Polyamino acid, preparation method for polyamino acid, and use of polyamino acid as biocompatible filling material

Abstract

The present invention provides a polyamino acid, a method for preparing the polyamino acid, and a use of the polyamino acid as a biocompatible filling material. The polyamino acid is prepared by ring-opening copolymerization of an N-carboxyanhydride, and has similar composition and sequence and similar physical and chemical properties to collagen.

Description

Polyamino acid, preparation method thereof and use as biocompatible filling material

[0001] This application claims priority to Chinese patent application No. 202410263708.X filed on March 7, 2024. The contents of the above-mentioned Chinese patent application disclosure are hereby incorporated by reference in their entirety as a part of this application. Technical Field

[0002] The present disclosure relates to the field of polymers, and in particular to a polyamino acid, a method for preparing the polyamino acid, and use of the polyamino acid as a biocompatible filling material. Background Art

[0003] In modern life, with the pursuit of beauty and the advancement of technology, more and more people are seeking to improve their appearance and retain their youth through medical aesthetics (medical aesthetics). Filling with fillers to improve facial depressions or reduce or even eliminate wrinkles is a popular medical aesthetics method. Common medical aesthetics filler methods include hyaluronic acid fillers, autologous fat fillers, and collagen fillers. Collagen fillers are popular among medical aesthetics enthusiasts due to their excellent biocompatibility, moisturizing, wrinkle-resistant, repairing, and body-shaping properties.

[0004] Collagen is the most important type of protein in animals, and has many functions, including structural support, tissue repair, and physical protection. The most characteristic feature of collagen's unique structural structure - the helical segment - is the amino acid sequence - [glycine (Gly) -XY] n -, where X and Y can represent any amino acid, but typically X is proline (Pro) and Y is hydroxyproline (Hyp). Hyp is crucial for the proper formation of collagen because its trans hydroxyl group can stabilize the secondary helical structure of the collagen molecule by forming intramolecular hydrogen bonds.

[0005] The collagen currently used in medical treatment mainly includes natural collagen extracted from animal tissue (animal-derived collagen) and recombinant humanized collagen obtained through biosynthesis. The production capacity of animal-derived collagen is limited and its immunogenicity cannot be eliminated. The production of recombinant humanized collagen requires a large number of screening for suitable strains, which is a complex process and high cost. Moreover, the synthetic recombinant humanized collagen does not contain Hyp, which is crucial for the correct formation of collagen, and its composition is significantly different from that of natural collagen.

[0006] Therefore, there is a need to develop a polyamino acid product that can be chemically synthesized, has a composition and / or properties similar to natural collagen, and can avoid problems such as immunogenicity and endotoxins. Summary of the Invention

[0007] To solve the above and other problems, the inventors designed and synthesized a polyamino acid, which is a glycine (Gly)-proline (Pro)-hydroxyproline (Hyp) terpolymer.

[0008] In one aspect, at least one embodiment of the present disclosure provides a polyamino acid having a structure shown in formula (I):

[0009] R1-(L1) x -(L2) y -(L3) z -R2

[0010] Formula (I)

[0011] in,

[0012] L1 is

[0013] L2 is

[0014] L3 is

[0015] R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0016] R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2;

[0017] R1 and R2 are independently H or a terminal group;

[0018] Each occurrence of x, y, and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0019] In some examples, the polyamino acid

[0020] L1 is

[0021] L2 is

[0022] L3 is

[0023] In some examples, the polyamino acid

[0024] L1 is

[0025] L2 is

[0026] L3 is

[0027] In some examples, in the polyamino acid, R A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x -(L2) y -(L3) z -R2.

[0028] In some examples, in the polyamino acid, R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x -(L2) y -(L3) z -R2, and at least some R A Selected from C 1-6 Alkanoyl, C 6-10 Aroyl.

[0029] In some examples, at least some of the polyamino acids have A -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is acetyl.

[0030] In some examples, in the polyamino acid, R A Each occurrence is independently selected from C 1-6 Alkanoyl and C 6-10 Aroyl.

[0031] In some examples, in the polyamino acid, R A It is acetyl.

[0032] In some examples, in the polyamino acid, the weight average molecular weight of the polyamino acid is 5 to 100 kg / mol.

[0033] On the other hand, the present disclosure also provides a method for preparing polyamino acids, comprising: reacting the compounds represented by the following formulas (II) to (IV):

[0034] in,

[0035] R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl,

[0036] R A1 Selected from H, C 1-6 Alkanoyl and C 6-10 Aroyl;

[0037] In the presence of a base, the polymer is polymerized in an organic solvent to obtain a polyamino acid represented by formula (I):

[0038] R1-(L1) x -(L2) y -(L3) z -R2

[0039] Formula (I)

[0040] in,

[0041] L1 is

[0042] L2 is

[0043] L3 is

[0044] R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0045] R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2;

[0046] R1 and R2 are independently H or a terminal group;

[0047] Each occurrence of x, y, and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0048] In some examples, R a 、R b and R c Both are H.

[0049] In some examples, R A1 is H, and R A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z -R2.

[0050] In some examples, the method further comprises further acylating the polymerized polyamino acid of formula (I) to obtain an at least partially acylated polyamino acid.

[0051] In some examples, at least some R A1 is H, and at least some R A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl, and at least some R A Selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2.

[0052] In some examples, R A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl, and R A Selected from C 1-6 Alkanoyl and C 6-10 Aroyl.

[0053] In some examples, R A1 and R A All are acetyl groups.

[0054] In some examples, the base is selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof.

[0055] In some examples, the organic solvent is selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

[0056] On the other hand, embodiments of the present disclosure also provide use of any of the above-mentioned polyamino acids as a biocompatible filling material.

[0057] In some examples, the biocompatible filling material is a medical cosmetic filling material. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, rather than limiting the present disclosure.

[0059] FIG1 shows the size exclusion chromatography (SEC) curves of products prepared by using N,N-diisopropylethylamine (DIPEA) as a base and adding different equivalents of raw materials according to one embodiment of the present disclosure;

[0060] FIG2 shows the SEC curves of products prepared by using 4-dimethylaminopyridine (DMAP) as a base and adding different equivalents of raw materials according to another embodiment of the present disclosure;

[0061] Figure 3 shows the SEC curves of products prepared using different bases according to another embodiment of the present disclosure, wherein blue represents the base is DIPEA, red represents the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and orange represents the base is DMAP;

[0062] FIG4 shows the SEC curve of a copolymer prepared by using acetylated hydroxyproline N-carboxyl lactam (AcHypNCA) to replace hydroxyproline N-carboxyl lactam (HypNCA) in full or 50% according to yet another embodiment of the present disclosure;

[0063] Figure 5 shows the glycine-proline-hydroxyproline terpolymer (GPO terpolymer, wherein G represents a glycine unit, P represents a proline unit, and O represents a hydroxyproline unit) prepared using DIPEA as a base and a Gly:Pro:Hyp:base raw material ratio of 25:25:25:1 (DIPEA-25-25-25). 1 H-NMR (D2O);

[0064] Figure 6 shows the GPO terpolymer made using DIPEA-40-30-30 1 H-NMR (D2O);

[0065] Figure 7 shows the GPO terpolymer made using DIPEA-100-100-100 1H-NMR (D2O) circular dichroism spectrum;

[0066] FIG8 shows the infrared absorption spectra of a GPO terpolymer prepared using DIPEA-25-25-25 (blue) and after hydrolysis (red);

[0067] Figure 9 shows the changes in the aqueous SEC curves of the GPO terpolymer (red) prepared using DBU as a base and a Gly:Pro:Hyp:base raw material ratio of 100:100:100:1 (DBU-100-100-100) and after the addition of NaOH (blue);

[0068] FIG10 shows the GPO terpolymer prepared using DIPEA-25-25-25 and the addition of NaOH thereto. 1 H-NMR (D2O) changes;

[0069] FIG11 is a bar graph showing a comparison of the cell adhesion ability of GPO terpolymers prepared using DIPEA-25-25-25 and dipolymers (GO-DIPEA: glycine-hydroxyproline dipolymer; PO: proline-hydroxyproline dipolymer) and hyaluronic acid and bovine Achilles tendon collagen, with phosphate-buffered saline (PBS) used as a control;

[0070] FIG12 is a bar graph showing a comparison of the cell adhesion ability of GPO terpolymers with different amino acid content ratios, bovine Achilles tendon collagen, and PBS as controls, wherein: 11-DIPEA-40-30-30; 12-DIPEA-30-35-35; 13-DIPEA-20-40-40; 14-DIPEA-40-40-20; 15-DIPEA-40-20-40; 16-DIPEA-40-10-50;

[0071] FIG13 is a bar graph showing a comparison of cell adhesion ability of GPO terpolymers with different degrees of acetylation and bovine Achilles tendon collagen and PBS as controls, wherein 1 # -GPO-DIPEA-45:20:35;2 # -GPO-DIPEA-45:25:30;3 # -GPO-DIPEA-45:27.5:27.5;4 # -GP(AcO)-BnNH2-40:30:30;5 # -GP(AcO)-DIPEA-40:30:30;6 # -GPO-DIPEA-50:15:35;7 #-GPO(AcO)-DIPEA-40:30:15:15, wherein AcO represents an acetylated hydroxyproline unit. DETAILED DESCRIPTION

[0072] For the purpose of the following detailed description, it should be understood that the present disclosure may adopt various alternative variations and step sequences, unless expressly provided otherwise. In addition, except in any operating examples, or when otherwise indicated, all numerals representing the amount of the components used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary according to the desired performance to be obtained by the present disclosure. At least, it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0073] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0074] Furthermore, it should be understood that any numerical range recited herein is intended to include all subranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0075] In this disclosure, unless otherwise expressly stated, the use of the singular includes the plural and the plural includes the singular. Furthermore, in this disclosure, unless otherwise expressly stated, the use of "or" means "and / or," even though "and / or" could be explicitly used in certain circumstances. Furthermore, in this disclosure, unless otherwise expressly stated, the use of "a" or "an" means "at least one." For example, "a" polymer, "a" composition, etc., refers to one or more of any of these items.

[0076] In recent years, medical cosmetology, especially cosmetic fillers, has become an increasingly popular way for beauty enthusiasts to improve their appearance. Collagen is an indispensable nutrient naturally present in the skin. Collagen is considered one of the suitable cosmetic filler materials due to its good biocompatibility, moisturizing, anti-wrinkle, repairing and shaping properties. Currently, the collagen commonly used for medical / cosmetic purposes mainly includes natural collagen extracted from animal tissue (animal-derived collagen) and recombinant humanized collagen obtained through biosynthesis. The shortcomings of animal-derived collagen are its limited production capacity and the inability to eliminate immunogenicity; the problem with recombinant humanized collagen is that its production requires a large number of screening for suitable strains, the process is complex and the cost is high; and the synthetic recombinant humanized collagen does not contain Hyp, which is crucial for the correct formation of collagen, and its composition is quite different from that of natural collagen.

[0077] The inventors of the present application have designed and synthesized a polyamino acid after extensive efforts, which is a glycine-proline-hydroxyproline terpolymer.

[0078] The polyamino acid provided in the present application is prepared by ring-opening copolymerization (chemical synthesis) of N-carboxylic anhydride (NCA), and has a similar composition and sequence as well as similar physicochemical properties to collagen (especially the helical segment therein). Compared with animal-derived collagen, the polyamino acid avoids immunogenicity; compared with recombinant humanized collagen, it avoids the problem of difficulty in removing endotoxins in bacterial expression; and contains an important Hyp in the sequence structure, in which the trans hydroxyl group of the side chain provides a stable PPII helical structure. The polyamino acid provided in the present application has strong cell adhesion, low cytotoxicity and immunogenicity, and the ability to provide nutrients to promote collagen regeneration, and can be used as an effective substitute (collagen-like) for animal-derived collagen and recombinant humanized collagen. In addition, the polyamino acid also has many advantages such as simple synthesis steps, low cost, high yield, and can be modified after polymerization to upgrade the product.

[0079] As described herein, the term "polyamino acid" refers to a compound composed of multiple amino acid molecules linked by peptide bonds. It can be divided into homopolymeric amino acids formed by the polymerization of a single amino acid monomer and copolymeric amino acids formed by the copolymerization of two or more amino acid monomers. The basic building blocks (monomer units) of polyamino acids are amino acid molecules, each composed of an amino group, a carboxyl group, and one or more side chains, and linked into linear chains by peptide bonds. Polyamino acids have low immunogenicity, good degradability and mechanical properties, and controllable performance, and are therefore widely used in fields such as drug controlled release, tissue engineering, and regenerative medicine.

[0080] In some examples, the polyamino acid provided herein can be a terpolymer. As used herein, the term "terpolymer" refers to a copolymer formed by copolymerization of three different amino acid monomers. For example, the terpolymer provided herein can be a non-branched linear copolymer, that is, the copolymer can have a main chain formed by connecting three different monomers in any order and ratio, and does not have a branched polyamino acid side chain (but can have other functional groups, such as hydroxyl or amino protecting groups, such as acyl groups, etc.) on the main chain. Additionally, or alternatively, the terpolymer provided herein can also be a branched copolymer, that is, the polymer can have a main chain formed by connecting one or more or all of the three different monomers in any order and ratio, and further have one or more polyamino acid side chains as substituted side groups on the monomeric unit in the main chain. Wherein, the one or more polyamino acid side chains can also further have one or more polyamino acid side chains as substituted side groups, thereby forming a dendrimer structure.

[0081] In some examples, the polyamino acid described in the present disclosure can be a glycine-proline-hydroxyproline terpolymer, also known as a Gly-Pro-Hyp terpolymer or a GPO terpolymer, the above terms being used interchangeably herein. For example, the polyamino acid described in the present disclosure can have a structure represented by the following formula (I):

[0082] R1-(L1) x -(L2) y -(L3) z -R2

[0083] Formula (I)

[0084] in,

[0085] L1 can be

[0086] L2 can be

[0087] L3 can be

[0088] R a 、R b and R c Can be independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0089] R A Each occurrence can be independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2)y -(L3) z -R2;

[0090] R1 and R2 can independently be H or a terminal group;

[0091] x, y, and z can each independently be a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0092] *Indicates the point of connection between the relevant group and other groups.

[0093] As used herein, the term "halogen" may also be referred to as a halogen atom or a halogen group, and refers to a monovalent group formed by the halogen atom fluorine (F), chlorine (Cl), bromine (Br) or iodine (I), i.e., -F, -Cl, -Br or -I.

[0094] As used herein, the term "alkyl" refers to a linear or branched monovalent hydrocarbon group formed by removing a hydrogen atom from a saturated aliphatic chain hydrocarbon, and its chemical formula is -C n H 2n+1 In some examples, the alkyl groups suitable for use in the present disclosure may include, but are not limited to, linear or branched alkyl groups having 1 to 20 carbon atoms, i.e., linear or branched C 1-20 Alkyl groups, such as C 1-18 Alkyl, C 1-16 Alkyl, C 1-12 Alkyl, C 1-10 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl, etc. Alternatively, alkyl groups suitable for use in the present disclosure may include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 2-ethylbutyl, and the like.

[0095] As used herein, the term "alkoxy" refers to an -O-alkyl group, the chemical formula of which is -OC n H 2n+1 In some examples, alkoxy groups suitable for use in the present disclosure may include, but are not limited to, alkoxy groups having 1 to 6 carbon atoms, i.e., C 1-6 Alkoxy groups include, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, tert-butoxy, and the like.

[0096] As used herein, the term "alkoxyalkyl" refers to an alkoxy-substituted alkyl group having the formula -C n H 2n -OC n H 2n+1 In some examples, alkoxyalkyl groups suitable for use in the present disclosure may include, but are not limited to, alkoxyalkyl groups having 1 to 6 carbon atoms, i.e., C 1-6Alkoxyalkyl groups include, but are not limited to, methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, tert-butoxymethyl, ethoxymethyl and the like.

[0097] As used herein, the term "acyl" refers to the remaining atomic group after removing one or more hydroxyl groups from an organic or inorganic oxygen-containing acid, and its general formula is RC(=O)-, wherein R can be H, alkyl or aryl. In some aspects, the acyl group described herein can be an alkanoyl group (i.e., R is H or alkyl), for example, C 1-6 Alkanoyl; In other aspects, the acyl group described herein may be an aroyl group (ie, R is an aryl group), such as C 6-10 Aroyl. In some examples, the acyl group described in the present disclosure may include, but is not limited to, formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl, benzoyl, naphthoyl, and the like.

[0098] As used herein, the term "end group" refers to a non-hydrogen group at the end of a polyamino acid molecular chain, i.e., a terminal group / capping group of the polyamino acid chain. The end group described in the embodiments of the present disclosure can be any terminal group commonly used in the art, including but not limited to hydroxyl, carboxyl, amino, amide, ester, etc., and the present disclosure does not impose specific limitations on this.

[0099] The symbols x, y and z in the above formula (I) represent the percentage of the number of glycine (Gly or G) units, the number of proline (Pro or P) units and the number of hydroxyproline (Hyp or O) units in the total number of monomer units in the polyamino acid molecular chain, respectively. Each occurrence of x, y and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y and z is 1.

[0100] In some embodiments, in the polyamino acid, L1 may be L2 can be

[0101] L3 can be

[0102] In other embodiments, in the polyamino acid, L1 may be L2 can be L3 can be

[0103] In other embodiments, the polyamino acid may have an unacylated branched structure, that is, in the structure of formula (I), R A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x-(L2) y -(L3) z -R2.

[0104] In other embodiments, the polyamino acid may have a branched structure that is at least partially acylated, that is, in the structure of formula (I), R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x -(L2) y -(L3) z -R2, and at least some R A Selected from C 1-6 Alkanoyl, C 6-10 For example, the polyamino acid may be a branched structure that is at least partially acetylated, i.e., in the structure of formula (I), at least some R A -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is acetyl.

[0105] In other embodiments, the polyamino acid may have a fully acylated non-branched structure, that is, in the structure of formula (I), R A Each occurrence is independently selected from C 1-6 Alkanoyl and C 6-10 For example, the polyamino acid may be a fully acetylated non-branched structure. That is, in the structure of formula (I), R A It is acetyl.

[0106] In some embodiments, any of the polyamino acids described above may have a suitable weight average molecular weight, for example, 5 to 100 kg / mol.

[0107] At least another embodiment of the present disclosure further provides a method for preparing polyamino acids, comprising: reacting compounds represented by the following formulas (II) to (IV):

[0108] in,

[0109] R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C1-6 Alkoxyalkyl,

[0110] R A1 Selected from H, C 1-6 Alkanoyl and C 6-10 Aroyl;

[0111] In the presence of a base, the polymer is polymerized in an organic solvent to obtain a polyamino acid represented by formula (I):

[0112] R1-(L1) x -(L2) y -(L3) z -R2

[0113] Formula (I)

[0114] in,

[0115] L1 is

[0116] L2 is

[0117] L3 is

[0118] R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl;

[0119] R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2;

[0120] R1 and R2 are independently H or a terminal group;

[0121] Each occurrence of x, y, and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

[0122] In some embodiments, R in the above formulas (I) to (IV) a 、R b and R c Both can be H.

[0123] In other embodiments, the method may include using the non-acylated monomer (IV) as a raw material to synthesize the polyamino acid.A1 can be H, and R in the above formula (I) A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z Furthermore, the method may further comprise further acylation of the polyamino acid of formula (I) obtained by polymerization to obtain at least partially acylated polyamino acid, for example, further acetylation of the polyamino acid of formula (I) to obtain at least partially acetylated polyamino acid.

[0124] In other embodiments, the method may include using a mixture of acylated and non-acylated monomers (IV) as a starting material to synthesize the polyamino acid. That is, at least some of R A1 is H, and at least some R A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl groups, so that the polyamino acid of formula (I) obtained by polymerization is at least partially acylated. That is, at least some of R A Selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2. For example, at least some R A1 (and at least some R A ) may be an acetyl group.

[0125] In other embodiments, the method may include using the acylated monomer (IV) as a raw material to synthesize the polyamino acid. A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl, and R in the above formula (I) A Selected from C 1-6 Alkanoyl and C 6-10 Aroyl. For example, R A1 and R A They may all be acetyl groups.

[0126] In some embodiments of the method, the base used can be selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline, and any mixture thereof. In addition, the organic solvent used can be selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone, and mixtures thereof.

[0127] At least another embodiment of the present disclosure further provides the use of any of the polyamino acids described herein as a biocompatible filling material. In some examples, the biocompatible filling material can be a medical and aesthetic filling material.

[0128] Example

[0129] The embodiments of the present disclosure will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained commercially. Unless otherwise indicated, all amounts listed are described in parts by weight based on gross weight. The present disclosure should not be construed as being limited to the specific examples described.

[0130] Example 1. Preparation of Gly-Pro-Hyp Terpolymer (GPO Terpolymer)

[0131] In a glove box, glycine-N-carboxylic anhydride (GlyNCA) (101 mg, 1.00 mmol, 100 equiv), L-proline-N-carboxylic anhydride (ProNCA) (141 mg, 1.00 mmol, 100 equiv), and L-hydroxyproline-N-carboxylic anhydride (HypNCA) (157 mg, 1.00 mmol, 100 equiv) were added to a 5 mL glass vial and dissolved in 1500 μL of anhydrous DMSO. N,N-diethylisopropylamine (DIPEA) (0.5 M × 20 μL, 0.01 mmol, 1 equiv) was then added and reacted for 30 min. The mixture was diluted to 10 mL with water, passed through a PD10 desalting column, and lyophilized to obtain the final product.

[0132] GPO terpolymers were synthesized in the same manner as above using the different base types and charges and different monomer ratios listed in Table 1 below, and representative molecular weights and dispersities were measured. Molecular weights and dispersities were determined using aqueous SEC coupled with a multi-angle light scattering / differential detector in 1× PBS (pH = 7.4). The refractive index (dn / dc) (658 nm) measured using a DIPEA-10-10-10 copolymer was 0.0762 ml / g.

[0133] Table 1. GPO copolymers synthesized with different base types and equivalents and different monomer ratios, as well as some representative molecular weights and dispersities

[0134] Figures 1-4 show the SEC curves of products obtained using DMAP (4-dimethylaminopyridine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), and DIPEA (N,N-diisopropylethylamine) as bases, and using different raw material types and ratios. Figures 1 and 4 were generated using a Cytiva Superdex 200 column, while Figures 2 and 3 used a Cytiva Superdex 75 column. These reaction conditions screened different organic base types and equivalent weights. Results from aqueous SEC analysis of the copolymer elution volume showed that higher base equivalent weights resulted in higher molecular weights, with DIPEA as the base producing the highest molecular weight.

[0135] The polyamino acid product prepared as above was tested using H NMR, CD, IR and hydrolysis.

[0136] Specifically, as shown in Figures 6-10, Figure 6 shows the GPO terpolymer made using DIPEA-40-30-30. 1 H-NMR (D2O); Figure 7 shows the GPO terpolymer made using DIPEA-100-100-100 1 H-NMR (DO) circular dichroism spectrum; Figure 8 shows the infrared absorption spectrum of the GPO terpolymer (blue) prepared using DIPEA-25-25-25 and its hydrolysis (red); Figure 9 shows the changes in the aqueous phase SEC curve of the GPO terpolymer (red) prepared using DBU as a base and a Gly:Pro:Hyp:base raw material ratio of 100:100:100:1 (DBU-100-100-100) and the addition of NaOH thereto (blue); Figure 10 shows the changes in the aqueous phase SEC curve of the GPO terpolymer (red) prepared using DIPEA-25-25-25 and its hydrolysis (red) after adding NaOH thereto; 1 H-NMR (D2O) changes.

[0137] As shown in the figures above, the synthesized copolymers with HypNCA components have branched structures with PPII helical conformations. These copolymers are stable in water for 4 days, and the ester bonds can be hydrolyzed by adding alkali.

[0138] Example 2. Cell Adhesion Performance Test of Synthesized GPO Terpolymer

[0139] The cell adhesion performance of the GPO terpolymer synthesized in Example 1 was tested, using PBS, hyaluronic acid, and bovine Achilles tendon collagen as controls. The specific testing method is as follows:

[0140] Cell adhesion was tested using the BALB / 3T3 cell line. The test sample was dissolved in dH2O at a concentration of 200 μg / mL. Bovine Achilles tendon collagen was dissolved in 0.5 M acetic acid at a concentration of 200 μg / mL. The mixture was stirred for 48 hours and centrifuged at 12,000 rpm to obtain the supernatant.

[0141] Take a 96-well plate and add 100 μL of the prepared test sample solution to each well. Seal and coat the plate in a dark place at 4°C for at least 11 hours. Remove the coated plate and let it sit at room temperature for 1 hour. Discard the coating solution, wash the plate with PBS, and block it with 0.1% BSA solution at room temperature for 1 hour. Wash again with PBS. Adjust the BALB / 3T3 cell concentration to 2-5 10 using complete culture medium. 5 After 100 μL of the solution was added to each well, the plate was incubated in a 5% CO2 incubator for 1 hour. The plate was then removed. The plate was washed four times with PBS, the PBS discarded, and the plate was incubated with 10% CCK8 solution for another hour. The plate was then read using a microplate reader at 450 nm.

[0142] Figure 11 shows a comparison of the cell adhesion properties of GPO terpolymers with those of GH and PH dipolymers; Figure 12 shows the cell adhesion properties of GPO terpolymers with different amino acid contents, with PBS, hyaluronic acid, and / or bovine Achilles tendon collagen used as controls. As shown in Figures 11 and 12, the GPO terpolymer (DIPEA-25-25-25) was significantly higher than the dipolymer and comparable to hyaluronic acid, showing stronger cell adhesion. Furthermore, the cell adhesion capacity of the GPO terpolymer decreased with decreasing glycine content, with a proline to hydroxyproline content of 1:1 being optimal.

[0143] Example 3. Synthesis of acylated polyamino acids and testing of their cell adhesion properties

[0144] Using a method similar to that described in Example 1, copolymer 1 was prepared according to the composition and initiator (base) listed in Table 2 below. # ~7 # .

[0145] Table 2. Composition of GPO copolymers and bases used

[0146] The cell adhesion properties of the prepared GPO copolymers were tested using the same method used in Example 2. Figure 13 shows a comparison of the cell adhesion capabilities of GPO terpolymers with varying degrees of acetylation, bovine Achilles tendon collagen, and a PBS control. As shown in Figure 13, the copolymers with varying degrees of acylation also exhibited good cell adhesion, approximately 70% of that achieved with bovine Achilles tendon and 90% of that achieved with hyaluronic acid.

[0147] It can be seen that the polyamino acid provided by the present disclosure has a similar composition and sequence to collagen, and has strong cell adhesion similar to collagen, and is therefore suitable for use as a biocompatible filling material, especially a filling material for medical aesthetics.

[0148] The foregoing description is merely an exemplary embodiment of the present disclosure and is not intended to limit the scope of protection of the present disclosure. The scope of protection of the present disclosure is determined by the appended claims.

Claims

1. A polyamino acid having a structure represented by formula (I): R1-(L1) x -(L2) y -(L3) z -R2 Formula (I) in, L1 is L2 is L3 is R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl; R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2; R1 and R2 are independently H or a terminal group; Each occurrence of x, y, and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

2. The polyamino acid according to claim 1, wherein L1 is L2 is L3 is 3. The polyamino acid according to claim 1 or 2, wherein L1 is L2 is L3 is 4. The polyamino acid according to any one of claims 1 to 3, wherein R A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x -(L2) y -(L3) z -R2.

5. The polyamino acid according to any one of claims 1 to 3, wherein R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2, provided that at least some R A -(L1) x -(L2) y -(L3) z -R2, and at least some R A Selected from C 1-6 Alkanoyl, C 6-10 Aroyl.

6. The polyamino acid according to any one of claims 1 to 3 and 5, wherein At least some R A -(L1) x -(L2) y -(L3) z -R2, and at least some R A It is acetyl.

7. The polyamino acid according to any one of claims 1 to 3, wherein R A Each occurrence is independently selected from C 1-6 Alkanoyl and C 6-10 Aroyl.

8. The polyamino acid according to any one of claims 1 to 3 and 7, wherein R A It is acetyl.

9. The polyamino acid according to any one of claims 1 to 8, wherein The weight average molecular weight of the polyamino acid is 5 to 100 kg / mol.

10. A method for preparing a polyamino acid, comprising: The compounds represented by the following formulas (II) to (IV): in, R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl, R A1 Selected from H, C 1-6 Alkanoyl and C 6-10 Aroyl; In the presence of a base, the polymer is polymerized in an organic solvent to obtain a polyamino acid represented by formula (I): <h2 style=";text-align:left;direction:ltr">R1-(L1)<h2 style=";text-align:left;direction:ltr"> x <h2 style=";text-align:left;direction:ltr"> -(L2)<h2 style=";text-align:left;direction:ltr"> y <h2 style=";text-align:left;direction:ltr"> -(L3)<h2 style=";text-align:left;direction:ltr"> z <h2 style=";text-align:left;direction:ltr"> -R2 Formula (I) in, L1 is L2 is L3 is R a 、R b and R c independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkoxyalkyl; R A Each occurrence is independently selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2; R1 and R2 are independently H or a terminal group; Each occurrence of x, y, and z is independently a fraction greater than 0 and less than 1, provided that the sum of all x, y, and z is 1.

11. The method according to claim 10, wherein: R a 、R b and R c Both are H.

12. The method according to claim 10 or 11, wherein: R A1 is H, and R A Each occurrence is independently selected from H and -(L1) x -(L2) y -(L3) z -R2.

13. The method according to any one of claims 10 to 12, further comprising further acylating the polyamino acid of formula (I) obtained by polymerization to obtain an at least partially acylated polyamino acid.

14. The method according to claim 10 or 11, wherein: At least some R A1 is H, and at least some R A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl, and at least some R A Selected from H, C 1-6 Alkanoyl, C 6-10 Aroyl and -(L1) x -(L2) y -(L3) z -R2.

15. The method of claim 10, wherein: R A1 Selected from C 1-6 Alkanoyl and C 6-10 Aroyl, and R A Selected from C 1-6 Alkanoyl and C 6-10 Aroyl.

16. The method according to claim 10 or 15, wherein: R A1 and R A All are acetyl groups.

17. The method according to any one of claims 10 to 16, wherein The base is selected from potassium hydride, sodium hydride, potassium methoxide, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, triazabicyclo[4.4.0]dec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-diisopropylethylamine, 4-dimethylaminopyridine, aniline and any mixture thereof.

18. The method according to any one of claims 10 to 17, wherein The organic solvent is selected from dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, acetone and a mixture thereof.

19. Use of the polyamino acid according to any one of claims 1 to 9 or the polyamino acid prepared by the method according to any one of claims 10 to 18 as a biocompatible filling material.

20. The use according to claim 19, wherein The biocompatible filling material is a medical and cosmetic filling material.