Composition and methods for a matrix filler

Abstract

The current invention involves a method of mixing POP with a polysaccharide to obtain an improved injectable tissue matrix. POP is a partially ordered polypeptide and a polysaccharide, in this instance, comprises at least one glycosaminoglycan. The invention involves mixing these compounds in an injectable tissue matrix to increase volume retention, vascularization, and remodeling immune response, all of which are characteristics that would be useful for an injectable dermal filler, and in plastic and reconstructive procedures.

Description

COMPOSITION AND METHODS FOR A MATRIX FILLERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 674,573, filed on July 23, 2024, the content of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 2304430 awarded by the National Science Foundation. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (INSO_005_01WO_SeqList_ST26.xml; Size: 156,705 bytes; and Date of Creation: July 18, 2025) are herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The present disclosure relates to the field of plastic and reconstructive procedures, in particular facial injections.BACKGROUND

[0005] Existing dermal fillers made of synthetic polymers, including hyaluronic acid-based products (e.g., Juvederm®), have several limitations as they are designed to be temporary, resorbable and do not integrate into surrounding tissue. They are, therefore, not recommended for use in reconstructive procedures such as fat grating, a process of rebuilding tissue using liposuction fat, but are instead primarily used by surgeons and consumers for facial injections. Given the growing popularity of routine facial injections, there is a need to improve upon commonly used dermal filler products to improve their longevity as well as to expand the potential use of these products in plastic and reconstructive procedures.SUMMARY

[0006] The present disclosure relates generally to injectable tissue matrices.

[0007] According to an embodiment, the present disclosure further relates to a matrix filler comprising a partially ordered polypeptide and a polysaccharide, wherein the partially orderedpolypeptide and the polysaccharide are present in a ratio of about 95:5 to about 5:95 (partially ordered polypeptide:polysaccharide). In another embodiment, the partially ordered polypeptide comprises a plurality of disordered domains and a plurality of ordered domains. In another embodiment, each ordered domain independently comprises a polyalanine motif or a polyproline motif. In another embodiment, each ordered domain independently comprises a polyalanine motif. In another embodiment, each ordered domain independently comprises an alpha helix. In another embodiment, the polyalanine motif comprises (A)m(SEQ ID NO: 29), wherein m is an integer from 5 to 50. In another embodiment, the polyalanine motif comprises one or more of: (A)n(SEQ ID NO: 3); K(A)nK (SEQ ID NO: 4), D(A)nK (SEQ ID NO: 5), GD(An)K (SEQ ID NO: 6), or GK(An)K (SEQ ID NO: 7), wherein n is an integer from 2 to 100. In another embodiment, the polyalanine motif comprises one or more of: (A)25 (SEQ ID NO: 8), K(A)25K (SEQ ID NO: 9), D(A)25K (SEQ ID NO: 10), GD(A25)K (SEQ ID NO: 11), or GK(A2S)K (SEQ ID NO: 12). In another embodiment, each disordered domain independently comprises a PG or GP motif. In another embodiment, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1. In another embodiment, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 2), wherein each X is independently Vai or Ala, and wherein n is an integer from 1 to 50. In another embodiment, a ratio of Ala to Vai in the amino acid sequence of (GXGVP)n (e.g., SEQ ID NO: 27 or SEQ ID NO: 28) ranges from 10: 1 to 1 : 10. In another embodiment, the polysaccharide is hydrophilic. In another embodiment, the polysaccharide comprises at least one glycosaminoglycan. In another embodiment, the at least one glycosaminoglycan comprises one or more residue of a heparin sulfate, a chondroitin sulfate, a dermatan sulfate, and a hyaluronic acid. In another embodiment, the at least one glycosaminoglycan comprises hyaluronic acid. In another embodiment, the hyaluronic acid is crosslinked. In another embodiment, the hyaluronic acid is a crosslinked hyaluronic acid present in an amount of about 1 mg / mL to about 500 mg / ml. In another embodiment, the crosslinked hyaluronic acid is present in an amount of about 1 mg / mL to about 150 mg / ml. In another embodiment, the partially ordered polypeptide and the hyaluronic acid are present in a ratio of 95:5, 90: 10, 70:30, 50:50, 30:70, 10:90, or 5:95 (partially ordered polypeptide:hyaluronic acid).

[0008] In another embodiment, the partially ordered polypeptide has a transition temperature of heating (Tt-heating) and a transition temperature of cooling (Tt-cooling). In another embodiment, the Tt-heating is dependent on the concentration of the partially ordered polypeptide in the composition. In another embodiment, the Tt-heating and the Tt-coolingrange from about 10 °C to about 45 °C. In another embodiment, the partially ordered polypeptide forms a solid aggregate above the Tt-heating. In another embodiment, the solid aggregate resolubilizes when cooled to below the Tt-cooling. In another embodiment, the solid aggregate is a stable three-dimensional matrix including the polysaccharide. In another embodiment, the solid aggregate comprises a plurality of micropores. In another embodiment, the composition comprises between about 200 pM and about 2.5 mM of the partially ordered polypeptide. In another embodiment, the composition is a liquid or semisolid. In another embodiment, the composition is shaped. In another embodiment, the composition further comprises a surfactant, stabilizer, buffer, linker, non-steroidal anti-inflammatory drug, or anesthetic. In another embodiment, the anesthetic is lidocaine.

[0009] According to an embodiment, the present disclosure further relates to a method of forming a matrix filler composition in a subject, the method comprising: mixing an amount of a partially ordered polypeptide and an amount of polysaccharide to form a composition, thereby forming a matrix filler composition, and administering an amount of the matrix filler composition to the subject. In another embodiment, the matrix filler composition is below the Tt-heating of the partially ordered polypeptide and the matrix filler composition forms a solid after exposure to the subject’s body temperature, which is above the Tt-heating of the partially ordered polypeptide. In another embodiment, the matrix filler composition is administered to a subject when the mixture is above the Tt-heating of the partially ordered polypeptide and the mixture forms a solid prior to administration to the subject.

[0010] According to an embodiment, the present disclosure further relates to a method of forming an autologous fat graft in a subject, the method comprising mixing an amount of a partially ordered polypeptide and an amount of polysaccharide to form a composition, thereby forming a matrix filler composition, and administering to the subject an amount of the composition. In another embodiment, the method further comprises mixing an amount of an adipose tissue with the matrix filler composition prior to administering the amount of the matrix filler composition to the subject. In another embodiment, the adipose tissue comprises an autologous fat. In another embodiment, the partially ordered polypeptide and the polysaccharide in vitro to form the matrix filler composition and implanting the mixture in the subject. In another embodiment, the method comprises adapting the mixture of the partially ordered polypeptide and the polysaccharide to a shape of an anatomical feature, then heating the mixture to the Tt-heating, to form a solid matrix filler composition. In another embodiment, the method comprises mixing the partially ordered polypeptide, the polysaccharide, and the adipose tissue in vitro to form a matrix filler composition and implanting the mixture in thesubject. In another embodiment, the adipose tissue comprises an autologous fat. In another embodiment, the method comprises administering to the matrix filler composition when the matrix filler composition is below the Tt-heating of the partially ordered polypeptide, wherein the matrix filler composition forms a solid after exposure to the subject’s body temperature, which is above the Tt-heating of the partially ordered polypeptide. In another embodiment, the method comprises heating the matrix filler composition above the Tt-heating of the partially ordered polypeptide in vitro to form a solid matrix filler composition, then implanting the solid matrix filler composition in the subject. In another embodiment, the administering comprises injecting the amount of the composition into the subject.

[0011] According to an embodiment, the present disclosure further relates to use of an amount of a partially ordered polypeptide and an amount of polysaccharide as a tissue matrix filler in a subject in need thereof.

[0012] According to an embodiment, the present disclosure further relates to a matrix composition, comprising a polypeptide comprising a plurality of disordered domains and a plurality of ordered domains, wherein each disordered domain independently comprises a PG or GP motif and each ordered domain independently comprises an alpha helix, and a polysaccharide comprising a crosslinked hyaluronic acid present at a molecular weight of about 1 kDa to about 3,000 kDa, wherein about 1 mg / mL to about 150 mg / mL of the matrix filler composition is administered to the subject and wherein the matrix composition augments a shape and / or volume of an anatomic region of the subject.DESCRIPTION OF THE DRAWINGS

[0013] FIG 1. is a flow diagram of a method of composing a tissue matrix filler to be administered to a subject to augment native tissue.

[0014] FIG 2. is a flow diagram of a process of preparing a composition comprising at least a partially ordered polypeptide (POP) solution and a polysaccharide solution.

[0015] FIG 3. shows images of the material products resulting from mixtures of 0 mM, 0.75 mM and 1.5 mM POP (or Fractomer) with a polysaccharide, which consisted of either a photocrosslinked hyaluronic acid (MeHA), an uncross-linked hyaluronic acid (HA) or a chondroitin sulfate (CS). Control samples of HA and CS are not shown since they were either entirely liquid or a viscous liquid without the inclusion of Fractomer.

[0016] FIG. 4 shows the effects of mixing Fractomer with HA and CS on the mechanical stiffness of the produced material.

[0017] FIGs. 5A-5D show scanning electron microscopy (SEM) images of (Fig. 5A) 0.75 mM Fractom er with 3% MeHA, (Fig. 5B) 1.5 mM Fractom er with 3% MeHA, (Fig. 5C) 0.75 mM Fractomer with un-crosslinked HA, and (Fig. 5D) 1.5 mM Fractomer with 3% Chondroitin Sulfate (CS). All samples were gold sputter coated for 400 s prior to imaging using the FEI Apreo SEM instrument located at Duke University.

[0018] FIGs. 6A-6D show the effects of injecting BL / 6 mice (n=5) with mixtures comprised of Fractomer and a hyaluronic acid (HA) at various ratios. Fig. 6A shows the volume retention at the injection site for each FractomerHA ratio after 9 weeks. Fig. 6B shows the variance in volume retention across each FractomerHA ratio over 9 weeks. Fig. 6C shows images of the injection site for select FractomerHA ratios at day 1 and after 9 weeks. Fig 6D shows histological H & E staining analysis of cell infiltration and immune response at injection sites for select Fractom er: HA ratios.

[0019] FIGs. 7A-7C compares cellular ingrowth of injections sites from BL / 6 mice injected with either 100% Fractomer, 50:50 FractomerHA or 100% HA. Fig. 7A compares vascular ingrowth measured from randomly identified regions of interest between Fractomer alone and HA alone injection sites (n=30). * for p<0.05 using Student’s T-test. Fig. 7B shows the total cell infiltration in the different injection groups measured by a cell counting algorithm in QuPath. Healthy subcutaneous fat was also quantified for comparison. Fig. 7C shows histological H & E staining analysis of cell infiltration and vascular growth (indicated by circles) for select injection groups.DETAILED DESCRIPTIONDefinitions

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0021] As used herein, the term "a," "an," "the" and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singularand plural unless otherwise indicated herein or clearly contradicted by the context. In addition, "a," "an," or "the" means "one or more" unless otherwise specified.

[0022] As used herein, the term "about" or "approximately" as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one embodiment, the term "about" refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term "about."

[0023] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 ... 2.0. If the end points are modified by the term "about," the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

[0024] As used herein, the terms "control," or "reference" are used herein interchangeably. A "reference" or "control" level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. "Control" also refers to control experiments or control cells.

[0025] As used herein, the terms "effective amount" or "therapeutically effective amount," refers to a substantially non-toxic, but sufficient amount of an agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0026] As used herein, the term "subject" refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non- human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0027] " Amino acid" as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0028] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tall domains, "Domains" are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. "Tertiary structure" refers to the complete three- dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three- dimensional structure formed by the noncovalent association of independent tertiary units. A "motif is a portion of a polypeptide sequence and includes at least two amino acids. A domain may be comprised of a series of motifs, which may be similar or different.

[0029] "Recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

[0030] "Sample" or "test sample" as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising an agent, cell, or partially ordered polypeptide (POP) as described herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile,digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid.

[0031] A "variant" can be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a substantially identical sequence. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity.

[0032] A variant can be a polypeptide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The polypeptide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0033] As used herein, "augmented" refers to the improvement of volume, shape, survival, improved blood supply, and reduction in necrosis of autologous adipose tissue or fat grafts. In some embodiments, an autologous adipose tissue or fat graft is augmented by the combination of the adipose tissue with a composition comprising a partially ordered polypeptide which leads to the enhanced long-term maintenance and survival of fat graft including conservation of the volume and shape of the fat graft, improved vascularization, enhanced survival, and reduction in necrosis.

[0034] As used herein, "shapeable" refers to the ability of the POP and composition discussed herein to be shaped or molded into various two- or three-dimensional shapes, areas, or volumes and the ability to maintain this shape, area, or volume over a prolonged period of time. Specific exemplary shapes or volumes include lips, cheeks, breasts, buttocks, hands, knees, 2- dimensional layers for placing under skin or skin grafts, or other irregular or indefinite shapes or volumes present in a subject's body.Matrix Filler Composition

[0035] Embodiments described herein are directed to an injectable or implantable matrix filler composition to augment tissue fillers. In embodiments, the composition comprises a recombinant partially ordered polypeptide (POP) or "Fractomer" and a polysaccharide. The polysaccharide is combined with POP to provide various polysaccharide:POP ratios as described herein to provide a matrix composition. As an example, 900 pL of polysaccharide combined with 100 pL of POP solution would be a 9: 1 ratio by volume or 90% polysaccharide by volume. In embodiments, the matrix composition improves the properties of the matrix composition compared to injections of the polysaccharide alone, including, but not limited to volume and / or shape.Partially ordered polypeptide

[0036] The term "Fractomer", “partially ordered polypeptide” and “POP” are used interchangeably herein to refer to the class of recombinant, artificial proteins that are designed to mimic native elastin. POP are thermally responsive, allowing them to be injected as a liquid, yet rapidly form a porous, solid network at body temperature. Alternatively, a POP can be prepared as a liquid, formed into a desired shape, and then heated to form a solid which is then implanted. Examples of POPs are described in International Patent Application Publication No. W02019006374A1, which is incorporated by reference herein in its entirety.

[0037] Each POP may include a plurality of disordered domains, and a plurality of structured domains. The POP may exhibit phase transition behavior by changing solubility and aggregate dissolution / formation with temperature.

[0038] The POP may include a plurality of disordered domains. The disordered domain may comprise any polypeptide that has minimal or no secondary structure as observed by CD and have phase transition behavior. The disordered domain may include an amino acid sequence of repeated amino acids, non-repeated amino acids, or a combination thereof.

[0039] The disordered domains and the structured domains of the POP can be arranged in any number of possible ways, in some embodiments, one or more disordered domains are positioned between at least two adjacent structured domains of the POP. In some embodiments, the POP includes a plurality of structured domains repeated in tandem and a plurality of disordered domains repeated in tandem, in some embodiments, the plurality of structured domains repeated in tandem are positioned C-terminal to the plurality of disordered domains repeated in tandem, in some embodiments, the plurality of structured domains repeated in tandem are positioned N-terminal to the plurality of disordered domains repeated in tandem. In some embodiments, the POP is arranged as [disordered domain]q- [structured domain]r -[disordered domain]s-[structured domain]t, wherein q, r, s, and t are independently an integer from 0 to 100, such as from 1 to 100, from 2 to 100, from 1 to 50 or from 2 to 50. In some embodiments, the POP is arranged as [disordered domain]q-[structured domain]r, wherein q and r are independently an integer from 1 to 100. in some embodiments, q, r, s, and t are independently an integer from 0 to 10, from 0 to 20, from 0 to 30, from 0 to 40, from 0 to 50, from 0 to 60, from 0 to 70, from 0 to 80, from 0 to 90, from 0 to 100, from 1 to 10, from 1 to 20, from 1 to 30, from 1 to 40, from 1 to 150, from 1 to 60, from 1 to 70, from 1 to 80, from 1 to 90 or from 1 to 100.

[0040] In some embodiments, the disordered domain independently comprises a PG or GP motif In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 2), wherein each X is independently Vai or Ala, and wherein n is an integer from 1 to 50. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (e.g., SEQ ID NO: 27 or SEQ ID NO: 28), wherein each X is independently Vai or Ala, and wherein n is an integer from 1 to 50, wherein a ratio of Ala to Vai in the amino acid sequence of (GXGVP)n ranges from 10:1 to 1 : 10. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (e.g., SEQ ID NO: 27 or SEQ ID NO: 28), wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif, wherein the polyalanine motif comprises (A)m(SEQ ID NO: 29) wherein m is an integer from 5 to 50. In some embodiments, the polyalanine motif comprises one or more of (A)n (SEQ ID NO: 3), K(A)nK (SEQ ID NO: 4), D(A)nK (SEQ ID NO: 5), GD(An)K (SEQ ID NO: 6), or GK(An)K (SEQ ID NO: 7), wherein n is an integer from 2 to 100. In some embodiments, the polyalanine motif comprises one or more of (A)25(SEQ ID NO: 8), K(A)25K (SEQ ID NO: 9), D(A)25K (SEQ ID NO: 10), GD(A25)K (SEQ ID NO: 11); or GK(A25)K (SEQ ID NO: 12).

[0041] A POP comprising structured domain of oligoalanine amino acids (from 5 to 500, but typically A2s) that form a-helices and are periodically inserted into an unstructured elastin-like polypeptide (ELP) that is composed of typically 80-120 total repeats of a (GXGVP)n pentapeptide motif (-30-50 kDa) (SEQ ID NO: 1), where X is any standard amino acid except proline. In some embodiments, the unstructured polypeptide or “disordered domain” is a (GXGVP)n motif (SEQ ID NO: 2), wherein X is Vai (SEQ ID NO: 25), or Ala (SEQ ID NO: 26), or mixture of Ala and Vai, as in SEQ ID NOS: 27 and 28, and wherein n isan integer from 1 to 50. In some embodiments, X is an alternating iteration of Ala and Vai in a ratio from 10: 1 to 1 : 10 (Ala: Vai). In some embodiments, X is an alternating iteration of Ala and Vai in a ratio of 1 : 1 (SEQ ID NO: 27) or 1 :4 (SEQ ID NO: 28). Fractomers are recombinantly synthesized in E. coli by overexpression of a plasmid-borne gene that encodes the Fractomer.

[0042] In some embodiments, about 20% to about 99%, such as about 25% to about 97%, about 35% to about 95% or about 50% to about 94% of the POP comprises disordered domains. At least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the POP may comprise disordered domains.

[0043] In some embodiments, the disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid and n is an integer greater than or equal to 1. In some embodiments, mis an integer from 1 to 500. In some embodiments, is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325,330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420,425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500. In some embodiments, may be less than 500, less than 400, less than 300, less than 200, or less than 100. In some embodiments, mis from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, or from 60 to 180. In some embodiments, m is 60, 120, or 180. In some embodiments, X is any amino acid except praline. In some embodiments, X is Vai, or Ala, or an alternating iteration of Ala and Vai. In some embodiments, X is Vai. In some embodiments, X is Ala. in some embodiments, X is an alternating iteration of Ala and Vai. In some embodiments, X is an alternating iteration of Ala and Vai in a ratio of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 2: 1, 3: 1, 4:1, 5: 1, 6: 1, 7:1, 8: 1, 9: 1, or 10: 1. In some embodiments, X is a mixture of Ala and Vai in a ratio of 1 :1 or 1 :4. In some embodiments, X is an alternating iteration of Ala and Vai in a ratio from 10: 1 to 1 : 10 (Ala:Val), such as from 5: 1 to 1 :5 or from 1 : 1 to 1 :4.Structured Domains

[0044] The POP may include a plurality of structured domains. The structured domain may have a secondary structure as observed by CD, such as, for example, an alpha helix. The structured domain may comprise at least one of a polyproline domain and a polyalanine domain, in some embodiments, the POP comprises alternating disordered domains andstructured domains. In some embodiments, the structured domain comprises only polyalanine domains. In some embodiments, the structured domain comprises only polyproline domains.

[0045] In some embodiments, about 4% to about 75%, such as about 5% to about 70%, about 6% to about 60% or about 7% to about 50% of the POP comprises structured domains. At least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the POP may comprise structured domains.

[0046] In some embodiments, the structured domain comprises a polyalanine domain. Each polyalanine domain may include at least 4 or at least 5 alanine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, including all values and ranges therein). Each polyalanine domain may have at least about 60% of the amino acids in an alpha-helical conformation. In some embodiments, the structured domain comprises a polyalanine domain (A)m(SEQ ID NO: 29), wherein m is an integer from 5 to 500. In some embodiments, the polyalanine domain comprises of one or more of: (A)25(SEQ ID NO: 8); K(A)25K (SEQ ID NO: 9); D(A)25K (SEQ ID NO: 10); GD(A25)K (SEQ ID NO: 11); or GK(A25)K (SEQ ID NO: 12).

[0047] In one embodiment, the POP comprises a plurality of disordered domains, each comprising a PG motif comprising an amino acid sequence selected from PG, P(X)nG (SEQ ID NO: 21), and (B)mP(X)nG(Z)p(SEQ ID NO: 22), or a combination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein B, X, and Z are independently any amino acid; and a plurality of structured domains, each comprising a polyalanine domain, each polyalanine domain comprising at least 5 alanine residues and having at least about 50% of the amino acids in an a-helical conformation; wherein the POP exhibits phase transition behavior. In one embodiment, at least one disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1. In another embodiments, at least about 60% of the amino acids in each polyalanine domain are in an a-helical conformation. In another embodiment, each polyalanine domain comprises an amino acid sequence of [Bp(A)qZr]n (SEQ ID NO: 23) or [(BAs)tZr]n (SEQ ID NO: 24), wherein B is Lys, Arg, Asp, or Glu; A is Ala; Z is Lys, Arg, Asp, or Glu; n is an integer from 1 to 50; p is an integer from 0 to 2; q is an integer from 1 to 50; r is an integer from 0 to 2; s is an integer from 1 to 5; and t is an integer from 1 to 50. In another embodiment, the structured domain comprises one or more of (A)25 (SEQ ID NO: 8); K(A)25K (SEQ ID NO: 9); D(A)25K (SEQ ID NO: 10); GD(A25)K (SEQ ID NO: 11); or GK(A2S)K (SEQ ID NO: 12). In another embodiment, about 4% to about 75% of the POP comprises structured domains.

[0048] In another embodiment, the Fractomer comprises a plurality of disordered domains; and a plurality of structured domains. In one embodiment, the Fractomer has the general structure of [(SEQ ID NO: l)n-a-helix]m(SEQ ID NO: 30), where n is an integer equal to or greater than 1, m is an integer equal to or greater than 1, and a-helix is any polyalanine based a-helix having about 5 to 50 Alanine residues. In another embodiment, the Fractomer has the structure [(SEQ ID NO: 2)n-GX1(A)25X1]m(SEQ ID NO: 31), where X1is K or D, n is an integer from 10 to 20, and m is an integer from 4 to 8 (e.g., [(SEQ ID NO: 2)n-(SEQ ID NO: 11 or 12)]m). In another embodiment, the Fractomer comprises one or more of the following structures:

[0049] M[(GVGVP)i5-GD(A25)K]6-GWP (SEQ ID NO: 13);

[0050] M[(GVGVP)i5-GD(A25)K]4-GWP (SEQ ID NO: 14);

[0051] M[(GVGVP)i5-GK(A25)K]6-GWP (SEQ ID NO: 15);

[0052] M[(GVGVP)i5-GK(A25)K]4-GWP (SEQ ID NO: 16);

[0053] M[(G[A1 : Vl]GVP)i6-GD(A25)K]6-GWP (SEQ ID NO: 17);

[0054] M[(G[A1 :Vl]GVP)i6-GD(A25)K]4-GWP (SEQ ID NO: 18);

[0055] M[(G[V4:Al]GVP)i5-GD(A25)K]6-GWP (SEQ ID NO: 19); or

[0056] M[(G[V4:Al]GVP)i5-GD(A25)K]4-GWP (SEQ ID NO: 20).

[0057] In one embodiment, the Fractomer comprises the following structures:

[0058] M[(GVGVP)i5-GD(A25)K]6-GWP (SEQ ID NO: 13); or

[0059] M[(G[V4:Al]GVP)i5-GD(A25)K]6-GWP (SEQ ID NO: 19).

[0060] A complete listing of exemplary sequences, sequence motifs, and POP constructs is provided herein.

[0061] The POP may also include amino acid derivatives that are not naturally occurring, such as a UV crosslinkable amino acid derivative. The non-native amino acid derivative can be used to introduce covalent crosslinks between different POPs and within the same POP. For example, POPs that include the UV crosslinkable amino acid derivative can be exposed to UV light, which can result in covalent crosslinks being formed between the amino acid derivative and a side chain of an amino acid of another POP or with a side chain of an amino acid of the same POP (having the amino acid derivative). The UV crosslinkable amino acid derivative may be any amino acid that has been functionalized with an azide group. In some embodiments, the amino acid derivative is para-azidophenylalanine.

[0062] The UV crosslinkable amino acid derivative may be included at varying amounts without affecting the POP's ability to transition at different temperatures. For example, the UV crosslinkable amino acid derivative may be included within the POP from about 0.1% to about20% (of the POP), such as from about 0.5% to about 15% or from about 1% to about 10% (of the POP).

[0063] The POP alone, without the presence of polysaccharide, may form aggregates, a three- dimensional matrix, a semi-solid or a gel. The following discussion of “aggregate” refers to the aggregate formed by POP alone.

[0064] The POP may demonstrate phase transition behavior by changing solubility and aggregate formation with temperature. The phase transition behavior of the POP may derive from the phase transition behavior of the disordered domains of the POP. "Phase transition" or "transition" may refer to the aggregation of a polypeptide, which occurs sharply at a specific temperature. The phase transition may be reversible, although the specific temperature of dissolution may be the same or different from the specific temperature of aggregation.

[0065] In some embodiments, the POP is soluble below a lower critical solution temperature (LOST). LOST is the temperature below which the polypeptide is miscible.

[0066] A transition temperature (Tt) is a temperature at which the POP changes from one state to another. States may include, for example, soluble polypeptides, gels, and aggregates of varying sizes and dimensions. The POP may have a transition temperature of heating (Tt- heating) and a transition temperature of cooling (Tt-cooling). In some embodiments, the transition temperature heating (Tt-heating) is concentration-dependent. In some embodiments, the transition temperature cooling (Tt-cooling) is concentration-independent. The Tt-heating may be primarily determined by the disordered domains. The Tt-cooling may be primarily determined by the structured domains.

[0067] Below the transition temperature (LCST or Tt), the POP may be highly soluble. Upon heating above the transition temperature, the POP may hydrophobically collapse and aggregate, forming a separate phase.

[0068] In some embodiments, the POP is soluble below a lower critical solution temperature (LCST). In some embodiments, the transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) are identical, or the transition temperature of heating (Tt-heating) is greater than the transition temperature of cooling (Tt-cooling). In some embodiments, the Tt-heating may be dependent on the concentration of the POP in the composition. The Tt-heating and the Tt-cooling may range from about 10 °C to about 45 °C. In some embodiments the Tt-heating and the Tt-cooling may range from about 10 °C to about 40 °C, about 15 °C to about 35 °C, and / or about 20 °C to about 30 °C. In some embodiments, the partially ordered polypeptide forms a solid aggregate above the Tt-heating.

[0069] The POP may phase transition at a variety of temperatures. The POP may have a transition temperature (Tt) from about 0 °C to about 100 °C, from about 10 °C to about 50 °C, or from about 20 °C to about 42 °C. The transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) may be identical. As used herein, temperatures may be "identical" when the temperatures are within 2.0 °C, 1.0 °C, 0.5 °C, or 0.1 °C of each other. In some embodiments, the transition temperature of heating (Tt-heating) is greater than the transition temperature of cooling (Tt-cooling). In embodiments where the POP has a Tt- heating greater than the Tt-cooling, the difference between the two transition temperatures may be referred to as a hysteresis, in some embodiments, the POP has a hysteresis of about 5 °C to about 70 °C, such as about 5 °C to about 60 °C or about 10 °C to about 50 °C.

[0070] The phase transition behavior of the POP may be utilized in purification of the POP according to a method referred to as "inverse transition cycling," in which the POP's reversible phase transition behavior is used to cycle the solution through soluble and insoluble phases, thereby removing contaminants. Phase transition may also be triggered using kosmotropic salts, such as, for example, ammonium sulfate or sodium chloride. The kosmotropic salt may be added to a solution comprising the POP, with the kosmotropic salt being added until the POP forms aggregates or is precipitated out of solution. The aggregates may be pelleted by centrifugation and resuspended in a second solution or buffer. Aggregates of the POP may resolubilize into solution once cooled below their Tt or when the kosmotropic salt is removed from the solution. In some embodiments, the POP is purified without any chromatographic purification. In some embodiments, the POP is generated recombinantly and purified from bacterial culture, such as, for example, from E. coli.

[0071] In some embodiments, the POP may form an aggregate when the temperature is greater than the Tt-heating. The aggregate may resolubilize when cooled to below a temperature less than the Tt-cooling.

[0072] The aggregate formed from a plurality of POPs may be a variety of sizes and dimensions. In some embodiments, the aggregate is a stable three-dimensional matrix. In some embodiments, the aggregate is fractal-like, in some embodiments, the aggregate is gel-like, in some embodiments, the aggregate is porous with a void volume, e.g., the nonprotein rich phase of the aggregate. In some embodiments, the void volume is tunable. For example, the aggregate may have a void volume from about 60% to about 90% (of the volume of the aggregate), in addition, the aggregate may comprise pores having a diameter of about 1 pm to about 100 pm, such as about 1 pm to about 10 pm, about 3 pm to about 5 pm, about 25 pm to about 60 pm, about 30 pm to about 50 pm, or about 3 pm to about 50 pm. In some embodiments, theaggregate may comprise pores having a diameter of about 1 pm to about 100 pm, such as about 1 pm to about 10 pm, about 3 pm to about 5 pm, about 25 pm to about 60 pm, about 30 pm to about 50 pm, or about 3 pm to about 50 pm.

[0073] In some embodiments, the solid aggregate is a stable three-dimensional matrix. This three-dimensional matrix may encapsulate the polysaccharide. In some embodiments, the solid aggregate comprises a plurality of micropores. In some embodiments, the composition comprises between about 200 pM and about 2.5 mM of the POP. In some embodiments, the composition comprises between about 300 pM and about 2.4 mM, about 400 pM and about 2.3 mM, about 500 pM and about 2.2 mM, about 600 pM and about 2.1 mM, about 700 pM and about 2.0 mM, about 800 pM and about 1.9 mM, about 900 pM and about 1.8 mM, about 1.0 mM and about 1.7 mM, about 1.1 mM and about 1.6 mM, about 1.2 mM and about 1.5 mM, or about 1.3 mM and about 1.4 mM.

[0074] The aggregate formed by a plurality of POPs may have advantageous properties that can arise from the structure of the POPs. For example, the aggregate may have physical, non- covalent crosslinks. These physical, non-covalent crosslinks may arise from helical bundling of the structured domain(s) interacting with each other. The aggregate may also have covalent crosslinks (e.g., chemical crosslinks) in addition to physical, non-covalent crosslinks. Covalent crosslinks can be included in the aggregate in order to increase their mechanical stability without altering their porous architecture, in some embodiments, the aggregate can be formed from a plurality of POPs and can then be further stabilized by covalent crosslinking (after the formation of the aggregate). Covalent crosslinks can be introduced via a UV crosslinkable amino acid derivative having an azide functionality as described herein. Further examples of crosslinks that can be incorporated into the aggregate include, but are not limited to, small molecule crosslinks and cysteine disulfide bridges. An example of a chemical, small molecule crosslink is tetrakis(hydroxymethyl)phosphonium chloride (TMPC), which can crosslink lysines within POPs.

[0075] In addition, the aggregate formed by a plurality of POPs may have solid-like properties that distinguish it from liquid-like coacervate structures. For example, the aggregate may have a storage modulus (C) that is greater than its loss modulus (G"), such as having a G' 2* greater, 5x greater, lOx greater, 15x greater, 20x greater, 25x greater, 30xgreater, 35x greater, 50x greater or lOOx greater than its G”. In some embodiments, the aggregate has a G' from 2x greater to lOOx greater than its G", such as from lOx greater to 5 Ox greater or from 20x greater to 35x greater than its G”.

[0076] Further provided are polynucleotides encoding the POPs described herein. A vector may include the polynucleotide encoding the POPs detailed herein. To obtain expression of a polypeptide, one may subclone the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription / translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. An example of a vector is pET24. Suitable bacterial promoters are well known in the art. Further provided is a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding a POP as described herein. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus species., and Salmonella. See Paiva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Retroviral expression systems can be used in the present invention.

[0077] The POP may be expressed recombinantly in a host cell according to one of skill in the art. The POP may be purified by any means known to one of skill in the art. For example, the POP may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, or affinity chromatography, or a combination thereof, in some embodiments, the POP is purified without chromatography, in some embodiments, the POP is purified using inverse transition cycling.

[0078] In other embodiments, the present disclosure further provides herein a scaffold comprising a plurality of POPs. The scaffold may be formed at a temperature greater than the transition temperature of the POP, such that the polypeptide forms an aggregate. The scaffold may be injectable.

[0079] Further provided in accordance with one embodiment is a cellular scaffold. A cellular scaffold includes the scaffold and a plurality of cells. The cells may include a variety of types. In some embodiments, the cells comprise stem cells, bacterial cells, or human tissue cells, or a combination thereof.

[0080] The scaffold may have low immunogenicity or low antigenicity or both. The scaffold may promote at least one of cell growth, cell recruitment, and cell differentiation, or a combination thereof. The scaffold, or cellular scaffold, may be suitable for cell transplantation, tissue regeneration, cell culture, and cell-based in vitro assays. In addition, the scaffold and / or cellular scaffold may promote the formation of vasculature, wound healing, or a combination thereof.

[0081] Further provided in accordance with one embodiment is a drug delivery composition. The drug delivery composition may include a plurality of POPs as detailed herein, selfassembled into an aggregate above the Tt-heating, and an agent encapsulated within the aggregate. In some embodiments, the POPs could have different structures.Polysaccharide

[0082] When a polysaccharide is introduced to the POP, the POPs may form an aggregate (when heating to or above the Tt-heating) that encapsulates the polysaccharide, forming a matrix filler composition. This aggregate may be used as filler in a subject in need thereof.

[0083] The tissue matrix composition may include a plurality of POPs as detailed herein, selfassembled into an aggregate above the Tt-heating, and a polysaccharide encapsulated within the aggregate. In some embodiments, the polysaccharide comprises at least one glycosaminoglycan. In some embodiments, the polysaccharide may be or may not be crosslinked. In some embodiments, the glycosaminoglycan comprises one or more residue of a heparin sulfate, a chondroitin sulfate, a dermatan sulfate, and a hyaluronic acid. In some embodiments, the glycosaminoglycan comprises hyaluronic acid, or a pharmaceutically acceptable salt thereof. In some embodiments, the hyaluronic acid is not crosslinked. In some embodiments, the hyaluronic acid is crosslinked. In some embodiments, the hyaluronic acid is physically crosslinked or chemically crosslinked. In some embodiments, wherein the hyaluronic acid is physically crosslinked by modifying at least one parameter selected from the group consisting of pH, temperature, ionic, strength conditions, and physiochemical interactions. In some embodiments, wherein the hyaluronic acid is chemically crosslinked by at least one chemical crosslinker selected from the group consisting of dicycloheyl carbodiimide, N-hydroxysuccinimide, benzotriazole-l-yl-oxy-tris-(dimethylamino)- phoponium hexafluorophosphate), l-hydroxy-7-azobenzotriazole (HO At), carbonyl dimidazole, 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), l-ethyl-3-(3-dimethlaminopropyl)carbodiimide hydrochloride (EDC), 1,4-butane diglycidyl ether, divinyl sulfone (DVS), gluraraldehyde, cyanogen bromide, octeylsuccinic anhydride, acid chloride, methacrylic anhydride, and sodium periodate.

[0084] In some embodiments, the molecular weight of hyaluronic acid is about 2.5 million Daltons (Da). In some embodiments, the molecular weight of hyaluronic acid is about 1,000 Da to about 3 million Da (e.g., 1,000 Da, 50,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da 500,000 Da, 600,000 Da, 700,000 Da, 800,000 Da, 900,000 Da, 1 million Da, 1.5 million Da, 2 million Da, 2.5 million Da, and 3 million Da). In some embodiments, themolecular weight of hyaluronic acid is about 1,000 Da to about 3 million Da, about 500,000 Da to about 3 million Da, about 1 million Da to about 3 million Da, about 1.5 million Da to about 3 million Da, about 2 million Da to about 3 million Da, or about 2.5 million Da to about 3 million Da.

[0085] In some embodiments, the polysaccharide is present in an amount of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL, including all values and ranges therein). In some embodiments, the polysaccharide is present in an amount of about 1 mg / mL to about 150 mg / mL. (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 mg / mL, including all values and ranges therein). In some embodiments, the polysaccharide is present in an amount of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the polysaccharide is present at a concentration In some embodiments, the polysaccharide is present at an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL including all ranges, subranges and values therebetween. In embodiments, the polysaccharide is hyaluronic acid.

[0086] In some embodiments, the partially ordered polypeptide and polysaccharide are present in a ratio of 95:5, 90: 10, 70:30, 50:50, 30:70, 10:90, or 5:95 (partially ordered polypeptide: polysaccharide), including all ranges, subranges and values therebetween. In embodiments, the polysaccharide is hyaluronic acid.

[0087] In some embodiments, the polysaccharide must be hydrophilic. In embodiments, the polysaccharide comprises at least one glycosaminoglycan. In embodiments, the at least one glycosaminoglycan comprises one or more residue of a heparin sulfate, a chondroitin sulfate, a dermatan sulfate, and a hyaluronic acid. In some embodiments, the polysaccharide includes hyaluronic acid. In one embodiment, the composition comprises about 10-90% by volume of polysaccharide (e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%, including all values and ranges therein). In oneembodiment the drug delivery composition comprises about 10-90% by volume of polysaccharide. In one embodiment the drug delivery composition comprises about 10-50% by volume of polysaccharide. In another embodiment, the composition comprises about 25-90% by volume of polysaccharide. In some embodiments, the composition comprises at least 25% by volume of polysaccharide. In other embodiments, the composition comprises at least 35% by volume of polysaccharide. In other embodiments, the composition comprises at least 45% by volume of polysaccharide. In yet other embodiments, the composition comprises at least50% by volume of polysaccharide. In other embodiments, the composition comprises at least60% by volume of polysaccharide. In other embodiments, the composition comprises at least70% by volume of polysaccharide. In other embodiments, the composition comprises at least80% by volume of polysaccharide. In other embodiments, the composition comprises at least90% by volume of polysaccharide.

[0088] In yet other embodiments, the POPs and or the drug delivery composition as detailed above can be formulated into a pharmaceutical composition in accordance with standard techniques well known to those skilled in the pharmaceutical art. Accordingly, a composition may comprise the POP or aggregate thereof and / or a POP or aggregate thereof and an effective amount of adipose tissue (e.g., lipoaspirate) along with one or more pharmaceutically acceptable carriers, excipients, or active pharmaceutical ingredients (APIs). The composition may be prepared for administration to a subject. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.MethodsSynthesis of Polymer Genes

[0089] All POPs were cloned. All polymers were cloned into a modified pet24 vector using a process known as recursive directional ligation by plasmid reconstruction (Pre-RDL). Singlestranded oligomers encoding the desired sequences were annealed into cassettes with CC and GG overhangs, allowing concatemerization and ligation into the pet24 vector. This was used to create a library of ELP and polyalanine cassettes which could be strung together through multiple cycles of Pre-RDL to form the final compositions. Plasmids were transfected into chemically competent EB5a cells for cloning and BL21(DE3) cells for protein expression.Expression and Purification of POPs

[0090] Protein expression is performed in E. coli using fermentation to achieve high density cell culture, followed by protein isolation and purification. Starter cultures of a semi-complex medium are inoculated from cryostocks of recombinant E. coli (BL21 derived) containing POPs producing genes and are grown overnight. The starter culture is used to inoculate a larger volume primary culture of the same medium, which is grown for 6 to 8 hours and then used to inoculate the fermenter containing the same medium. The E. coli culture is grown as a fed batch fermentation using a glucose feed as the provided carbon source, at a high agitation speed with supplied oxygen and pH control, in order to yield high density cell culture. The E. coli is induced to turn on protein expression during exponential phase of growth. The culture is grown under induction conditions to allow for high levels of protein production. The E. coli culture is harvested and remaining medium is removed by centrifugation. Resulting cell paste is resuspended with lx phosphate buffered saline (PBS) at a 5: 1 vol / wt ratio. The resuspension is run through two passes in a microfluidizer at 10,000 psi in order to lyse cells. Cell lysate is treated with 0.5% polyethyleneimine (PEI) to remove contaminating DNA and insoluble cell waste and centrifuged at 12,000 x g for lOmin at 4C to remove cell waste. The resulting supernatant containing the soluble protein target is collected. Protein is purified from this solution using four rounds of hot / cold centrifugation. In brief, the solution is heated at 45°C for 1 hour, causing the desired protein to aggregate and fall out of solution as an insoluble fraction. Once aggregated, the mixture is centrifuged at 5,000 x g for 10 minutes at 37°C in order to form a protein pellet of the target protein. The supernatant is discarded. The protein pellet is resuspended in lx PBS (5: 1 vol / wt) and incubated at 4°C, while shaking, for 4 hours in order to solubilize the protein back into solution. Once fully resuspended, the soluble protein solution is centrifuged at 12,000x g for 10 minutes at 4°C to remove insoluble waste. After centrifugation the cold supernatant containing the target protein is collected. The cycle of hot / cold centrifugation is repeated at least 3 times, achieving a 95% purity of our target protein, as determined by SDS-PAGE. The purified protein solution is then treated for endotoxin removal using a chemical treatment and filtration. Protein solution is brought into a salt free water background using dialysis with multiple buffer exchanges. The final, salt free, protein solution is filtered sterilized and then lyophilized for final storage.Forming a POP solution

[0091] A solution comprising partially ordered peptides (POPs) can be prepared. The solution may comprise POPs in a buffer solution. The buffer solution may comprise phosphate bufferedsaline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris-EDTA buffer solution, water, distilled water, and / or double distilled water. The POP used can include a plurality of disordered domains and a plurality of ordered domains, where each disordered domain independently may comprise a PG or GP motif and each ordered domain independently may comprise an alpha helix. In embodiments, each disordered domain may comprise an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X may be any amino acid except proline and n may be an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif. In embodiments, each disordered domain may comprise an amino acid sequence of (GXGVP)n (SEQ ID NO: 2), wherein each X may be independently Vai or Ala, and wherein n may be an integer from 2 to 50, from 10 to 40, from 15 to 35, and / or from 20 to 30. X may be an alternating iteration of Ala to Vai in a ratio from 10: 1 to 1 : 10. The polyalanine motif may comprises (A)m(SEQ ID NO: 29), wherein m may be an integer from 5 to 50. In embodiments, each disordered domain may comprise an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain may comprise a polyalanine motif. Each disordered domain may comprise an amino acid sequence of (GXGVP)n (SEQ ID NO: 2), X may be independently Vai or Ala, and n may be an integer from 2 to 50, from 10 to 40, from 15 to 35, and / or from 20 to 30. In either pentapeptide sequence, the polyalanine motif may comprise one or more of (A)n(SEQ ID NO: 3), K(A)nK (SEQ ID NO: 4), D(A)nK (SEQ ID NO: 5), GD(An)K (SEQ ID NO: 6), or GK(An)K (SEQ ID NO: 7), wherein n may be an integer from 2 to 100. In an example, the polyalanine motif may comprise one or more of (A)25 (SEQ ID NO: 8), K(A)25K (SEQ ID NO: 9), D(A)25K (SEQ ID NO: 10), GD(A25)K (SEQ ID NO: 11), or GK(A2S)K (SEQ ID NO: 12). In embodiments, the POP may have a transition temperature of heating (Tt-heating). The Tt-cooling may be dependent on the concentration of the partially ordered polypeptide in the solution. The Tt-heating and the Tt-cooling may range from about 10 °C to about 45 °C. The POP may form a solid aggregate above the Tt-heating.

[0092] The POP solution prepared may comprise POP in buffer solution at about 100 pM to about 3 mM, about 150 pM to about 2 mM, about 200 pM to about 1.5 mM, about 250 pM to about 1.25 mM, about 500 pm to about 1 mM, about 600 pM to about 900 pM, and / or about 700 pm to about 800 pM. In other words, the solution prepared at step 102 of method 100 may comprise about 0.1% by weight POP to about 100% by weight POP, about 0.1% by weight POP to about 85% by weight POP, and / or about 50% by weight POP to about 90% by weight POP. The solution can comprise a variety of concentrations of POPs, where higher concentrations result in greater rigidity and stability.

[0093] While the POP may form an aggregate above the Tt-heating due to physical crosslinks from helical domain swapping, this does not preclude the use of additional chemical crosslinking techniques to further modulate mechanical properties.

[0094] In embodiments, a crosslinker may be added to the POP solution. The crosslinker may be a covalent crosslinker. The covalent crosslinker may by a chemical crosslinker, a pH-based crosslinker, may utilize click chemistry, ultraviolet light, or a combination thereof. When ultraviolet light is used, a UV functionalized POP may be used. In embodiments, the chemical crosslinker may be an amine reactive crosslinker. In embodiments, the chemical crosslinker may be configured to crosslink residues, motifs, and the like of the POPs. To this end, the chemical crosslinker may comprise at least one crosslinker or a derivative thereof selected from the group consisting of tetrakis (hydroxymethyl) phosphonium chloride (THCP), NHS ester crosslinkers, NHS-mal eimide crosslinkers, NHS-pyridyldithiol crosslinkers, ( 1R, 85, 9 )- Bicyclo[6.1.0]non-4-yn-9-ylmethyl-7V-succinimidyl carbonate, dibenzyocyclooctyne-7V- hydroxysuccinimidyl ester, propargyl-A-hydroxysuccinimidyl ester, maleimide-PEG2- succinimidyl ester, Azido-dPEG®4-NHS ester, 3-(2-Pyridyldithio)propionic acid N- hydroxy succinimide ester, 3-Maleimidobenzoic acid Why droxy succinimide ester, methyltetrazine-NHS ester, bromoacetic acid N-hydroxysuccinimide ester, 0,0 ’-Bis[2-(W Succinimidyl-succinylamino)ethyl]polyethylene glycol, maleimide-PEGs-succinimidyl ester, iodoacetic acid Whydroxysuccinimide ester, alkyne-PEGs-TV-hydroxysuccinimidyl ester, maleimide-PEGe-succinimidyl ester, 6-maleimidohexanoic acid Whydroxysuccinimide ester, LC-SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxy-(6- amidocaproate)), azido-dPEG®s-NHS ester, PEG4-SPDP (PEGylated, long-chain SPDP crosslinker), SMPH (succinimydl-6-((b-maleimidoproprionamido)hexanoate), LC-SPDP (succinimidyl 6-[3(2-pyridyldithio)propionamido]hexanoate), O-[7V-(3- maleimidopropionyl)aminoethyl]-O’-[3-(7V-succinimidyloxy)-3-oxopropyl]triethylene glycol, O-[7V](3-Maleimidopropionyl)aminoethyl]-O’-[3-(7V-succinimidyloxy)-3- oxopropyl]heptacosaethylene glycol, SBAP (succinimidyl 3-bromoacetamido)propionate), SPDP-dPEG®4-NHS ester, acid-dPEG®s-NHS ester, 0,0 ’-Bis[2-(7V-succinimidyl- succinylamino)ethyl]polyethylene glycol, SMPT (4-succinimidyloxycarbonyl-alpha-methyl- alpha(2-pyridyldithio)toluene), phthalimidooxy-dPEG®4-NHS ester, acid-dPEG®9-NHS ester, Fmoc-N-amido-dPEG®8-NHS ester, SPDP-dPEG' x-NHS ester, and Fmoc-N-amido-dPEG®4- NHS ester. In embodiments, the crosslinking may be performed on ice. In embodiments, the crosslinking may be performed above Tt-heating of the POPs. In embodiments, the chemical crosslinker may be added to the solution at a ratio of the POP to chemical crosslinker of about1 to about 10, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 1 to about 1, about 2 to about 1, about 3 to about 1, about 4 to about 1, about 5 to about 1, about 10 to about 1, about 20 to about 1, about 30 to about 1, about 40 to about 1, about 50 to about 1, about 100 to about 1, about 200 to about 1, about 300 to about 1, about 400 to about 1, about 500 to about 1, about 1,000 to about 1, about 2,000 to about 1, about 3,000 to about 1, about 4,000 to about 1, about 5,000 to about 1, and / or about 10,000 to about1. In embodiments, the chemical crosslinker is added to the composition at a ratio of chemical crosslinker to the composition based on a number of available crosslinking sites on each partially ordered polypeptide, wherein each crosslinking site is a lysine residue, an aspartic acid residue, an amine reactive site, or a carboxylic group reactive site. In embodiments, the ratio of chemical crosslinker to the composition is about 6 to about 1. In embodiments, the crosslinking comprises adding a crosslinker to the composition and periodically inverting the composition.

[0095] In some embodiments, no crosslinker is added to the POP solution.

[0096] Additional variations, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.Forming a tissue matrix filler

[0097] In an exemplary embodiment, with reference to Fig. 1, method 100 describes a method of composing a tissue matrix filler to be administered to a subject to augment native tissue matrices. At process 102 of method 100, a composition comprising at least a POP solution and a polysaccharide solution is prepared. As will be described in more detail with reference to Fig.2, the composition may further comprise a third or more components that provide desirable biochemical effects (e.g., numbing).

[0098] At step 107 of method 100, the prepared composition is administered to a subject as a tissue matrix filler.

[0099] In some embodiments, the composition can be used as an autologous fat graft. In some embodiments, the composition is administered to the subject in a region of interest. In some embodiments, the POP and polysaccharide are mixed in vitro, shaped and then implanted in situ in the subject. In some embodiments the POP and polysaccharide composition are a shapeable liquid, semisolid or molded semisolid prior to administration.

[0100] In some embodiments, the composition is administered to a subject at an amount of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL). In some embodiments, thecomposition is administered to a subject at an amount of about 1 mg / mL to about 150 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 mg / mL). In some embodiments, the composition is administered to a subject at an amount of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the composition is administered at an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about13 mg / mL, about 14 mg / mL, about 1 5 mg / mL, about 1 5 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about148 mg / mL, about 149 mg / mL, or about 150 mg / mL including all ranges, subranges and values therebetween. In some embodiments, the composition is administered at about 15 mg / mL to about 35 mg / mL, about 20 mg / mL to about 35 mg / mL, about 25 mg / mL to about 35 mg / mL, about 15 mg / mL to 30 mg / mL, about 20 mg / mL to about 30 mg / mL, about 25 mg / mL to about 30 mg / mL, about 15 mg / mL to about 25 mg / mL, or about 20 mg / mL to about 25 mg / mL.

[0101] At step 108 of method 100, the administered composition is aggregated upon exposure to temperatures above Tt-heating (e.g., body temperature). In some embodiments, the composition is soluble below LCST. In some embodiments, the composition has a transition temperature of heating (Tt-heating) and a transition temperature of cooling (Tt-cooling). In some embodiments, the transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) may be identical. In some embodiments, the transition temperature of heating (Tt-heating) is greater than the transition temperature of cooling (Tt- cooling). In some embodiments, the Tt-heating may be dependent on the concentration of the POP in the composition. The Tt-heating and the Tt-cooling may range from about 10 °C to about 45 °C. In some embodiments the Tt-heating and the Tt-cooling may range from about 10 °C to about 40 °C, about 15 °C to about 35 °C, and / or about 20 °C to about 30 °C. In some embodiments, the composition forms a solid aggregate above the Tt-heating.

[0102] With reference now to Fig. 2, process 102 of method 100 will be described in further detail. As noted, above process 102 comprises preparing and mixing a solution of POP and a solution of polysaccharide to form a composition, wherein the composition may optionally comprise a third solution. The resulting composition, formed in vitro, may be referred to as a tissue matrix filler.

[0103] In some embodiments, the compositions described herein comprise at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris- EDTA buffer solution, water, distilled water, phosphate (e.g. sodium phosphate), acetate, and / or double distilled water. In some embodiments, the dried composition has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the POP solutions comprise at least one buffer at a concentration of about ImM to about IM (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0104] At step 103 of process 102, a solution of POP is prepared (referred to herein interchangeably as the first solution). In some embodiments, POP is added as a lyophilized power to a solution. In some embodiments, the lyophilizing comprises performing lyophilization for a predetermined time period, wherein the predetermined time period is about 24 hours to about 72 hours. In embodiments, the method further comprises rehydrating the dried composition, wherein the rehydrating comprises submerging the dried composition in an aqueous buffer for a predetermined time period and / or wherein the predetermined time period is about 1 minute to about 5 days, about 2 minutes to about 4 days, about 3 minutes to about 3 days, about 4 minutes to about 2 days, about 5 minutes to about 1 day, about 6 minutes to about 12 hours, about 7 minutes to about 6 hours, about 8 minutes to about 1 hour, about 9 minutes to about 30 minutes, and / or about 10 minutes to about 15 minutes. In embodiments, the drying and the rehydrating are performed iteratively. In embodiments, the composition is a resuspension of lyophilized partially ordered polypeptide in an aqueous buffer.

[0105] The POP solution prepared may comprise POP in buffer solution at about 100 pM to about 3 mM, about 150 pM to about 2 mM, about 200 pM to about 1.5 mM, about 250 pM to about 1.25 mM, about 500 pm to about 1 mM, about 600 pM to about 900 pM, and / or about 700 pm to about 800 pM. In other words, the solution prepared at step 107 of method 106 may comprise about 0.1% by weight POP to about 100% by weight POP, about 0.1% by weight POP to about 85% by weight POP, and / or about 50% by weight POP to about 90% by weightPOP. The solution can comprise a variety of concentrations of POPs, where higher concentrations result in greater rigidity and stability.

[0106] In some embodiments, the POP is present in solution at a concentration between about 200 pM and about 3 mM. In some embodiments, the POP solution is present at a concentration between about 300 pM and about 2.9 mM, about 400 pM and about 2.8 mM, about 500 pM and about 2.7 mM, about 600 pM and about 2.6 mM, about 700 pM and about 2.5 mM, about 800 pM and about 2.4 mM, about 900 pM and about 2.3 mM, about 1.0 mM and about 2.2 mM, about 1.1 mM and about 2.1 mM, about 1.2 mM and about 2.0 mM, about 1.3 mM and about 1.9 mM, about 1.4 mM and about 1.8 mM, or about 1.5 mM and about 1.7 mM. In some embodiments, the POP is present at a concentration of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL). In some embodiments, the POP is present at a concentration of about 1 mg / mL to about 150 mg / mL. (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg / mL). In some embodiments, the POP is present at a concentration of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the POP is present at a concentration of about 1 mg / mL, about2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, about 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL including all ranges, subranges and values therebetween.

[0107] In some embodiments, the POP solutions described herein comprises at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris- EDTA buffer solution, water, distilled water, phosphate (e.g. sodium phosphate), acetate, and / or double distilled water. In some embodiments, the dried POP has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the POP solutions comprise at least one buffer at a concentration of about ImM to about IM (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0108] At step 104 of method 102, a solution of polysaccharide is prepared (referred to herein interchangeably as the second solution). In some embodiments, the polysaccharide is added as a lyophilized power to a solution. In some embodiments, the solution of polysaccharide comprises hyaluronic acid. In some embodiments, the solution comprises between about 200 pM and about 3 mM of the polysaccharide. In some embodiments, the solution comprises a polysaccharide at a concentration of about 300 pM and about 2.9 mM, about 400 pM and about 2.8 mM, about 500 pM and about 2.7 mM, about 600 pM and about 2.6 mM, about 700 pM and about 2.5 mM, about 800 pM and about 2.4 mM, about 900 pM and about 2.3 mM, about 1.0 mM and about 2.2 mM, about 1.1 mM and about 2.1 mM, about 1.2 mM and about 2.0 mM, about 1.3 mM and about 1.9 mM, about 1.4 mM and about 1.8 mM, or about 1.5 mM and about 1.7 mM. In some embodiments, the polysaccharide is present in an amount of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL). In some embodiments, the polysaccharide is present in an amount of about 1 mg / mL to about 150 mg / mL. (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 mg / mL). In some embodiments, the polysaccharide is present in an amount of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the polysaccharideis present at an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, about 100 mg / mL, 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about149 mg / mL, or about 150 mg / mL, including all ranges, subranges and values therebetween.

[0109] In some embodiments, the polysaccharide solutions described herein comprises at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate,Tris-EDTA buffer solution, water, distilled water, phosphate (e.g. sodium phosphate), acetate, and / or double distilled water. In some embodiments, the dried polysaccharide has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the polysaccharide solutions comprise at least one buffer at a concentration of about ImM to about IM (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0110] At optional step 105 of process 102, a third solution may be prepared. The third solution may comprise a buffer, a stabilizer, a surfactant, a linker, a non-steroidal anti-inflammatory drug (NS AID), a local anesthetic or a combination there of. In some embodiments, the third solution comprises at least one surfactant. Surfactants include but are not limited to polysorbate 80, polysorbate 20, and poloxamer 188. In some embodiments, the third solution comprises at least one surfactant at a concentration of about 5% to about 20% weight by volume (w / v). In some embodiments, the surfactant is present at a concentration of about 10% w / v to about 20% w / v, about 15% w / v to about 20% w / v, about 5% w / v to about 15% w / v, about 10% w / v to about 15% w / v, or about 5% w / v to about 10%w / v. In some embodiments, the surfactant is present at a concentration of about 1% w / v to about 20% w / v (e.g., 1, 5, 10, 15 and 20% w / v). In some embodiments, the surfactant is present at a concentration of about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v or about 20% w / v, including all ranges, subranges and values therebetween.[OHl] In some embodiments, the third solution described herein comprises at least one stabilizer. Stabilizers include but are not limited to sucrose, sorbitol, glucose, glycine, glycerine, or sodium chloride. Herein, a “stabilizer” refers to any reagent or chemical that is used in organic materials subject to mechanical stress, UV radiation, discoloration, or effects such as oxidation, chain scission, or uncontrolled recombination and cross-linking reactions, to protect the organic materials from deleterious effects and degradation. In some embodiments, the stabilizer is present at a concentration of about 25 mM to about 125 mM (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, and 125mM, or any value or range therein). In some embodiments, the at least one stabilizer is present at a concentration of about 25 mM to about 100 mM, about 25 mM to about 75 mM, about 25 mM to about 50 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, about50 mM to about 75 mM, about 75 mM to about 125 mM, or about 75 mM to about 100 mM. In some embodiments, the at least one stabilizer is present at a concentration about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, about 106 mM, about 107 mM, about 108 mM, about 109 mM, about 110 mM, about 111 mM, about 112 mM, about 113 mM, about 114 mM, about 115 mM, about 116 mM, about 117 mM, about 118 mM, about 119 mM, about 120 mM, about 121 mM, about 122 mM, about 123 mM, about 124 mM, or about 125 mM, including all ranges, subranges and values therebetween. In some embodiments, the at least one stabilizer is present at 100 mM. In some embodiments, at least one stabilizer is present at about 90 mM to about 110 mM, about 95 mM to about 110 mM, about 100 mM to about 110 mM, about 105 mM to about 110 mM, about 90 mM to about 105 mM, about 95 mM to about 105 mM, about 100 mM to about 105 mM, about 90 mM to about 100 mM, or about 95 mM to about 100 mM.

[0112] In some embodiments, the at least one stabilizer of the third solution is present at a concentration of about 100 mM to about 1 M, about 200 mM to about 1 M, about 300 mM to about 1 M, about 400 mM to about 1 M, about 500 mM to about 1 M, about 600 mM to about 1 M, about 700 mM to about 1 M, about 800 mM to about IM, about 900 mM to about IM, about 1 mM to about 900 mM, about 100 mM to about 900 mM, about 200 mM to about 900 mM, about 300 mM to about 900 mM, about 400 mM to about 900 mM, about 500 mM to about 900 mM, about 600 mM to about 900 mM, about 700 mM to about 900 mM, about 800mM to about 900 mM, about 1 mM to about 800 mM, about 100 mM to about 800 mM, about 200 mM to about 800 mM, about 300 mM to about 800 mM, about 400 mM to about 800 mM, about 500 mM to about 800 mM, about 600 mM to about 800 mM, about 700 mM to about 800 mM, about 1 mM to about 700 mM, about 100 mM to about 700 mM, about 200 mM to about 700 mM, about 300 mM to about 700 mM, about 400 mM to about 700 mM, about 500 mM to about 700 mM, about 600 mM to about 700 mM, about 1 mM to about 600 mM, about 100 mM to about 600 mM, about 200 mM to about 600 mM, about 300 mM to about 600 mM, about 400 mM to about 600 mM, about 500 mM to about 600 mM, about 1 mM to about 500 mM, about 100 mM to about 500 mM, about 200 mM to about 500 mM, about 300 mM to about 500 mM, about 400 mM to about 500 mM, about 1 mM to about 400 mM, about 100 mM to about 400 mM, about 200 mM to about 400 mM, about 300 mM to about 400 mM, about 1 mM to about 300 mM, about 100 mM to about 300 mM, about 200 mM to about 300 mM, about 1 mM to about 200 mM, about 100 mM to about 200 mM, or about 1 mM to about 100 mM.

[0113] In some embodiments, the third solution described herein comprises at least one crosslinker. In some embodiments, the cross-linker is a chemical cross-linker. In some embodiments, the third solution comprises at least one cross-linker at a concentration of about 1 pM to about 100 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 mM, or any value or range therein). In some embodiments, the at least cross-linker is present at a concentration of about 100 pM to about 100 mM, about 500 pM to about 100 mM, about 1 mM to about 100 mM, about 10 mM to about 100 mM, about 15 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, about 30 mM to about 100 mM, about 35 mM to about 100 mM, about 45 mM to about 100 mM, about 50 mM to about 100 mM, about 55 mM to about 100 mM, about 60 mM to about 100 mM, about 65 mM to about 100 mM, about 70 mM to about 100 mM, about 75 mM to about 100 mM, about 80 mM to about 100 mM, about 85 mM to about 100 mM, about 90 mM to about 100 mM, about 95 mM to about 100 mM, 1 pM to about 75 mM, about 100 pM to about 75 mM, about 500 pM to about 75 M, about 1 mM to about 75mM, about 10 mM to about 75 mM, about 15 mM to about 75 mM, about 20 mM to about75 mM, about 25 mM to about 75 mM, about 30 mM to about 75 mM, about 35 mM to about75 mM, about 45 mM to about 75 mM, about 50 mM to about 75 mM, about 55 mM to about75 mM, about 60 mM to about 75 mM, about 65 mM to about 75 mM, about 70 mM to about75 mM, 1 pM to about 50 mM, about 100 pM to about 50 mM, about 500 pM to about 50 M, about 1 mM to about 50 mM, about 10 mM to about 50 mM, about 15 mM to about 50 mM,about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 50 mM, about 45 mM to about 50 mM, 1 pM to about 25 mM, about 100 pM to about 25 mM, about 500 pM to about 25 M, about 1 mM to about 25 mM, about 10 mM to about 25 mM, about 15 mM to about 25 mM, about 20 mM to about 25 mM, 1 pM to about 1 mM, about 100 pM to about 1 mM, about 500 pM to about 1 M, 1 pM to about 500 pM, or about 100 pM to about 500 pM. In some embodiments, the third solution described herein does not comprise a cross-linker.

[0114] In some embodiments, the third solution described herein comprises at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris- EDTA buffer solution, water, distilled water, phosphate (e.g. sodium phosphate), acetate, and / or double distilled water. In some embodiments, the dried composition has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the polysaccharide solutions comprise at least one buffer at a concentration of about ImM to about IM (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM, or any value or range therein). In some embodiments, the at least one stabilizer is present at a concentration of about 100 mM to about 1 M, about 200 mM to about 1 M, about 300 mM to about 1 M, about 400 mM to about 1 M, about 500 mM to about 1 M, about 600 mM to about 1 M, about 700 mM to about 1 M, about 800 mM to about IM, about 900 mM to about IM, about 1 mM to about 900 mM, about 100 mM to about 900 mM, about 200 mM to about 900 mM, about 300 mM to about 900 mM, about 400 mM to about 900 mM, about 500 mM to about 900 mM, about 600 mM to about 900 mM, about 700 mM to about 900 mM, about 800 mM to about 900 mM, about 1 mM to about 800 mM, about 100 mM to about 800 mM, about 200 mM to about 800 mM, about 300 mM to about 800 mM, about 400 mM to about 800 mM, about 500 mM to about 800 mM, about 600 mM to about 800 mM, about 700 mM to about 800 mM, about 1 mM to about 700 mM, about 100 mM to about 700 mM, about 200 mM to about 700 mM, about 300 mM to about 700 mM, about 400 mM to about 700 mM, about 500 mM to about 700 mM, about 600 mM to about 700 mM, about 1 mM to about 600 mM, about 100 mM to about 600 mM, about 200 mM to about 600 mM, about 300 mM to about 600 mM, about 400 mM to about 600 mM, about 500 mM to about 600 mM, about 1 mM to about 500 mM, about 100 mM to about 500 mM, about 200 mM to about 500 mM, about 300 mM to about 500 mM, about 400 mM to about 500 mM, about 1 mM to about 400 mM, about 100 mM to about 400 mM, about 200 mM to about 400mM, about 300 mM to about 400 mM, about 1 mM to about 300 mM, about 100 mM to about 300 mM, about 200 mM to about 300 mM, about 1 mM to about 200 mM, about 100 mM to about 200 mM, or about 1 mM to about 100 mM. In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0115] In some embodiments, the third solution described herein comprises at least one NSAID. In some embodiments, the third solution comprise at least one NSAID at a concentration of about 0.01 mg / mL to about 0.05 mg / mL (e.g., 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, and 0.05 mg / mL, or any value or range therein). In some embodiments, an NSAID is present at a concentration of about 0.015 mg / mL to about 0.05 mg / mL, about 0.02 mg / mL to about 0.05 mg / mL, about 0.025 mg / mL to about 0.05 mg / mL, about 0.03 mg / mL to about 0.05 mg / mL, about 0.035 mg / mL to about 0.05 mg / mL, about 0.04 mg / mL to about 0.05 mg / mL, about 0.045 mg / mL to about 0.05 mg / mL, about 0.01 mg / mL to about 0.045 mg / mL, about 0.015 mg / mL to about 0.045 mg / mL, about 0.02 mg / mL to about 0.045 mg / mL, about 0.025 mg / mL to about 0.045 mg / mL, about 0.03 mg / mL to about 0.045 mg / mL, about 0.035 mg / mL to about 0.045 mg / mL, about 0.04 mg / mL to about 0.045 mg / mL, about 0.01 mg / mL to about 0.04 mg / mL, about 0.015 mg / mL to about 0.04 mg / mL, about 0.02 mg / mL to about 0.04 mg / mL, about 0.025 mg / mL to about 0.04 mg / mL, about 0.03 mg / mL to about 0.04 mg / mL, about 0.035 mg / mL to about 0.04 mg / mL, about 0.01 mg / mL to about 0.035 mg / mL, about 0.015 mg / mL to about 0.035 mg / mL, about 0.02 mg / mL to about 0.035 mg / mL, about 0.025 mg / mL to about 0.035 mg / mL, about 0.03 mg / mL to about 0.035 mg / mL, about 0.01 mg / mL to about 0.03 mg / mL, about 0.015 mg / mL to about 0.03 mg / mL, about 0.02 mg / mL to about 0.03 mg / mL, about 0.025 mg / mL to about 0.03 mg / mL, about 0.01 mg / mL to about 0.025 mg / mL, about 0.015 mg / mL to about 0.025 mg / mL, about 0.02 mg / mL to about 0.025 mg / mL, about 0.01 mg / mL to about 0.02 mg / mL, about 0.015 mg / mL to about 0.02 mg / mL, or about 0.01 mg / mL to about 0.015 mg / mL.

[0116] In some embodiments, the third solution described herein comprises at least one local anesthetic. In some embodiments, the local anesthetic is lidocaine. In some embodiments, the third solution comprise at least one local anesthetic at a concentration of about 0.1 mg / mL to about 1 mg / mL (e.g., 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 mg / mL, or any value or range therein). In some embodiments, a local anesthetic is present at a concentration of about 0.2 mg / mL to about 1 mg / mL, about 0.3 mg / mLto about 1 mg / mL, about 0.4 mg / mL to about 1 mg / mL, about 0.5 mg / mL to about 1 mg / mL, about 0.6 mg / mL to about 1 mg / mL, about 0.7 mg / mL to about 1 mg / mL, about 0.8 mg / mL to 1 mg / mL, about 0.9 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 0.9 mg / mL, about 0.2 mg / mL to about 0.9 mg / mL, about 0.3 mg / mL to about 0.9 mg / mL, about 0.4 mg / mL to about 0.9 mg / mL, about 0.5 mg / mL to about 0.9 mg / mL, about 0.6 mg / mL to about 0.9 mg / mL, about 0.7 mg / mL to about 0.9 mg / mL, about 0.8 mg / mL to 0.8 mg / mL, about 0.1 mg / mL to about 08. mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.3 mg / mL to about 0.8 mg / mL, about 0.4 mg / mL to about 0.8 mg / mL, about 0.5 mg / mL to about 0.8 mg / mL, about 0.6 mg / mL to about 0.8 mg / mL, about 0.7 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.7 mg / mL, about 0.2 mg / mL to about 0.7 mg / mL, about 0.3 mg / mL to about 0.7 mg / mL, about 0.4 mg / mL to about 0.7 mg / mL, about 0.5 mg / mL to about 0.7 mg / mL, about 0.6 mg / mL to about 0.7 mg / mL, about 0.1 mg / mL to about 0.6 mg / mL, about 0.2 mg / mL to about 0.6 mg / mL, about 0.3 mg / mL to about 0.6 mg / mL, about 0.4 mg / mL to about 0.6 mg / mL, about 0.5 mg / mL to about 0.6 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.2 mg / mL to about 0.5 mg / mL, about 0.3 mg / mL to about 0.5 mg / mL, about 0.4 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 0.4 mg / mL, about 0.2 mg / mL to about 0.4 mg / mL, about 0.3 mg / mL to about 0.4 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.2 mg / mL to about 0.3 mg / mL, or about 0.1 mg / mL to about 0.2 mg / mL. In some embodiments, the third solution comprises at least one local anesthetic at a concentration of about 0.3% w / v. In some embodiments, the local anesthetic is present at a concentration of about 0.2% w / v to about 0.5% w / v, about 0.25% w / v to about 0.5% w / v, about 0.3% w / v to about 0.5% w / v, about 0.2% w / v to about 0.45% w / v, about 0.25% w / v to about 0.45% w / v, about 0.3% w / v to about 0.45% w / v, about 0.2% w / v to about 0.4% w / v, about 0.25% w / v to about 0.4% w / v, about 0.3% w / v to about 0.4% w / v, about 0.2% w / v to about 0.35% w / v, about 0.25% w / v to about 0.35% w / v, about 0.3% w / v to about 0.35% w / v, about 0.2% w / v to about 0.3% w / v, or about 0.2.5% w / v to about 0.45% w / v.

[0117] At step 106 of process 102, solutions of POP and polysaccharide and, optionally, third solution, are mixed below the Tt-heating of the POP to form a composition. In some embodiments, solutions of POP and polysaccharide are mixed, wherein the polysaccharide is hyaluronic acid. In some embodiments, the POP is added to the mixture concurrently with the polysaccharide. In some embodiments, the POP is added to the mixture before the polysaccharide. In other embodiments, the POP is added to the mixture after the polysaccharide. In some embodiments, the POP is mixed at higher levels with the polysaccharide. In some embodiments, the POP is mixed at the same level with thepolysaccharide. In some embodiments, the POP is mixed at a lower level with the polysaccharide. In some embodiments, only the POP is in the mixture. In some embodiments, the POP and polysaccharide are present in a ratio of 95:5, 90: 10, 70:30, 50:50, 30:70, 10:90, or 5:95 (POP:polysaccharide), including all ranges, subranges and values therebetween. In some embodiments, a third solution can be added to the mixture. In some embodiments, the third solution is added to the mixture concurrently with the POP, polysaccharide or POP / polysaccharide solution. In some embodiments, the third solution is added to the mixture before the POP, polysaccharide or POP / polysaccharide solution. In some embodiments, the third solution is added to the mixture after the POP, polysaccharide or POP / polysaccharide solution. The third solution can be present in a ratio of 95:5, 90: 10, 70:30, 50:50, 30:70, 10:90, or 5:95 (POP / polysaccharide:third solution), including all ranges, subranges and values therebetween.

[0118] In some embodiments, the POP solution is mixed with a polysaccharide solution below the Tt-heating of the POP to form a composition and is then administered to a subject. In some embodiments, a third solution can be mixed with the POP solution or the POP solution in combination with the polysaccharide solution to form a composition below the Tt-heating of the POP. In some embodiments, solutions of POP and polysaccharide are mixed, wherein the polysaccharide is hyaluronic acid. In some embodiments no polysaccharide is added to the mixture.

[0119] As stated above with reference to step 107 of method 100, the composition is administered to the subject in a region of interest. In some embodiments, the POP is administered concurrently with the polysaccharide. In some embodiments, the POP is administered prior to the polysaccharide. In some embodiments, the POP is administered after the polysaccharide. In some embodiments, the POP is not administered with a polysaccharide. In some embodiments, the third solution is administered concurrently with the POP or POP / polysaccharide solution. In some embodiments, the third solution is administered before the POP or POP / polysaccharide solution. In some embodiments, the third solution is administered after the POP or POP / polysaccharide solution.Uses of a matrix filler compositions described herein

[0120] The method described herein comprises the use of an amount of a matrix filler composition of the present disclosure in a subject in need thereof.

[0121] In some embodiments, the compositions described herein can be administered to augment shapes or volumes in a region of interest of a subject. Specific exemplary shapes orvolumes that could be augmented include breasts, buttocks, hands, knees, 2-dimensional layers for placing under skin or skin grafts, or other irregular or indefinite shapes or volumes present in a subject's body.

[0122] In some embodiments, the matrix filler compositions described herein retain shape at least 2-times longer than otherwise similar compositions comprising polysaccharide or hyaluronic alone. In some embodiments, compositions comprising the POP retains shape at least 3-times, 4-times, 5-times, 6-times, 7-times, 8-time, 9-times, or 10-times longer, or any value or range therein. In some embodiments, the compositions described herein comprising the POP retain shape within a 10% deviation over a period of time. In some embodiments, the period of time comprises about 1 weeks about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 moths, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months, or any value or range therein.

[0123] In some embodiments, the matrix filler compositions retain volume at least 2-times longer than the compositions comprising polysaccharide or hyaluronic alone. In some embodiments, the compositions comprising the POP retain volume at least 3-times, 4-times, 5- times, 6-times, 7-times, 8-time, 9-times, or 10-times longer, or any value or range therein. In some embodiments, the compositions described herein comprising the POP retain volume within a 10% deviation over a period of time. In some embodiments, the period of time comprises about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 moths, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months, or any value or range therein. In some embodiments, the matrix filler comprises a preadministration volume and a post-administration volume.

[0124] In some embodiments, an autologous fat graft is augmented by the combination of the adipose tissue with a POP which leads to the enhanced long-term maintenance and survival of fat graft including conservation of the volume and shape of the fat graft, improved vascularization, enhanced survival, and reduction in necrosis.

[0125] In some embodiments, a dermal tissue filler is augmented for use in, but not limited to, correcting moderate-to-severe facial wrinkles and skin folds, increasing fullness of lips, cheeks,chin, under-eye hollows, jawline and back of hand, restoring facial fat loss, and correcting acene scars on the cheek.

[0126] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.EXAMPLESEXAMPLE 1 - Mechanical Evaluation of partially ordered polypeptide (POP) and Polysaccharide Mixtures

[0127] Material properties resulting from mixtures of POP and a polysaccharide were evaluated. The assessed mixtures comprised either 0.75 mM (37.5 mg / mL) POP or 1.5 mM (75 mg / mL) POP and a polysaccharide, which included either a photo-crosslinked hyaluronic acid (MeHA), an uncrossed hyaluronic acid (HA), or chondroitin sulfate (CS). MeHA was present in the mixture at a concentration of 3% w / v (30 mg / mL) and HA was present at a concentration of 2.5% w / v (25 mg / mL). Starting solutions of POP and polysaccharides were suspended together in lx PBS to achieve the indicated concentrations. No lidocaine was used in these experiments. The photoinitiator used with the MeHA was lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) at a 1 mM final concentration. Only lx PBS (no photoinitiator) was used with the uncrosslinked HA and CS.

[0128] The mechanical stiffness of the mixtures was evaluated using monotonic compression. Matrices were formed from the mixtures by injecting a volume of the POP and polysaccharide mixtures into a pre-defined cylindrical mold. Once filled, each mold was placed in a 37°C incubator for 5 minutes to allow for aggregation, or phase transition, of the POP to occur. Each aggregate was removed and placed on a TA Instrument RSA III microstain analyzer. An 8 mm pressure plate was then lowered into initial contact with the top of the aggregate as determined by a force read of 0.1-1. ON on the instrument. The compression of the aggregates was carried out to a strain of 30% over the course of three minutes. The Young’s modulus was then calculated from the 0-5% strain range, determined to be the linear viscoelastic region. Images of the resulting material products are shown in Fig. 3. The stiffness of these compositions was quantified in Fig. 4 and Table 2. As shown, the MeHA at 3% w / v showed the highest stiffness at 1558.0 ± 282.7 Pa, while MeHA + 0.75 mM POP and MeHA + 1.5 mM POP had measures of 1023.0 ± 253.4 Pa and 1137.3 ± 177.3 Pa, respectively. Scanning electron microscopy images for the mixtures with the highest stiffness are shown in Figs. 5A-5D.Table 1. Compression testing of the polysaccharide mixtures with Fractomer at variable concentrations.EXAMPLE 2 - Biological Evaluation of POP and Polysaccharide Mixtures

[0129] Additional studies were performed to assess the biocompatibility of POP versus Juvederm®, a predominantly HA product, alone as well as together on tissue physiology. To this end, POP and Juvederm® were mixed in a range of ratios including 100% POP:0% Juvederm®, 90: 10, 70:30, 50:50, 30:70, and 0:100. Fractomer solution at a concentration of 0.75 mM (37.5 mg / mL) was added in appropriate volumes to each mixture. Similarly, Juvederm® solutions at a concentration of 2.5% w / v (25 mg / mL) were added in appropriate volumes to each mixture. For each mixture, 200 pL was injected into the hind flank of BL / 6 mice (n=5) and the volumes at the injection sites were monitored over 9 weeks using regularelectronic caliper measurements. The Juverderm® alone injection consisted of 200 pL of the Juvederm® solution, while varying amounts were used for the different ratio mixtures to achieve 200 pL per injection. All groups had statistically equivalent volume retention after the 9 weeks (Fig. 6A), however, higher POP content was associated with lower variance in the final volumes (Fig. 6B). Also, after 9 weeks, tissue at the injection sites was harvested for histological analysis of cell infiltration and immune response. Surprisingly, the POP injections showed almost no swelling and minimal variation in murine models (Fig. 6C), both of which are desirable attributes for a dermal filler.

[0130] To further investigate the observed lack of swelling, histological analyses were performed on select injection groups including: (1) 100% POP:0% Juvederm®; (2) 50% POP:50% Juvederm®; and (3) 0% POP: 100% Juvederm®. Results are shown in Fig. 6D. As shown, the lack of swelling was likely driven by an overall reduced immune response to POP compared to Juvederm®.

[0131] Moreover, a study was conducted to assess vascular ingrowth among injection groups 1 and 3. Randomly identified regions from the injection sites were quantitatively assessed for vascular growth. The POP only group (1) demonstrated a statistically 3-fold higher blood vessel growth than the Juvederm® only group (3) (Fig. 7A). Another study utilized a cell counter algorithm on H&E stained slides to determine the total cell infiltration in injection groups 1, 2 and 3. Healthy subcutaneous fatwas quantified for a comparison. Results are shown in Fig. 7B. Surprisingly, similar cell infiltration numbers were observed in the POP only and Juvederm® only groups. Additional imaging analysis was conducted to assess the cell infiltrate and inflammatory responses. Results are shown in Fig. 7C. Although, cell infiltration numbers were similar for all injection groups, the composition of the infiltrate substantially differed between inj ections groups 1 and 2 versus group 3. The POP only inj ection tended to have higher infiltration of macrophages, fibroblast, and endothelial cells, indicative of a remodeling response, in comparison to the Juvederm® only injection which showed a predominantly neutrophil driven acute inflammatory response. Interestingly, injection group 2, primarily resembled the favorable POP only remodeling response. This observation was consistent regardless of the POP ratio mixture (data not shown). None of the injection groups showed fibrosis or encapsulation.

[0132] POP only injections demonstrated a surprising level of vascularization in comparison to Juvederm®, one of the most well recognized commercially available soft tissue repair matrices. Ultimately, the incorporation of POP alone or in combination with a polysaccharide like HA, the main component of Juvederm®, improved volume retention, vascularization, andremodeling responses in comparison to Juvederm® / HA alone. These findings indicate that POP injections alone or POP mixed with a very small amount of HA innovate upon the existing injectable dermal filler space.

[0133] Exemplary Sequences of Motifs and POP Constructs:

[0134] SEQ ID NO: 1

[0135] [GXGVP]n

[0136] where X is any amino acid except proline and where n is an integer equal to or greater than 1.

[0137] SEQ ID NO: 2

[0138] [GXGVP]n

[0139] where X is Ala or Vai and where n is an integer equal to or greater than 1.

[0140] SEQ ID NO: 3

[0141] (A)n

[0142] where n is an integer from 2 to 100.

[0143] SEQ ID NO: 4

[0144] K(A)nK

[0145] where n is an integer from 2 to 100.

[0146] SEQ ID NO: 5

[0147] D(A)nK

[0148] where n is an integer from 2 to 100.

[0149] SEQ ID NO: 6

[0150] GD(An)K

[0151] where n is an integer from 2 to 100.

[0152] SEQ ID NO: 7

[0153] GK(An)K

[0154] where n is an integer from 2 to 100.

[0155] SEQ ID NO: 8

[0156] (A)25

[0157] SEQ ID NO: 9

[0158] K(A)25K

[0159] SEQ ID NO: 10

[0160] D(A)25K

[0161] SEQ ID NO: 11

[0162] GD(A25)K

[0163] SEQ ID NO: 12

[0164] GK(A25)K

[0165] SEQ ID NO: 13

[0166] M[(GVGVP)i5-GD(A25)K]6-GWP

[0167] SEQ ID NO: 14

[0168] M[(GVGVP)i5-GD(A25)K]4-GWP

[0169] SEQ ID NO: 15

[0170] M[(GVGVP)i5-GK(A25)K]6-GWP

[0171] SEQ ID NO: 16

[0172] M[(GVGVP)i5-GK(A25)K]4-GWP

[0173] SEQ ID NO: 17

[0174] M[(G[A1 : Vl]GVP)i6-GD(A25)K]6-GWP

[0175] MGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGWP

[0176] SEQ ID NO: 18

[0177] M[(G[A1 : Vl]GVP)i6-GD(A25)K]4-GWP

[0178] MGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVP GVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAA AAAAAAAAAKGWP

[0179] SEQ ID NO: 19

[0180] M[(G[V4:Al]GVP)i5-GD(A25)K]6-GWP

[0181] MGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAG VPGVGVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGV GVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPG VGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPG VGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVP GVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVP GVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGV PGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVPGVGV PGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVG VPGAGVPGDAAAAAAAAAAAAAAAA AAAAAAAAAKGWP

[0182] SEQ ID NO: 20

[0183] M[(G[V4:Al]GVP)i5-GD(A25)K]4-GWP

[0184] MGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAG VPGVGVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGV GVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGV GVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPG VGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPG VGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVP GVGVPGVGVP GAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGWP

[0185] SEQ ID NO: 21

[0186] P(X)nG

[0187] where n is an integer from 1 to 15 and X is any amino acid.

[0188] SEQ ID NO: 22

[0189] (B)mP(X)nG(Z)p

[0190] where m, n, and p are independently an integer from 1 to 15 and B, X, and Z are independently any amino acid.

[0191] SEQ ID NO: 23

[0192] [Bp(A)qZr]n

[0193] where B is Lys, Arg, Asp, or Glu, Z is Lys, Arg, Asp, or Glu, n is an integer from 1 to 50, p is an integer from 0 to 2, q is an integer from 1 to 50, and r is an integer from 0 to 2.

[0194] SEQ ID NO: 24

[0195] [(BAs)tZr]n

[0196] where B is Lys, Arg, Asp, or Glu, Z is Lys, Arg, Asp, or Glu, n is an integer from 1 to 50, r is an integer from 0 to 2, s is an integer from 1 to 5, and t is an integer from 1 to 50.

[0197] SEQ ID NO: 25

[0198] [GVGVP]n

[0199] where n is an integer from 1 to 50.

[0200] SEQ ID NO: 26

[0201] [GAGVP]n

[0202] where n is an integer from 1 to 50.

[0203] SEQ ID NO: 27

[0204] [G[Al:Vl]GVP]n

[0205] where n is an integer from 1 to 50. When n is 1, SEQ ID NO: 27 is GAGVPGVGVP.

[0206] SEQ ID NO: 28

[0207] [G[V4:Al]GVP]n

[0208] where n is an integer from 1 to 50. When n is 1, SEQ ID NO: 28 isGVGVPGVGVPGVGVPGVGVPGAGVP.

[0209] SEQ ID NO: 29

[0210] (A)m

[0211] where m is an integer from 5 to 50.

[0212] SEQ ID NO: 30

[0213] [(SEQ ID NO: l)n-a-helix]m

[0214] where n is an integer equal to or greater than 1, m is an integer equal to or greater than 1, and a-helix is any polyalanine based a-helix having about 5 to 50 alanine residues.

[0215] SEQ ID NO: 31

[0216] [(SEQ ID NO: 2)n-GX1(A)25X1]m

[0217] where X1is K or D, n is an integer from 10 to 20, and m is an integer from 4 to 8.

Claims

CLAIMS1. A matrix filler composition, comprising: a partially ordered polypeptide; and a polysaccharide; wherein the partially ordered polypeptide and the polysaccharide are present in a ratio of about 95:5 to about 5:95 (partially ordered polypeptide:polysaccharide).

2. The matrix filler composition of claim 1, wherein the partially ordered polypeptide comprises a plurality of disordered domains and a plurality of ordered domains.

3. The matrix filler composition of claims 1, wherein each ordered domain independently comprises a polyalanine motif or a polyproline motif.

4. The matrix filler composition of claims 2 or 3, wherein each ordered domain independently comprises a polyalanine motif.

5. The matrix filler composition of any one of claims 2-4, wherein each ordered domain independently comprises an alpha helix.

6. The matrix filler composition of claims 3 or 4, wherein the polyalanine motif comprises (A)m, wherein m is an integer from 5 to 50.

7. The matrix filler composition of any one of claims 3-6, wherein the polyalanine motif comprises one or more of:(A)n (SEQ ID NO: 3);K(A)nK (SEQ ID NO: 4);D(A)nK (SEQ ID NO: 5);GD(An)K (SEQ ID NO: 6); orGK(An)K (SEQ ID NO: 7), wherein n is an integer from 2 to 100.

8. The matrix filler composition of claim 7, wherein the polyalanine motif comprises one or more of:(A)25(SEQ ID NO: 8);K(A)25K (SEQ ID NO: 9);D(A)25K (SEQ ID NO: 10);GD(A25)K (SEQ ID NO: 11); orGK(A25)K (SEQ ID NO: 12).

9. The matrix filler composition of any one of claims 2-8, wherein each disordered domain independently comprises a PG or GP motif.

10. The matrix filler composition of any one of claims 2-9, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n, wherein X is any amino acid except proline and n is an integer greater than or equal to 1.

11. The matrix filler composition of any one of claims 2-10, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n, wherein each X is independently Vai or Ala, and wherein n is an integer from 1 to 50.

12. The matrix filler composition of claim 11, wherein a ratio of Ala to Vai in the amino acid sequence of (GXGVP)n ranges from 10: 1 to 1 : 10.

13. The matrix filler composition of claim 1, wherein the polysaccharide is hydrophilic.

14. The matrix filler composition of claim 1 or 13, wherein the polysaccharide comprises at least one glycosaminoglycan.

15. The matrix filler composition of claim 14, wherein the at least one glycosaminoglycan comprises one or more residue of a heparin sulfate, a chondroitin sulfate, a dermatan sulfate, and a hyaluronic acid.

16. The matrix filler composition of claim 14, wherein the at least one glycosaminoglycan comprises hyaluronic acid.

17. The matrix filler composition of claim 16, wherein the hyaluronic acid is crosslinked.

18. The matrix filler composition of claims 16 or 17, wherein the hyaluronic acid is a crosslinked hyaluronic acid present in an amount of about 1 mg / mL to about 500 mg / ml.

19. The matrix filler composition of claims 17 or 18, wherein the crosslinked hyaluronic acid is present in an amount of about 1 mg / mL to about 150 mg / ml.

20. The matrix filler composition of any one of claims 16-19, wherein the partially ordered polypeptide and the hyaluronic acid are present in a ratio of 95:5, 90: 10, 70:30, 50:50, 30:70, 10:90, or 5:95 (partially ordered polypeptide:hyaluronic acid).

21. The matrix filler composition of claim 1, wherein the partially ordered polypeptide has a transition temperature of heating (Tt-heating) and a transition temperature of cooling (Tt- cooling).

22. The matrix filler composition of claim 21, wherein the Tt-heating is dependent on the concentration of the partially ordered polypeptide in the composition.

23. The matrix filler composition of claims 21 or 22, wherein the Tt-heating and the Tt- cooling range from about 10 °C to about 45 °C.

24. The matrix filler composition of any one of claims 21-23, wherein the partially ordered polypeptide forms a solid aggregate above the Tt-heating.

25. The matrix filler composition of claims 24, wherein the solid aggregate resolubilizes when cooled to below the Tt-cooling.

26. The matrix filler composition of claims 24 or 25, wherein the solid aggregate is a stable three-dimensional matrix including the polysaccharide.

27. The matrix filler composition of any one of claims 24-26, wherein the solid aggregate comprises a plurality of micropores.

28. The matrix filler composition of any one of claims 1-27, wherein the composition comprises between about 200 pM and about 2.5 mM of the partially ordered polypeptide.

29. The matrix filler composition of any one of claims 1-28, wherein the composition is a liquid or semisolid.

30. The matrix filler composition of any one of claims 1-29, wherein the composition is shaped.

31. The matrix filler composition of claim 1, wherein the composition further comprises a surfactant, stabilizer, buffer, linker, non-steroidal anti-inflammatory drug, or anesthetic.

32. The matrix filler composition of claim 31, wherein the anesthetic is lidocaine.

33. A method of forming a matrix filler composition in a subject, the method comprising: mixing an amount of a partially ordered polypeptide and an amount of polysaccharide to form a composition, thereby forming a matrix filler composition, and administering an amount of the matrix filler composition to the subject.

34. The method of claims 33, wherein the matrix filler composition is below the Tt-heating of the partially ordered polypeptide and the matrix filler composition forms a solid after exposure to the subject’s body temperature, which is above the Tt-heating of the partially ordered polypeptide.

35. The method of claims 33, wherein the matrix filler composition is administered to a subject when the mixture is above the Tt-heating of the partially ordered polypeptide and the mixture forms a solid prior to administration to the subject.

36. A method of forming an autologous fat graft in a subject, the method comprising: mixing an amount of a partially ordered polypeptide and an amount of polysaccharide to form a composition, thereby forming a matrix filler composition; and administering to the subject an amount of the composition.

37. The method of claim 36, wherein the method further comprises mixing an amount of an adipose tissue with the matrix filler composition prior to administering the amount of the matrix filler composition to the subject.

38. The method of claim 37, wherein the adipose tissue comprises an autologous fat.

39. The method of claims 36, comprising mixing the partially ordered polypeptide and the polysaccharide in vitro to form the matrix filler composition and implanting the mixture in the subject.

40. The method of claims 36, comprising adapting the mixture of the partially ordered polypeptide and the polysaccharide to a shape of an anatomical feature, then heating the mixture to the Tt-heating, to form a solid matrix filler composition.

41. The method of claims 37, comprising mixing the partially ordered polypeptide, the polysaccharide, and the adipose tissue in vitro to form a matrix filler composition and implanting the mixture in the subject.

42. The method of claim 41, the adipose tissue comprises an autologous fat.

43. The method of claim 36, comprising administering to the matrix filler composition when the matrix filler composition is below the Tt-heating of the partially ordered polypeptide, wherein the matrix filler composition forms a solid after exposure to the subject’s body temperature, which is above the Tt-heating of the partially ordered polypeptide.

44. The method of claim 36, comprising heating the matrix filler composition above the Tt- heating of the partially ordered polypeptide in vitro to form a solid matrix filler composition, then implanting the solid matrix filler composition in the subject.

45. The method of claim 36, wherein the administering comprises injecting the amount of the composition into the subject.

46. Use of an amount of a partially ordered polypeptide and an amount of polysaccharide as a tissue matrix filler in a subject in need thereof.

47. A matrix composition, comprising: a polypeptide comprisinga plurality of disordered domains and a plurality of ordered domains, wherein each disordered domain independently comprises a PG or GP motif and each ordered domain independently comprises an alpha helix; and a polysaccharide comprising a crosslinked hyaluronic acid present at a molecular weight of about 1 kDa to about 3,000 kDa; wherein about 1 mg / mL to about 150 mg / mL of the matrix filler composition is administered to the subject; and wherein the matrix composition augments a shape and / or volume of an anatomic region of the subject.

48. The matrix filler composition of claim 1, wherein the partially ordered polypeptide and the polysaccharide are present in a ratio of about 90: 10 to about 50:50 (partially ordered polypeptide:polysaccharide).

49. The matrix filler composition of claim 1, comprising about 10% to about 50% polysaccharide.

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