Bioactive synthetic copolymers, bioactive polymers and related methods

Abstract

Bioactive synthetic copolymers having a poly(norbornene) backbone comprising one or more repeating units represented by general formula (I) and one or more repeating units represented by general formula (II) are provided. Also provided are bioactive polymers, materials comprising the bioactive synthetic copolymers, methods for preparing the bioactive synthetic copolymers, and methods for preparing the bioactive polymers. JPEG2023554220000044.jpg79158

Description

[Technical Field]

[0001] The present disclosure relates generally to bioactive synthetic copolymers, bioactive polymers, and materials comprising said bioactive synthetic copolymers. The present disclosure also relates to methods of preparing said bioactive synthetic copolymers, bioactive polymers, and materials. [Background technology]

[0002] background A better understanding of the biology and physiology of living organisms over the years has led to an appreciation of the possibility of using alternative materials to augment or replace existing functions in biological systems.

[0003] However, it is often difficult to identify suitable materials that meet both the mechanical and biological requirements to function desirably in or with biological systems.

[0004] This is because bioactive molecules with desirable biological attributes (e.g., collagen, chitosan, etc.) often lack the mechanical strength required for useful biomedical applications. For example, many such bioactive molecules are highly hygroscopic and exist as gels when they absorb water, which makes them too weak on their own for use in weight-bearing biomedical applications such as implantable devices.

[0005] On the other hand, synthetic materials with excellent mechanical properties lack the biological attributes required for their proper use in applications requiring constant interaction with biological systems. For example, many such synthetic materials can induce a foreign body reaction (FBR) when inserted into the human body. This can lead to inflammation and other types of undesirable immune responses being elicited around the implantation site.

[0006] Combining these different materials in the hope that the resulting material can achieve both the desired biological and mechanical properties is also difficult because bioactive molecules such as peptides and carbohydrates are often incompatible with synthetic polymers due to the hydrophilic nature of the former and the hydrophobic nature of the latter.

[0007] That is, physically blending two different materials together often results in phase separation of the two mutually incompatible materials, rendering the resulting overall material ineffective.

[0008] Their inherent differences in hydrophilicity also make it extremely difficult to chemically synthesize bioactive polymers from these materials, especially when their molecular weights are relatively high. This is in addition to the various complex chemical hurdles (e.g., potentially high intramolecular reactivity, unwanted chemical leaching of by-products, etc.) that must be overcome when attempting to chemically combine these two chemically distinct types of materials together.

[0009] In view of the above, there is a need to address or at least ameliorate the above-mentioned problems, and in particular to provide bioactive synthetic copolymers, bioactive polymers, materials comprising said bioactive synthetic copolymers, and related methods that address or at least ameliorate the above-mentioned problems. Summary of the Invention

[0010] overview In one aspect, one or more repeat units represented by general formula (I) and one or more repeat units represented by general formula (II):

[0011] [ka]

[0012] (In the formula, R 1is optionally substituted alkyl; R 2 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y 1 comprises a synthetic polymer or a portion thereof; and Z 1 and Z 2 are each independently a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0013] In one embodiment, the molecular weight of general formula (I) does not differ from the molecular weight of general formula (II) by more than 30% of the molecular weight of general formula (II).

[0014] In one embodiment, L is heteroalkylene having from 20 to 300 carbon atoms.

[0015] In one embodiment, L is polyethylene glycol (PEG).

[0016] In one embodiment, L is a polyethylene glycol (PEG) having a number average molecular weight between 500 and 7,000.

[0017] In one embodiment, R 1 is C1-C4 alkyl, and R 2 is C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C1-C 20 Alkoxyalkyl, C2-C 20 Alkylcarbonyl or C3-C 20 alkylcarbonylalkyl.

[0018] In one embodiment, R 1 is a straight or branched C-C alkyl substituent independently selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or t-butyl, and R 2 is methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, straight or branched C1-C alkyl esters independently selected from methylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, or decyl; 20 It is an alkyl substituent.

[0019] In one embodiment, Z 1 and Z 2 Both are CR a R b where R a and R b are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0020] In one embodiment, X comprises a protein, peptide, or carbohydrate selected from the group consisting of a peptide sequence, a laminin-derived peptide, an integrin-binding peptide, a cell-penetrating peptide, a collagen mimetic, a collagen fragment, heparin sulfate, a glycosaminoglycan (GAG), and derivatives thereof.

[0021] In one embodiment, X is RGD, SRGDS, RGDS, A5G81(AGQWHRVSVRWGC), SVVYGLR, (IRIK)2, (IKKI)3, heparin oligosaccharide DP8, DP10, DP12, DP14, DP16, DGEA, (PHypG) n Type array, (PGHyp) n Type array, (HypGP) n Type array, (HypPG) n Type sequence, (GHypP) n Type array, (GPHyp) n The amino acid sequence is selected from the group consisting of hydroxybenzoates and hyaluronic acid.

[0022] In one embodiment, X comprises an antibiotic, antimicrobial, antimicrobial moiety, blood thinner, or anti-inflammatory agent.

[0023] In one embodiment, X comprises an antibiotic, antimicrobial, antibacterial, blood thinner or anti-inflammatory agent selected from the group consisting of penicillin, amoxicillin, amphotericin, ciprofloxacin (CIF), atorvastatin, aspirin, streptomycin, ribostamycin and gentamicin.

[0024] In one embodiment, Y 1 is represented by the general formula (III):

[0025] [ka]

[0026] (In the formula, A is selected from a single bond, oxy, carbonyl, oxycarbonyl, carboxyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, or optionally substituted alkoxycarbonylalkyl; B is optionally present as a ring selected from 1,2,3-triazole or succinimide; R 5 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; Y 2 is selected from the group consisting of polypropylene (PP), polyester, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylate, poly(meth)acrylate, polyamide (PA), and portions thereof; and T is an end group selected from the group consisting of hydrogen, halogen, hydroxyl, amino, acyl, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, or optionally substituted alkoxycarbonylalkyl).

[0027] In one embodiment, Y 1 is represented by the following general formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IIIf):

[0028] [ka]

[0029] is selected from.

[0030] In one aspect, there is provided a method of preparing a bioactive synthetic copolymer disclosed herein, the method comprising: One or more bioactive polymers represented by general formula (IV) are polymerized with one or more synthetic polymers represented by general formula (V) in the presence of a catalyst to produce a bioactive synthetic copolymer:

[0031] [ka]

[0032] (In the formula, R 1 is optionally substituted alkyl; R 2is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y 1 comprises a synthetic polymer or a portion thereof; and Z 1 and Z 2 are each independently a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0033] In one embodiment, the catalyst comprises a ruthenium complex.

[0034] In one embodiment, the method comprises ring-opening metathesis polymerization (ROMP).

[0035] In one aspect, for preparing the copolymers disclosed herein, a bioactive polymer represented by the general formula (IV):

[0036] [ka]

[0037] (In the formula, R 1 is optionally substituted alkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; and Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0038] In one aspect, there is provided a method of preparing a bioactive polymer disclosed herein, the method comprising: (i) General formula (VI):

[0039] [ka]

[0040] (In the formula, Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R care each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; (ii) reacting said dicarboxylic acid anhydride having the general formula (VI) with a diamine R 4 R 3 NLR 1 -NH2 to form a compound of general formula (VII):

[0041] [ka]

[0042] (In the formula, R 1 is optionally substituted alkyl; R 3 and R 4 are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, where R 3 and R 4 at least one of is H; L is heteroalkylene; and Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; (iii) reacting said amine having general formula (VII) with an acid-containing biologically active moiety XC(=O)OH to obtain a biologically active macromolecule, wherein X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof.

[0043] In one embodiment, the method further comprises purifying the amine having general formula (VII) to remove impurities prior to the step of reacting the amine having general formula (VII) with XC(=O)OH.

[0044] In one embodiment, the purification step comprises a double neutralization step.

[0045] In one embodiment, the dual neutralization step comprises a first acid wash step and a second base wash step.

[0046] In one aspect, there is provided a material comprising the copolymers disclosed herein for use in medicine.

[0047] In one embodiment, the material is part of an apparatus selected from the group consisting of a wound dressing, a skin scaffold, a bone scaffold, an organoid scaffold, an implant, and a medical device.

[0048] definition As used herein, the term "polymer" refers to a chemical compound containing repeating units and is created through the process of polymerization. The units that make up a polymer are typically derived from monomers and / or macromonomers. A polymer typically contains many repeating constitutional units.

[0049] As used herein, the term "monomer" or "macromonomer" refers to a chemical entity that can be covalently bonded to one or more such substances to form a polymer.

[0050] As used herein, the term "bioactivity" refers broadly to a property that has a biological effect, preferably a desirable or positive biological effect on a living organism, tissue, or cell.

[0051] As used herein, the term "biocompatible" refers broadly to the property of being compatible with a biological system or portion of a biological system without substantially or significantly eliciting an adverse physiological response, such as a toxic response, an immune response, injury, etc. Such biological systems or portions include blood, cells, tissues, organs, etc.

[0052] The term "bond" refers to the linkage between atoms in a compound or molecule. A bond can be a single, double, or triple bond.

[0053] In the definitions of many of the substituents below, it is stated that "the group may be a terminal group or a bridging group." This is intended to mean that the use of the term encompasses situations in which the group is a terminal group / moiety, as well as situations in which the group is a linker between two other parts of a molecule. Using the term "alkyl" having one carbon atom as an example, it will be understood that when present as a terminal group, the term "alkyl" having one carbon atom may mean -CH3, when present as a bridging group, the term "alkyl" having one carbon atom may mean -CH2-, etc.

[0054] The term "alkyl" as a group or part of a group refers to a straight or branched chain aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Examples of suitable straight and branched alkyl substituents are methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2 -trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, etc. The group can be a terminal group or a bridging group.

[0055] The term "alkenyl" as a group or part of a group means an aliphatic hydrocarbon group which contains at least one carbon-carbon double bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain multiple double bonds, and the orientation about each double bond is independently E or Z. Exemplary alkenyl groups are ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl , 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, etc. The group can be a terminal group or a bridging group.

[0056] The term "alkynyl" as a group or part of a group means an aliphatic hydrocarbon group which contains at least one carbon-carbon triple bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain more than one triple bond. Exemplary alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl, etc. The group may be a terminal group or a bridging group.

[0057] The term "heteroalkylene," as used herein, refers to an alkylene having one or more -CH2- replaced with a heteroatom selected from O, NR, Si, P, or S, where R is hydrogen or alkyl as defined herein. The term "heteroalkylene" can be linear, branched, or cyclic and can contain up to 500 carbon atoms.

[0058] The term "alkoxy" as used herein refers to a straight or branched chain alkyloxy group. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

[0059] As used herein, the term "alkoxyalkyl" is intended to refer broadly to a group containing -RO-R', where R and R' are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0060] As used herein, the term "alkylcarbonyl" is intended to refer broadly to a group containing -RC(=O)-, where R is alkyl as defined herein. The group may be a terminal group or a bridging group.

[0061] As used herein, the term "alkylcarbonylalkyl" is intended to refer broadly to a group containing -RC(=O)-R', where R and R' are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0062] As used herein, the term "carboxylalkyl" is intended to refer broadly to a group containing -C(=O)-OR, where R is alkyl as defined herein. The group may be a terminal group or a bridging group.

[0063] As used herein, the term "oxycarbonylalkyl" is intended to refer broadly to a group containing -OC(=O)-R, where R is alkyl as defined herein. The group can be a terminal group or a bridging group.

[0064] As used herein, the term "alkylcarboxylalkyl" is intended to refer broadly to a group containing -RC(=O)-O-R', where R and R' are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0065] As used herein, the term "alkoxycarbonylalkyl" is intended to refer broadly to a group containing -ROC(=O)-R', where R and R' are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0066] As used herein, the term "oxy" is intended to refer broadly to groups containing --O--.

[0067] As used herein, the term "carbonyl" is intended to refer broadly to groups containing -C(=O)-.

[0068] As used herein, the term "oxycarbonyl" is intended to refer broadly to groups containing -OC(=O)-.

[0069] As used herein, the term "carboxyl" is intended to refer broadly to groups containing -C(=O)-OR, where R is hydrogen or an organic group.

[0070] The term "halogen" refers to chlorine, fluorine, bromine or iodine. The term "halo" refers to chloro, fluoro, bromo or iodo.

[0071] The term "amine group" and the like are intended to refer broadly to groups containing -NR, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0072] The term "amide group" and the like are intended to refer broadly to groups containing -C(=O)NR, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0073] The term "optionally substituted," when used to describe a chemical structure or moiety, means that one or more of its hydrogen atoms has been substituted with an alkyl, alkyloxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amido (-C(O)NH-alkyl- or -alkylNHC(O)alkyl), amine (such as alkylamino, arylamino, arylalkylamino), aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH, as well as CONH-alkyl, CONH refers to a chemical structure or moiety that is optionally substituted with a chemical moiety or functional group such as, for example, -NHCONH-aryl, -CONH-arylalkyl, carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halo, haloalkyl (e.g., -CCl3, -CF3, -C(CF3)3), heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphodiester, sulfide, sulfonamide (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl, and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl, thioether), or urea (-NHCONH-alkyl-).

[0074] As used herein, the term "micro" should be interpreted broadly to include dimensions from about 1 micron to about 1000 microns.

[0075] As used herein, the term "nano" should be interpreted broadly to include dimensions of less than about 1000 nm, less than about 500 nm, less than about 100 nm, or less than about 50 nm.

[0076] The terms "coupled" or "connected," as used in this description, are intended to cover both direct connections or connections through one or more intermediary means, unless otherwise stated.

[0077] The term "associated" as used herein when referring to two elements refers to a broad relationship between the two elements, including, but not limited to, a physical, chemical, or biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other, or element A may contain element B, or vice versa.

[0078] The term "adjacent," as used herein when referring to two elements, refers to the proximity of one element to another, and may be, but is not limited to, elements that contact each other, or may further include elements that are separated by one or more additional elements disposed between them.

[0079] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0080] Furthermore, in the description herein, the word "substantially," whenever used, is understood to include, but not be limited to, "entirely," "completely," etc. Furthermore, terms such as "comprising," "comprise," and the like, whenever used, are intended to be open-ended descriptive language in that they broadly include the elements / components listed after such term, in addition to other components not explicitly recited. For example, when using "comprising," a reference to "one" feature is also intended to be a reference to "at least one" of that feature. Terms such as "consisting," "consist," and the like may, in appropriate context, be considered subsets of terms such as "comprising," "comprise," and the like. Thus, in embodiments disclosed herein using terms such as "comprising," "comprise," and the like, it will be understood that these embodiments provide teachings of corresponding embodiments using terms such as "consisting," "consist," and the like. Additionally, the terms "about," "approximately," and the like, whenever used, typically refer to a reasonable variation, for example, a + / - 5% variation of the disclosed value, or a 4% variation of the disclosed value, or a 3% variation of the disclosed value, a 2% variation of the disclosed value, or a 1% variation of the disclosed value.

[0081] Furthermore, in the description herein, specific values ​​may be disclosed within a range. The values ​​indicating the end points of the range are intended to exemplify a preferred range. Whenever a range is described, it is intended that the range covers and teaches not only the individual numerical values ​​within the range, but also all possible subranges. That is, the end points of the range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., as well as individual values ​​within the range such as 1%, 2%, 3%, 4%, 5%, etc. It should be understood that the individual numerical values ​​within a range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it is intended that the range also covers and teaches values ​​up to two additional decimal places or significant digits (where appropriate) from the indicated numerical endpoint. For example, a description of a range of 1% to 5% is intended to specifically disclose 1.00% to 5.00% and also 1.0% to 5.0% and all intermediate values ​​therein (1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure intent is applicable to any depth / breadth of range.

[0082] Furthermore, when describing some embodiments, the present disclosure may disclose a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be understood that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may also be possible. The particular order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the method and / or process disclosed herein should not be limited to steps performed in the order described. The order of steps may be varied and still be within the scope of the present disclosure.

[0083] Furthermore, while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, it will be understood that one or more of these features / characteristics may also be waived in other alternative embodiments, and the present disclosure provides support for such waiver and these related alternative embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0084] Description of the Embodiments Exemplary, non-limiting embodiments of bioactive synthetic copolymers, bioactive polymers for preparing the bioactive synthetic copolymers, materials comprising the bioactive synthetic copolymers, and related methods are disclosed below.

[0085] bioactive synthetic copolymers One or more repeating units represented by general formula (I) and one or more repeating units represented by general formula (II):

[0086] [ka]

[0087] (In the formula, R 1 is optionally substituted alkyl; R 2 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y 1 comprises a synthetic polymer or a portion thereof; and Z 1 and Z 2 are each independently a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0088] In various embodiments, the repeating unit and / or moiety X represented by general formula (I) is bioactive, biocompatible, and / or biodegradable. In various embodiments, the repeating unit and / or moiety Y represented by general formula (II) is 1 has good mechanical strength / hardness. In various embodiments, the repeating units and / or moieties Y represented by general formula (II) 1has higher mechanical strength than the repeating units and / or moieties X represented by general formula (I). Advantageously, the presence of repeating units represented by general formulas (I) and (II) in the bioactive synthetic copolymer confers both bioactivity and mechanical strength to the copolymer, leading to a mechanically strong bioactive copolymer. In various embodiments, the copolymer may also be biocompatible and / or biodegradable. Thus, in various embodiments, the copolymer may be classified as a biomaterial. Advantageously, due to the presence of the synthetic bioactive side chains, the bioactive synthetic copolymer may also have higher thermal stability than conventional biomolecules such as peptides, proteins, carbohydrates, or glycosaminoglycans. Even more advantageously, the thermal stability of the bioactive synthetic copolymer allows embodiments of the copolymer to be suitable for high-temperature processing, even harsh material processing such as melt extrusion above 200°C, making the copolymer ideal / attractive for use in applications such as biomedical devices. In various embodiments, the synthetic polymer is substantially or completely non-bioactive, or at least less bioactive than the bioactive moiety.

[0089] In various embodiments, L is a polymeric linker that connects the biologically active moiety X to the poly(norbornene) backbone. Advantageously, L is selected based on the size of the biologically active moiety X and the size of Y. 1 The molecular weight and / or length of the polymer linker L is designed to be adjustable and / or customizable based on the size of the synthetic polymers present in the copolymer. The molecular weight and / or length of the polymer linker L is determined by the size and / or length of the biologically active moieties X and Y, depending on the application for which the copolymer is to be used. 1The molecular weight and / or length of the synthetic polymer selected for the scaffold can be customized to suit the application. For example, in skin scaffolds, shorter synthetic polymer (e.g., PCL or PLA) side chains are preferred for fast degradation, while in bone scaffolds, longer synthetic polymer (e.g., PCL or PLA) side chains are selected for slower degradation in the body. Without being bound by theory, it is believed that bone tissue is expected to grow slower than skin tissue, and therefore bone scaffolds must remain intact in the body for a long period of time for bone tissue to regenerate, and cannot degrade quickly. For example, for applications in bandages or non-biodegradable nonwoven fibers that require thermal stability and / or mechanical strength properties, low molecular weights are preferred for synthetic polymers due to their poor solubility in common solvents. In various embodiments, synthetic polymers with low molecular weights include synthetic polymers with molecular weights of about 5,000 or less, for example, when the synthetic polymer is highly insoluble, such as polyamide (PA). In other embodiments, synthetic polymers having molecular weights of about 10,000 or less may be used / acceptable, for example, when the synthetic polymer has low insolubility.

[0090] In various embodiments, the molecular weight and / or length of the polymer linker L is selected so that the overall molecular size of the repeating unit represented by general formula (I) is similar / comparable to the molecular size of the repeating unit represented by general formula (II). For example, PCL having a molecular weight of 4,000 is used as a polymer linker Y 1 is selected as the synthetic polymer of choice for X, and a peptide having a molecular weight of about 400 to about 500 is selected as the biologically active moiety X, then L may be designed to comprise a molecular weight of about 3,400. It will be understood that in various embodiments, it is the length of L that is adjusted to match the molecular weight of general formula (I) with the molecular weight of general formula (II).

[0091] In various embodiments, the molecular weight of the compound of general formula (I) is equivalent / substantially similar to the molecular weight of the compound of general formula (II). In various embodiments, the molecular weight of the compound of general formula (I) does not differ from the molecular weight of the compound of general formula (II) by more than 30% of the molecular weight of the compound of general formula (II), or vice versa. For example, the molecular weight of the compound of general formula (I) can be at most about 30% more or at most 30% less than the molecular weight of the compound of general formula (II), or vice versa. The molecular weight of the compound of general formula (I) cannot differ from the molecular weight of the compound of general formula (II) by more than about 30%, more than about 25%, more than about 20%, more than about 15%, more than about 10%, more than about 5%, more than about 4%, more than about 3%, more than about 2%, or more than about 1%, or vice versa. In various embodiments, the molecular weight of the compound of general formula (I) does not differ from the molecular weight of the compound of general formula (II) by more than about 20% of the molecular weight of the compound of general formula (II), or vice versa. For example, the molecular weight of general formula (I) can be up to about 20% more or up to 20% less than the molecular weight of general formula (II), or vice versa. Advantageously, the repeating unit bearing the biologically active moiety has a molecular size / weight / length that is similar to that of the repeating unit bearing the synthetic polymer, thereby extending the length of the biologically active moiety X, thereby allowing X to be "visible," available for binding to cells, or accessible to its target physiological site for desired biological activity, i.e., not buried in the sea / matrix of the synthetic polymer.

[0092] In various embodiments, the molecular weight of general formula (I) is about 15,000, about 14,000, about 13,000, or at least about 12,000. In various embodiments, the molecular weight of general formula (I) is from about 100 to about 15,000, from about 200 to about 14,000, from about 300 to about 13,000, from about 400 to about 12,000, from about 500 to about 11,000, from about 1,000 to about 10,000, from about 1,500 to about 9,500, from about 2,000 to about 9,000, from about 2,500 to about 8,500, from about 3,000 to about 8,000, from about 3,500 to about 7,500, from about 4,000 to about 7,000, from about 4,500 to about 6,500, from about 5,000 to about 6,000, or about 5,500. In various embodiments, when X comprises a longer peptide containing more than 10 amino acids and the molecular weight of L is about 6,000, then the molecular weight of general formula (I) is greater than about 7,000.

[0093] In various embodiments, the molecular weight of general formula (II) is from about 100 to about 15,000, from about 200 to about 14,000, from about 300 to about 13,000, from about 400 to about 12,000, from about 500 to about 11,000, from about 1,000 to about 10,000, from about 1,500 to about 9,500, from about 2,000 to about 9,000, from about 2,500 to about 8,500, from about 3,000 to about 8,000, from about 3,500 to about 7,500, from about 4,000 to about 7,000, from about 4,500 to about 6,500, from about 5,000 to about 6,000, or about 5,500.

[0094] In various embodiments, the total molecular weight of general formula (I) and general formula (II) is kept to about 300,000, about 300,000 or less, about 200,000 or less, about 100,000 or less, about 90,000 or less, about 80,000 or less, about 70,000 or less, about 60,000 or less, about 50,000 or less, about 45,000 or less, about 40,000 or less, about 35,000 or less, about 30,000 or less, about 25,000 or less, about 20,000 or less, or about 15,000 or less to facilitate copolymerization.

[0095] In various embodiments, L is hydrophilic. Because L is adjustable, the hydrophilicity of the repeating unit represented by general formula (I), and also the overall hydrophilicity of the bioactive synthetic copolymer, can be adjusted as desired. Advantageously, the presence of L increases the hydrophilicity of the repeating unit represented by general formula (I), and also the overall hydrophilicity of the bioactive synthetic copolymer. Even more advantageously, the presence of L increases the hydrophilicity of the bioactive synthetic copolymer, thus softening the hydrophobic synthetic polymer chain and making the copolymer less rigid after processing. It will be understood by those skilled in the art that because individual bioactive moieties are generally hydrophilic, while synthetic polymers are generally hydrophobic, bioactive moieties and synthetic polymers are typically incompatible with each other. Advantageously, L in the repeating unit represented by general formula (I) can also be used to extend the chain length of the bioactive moiety X attached to the end of L.

[0096] In various embodiments, L is amorphous. Advantageously, the presence of L increases the amorphousness and / or decreases the crystallinity of the bioactive synthetic copolymer, making the copolymer useful for creating softer or less rigid plastics, such as polystyrene-based materials.

[0097] In various embodiments, L is heteroalkylene having at least 20 carbon atoms, at least 30 carbon atoms, at least 40 carbon atoms, at least 50 carbon atoms, at least 60 carbon atoms, at least 70 carbon atoms, at least 80 carbon atoms, at least 90 carbon atoms, at least 100 carbon atoms, at least 150 carbon atoms, at least 200 carbon atoms, at least 250 carbon atoms, or at least 300 carbon atoms. 20 -C 300 It is a heteroalkylene or heteroalkylene having from 20 carbon atoms to 300 carbon atoms.

[0098] In various embodiments, L has a number average molecular weight between about 500 and about 7,000. L can have a number average molecular weight of about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, or about 7,000. In various embodiments, when X includes a small biologically active moiety, the molecular weight of L can be adjusted to about 7,000 so that the total molecular weight of General Formula (I) and General Formula (II) is kept below about 10,000. In various embodiments, the number average molecular weight of L is from about 1,000 to about 6,000.

[0099] In various embodiments, the heteroatom in L is O. In various embodiments, L is a polyalkylene glycol. In various embodiments, L is a poly(C2-C4 alkylene glycol). L may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), polybutylene glycol (PBG), and the like. Advantageously, the use of a polyalkylene glycol such as PEG can increase the hydrophilicity of the macromonomer and the resulting copolymer. In various embodiments, a polyalkylene glycol such as PEG is used as a spacer, linker, or linking group in the overall polymer instead of as a terminal group.

[0100] In various embodiments, L is a polyalkylene glycol having at least about 10 repeating units, at least about 15 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 30 repeating units, at least about 40 repeating units, at least about 50 repeating units, at least about 60 repeating units, at least about 70 repeating units, at least about 80 repeating units, at least about 90 repeating units, at least about 100 repeating units, at least about 150 repeating units, at least about 200 repeating units, or at least about 250 repeating units. In various embodiments, L comprises from about 10 monomers / repeating units to about 250 monomers / repeating units. Unlike conventional polymers that use short PEG chains, embodiments of the bioactive synthetic copolymers disclosed herein incorporate long polyalkylene glycol chains of at least 21 repeating units in L.

[0101] In various embodiments, L is PEG 500 , PEG 600 , PEG 700 , PEG 800 , PEG 900 , PEG 1000 , PEG 1100 , PEG 1200 , PEG 1300 , PEG 1400 , PEG 1500 , PEG 2000 , PEG 2500 , PEG 3000 , PEG 3500 , PEG 4000 , PEG 4500 , PEG 5000 , PEG 5500 , PEG 6000 , PEG 6600 and mixtures thereof.

[0102] In various embodiments, X has the following configuration: -R 1 -L-NR 3 -C(=O)-X is attached to the poly(norbornene dicarboximide) backbone through the carboxylic acid functionality. Advantageously, by linking X through the carboxylic acid functionality, the amine end groups in X are completely free to deliver their biological activity, thus ensuring the bioavailability of X. It will be appreciated that since the amine groups confer biological activity, it may not be desirable to use up the amine groups in the biologically active moiety for polymer attachment.

[0103] In various embodiments, X is a peptide / amide bond, i.e., -NR 3 The amide bond is attached to the poly(norbornene dicarboximide) backbone via -C(=O)-. Advantageously, the bioactive synthetic copolymers disclosed herein are significantly stronger and / or more stable than conventional polymers containing ester linkages. Without being bound by theory, it is believed that the amide bond is stronger than the ester bond because the ester bond is more susceptible to hydrolysis, which can release the bioactive moiety into the bloodstream and lead to premature metabolism of the bioactive moiety.

[0104] In various embodiments, one or more of the H atoms in the alkyl, alkenyl, alkynyl, alkoxyalkyl, alkylcarbonyl, and alkylcarbonylalkyl are optionally replaced by hydroxy, hydroxyalkyl, halogen, haloalkyl, cyano, cyanoalkyl, and nitro.

[0105] In various embodiments, R 1 is C1-C 20 alkyl. C-C 20Alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, R can be a straight chain or branched substituent selected from 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, and the like. 1 can be a straight chain or branched C1-C4 alkyl substituent. In various embodiments, R 1 The length of R is the same as the length of the repeating unit in L. For example, if L is poly(butylene glycol), then R 1 is butyl. In another example, when L is poly(ethylene glycol), then R 1 is ethyl. In various embodiments, R 1 It will be appreciated that is carefully designed to fit L.

[0106] In various embodiments, R 3 is H, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 alkynyl.

[0107] In various embodiments, Z 1 and Z 2 are each independently CH2, O, NH, SiR a Rb , PR a or S. The poly(norbornene) backbone may be selected from the group consisting of poly(norbornene-imide), poly(norbornene-dicarboximide), poly(5-norbornene-2,3-dicarboximide), poly(7-oxanorbornene), poly(oxanorbornene-imide), poly(oxanorbornene-dicarboximide), and the like. In various embodiments, Z 1 and Z 2 are each independently a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b , and R c are each independently H, C1-C 20 Alkyl, C1-C 20 Alkenyl and C1-C 20 In various embodiments, Z is selected from the group consisting of alkynyl. 1 is CH. In various embodiments, Z 2 is CH2.

[0108] In various embodiments, X comprises a biologically active moiety selected from a protein, a peptide, a carbohydrate, a therapeutic / drug molecule, and a derivative thereof. In various embodiments, the protein, peptide, carbohydrate, or therapeutic / drug molecule, or a derivative thereof, comprises a protein, peptide, carbohydrate, or therapeutic / drug molecule that is optionally modified or modified to contain one carboxylic acid terminal group. In some embodiments, the biologically active moiety contains only one carboxylic acid terminal group.

[0109] In various embodiments, the biologically active moiety comprises a monocarboxylic acid. Advantageously, the use of a biologically active moiety with a monocarboxylic acid end group avoids the possibility of undesired cross-linking that would otherwise occur if there were two or more carboxylic acids. Accordingly, in various embodiments, the biologically active moiety X is substantially free of two or more carboxylic acid end groups, for example, dicarboxylic acids or tricarboxylic acids.

[0110] In various embodiments, X comprises a protein or peptide. X can be a peptide sequence, a laminin-derived peptide, an integrin-binding peptide, a cell-penetrating peptide, a collagen mimetic, or a collagen fragment. In various embodiments, X comprises 2 to 50 amino acid residues, 2 to 40 amino acid residues, or 2 to 20 amino acid residues of any sequence. In various embodiments, X comprises 7 amino acid residues, 6 amino acid residues, 5 amino acid residues, 4 amino acid residues, or 3 amino acid residues, including 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 25 amino acid residues, 20 amino acid residues, 15 amino acid residues, 10 amino acid residues, 9 amino acid residues, 8 amino acid residues, or 3 amino acid residues of any sequence. The amino acid residues may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, glycine, serine, threonine, serine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid. In various embodiments, X is a peptide sequence containing 3 to 20 naturally occurring amino acids. X may be an integrin-binding peptide selected from the group consisting of arginine-glycine-aspartic acid (RGD), SRGDS, and RGDS; a laminin-derived peptide A5G81 (AGQWHRVSVRWGC); an osteopontin-derived peptide SVVYGLR; or a cell-permeable / antimicrobial peptide selected from (IRIK)2 or (IKKI)3. In various embodiments, X is a collagen sequence containing 3 to 20 units of glycine (G), proline (P) and hydroxyproline (Hyp) in any sequence or permutation. X is (PHypG) n Type array, (PGHyp) n Type array, (HypGP) n Type array, (HypPG) n Type sequence, (GHypP) n Type array, (GPHyp) n The collagen fragment may have a type 2 sequence or a collagen mimetic DGEA.

[0111] In various embodiments, X comprises a carbohydrate. In various embodiments, X comprises a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. In various embodiments, X comprises 2 to 50 saccharide units, 2 to 40 saccharide units, 2 to 20 saccharide units, or 10 to 14 saccharide units. In various embodiments, X comprises 50 saccharide units, 40 saccharide units, 30 saccharide units, 25 saccharide units, 20 saccharide units, 15 saccharide units, 14 saccharide units, 13 saccharide units, 12 saccharide units, 11 saccharide units, 10 saccharide units, 9 saccharide units, 8 saccharide units, 7 saccharide units, 6 saccharide units, 5 saccharide units, 4 saccharide units, 3 saccharide units, or 2 saccharide units. X can be heparan sulfate (HS) or glycosaminoglycan (GAG). In various embodiments, X is a heparan sulfate / oligosaccharide selected from the group consisting of DP8, DP10, DP12, DP14, and DP 16. In various embodiments, X is hyaluronic acid, the simplest form of glycosaminoglycan (GAG).

[0112] In various embodiments, X is chemically bonded to the remainder of general formula (I) via its hydroxy group. For example, when X is a carbohydrate / sugar, an oxidation and / or reductive amination reaction can be performed on the hydroxy of the carbohydrate to link X to general formula (I). The -CHOH on the sugar can be oxidized to -C(=O)H, which subsequently undergoes reductive amination using the -NH terminus on L to create a peptide bond.

[0113] In various embodiments, X comprises a carbohydrate / sugar that contains or has been modified to contain a carboxylic acid terminal group. Modification by one or more chemical reactions, such as oxidation, can be performed on the carbohydrate / sugar to create a carboxylic acid group. In various embodiments, the modification is performed on a hydroxyl group originally present in the carbohydrate / sugar. In various embodiments, the —CHOH on the carbohydrate / sugar is completely oxidized to —C(═O)OH, which subsequently reacts with the —NH terminus on L to create a peptide bond linking the carbohydrate / sugar to the remainder of general formula (I): XC(═O)—NH-L—. However, it will be understood that if a carboxylic acid is naturally present in the carbohydrate / sugar, no modification to the carbohydrate / sugar is / may not be required.

[0114] In various embodiments, X comprises a therapeutic / drug molecule. In various embodiments, X comprises an antibiotic, antimicrobial, antibacterial, blood thinner, or anti-inflammatory agent. X can be penicillin, amoxicillin, amphotericin, ciprofloxacin (CIF), atorvastatin, aspirin, or an aminoglycoside molecule selected from streptomycin, ribostamycin, or gentamicin. It will be understood that X can be any therapeutic or drug molecule containing a carboxylic acid group.

[0115] In various embodiments, X is chemically bonded to the remainder of general formula (I) through one of its chemical moieties selected from the group consisting of -COOH, -CHOH, -CHNH, and =CHNH. For example, -CHNH or =CHNH on a drug molecule can be attached to a small dicarboxylic acid before reacting with the -NH terminus on L to create a peptide bond linking the drug molecule to the remainder of general formula (I): XC(=O)-NH-L-.

[0116] In various embodiments, X comprises a therapeutic / drug molecule that contains or has been modified to contain a carboxylic acid terminal group. Modification by one or more chemical reactions, such as oxidation, can be performed on the therapeutic / drug molecule to create a carboxylic acid group. In various embodiments, the modification is performed on a hydroxyl group originally present in the therapeutic / drug molecule. For example, in various embodiments, when X is ribostamycin or gentamicin, the —CHOH on the drug molecule is fully oxidized to —C(═O)OH, which subsequently reacts with the —NH terminus on L to create a peptide bond linking the drug molecule to the remainder of general formula (I): XC(═O)—NH-L—. However, it will be understood that if a carboxylic acid is already present in the therapeutic / drug molecule, no modification to the therapeutic / drug molecule is / may not be required.

[0117] In various embodiments, the biologically active moiety is modified or has been modified to contain one carboxylic acid terminal group. For example, if a carboxylic acid terminal group is not present in the carbohydrate or therapeutic / drug molecule, the carbohydrate or therapeutic / drug molecule can be modified to add a carboxylic acid to one of the termini of the carbohydrate or therapeutic / drug molecule. The modification can include an oxidation reaction to convert a hydroxy group in the carbohydrate to a carboxylic acid.

[0118] In various embodiments, the repeat unit represented by general formula (I) is present in an amount of about 1 mol% to about 100 mol%, about 2 mol% to about 99 mol%, about 3 mol% to about 98 mol%, about 4 mol% to about 97 mol%, about 5 mol% to about 96 mol%, about 10 mol% to about 95 mol%, about 15 mol% to about 90 mol%, about 20 mol% to about 85 mol%, about 25 mol% to about 80 mol%, about 30 mol% to about 75 mol%, about 35 mol% to about 70 mol%, about 40 mol% to about 65 mol%, about 45 mol% to about 60 mol%, or about 50 mol% to about 55 mol% of the copolymer. In various embodiments, the repeat unit represented by general formula (I) is present in an amount of about 1 mol% to about 10 mol% of the copolymer. In various embodiments, the biologically active moiety is about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, or about 10 mol% of the biologically active synthetic copolymer.

[0119] In various embodiments, R 2 is C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxyalkyl, C2-C 20 Alkylcarbonyl or C3-C 20 alkylcarbonylalkyl. C1-C 20Alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2 The substituents may be straight chain or branched and may be selected from 1,2,3-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, and the like.

[0120] In various embodiments, Y 1 is represented by the general formula (III):

[0121] [ka]

[0122] (In the formula, A is selected from a single bond, oxy, carbonyl, oxycarbonyl, carboxyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, or optionally substituted alkoxycarbonylalkyl; B is optionally present as a ring selected from 1,2,3-triazole or succinimide; R 5is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; Y 2 is selected from the group consisting of polypropylene (PP), polyester, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylate, poly(meth)acrylate, polyamide (PA), and portions thereof; and T is an end group selected from the group consisting of hydrogen, halogen, hydroxyl, amino, acyl, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, and optionally substituted alkoxycarbonylalkyl).

[0123] In various embodiments, Y 2 is a polyacrylate containing one or more monomers selected from the group consisting of methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, hexyl acrylate, cyclohexyl acrylate, 2-ethylhexyl acrylate, benzyl acrylate, and phenyl acrylate. 2 can be poly(methyl acrylate), poly(ethyl acrylate), poly(butyl acrylate), or poly(2-ethylhexyl acrylate). In various embodiments, Y 2is a poly(meth)acrylate containing one or more monomers selected from the group consisting of methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate, and phenyl methacrylate. 2 can be poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) and poly(butyl methacrylate) or poly(2-ethylhexyl methacrylate).

[0124] In various embodiments, A is selected from a single bond, oxy, carbonyl, or oxycarbonylalkyl. A can be a single bond, O, C(=O), or OC(=O)-R, where R is an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl. In various embodiments, R is methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, A is a straight chain or branched alkyl substituent selected from 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, etc. In various embodiments, A is selected from a single bond, O, C(═O) or OC(═O)—C1-C6 alkyl.

[0125] In various embodiments, B is absent. In various embodiments, B is present as a ring selected from 1,2,3-triazole or succinimide. Advantageously, 1,2,3-triazole is suitable for connectivity with the present system due to the chemistry used. For example, azide-alkyne click chemistry can be used to synthesize norbornene dicarboximide into a synthetic polymer Y. 2 Advantageously, succinimides are suitable for compatibility with this system due to the chemistry used. For example, addition of maleic anhydride onto a vinyl-terminated polyolefin forms succinic anhydride, which then reacts with the amine terminus created on norbornene dicarboximide (via hexamethylenediamine (HMDA) or a similar diamine) to convert norbornene dicarboximide to the synthetic polymer Y. 2 This forms a succinimide which links to

[0126] In various embodiments, R 5 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. 5 represents a single bond or an alkyl group such as ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2- It can be a straight chain or branched alkenyl substituent selected from butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, and the like. 5is selected from a single bond or C2-C6 alkenyl.

[0127] In various embodiments, Y 2 is selected from the group consisting of polypropylene (PP), polyester, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylate, poly(meth)acrylate, polyamide (PA), and portions thereof. In various embodiments, Y 2 include one or more of the following properties: bioabsorbability; inertness; long shelf life; mechanical strength; impact resistance; thermal stability; elasticity; elastic recovery; smoothness; biodegradability; lightweight; and low or no toxicity.

[0128] In various embodiments, Y 2 is substantially free of polyalkylene glycols such as polyethylene glycol.

[0129] In various embodiments, T is a terminal group selected from the group consisting of hydrogen, halogen, hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkylcarboxylalkyl, and optionally substituted alkoxycarbonylalkyl. T ​​can be a halogen selected from H, OH, Cl, F, Br, I, C-C alkyl, C-C alkyl-C(=O)-O-C-C alkyl, or C-C alkyl-OC(=O)-C-C alkyl.

[0130] In various embodiments, Y 1 is represented by the following general formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IIIf):

[0131] [ka]

[0132] where n≧1; and m≧1.

[0133] In various embodiments, the total molecular weight of general formula (II) is kept below about 15,000 or below about 10,000. It will be understood that if the total molecular weight of general formulas (I) and (II) is too high, copolymerization may become inefficient. In various embodiments, when the bioactive synthetic copolymer is used for applications requiring fast biodegradation, the molecular weight of general formula (II) is kept low by adjusting the value of n and / or m.

[0134] In various embodiments, R 2 -Y 1 is the following:

[0135] [ka]

[0136] where n≧1; and m≧1.

[0137] In various embodiments, the ratio of the number of repeating units represented by general formula (I) to the number of repeating units represented by general formula (II) in the bioactive synthetic copolymer is from about 1:1 to about 1:100, from about 1:2 to about 1:99, from about 1:3 to about 1:98, from about 1:4 to about 1:97, from about 1:5 to about 1:96, from about 1:6 to about 1:95, from about 1:7 to about 1:90, from about 1:8 to about 1:85, from about 1:9 to about 1:80, from about 1:10 to about 1:75, from about 1:15 to about 1:70, from about 1:20 to about 1:65, from about 1:25 to about 1:60, from about 1:30 to about 1:55, from about 1:35 to about 1:50, or from about 1:40 to about 1:45. In various embodiments, the ratio of the number of repeating units represented by general formula (I) to the number of repeating units represented by general formula (II) in the bioactive synthetic copolymer is about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, or about 1:50.

[0138] In various embodiments, the number of repeating units represented by general formula (I) in the copolymer is from about 10 to about 1,000. In various embodiments, the number of repeating units represented by general formula (II) in the copolymer is from about 10 to about 1,000. In various embodiments, for bone scaffolding, the PLA side chains include from about 50 to about 60 lactide units.

[0139] In various embodiments, the bioactive synthetic copolymer has a number average molecular weight (Mn) of about 1,000 to about 300,000, 2,000 to about 250,000, about 3,000 to about 200,000, about 4,000 to about 150,000, about 5,000 to about 100,000, about 10,000 to about 90,000, about 20,000 to about 80,000, about 30,000 to about 70,000, about 40,000 to about 60,000, or about 50,000.

[0140] In various embodiments, the bioactive synthetic copolymer has a polydispersity index (PDI) of about 1.0 to about 10.0. In various embodiments, the PDI of the bioactive synthetic copolymer is about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In various embodiments, the bioactive synthetic copolymer has a polydispersity index (PDI) of about 1.0 to about 3.0, about 1.05 to about 2.95, about 1.1 to about 2.9, about 1.2 to about 2.8, about 1.4 to about 2.6, about 1.6 to about 2.4, about 1.8 to about 2.2, or about 2.0. In various embodiments, the PDI of the bioactive synthetic copolymer is 1.50 or less.

[0141] In various embodiments, one or more repeating units represented by general formula (I) and one or more repeating units represented by general formula (II) are designed to be linked to the poly(norbornene) backbone via at least a covalent interaction. In various embodiments, each repeating unit represented by general formula (I) is covalently attached to the poly(norbornene) backbone, and / or each repeating unit represented by general formula (II) is covalently attached to the poly(norbornene) backbone. Advantageously, because the bioactive moiety (in general formula (I)) is covalently attached to the bioactive synthetic polymer, the bioactivity is localized. In various embodiments, the bioactive moiety, such as a biomolecule, does not leach from the polymer, thereby preventing undesirable / unwanted side effects caused by the biomolecule entering the circulatory system and / or reaching unintended parts of the body's system. Embodiments of the bioactive synthetic copolymer thus overcome the problems faced by conventional biomolecules administered as drugs, which may be prematurely metabolized before a therapeutic effect is achieved. In various embodiments, the biologically active moiety, such as a drug molecule, does not leach into media that may leak into the environment if disposal is improperly managed.

[0142] It will be appreciated that other interactions, such as van der Waals interactions, may also be present within the copolymer.

[0143] In various embodiments, the bioactive synthetic copolymer comprises a brush, bottle-brush, block, comb, or graft copolymer structure. In various embodiments, the repeating units can be randomly distributed / arranged within the polymer.

[0144] In various embodiments, one or more repeat units represented by general formula (I) comprise two or more different types of biologically active moieties X. In various embodiments, one or more repeat units represented by general formula (I) comprise 2, 3, 4, 5, 6, 7, or 8 different types of biologically active moieties X. For example, within a bioactive synthetic copolymer, there can be a repeat unit represented by general formula (I) that comprises a peptide as X and a repeat unit represented by general formula (I) that comprises a carbohydrate as X. Advantageously, in various embodiments, the bioactive synthetic copolymer confers two or more different types of biological activity.

[0145] In various embodiments, one or more repeat units represented by general formula (II) are selected from two or more different types of synthetic polymers Y 2 In various embodiments, one or more repeat units represented by general formula (II) include 2, 3, 4, 5, 6, 7, or 8 different types of synthetic polymers Y 2 Includes.

[0146] In various embodiments, the bioactive synthetic copolymer is a random polymer or a block copolymer. In some embodiments, the block polymer is a diblock or triblock polymer. For example, the copolymer can have or be composed of two or three different polymer blocks. In some embodiments, the multiblock copolymer contains more than three polymer blocks. The blocks can be randomly distributed / arranged within the polymer.

[0147] In various embodiments, the bioactive synthetic copolymer is selected from one of the following: a PCL-(GPHyp)3 copolymer comprising (GPHyp)3 in general formula (I) and PCL in general formula (II); a PA-DGEA copolymer comprising DGEA in general formula (I) and PA in general formula (II); a PS-ciprofloxacin copolymer comprising ciprofloxacin in general formula (I) and PS in general formula (II); a PLA-RGD copolymer comprising RGD in general formula (I) and PLA in general formula (II); a PLGA-(GPHyp)3 copolymer comprising (GPHyp)3 in general formula (I) and PLGA in general formula (II); and a PMMA-(GPHyp)3 copolymer comprising (GPHyp)3 in general formula (I) and PMMA in general formula (II).

[0148] Advantageously, the bioactive synthetic copolymers disclosed herein are highly customizable. Depending on the intended use of the bioactive synthetic copolymer, X can be selected to have the desired biological activity and Y to have the desired physical attributes. 2 can be selected to ultimately obtain a bioactive synthetic copolymer having the desired repeating units represented by general formulas (I) and (II).

[0149] In various embodiments, the bioactive synthetic copolymer is blended with a base polymer for further use. In various embodiments, the base polymer is a synthetic polymer Y used in general formula (II): 2 In various embodiments, a medical grade polymer is used for the matrix, while a low molecular weight synthetic polymer is used in the synthetic side chains of the bioactive synthetic copolymer. Advantageously, the bioactive synthetic polymer embodiment allows for the blending of biomolecules into the base material of the same synthetic polymer as the synthetic polymer side arms of the copolymer without phase separation.

[0150] method A method for preparing a bioactive synthetic copolymer is provided, the method comprising: polymerizing one or more bioactive polymers represented by general formula (IV) with one or more synthetic polymers represented by general formula (V) to obtain a bioactive synthetic copolymer:

[0151] [ka]

[0152] (In the formula, R 1 is optionally substituted alkyl; R 2 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y 1 comprises a synthetic polymer; and Z 1 and Z 2 are each independently a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0153] Advantageously, in various embodiments, the methods for preparing bioactive synthetic copolymers disclosed herein are also modular methods for designing bioactive synthetic copolymers.

[0154] Also provided is a modular method for designing bioactive synthetic copolymers, the method comprising: selecting one or more polymers from a first module based on a desired biological activity, the first module consisting of a library of norbornene dicarboximide-containing bioactive polymers represented by general formula (IV) with known biological activity; selecting one or more polymers from a second module based on a desired physical attribute, the second module consisting of a library of norbornene dicarboximide-containing synthetic polymers represented by general formula (V) with known physical attribute; and polymerizing the one or more polymers selected from the first module with the one or more polymers selected from the second module to obtain a bioactive synthetic copolymer.

[0155] [ka]

[0156] Advantageously, the methods disclosed herein allow for the rapid customization and rapid development / construction of bioactive synthetic copolymers with desired biological activity and physical properties.

[0157] In various embodiments, the polymerization reaction comprises one or more olefin metathesis chain-growth polymerization steps. The olefin metathesis chain-growth polymerization can be ring-opening metathesis polymerization (ROMP). In various embodiments, the ROMP reaction occurs at a reactive portion of a macromonomer, for example, at an olefin / alkene / C=C portion. ROMP can include many different approaches, including "arm-first" ROMP, "brush-first" ROMP, "graft-to" ROMP, "graft-from" ROMP, "graft-through" ROMP, or a combination thereof. Advantageously, ROMP allows for the rapid development / construction of well-defined synthetic polymers with desired biological activity. In various embodiments, depending on the target application, appropriate synthetic polymers and biomolecules with desired biological activity can be selected and copolymerized together using ROMP.

[0158] In various embodiments, the polymerization reaction is carried out in the presence of a polymerization initiator / catalyst / accelerator. In various embodiments, the polymerization initiator / catalyst / accelerator comprises a metal complex. The metal complex can be a ruthenium (Ru), molybdenum (Mo), or tungsten (W) complex. In various embodiments, ROMP is carried out in the presence of a ruthenium complex. Advantageously, compared to other transition metals (e.g., W and Mo), Ru is more stable in the presence of polar functional groups, making Ru a suitable olefin metathesis catalyst for ROMP reactions involving biologically active moieties selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules, and their derivatives. In various embodiments, Ru is air-stable (i.e., stable in air) and thermally stable (i.e., stable at high temperatures), while being commercially available on a large scale, allowing ROMP to be carried out at high temperatures. The ruthenium complex may comprise a Grubbs catalyst selected from a first generation Grubbs catalyst, a second generation Grubbs catalyst, a Hoveyda-Grubbs catalyst, a third generation Grubbs catalyst, or derivatives thereof.

[0159] In various embodiments, R 1 , R 2, R 3 , L, X, Y 1 , Z 1 and Z 2 contains one or more features and / or shares one or more properties similar to those described above.

[0160] In various embodiments, the polymerization reaction includes: a) mixing one or more bioactive polymers represented by general formula (IV) with one or more synthetic polymers represented by general formula (V) to obtain a solution; b) adding a catalyst to the solution from a); and c) precipitating the bioactive synthetic copolymer.

[0161] In various embodiments, step a) and / or step b) are performed or undertaken at a temperature ranging from about 20° C. to about 100° C. The temperature at which step a) and step b) are performed may be independently selected from temperatures of about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C., about 90° C., or about 100° C.

[0162] In various embodiments, step a) and / or step b) are performed or engaged for a period of time ranging from about 30 minutes to about 3 days. The period of time for performing step a) and step b) may be independently selected from a period of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 20 hours, 1 day, 2 days, or 3 days.

[0163] In various embodiments, step a) and / or step b) are carried out in the presence of an organic solvent. The organic solvent can be a protic solvent, an aprotic solvent, or a combination thereof. In various embodiments, the organic solvent for step a) and step b) is independently selected from the group consisting of tetrahydrofuran (THF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), formic acid, acetic acid, etc., and combinations thereof. In various embodiments, protic solvents such as formic acid and / or acetic acid are preferred, particularly for PA-based materials (e.g., Y 1(wherein the formula IIIf is included) can be used. In various embodiments, the organic solvent used is the same for steps a) and b). It should be understood that the type of solvent used depends on the type of reactants used and is not limited to the above.

[0164] In various embodiments, step c) is carried out in a mixture of organic solvents. The mixture of organic solvents may contain one or more aprotic organic solvents and one or more protic organic solvents. In various embodiments, the mixture of organic solvents for step c) is selected from the group consisting of tetrahydrofuran (THF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), ethyl vinyl ether, methanol, ethanol, butanol, etc., and combinations thereof. It should be understood that the type of solvent used depends on the type of reactants used and is not limited to the above.

[0165] Advantageously, by conducting the polymerization under the above-described carefully designed / controlled conditions, embodiments of the methods disclosed herein have successfully overcome widely varying and / or opposing properties of the individual components (e.g., L, X, Y components) to construct the bioactive synthetic copolymers disclosed herein.

[0166] Also provided is a method for preparing a bioactive homopolymer, the method comprising: polymerizing one or more bioactive macromolecules represented by general formula (IV) to obtain a bioactive synthetic homopolymer:

[0167] [ka]

[0168] (In the formula, R 1 is optionally substituted alkyl; R 3is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules, and derivatives thereof; and Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0169] Also provided is a method for preparing a synthetic homopolymer, the method comprising: polymerizing one or more synthetic polymers represented by general formula (V) to obtain a synthetic homopolymer:

[0170] [ka]

[0171] (In the formula, R 2 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, or optionally substituted alkylcarbonylalkyl; Y 1 comprises a synthetic polymer; and Z 2 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R care each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0172] bioactive polymer Also provided is a bioactive polymer represented by general formula (IV) for preparing the copolymers disclosed herein:

[0173] [ka]

[0174] (In the formula, R 1 is optionally substituted alkyl; R 3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is heteroalkylene; X comprises a biologically active moiety selected from proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; and Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

[0175] In various embodiments, R 1 , R 3 , L, X and Z 1 contains one or more features and / or shares one or more properties similar to those already described above.

[0176] In various embodiments, the bioactive polymer undergoes self-polymerization or copolymerization, and in various embodiments, the bioactive polymer also behaves as a bioactive macromonomer.

[0177] In various embodiments, X has the following configuration: -R 1 -L-NR 3 The linkage to norbornene dicarboximide is via the carboxylic acid functionality in -C(=O)-X. Advantageously, by linking X through the carboxylic acid functionality, the amine end group in X is completely free to deliver its biological activity, thus ensuring the bioavailability of X. It will be appreciated that since the amine group confers biological activity, it may not be desirable to use up the amine groups in the biologically active moiety for polymer attachment.

[0178] In various embodiments, X is a peptide / amide bond, i.e., -NR 3 The amide bond is bonded to norbornene dicarboximide via -C(=O)-. Advantageously, the bioactive polymers disclosed herein are significantly stronger and / or more stable than conventional polymers containing ester bonds. Without being bound by theory, it is believed that the amide bond is stronger than the ester bond because the ester bond is more susceptible to hydrolysis, which can release the bioactive moiety into the bloodstream and lead to early metabolism of the bioactive moiety.

[0179] Also provided is a method for preparing the bioactive polymers disclosed herein, the method comprising: (i) a compound of general formula (VI):

[0180] [ka]

[0181] (In the formula, Z 1 is CR a R b , O, N.R. c , SiR a R b , PRa or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; (ii) reacting said dicarboxylic acid anhydride having the general formula (VI) with a diamine R 4 R 3 NLR 1 -NH2 to form a compound of general formula (VII):

[0182] [ka]

[0183] (In the formula, R 1 is optionally substituted alkyl; R 3 and R 4 are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, where R 3 and R 4 at least one of is H; L is heteroalkylene; Z 1 is CR a R b , O, N.R. c , SiR a R b , PR a or S, where R a , R b and R c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; and (iii) reacting said amine having general formula (VII) with an acid-containing biologically active moiety XC(=O)OH to obtain a biologically active macromolecule, where X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof.

[0184] In various embodiments, R 1 , R 3 , L, X and Z 1 contains one or more features and / or shares one or more properties similar to those described above.

[0185] In various embodiments, R 3 and R 4 are each independently H, C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 alkynyl, wherein R 3 and R 4 At least one of is H.

[0186] In various embodiments, step (ii) comprises the step of providing a diamine R to attach X to the norbornene dicarboxylic acid anhydride. 4 R 3 NLR 1 The diamines used may be commercially available. Advantageously, the method is a simple reaction and does not require poly(ethylene glycol) aminocarboxylic acids, which are not commercially available and are difficult to prepare synthetically. Accordingly, in various embodiments, the method does not require tedious multi-step and / or low-yield synthetic procedures. In various embodiments, the diamine R 4 R 3 NLR 1 -NH2 is an amine having the general formula (VII) attached to norbornene dicarboximide on two ends (otherwise, diamine R 4 R 3 NLR 1 Use a slight excess to ensure that the -NH2 is the linker instead of the terminal group.

[0187] In various embodiments, the diamine is a heteroalkylenediamine where L is heteroalkylene.20 -C 300 Heteroalkylene, i.e., heteroalkylene having 20 carbon atoms to 300 carbon atoms. In various embodiments, L has a number average molecular weight between about 500 and about 7,000. In various embodiments, the heteroatom in L is O. In various embodiments, L is a polyalkylene glycol. In various embodiments, the diamine is a poly(ethylene glycol) diamine, where L is poly(ethylene glycol). In various embodiments, L is PEG. 500 , PEG 600 , PEG 700 , PEG 800 , PEG 900 , PEG 1000 , PEG 1100 , PEG 1200 , PEG 1300 , PEG 1400 , PEG 1500 , PEG 2000 , PEG 2500 , PEG 3000 , PEG 3500 , PEG 4000 , PEG 4500 , PEG 5000 , PEG 6000 and mixtures thereof.

[0188] In various embodiments, the method further comprises purifying the amine having general formula (VII) to isolate the product and / or remove impurities prior to step (iii). In various embodiments, the purification step comprises washing with at least one of an acid or a base. The purification step can comprise washing with at least one of an acid or a base at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least eight times to neutralize the amine having general formula (VII). In various embodiments, the purification step comprises a double neutralization step. In one embodiment, the double neutralization step removes unreacted diamine R. 4 R 3 NLR 1The diamine R comprises a first step of washing with an acid to remove —NH and a second step of washing with a base to neutralize the amine having the general formula (VII). 4 R 3 NLR 1It will be appreciated that because —NH is basic, adding an acid to the diamine will neutralize the diamine for removal from the amine having general formula (VII). It will also be appreciated that a first step of washing with acid may protonate the amine having general formula (VII) at the amine terminus, while a subsequent second step of washing with base or excess base will convert the protonated form to its free amine form. The acid used for the first neutralization step may be selected from the group consisting of HCl, HNO, HSO, and HPO. The base used for the second neutralization step may be selected from the group consisting of NaOH, KOH, NHOH, and Ca(OH). In various embodiments, the second neutralization step comprises washing with base at least once, at least twice, at least three times, or at least four times to fully extract the amine having general formula (VII) for maximized yield. In one embodiment, the second neutralization comprises washing with base twice. Without being bound by theory, it is believed that up to 30% of the protonated form of the amine having general formula (VII) may be present in the aqueous phase during extraction. In various embodiments, the base washing step includes washing the aqueous phase once with base and washing the organic phase once with base to completely extract the amine having general formula (VII) from both the aqueous and organic phases. Advantageously, by using a double neutralization step after coupling to obtain free amine ends, the method eliminates the need for any additional steps, such as a protection / deprotection step. Those skilled in the art will understand that the use of diamines, particularly poly(ethylene glycol) diamines, is extremely difficult and typically requires protection of one amine end for coupling to norbornene dicarboxylic acid anhydride. Indeed, in various embodiments, polyalkylene glycols such as PEG are used as spacers, linkers, or linking groups in the overall polymer, instead of as end groups. That is, it may seem intuitive to consider protecting one amine end of a PEG diamine and coupling it to norbornene dicarboxylic acid anhydride. The protecting group can then be removed to expose the amine terminus for further reaction.However, this would add an extra step to the reaction and may therefore be undesirable. Embodiments of the present disclosure successfully overcome this problem in the synthesis and purification process by performing a double neutralization step after conjugation to provide a free amine terminus for further conjugation to the peptide.

[0189] Materials containing bioactive synthetic copolymers - Patent Application 20070122997 Also provided is a material comprising the copolymer disclosed herein for use in medicine.In various embodiments, the material is part of or used on a device selected from the group consisting of wound dressings, skin scaffolds, bone scaffolds, organoid scaffolds, implants, and medical devices.For example, the material can be a scaffold for tissue regeneration, comprising the bioactive synthetic copolymer disclosed herein.The material can be a material suitable for increasing the biocompatibility of polyamides used in medical devices through collagen stimulation.The material can be an antibacterial polystyrene material suitable for use in tissue and serum handling devices.The material can be a polylactide scaffold suitable for stimulating tissue regeneration.The material can be a poly(lactic acid-co-glycolic acid) scaffold suitable for stimulating cartilage tissue regeneration.The material can also be a poly(methyl methacrylate) material for use in medical implants.

[0190] In various embodiments, the material is processed / printed / 3D printed via electrospinning, melt extrusion, hot melt extrusion, injection molding, fused filament fabrication, fused deposition modeling, additive manufacturing, meltblowing, and the like.

[0191] In various embodiments, the material or bioactive synthetic copolymer, when used on / in the human or animal body, is compatible with a biological system or part of a biological system without substantially or significantly eliciting an adverse physiological response, such as a toxic reaction / response, an immune reaction / response, injury, etc. In various embodiments, the polymer is substantially free of substances that elicit an adverse physiological response.

[0192] Also provided is a method of accelerating / stimulating / promoting cell growth or tissue regeneration, such as bone or skin tissue regeneration, or wound healing, which method comprises administering / applying to the human or animal body a bioactive copolymer or material disclosed herein.

[0193] Also provided is the use of the bioactive synthetic copolymers or materials disclosed herein in the manufacture of a medicament for accelerating / stimulating / promoting cell growth or tissue regeneration, or wound healing, such as the regeneration of bone or skin tissue.

[0194] Also provided is the use of the bioactive synthetic copolymers or materials disclosed herein for biofilm eradication.

[0195] In various embodiments, the bioactive synthetic copolymer is substantially free of stem cells and / or growth factors. In various embodiments, the bioactive synthetic copolymer is non-biofouling.

[0196] In various embodiments, the biologically active moiety is directly chemically linked to the copolymer. In various embodiments, the biologically active moiety is not encapsulated in a polymer matrix.

[0197] In various embodiments, the biologically active moiety (eg, a peptide) is not attached to the norbornene dicarboximide via an aminobutyric acid spacer.

[0198] In various embodiments, the bioactive moiety comprises a structurally well-defined collagen having a specific sequence, hi various embodiments, the bioactive moiety is substantially free of animal-derived collagens that have a broad molecular weight distribution and / or an ill-defined structure and / or are known to elicit a negative immune response in the human body.

[0199] In various embodiments, polyethylene glycol is not used as a monomer as such, for example, in various embodiments, ethylene glycol units are not present as end groups in the copolymer / polymer.

[0200] Embodiments of the bioactive synthetic polymers and / or methods disclosed herein do not involve any release of bioactive molecules, such as drug molecules, from the copolymer upon activation, such as photoactivation. Embodiments of the bioactive synthetic polymers are substantially free of photocleavable groups. [Brief explanation of the drawings]

[0201] [Figure 1] FIG. 1 is a schematic diagram 100 of a bioactive synthetic polymer according to various embodiments disclosed herein. [Figure 2] Figure 2 shows thermogravimetric analysis (TGA) graphs of pure RGD peptide ("RGD(PURE)"), NBPEG3400RGD macromonomer ("NB-PEG3400RGD"), NBPCL macromonomer ("NB-PCL"), and PCL-RGD ROMP copolymer ("PCL_PEG3400_RGD"). [Figure 3] Figure 3 is a graph showing the biocompatibility of PCL-peptide-based materials prepared according to various embodiments disclosed herein compared to a control. Results are from a cell viability study of human fibroblasts (Hs27) on PCL-peptide-based materials over a 72-hour period, in which macromonomers of three peptides (SRGDS, (GPHyp)3, and DGEA) were copolymerized with macromonomers of PCL. Comparative examples are the commercially available bandages Allevyn (i.e., a polyurethane-based bandage) and Acticoat (a silver nanoparticle-based bandage). [Figure 4] 4 is a graph showing the BMP-2-induced ALP activity of PCL-peptide based materials after 72 hours. Commercially available PCL is used as a control. [Figure 5]FIG. 5 shows thermogravimetric analysis (TGA) graphs of NBPEG3400(GPHyp)3 macromonomer ("NB-PEG-GPHP"), PA6 ROMP polymer ("PA6-homopoly"), and PA6-(GPHyp)3 ROMP copolymer ("PA6-GPHP"). [Figure 6] Figure 6 shows the cell proliferation results from a cell viability test of human fibroblasts (Hs27) cultured on polyamide 6 (PA6)-based electrospun sheets using a Luminescent Cell Viability Assay (CellTiter-Glo). The PA-collagen materials were PA6-(PHypG)3, PA6-(GPHyp)3, and PA6-DGEA, where (PHypG)3 and (GPHyp)3 are both collagen fragments, and DGEA is a collagen mimetic. The controls used were poly(norbornene dicarboximide) with PA6 side chains; and poly(norbornene dicarboximide) with PA6 and mPEG5000 side chains. PA6-homopoly refers to poly(norbornene dicarboximide) with PA6 side chains; PA6-mPEG refers to poly(norbornene dicarboximide) with PA6 and mPEG5000 side chains; PA6-PHPG refers to PA6-(PHypG)3 copolymer; and PA6-GPH refers to PA6-(GPHyp)3 copolymer. [Figure 7] Figure 7 is a graph showing cell proliferation results from cell viability testing of human fibroblasts (Hs27) cultured on poly(lactide) (PLA)-based electrospun sheets using a Luminescent Cell Viability Assay (CellTiter-Glo). The bioactive synthetic copolymer is PLA-RGD, and the commercially available base polymer PLA is used as a control ("PLA bulk"). [Figure 8]Figure 8 shows the biocompatibility results from cell viability testing of human fibroblasts (Hs27) cultured on poly(lactic-co-glycolic acid) (PLGA)-based electrospun sheets using the Luminescent Cell Viability Assay (CellTiter-Glo). The bioactive synthetic copolymer used was PLGA-RGD. The controls used were the commercially available base polymer PLGA (PLGA-Bulk), poly(norbornene dicarboximide) with PLGA side chains, and poly(norbornene dicarboximide) with PLGA and mPEG5000 side chains. PLGA-homo refers to poly(norbornene dicarboximide) with PLGA side chains; PLGA-mPEG refers to poly(norbornene dicarboximide) with PLGA and mPEG5000 side chains. [Figure 9] Figure 9 is a graph showing biocompatibility results from cell viability testing of human fibroblasts (Hs27) cultured on poly(methyl methacrylate) (PMMA)-based electrospun sheets using the Luminescent Cell Viability Assay (CellTiter-Glo). The bioactive synthetic copolymer is PMMA-(GPHyp)3 ("PMMA-GPHP"). The controls used were the commercially available base polymer PMMA (PMMA-bulk), poly(norbornene dicarboximide) with PMMA side chains; and poly(norbornene dicarboximide) with PMMA and mPEG5000 side chains. PMMA-homo refers to poly(norbornene dicarboximide) with PMMA side chains; PMMA-mPEG refers to poly(norbornene dicarboximide) with PMMA and mPEG5000 side chains. [Example]

[0202] Exemplary embodiments of the present disclosure will be better understood and readily apparent to those skilled in the art in conjunction with the following examples, tables, and, where applicable, drawings. It should be understood that other modifications, including structural and chemical changes, may be made without departing from the scope of the present invention. The exemplary embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of the disclosure.

[0203] Example 1: Modular approach to constructing bioactive synthetic polymers A general strategy for the construction of bioactive macromonomers containing either peptides, carbohydrates, or drug molecules has been developed. A simple two-step synthesis allows for the rapid construction of a broad library of bioactive macromonomers of various chain lengths, enabling the rapid development of synthetic polymers with the desired biological activity required for targeted applications. By matching bioactive macromonomers with synthetic polymer macromonomers with desired physical properties, this library allows for a modular approach to constructing desired macromolecules suitable for a variety of applications. Rapid polymer customization can thus be achieved.

[0204] We have developed a modular building block system for designing / constructing desired bioactive materials, as shown in Scheme 1. Once the target medical application is identified, a "plug-and-play" approach (Scheme 1) can be used to create the desired bioactive synthetic material that not only has therapeutic efficacy but also possesses the necessary mechanical properties for easy storage and handling.

[0205] [ka]

[0206] Using a modular approach, macromonomers consisting of bioactive molecules at the monomer termini can be created and copolymerized with other synthetic polymers to create bioactive synthetic polymers with targeted biological activity. Desired polymers are highly customizable using the strategies developed according to various embodiments disclosed herein by switching the bioactive molecule to any peptide or carbohydrate bearing a carboxylic acid group. This allows for the rapid synthesis of bioactive polymers once a target application is identified. The bioactive polymers created can have properties ranging from skin cell regeneration, bone cell regeneration, antibacterial activity, cartilage tissue regeneration, wound healing, collagen production, and anti-inflammation to cholesterol synthesis inhibition (e.g., using atorvastatin as the drug) and can be made to be mechanically robust or biodegradable, depending on the needs. Modular synthesis therefore makes tailoring polymer properties to applications much simpler and more effective.

[0207] Example 2: Method for preparing bioactive synthetic copolymers A method for preparing bioactive synthetic copolymers according to various embodiments disclosed herein involves separately creating macromonomers and synthetic polymers of bioactive molecules and then using ring-opening metathesis polymerization (ROMP) techniques to link these otherwise incompatible molecules together. The result is a brush polymer bearing both the bioactive molecule and the synthetic polymer for the material's overall mechanical strength (Scheme 2). By separately creating the macromonomers, the inventors can build a library of macromonomers with various properties from which clinicians or medical technology companies can choose, allowing materials with desired therapeutic effects to be easily and quickly constructed to suit the targeted application. By separately creating the macromonomers, the inventors can also build libraries of macromonomers and final copolymers for rapid testing of efficacy in biomedical laboratories. Various combinations of these macromonomers (MMs) can also generate libraries of well-defined brush copolymers containing various bioactive molecules for rapid screening of bioactivity in the laboratory.

[0208] In the following examples, brush polymers containing pendant arms of synthetic polymers and bioactive molecules tethered to polyethylene glycol (PEG) moieties were created. The synthetic polymers can include polyesters such as poly(caprolactone) (PCL), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), polyacrylates, poly(meth)acrylates such as poly(methyl methacrylate) (PMMA), and polyamides (PA). The bioactive molecules can include biomolecules selected from peptide sequences of 3 to 20 amino acid residues, any combination of the 20 natural amino acids, carbohydrates such as glycosaminoglycans, or drug molecules containing a carboxylic acid terminus, such as certain antibiotics. Biomolecules can also include collagen-mimetic peptides of any sequence of 3 to 20 amino acid residues, such as DGEA, (Gly-Pro-Hyp)3, and (Pro-Hyp-Gly)3. Depending on the application, suitable synthetic polymers and biomolecules with desired biological activity can be selected and copolymerized together using the brush polymer technology disclosed herein via ring-opening metathesis polymerization.

[0209] The resulting polymers exhibit the bioactivity of the biomolecules involved while possessing much better physical and mechanical properties for easier material handling and processability. For example, improvements in cell viability or proliferation over controls were observed for both PA-collagen and PLA-RGD copolymers.

[0210] The polymer can subsequently be blended with a similar polymer to that on the pendant arm to create a bioactive material for use in biomedical devices such as catheters, wound dressings, tissue scaffolds, plastic surgery implants, prosthetic components, cartilage joint implants, and the like.

[0211] A synthetic route for preparing bioactive synthetic polymers according to various embodiments disclosed herein is illustrated in Scheme 2.

[0212] [ka]

[0213] In the following examples, six types of synthetic polymers were selected for the synthetic polymer side chains on the brush polymers. The exact polymer chosen will depend on the nature of the biomedical device being fabricated, such as whether properties such as biodegradability, flexibility, or impact resistance are required in the device material.

[0214] Example 3: Bioactive Macromonomers and Methods of Synthesis A general strategy for the synthesis of bioactive macromonomers according to various embodiments disclosed herein was developed. Polyethylene glycol diamines of various chain lengths (e.g., M W =1,000-6,000) is reacted with cis-norbornene-exo-2,3-dicarboxylic anhydride to produce the primary macromonomer entity, i.e., a macromonomer entity containing norbornene dicarboximide and polyethylene glycol (NBPEG) (Scheme 3.1). Once the NBPEG is produced, various peptides, carbohydrates, or drug molecules can then be reacted with these NBPEG chains to produce bioactive macromonomers with desired therapeutic properties.

[0215] Using the macromonomer body, any peptide, carbohydrate, or drug molecule (R) can be used by condensation reaction using the carboxylic acid terminus on the bioactive molecule to form a peptide / amide bond between the amine group on NBPEG and the carboxylic acid terminus of the carbohydrate or peptide (Scheme 3.2). Examples of drug molecules include antibiotics such as amoxicillin or ciprofloxacin. In various embodiments, R is a copolymer of antimicrobial peptides (IRIK)2 or (IKKI)3; heparin oligosaccharides DP10, DP12, DP14; COL or RGD, where COL is DGEA, (GPHyp) n or (PHypG) nIn various embodiments, R is a carbohydrate or drug molecule having a COH group or a peptide sequence of 3 to 20 amino acid residues formed from the 20 naturally occurring amino acids. In various embodiments, R is DP12, DP14, COL, or RGD, where COL is DGEA or (GPHyp) n If the carbohydrate does not have a carboxylic acid group, modification of the carbohydrate to include one may be necessary. Alternatively, amine substitution or reductive amination reactions can also be performed on hydroxy or carbonyl groups of the carbohydrate.

[0216] [ka]

[0217] [ka]

[0218] Example 4: Synthetic Macromonomers and Synthesis Methods Schemes 4.1 to 4.5 show the synthetic macromonomers of PCL, PLA, PLGA, PS, PMMA and PA.

[0219] PLA, PLGA, and PCL are produced using ring-opening polymerization on a norbornene dicarboximide linker bearing a terminal hydroxyl group. Briefly, cis-norbornene-exo-2,3-dicarboxylic anhydride is reacted with 3-amino-1-propanol to produce an initiator molecule. This initiator is then reacted with ε-caprolactone (or D,L-lactide for PLA formation; D,L-lactide and glycolide for PLGA formation) in the presence of a Sn(Oct)2 catalyst to form PCL chains on the norbornene dicarboximide linker, N-[3-hydroxypropyl]-cis-5-norbornene-exo-2,3-dicarboximide (NPH), to produce a PCL macromonomer (NB-PCL) (Scheme 4.1) (or NB-PLA macromonomer).

[0220] [ka]

[0221] The PLA macromonomer is synthesized using ring-opening polymerization. cis-norbornene-exo-2,3-dicarboxylic anhydride is first reacted with 3-amino-1-propanol to provide the initiator molecule. This alcohol initiator is then stirred with D,L-lactide in the presence of a Sn(Oct)2 catalyst to yield the NB-PLA macromonomer (Scheme 4.2).

[0222] [ka]

[0223] Poly(lactic-co-glycolic acid) (PLGA) macromonomers are synthesized in two steps via a cis-norbornene-exo-2,3-dicarboximide aminopropanol initiator molecule (Scheme 4.3).

[0224] [ka]

[0225] [ka]

[0226] PS is prepared by atom transfer radical polymerization (ATRP), in which azide termini are formed at the polymer chain ends after the polymerization reaction, so that a norbornene dicarboximide linker can be "clicked" onto the polymer to create the PS (NB-PS) macromonomer (Schemes 4.4a to 4.4c).

[0227] PMMA (NB-PMMA) macromonomers are prepared using atom transfer radical polymerization (ATRP). N-(hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH) is first reacted with 2-bromoisobutyryl bromide to give a norbornenyl-functionalized ATRP initiator. The NB-PMMA macromonomer is then synthesized by direct polymer growth from the norbornenyl-functionalized ATRP initiator using a CuBr / TMEDA catalyst system (Scheme 4.4d).

[0228] Polyamide (PA) macromonomers can be produced by the ring-opening polymerization of ε-caprolactam over N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) under reflux conditions using HO and HPO as catalysts (Scheme 4.5). NCP served as the initiator for ε-caprolactam ROP.

[0229] [ka]

[0230] Example 5: Ring-opening metathesis polymerization catalyst Using both bioactive macromonomers and synthetic polymer (PCL, PLA, PLGA, PS, PMMA, or PA) macromonomers, the final bioactive copolymers are prepared by ROMP using Grubbs-type catalysts 1 or 2 (Scheme 5).

[0231] [ka]

[0232] Example 6: Examples of bioactive synthetic copolymers Figure 1 shows a bioactive synthetic copolymer 100 designed in accordance with various embodiments disclosed herein. The bioactive synthetic copolymer 100 comprises a poly(norbornene dicarboximide) backbone 102, pendant arms of synthetic polymers 104a, 104b, and 104c, and pendant arms of bioactive molecules 106a, 106b, and 106c tethered to PEG chains 108a, 108b, and 108c. As shown in the schematic diagram, the pendant arms are attached to the poly(norbornene dicarboximide) backbone 102. 106a, 106b, and 106c can be the same or different types of bioactive moieties.

[0233] Examples of synthetic polymers include polyesters such as poly(caprolactone) (PCL), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), polyacrylates, poly(meth)acrylates such as poly(methyl methacrylate) (PMMA), and polyamides (PA). Examples of bioactive molecules include peptide sequences of 3 to 20 amino acid residues formed from the 20 naturally occurring amino acids, DGEA, collagen-mimetic peptides of 3 to 20 amino acid residues in any sequence such as (Gly-Pro-Hyp)3 and (Pro-Hyp-Gly)3, carbohydrates such as glycosaminoglycans, or drug molecules containing a carboxylic acid terminus, such as certain antibiotics.

[0234] Depending on the target application, bioactive macromonomers can be matched with various types of synthetic polymers to create materials with different physical properties.

[0235] An example would be a bioactive macromonomer containing RGD, a peptide sequence that can bind to integrins for cell attachment, migration, and proliferation. Therefore, a macromonomer of RGD is created (Scheme 6).

[0236] [ka]

[0237] RGD macromonomers can be paired with biodegradable macromonomers to create skin scaffolds that will degrade in the human body after the patient's own skin takes over. This macromonomer can also be copolymerized with heparan sulfate-containing macromonomers and polycaprolactone-containing macromonomers to create triblock copolymers that enable bone tissue regeneration for use as bioresorbable bone scaffolds. This modular approach to polymer construction allows for the rapid creation of mechanically strong therapeutic materials based on patient needs by matching various bioactive macromonomers with synthetic macromonomers. The dosage of the therapeutic agent (bioactive macromonomer) can also be tailored to suit the patient's needs by adjusting the ratio of macromonomers during polymerization.

[0238] RGD can be replaced with any peptide sequence via its acid terminus, or any carbohydrate or any drug molecule such as amoxicillin or ciprofloxacin with a carboxylic acid functionality.

[0239] For example, glycosaminoglycans (GAGs), such as heparan sulfate (HS) chains of between five and ten disaccharide units, can be used as bioactive moieties. Without being bound by theory, HS chains are believed to be active against bone morphogenetic proteins (BMPs), particularly BMP-2, which can transdifferentiate myoblasts into osteoblasts. Without being bound by theory, DP12, an HS fragment with six disaccharide units, is believed to have the highest binding affinity for BMP-2. In vitro experiments using BMP-2 complexed with DP12 demonstrated greater osteogenic differentiation in cells, while in vivo experiments using a rat model demonstrated that DP12 enhanced bone tissue regeneration compared to a control collagen sponge in a polycaprolactone (PCL) tube. Therefore, PCL copolymers with DP12 macromonomers can be created, which can then be added to the base polymer PCL and processed into whole-bone implants. By chemically linking DP12 to the PCL itself before blending the polymer into the base polymer PCL, the present disclosure advantageously demonstrates that it is possible to localize the GAG ​​on the implant to prevent undesirable side effects such as bone tissue regeneration anywhere else in the body except the implantation site. GAGs are also known to enhance keratinocyte regeneration, so in addition to being used as bone scaffolds, DP14-PCL / PCL blends can also be used to create skin scaffolds.

[0240] In addition to GAGs, peptides such as integrin binders or collagen fragments, which are useful for regenerating skin and bone tissue, can also be used. Extracellular peptides such as RGD can function as integrin binders to promote cell attachment, migration, and proliferation.

[0241] In addition to RGD peptides, materials can also be engineered with collagen fragments and mimetics. Bone is a mineralized collagenous tissue that remodels itself throughout the human life cycle to adapt to mechanical stress and maintain the integrity of skeletal tissue. Current bone scaffolds are typically made of collagen sponge, sometimes mineralized with some calcium phosphate ceramic, such as tricalcium phosphate or hydroxyapatite. Collagen's biocompatibility and similarity to bone tissue make it an ideal scaffold material for bone. Without being bound by theory, it is believed that the use of collagen fragments or collagen mimetics (COL) in PCL scaffolds helps increase the biocompatibility and biomimetic properties of the overall PCL-based scaffold material. Some potential collagen mimetics that can be used include DGEA and collagen fragments with various lengths of glycine, proline, and hydroxyproline sequences. Without being bound by theory, DGEA is believed to support mesenchymal stem cell adhesion and osteoblast differentiation. Furthermore, without being bound by theory, collagen is also believed to be an excellent skin scaffold material because the extracellular matrix (ECM) is primarily a collagenous material. Other ECM peptides that have been studied include the laminin-derived peptide A5G81, which has been reported to promote wound healing in rats.

[0242] In addition to tissue-regenerating biomolecules, cell-penetrating peptides such as (IRIK)2 and (IKKI)3 can also be used as bioactive moieties for incorporation into non-biofouling materials. Biofouling is a serious problem in biomedical devices such as catheters, intestinal stents, and even wound dressings. The ability to target biofilm-forming bacteria such as Pseudomonas aeruginosa while not being toxic to humans makes such peptides attractive candidates for biomedical device materials.

[0243] Drug molecules, such as antibiotics, can also be incorporated into brush polymers. In theory, any drug molecule with a carboxylic acid end group could be used to create these bioactive synthetic polymers. Some drug molecules that have been successfully polymerized include amoxicillin and ciprofloxacin. Brush polymers have also been created for use in antibacterial devices.

[0244] In summary, by using a modular approach to polymer design and synthesis, we have developed a general strategy for creating bioactive macromonomers for the rapid construction of bioactive synthetic polymers. Such a modular approach to therapeutic materials synthesis allows for therapeutic customization to suit each patient's needs, thus moving closer to the ideal situation of personalized medicine.

[0245] In summary, a series of synthetic polymers, including polystyrene, polyacrylate, poly(meth)acrylate, poly(lactide), poly(lactic-co-glycolic acid), poly(ε-caprolactone), and polyamides, bearing PEGylated biomolecules as side chains on a poly(norbornene dicarboximide) backbone were developed via ROMP technology. The biocompatibility, bone growth factor, and skin cell viability of some of these polymers were also tested, demonstrating the materials' ability to withstand harsh material processing temperatures without loss in bioactivity. The general strategy presented here forms a way to create bioactive synthetic polymers for use as bioadditives in biomedical device materials, where bioadditives can be blended with base polymers of the same type as the polymer side chains on the poly(norbornene dicarboximide) backbone. The side chains of the synthetic polymer help make the biomolecules more compatible with the base synthetic polymer, allowing them to be blended together without phase separation. The formation of brush polymers also allows the biomolecules to have better structural integrity compared to the natural biomolecules themselves, which tend to be highly hygroscopic, leading to their poor handling and processability as materials.

[0246] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions, PS (NB-PS) and PCL (NPH-PCL), macromonomer synthesis, bioactive macromonomer synthesis, and catalyst 2 synthesis were performed in a vacuum atmosphere glovebox under nitrogen atmosphere. NBPEG and NB amino alcohol condensation reactions to obtain N-(hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH), N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP), and N-(hydroxydecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (NDH) were performed in a fume hood under atmospheric conditions. All solvents used in the glovebox were anhydrous and as purchased. Grubbs second-generation catalyst (catalyst 1) was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma Aldrich (6,000). i Pr2EtN was purchased from Sigma Aldrich, cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar, DP12 was purchased from Iduron, and all purchased reagents were used without further purification.

[0247] 1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as the solvent for all biomolecular macromonomers. CDCl3 was used as the solvent for the PCL macromonomer. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, with a flow rate of 1.0 ml / min at 40 °C. Polystyrene was used as the calibration standard. TGA / DSC measurements were performed using a TA Instruments SDT2960 simultaneous DSC-TGA.

[0248] Synthesis of (H2IMes)(pyr)2(Cl)2RuCHPh (catalyst 2) Pyridine (2 mL) was added to catalyst 1 (0.5 g, 0.59 mmol) in a 20 mL vial with a screw cap. The reaction was stirred at room temperature for 15 minutes, during which time a color change from red to green was observed. Hexane (16 mL) was added to the green solution, and a green solid began to precipitate. The green precipitate was vacuum filtered, washed with hexane (4 × 10 mL), and dried under vacuum to give catalyst 2 as a green powder.

[0249] Synthesis of N-(hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH) A round-bottom flask was charged with cis-5-norbornene-exo-2,3-dicarboxylic anhydride (0.985 g, 6.0 mmol) and 3-amino-1-propanol (0.473 g, 6.3 mmol). 30 mL of toluene was added to the flask, followed by triethylamine (84 μL, 0.60 mmol). A Dean-Stark trap was attached to the flask, and the reaction mixture was heated to reflux (135°C) for 4 hours. The reaction mixture was then cooled and concentrated in vacuo to give a pale yellow oil. The residue was diluted with 30 mL of dichloromethane and washed with 0.2 M HCl (20 mL) and saturated NaCl (20 mL). The organic layer was dried over NaSO, concentrated in vacuo, and dried overnight in a vacuum oven to give 1.22 g of a white solid. 1 H NMR (500MHz, CDCl3): δ6.27(t, J=2.0Hz, 2H), 3.64(t, J=6.4Hz, 2H), 3.53(q, J=6.1Hz, 2 H), 3.26(s, 2H), 2.71(m, 2H), 2.60(m, 1H), 1.84-1.70(m, 2H), 1.55(m, 1H), 1.24(d, 1H).

[0250] Synthesis of NPH-PCL macromonomers by ROP NPH-PCL macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, ε-CL (0.5 ml, 0.52 mol) was added to a 20 ml scintillation vial containing NPH initiator (0.05 g, 0.23 mmol) dissolved in toluene (1 ml). Sn(Oct)2 (0.0037 g, 9.1 μmol) was added to the mixture, and the resulting solution was stirred at 110 °C for 90 minutes and precipitated in methanol. The methanol solution was then placed in a freezer overnight to obtain a white precipitate, which was filtered and washed with methanol. The residue was then dried under vacuum overnight. GPC analysis (THF): M n = 5,613, PDI = 1.08, yield 0.4478g.

[0251] A standard solution of Sn(Oct)2 with a concentration of 91 μmol / ml was prepared and used for the ROP reaction.

[0252] Synthesis of NPH-PLA macromonomer by ROP NPH-PLA macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, a flame-dried 25 mL Schlenk tube was charged with NPH initiator (110 mg, 0.50 mmol), D,L-lactide (864 mg, 6.0 mmol), Sn(Oct)2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times and then immersed in an oil bath at 130 °C. After 2.5 h, the contents were cooled to room temperature, diluted with dichloromethane, and precipitated twice into cold methanol. The macromonomers were isolated by decanting the supernatant and drying under vacuum overnight. GPC analysis (THF): M n = 2,471, PDI = 1.20, yield 0.600 g. 1 H NMR(CDCl3):δ6.28(br t, 2H), 5.27-5.08(m), 4.35(m, 1H), 4.19-4.02(m, 2H), 3.62-3.44(m, 2H), 3.27(s, 2H), 2.69(m, 2H), 1.97-1.47(m), 1.19(d, 1H).

[0253] Synthesis of N-(hydroxydecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (NDH) A round-bottom flask was charged with cis-5-norbornene-exo-2,3-dicarboxylic anhydride (0.95 g, 5.8 mmol) and 10-amino-1-decanol (1.0 g, 5.8 mmol). 20 mL of toluene was added to the flask, followed by triethylamine (80 μL, 0.58 mmol). Heating resulted in a homogeneous solution. A Dean-Stark trap was attached to the flask, and the reaction mixture was heated to reflux (135° C.) for 4 hours. The reaction mixture was then cooled and concentrated in vacuo to give an off-white solid. This residue was dissolved in 20 mL of CHCl and washed with 0.1 N HCl (10 mL) and saturated NaCl (10 mL). The organic layer was dried over MgSO and concentrated in vacuo to give 1.96 g of a colorless, viscous oil. 1H NMR (500MHz, CDCl3): δ1.20-1.28(m, 13H), 1.49-1.56(m, 5H), 2.65(d, J=1.5Hz, 2H), 3.2 6(t, J=1.5Hz, 2H)), 3.44(t, J=7.5Hz, 2H), 3.62(t, J=6.5Hz, 2H), 6.27(t, J=2.0Hz, 2H).

[0254] Synthesis of N-(pentynoyldecanyl)-cis-5-norbornene-exo-2,3-dicarboximide To a round-bottom flask were added N-(hydroxydecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (NDH) (0.80 g, 2.5 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (0.58 g, 3.0 mmol), and 4-dimethylaminopyridine (DMAP) (0.10 g, 0.82 mmol), followed by 10 mL of CHCl. ​​Pentynoic acid (0.25 g, 2.5 mmol) was added via syringe as a solution in 5 mL of CHCl. ​​The reaction mixture was allowed to stir overnight at room temperature. The reaction mixture was washed with water (2 × 20 mL) and saturated NaCl (20 mL) and dried over MgSO. The solvent was evaporated and the remaining residue was purified by silica gel chromatography (ethyl acetate / hexane, 1:9 v / v) to give 0.88 g of the product as a colorless oil (88% yield). 1 H NMR (500MHz, CDCl3): δ1.21-1.33(m, 13H), 1.49-1.54(m, 3H), 1.62(t, J=7.5Hz, 2H), 1.97(t, J=2.5Hz, 1H), 2.48-2.57 (m, 4H), 2.67(d, J=1.5Hz, 2H), 3.27(t, J=1.5Hz, 2H), 3.45(t, J=7.5Hz, 2H), 4.09(t, J=7Hz, 2H), 6.28(t, J=2.0Hz, 2H).

[0255] Synthesis of NB-PS macromonomers via ATRP-click NB-PS macromonomers with various degrees of polymerization (DP) were prepared using ATRP and click reactions. As an example, CuBr (0.1435 g, 1 mmol) was weighed into a 20 ml scintillation vial in a glovebox. Styrene (prefiltered through basic Al2O3, 11.5 ml, 100 mmol) was added, followed by methyl-2-bromopropionate (112 μl, 1 mmol) and PMDETA (209 μl, 1 mmol). The mixture was heated at 80 °C for 1 h and added dropwise to stirred MeOH (400 ml), yielding a white precipitate (ppt) in a deep blue solution. The ppt was filtered to yield a bluish-white solid, which was redissolved in a minimum amount of CHCl2, reprecipitated in MeOH, and filtered. This redissolution, precipitation, and filtration process was repeated until a pure white solid of PS-Br was obtained. The solid was then dried overnight in a vacuum oven. GPC analysis (THF): M n =2,523, PDI=1.18.

[0256] PS-Br (0.5 mmol) and NaN3 (2.5 mmol) were added to a 20 ml scintillation vial in a glove box, followed by DMF (10 ml), and the mixture was stirred for 48 hours to give a colorless solution with a white precipitate of NaBr. The mixture was added to a beaker of stirred MeOH in a fume hood. The white precipitate was filtered, washed with MeOH, and dried in a vacuum oven to give the PS-N3 prepolymer.

[0257] In a 20 ml scintillation vial, PS-N3 prepolymer (0.1 mmol), N-(pentyloyldecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (0.15 mmol), and CuBr (0.01 mmol) were added. THF (2 ml) and PMDETA (0.01 mmol) were added, and the mixture was stirred at 50 °C overnight. MeOH was added to the cooled reaction mixture to give a white precipitate, which was filtered, washed with MeOH, and then dried in a vacuum oven to give the NB-PS macromonomer. 1H NMR(CDCl3):7.10-6.46(m), 6.28(s, 2H), 5.04-4.94(m, 1H), 4.13-4.0(m, 2H), 3.5 1-3.40(m, 5H), 3.27(s, 2H), 2.91-2.86(m, 2H), 2.67-2.56(m, 2H), 0.92(brs, 3H).

[0258] Synthesis of norbornenyl-functionalized ATRP initiators A round-bottom flask was charged with N-(hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH) (0.66 g, 3.0 mmol). Dichloromethane (12 mL) was added to the flask, followed by triethylamine (0.63 mL, 4.5 mmol). The reaction flask was submerged in an ice-water bath, and 2-bromoisobutyryl bromide (0.55 mL, 4.5 mmol) was added dropwise to the reaction mixture. Upon completion of the addition, the reaction mixture was allowed to stir overnight at room temperature. The reaction mixture was washed with 0.1 M HCl (15 mL), saturated NaHCO solution (15 mL), and saturated NaCl (2 × 15 mL). The organic layer was dried over NaSO and concentrated in vacuo. The residue was purified by silica gel chromatography (dichloromethane) to give the product as a pale yellow solid (0.80 g, 72%). 1 H NMR (500MHz, CDCl3): δ6.28(t, J=1.8Hz, 2H), 4.17(t, J=6.5Hz, 2H), 3.61(t, J=7.1Hz, 2H), 3.28(s, 2H), 2.69(d, J=1.8Hz, 2H), 1.99-1.96(m, 8H), 1.52(m, 1H), 1.21(d, J=9.9Hz, 1H).

[0259] Synthesis of NB-PMMA macromonomer by ATRP NB-PMMA macromonomers with various degrees of polymerization (DP) were prepared using ATRP. For example, a 25 mL Schlenk tube was charged with norbornenyl-functionalized ATRP initiator (53 mg, 0.143 mmol), MMA (1.06 mL, 10.0 mmol), anisole (1.0 mL), and TMEDA (0.011 mL, 0.072 mmol). The solution was degassed by three freeze-pump-thaw cycles. During the final cycle, the Schlenk tube was filled with nitrogen, and CuBr (10.3 mg, 0.072 mmol) was quickly added to the frozen reaction mixture. The Schlenk tube was sealed, evacuated, and backfilled with nitrogen three times. The Schlenk tube was thawed to room temperature, and polymerization was carried out in an oil bath at 70 °C for 3 h. The mixture was filtered through neutral alumina, precipitated in MeOH, and filtered. The solid was then dried overnight in a vacuum oven. GPC analysis (THF): M n =5,158, PDI=1.13. 1 H NMR (CDCl3): δ6.30(s, 2H), 4.17(m, 2H), 3.76(m), 3.65-3.59(m), 3.28(s, 2H), 2.72(s, 2H), 2.00-1.69(m), 1.07-0.75(m).

[0260] Synthesis of N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) Cis-5-norbornene-exo-2,3-dicarboxylic anhydride (4.0 g, 24.3 mmol) and 6-aminohexanoic acid (3.3 g, 25.3 mmol) were weighed into a round-bottom flask. To the solid mixture, toluene (50 mL) and EtN (410 μL, 2.92 mmol) were added. The flask was equipped with a Dean-Stark trap and heated to reflux for 4 h. The mixture was then allowed to cool to room temperature, diluted with CHCl (50 mL), and washed with 1 M aqueous HCl (2 × 20 mL). The organic layer was washed with saturated aqueous NaCl (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give NCP as a pale yellow solid. 1H NMR (500MHz, CD3OD, 25℃) δ6.26(t, 2H, J=2.0Hz), 3.44(m, 2H), 3.25(m, 2H), 2.66(d, 2H, J=1.0Hz) ), 2.32 (t, 2H, J=7.2Hz), 1.63 (m, 2H), 1.55 (m, 2H), 1.46-1.51 (m, 1H), 1.33 (m, 2H), 1.19 (d, 1H).

[0261] Synthesis of NCP-PA6 macromonomer by ROP NCP-PA6 macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, ε-caprolactam (2.56 g, 12 mmol) was weighed into a 50 ml round-bottom flask (RBF) equipped with a nitrogen inlet and containing NCP initiator (0.2 g, 0.6 mmol). H3PO3 (0.081 g) in deionized HO (5 ml) was added to the mixture, and the resulting mixture was heated to 170 °C for 30 minutes and maintained at 240 °C for 4 hours. The HO was removed by distillation, and the reaction mixture was heated under vacuum at 240 °C for an additional 2 hours. The beige solid was precipitated from MeOH and washed repeatedly. After drying overnight in a vacuum oven, NCP-PA6 was obtained. 1 H NMR [500MHz, DCO2D / CD2Cl2(1:4), ]:δ6.42(br, PA6), 6.28(s, 2H, NCP), 3.42(s, 6H, NCP), 3.14-3.12(m, PA6), 2.67(s, 2H, NCP), 2.14-2.12(m, PA6), 1.56-1.53(m, PA6), 1.46-1.44(m, PA6), 1.29-1.25(m, PA6).

[0262] Synthesis of N-BPEG macromonomers (H2N-PEG-NH2 for 1000, 3,400, and 6000) PEG diamine (1 g) and cis-norbornene-exo-2,3-dicarboxylic anhydride (1 equivalent) were added to a 100 mL RBF, followed by toluene (50 mL). Triethylamine (1 equivalent) was added, and the mixture was stirred overnight at reflux with a Dean-Stark trap attached for water removal. The resulting solution was evaporated to dryness, and dichloromethane (40 mL) was added, followed by 0.1 M HCl (40 mL). The organic layer was extracted and washed with 0.1 M NaOH (50 mL). 0.1 M NaOH (50 mL) was added to the aqueous fraction from the acid wash, followed by CHCl (30 mL). The organic layers were extracted, combined, washed with saturated NaCl, and dried over NaSO. The materials were evaporated to dryness to yield a pale orange oil for PEG diamine 1,000 and a beige solid for NBPEG and PEG diamines 3,400 and 6,000. 1 H NMR (MeOD): δ = 6.36 (t, 2H, NB), 3.67 (s, PEG), 3.21 (s, 2H, NB), 2.74 (s, 2H, NB), 1.92 (s, 2H).

[0263] NBPEG as a representative preparation for PEG 1000, 3,400, and 6,000 1000 Synthesis of RGD RGD (with one carboxylic acid terminus on the aspartic acid protected with OMe) (0.0937 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a 4 ml vial in a glove box. i PrEtN (91 μL, 0.52 mmol) was added and the mixture was stirred (A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) in a 20 ml vial at 40° C., after which the RGD solution from (A) was added to obtain solution (B). Solution B was then dissolved in NBPEG in a 40 ml vial. 1000(0.25 g, 0.218 mmol) and stirred at room temperature overnight. The resulting mixture was then evaporated to dryness, and the oil was added to diethyl ether (50 ml). The diethyl ether solution was chilled in a freezer for 48 hours and decanted. MeOH (5 ml) was added to the residue to give an orange solution with a white ppt. The mixture was passed through a syringe filter, and the clear filtrate was evaporated to dryness to give an orange oil of RGDPEGNB in ​​95% yield. 1 H NMR (MeOD): δ=7.74(dd), 7.35-7.42(m), 6.32(t), 4.39(s), 4.20(s), 3.63(brs), 3.60(d), 3.17(t), 2.70(d). MALDI-MS:661.3([M-NB]+2H + ).

[0264] NBPEG as a representative preparation for PEG 1000, 3,400, and 6,000 1000 Synthesis of DGEA DGEA (with carboxylic acids on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. i PrEtN (91 μl, 0.52 mmol) was added, and the mixture was stirred as solution A. HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a yellow oily mixture. The mixture was dispersed in EtO, and the solution was placed in a freezer for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a yellow suspension. Filtration and solvent evaporation gave the yellow oily product NB-PEG-DGEA (0.28 g, 73% yield).

[0265] 1H NMR (500MHz, CD3OD, 25℃): δ7.80(d, 1H), 7.71(d, 1H), 7.44-7.38(m, 2H), 6.33(s, 2H), 4.40(s, 2H), 4.22(s, 1H), 3.95(s, 1H), 3.68(m , 6H), 3.64(m, 84H), 3.57(m, 4H), 3.18(s, 2H), 2.82(s, 2H), 2.72(s, 2H), 2.47(m, 2H), 2.14(m, 1H), 1.96(m, 1H), 1.48-1.41(dd, 2H).

[0266] NBPEG as a representative preparation for collagen fragments of glycine, proline, and hydroxyproline with various sequences and chain lengths up to n=6, PEG 1000, 3,400, and 6,000. 1000 Synthesis of (GPHyp)3 (GPHyp)3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. i PrEtN (91 μl, 0.52 mmol) was added and the mixture was stirred (solution A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a beige mixture. The mixture was dispersed in EtO and frozen for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a beige suspension. Filtration and solvent evaporation gave the beige oily product NB-PEG-(GPHyp) (0.22 g, 50% yield).

[0267] 1 H NMR (500MHz, CD3OD, 25℃): δ6.33(s, 2H), 4.73-.4.44(br, 4H), 3.65(m, 84H), 3 .57(m, 4H), 3.18(s, 2H), 2.72(s, 2H), 2.39-1.80(br, 8H), 1.44-1.37(dd, 2H).

[0268] NBPEG 3400 Synthesis of DP12 DP12 (0.0211 g, 8.5 μmol) was dissolved in MeOH (1.5 ml) in an 8 ml scintillation vial. i PrEtN (3 μl, 17 μmol) was added and the mixture was stirred (Solution A). HOBT (0.0032 g, 8.5 μmol) and HBTU (0.0012 g, 8.5 μmol) were dissolved in MeOH (2.5 ml) at 40° C., after which Solution A was added to obtain Suspension B. Suspension B was then dissolved in NBPEG. 3400 NH (0.025 g, 7.05 μmol) was added and stirred at room temperature for 24 hours. The resulting pale yellow mixture was then concentrated by solvent evaporation to give a beige mixture. The mixture was dispersed in EtO, and the mixture was placed in a freezer for 84 hours. The EtO was decanted, and MeOH was added to the residue to give a pale yellow solution, from which filtration and subsequent solvent evaporation gave an orange oil.

[0269] 1 H NMR (500MHz, CD3OD, 25℃): δ7.81(dd, 4H), 7.44-7.52(m), 6.36(t, 2H), 4.28(br, 4H), 3.67(s, 304H), 3.21(s, 2H), 2.77(s, 2H), 1.48-1.32(m, 10H).

[0270] NPH-PCL macromonomer and NBPEG as representative preparations for PCL-peptide type copolymers 3400 Typical procedure for ROMP of RGD macromonomers NBPEG 3400RGD macromonomer (0.2 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NPH-PCL (0.05 g). THF (0.021 M with respect to NPH-PCL) was added, and the mixture was stirred at room temperature until a clear solution was obtained. A solution of catalyst 1 or 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the reaction was stirred at 30 ° C. for 2 hours. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 hour to obtain a white ppt. The mixture was centrifuged, and the mother liquor was decanted. The residue was resuspended in methanol, centrifuged, and the mother liquor was decanted again to wash the residue. Three MeOH washes were performed, and the final residue was dried overnight in a vacuum oven.

[0271] GPC analysis of PCL-RGD copolymer (THF): n =140,000, PDI=1.21.

[0272] NPH-PLA macromonomer and NBPEG as representative preparations for PLA-peptide type copolymers 1000 Typical procedure for ROMP of RGD macromonomers NBPEG 1000 RGD macromonomer (0.1 equivalents) is weighed into a 4 ml scintillation vial, followed by the addition of NPH-PLA (0.05 g). THF (0.05 M with respect to NPH-PCL) is added, and the mixture is stirred at room temperature until a clear solution is obtained. A solution of catalyst 2 in THF (1.25 mol%) is added to the solution, and the reaction is stirred for 1 hour. Ethyl vinyl ether is added to the reaction mixture, followed by MeOH (3 ml), and the mixture is placed in a freezer for 1 hour to obtain a sticky solid. The mother liquor is decanted, and the residue is washed repeatedly with MeOH, then dried in a vacuum oven. GPC analysis (THF): M n =76,681, PDI=1.44.

[0273] NB-PS macromonomer and NBPEG as representative preparations for PS-peptide type copolymers1000 Typical procedure for ROMP of RGD macromonomers NBPEG 1000 RGD (0.1 equivalents) was weighed into a 4 ml glass vial, followed by the addition of NB-PS (0.05 g). THF (0.6 ml) was added, and the mixture was stirred at 25 °C until a clear solution was obtained. A solution of catalyst 1 or 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the reaction was stirred for 1 hour. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 hour to obtain a white precipitate. The mixture was filtered, and the residue was repeatedly washed with MeOH and then dried in a vacuum oven.

[0274] GPC analysis of PS-RGD copolymer (THF): n =33,484, PDI=1.41.

[0275] NB-PMMA macromonomer and NBPEG as representative preparations for PMMA-peptide type copolymers 1000 Typical procedure for ROMP of RGD macromonomers NBPEG 1000 RGD macromonomer (0.1 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NB-PMMA (0.05 g). THF (0.05 M with respect to NB-PMMA) was added, and the mixture was stirred at room temperature until a clear solution was obtained. A solution of catalyst 2 in THF (1.25 mol%) was added to the solution, and the reaction was stirred for 1 hour. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 hour to obtain a white precipitate. The residue was resuspended in methanol, centrifuged, and then the mother liquor was decanted again to wash the residue. Three washes with MeOH were performed, and the final residue was dried overnight in a vacuum oven.

[0276] GPC analysis of PMMA-RGD copolymer (THF): n =68,512, PDI=1.72.

[0277] NCP-PA6 macromonomer and NBPEG as representative preparations for PA-peptide type copolymers 3400 Typical Procedure for ROMP of DGEA Macromonomers NBPEG 3400 DGEA macromonomer (0.2 equivalents) was weighed into a 4 ml glass vial, followed by the addition of NCP-PA6 (0.12 g). CH3CO2H (0.021 M with respect to NCP-PA6) was added, and the mixture was stirred at 80 °C until a clear solution was obtained. Catalyst 2 (1.25 mol%, 0.05 M in CHCl2) was added to the solution, and the reaction was stirred at 80 °C for 24 hours. Ethyl vinyl ether was added to the reaction, followed by the addition of MeOH. The mixture was placed in a freezer for 1 day, yielding a beige ppt. The suspension was centrifuged, and the mother liquor was decanted. The residue was repeatedly washed with MeOH and then dried in a vacuum oven to yield a beige solid product. 1 H NMR [500MHz, DCO2D / CD2Cl2(1:4)]: δ6.42(br, PA6), 3.60(s, PEG), 3.19-3.13(m, PA 6), 2.15-2.12(m, PA6), 1.62-1.56(m, PA6), 1.48-1.42(m, PA6), 1.29-1.23(m, PA6).

[0278] Example 7: Examples of bioactive synthetic copolymers - Poly(ε-caprolactone)-biomolecular copolymers as bioadditives for human skin and bone tissue regeneration A series of poly(ε-caprolactone) (PCL) copolymers bearing various PEGylated biomolecules, such as collagen mimetics (COLs), integrin-binding peptides, and glycosaminoglycans (GAGs), were synthesized and characterized. Such copolymers can be used to create tissue regeneration scaffolds for either bone or skin regeneration in humans.

[0279] PCL is the synthetic polymer of choice in this example due to its ability to biodegrade in the human body without causing local acidity like poly(lactic acid) (PLA), making the material biocompatible. Incorporation of biomolecules such as heparin oligosaccharide DP12 into PCL would be desirable for a bone scaffolding material that would allow for the regeneration of bone tissue, while the material itself would eventually biodegrade within the body.

[0280] However, a problem with DP12 is that it is highly hygroscopic, and there is no way to control the uniformity of the coating before use, making it less than ideal to coat it onto a PCL tube before implantation. Furthermore, because GAGs are highly water-soluble, they have a high tendency to leach into the body upon implantation and not remain on the implant itself, providing the BMP binding required for bone regeneration. Therefore, in this example, a PCL copolymer containing a DP12 macromonomer was created, which could then be added to a PCL base polymer and processed into a whole-bone implant. By chemically linking DP12 to the PCL itself before blending the polymer into the PCL base polymer, the present disclosure advantageously demonstrated that it is possible to localize the GAG ​​onto the implant, preventing undesirable side effects, such as bone regeneration, anywhere in the body except at the implantation site.

[0281] In addition to GAGs, peptides such as integrin binders or collagen fragments are also useful for bone tissue regeneration. In fact, these biomimetic molecules (GAGs, integrin binders, collagen fragments) are not only useful for bone tissue regeneration, but also for skin tissue regeneration. Therefore, polymers synthesized according to various embodiments disclosed herein not only function as bone scaffolds, but they can also be used in skin scaffolds to enable skin tissue regeneration in patients with extensive wounds, such as burn patients.

[0282] Extracellular peptides such as RGD can function as integrin binders to promote cell attachment, migration, and proliferation. The RGD sequence is mostly found in native collagen, but it is often inaccessible for integrin binding until the collagen is denatured. Therefore, it would be useful to isolate the RGD sequence from collagen and apply it directly to tissue regeneration products. RGD can induce cell proliferation and angiogenesis through its integrin-binding ability, so it can be advantageously used in skin and bone tissue regeneration products. However, like many biomolecules, it is highly hygroscopic. In fact, it is more hygroscopic than DP12, and exposure to moist air for 5–10 minutes quickly transforms it from a crystalline solid into a liquid. Without immobilizing RGD on a synthetic polymer to increase its ease of handling, applying the peptide to the repair site, especially in bone defects, is extremely difficult.

[0283] Bone is a mineralized collagenous tissue that remodels itself throughout the human life cycle to adapt to mechanical stress and maintain the integrity of skeletal tissue. Current bone scaffolds are typically made of collagen sponge, sometimes mineralized with some calcium phosphate ceramics such as tricalcium phosphate or hydroxyapatite. The biocompatibility of collagen and its similarity to bone tissue make it a desirable scaffold material for bone. In this example, collagen fragments or collagen mimics (COL) were also used in the PCL scaffold to increase the biocompatibility and biomimetic properties of the overall PCL-based scaffold. Several collagen mimics, such as DGEA and collagen fragments with various lengths of glycine, proline, and hydroxyproline sequences, have been used as bioactive moieties (see Scheme 7). Without being bound by theory, DGEA is thought to support the adhesion and differentiation of mesenchymal stem cells into osteoblasts. Because the extracellular matrix (ECM) is primarily collagenous, collagen is not only a good bone scaffold material but also an excellent skin scaffold material. Therefore, the same PCL-COL polymer can also be used as a bioadditive to a PCL scaffold matrix for application in skin scaffolds.

[0284] [ka]

[0285] thermal stability The thermal stability of the biomacromonomer NBPEGRGD and NBPCL-PEGRGD ROMP polymers was measured and compared. Pure RGD exhibited thermal decomposition at 181 °C, while the macromonomer NBPEG 3400RGD exhibits two phases of mass loss at 220°C (RGD loss) and 398°C (PEG loss). However, when copolymerized with NBPCL macromonomer, the overall bioactive synthetic polymer exhibits only one significant mass loss at 393°C, indicating an overall improvement in the stability of the RGD moieties on the synthetic polymer. Indeed, the thermal stability of the NBPCL macromonomer also contributes to the overall improvement of the stability of the NBPCL macromonomer in the overall bioactive synthetic polymer product. 3400 This is improved by including RGD macromonomers (Figure 2).

[0286] Biocompatibility Copolymers were created using PCL as the synthetic polymer and a range of peptides with different properties as bioactive macromonomers: collagen fragment (GPHp)3: GPHP; collagen mimetic: DGEA; and integrin-binding peptides: SRGDS and RGD. The copolymers were subsequently blended with medical-grade PCL, 3D printed into sheets, and tested for cell viability and biocompatibility against Allevyn, the most commonly used commercial wound dressing in hospitals.

[0287] As shown in Figure 3, all materials designed according to various embodiments disclosed herein demonstrated better cell viability than the commercially available bandage after a 72-hour test period using human dermal fibroblasts (Hs27).

[0288] An alkaline phosphatase (ALP) assay to check for osteoblast activity was performed on the materials to determine their compatibility with BMP-2, a bone growth factor required for bone tissue growth, compared to pure PCL, a commonly used material for bone scaffolds. Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. The osteoinductivity of BMP-2 can be measured in vitro using the pluripotent myoblast C2C12 cell line. PCL-RGD exhibited superior ALP activity compared to PCL. At a 20% blend in pure PCL, PCL-RGD exhibited four-fold higher activity after 72 hours of incubation. PCL-(GPHyp) also showed improved activity compared to PCL (Figure 4). The ALP assay demonstrates the ability of PCL peptide materials, such as RGD and GPHyp, to promote cellular osteogenic activity compared to BMP-2 and pure PCL controls.

[0289] In summary, we have developed a series of polymers with bioresorbable PCL side chains and bioactive molecules such as heparan sulfate DP12, collagen mimetics or fragments, and integrin binders such as RGD. These copolymers will enhance the regeneration of both bone and skin tissues and serve as useful bioactive components for tissue regenerating scaffolds. A common strategy for creating scaffolds would be through blending such bioactive components with a substrate of similar properties, i.e., medical-grade PCL itself.

[0290] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions, PCL macromonomer (NPH-PCL) synthesis, and bioactive macromonomer synthesis were carried out in a vacuum atmosphere glove box under a nitrogen atmosphere. NBPEG and NPH synthesis were carried out in a fume hood under atmospheric conditions according to the procedures provided in Example 6. All solvents used in the glove box were anhydrous and used as purchased. Grubbs second-generation catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). HOBT, HBTU, i Pr2EtN was purchased from Sigma-Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. Heparin oligosaccharide DP12 was purchased from Iduron. All purchased reagents were used without further purification.

[0291] 1 H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as the solvent for all biomolecular macromonomers. CDCl was used as the solvent for the PCL macromonomer. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, with a flow rate of 1.0 ml / min at 40 °C.

[0292] The synthesis of NBPEG and NBPEGRGD is described in Example 6.

[0293] For BMP-2 binding studies, scaffolds were sterilized using 100% ethanol, then rinsed in sterile water and transferred to a 24-well plate. BMP-2 (50 ng in 100 μL PBS) was added directly to the top of each scaffold and incubated at room temperature for 20 minutes. BMP-2 alone was added directly to empty wells. Cells were plated at 2 × 104 cells / cm2 in 1 mL of 5% FCS medium. 2 The cells were added directly onto the scaffolds and into the surrounding wells at 100°C. The cells were incubated for 72 hours (37°C, 5% CO2) before the ALP assay.

[0294] Synthesis of NPH-PCL macromonomers by ROP NPH-PCL macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, ε-CL (0.5 ml, 0.52 mol) was added to a 20 ml scintillation vial containing NPH initiator (0.05 g, 0.23 mmol) dissolved in toluene (1 ml). Sn(Oct)2 (0.0037 g, 9.1 μmol) was added to the mixture, and the resulting solution was stirred at 110 °C for 90 minutes and precipitated in methanol. The methanol solution was then placed in a freezer overnight to obtain a white precipitate, which was filtered and washed with methanol. The residue was then dried under vacuum overnight. GPC analysis (THF): M n = 5,613, PDI = 1.08, yield 0.4478g.

[0295] A standard solution of Sn(Oct)2 with a concentration of 91 μmol / ml was prepared and used for the ROP reaction.

[0296] NBPEG as a representative preparation for PEG 1000, 3,400, and 6,000 1000 Synthesis of DGEA DGEA (with carboxylic acids on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. iPrEtN (91 μl, 0.52 mmol) was added, and the mixture was stirred as solution A. HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a yellow oily mixture. The mixture was dispersed in EtO, and the solution was placed in a freezer for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a yellow suspension. Filtration and solvent evaporation gave the yellow oily product NB-PEG-DGEA (0.28 g, 73% yield).

[0297] 1 H NMR (500MHz, CD3OD, ): δ7.80(d, 1H), 7.71(d, 1H), 7.44-7.38(m, 2H), 6.33(s, 2H), 4.40(s, 2H), 4.22(s, 1H), 3.95(s, 1H), 3.68(m, 6H), 3.64(m, 84H), 3.57(m, 4H), 3.18(s, 2H), 2.82(s, 2H), 2.72(s, 2H), 2.47(m, 2H), 2.14(m, 1H), 1.96(m, 1H), 1.48-1.41(dd, 2H).

[0298] NBPEG as a representative preparation for collagen fragments of glycine, proline, and hydroxyproline with various sequences and chain lengths up to n=6, PEG 1000, 3,400, and 6,000. 1000 Synthesis of (GPHyp)3 (GPHyp)3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. iPrEtN (91 μl, 0.52 mmol) was added and the mixture was stirred (solution A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a beige mixture. The mixture was dispersed in EtO and frozen for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a beige suspension. Filtration and solvent evaporation gave the beige oily product NB-PEG-(GPHyp) (0.22 g, 50% yield).

[0299] 1 H NMR (500MHz, CD3OD): δ6.33(s, 2H), 4.73-.4.44(br, 4H), 3.65(m, 84H), 3.5 7(m, 4H), 3.18(s, 2H), 2.72(s, 2H), 2.39-1.80(br, 8H), 1.44-1.37(dd, 2H).

[0300] NBPEG 3400 Synthesis of DP12 DP12 (0.0302 g, 8.5 μmol) was dissolved in MeOH (1.5 ml) in an 8 ml scintillation vial. i PrEtN (3 μl, 17 μmol) was added and the mixture was stirred (Solution A). HOBT (0.0032 g, 8.5 μmol) and HBTU (0.0012 g, 8.5 μmol) were dissolved in MeOH (2.5 ml) at 40° C., after which Solution A was added to obtain Suspension B. Suspension B was then dissolved in NBPEG. 3400 NH (0.025 g, 7.05 μmol) was added and stirred at room temperature for 24 hours. The resulting pale yellow mixture was then concentrated by solvent evaporation to give a beige mixture. The mixture was dispersed in EtO, and the mixture was placed in a freezer for 84 hours. The EtO was decanted, and MeOH was added to the residue to give a pale yellow solution, from which filtration and subsequent solvent evaporation gave an orange oil.

[0301] 1 H NMR (500MHz, CD3OD): δ7.81(dd, 4H), 7.44-7.52(m), 6.36(t, 2H), 4.28(br, 4H), 3.67(s, 304H), 3.21(s, 2H), 2.77(s, 2H), 1.48-1.32(m, 10H).

[0302] NPH-PCL macromonomer and NBPEG as representative preparations for PCL-peptide type copolymers 3400 Typical procedure for ROMP of RGD macromonomers NBPEG 3400 RGD macromonomer (0.2 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NPH-PCL (0.05 g). THF (0.021 M with respect to NPH-PCL) was added, and the mixture was stirred at room temperature until a clear solution was obtained. A solution of catalyst 1 or 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the reaction was stirred at 30 ° C. for 2 hours. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 hour to obtain a white ppt. The mixture was centrifuged, and the mother liquor was decanted. The residue was resuspended in methanol, centrifuged, and the mother liquor was decanted again to wash the residue. Three MeOH washes were performed, and the final residue was dried overnight in a vacuum oven.

[0303] GPC analysis of PCL-RGD copolymer (THF): n =140,000, PDI=1.21.

[0304] Typical Procedure for ROMP of NPH-PCL Macromonomer as a Representative Preparation for PCL Homopolymer NPH-PCL macromonomer (0.63 g) was weighed into a 10 ml scintillation vial, followed by the addition of THF (0.021 M with respect to NPH-PCL), and the mixture was stirred at 27 °C until a clear solution was obtained. A solution of catalyst 1 or 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the reaction was stirred at 27 °C for 2 h. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (5 ml), and the mixture was placed in a freezer for 1 day to obtain a white ppt. The mixture was filtered and washed repeatedly with MeOH, and the final product was dried overnight in a vacuum oven.

[0305] 1 H NMR (500MHz, CDCl3): δ4.07-.4.04(m, PCL), 2.32-2.29(m, PCL), 1.65-1.62(m, PCL), 1.37-1.31(m, PCL).GPC analysis (THF): M n =225,000, PDI=1.09. TGA:315.7℃

[0306] NPH-PCL macromonomer and NB-mPEG as representative preparations for PCL-mPEG type copolymers 5000 Typical Procedure for ROMP of Macromonomers NB-mPEG 5000 Macromonomer (0.1 eq.) was weighed into a 10 ml scintillation vial, followed by the addition of NPH-PCL (0.5 g). THF (0.021 M with respect to NPH-PCL) was added, and the mixture was stirred at 45°C until a clear solution was obtained. A solution of catalyst 1 or 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the reaction was stirred at 45°C for 2 hours. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (5 ml), and the mixture was placed in a freezer for 1 day to obtain a white ppt. The mixture was filtered and repeatedly washed with MeOH, and the final product was dried overnight in a vacuum oven.

[0307] 1H NMR (500MHz, CDCl3): δ4.07-.4.04(m, PCL), 3.64(s, PEG), 2.32-2.28(m, PCL), 1.65-1.62(m, PCL), 1.38-1.32(m, PCL). GPC analysis (THF): M n =171,000, PDI=1.21. TGA:316.4℃

[0308] Example 8: Examples of bioactive synthetic copolymers - Polyamide-Peptide Brush Polymers for Use as Bioadditives in Biomedical Devices A series of polyamide (PA) copolymers bearing various PEGylated biomolecules, such as collagen mimetics and integrin-binding peptides, were synthesized and characterized using ring-opening metathesis polymerization. The brush polymers can be blended with similar polymers on the pendant arms to create bioactive materials for use in biomedical devices such as catheters, plastic surgery implants, prosthetic components, and cartilage joint implants.

[0309] This example reports another type of bioactive brush polymer using polyamide (PA) and a collagen mimetic for use in polyamide-based biomedical devices.

[0310] Polyamide (PA) is the synthetic polymer of choice in this example. Polyamides (PA), such as PA6, PA12, and PA6,6, are silk-like thermoplastics that have found important biomedical applications, such as in tubing, surgical guides, prosthetics, sutures, and ligament and tendon repair. PA is believed to have the least microbial contamination compared to other materials. PA-type polymers can be blended with a wide variety of additives to achieve many different property variations, allowing devices to be fabricated using a wide variety of material processing methods, such as melt extrusion, 3D printing, and injection molding. However, polyamide chains polymerize under harsh conditions of high temperature and reduced pressure. This polymer is also insoluble in most solvents, which increases the difficulty of preparing this material.

[0311] PA-based materials containing collagen fragments and mimetics were created using ring-opening metathesis polymerization (ROMP) technology. Collagen's biocompatibility and similarity to human tissue make it an ideal material for biomedical devices. However, like many biomolecules, it is highly hygroscopic. Creating implants or biomedical devices for insertion into the human body is nearly impossible without immobilizing collagen fragments or mimetics to synthetic polymers to increase their ease of handling. Cross-linked collagens are often used in wound care products, but they are also highly hygroscopic and form gels upon absorbing moisture, making them too weak on their own for use as implantable devices. Furthermore, full-length human collagen requires complex synthesis and often exhibits poor solubility in buffers. Short collagen-mimetic peptide sequences or fragments containing only a small portion of the critical peptide sequence have been used to elicit biological responses similar to their full-length collagen counterparts. Collagen mimetics such as DGEA (Asp-Gly-Glu-Ala) and collagen fragments with varying lengths of glycine, proline, and hydroxyproline sequences have been incorporated into synthetic polymers. DGEA can promote cell adhesion, spreading and osteogenic differentiation, which would be advantageous for applications in both skin and cartilage bone regeneration.

[0312] On the other hand, polyamides, which are FDA-approved polymers for use in biomedical devices, still induce an inflammatory response in the host body because they are ultimately foreign bodies. Foreign body reactions (FBRs) can be triggered by polyamides, leading to inflammation around the implantation site. Without being bound by theory, it is believed that the use of collagen fragments or collagen mimetics (COLs) in polyamide polymer materials can help increase the biocompatibility and biomimetic properties of polyamide-based implants or devices overall. Some collagen mimetics that could be used include DGEA and collagen fragments with various lengths of glycine, proline, and hydroxyproline sequences in any order. Indeed, because the extracellular matrix (ECM) and bone are primarily collagenous materials, collagen fragments make excellent skin and bone regeneration materials. Bone, in particular, is mineralized collagen, while articular cartilage is primarily collagen fibers, glycosaminoglycans, and proteoglycans. Therefore, the use of collagen-modified polyamides in joint implants may be particularly useful in aiding joint healing by stimulating collagen regeneration at the implantation site. This very property also makes it suitable for use in plastic surgery implants where cartilaginous bone is required, such as in rhinoplasty implants.

[0313] Using both the biomacromonomer and the synthetic PA macromonomer, the final bioactive polymer is prepared by ROMP using a Grubbs-type catalyst (Scheme 8).

[0314] [ka]

[0315] Upon synthesis, metal catalyst removal and characterization, the bioactive polymer is blended with a medical grade PA of choice, depending on the application, processed into relevant shapes by either fused filament fabrication (fff) or fused deposition modelling (FDM) type 3D printing, melt extrusion, melt blowing or electrospinning, and tested for biocompatibility. TGA-DSC analysis of the synthesized copolymers is typically performed to determine the TGA of the material prior to processing. g and decomposition temperature before processing the material.

[0316] thermal stability The thermal stability of the biomacromonomer NB-PEG-(GPHyp) and PA6 ROMP polymer was measured and compared (Figure 5). As can be seen, the copolymer PA6-(GPHyp) only decomposes above 450°C. Such high thermal stability allows various material processing methods, such as FFF or FDM type 3D printing, to be performed on materials designed according to various embodiments disclosed herein for implant fabrication.

[0317] Biocompatibility Biocompatibility testing using human fibroblasts, Hs27, was performed on three PA-collagen materials: PA6-(GPHyp)3, PA6-(PHypG)3, and PA6-DGEA (where (GPHyp)3 and (PHypG)3 are both collagen fragments, and DEGA is a collagen mimetic). The bioactive polymers were blended with medical-grade PA12, electrospun into fibrous sheets, sterilized with 70% EtOH, dried, and incubated with human dermal fibroblasts (Hs27) for 72 hours and checked for cell viability using the Celltitre-Glo assay. Cell viability data (Figure 6) demonstrate that materials designed according to various embodiments disclosed herein exhibited excellent biocompatibility with collagen, PA6-homopolymer, and PA6-mPEG. 5000It can be seen that not only were they able to maintain better cell viability than the control without (GPHyp)3, but they even showed an increased amount of viable cells, suggesting cell proliferation even at 72 hours. This is despite only 2% (GPHyp)3 in the copolymer blended with PA12 at a 1:9 ratio (0.2% (GPHyp)3 in the polymer formulation). In fact, the material significantly improved cell viability over pure medical-grade PA12 (Rilsamid®), demonstrating the importance of bioactive polymers in improving the biocompatibility of commercially available medical-grade polymers intended for the manufacture of biomedical devices. Tests were performed in triplicate. Tests with different blend ratios and more cell assays are ongoing to reconfirm this cell regeneration ability of the material. Nevertheless, as can be seen, the preliminary results are encouraging.

[0318] In summary, a series of brush polymers bearing polyamide 6 and PEGylated biomolecules as side chains on a poly(norbornene dicarboximide) backbone were developed via ROMP technology. The general strategy presented herein forms a way to create bioactive synthetic polymers for use as bioadditives in materials for biomedical devices, in which bioadditives can be blended with base polymers of the same type as the polymer side chains on the poly(norbornene dicarboximide) backbone. In this case, the base polymer is a polyamide. The synthetic PA6 side chains help make the biomolecules more compatible with the base polymer PA12, allowing them to be blended together without phase separation. The brush polymer formation also allows the biomolecules to have better structural integrity compared to the native biomolecules themselves, which tend to be highly hygroscopic, resulting in their poor handling and processability as materials. Preliminary cell viability tests showed an improvement in cell viability from the PA6 collagen material compared to the PA6 polymer without the biomolecule, and a significantly greater improvement in cell viability compared to pure medical-grade PA12. Superior human dermal fibroblast proliferation was observed for the PA-collagen material compared to pure PA12.

[0319] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions and bioactive macromonomer synthesis were carried out in a Vacuum Atmosphere glove box under nitrogen atmosphere. PA6 macromonomer synthesis was carried out on the bench under positive N2 flow. Reactions to obtain NBPEG and N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) were carried out in a fume hood under atmospheric conditions according to the procedure provided in Example 6. All solvents used were anhydrous and used as purchased. Grubbs catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). HOBT, HBTU, i PrEtN and 2,2,2-trifluoroethanol were purchased from Sigma-Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. Medical-grade PA12 (Rilsamid®) for blending was purchased from Arkema. All purchased reagents were used without further purification.

[0320] 1 H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using CD3OD as the solvent for all biomolecular macromonomers. CD3OD / CD2Cl2 (1:4) was used as the solvent for polyamide peptide macromonomers.

[0321] Synthesis of N-BPEG macromonomers (H2N-PEG-NH2 for 1000, 3,400, and 6000) PEG diamine (1 g) and cis-norbornene-exo-2,3-dicarboxylic anhydride (1 equivalent) were added to 100 mL of RBF, followed by toluene (50 mL). Triethylamine (1 equivalent) was added, and the mixture was stirred overnight at reflux with a Dean Stark trap attached for water removal. The resulting solution was evaporated to dryness, and dichloromethane (40 mL) was added, followed by 0.1 M HCl (40 mL). The organic layer was extracted and washed with 0.1 M NaOH (50 mL). 0.1 M NaOH (50 mL) was added to the aqueous fraction from the acid wash, followed by CHCl (30 mL). The organic layers were extracted, combined, washed with saturated NaCl, and dried over NaSO. The materials were evaporated to dryness to yield a pale orange oil for PEG diamine 1,000 and a beige solid for NBPEG and PEG diamines 3,400 and 6,000. 1 H NMR (500MHz, MeOD): δ = 6.36 (t, 2H, NB), 3.67 (s, PEG), 3.21 (s, 2H, NB), 2.74 (s, 2H, NB), 1.92 (s, 2H).

[0322] NBPEG as a representative preparation for PEG 1000-6,000 1000 Synthesis of DGEA DGEA (with carboxylic acids on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. iPrEtN (91 μl, 0.52 mmol) was added, and the mixture was stirred as solution A. HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a yellow oily mixture. The mixture was dispersed in EtO, and the mixture was placed in a freezer for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a yellow suspension. Filtration and solvent evaporation gave the yellow oily product NB-PEG-DGEA (0.28 g, 73% yield). 1 H NMR (500MHz, CD3OD): δ7.80(d, 1H), 7.71(d, 1H), 7.44-7.38(m, 2H), 6.33(s, 2H), 4.40(s, 2H), 4.22(s, 1H), 3.95(s, 1H), 3.68(m, 6 H), 3.64(m, 84H), 3.57(m, 4H), 3.18(s, 2H), 2.82(s, 2H), 2.72(s, 2H), 2.47(m, 2H), 2.14(m, 1H), 1.96(m, 1H), 1.48-1.41(dd, 2H).

[0323] NBPEG as a representative preparation of collagen fragments of glycine, proline, and hydroxyproline with various sequences and chain lengths up to n=6, PEG 1000-6,000 1000 (GPHyp) n Synthesis of (n=3) (GPHyp)3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. iPrEtN (91 μl, 0.52 mmol) was added and the mixture was stirred (solution A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a beige mixture. The mixture was dispersed in EtO and placed in a refrigerator for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a beige suspension. Filtration and solvent evaporation gave the beige oily product NB-PEG-(GPHyp) (0.22 g, 50% yield). 1 H NMR (500MHz, CD3OD): δ6.33(s, 2H), 4.73-4.44(br, 4H), 3.65(m, 84H), 3.57 (m, 4H), 3.18(s, 2H), 2.72(s), 2H), 2.39-1.80(br, 8H), 1.44-1.37(dd, 2H).

[0324] Synthesis of N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) Cis-5-norbornene-exo-2,3-dicarboxylic anhydride (4.0 g, 24.3 mmol) and 6-aminohexanoic acid (3.3 g, 25.3 mmol) were weighed into a round-bottom flask. To the solid mixture was added toluene (50 mL) and EtN (410 μL, 2.92 mmol). The flask was equipped with a Dean-Stark trap and heated to reflux for 4 h. The mixture was then cooled to room temperature, diluted with CHCl (50 mL), and washed with 1 M aqueous HCl (2 × 20 mL). The organic layer was washed with saturated aqueous NaCl (20 mL), dried (NaSO), filtered, and concentrated under reduced pressure to give NCP as a pale yellow solid. 1H NMR (500MHz, CD3OD, 25℃) δ6.26(t, 2H, J=2.0Hz), 3.44(m, 2H), 3.25(m, 2H), 2.66(d, 2H, J=1.0Hz) ), 2.32 (t, 2H, J=7.2Hz), 1.63 (m, 2H), 1.55 (m, 2H), 1.46-1.51 (m, 1H), 1.33 (m, 2H), 1.19 (d, 1H).

[0325] Synthesis of NCP-PA6 macromonomer by ROP NCP-PA6 macromonomers with various degrees of polymerization (DP) were prepared by ROP. As an example, ε-caprolactam (2.56 g, 12 mmol) was weighed into a 50 ml rbf containing NCP initiator (0.2 g, 0.6 mmol) under positive N pressure. H3PO3 (0.081 g) in deionized HO (5 ml) was added to the mixture, and the resulting mixture was heated to 170 °C for 30 min and maintained at 240 °C for 4 h. The HO was removed by distillation, and the reaction mixture was heated under vacuum at 240 °C for an additional 2 h. A beige solid was precipitated from MeOH, filtered, and the residue was repeatedly washed with MeOH and dried overnight in a vacuum oven to obtain NCP-PA6. 1 H NMR [500MHz, DCO2D / CD2Cl2(1:4)]: δ6.42(br, PA6), 6.28(s, 2H, NCP), 3.42(s, 6H, NCP), 3.14-3.12(m, P A6), 2.67(s, 2H, NCP), 2.14-2.12(m, PA6), 1.56-1.53(m, PA6), 1.46-1.44(m, PA6), 1.29-1.25(m, PA6).

[0326] NCP-PA6 macromonomer and NBPEG as representative preparations for PA-peptide type copolymers 3400 Typical Procedure for ROMP of DGEA Macromonomers NBPEG 3400DGEA macromonomer (0.2 equivalents) was weighed into a 4 ml glass vial, followed by the addition of NCP-PA6 (0.12 g). CH3CO2H (0.021 M with respect to NCP-PA6) was added, and the mixture was stirred at 80 °C until a clear solution was obtained. A solution of catalyst 2 in CHCl2 (1.25 mol%, 0.05 M) was added to the solution. The mixture was then stirred at 80 °C for 24 hours. Ethyl vinyl ether was added to the reaction, followed by MeOH. The mixture was placed in a freezer for 1 day, resulting in a beige ppt. The suspension was centrifuged, and the mother liquor was decanted. The residue was repeatedly washed with MeOH and then dried in a vacuum oven to yield a beige solid product of PA6-DGEA copolymer. 1 H NMR [500MHz, DCO2D / CD2Cl2(1:4), 500MHz, 25℃]: δ6.42(br, PA6), 3.60(s, PEG), 3.19-3.13 (m, PA6), 2.15-2.12(m, PA6), 1.62-1.56(m, PA6), 1.48-1.42(m, PA6), 1.29-1.23(m, PA6).

[0327] Example 9: Examples of bioactive synthetic copolymers - Antibiotic-containing polystyrene for use in tissue and serum handling devices - Patent Application 20070122999 Brush polymers containing PEGylated antibiotics and polystyrene were engineered for use as antimicrobial additives in medical-grade polystyrene to create non-leaching antibiotic-containing tissue handling devices, such as tissue culture plates and serum tubes. Such devices are typically made of medical-grade polystyrene, and antibiotics are usually added to the medium in which the tissue or serum is held in or coated onto the device, which tends to be a costly approach.

[0328] This example reports the development of antibiotic-containing polystyrene for use in tissue and serum handling devices such as tissue culture plates and serum sample tubes.

[0329] Polystyrene (PS) is the synthetic polymer of choice in this example due to its low cost and ease of sterilization by common sterilization techniques such as ethylene oxide, UV, and gamma irradiation. PS is particularly stable to gamma and electron beam irradiation, among other common medical device polymers, making it a very popular material for tissue handling devices, as these two sterilization techniques are the most effective methods for sterilization before use. Furthermore, the polymer's high transparency allows for its use in tissue and serum handling devices, allowing visual inspection of the contents from the outside of the device.

[0330] In this strategy, antibiotics are tethered onto polyethylene glycol (PEG) chains bearing norbornene-exo-dicarboximide (NB) moieties to create biomacromonomers, which are then subjected to ROMP with the norbornene-exo-dicarboximide synthetic macromonomers bearing polystyrene to create the final antibiotic-loaded polystyrene bioadditive, which can then be blended into the base medical-grade polystyrene for device fabrication.

[0331] In penicillin-class antibiotics, the mechanism of action resides in the β-lactam ring, which binds to the enzyme transpeptidase and prevents bacteria from forming crosslinks in their cell walls. Crosslinks in peptidoglycan are required for cell wall formation in bacterial cells. By inhibiting cell wall production, bacterial cells are rapidly killed. Therefore, the β-lactam ring of penicillin must remain exposed to the bacterial cell for its antibacterial effect. Penicillin was selected to connect through its carboxylic acid end, which is quite far from the β-lactam ring, thus allowing its access to the bacterial cell. When bacterial cells come into contact with the penicillin-containing polystyrene, the cells bind to the penicillin, and their cell walls are disrupted as a result of this contact, thus killing the bacterial cells.

[0332] Ciprofloxacin (CIF) is a broad-spectrum fluoroquinolone antibiotic, particularly active against Gram-negative bacteria such as Pseudomonas aeruginosa. The fluoroquinolone ring binds to the essential bacterial enzyme DNA gyrase, preventing bacterial cells from replicating. By tethering CIF through its carboxyl terminus and exposing its fluoroquinolone ring in the side arms of the brush polymer (Scheme 3.2), the CIF binding site becomes available for bacterial cell binding when the cells come into contact with the polymer surface, thereby killing bacterial cells present in the sample container.

[0333] Aminoglycosides are broad-spectrum antibiotics commonly used as anti-infectives in clinical settings. They are bactericidal and contain hydrophilic sugar units with multiple hydroxy and amino functional groups. Aminoglycoside antibiotics include streptomycin, ribostamycin, and gentamicin. These can be attached to NBPEG moieties via the -CHOH (strep), -CHNH (rib), or -CH(CH)NH (gen) groups on the antibiotic molecule, leaving the attachment site on the molecule exposed to bacterial cell binding. Such antibiotics bind to bacterial ribosomal subunits, preventing them from synthesizing proteins essential for growth. Polymers bearing these antibiotics, apart from penicillin, are useful for tissue culture devices because they are part of the standard antibiotic recipe for cell culture media. Once antibiotic-containing macromonomers are synthesized, they can be copolymerized with polystyrene-bearing macromonomers using ROMP technology to create the desired brush polymers of polystyrene and antibiotic held together by a norbornene dicarboximide backbone (Scheme 9). This antibiotic-containing polystyrene brush polymer is then used as a bioadditive to blend into base medical grade polystyrene for medical device manufacturing.

[0334] [ka]

[0335] In summary, antibiotic-conjugated polystyrene copolymers are developed for the creation of antibiotic-loaded polystyrene tissue handling devices, in which the antibiotic is covalently bound to the polystyrene and cannot leach from the material. This is achieved by using ring-opening metathesis techniques to polymerize PEGylated antibiotics on norbornene dicarboximide linkers and polystyrene on norbornene dicarboximide linkers. The result is a brush polymer with pendant PEGylated antibiotics and polystyrene, in which the active group on the antibiotic molecule is exposed to bacterial cell binding for bactericidal effect.

[0336] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions and bioactive macromonomer synthesis were carried out in a Vacuum Atmosphere glove box under a nitrogen atmosphere. NBPEG and NBPS synthesis were carried out in a fume hood under atmospheric conditions according to the procedures provided in Example 6. All solvents used in the glove box were anhydrous and used as purchased. Grubbs second-generation catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. PEG diamines were purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). Amoxicillin, ciprofloxacin, ribostamycin, HOBT, HBTU, i Pr2EtN was purchased from Sigma Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. All purchased reagents were used without further purification.

[0337] 1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as the solvent for all biomolecular macromonomers. CDCl was used as the solvent for the PS macromonomer. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, and a flow rate of 1.0 ml / min was used at 40 °C.

[0338] The synthesis of NBPEG and NB-PS is described in Example 6.

[0339] NBPEG as a representative preparation for PEG 1,000-6,000 1000 Synthesis of CIF Ciprofloxacin (0.0866 g, 0.26 mmol) was suspended in MeOH (2.5 ml) in a 4 ml vial in a glove box. i PrEtN (91 μL, 0.52 mmol) was added and the mixture was stirred (A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) in a 20 ml vial at 40 °C, after which the CIF solution from (A) was added to obtain a suspension (B). Suspension B was then dissolved in NBPEG in a 40 ml vial. 1000 (0.25 g, 0.218 mmol) was added, and the mixture was stirred at room temperature overnight to give a pale yellow solution with a white suspension. The mixture was then evaporated to dryness, and the oil was added to diethyl ether (50 ml). The diethyl ether solution was chilled in a freezer for 48 hours and decanted to give a white sticky residue. MeOH (3 ml) was added to the residue, and the mixture was added to diethyl ether in an Erlenmeyer flask, which was again placed in the freezer for another 48 hours to give a yellow oil at the bottom of the flask. The ether was decanted, and MeOH (3 ml) was added to give a yellow solution with a white precipitate. The mixture was passed through a syringe filter, and the clear filtrate was evaporated to dryness to give a yellow oil of NBPEGCIF. 1H NMR (500MHz, MeOD): δ = 7.76 (ddd, 2H), 7.44-7.37 (m), 6.36 (t, 2H), 3.67 (br s, 82H), 3.21 (t, 2H), 2.75 (d, 2H), 2.73 (s, 5H), 1.25 (d, 1H).

[0340] Representative synthesis of PS antibiotic polymers by ROMP using NBPEGCIF as an example NBPEG 1000 CIF macromonomer (0.1 eq) was weighed into a 4 ml glass vial, followed by the addition of NB-PS (0.050 g, 0.030 mmol). THF (0.05 M with respect to NB-PS) was added, and the mixture was stirred at room temperature until a clear solution was obtained. A solution of catalyst 2 (mentioned in Example 5) in THF (1.25 mol%, 0.05 M) was added to the solution, and the mixture was stirred at room temperature for 2 hours, and the reaction was stopped by adding ethyl vinyl ether. The polymer solution was precipitated in methanol. The polymer mixture was centrifuged, and the supernatant was decanted. The residue was repeatedly washed with MeOH and then dried under vacuum to obtain a white powder polymer. 1 H NMR (500MHz, CDCl3): δ6.99-7.15 (m, 3H, Ph), 6.4-6.8 (m, 2H, Ph), 3.60 (s, 4H, PEG), 1.93 (quintet, 1H, PS), 1.42 (t, 2H, PS). GPC analysis (THF): M n =30,056, PDI=1.36

[0341] Example 10: Examples of bioactive synthetic copolymers - Polylactide-biomolecular copolymers as bioadditives for human skin and bone tissue regeneration A series of brush copolymers containing polylactide (PLA) side chains and PEGylated biomolecules, including integrin-binding peptides, collagen mimetics or fragments (COLs), and glycosaminoglycans (GAGs), were synthesized via ring-opening metathesis polymerization (ROMP). These copolymers can be utilized as bioadditives in scaffolding materials for either human skin or bone tissue regeneration.

[0342] The biomolecules used in this example include heparin oligosaccharides (HS) DP12, DP14, integrin-binding peptides such as RGD, collagen fragments with repeating units of glycine, proline, and hydroxyproline (G, P, Hyp) of various sequences and lengths, and collagen mimetic DGEA.

[0343] Polylactide (PLA) is the synthetic polymer of choice for this example because it degrades under physiological conditions to form non-toxic lactic acid, which is also present in the human body, making it a bioabsorbable polymer. Due to its biocompatibility and good processability, PLA and its copolymers are commonly used in medical implants and tissue engineering. Bioactive molecules that can promote the regeneration of skin or bone tissue are then incorporated into the final polymer via copolymerization in our approach.

[0344] Integrin-binding peptides, such as RGD (Arg-Gly-Asp), found in several extracellular matrix proteins, have been identified as an important motif for cell recognition and cell adhesion. Immobilization of RGD onto scaffolds has been shown to enhance cell attachment, migration, and proliferation. RGD can also promote osteogenic differentiation and mineralization, thereby inducing bone regeneration. Due to the hygroscopic nature of RGD, it is difficult to handle and administer by itself. Therefore, covalently attaching the peptide to a synthetic PLA polymer increases its stability and ease of handling. These RGD-containing polymers can then be blended with substrates to create scaffolds for skin or bone regeneration.

[0345] In addition to RGD peptides, polymers containing collagen mimetics or fragments have also been synthesized. Collagen is the most abundant protein in the extracellular matrix and is widely used in biomaterials to increase biocompatibility and promote tissue regeneration. However, full-length human collagen requires complex synthesis and often exhibits poor solubility in buffer solutions. Short collagen mimetic peptide sequences or fragments containing only a small portion of the critical peptide sequence have been used to elicit biological responses similar to their full-length collagen counterparts. Collagen mimetics such as DGEA (Asp-Gly-Glu-Ala) and collagen fragments with various lengths of glycine, proline, and hydroxyproline sequences have been incorporated into synthetic polymers. Without being bound by theory, DGEA is thought to promote cell adhesion, spreading, and osteogenic differentiation, which would be advantageous for applications in both skin and bone regeneration.

[0346] Heparan sulfate (HS) is a GAG with repeating disaccharide units extensively modified with sulfate groups. Without being bound by theory, HS chains of between 5 and 10 disaccharide units are believed to be most active for bone morphogenetic protein (BMP) binding. Without being bound by theory, HS is believed to directly regulate BMP-2-mediated myoblast differentiation into osteoblasts. In particular, the HS fragment with six disaccharide units (DP12) is believed to have the highest binding affinity for BMP-2. In vitro studies of BMP-2 complexed with DP12 demonstrated enhanced osteogenic differentiation in cells, while in vivo work using a rat model revealed improved bone tissue regeneration using DP12 compared to a collagen sponge control in a polycaprolactone (PCL) tube. HS interacts with angiogenic factors and induces angiogenesis. Angiogenesis is important in tissue scaffolds for delivering oxygen and nutrients throughout engineered tissues. Furthermore, HS can interact with growth factors that stimulate epithelial repair and promote wound healing. PLA containing HS molecules, such as DP12 or DP14, would be desirable for use in scaffolding materials to enable skin or bone tissue regeneration. However, HS molecules are highly hygroscopic and cannot be simply coated onto polymers. The high water solubility of HS also means that it may leach into the body and not remain at the desired site where tissue regeneration is needed. In this approach, macromonomers containing HS molecules are copolymerized with PLA macromonomers to prepare bioactive copolymers that can be blended with the base polymer PLA to produce skin scaffolds or bone implants. This ensures that the HS molecules are localized at the implantation site and do not cause undesirable effects in other parts of the body.

[0347] The final brush copolymer is prepared via copolymerization of PLA macromonomers with bioactive macromonomers by ROMP using a Grubbs-type catalyst (Scheme 10).

[0348] [ka]

[0349] Biocompatibility To demonstrate the biocompatibility of the polymers, the materials were tested on human fibroblast cells in vitro. A bioactive synthetic polymer (PLA-RGD) was blended with commercially available PLA as the substrate and electrospun into thin sheets. The commercially available base polymer, PLA (PLA-bulk), was used as a control for this study. The sheets were then tested on human fibroblast cells, Hs27, and all tested materials demonstrated good biocompatibility with high cell viability after 72 hours (Figure 7). Preliminary data showed >100% cell viability for the bioactive synthetic polymers designed according to various embodiments disclosed herein, indicating cell proliferation (cell growth) versus cell death (<100%). This demonstrates the low toxicity of the materials to human fibroblast cells. Furthermore, the bioactive polymer-containing PLA showed improvement in cell viability compared to the base polymer PLA, demonstrating their ability to enhance the biocompatibility of pure PLA itself. Optimization of biomolecule concentrations and blend ratios in the bioactive synthetic polymer is underway to obtain the best tissue regeneration results for this material.

[0350] In summary, we synthesized a series of brush copolymers with biodegradable PLA side chains and bioactive molecules such as integrin-binding peptides, collagen mimetics or fragments (COLs), and heparan sulfate (HS). These bioactive polymers can be blended with substrates such as medical-grade PLA to create scaffolding materials for use in skin or bone regeneration.

[0351] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions and bioactive macromonomer synthesis were performed in a Vacuum Atmosphere glove box under a nitrogen atmosphere. PLA macromonomer (NPH-PLA) synthesis was performed using standard Schlenk line techniques under a nitrogen atmosphere. NBPEG and NPH syntheses were performed in a fume hood under atmospheric conditions according to the procedures provided in Example 6. All solvents used were anhydrous and used as purchased. Grubbs second-generation catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. Catalyst 2 ((HIMes)(pyr)(Cl)RuCHPh) was synthesized according to the procedures provided in Example 6. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). HOBT, HBTU, i Pr2EtN was purchased from Sigma-Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. All purchased reagents were used without further purification. Heparin oligosaccharides DP12 and DP14 were purchased from Iduron.

[0352] 1 H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD or DO as the solvent for all biomolecular macromonomers. CDCl was used as the solvent for PCL macromonomers and ROMP polymers. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, and a flow rate of 1.0 ml / min was used at 40 °C.

[0353] The synthesis of NBPEG, NBPEGRGD, NBPEG(DGEA), NBPEG(GPHyp)3, and NBPEGDP12 is described in Example 6.

[0354] Synthesis of NPH-PLA macromonomer by ROP NPH-PLA macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, a 25 mL Schlenk tube was charged with NPH initiator (110 mg, 0.50 mmol), D,L-lactide (864 mg, 6.0 mmol), Sn(Oct)2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times and then immersed in an oil bath at 130 °C. After 2.5 h, the contents were cooled to room temperature, diluted with dichloromethane, and precipitated twice into cold methanol. The mother liquor was decanted, and the residue was washed with MeOH and dried in a vacuum oven.

[0355] 1 H NMR(CDCl3):δ6.28(br t, 2H), 5.27-5.08(m), 4.35(m, 1H), 4.19-4.02(m, 2H), 3.62-3.44(m, 2H), 3.27(s, 2H), 2.69(m, 2H), 1.97-1.47(m), 1.19(d, 1H).

[0356] GPC analysis (THF): M n = 2,471, PDI = 1.20, yield 0.600 g.

[0357] NBPEG 3400 Synthesis of DP14 DP14 (0.0285 g, 8.4 μmol) was dissolved in MeOH / DMF (0.5 ml / 1.0 ml) in an 8 ml scintillation vial. i PrEtN (2.9 μl, 16.8 μmol) was added and the mixture was stirred (Solution A). HOBt (0.0011 g, 8.4 μmol) and HBTU (0.0032 g, 8.4 μmol) were dissolved in MeOH (2.5 ml) at 40° C., after which Solution A was added to obtain Suspension B. Suspension B was then treated with NBPEG. 3400NH2 (0.025 g, 7.03 μmol) was added and stirred at room temperature for 24 hours. The resulting pale yellow mixture was centrifuged to remove insoluble impurities. The supernatant was concentrated by solvent evaporation, then precipitated in cold Et2O, and the mixture was placed in a freezer for 24 hours. Filtration gave an orange powder product (43.5 mg, 90% yield).

[0358] 1 H NMR (500MHz, D2O): 6.36 (t, 2H), 4.30 (br, 4H), 3.70 (s, 340H), 2.85 (s, 2H), 2.71 (s, 2H), 1.40-1.32 (m, 10H).

[0359] NPH-PLA macromonomer and NBPEG as representative preparations for PLA-peptide type copolymers 1000 Typical procedure for ROMP of RGD macromonomers NBPEG 1000 RGD macromonomer (0.1 equivalents) is weighed into a 4 ml scintillation vial, followed by addition of NPH-PLA (0.05 g). THF (0.05 M with respect to NPH-PLA) is added, and the mixture is stirred at room temperature until a clear solution is obtained. A solution of catalyst 2 in THF (1.25 mol%) is added to the solution, and the reaction is stirred for 1 hour. Ethyl vinyl ether is added to the reaction mixture, followed by addition of MeOH (3 ml), and the mixture is placed in a freezer for 1 hour to obtain a sticky solid. The mother liquor is decanted, and the residue is repeatedly washed with MeOH and then dried in a vacuum oven. Regarding PLA-RGD copolymer, 1 H NMR (500MHz, CDCl3): δ5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA). GPC analysis (THF): M n =76,700, PDI=1.44.

[0360] NPH-PLA macromonomer and NBPEG as representative preparations for PLA-COL type copolymers 1000Typical procedure for ROMP of (GPHyp)3 macromonomer NBPEG 1000 (GPHyp)3 macromonomer (0.1 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NPH-PLA (0.05 g). THF (0.05 M with respect to NPH-PLA) was added, and the mixture was stirred at 45 °C. A solution of catalyst 2 in THF (1.25 mol%) was added to the solution, and the reaction was stirred at 45 °C for 2 h. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 h to obtain a sticky solid. The mother liquor was decanted, and the residue was washed repeatedly with MeOH and then dried in a vacuum oven. 1 H NMR (500MHz, CDCl3): δ5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA). GPC analysis (THF): M n =97,262, PDI=1.14.

[0361] NPH-PLA macromonomer and NBPEG as representative preparations for PLA-HS type copolymers 3400 Typical procedure for ROMP of DP14 macromonomer NBPEG 3400 DP14 macromonomer (0.1 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NPH-PLA (0.03 g). THF (0.02 M with respect to NPH-PLA) was added, and the mixture was stirred at 45 °C. A solution of catalyst 2 in THF (1.25 mol%) was added to the solution, and the reaction was stirred at 45 °C for 2 h. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 h to obtain a sticky solid. The mother liquor was decanted, and the residue was washed repeatedly with MeOH and then dried in a vacuum oven. 1 H NMR (500MHz, CDCl3): δ5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA).

[0362] Example 11: Examples of bioactive synthetic copolymers PLGA-peptide and -oligosaccharide polymers for cartilage tissue regeneration A series of poly(lactic-co-glycolic acid) (PLGA) peptide and oligosaccharide brush polymers were prepared by ring-opening metathesis polymerization. Extracellular matrix (ECM) peptides such as RGD, collagen fragments, and oligosaccharides such as heparin oligosaccharides were PEGylated and linked to PLGA as side chains on a poly(norbornene-exo-2,3-dicarboximide) backbone via ring-opening metathesis polymerization. The resulting brush polymers can be used as bioadditives for PLGA-based cartilage tissue regeneration materials. Preliminary in vitro testing on bioactive PLGA demonstrated excellent cell viability with some cell proliferation at 72 hours.

[0363] The biomolecules used in this example include ECM peptides such as RGD, collagen fragments, and collagen mimetics, which are known to regenerate cartilage tissue. The synthetic polymer of choice in this example is PLGA, a bioabsorbable polymer with properties between those of PLA and poly(glycolic acid) (PGA). The resulting overall polymer has been shown to be a thermally stable, bioactive polymer that is osteoinductive for use in cartilage implants.

[0364] Using bioactive synthetic polymer technology according to various embodiments disclosed herein, collagen-containing synthetic polymers are created that allow collagen to be incorporated into synthetic materials without loss of functionality of these collagen fragments. Furthermore, the PLGA side chains in these bioactive synthetic polymers help increase the thermal stability of collagen, allowing for efficient blending of otherwise hygroscopic collagen into the hydrophobic base polymer PLGA. The overall PLGA material is not only bioactive, but also thermally stable and mechanically strong for material processing and use in meniscal cartilage implants.

[0365] PLGA was chosen as the synthetic polymer of choice due to its better control of polymer crystallinity, melting point, and load-bearing capacity compared to its homopolymer counterparts, PGA and PLA (where PGA is more crystalline and has a higher melting point than PLA). However, despite its apparent biocompatibility, PLGA is non-osteoinductive. Therefore, it is necessary to introduce stimuli into PLGA by copolymerizing it with bioactive macromonomers that are osteoinductive or osteoconductive. The overall material is then a mechanically strong, thermally stable, osteoinductive polymer for use in cartilage implants. PLGA is also biodegradable, allowing the patient's own cartilage to take over the synthetic material after the material degrades in the body. The polymer's by-products are lactic acid and glycolic acid, both of which are non-toxic to humans.

[0366] To enhance the softness and hydrophilicity of the material, polyethylene glycol (PEG) is introduced into the bioactive synthetic polymer chain. This same strategy can be used to adjust the softness / hardness of the material. By adjusting the PEG to PLGA side chain content in the bioactive synthetic polymer and the ratio of PLGA-COL bioactive synthetic polymer to PLGA-based material, the overall hardness of the material can be adjusted. This is particularly important for articular cartilage implants. To further enhance the strength of articular cartilage implants, lattice designs can be used / created in additive manufacturing (AM) of the scaffold. The strength of the material can be greatly enhanced through lattice designs using AM, while maintaining the porosity of the material for enhanced osseointegration in the scaffold and a lighter overall scaffold weight.

[0367] In addition to ECM peptides, heparan sulfate mimetics, such as highly sulfated glycosaminoglycans, can also be used to provide the necessary stimulation required for articular cartilage regeneration. Glycosaminoglycans (GAGs) are heterogeneous polysaccharides found widely in mammalian tissues. Heparan sulfate (HS) is a highly sulfated GAG with enormous structural diversity that can interact with a large number of proteins to regulate many physiological processes. Proteins that interact with HS include growth factors (GFs), chemokines, enzyme inhibitors, extracellular matrix proteins, and membrane-bound receptors. HS enhances the efficacy of key GFs involved in cell proliferation and differentiation, including the bone morphogenetic protein BMP-2, which is important in bone growth, and vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), which are important for angiogenesis. Incorporating such oligosaccharides into PLGA introduces BMP-binding properties into the polymer, allowing the material to be used in cartilage implants. Through a combination of advanced chemical synthesis for material design and additive manufacturing for scaffold design and fabrication, various types of bone scaffolds can be created for regenerating different parts of bone as needed. Alternatively, scaffolds can also be fabricated by other material processing methods such as melt extrusion, injection molding, and electrospinning.

[0368] Using both biomacromonomers and synthetic macromonomers, the final bioactive synthetic polymers are prepared by ROMP using a Grubbs-type catalyst (Scheme 11).

[0369] [ka]

[0370] Bioactive polymers are characterized using TG-DSC analysis to confirm the polymer's melting point and decomposition temperature. ICP-MS is also performed prior to material processing to ensure that metal residues from the ruthenium catalyst are minimized to below the ISO 10993 guidelines for metal catalysts in biomedical devices. Once these parameters are confirmed, the material can be processed into prototypes for in vitro testing to confirm the material's biocompatibility and cell viability.

[0371] Biocompatibility To demonstrate the biocompatibility of the polymer, the material was tested in vitro on human fibroblasts. A bioactive synthetic polymer (PLGA-RGD) was blended with commercially available PLGA as a base material and electrospun into thin sheets. The commercially available base polymers PLGA (PLGA-Bulk), PLGA ROMP homopolymer (PLGA-homo), and PLGA-mPEG were used. 5000 was used as a control for this study. The sheets were then tested on human fibroblast cells Hs27, and all tested materials demonstrated good biocompatibility with high cell viability after 72 hours (Figure 8). Preliminary data showed >100% cell viability for the bioactive synthetic polymers designed according to various embodiments disclosed herein, indicating cell proliferation (cell growth) versus cell death (<100%). This demonstrates the low toxicity of the materials to human fibroblast cells. Furthermore, all bioactive polymer-containing PLGAs showed improved cell viability compared to the base polymer PLGA, indicating their ability to enhance the biocompatibility of pure PLGA itself. Optimization of the biomolecule concentration and blend ratio in the bioactive synthetic polymer to obtain the best tissue regeneration results for this material is ongoing.

[0372] In summary, we developed a series of biodegradable polymers with biodegradable synthetic polymer side chains (PLGA) and bioactive side chains of extracellular matrix peptides or sulfated glycosaminoglycans for cartilage tissue regeneration. The materials can be processed by a wide variety of material processing methods, including melt extrusion, FFF or FDM-type 3D printing, and electrospinning. Preliminary in vitro studies demonstrated good cell viability and proliferation without the introduction of stem cells or growth factors.

[0373] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions and bioactive macromonomer synthesis were performed in a Vacuum Atmosphere glovebox under a nitrogen atmosphere. PLGA macromonomer (NPH-PLGA) synthesis was performed using standard Schlenk line techniques under a nitrogen atmosphere. NBPEG and NPH syntheses were performed in a fume hood under atmospheric conditions according to the procedures provided in Example 6. All solvents used in the glovebox were anhydrous and used as purchased. Grubbs second-generation catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). HOBT, HBTU, i Pr2EtN was purchased from Sigma Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. All purchased reagents were used without further purification.

[0374] 1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as the solvent for all biomolecular macromonomers. CDCl was used as the solvent for PLGA macromonomers. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, and a flow rate of 1.0 ml / min was used at 40 °C.

[0375] The synthesis of NBPEG and NBPEG(peptide) is described in Example 6.

[0376] Synthesis of NPH(PLGA) macromonomer NPH-PLGA macromonomers with various degrees of polymerization (DP) were prepared by ROP. For example, a 25 mL Schlenk tube was charged with NPH initiator (55 mg, 0.25 mmol), D,L-lactide (864 mg, 6.0 mmol), glycolide (174 mg, 1.5 mmol), Sn(Oct)2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times and then immersed in an oil bath at 125 °C. After 3 h, the contents were cooled to room temperature, diluted with dichloromethane, and precipitated into cold MeOH. The mother liquor was decanted, and the residue was washed with MeOH and then dried in a vacuum oven.

[0377] 1 H NMR (500MHz, CDCl3): δ6.28(br t, 2H), 5.27-5.08(m, PLA), 4.85-4.65(m, PGA) 4.35(m, 1H), 4.19-4.02(m, 2H), 3.62-3.44(m, 2H), 3.27(s, 2H), 2.69(m, 2H), 1.97-1.47(m, PLA), 1.19(d, 1H). GPC analysis (THF): M n =4,336, PDI-1.27.

[0378] NBPEG as a representative preparation for collagen fragments of glycine, proline, and hydroxyproline with various sequences and chain lengths up to n=6, PEG 1000, 3,400, and 6,000. 1000 Synthesis of (GPHyp)3 (GPHyp)3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in a glove box. i PrEtN (91 μl, 0.52 mmol) was added and the mixture was stirred (solution A). HOBT (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) at 40 °C, followed by the addition of solution A to obtain suspension B. Suspension B was then added to NBPEGNH (0.25 g, 0.218 mmol) and stirred at room temperature for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to obtain a beige mixture. The mixture was dispersed in EtO and frozen for 48 h. The EtO layer was removed, and MeOH was added to the residue to obtain a beige suspension. Filtration and solvent evaporation gave the beige oily product NB-PEG-(GPHyp) (0.22 g, 50% yield).

[0379] 1 H NMR (500MHz, CD3OD): δ6.33(s, 2H), 4.73-.4.44(br, 4H), 3.65(m, 84H), 3.5 7(m, 4H), 3.18(s, 2H), 2.72(s, 2H), 2.39-1.80(br, 8H), 1.44-1.37(dd, 2H).

[0380] Examples include NPH(PLGA) and NB-PEG. 3.4k PLGA-PEG by ROMP using (GPHyp)3 3.4K Representative synthesis of (GPHyp)3 polymer NBPEG 3.4K(GPHyp)3 macromonomer (0.1 equivalent) was weighed into a 4 ml glass vial, followed by the addition of NPH(PLGA) (0.050, 0.012 mmol). THF (NPH (0.05 M relative to PLGA)) was added, and the mixture was stirred at 40 °C until a clear solution was obtained. A solution of catalyst 2 in THF (1.25 mol%, 0.05 M) was added to the solution, and the mixture was stirred at 40 °C for 2 h. The reaction was terminated by the addition of ethyl vinyl ether. The polymer solution was precipitated in methanol. The polymer mixture was centrifuged, and the supernatant was decanted. The residue was washed repeatedly with MeOH and then dried under vacuum. The resulting polymer is a white powder.

[0381] 1 H NMR (500MHz, CDCl3): δ5.20 (m, 1H, PLA), δ4.8 (m, 2H, PGA), δ3.63 (s, 4H, PEG), δ1.56 (d, 3H, PLA). GPC analysis (THF): M n =65,166, PDI=1.23.

[0382] Example 12: Examples of bioactive synthetic copolymers PMMA-Peptide Copolymers for Use as Bioadditives in Medical Implants A series of brush copolymers containing biomolecules tethered onto poly(methyl methacrylate) (PMMA) side chains and PEG moieties were synthesized via ring-opening metathesis polymerization (ROMP). The biomolecules could include collagen fragments or collagen-mimetic peptides of any sequence of 3 to 20 amino acid residues, such as DGEA, (Gly-Pro-Hyp)3, and (Pro-Hyp-Gly)3. These brush polymers can be blended with the base polymer PMMA to create bioactive materials for use in biomedical implants such as bone cements, bone implants, and craniofacial implants.

[0383] This example reports the straightforward synthesis of brush copolymers containing collagen mimetics tethered onto PMMA side chains and PEG moieties for use as bioactive polymers in PMMA-based biomedical implants.

[0384] PMMA is the synthetic polymer of choice in this example because it is a biocompatible, non-degradable, and lightweight thermoplastic with good mechanical strength. It was the first synthetic polymer used in biomedical applications and is now used in a variety of medical implants, including intraocular lenses, rhinoplasty, dentistry, and orthopedics. PMMA is also currently the most widely used alloplastic implant material for craniomaxillofacial reconstruction. PMMA polymers are often modified with varying amounts of additives or fillers to achieve desired properties in the final material. PMMA-based implant materials can be manufactured using traditional molding methods, such as injection molding or extrusion, and also 3D printing. With rapid advances in 3D printing technology, PMMA is increasingly being utilized in patient-specific biomedical applications for the production of customized medical implant structures.

[0385] In this example, PMMA brush copolymers bearing collagen fragments or mimetics were synthesized using ROMP. Collagen is the most abundant protein in the extracellular matrix and is widely used in biomaterials to increase biocompatibility and promote tissue regeneration. However, full-length human collagen requires complex synthesis and often exhibits poor solubility in buffer solutions. Short collagen-mimetic peptide sequences or fragments containing only a small portion of the critical peptide sequence can be used to elicit biological responses similar to their full-length collagen counterparts. However, like many biomolecules, these peptides are highly hygroscopic. Creating implants for insertion into the human body is difficult without immobilizing collagen fragments or mimetics on synthetic polymers to increase their ease of handling. Furthermore, PMMA medical implants are foreign to the body and can trigger a host immune response, leading to tissue inflammation. PMMA itself also does not support osseointegration of the structure with other structures it comes into contact with. Therefore, without being bound by theory, it is believed that incorporating collagen fragments or collagen mimetics (COL) into PMMA polymers will help increase the biocompatibility and biomimetic properties of the material. Some collagen mimetics that may be used include DGEA (Asp-Gly-Glu-Ala) and collagen fragments with various lengths of glycine, proline, and hydroxyproline sequences in any order. Without being bound by theory, DGEA is believed to promote cell adhesion, osteogenic differentiation, and osseointegration, which would be advantageous for applications in bone or craniofacial implants.

[0386] The final brush copolymer is prepared by ROMP using a Grubbs-type catalyst (Scheme 12).

[0387] [ka]

[0388] Bioactive PMMA polymers can be blended with medical grade PMMA and fabricated into suitable shapes either by extrusion, 3D printing or electrospinning and tested for biocompatibility.

[0389] Biocompatibility To demonstrate the biocompatibility of the polymer, the material was tested in vitro on human fibroblasts. The bioactive synthetic polymer (PMMA-GPHyp) was blended with commercially available PMMA as a base material and electrospun into thin sheets. The commercially available base polymers PMMA (PMMA-bulk), PMMA ROMP homopolymer (PMMA-homo), and PMMA-mPEG were used. 5000 was used as a control for this study. The sheets were then tested on human fibroblast cells Hs27, and all tested materials showed good biocompatibility with high cell viability after 72 hours (Figure 9). This indicates the low toxicity of materials designed according to various embodiments disclosed herein to human fibroblast cells. Furthermore, bioactive polymer-containing PMMA showed improvement in cell viability compared to the base polymer PMMA, indicating their ability to enhance the biocompatibility of pure PMMA itself. Optimization of the biomolecule concentration and blend ratio in the bioactive synthetic polymer will be performed in the future to improve the biocompatibility of the material.

[0390] In summary, a series of brush copolymers with PMMA side chains and peptide molecules such as collagen mimetics or fragments (COLs) were synthesized. These bioactive polymers can be used as bioadditives to create implant materials for use in orthopedics or cranioplasty.

[0391] Experimental procedure General Procedure Ring-opening metathesis polymerization (ROMP) reactions and bioactive macromonomer synthesis were carried out in a Vacuum Atmosphere glovebox under a nitrogen atmosphere. PMMA macromonomer (NB-PMMA) synthesis was carried out using standard Schlenk techniques under a nitrogen atmosphere. NBPEG and norbornenyl-functionalized ATRP initiator syntheses were carried out in a fume hood under atmospheric conditions according to the procedures provided in Example 6. All solvents used in the glovebox were anhydrous and used as purchased. Grubbs second-generation catalyst was purchased from Sigma-Aldrich, and peptides were purchased from Biomatik Inc. Catalyst 2 was synthesized according to the procedures provided in Example 6. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma-Aldrich (6,000). HOBT, HBTU, i Pr2EtN was purchased from Sigma Aldrich, and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. All purchased reagents were used without further purification.

[0392] 1 H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD or DO as the solvent for all biomolecular macromonomers. CDCl was used as the solvent for PMMA macromonomers and ROMP polymers. Gel permeation chromatography was performed on a Waters Aquity APC System equipped with Acquity APC XT45, XT200, and XT450 columns and an Acquity RI detector. THF was used in sample preparation, and a flow rate of 1.0 ml / min was used at 40 °C.

[0393] The synthesis of NBPEG, NBPEG(DGEA), and NBPEG(GPHyp)3 is described in Example 6.

[0394] Synthesis of NB-PMMA macromonomer by ATRP NB-PMMA macromonomers with various degrees of polymerization (DP) were prepared by ATRP. A 25 mL Schlenk tube was charged with norbornenyl-functionalized initiator (53 mg, 0.143 mmol), MMA (1.06 mL, 10.0 mmol), anisole (1.0 mL), and TMEDA (0.011 mL, 0.072 mmol). The solution was degassed by three freeze-pump-thaw cycles. During the final cycle, the Schlenk tube was filled with nitrogen, and CuBr (10.3 mg, 0.072 mmol) was quickly added to the frozen reaction mixture. The Schlenk tube was sealed, evacuated, and backfilled with nitrogen three times. The Schlenk tube was thawed to room temperature, and polymerization was carried out in an oil bath at 70 °C for 3 h. The mixture was filtered through neutral alumina, precipitated in MeOH, and filtered. The white solid was washed with MeOH and then dried overnight in a vacuum oven. 1 H NMR (CDCl3): δ6.30(s, 2H), 4.17(m, 2H), 3.76(m), 3.65-3.59(m, PMMA), 3.28(s, 2H), 2.72(s, 2H), 2.00-1.69(m, PMMA), 1.07-0.75(m, PMMA). GPC analysis (THF): M n =5,158, PDI=1.13.

[0395] NB-PMMA macromonomer and NBPEG as representative preparations for PMMA-peptide copolymers for PEG with molecular weights of 1,000 to 6,000 3400 Typical procedure for ROMP of (GPHyp)3 macromonomer NBPEG 3400(GPHyp)3 macromonomer (0.1 equivalents) was weighed into a 4 ml scintillation vial, followed by the addition of NB-PMMA (0.05 g). THF (0.02 M with respect to NB-PMMA) was added, and the mixture was stirred at 45 °C until a clear solution was obtained. A solution of catalyst 2 (1.25 mol%) in THF was added to the solution, and the reaction was stirred at room temperature for 2 h. Ethyl vinyl ether was added to the reaction mixture, followed by MeOH (3 ml), and the mixture was placed in a freezer for 1 h, resulting in a white precipitate. The mother liquor was decanted, and the residue was washed repeatedly with MeOH and then dried in a vacuum oven.

[0396] 1 H NMR (500 MHz, CDCl): 3.65-3.59 (m, PMMA and PEG), 2.00-1.69 (m, PMMA), 1.07-0.75 (m, PMMA). GPC analysis (THF): M n =65,600, PDI=1.37.

[0397] Purpose The present disclosure provides a novel modular synthetic method for rapidly generating bioactive macromonomers for the construction of bioactive copolymers using optimal synthetic polymers. Bioactive macromonomers can be easily copolymerized with other synthetic copolymers to form bioactive polymers with desired physical and mechanical properties. Advantageously, there is increased stability of the bioactive molecule upon attachment to the polymer linker. Embodiments of the strategy disclosed herein allow for the use of any peptide, carbohydrate, or drug molecule in polymer synthesis without loss of biological activity. Embodiments of the strategy disclosed herein also allow for the rapid construction of bioactive macromonomer libraries. Any bioactive molecule bearing a carboxylic acid group may be used. In summary, the present disclosure provides a highly versatile strategy for customizing biomedical materials.

[0398] Embodiments of the methods disclosed herein allow for the pairing of macromonomers with synthetic polymers of choice to create bioactive polymers that possess both the mechanical and physical properties of the synthetic polymer and the biological activity of the bioactive molecule.

[0399] An embodiment of the method disclosed herein is a simple strategy to create various types of bioactive polymers that are chemically conjugated, instead of physically blending, bioactive molecules into synthetic polymers.

[0400] Advantageously, non-cellular or growth factor-based bioactivity is provided on the polymers disclosed herein. Bioactive synthetic polymer embodiments disclosed herein possess both bioactivity that enhances therapeutic effects such as tissue regeneration and biofilm eradication, as well as polymer-like structural integrity and mechanical strength. Bioactive synthetic polymer embodiments disclosed herein allow biomolecules to be blended into synthetic polymer base materials, such as the synthetic polymer side arms of copolymers, without phase separation. Method embodiments disclosed herein allow synthetic polymers to become biocompatible with human tissue upon modification with biomolecules. Method embodiments disclosed herein allow a wide range of biomolecules to be used to achieve any desired therapeutic effect. Method embodiments disclosed herein also allow a wide range of synthetic polymers to be used to achieve the various mechanical and physical properties required in materials for targeted biodevices.

[0401] Embodiments of the bioactive synthetic polymers disclosed herein may be used as bioadditives for biomedical devices, allowing the device material itself to provide a therapeutic effect.

[0402] Embodiments of the methods disclosed herein use non-cell-based or growth factor-based therapies, which allow for a long shelf life of materials such as devices or scaffolds and prevent undesired or uncontrolled biological activity (e.g., tissue regeneration).

[0403] Embodiments of the bioactive synthetic polymers disclosed herein can be used as bioadditives for skin or bone scaffolds to create the stimulation needed for the regeneration of skin or bone tissue.

[0404] Embodiments of the bioactive synthetic polymers disclosed herein can be used in bone scaffolds, resulting in PCL that is more "bone-like" and more biocompatible. Studies have shown that bone cells do not bind to PCL, but only begin to bind to PCL after collagen is coated onto it.

[0405] The present disclosure also provides bioactive polyamide-peptide brush polymers that possess both structural integrity and mechanical strength, as well as biocompatibility and wound-healing-enhancing bioactivity. Polyamide-peptide brush polymer embodiments can be blended into polymers similar to synthetic side chains as bioadditives to create materials for use in medical devices such as catheters, plastic surgery implants, prosthetic components, and cartilage joint implants. Advantageously, bioactive polyamide-peptide brush polymer embodiments disclosed herein can be sterilized by heat before implantation and have a long shelf life. Because bioactive polyamide-peptide brush polymer embodiments disclosed herein are thermally stable, they enable product customization via 3D printing. Bioactive polyamide-peptide brush polymer embodiments disclosed herein improve the biocompatibility of polyamides, which can cause inflammatory responses in the body.

[0406] The present disclosure also provides bioactive polystyrenes that are engineered to be biocidal while still possessing the structural integrity and mechanical strength of polymers. In various embodiments, the antibiotic is covalently attached to the polymer, thus preventing leaching of the antibiotic into the medium, which could leak into the environment if disposal is improperly managed. In various embodiments, the active site on the antibiotic molecule remains exposed on the polymer chain, allowing for bacterial cell penetration or bacterial RNA binding. Bioactive synthetic polymer embodiments disclosed herein allow antibiotics to be blended into synthetic polymer base materials, such as the synthetic polymer side arms of the copolymer, without phase separation. Antibiotic-polystyrene copolymer embodiments can be used as bioadditives for biomedical devices to provide a biocidal effect on the device without the need for additional agents.

[0407] The present disclosure also provides bioactive poly(lactic-co-glycolic acid) that can be used as a bioadditive in the manufacture of cartilage implant materials. For example, bioactive poly(lactic-co-glycolic acid) can be an acellular, biodegradable cartilage scaffold capable of binding chondrocytes for cartilage regeneration. Polymer embodiments disclosed herein incorporate acellular implant materials, thus offering lower regulatory barriers and a faster path to market. In various embodiments, bioactivity is localized because biomolecules are covalently attached to the synthetic polymer and cannot leach out. This prevents premature metabolism of sulfated sugars or unintended BMP binding elsewhere in the body. In various embodiments, biomolecules attached to the polymer can bind to BMP while remaining immobilized on the scaffold, instead of leaching to other parts of the body or being prematurely metabolized due to undesirable side effects. Bioactive poly(lactic-co-glycolic acid) embodiments may more closely resemble polymers used in substrates for device fabrication, allowing for effective blending of biomolecules into the primary polymer matrix. In various embodiments, phase separation of bioactive poly(lactic-co-glycolic acid) is unlikely. Advantageously, in various embodiments, biomolecules exhibit improved thermal stability upon conjugation to the polymer, which allows for material processing. For example, the polymer can be 3D printed by filament melt fabrication, fused deposition modeling, and / or customized into scaffolds. Embodiments of the bioactive synthetic polymers disclosed herein allow peptides and oligosaccharides to be blended into synthetic polymer base materials, such as the synthetic polymer side arms of copolymers, without phase separation.

[0408] The present disclosure also provides PMMA-peptide brush polymers that can be blended with the base polymer PMMA as bioadditives to create materials for use in medical devices such as orthopedic or cranial implants. The PMMA-peptide brush polymer embodiments disclosed herein enable customization of implants and pre-operative manufacturing via 3D printing, thus improving "fit" and reducing surgical time. The PMMA-peptide brush polymer embodiments disclosed herein also improve the biocompatibility of PMMA and reduce inflammatory responses in the body. In various embodiments, the biomolecules are covalently attached to the synthetic polymer and cannot leach out.

[0409] It will be understood by those skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, features of the various exemplary embodiments described herein may be mixed, combined, substituted, incorporated, adopted, modified, included, etc. across the various exemplary embodiments. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

Claims

1. One or more repeating units represented by general formula (I) and one or more repeating units represented by general formula (II): 【Chemistry 1】 (In the formula, R １ is an optionally substituted alkylene; R ２ is selected from a single bond, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted alkyleneoxyalkylene, optionally substituted alkylenecarbonyl, or optionally substituted alkylenecarbonylalkylene; R ３ is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is a polyalkylene glycol; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y １ comprises a synthetic polymer; and Z １ and Z ２ are each independently CR ａ R ｂ , O., N.R. ｃ , SiR ａ R ｂ , P.R. ａ or S, where R ａ , R ｂ and R ｃ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

2. 2. The copolymer of claim 1, wherein the molecular weight of general formula (I) does not differ from the molecular weight of general formula (II) by more than 30% of the molecular weight of general formula (II).

3. 3. The copolymer of claim 1, wherein L is a polyalkylene glycol having from 20 to 300 carbon atoms.

4. 4. The copolymer of claim 1, wherein L is polyethylene glycol (PEG).

5. 5. The copolymer of claim 1, wherein L is a polyethylene glycol (PEG) having a number average molecular weight between 500 and 7,000.

6. R １ is C １ -C ４ alkylene, and R ２ is C １ -C ２０ Alkylene, C ２ -C ２０ Alkenylene, C ２ -C ２０ Alkynylene, C １ -C ２０ Alkyleneoxy, C １ -C ２０ Alkyleneoxyalkylene, C ２ -C ２０ Alkylenecarbonyl or C ３ -C ２０ 6. The copolymer of claim 1, wherein the alkylene group is selected from alkylenecarbonylalkylene.

7. R １ are linear or branched C independently selected from methanediyl, ethanediyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, 2-methylpropane-1,3-diyl, 1-methylpropane-1,3-diyl, and 1,1-dimethylethanediyl. １ -C ４ is an alkylene substituent, and R ２ Methanediyl, ethanediyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, 2-methylpropane-1,3-diyl, 1-methylpropane-1,3-diyl, 1,1-dimethylethanediyl, hexane-1,6-diyl, 1,2-dimethylpropane-1,3-diyl, 1,1-dimethylpropane-1,3-diyl, pentane-1,5-diyl, 3-methylbutane-1,4-diyl 4-methylpentane-1,5-diyl, 1-methylpentane-1,5-diyl, 2-methylpentane-1,5-diyl, 3-methylpentane-1,5-diyl, 2,2-dimethylbutane-1,4-diyl, 3,3-dimethylbutane-1,4-diyl, 1,2-dimethylbutane-1,4-diyl, 1,3-dimethylbutane-1,4-diyl, 1,2,2-trimethylpropane-1,3-diyl, 1,1,2-trimethylpropane-1,3-diyl, Methylpropane-1,3-diyl, 2-ethylpentane-1,5-diyl, 3-ethylpentane-1,5-diyl, heptane-1,7-diyl, 1-methylhexane-1,6-diyl, 2,2-dimethylpentane-1,5-diyl, 3,3-dimethylpentane-1,5-diyl, 4,4-dimethylpentane-1,5-diyl, 1,2-dimethylpentane-1,5-diyl, 1,3-dimethylpentane-1,5-diyl linear or branched C independently selected from 1,4-dimethylpentane-1,5-diyl, 1,2,3-trimethylbutane-1,4-diyl, 1,1,2-trimethylbutane-1,4-diyl, 1,1,3-trimethylbutane-1,4-diyl, 5-methylheptane-1,7-diyl, 1-methylheptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, and decane-1,10-diyl; １ -C ２０ 7. The copolymer of claim 1, wherein the substituent is alkylene.

8. Z １ and Z ２ Both are CR ａ R ｂ where R ａ and R ｂ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

9. 9. The copolymer of any one of claims 1 to 8, wherein X comprises a protein, peptide, or carbohydrate selected from the group consisting of a peptide sequence, a laminin-derived peptide, an integrin-binding peptide, a cell-penetrating peptide, a collagen mimetic, a collagen fragment, heparan sulfate, a glycosaminoglycan (GAG), and derivatives thereof.

10. X is RGD, SRGDS, RGDS, A5G81 (AGQWHRVSVRWGC), SVVYGLR, (IRIK) ２ , (IKKI) ３ , heparin oligosaccharides DP8, DP10, DP12, DP14, DP16, DGEA, (PHypG) ｎ Type sequence, (PGHyp) ｎ Type sequence, (HypGP) ｎ Type sequence, (HypPG) ｎ Type sequence, (GHypP) ｎ Type sequence, (GPHyp) ｎ 10. The copolymer of claim 1, wherein the copolymer is selected from the group consisting of a hydroxybenzoate sequence and hyaluronic acid.

11. 9. The copolymer of claim 1, wherein X comprises an antibiotic, antimicrobial, antibacterial, blood thinner, or anti-inflammatory agent.

12. 12. The copolymer of claim 11, wherein X comprises an antibiotic, antimicrobial, antibacterial, blood thinner, or anti-inflammatory agent selected from the group consisting of penicillin, amoxicillin, amphotericin, ciprofloxacin (CIF), atorvastatin, aspirin, streptomycin, ribostamycin, and gentamicin.

13. Y １ is represented by the general formula (III): 【Chemistry 2】 (In the formula, A is selected from a single bond, oxy, carbonyl, oxycarbonyl, carbonyloxy, optionally substituted alkyleneoxy, optionally substituted alkyleneoxyalkylene, optionally substituted alkylenecarbonyl, optionally substituted alkylenecarbonylalkylene, optionally substituted carbonyloxyalkylene, optionally substituted oxycarbonylalkylene, optionally substituted alkylenecarbonyloxyalkylene, or optionally substituted alkyleneoxycarbonylalkylene; B is optionally present as a ring selected from 1,2,3-triazole or succinimide; R ５ is selected from a single bond, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted alkyleneoxyalkylene, optionally substituted alkylenecarbonyl, or optionally substituted alkylenecarbonylalkylene; Y ２ is selected from the group consisting of polypropylene (PP), polyester, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylate, poly(meth)acrylate, and polyamide (PA); and 13. The copolymer of any one of claims 1 to 12, represented by the formula: T is an end group selected from the group consisting of hydrogen, halogen, hydroxyl, amino, acyl, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, or optionally substituted alkoxycarbonylalkyl.

14. Y １ has the following general formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IIIf): 【Transformation 3】 wherein n≧1; and m≧1. and the total molecular weight of the repeating units represented by general formula (II) is 15,000 or less.

15. 15. A method for preparing the bioactive synthetic copolymer of any one of claims 1 to 14, comprising: One or more bioactive polymers represented by general formula (IV) are polymerized with one or more synthetic polymers represented by general formula (V) in the presence of a catalyst to produce a bioactive synthetic copolymer: 【Chemistry 4】 (In the formula, R １ is an optionally substituted alkylene; R ２ is selected from a single bond, optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted alkyleneoxyalkylene, optionally substituted alkylenecarbonyl, or optionally substituted alkylenecarbonylalkylene; R ３ is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is a polyalkylene glycol; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; Y １ comprises a synthetic polymer; and Z １ and Z ２ are each independently CR ａ R ｂ , O., N.R. ｃ , SiR ａ R ｂ , P.R. ａ or S, where R ａ , R ｂ and R ｃ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

16. 16. The method of claim 15, wherein the catalyst comprises a ruthenium complex.

17. 17. The method of any one of claims 15 to 16, wherein the method comprises ring-opening metathesis polymerization (ROMP).

18. For preparing the copolymer of any one of claims 1 to 14, a bioactive macromolecule represented by general formula (IV): 【Transformation 5】 (In the formula, R １ is an optionally substituted alkylene; R ３ is selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; L is a polyalkylene glycol; X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof; and Z １ is CR ａ R ｂ , O., N.R. ｃ , SiR ａ R ｂ , P.R. ａ or S, where R ａ , R ｂ and R ｃ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl.

19. 20. A method for preparing the bioactive polymer of claim 18, comprising: (i) General formula (VI): 【Transformation 6】 (In the formula, Z １ is CR ａ R ｂ , O., N.R. ｃ , SiR ａ R ｂ , P.R. ａ or S, where R ａ , R ｂ and R ｃ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; (ii) reacting the dicarboxylic acid anhydride having the general formula (VI) with a diamine R ４ R ３ N-L-R １ -NH ２ to react with a compound of the general formula (VII): 【Transformation 7】 (In the formula, R １ is an optionally substituted alkylene; R ３ and R ４ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, where R ３ and R ４ at least one of is H; L is a polyalkylene glycol; and Z １ is CR ａ R ｂ , O., N.R. ｃ , SiR ａ R ｂ , P.R. ａ or S, where R ａ , R ｂ and R ｃ are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; (iii) reacting said amine having general formula (VII) with an acid-containing biologically active moiety X—C(═O)OH to obtain a biologically active macromolecule, wherein X comprises a biologically active moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof. A method comprising:

20. 20. The method of claim 19, wherein the method further comprises purifying the amine having the general formula (VII) to remove impurities prior to the step of reacting the amine having the general formula (VII) with X-C(=O)OH.

21. 21. The method of claim 20, wherein the purification step comprises a double neutralization step.

22. 22. The method of claim 21, wherein the dual neutralization step comprises a first step of washing with an acid and a second step of washing with a base.

23. 15. A material comprising a copolymer according to any one of claims 1 to 14 for use in medicine.

24. 24. The material of claim 23, wherein the material is part of an apparatus selected from the group consisting of a wound dressing, a skin scaffold, a bone scaffold, an organoid scaffold, an implant, and a medical device.

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