid and had a uniformly distributed turbidity throughout the membrane.

[0422] Example 45 (AcHA / Collagen)

[0423] A mixture of acetylated hyaluronate (AcHA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared, dried, and visually inspected. Example 45 was prepared using the method described in Example 21, except that acetylated sodium hyaluronate (Huaxi Biotechnology Co., Ltd.) was used instead of polymer 1. A 5 wt % AcHA solution was prepared by dissolving 0.25 g of acetylated hyaluronic acid (AcHA) in 4.75 g of MQ water. A 5 wt % solution of a recombinant collagen fragment containing SEQ ID NO: 1 was prepared by dissolving 0.25 g of collagen in 4.75 g of water. The rest of the method was the same as described in Example 21. The solution was transparent, indicating that the polymer was miscible in the solution. The film was prepared using the same method described in Example 9, except that an aluminum pan was used. The dried film was visually turbid and had a uniformly distributed turbidity throughout the film.

[0424] Example 46 (ZnHA / Collagen)

[0425] A mixture of zinc hyaluronate (ZnHA) and collagen (a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and dried and visually inspected. Example 46 was prepared using the method described in Example 21, except that zinc hyaluronate (ZnHA) (molecular weight of 170 kDa; Huaxi Biotechnology Co., Ltd.) was used instead of polymer 1. A 5 wt % ZnHA solution was prepared by dissolving 0.25 g of zinc hyaluronate (ZnHA) in 4.75 g of MQ water. A 5 wt % solution of a recombinant collagen fragment containing SEQ ID NO: 1 was prepared by dissolving 0.25 g of collagen in 4.75 g of water. The rest of the method was the same as described in Example 21. The solution was turbid, indicating aggregation of particles. The membrane was prepared using the same method as described in Example 9, except that an aluminum pan was used. The dried membrane was transparent, indicating that the polymers were miscible.

[0426] Example 47 (HA / collagen salt doping study)

[0427] Prepare hyaluronic acid and collagen (recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1) solution, dry, and visually inspect. Use the method as described in Example 21 to prepare Example 47, except that a solution containing a recombinant collagen fragment with a molecular weight of 50kDa with SEQ ID NO:1 is prepared in a NaCl solution. Prepare 5 wt% collagen solution by dissolving 0.25g in 4.75g 0.05mM NaCl solution. Prepare 5 wt% 50kDa HA solution using the same method described in Example 41. The two solutions are mixed in a 50mL conical tube with a 1:1 (w / w) ratio and mixed at 10RPM for 4 hours. The solution is transparent, indicating that the polymer is miscible in a solution with a higher salt content. Use the same method as described in Example 9 to prepare a film, except that an aluminum pan is used. The dried film is turbid.

[0428] Example 48 (HA / Collagen Dialysis)

[0429] Prepare hyaluronic acid and collagen (recombinant collagen fragment with SEQ ID NO:1 molecular weight of 50kDa) solution, dry, and visually inspect.Use the method described in Example 21 to prepare Example 48, except that hyaluronic acid and collagen solution are dialyzed.5 % by weight collagen solution of the recombinant collagen fragment comprising SEQ ID NO:1 is prepared by dissolving 0.25g recombinant collagen in 4.75g MQ water.5 % by weight 50kDa HA solution is prepared by dissolving 0.25g hyaluronic acid powder in 4.95g MQ water.Two solutions are mixed in 50mL conical tubes with 1:1 (w / w) ratio and mixed for 4 hours at 10RPM.Then, 9g of the combined solution is loaded into 3.5-5kDa molecular weight cutoff (MWCO) membrane of 10ml volume.The dialysate reservoir is prepared as a Nalgene bottle containing 500ml MQ water and a stirring bar for keeping the surrounding solution gently rotating. The film containing solution is immersed in the dialysate reservoir and at room temperature dialyzed for 16 hours.Solution is removed from the film and transferred to the 50ml conical tube.Solution is transparent, shows that polymer is miscible in the solution of lower salt content.Use the same method preparation film described in Example 9, different is to use aluminum tray.Exsiccant film is turbid visually, and has uniformly distributed turbidity in whole film.

[0430] Example 49 (HA and hydrolyzed recombinant collagen fragments-5 mg / mL papain)

[0431] Prepare hyaluronic acid and hydrolyzed collagen solution (hydrolyzed fragment of recombinant collagen fragment with molecular weight of 50kDa of SEQ ID NO:1), dry, and visually inspect. Prepare 5 wt % solution of recombinant collagen fragment containing amino acid sequence of SEQ ID NO:1 by dissolving 0.25g collagen in 4.75g water. Prepare 10 wt % papain solution by dissolving 100mg papain (200TU / mg, derived from BIO-CAT) in 900μl water. Prepare 10 wt % papain solution by adding 250μL of 10 wt % papain solution to 4.75ml of 5 wt % recombinant collagen solution and incubate at 60°C for 2 hours for enzymatic hydrolysis, then incubate at 90°C for 10 minutes to inactivate enzyme activity to prepare the recombinant fragment solution of hydrolysis. Afterwards, the hydrolyzed solution was centrifuged at 3214 relative centrifugal forces (RCF) for 20 minutes. Collect supernatant to mix with following HA solution. 5 wt% recombinant collagen fragment and HA solution was prepared by dissolving 0.25g 50kDa HA in 4.75g water. The two solutions were mixed in a 50mL conical tube at a 1:1 (w / w) ratio and mixed at 5RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The final solution was transparent. The film was prepared using the method described in Example 9, except that a silica gel mold was used. The dried film was visually transparent, indicating that the polymer was completely miscible.

[0432] Example 50 (PVP and hydrolyzed recombinant collagen fragments-5 mg / mL papain)

[0433] Prepare polyvinyl pyrrolidone and hydrolyzed recombinant collagen solution (hydrolyzed fragment of recombinant collagen fragment with molecular weight of 50kDa of SEQ ID NO:1), dry, and visually inspect. Example 50 was prepared using the method described in Example 49, except that polyvinyl pyrrolidone (molecular weight of 40kDa; Sigma) was used instead of polymer 1. A 5 wt% 40kDa PVP solution was prepared by dissolving 0.25g 40kDa PVP in 4.75g water. The hydrolyzed recombinant collagen solution was prepared using the method described in Example 43. The two solutions were mixed in a 50mL conical tube at a ratio of 1:1 (w / w) and mixed at 5RPM for 2 hours. The conical tube was placed vertically on a shelf overnight to evaluate the solution stability. The final solution was transparent. The film was prepared using the method described in Example 9, except that a silica gel mold was used. The dried film was visually transparent, indicating that the polymer was miscible in solution and solid.

[0434] Example 51 (HA and blend of recombinant collagen fragments and hydrolyzed recombinant collagen fragments - 0.1 mg / mL papaya Protease

[0435] Prepare hyaluronic acid, the recombinant collagen fragment of 50kDa with the molecular weight of SEQ ID NO:1 and the solution of the recombinant collagen fragment of partial hydrolysis, dry and visual inspection. Prepare 5 weight % solution of the recombinant collagen fragment containing the amino acid sequence of SEQ ID NO:1 by dissolving 0.25g recombinant collagen fragment in 4.75g water. Prepare the blend of recombinant collagen fragment and the recombinant collagen fragment solution of hydrolysis by partially hydrolyzing 5 weight % collagen solution. Prepare 1 weight % papain solution by dissolving 10mg papain (200TU / mg, derived from BIO-CAT) in 990μl water. Prepare 1 weight % papain solution by adding 50μl of 1 weight % papain solution to 4.95ml of 5 weight % recombinant collagen solution and incubate at 60°C for 2 hours to carry out enzymatic hydrolysis, then incubate at 90°C for 10 minutes to inactivate the enzyme activity to prepare the solution of the recombinant fragment of partial hydrolysis (polymer 2). A 5 wt% 50 kDa HA (Polymer 1) solution was prepared by dissolving 0.25 g of 50 kDa HA in 4.75 g of water. The two solutions were mixed in a 50 mL conical tube at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The final solution was transparent, indicating that the polymer was miscible in the solution. Films were prepared using the method described in Example 9, except that a silica gel mold was used. The dried film was visually turbid and had evenly distributed turbidity throughout the film.

[0436] Example 52 (PVP and a blend of recombinant collagen fragments and hydrolyzed recombinant collagen fragments - 0.1 mg / mL papaya Protease

[0437] Solutions of polyvinyl pyrrolidone, a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1, and a partially hydrolyzed recombinant collagen fragment were prepared, dried, and visually inspected. Example 52 was prepared using the method described in Example 51, except that polyvinyl pyrrolidone (molecular weight of 40 kDa; Sigma) was used instead of polymer 1. A 5 wt% 40 kDa PVP solution was prepared by dissolving 0.25 g 40 kDa PVP in 4.75 g water. A blend of recombinant collagen fragment and hydrolyzed recombinant collagen fragment solution was prepared using the method described in Example 51. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a shelf overnight to assess solution stability. The final solution was transparent. The film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually transparent, indicating that the polymer was miscible in solution and solid.

[0438] Example 53 (Biphasic HA and a blend of recombinant collagen fragments and hydrolyzed recombinant collagen fragments - 0.1 mg / mL Papain)

[0439] A solution of a blend of biphasic cross-linked hyaluronic acid (comprising cross-linked HA and uncross-linked HA) and a recombinant collagen fragment having a molecular weight of 50 kDa and partially hydrolyzed recombinant collagen fragments with SEQ ID NO: 1 was prepared and visually inspected. Example 53 was prepared using the method described in Example 51, except that 2.4% (w / v) biphasic cross-linked HA (Shandong Runxin Biotechnology Co., Ltd. (Qufu City, Shandong Province, China)) with a particle size of 0.10-0.25 mm was used instead of polymer 1. A solution containing a blend of a recombinant collagen fragment having a molecular weight of 50 kDa and a hydrolyzed recombinant collagen fragment with SEQ ID NO: 1 was prepared using the method described in Example 51. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and vortexed for 3 minutes and mixed at 5 RPM for 3 hours. The conical tube was placed vertically on a shelf overnight to evaluate the solution stability. The solution was visually slightly turbid due to the visible texture of the cross-linked HA particles.

[0440] Example 54 (CMC sodium and a blend of recombinant collagen fragments and hydrolyzed recombinant collagen fragments - 0.1 mg / mL wood Guarana

[0441] Sodium carboxymethylcellulose and a blend of a recombinant collagen fragment with a molecular weight of 50 kDa and a partially hydrolyzed recombinant collagen fragment having SEQ ID NO: 1 were prepared, dried and visually inspected. Example 54 was prepared using the method described in Example 51, except that sodium carboxymethylcellulose (molecular weight of about 250 kDa and degree of substitution of 1.22; Sigma) was used instead of polymer 1. A 5 wt% sodium carboxymethylcellulose solution was prepared by dissolving 0.5 g of sodium carboxymethylcellulose in 9.5 g of DI water and mixing at 5 RPM in a conical tube for 16 hours. A blend of recombinant collagen fragments and hydrolyzed recombinant collagen fragment solutions was prepared using the method described in Example 51. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a shelf overnight to evaluate solution stability. The final solution was transparent, indicating that the polymer was miscible in the solution. The membrane was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0442] Example 55 (sodium carboxymethylcellulose and SEQ ID NO: 1007)

[0443] Sodium carboxymethylcellulose and a collagen polymer solution having the amino acid sequence shown in SEQ ID NO: 1007 were prepared, dried, and visually inspected. SEQ ID NO: 1007 has 98% identity with SEQ ID NO: 1. A 5 wt% solution of a recombinant collagen polymer having the amino acid sequence shown in SEQ ID NO: 1007 was prepared by dissolving 0.5 g of recombinant collagen fragment in 9.5 g of water. A 5 wt% sodium carboxymethylcellulose solution was prepared by dissolving 0.5 g of sodium carboxymethylcellulose (molecular weight of about 250 kDa and degree of substitution of 1.22; Sigma) in 9.5 g of DI water and mixing at 5 RPM in a conical tube for 16 hours. The two solutions were mixed in a 50 mL conical tube at a ratio of 1: 1 (w / w) and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a shelf overnight to evaluate the solution stability. The final solution was transparent, indicating that the polymer was miscible in the solution. The membrane was prepared using the method described in Example 9, except that a silicone mold was used. The dried film was visually hazy with a uniform distribution of haze throughout the film.

[0444] Example 56 (PVP and SEQ ID NO: 1007)

[0445] Polyvinyl pyrrolidone and collagen polymer solutions having the amino acid sequence shown in SEQ ID NO: 1007 were prepared, dried, and visually inspected. SEQ ID NO: 1007 has 98% identity with SEQ ID NO: 1. A 5 wt% solution containing a recombinant collagen fragment having the amino acid sequence shown in SEQ ID NO: 1007 was prepared by dissolving 0.5 g of the recombinant collagen fragment in 9.5 g of water. A 5 wt% 40 kDa PVP solution was prepared by dissolving 0.25 g of 40 kDa PVP in 4.75 g of water. The two solutions were mixed in a 50 mL conical tube at a 1: 1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a shelf overnight to evaluate the solution stability. The final solution was transparent. The membrane was prepared using the method described in Example 9, except that a silicone mold was used. The dried membrane was visually transparent, indicating that the polymer was miscible in solution and in the solid state.

[0446] Example 57 (Biphasic HA and SEQ ID NO: 1007)

[0447] A solution of a blend of biphasic cross-linked hyaluronic acid (comprising cross-linked HA and uncross-linked HA) and a recombinant collagen fragment having an amino acid sequence shown in SEQ ID NO: 1007 and a hydrolyzed recombinant collagen fragment was prepared and visually inspected. SEQ ID NO: 1007 has 98% identity to SEQ ID NO: 1. 2.4% (w / v) biphasic cross-linked HA with a particle size of 0.10-0.25 mm purchased from Shandong Runxin Biotechnology Co., Ltd. (Qufu City, Shandong Province, China) was used as polymer 1. A 5% by weight solution of a recombinant collagen fragment having an amino acid sequence shown in SEQ ID NO: 1007 was prepared by dissolving 0.5g of the recombinant collagen fragment in 9.5g of water. The two solutions were mixed in a 50mL conical tube at a ratio of 1: 1 (w / w) and vortexed for 3 minutes and mixed at 5RPM for 3 hours. The conical tube was placed vertically on a shelf overnight to evaluate solution stability. The solution was visually transparent. Additionally, the same conical tube was placed upright on a shelf in a 4°C refrigerator overnight and the solution remained transparent, indicating that the polymers were miscible in the solution.

[0448] Example 58 (Different ratios of HA:SEQ ID NO: 1)

[0449] Hyaluronic acid (HA) and collagen (recombinant collagen fragment with a molecular weight of 50kDa of SEQ ID NO: 1) mixture was prepared and dried, and visually inspected. 5% by weight 50kDaHA solution was prepared by dissolving 2.5g HA in 47.5g DI water. 5% (w / v) collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 2.5g recombinant collagen fragment in 47.5g water. The two solutions were mixed in a 50ml conical tube with HA: collagen ratios of 1: 9, 1: 3, 2: 3, 1: 1, 3: 2, 3: 1 and 9: 1 (w / w) and 10g total solution weight and mixed at 5RPM for 3 hours. The conical tube was placed vertically on a shelf overnight to assess solution stability. The final solution was transparent, indicating that all ratios were miscible in solution. Films were prepared using the same method as described in Example 9, except that a silicone mold was used. The dried film with a HA:collagen ratio of 1:9 was visually clear. The dried film with a HA:collagen ratio of 1:3 was visually clear to slightly turbid. The dried films of the remaining ratios were visually turbid with uniformly distributed turbidity throughout the film, indicating that the miscibility in the solid state is composition-dependent. In addition, aliquots of the HA:collagen solution mixtures of the seven ratios were placed in a 4°C refrigerator overnight, and the solutions remained clear.

[0450] Example 59 (HA: 5% hydroxylation)

[0451] A mixture of hyaluronic acid and a 5% hydroxylated recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 59 was prepared using the method described in Example 27, except that polymer 2 was replaced with 5.41% hydroxylated collagen (molecular weight 50 kDa). A 2 wt% 50 kDa HA solution (polymer 1) was prepared by dissolving 0.2 g HA in 9.8 g MQ water. The solid content of 5% hydroxylated collagen was 12% and the pH was about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and purified by centrifugation at 15,000 RPM for 10 min. The supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 15 ml Falcon tube at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was transparent, indicating that the polymers were miscible in the solution. The membranes were prepared using the method described in Example 9, except that they were performed in a silicon mold. The dried membranes were visually turbid.

[0452] Example 60 (HA: 12% hydroxylation)

[0453] A mixture of hyaluronic acid and a 12% hydroxylated recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 60 was prepared using the method described in Example 27, except that 12.47% hydroxylated collagen with a molecular weight of 50 kDa was used as polymer 2. A 2 wt% 50 kDa HA solution (polymer 1) was prepared by dissolving 0.2 g HA in 9.8 g MQ water. The solid content of 12% hydroxylated collagen was 3% and the pH was about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and purified by centrifugation at 15,000 RPM for 10 min. The supernatant of the hydroxylated collagen solution and the HA solution were in a 1:1 (w / w) ratio in a 15 ml Falcon tube and mixed at 10 RPM for 16 hours. The solution was transparent, indicating that the polymers were miscible in the solution. The membranes were prepared using the method described in Example 9, except that they were performed in a silicone mold. The dried membranes were visually turbid.

[0454] Example 61 (HA: 5% hydroxylation in HCl)

[0455] A mixture of hyaluronic acid and a 5% hydroxylated recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 61 was prepared using the method described in Example 27, except that 5.41% hydroxylated collagen with a molecular weight of 50 kDa was used as polymer 2. A 2 wt% 50 kDa HA solution (polymer 1) was prepared by dissolving 0.2 g HA in 9.8 g MQ water. The solid content of 5% hydroxylated collagen was 12% and the pH was about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and the pH was adjusted to about 2.2 with 12 N HCl. The collagen solution was purified by centrifugation at 15,000 RPM for 10 minutes. The supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 15 ml Falcon tube at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was turbid. The collagen and HA solutions were neutralized using 10N NaOH. The solution was transparent. The membranes were prepared using the method described in Example 9, except that they were performed in a silicon mold. The dried membranes were visually turbid.

[0456] Example 62 (HA: 12% hydroxylation in HCl)

[0457] A mixture of hyaluronic acid and a 12% hydroxylated recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared and visually inspected. The recombinant collagen fragment was hydroxylated by the method described in International Application No. WO2021 / 163485, and the hydroxylation percentage was measured by the method described in International Application No. WO2021 / 163485, the disclosure of which is incorporated herein in its entirety. Example 62 was prepared using the method described in Example 27, except that 12.47% hydroxylated collagen with a molecular weight of 50 kDa was used as polymer 2. A 2 wt% 50 kDa HA solution (polymer 1) was prepared by dissolving 0.2 g HA in 9.8 g MQ water. The solid content of 12% hydroxylated collagen was 3% and the pH was about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and the pH was adjusted to about 2.2 with 12N HCl. The collagen solution was purified by centrifugation at 15,000 RPM for 10 minutes. The supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 15 ml Falcon tube at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was turbid. The collagen and HA solutions were neutralized using 10N NaOH. The solution was transparent. The membranes were prepared using the method described in Example 9, except that they were performed in a silicon mold. The dried membranes were visually turbid.

[0458] Example 63 (SEQ ID NO: 1 Foam Preparation)

[0459] A stock solution of collagen (SEQ ID NO: 1) in water was prepared at a weight concentration of 16.7% (w / w). Baymedix FD103 and AD111 is each a polyurethane aqueous dispersion. Based on the weight of the polyurethane dispersion, a stock solution of Baymedix (Bayer MaterialScience, Pittsburgh, PA) FD103:AD111 is prepared at a ratio of 70:30. A stock solution of Rheolate 208 (Elementis, London, UK) in water is prepared at a weight concentration of 10% (w / w). 9.16g of recombinant collagen stock solution and 11.50g of Baymedix FD103:AD111 stock solution are mixed in a 50mL beaker with an overhead mixer. Afterwards, 1.55g of Rheolate 208 stock solution is added and mixed. Then 0.72g of ChemTex (Cumberland, RI) 2216, 0.65g of ChemTex 2317 and 0.03g of ChemTex 2243 are added and the resulting mixture is further mixed. The resulting mixture is mechanically foamed by vigorous mixing with an overhead mixer. After foaming, the foamed mixture was coated on a glass plate with a gap setting of 600 μm. The coating was dried at 75° C. for 30 minutes, and a porous foam sheet was obtained after drying.

[0460] Example 64 (Collagen Release Kinetics)

[0461] The release kinetics of solutions (A) recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and (B) a formulation of recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid were studied. Fig.11A , Fig. 11B , Fig. 12A and Fig. 12B Shown in.

[0462] analyze

[0463] Standard Curve: Average the triplicate absorbance values ​​obtained from each sample and dilution and subtract them from the blank (1X PBS + 0.5% Sodium Benzoate) value to obtain the mean absorbance value (MAV) from collagen alone in each sample / dilution. Plot the MAVs of the serial dilutions from 10 mg / mL to 0.5 mg / mL against their corresponding concentrations. Do the same for the 0.08-0.02 mg / mL dilutions. Draw a linear trend line for the 0.08-0.02 mg / mL range. Obtain the equation y = 0.7033x - 0.0131, R 2 The value is 0.9936. This equation was used to calculate the protein concentration of the control (A) and formulation (B) test samples from their corresponding MAVs ( Fig.11A The collagen mass of each sample was calculated by multiplying the total volume of solution present outside during the diffusion experiment (90 mL, 89.8 mL, 89.6 mL, or 89.4 mL).

[0464] The concentration was plotted against the time point to obtain the release trend. Finally, two such trends were obtained, one for the solution (A) and the other for the formulation (B). Fig. 11B ). As shown, the protein is gradually released from solution A and formulation B containing HA. The rate of protein release from formulation B appears to be slightly slower than the rate of release from the solution without HA. This suggests that there is a weak but measurable interaction between HA and collagen, probably because the solutions are miscible.

[0465] Micro BCA assay

[0466] The Micro-BCA™ Protein Assay Kit protocol is referenced for analysis using the Micro-BCA assay.

[0467] analyze

[0468] The triplicate absorbance values ​​obtained from each sample and dilution were averaged and subtracted from the blank (1X PBS + 0.5% sodium benzoate) value to obtain the mean absorbance value (MAV) from the collagen alone in each sample / dilution. The standard deviation and error were calculated for each value.

[0469] The MAV of the serial dilutions from 10 mg / mL to 0.5 mg / mL were plotted against their corresponding concentrations. The same procedure was performed for the dilutions from 0.08 to 0.02 mg / mL. A linear trend line was drawn for the range of 0.08 to 0.02 mg / mL. The equation y = 13.312x + 0.0692, R 2 The value is 0.9918. This equation was used to calculate the protein concentration of the control (A) and formulation (B) test samples from their corresponding MAVs ( Fig. 12A The mass of collagen for each sample was calculated by multiplying the total volume of solution present outside during the diffusion experiment (90 mL, 89.8 mL, 89.6 mL, or 89.4 mL). The concentration was plotted against the time point to obtain a trend in the amount of protein released from the solution / formulation. Ultimately two such trends were obtained, one for the solution (A) and one for the formulation (B) ( Fig. 12B ). As shown, the protein diffuses gradually from the solution (A) and formulation (B). The protein appears to diffuse more slowly from formulation (B), indicating an attractive force between HA and collagen.

[0470] Example 65 (Fluorescent labeling for collagen release kinetics)

[0471] The release kinetics of the control solution (A) recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and the formulation (B) recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid were fluorescently labeled to display the release kinetics data. Fig.13A and Fig. 13B Shown in.

[0472] analyze

[0473] All dilutions from the standard curves and time points within (A) and (B) were pipetted into a 96-well plate. Three technical replicates of 100 μL each were pipetted. Fluorescence intensity was measured by setting the excitation and emission wavelengths to 680 nm and 702 nm.

[0474] Standard curve: The three intensity values ​​obtained from each sample and dilution were averaged to obtain the mean intensity value (MIV). The MIV of the serial dilutions of 0.0625 mg / mL, 0.03125 mg / mL, 0.015625 mg / mL, 0.0078125 mg / mL, 0.00390625 mg / mL were plotted against their corresponding concentrations. A linear trend line was drawn ( Fig.13A ). The equation y = 72137x + 253.72 was obtained with an R2 value of 0.9967. This equation was used to calculate the protein concentration of the test samples of solution (A) and formulation (B) from their corresponding MIV. The unlabeled collagen concentration was calculated by multiplying the labeled concentration value by 50. These concentrations were plotted against time points to give a trend in the amount of protein retained inside the solution / formulation ( Fig. 13B ).like Fig. 13B As shown, collagen diffuses at a gradual rate from solution (A) and formulation (B). Collagen appears to diffuse more slowly from the formulation containing HA (formulation (B)), indicating an attractive force between HA and collagen.

[0475] Example 66 (Collagen Release Kinetics / Cross-linked HA)

[0476] The release kinetics of solutions (A) 1% solution of recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and (B) a formulation of recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and cross-linked hyaluronic acid were studied. Fig.14A and Fig. 14B Shown in.

[0477] analyze

[0478] The triplicate absorbance values ​​obtained from each sample and dilution were averaged and subtracted from the blank (1X PBS+ value to obtain the mean absorbance value (MAV) from the collagen alone in each sample / dilution. The standard deviation and error were calculated for each value.

[0479] The MAVs of the serial dilutions from 0.2 mg / mL to 0.01 mg / mL were plotted against their corresponding concentrations. A linear trend line was drawn for the 0.2-0.01 mg / mL range ( Fig.14A ). The equation y = 13.947x was obtained, and the R2 value was 0.9921. The trend line was highly consistent with the expectation and was suitable for converting the absorbance values ​​into concentrations for diffusion experiments.

[0480] This equation was used to calculate the collagen concentration of the control (A) and formulation (B) test samples from their corresponding MAVs. The collagen mass of each sample was calculated by multiplying by the total solution volume present outside during the diffusion experiment (95 mL, 94 mL, 93 mL, or 92 mL).

[0481] The concentration was plotted against the time point to obtain the trend of the amount of collagen released from the solution / formulation ( Fig. 14B Finally, two such trends were obtained, one for the collagen solution (A) and the other for the collagen / HA preparation (B). Figure 7 As shown in Figure B, the rate of collagen release from the control solution (A) is similar to the previous control example, however, the release of collagen from the cross-linked HA / collagen formulation (B) is much slower. For example, the amount of protein released from formulation (B) in 8 days only requires 3 days on average to diffuse from the control solution (A). After 19 days, collagen is completely released from formulation (B).

[0482] Embodiment 67

[0483] A polyethylene glycol (PEG) and collagen mixture was prepared and dried and visually inspected. Example 67 was prepared using the method described in Example 7, except that polyethylene glycol (molecular weight 8 kDa; Sigma) was used instead of polymer 1. The final solution was visually clear, indicating that the two polymers were miscible in solution. The membrane was prepared using the method described in Example 9. The dried membrane separated into two phases, with small broken pieces of brittle and opaque material surrounded by a circle of brown and transparent material.

[0484] Embodiment 68

[0485] Hyaluronic acid and collagen mixtures were prepared and visually inspected. Example 68 was prepared using the method described in Example 20, except that the hyaluronic acid and collagen mixture from Geltor was used. The powder was substituted for polymer 1. A 2 wt% 50 kDa HA solution was prepared using the method described in Example 20. The two solutions were mixed together in a 1:1 (w / w) ratio in a 15 ml conical tube and mixed on a stir plate at 10 RPM for 16 hours. The final solution was visually clear. Films were prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was clear with a milky white ring along the edge of the film, which was likely the preservative.

[0486] This example demonstrates that The powder and the solution of 50 kDa HA were miscible.

[0487] Embodiment 69

[0488] A series of in vivo experiments were carried out using the chicken embryo chorioallantoic membrane (CAM) assay. T-HEp3 cells were inoculated on the CAM with type I, type III or type IV collagen or with Dulbecco's phosphate buffered saline (DPBS) (as vehicle control). For tumor cell growth, T-HEp3 cells co-injected with recombinant collagen fragments and vehicle control were observed. In nude mice, T-HEp3 cells were injected into the right flank and T-HEp3 cells were injected contralaterally with type III collagen. Tumor growth was observed.

[0489] Mice were orthotopically injected with 4T1 or D2A1 cells and co-injected with recombinant collagen fragments. Phosphorylated histone H3 protein levels were observed to determine cell proliferation. Nuclear dormancy markers were observed, and live cell imaging of T-HEp3 cells expressing the CDK2 cell cycle sensor was performed to determine the induction of dormancy.

[0490] It should be understood that the detailed description section, rather than the summary section and the abstract section, is intended to be used to interpret the claims. The summary section and the abstract section may set forth one or more but not all exemplary embodiments of the invention contemplated by the inventors, and are therefore not intended to limit the invention and the appended claims in any way.

Claims

1. A method for treating cancer in a subject in need thereof, the method comprising contacting the surface of cells of the subject with an effective amount of a recombinant collagen fragment, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The method of claim 1, wherein the recombinant collagen fragment is non-hydroxylated. The method according to claim 1 , wherein the recombinant collagen fragment is hydroxylated. The method according to claim 1 , wherein the collagen fragment has an amino acid sequence shown in SEQ ID NO:

1. The method of claim 4 , wherein the recombinant collagen fragment is non-hydroxylated. The method of claim 4 , wherein the recombinant collagen fragment is hydroxylated.

7. A method for treating cancer in a subject in need thereof, the method comprising contacting a cell surface of the subject with an effective amount of a recombinant collagen fragment having an amino acid sequence shown in any one of SEQ ID NOs: 2-972.

8. The method of any one of claims 1 to 7, wherein contacting the cell surface with the fragment induces cell quiescence.

9. The method of any one of claims 1 to 8, wherein the cancer is characterized by the presence of solid tumors.

10. The method of claim 9, wherein the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the foregoing cancers.

11. The method of claim 9 or 10, wherein the subject has not undergone resection of a tumor.

12. The method of claim 9 or 10, wherein the subject has undergone resection of a primary tumor.

13. The method of claim 13, wherein the cell surface of the subject comprises residual cancer cells after resection of the primary tumor.

14. The method of any one of claims 9 to 13, wherein contacting the cell surface with the fragment maintains cell quiescence.

15. A method for inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

16. The method of claim 15, wherein the recombinant collagen fragment is non-hydroxylated.

17. The method of claim 15, wherein the recombinant collagen fragment is hydroxylated. The method according to claim 15 , wherein the collagen fragment has the amino acid sequence shown in SEQ ID NO:

1.

19. The method of claim 18, wherein the recombinant collagen fragment is non-hydroxylated.

20. The method of claim 18, wherein the recombinant collagen fragment is hydroxylated.

21. A method for inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having an amino acid sequence shown in any one of SEQ ID NOs: 2-972.

22. The method according to any one of claims 1 to 21, wherein the fragment is formulated in a pharmaceutically acceptable composition.

23. The method of any one of claims 1 to 21, wherein the fragment is formulated in a therapeutic biomaterial.

24. The method of claim 23, wherein the therapeutic biomaterial is a protein polyurethane alloy.

25. The method of claim 24, wherein the protein is dissolved in the polyurethane.

26. The method of claim 24 or 25, wherein when the alloy is exposed to water or a buffer solution, the protein migrates out of the alloy to contact the cell surface.

27. The method of any one of claims 23 to 26, wherein the alloy comprises from about 10 wt% to about 50 wt% of the fragment and from about 50 wt% to about 90 wt% of the polyurethane.

28. The method of any one of claims 23 to 26, wherein the alloy comprises about 20 wt% to about 35 wt% of the protein and about 65 wt% to about 80 wt% of the polyurethane.

29. The method of any one of claims 23 to 28, wherein the alloy is free or substantially free of particles of the recombinant collagen fragments having an average diameter greater than 1 micron.

30. A therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

31. The biomaterial of claim 30, wherein the recombinant collagen fragment is non-hydroxylated.

32. The biomaterial of claim 30, wherein the recombinant collagen fragment is hydroxylated.

33. The biomaterial according to claim 30, wherein the collagen fragment has the amino acid sequence shown in SEQ ID NO:

1.

34. The biomaterial of claim 33, wherein the recombinant collagen fragment is non-hydroxylated.

35. The biomaterial of claim 33, wherein the recombinant collagen fragment is hydroxylated.

36. A therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has a sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-972 or the amino acid sequence shown in any one of SEQ ID NOs: 974-1002.

37. A therapeutic biomaterial comprising a recombinant collagen fragment and a pharmaceutically acceptable scaffold, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and the therapeutic biomaterial is used to treat cancer in a subject in need thereof.

38. The biomaterial for use according to claim 37, wherein the cancer is characterized by the presence of solid tumors.

39. The biomaterial for use according to claim 37 or 38, wherein the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer or any combination of the above cancers.

40. The biomaterial according to any one of claims 37 to 39, wherein the biomaterial is a protein polyurethane alloy.

41. The biomaterial of claim 40, wherein the protein is dissolved in the polyurethane.

42. The biomaterial of claim 40 or 41, wherein when the alloy is exposed to water or a buffer solution, the protein migrates out of the alloy to contact the cell surface.

43. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of hyaluronic acid and the recombinant collagen fragment.

44. The biomaterial of claim 43, wherein the recombinant collagen fragments are mixed with the hyaluronic acid.

45. The biomaterial of claim 44, wherein the hyaluronic acid scaffold is cross-linked.

46. ​​The biomaterial according to claim 44, wherein the hyaluronic acid is a biphasic cross-linked hyaluronic acid comprising cross-linked HA and uncross-linked HA.

47. The biomaterial of any one of claims 37 to 46, wherein when the alloy is exposed to water or a buffer solution, the recombinant collagen fragments migrate out of the pharmaceutically acceptable scaffold to contact the cell surface.

48. The biomaterial according to any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of polyvinyl pyrrolidone and the recombinant collagen fragment.

49. The biomaterial according to any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of polyacrylamide and the recombinant collagen fragment.

50. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of poly(ethylene oxide) and the recombinant collagen fragment.

51. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of poly(2-oxazoline) and the recombinant collagen fragment.

52. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of polyethyleneimine and the recombinant collagen fragment.

53. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of carboxymethyl cellulose and the recombinant collagen fragment.

54. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of chondroitin sulfate and the recombinant collagen fragment.

55. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of acetylated hyaluronate and the recombinant collagen fragment.

56. The biomaterial of any one of claims 30 to 39, wherein the pharmaceutically acceptable scaffold comprises a mixture of zinc hyaluronate and the recombinant collagen fragment.

57. The biomaterial of any one of claims 30 to 56, wherein the biomaterial is directly attached to the cancer cell.

58. Use of a biomaterial according to any one of claims 30 to 57 for treating wounds resulting from the excision of a primary tumour.

59. Use of a biomaterial according to any one of claims 30 to 57 for maintaining cell dormancy.

60. A method for treating cancer in a subject in need thereof, the method comprising contacting the surface of cells of the subject with a therapeutic biomaterial comprising an effective amount of a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

61. A method for inhibiting the proliferation of cancer cells, the method comprising contacting the cancer cells with a therapeutic biomaterial comprising a recombinant collagen fragment, wherein the recombinant collagen fragment has the amino acid sequence shown in SEQ ID NO:

973.

62. A method of inducing cell dormancy in a subject in need thereof, the method comprising contacting a cell surface of the subject with a therapeutic biomaterial comprising recombinant collagen having the amino acid sequence shown in SEQ ID NO:

973.

63. The method of any one of claims 60 to 62, wherein the recombinant collagen is non-hydroxylated.

64. The method of any one of claims 60 to 62, wherein the recombinant collagen is hydroxylated.

65. The method of any one of claims 60 to 64, wherein the cancer is characterized by the presence of solid tumors.

66. The method of claim 65, wherein the cancer is skin cancer, colon cancer, oral mucosal cancer, rectal cancer, esophageal cancer, thyroid cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, lung cancer, brain cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer, bone cancer, or a combination of any of the foregoing cancers.

67. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial is a protein polyurethane alloy.

68. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises polyurethane, hyaluronic acid, or gelatin.

69. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises polyvinylpyrrolidone.

70. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises polyacrylamide.

71. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises poly(ethylene oxide).

72. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises poly(2-oxazoline).

73. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises polyethyleneimine.

74. The method of any one of claims 60 to 69, wherein the therapeutic biomaterial comprises carboxymethylcellulose.

75. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises chondroitin sulfate.

76. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises acetylated hyaluronate.

77. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises acetylated hyaluronate.

78. The method of any one of claims 60 to 66, wherein the therapeutic biomaterial comprises zinc hyaluronate.

79. The method of any one of claims 60 to 78, wherein the therapeutic biomaterial is directly attached to the cancer cell.

80. The method of any one of claims 1 to 29 and 60 to 79, wherein the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5.

81. The biomaterial of any one of claims 30 to 57, wherein the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5.

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