BIOACTIVE POLYETHYLENE COPOLYMER, POLYETHYLENE MACROMOLECULE AND RELATED METHODS THEREOF

Abstract

There is provided a bioactive polyethylene copolymer with 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). Also provided are a polyethylene macromolecule, a material comprising said bioactive polyethylene copolymer, a method of preparing said bioactive polyethylene copolymer and a method of preparing said polyethylene macromolecule.

Claims

1. A bioactive polyethylene copolymer with 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): ##STR00057## wherein R.sup.1 is optionally substituted alkyl; R.sup.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.sup.3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; L is heteroalkylene; X comprises a bioactive moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic/drug molecules and derivatives thereof, Y comprises polyethylene or parts thereof, and Z.sup.1 and Z.sup.2 are each independently selected from CR.sup.aR.sup.b, O, NR.sup.c, SiR.sup.aR.sup.b, PR.sup.a or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

2. The copolymer of claim 1, wherein Y is represented by general formula (III): ##STR00058## wherein A is optionally present as NR.sup.c, wherein R.sup.c is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; B is optionally present as a 5-membered or 6-membered heterocyclic ring having at least one N heteroatom in the ring; R.sup.5 is selected from an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl; T is a terminal group selected from the group consisting of hydrogen and methyl; and n is from 10 to 350.

3. The copolymer of claim 2, wherein n is from 20 to 250.

4. The copolymer of claim 2, wherein B is present and represented by the following structure: ##STR00059## wherein R.sup.6a, R.sup.6b, R.sup.6c and R.sup.6d are each independently selected from the group consisting of C, CR.sup.a, CR.sup.aR.sup.b, N, NR.sup.c, O or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl; and R.sup.7a, R.sup.7b and R.sup.7c are optionally present as O, S, F, Cl, Br, I, CR.sup.aR.sup.b, CR.sup.aR.sup.bR.sup.c, OH, SH, NH.sub.2 or NR.sup.c.

5. The copolymer of claim 1, wherein Y is selected from the following general formulae (IIIa), (IIIb) or (IIIc): ##STR00060## wherein R.sup.5 is selected from the group consisting of C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxyalkyl, C.sub.2-C.sub.20 alkylcarbonyl and C.sub.3-C.sub.20 alkylcarbonylalkyl; R.sup.6a and R.sup.6d are each independently selected from the group consisting of C, CR.sup.a, CR.sup.aR.sup.b, N, NR.sup.c, O or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl; R.sup.7a is optionally present as O, S, F, Cl, Br, I, CR.sup.aR.sup.b, CR.sup.aR.sup.bR.sup.c, OH, SH, NH.sub.2 or NR.sup.c; T is a terminal group selected from the group consisting of hydrogen and methyl; and n is from 10 to 350.

6. The copolymer of claim 1, wherein Y is selected from the following general formulae (IIId), (IIIe) or (IIIf): ##STR00061## wherein R.sup.5 is selected from the group consisting of C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxyalkyl, C.sub.2-C.sub.20 alkylcarbonyl and C.sub.3-C.sub.20 alkylcarbonylalkyl; T is a terminal group selected from the group consisting of hydrogen and methyl; and n is from 10 to 350.

7. The copolymer of claim 1, wherein the repeating unit represented by general formula (I) is in an amount of from 1 to 100 molar % relative to the copolymer.

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

9. The copolymer of claim 1, wherein L is heteroalkylene having from 20 carbon atoms to 300 carbon atoms.

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

11. (canceled)

12. The copolymer of claim 1, wherein R.sup.1 is C.sub.1-C.sub.4 alkyl and R.sup.2 is selected from C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxyalkyl, C.sub.2-C.sub.20 alkylcarbonyl or C.sub.3-C.sub.20 alkylcarbonylalkyl, optionally wherein R.sup.1 is straight or branched C.sub.1-C.sub.4 alkyl substituents independently selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or t-butyl, and R.sup.2 is straight or branched C.sub.1-C.sub.20 alkyl substituents independently selected from 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 or decyl.

13. (canceled)

14. The copolymer of claim 1, wherein Z.sup.1 and Z.sup.2 are both CR.sup.aR.sup.b wherein R.sup.a and R.sup.b are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

15. The copolymer of claim 1, wherein X comprises protein, peptide or carbohydrate selected from the group consisting of peptide sequence, laminin-derived peptide, integrin binding peptide, cell-penetrating peptide, collagen sequence, collagen mimics, collagen fragment, heparin sulfate, glycosaminoglycans (GAGs) and derivatives thereof.

16. The copolymer of claim 1, wherein X is selected from the group consisting of RGD, SRGDS (SEQ ID NO: 1), RGDS (SEQ ID NO: 2), A5G81 (AGQWHRVSVRWGC) (SEQ ID NO: 3), SVVYGLR (SEQ ID NO: 4), (IRIK).sub.2 (SEQ ID NO: 6), (IKKI).sub.3 (SEQ ID NO: 7), DGEA (SEQ ID NO: 5), (PHypG).sub.ntype sequence, (PGHyp).sub.n type sequence, (HypGP).sub.n type sequence, (HypPG).sub.n type sequence, (GHypP).sub.n type sequence, (GPHyp).sub.n type sequence, heparin oligosaccharide DP8, DP10, DP12, DP14, DP16 and hyaluronic acid.

17. A method of preparing a bioactive polyethylene copolymer of claim 1, the method comprising: polymerising one or more bioactive macromolecules represented by general formula (IV) with one or more polyethylene macromolecules represented by general formula (V) in the presence of a catalyst to obtain the bioactive polyethylene copolymer: ##STR00062## wherein R.sup.1 is optionally substituted alkyl; R.sup.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.sup.3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; L is heteroalkylene; X comprises a bioactive moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic/drug molecules and derivatives thereof, Y comprises polyethylene or parts thereof, and Z.sup.1 and Z.sup.2 are each independently selected from CR.sup.aR.sup.b, O, NR.sup.c, SiR.sup.aR.sup.b, PR.sup.a or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

18. The method according to claim 17, wherein the catalyst comprises a ruthenium complex.

19. The method according to claim 17, wherein the method comprises ring opening metathesis polymerisation (ROMP).

20. (canceled)

21. The method according to claim 17, wherein the method further comprises, prior to polymerising, preparing a polyethylene macromolecule represented by general formula (VIII) by: ##STR00063## (i) providing a dicarboxylic anhydride having general formula (IX): ##STR00064## wherein Z.sup.2 is selected from CR.sup.aR.sup.b, O, NR.sup.c, SiR.sup.aR.sup.b, PR.sup.a or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl; and (ii) reacting said dicarboxylic anhydride having general formula (IX) with an amine to obtain the polyethylene macromolecule, the amine is represented by general formula (X): ##STR00065## wherein R.sup.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; A is optionally present as NR.sup.c, wherein R.sup.c is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; B is optionally present as a 5-membered or 6-membered heterocyclic ring having at least one N heteroatom in the ring; R.sup.5 is selected from the group consisting of C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxyalkyl, C.sub.2-C.sub.20 alkylcarbonyl and C.sub.3-C.sub.20 alkylcarbonylalkyl; T is a terminal group selected from the group consisting of hydrogen and methyl; n is from 10 to 350, optionally wherein the method further comprises, prior to (ii), (a-i) providing a polyethylene having general formula (XIa) or (XIb): ##STR00066## wherein R.sup.6a and R.sup.6d are each independently selected from the group consisting of C, CR.sup.a, CR.sup.aR.sup.b, N, NR.sup.c, O or S, wherein R.sup.a, R.sup.b, and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl; R.sup.7a is optionally present as O, S, F, Cl, Br, I, CR.sup.aR.sup.b, CR.sup.aR.sup.bR.sup.c, OH, SH, NH.sub.2 or NR.sup.c; R.sup.8 and R.sup.9 are each independently selected from the group consisting of C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynl, C.sub.1-C.sub.20 alkoxy, C.sub.1-C.sub.20 alkoxyalkyl, C.sub.2-C.sub.20 alkylcarbonyl and C.sub.3-C.sub.20 alkylcarbonylalkyl; T is a terminal group selected from the group consisting of hydrogen and methyl; n is from 10 to 350; and (b-i) reacting said polyethylene having general formula (XIa) or (XIb) with a diamine H.sub.2NR.sup.2NH.sub.2 or ammonia NH.sub.3 to obtain the amine having general formula (X), optionally wherein at least one of (ii) and (b-i) is performed in the presence of an organic solvent and/or a base, and optionally wherein the organic solvent comprises an aromatic solvent; and the base comprises a tertiary amine.

22.-24. (canceled)

25. A material comprising a copolymer of claim 1 for use in medicine.

26. The material according to claim 25, wherein the material is part of an apparatus selected from the group consisting of consumer care products, wound dressing, skin scaffold, bone and bone marrow organoid scaffold, cartilage implant, joint implant and medical device.

Description

BRIEF DESCRIPTION OF FIGURES

[0325] FIG. 1 is a schematic diagram 100 of a bioactive polyethylene copolymer in accordance with various embodiments disclosed herein.

[0326] FIG. 2 shows the thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) graphs of pure RGD peptide (RGD(PURE)), NBPEG.sub.1000RGD macromonomer (NB PEG1000RGD), NBPE homopolymer (PE) and succinic acid-terminated polyethylene (SAt-PE)-PEGRGD copolymer ((RGD)ROMP).

[0327] FIG. 3A is a graph showing the biocompatibility of PE-peptide based materials prepared in accordance with various embodiments disclosed herein, relative to a control. The results were obtained from cell viability tests on human keratinocytes cultured on a human de-epidermised dermis model. 3D printed sheets of bioactive PE blended with medical grade polypropylene were applied to the skin models for 24 and 48 h, where macromonomers of 6 peptides (SRGDS, RGDS, (IRIK).sub.2, (GPHyp).sub.3, DGEA and RGD) have been copolymerized with macromonomers of PE. Comparative examples are commercial dressings Allevyn (i.e. polyurethane foam) and Acticoat (i.e. silver-based polypropylene non-woven dressing). Control used include poly(norbornene dicarboximide) with PE and mPEG.sub.1000 as side chains.

[0328] FIG. 3B is a graph showing the biocompatibility of PE-peptide based materials prepared in accordance with various embodiments disclosed herein, relative to a control. The results were obtained from cell viability tests on a human de-epidermised dermis model, as ex vivo human skin models where macromonomers of 6 peptides (SRGDS, RGDS, (IRIK).sub.2, (GPHyp).sub.3, DGEA and RGD) have been copolymerized with macromonomers of PE, and the bioactive copolymers blended with polypropylene, extruded into filaments and 3D printed into sheets, then applied to the human skin models. Comparative examples are commercial dressings Allevyn (i.e. polyurethane foam) and Acticoat (i.e. silver-based polypropylene non-woven dressing). Control used include poly(norbornene dicarboximide) with PE and mPEG.sub.1000 as side chains.

[0329] FIG. 4 shows cross sectional hematoxylin and eosin (H&E) staining images of human skin samples obtained from preliminary ex vivo wound closure tests after 3 days of material application. Comparative example is commercial dressing Allevyn (i.e. polyurethane-based dressing). Control used include poly(norbornene dicarboximide) with PE and mPEGooo as side chains.

[0330] FIG. 5 shows cross sectional hematoxylin and eosin (H&E) staining images of human skin samples obtained from preliminary ex vivo wound closure tests after 3 days of application of PE-peptide based materials. The PE-peptide based materials are macromonomers of PE copolymerized with 6% of RGD macromonomer, and the final bioactive polyethylene copolymer blended with medical grade polypropylene at 0.1%, 1% and 3% blending ratios.

[0331] FIG. 6 shows the thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) graphs of a bioactive amine-terminated polyethylene (PE)-RGD copolymer.

[0332] FIG. 7 is a graph showing the biocompatibility of PE-peptide based materials prepared in accordance with various embodiments disclosed herein, relative to commercial wound dressings Acticoat and Allevyn. The results were obtained from Hs27 human skin fibroblasts viability tests on PERGD copolymer blended with PLA at 1:4 ratio. PERGD copolymer blended with medical grade PLA was electrospun into nanofibers for biocompatibility tests with Hs27 human fibroblasts grown in DMEM w/10% FBS and 1% Pen/Strep.

EXAMPLES

[0333] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, and chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

Example 1: Modular Approach to Constructing Bioactive Polyethylene Copolymer

[0334] A general strategy for construction of bioactive macromonomers containing either peptides, carbohydrates or drug molecules has been developed. The simple 2-step synthesis allows for rapid buildup of a wide library of bioactive macromonomers of various chain length, allowing for quick development of synthetic polymers (i.e polyethylene) with desired bioactivities as required in targeted application. A modular approach to construct the desired polymers to suit various applications by matching the bioactive macromonomer with macromonomers of synthetic polymers with desired physical properties (i.e. polyethylene), is made possible using this library. Rapid polymer customization can thus be achieved.

[0335] A modular building block system to designing/constructing desired bioactive materials has been developed as shown in Scheme 1. Once the targeted medical application has been identified, a plug and play approach (Scheme 1) can be used to create the desired bioactive polyethylene material which not only possess therapeutic effects but also, necessary mechanical properties for easy storage and handling.

[0336] By using the modular approach, a macromonomer consisting of a bioactive molecule at the terminal of the monomer is created and can be copolymerized with other synthetic polymers (i.e. polyethylene) to create bioactive polyethylene copolymers with targeted bioactivities. The desired polymer is highly customizable using the strategy developed in accordance with various embodiments disclosed herein, by switching the bioactive molecule for any peptide or carbohydrate that bears a carboxylic acid group. This allows for 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, antimicrobial activity, cartilage tissue regeneration, wound healing, collagen production, anti-inflammatory to cholesterol synthesis inhibition (for e.g., using atorvastatin as drug) and can be made to be mechanically tough, depending on the needs. The modular synthesis therefore makes application matching to polymer properties much simpler and effective.

Example 2: Method of Preparing Bioactive Polyethylene Copolymer

[0337] The method of preparing a bioactive polyethylene copolymer in accordance with various embodiments disclosed herein involve creating macromonomers of the bioactive molecules and polyethylene separately and using ring opening metathesis polymerization (ROMP) techniques to link these otherwise mutually incompatible molecules together. The result is a brush polymer bearing both the bioactive molecule and the polyethylene for overall mechanical strength of the material (Scheme 2). By creating the macromonomers separately, the inventors are able to build a library of macromonomers with different properties for clinicians or medtech companies to choose from, and the material with desired therapeutic effects can be constructed easily and rapidly, to suit the targeted application. By creating the macromonomers separately, the inventors are also able to build a library of macromonomers and the eventual copolymers, for rapid testing of efficacy in the biomedical laboratory. Different combinations of these macromonomers (MMs) can also generate a library of well-defined brush copolymers containing different bioactive molecules for rapid screening of bioactivity in laboratory.

[0338] In the following examples, brush polymers containing pendant arms of polyethylene and bioactive molecules tethered on polyethylene glycol (PEG) moieties, have been created. Bioactive molecules may include biomolecules selected from peptide sequences of any combination of the 20 natural amino acids, from 3-20 amino acid residues, carbohydrates such as glycosaminoglycans or drug molecules containing a carboxylic acid terminal such as certain antibiotics. Biomolecules may also include collagen mimic peptides from 3-20 amino acid residues in any sequence, such as DGEA, (Gly-Pro-Hyp).sub.3 and (Pro-Hyp-Gly).sub.3. Depending on the application, the biomolecule with the bioactivity of interest can be chosen, and copolymerized together with polyethylene using the brush polymer technology as disclosed herein by ring opening metathesis polymerization.

[0339] The resultant polymer shows bioactivity of the biomolecule involved while having much better physical and mechanical properties for good material handling and processability.

[0340] The bioactive polyethylene copolymers can be subsequently blended with polymers similar to that on the pendant arms (e.g., polyethylene or polypropylene) to create bioactive materials for use in biomedical devices such as catheters, wound dressings, tissue scaffolds, plastic surgery implants, prosthetic parts, cartilage joint implants etc.

[0341] The synthetic route for preparing a bioactive polyethylene copolymer in accordance with various embodiments disclosed herein is illustrated in Scheme 2.

##STR00044##

Example 3: Bioactive Macromonomers and Method of Synthesis

[0342] A general strategy for the synthesis of bioactive macromonomers in accordance with various embodiments disclosed herein has been developed. Polyethylene glycol diamine of various chain lengths (e.g., M.sub.W=1,000-6,000) are reacted with cis-norbornene-exo-2,3-dicarboxylic anhydride to create the main macromonomer body, i.e. macromonomer body containing norbornene dicarboximide and polyethylene glycol (NBPEG) (Scheme 3.1). Once NBPEG is created, various peptides, carbohydrates or drug molecules are then reacted with these NBPEG chains to create the bioactive macromonomer with desired therapeutic properties.

[0343] With the main body of macromonomer, any peptides, carbohydrates or drug molecules (R) can be used using the carboxylic acid terminus on the bioactive molecule by means of condensation reaction to form peptide/amide bonds between the amine group on NBPEG-NH.sub.2 and carboxylic acid terminal of the carbohydrate or peptide (Scheme 3.2). Examples of drug molecules include antibiotics such as amoxicillin or ciprofloxacin. In various embodiments, R is copolymers of antimicrobial peptides (IRIK).sub.2 or (IKKI).sub.3; heparin oligosaccharides DP10, DP12, DP14, extracellular matrix peptides COL or RGD, where COL may be DGEA, (GPHyp).sub.n or (PHypG).sub.n, or (PGHyp).sub.n. In various embodiments, R is carbohydrates or drug molecules with a CO.sub.2H group or peptide sequences of 3-20 amino acid residues, formed from 20 natural amino acids. In various embodiments, R is DP12, DP14, COL or RGD, where COL may be DGEA or (GPHyp).sub.n or any combinations of P, Hyp and G. Should the carbohydrate not possess a carboxylic acid group, modification of the carbohydrate to include one may be necessary. Alternatively, amine substitution reactions or reductive amination reactions can also be done on the carbohydrate's hydroxy or carbonyl groups.

##STR00045##

##STR00046##

Example 4: Polyethylene Macromonomers and Method of Synthesis

[0344] Polyethylene (PE) macromonomers may be created with a succinic acid terminus or a primary amine terminus.

[0345] To create PE macromonomers with a succinic acid terminus, vinyl-terminated polyethylene of low molecular weight and polydispersity is reacted with maleic anhydride to obtain a succinic acid-terminated polyethylene (SA-t-PE). This PE can then be reacted with hexamethylenediamine (HMDA) and finally, cis-norbornene-exo-2,3-dicarboxylic anhydride, to create the desired PE macromonomer (Scheme 4.1).

[0346] To obtain a primary amine terminus on polyethylene (PE) for macromonomer formation, hydroaminomethylation of vinyl terminated PE is carried out in a one pot, two-step process where it is first converted into a linear carbonyl via hydroformylation, followed by reductive amination in the presence of hexamethylenediamine (HMDA) (Scheme 4.2). Upon obtainment of the amine terminated PE, condensation with cis-norbornene-exo-2,3-dicarboxylic anhydride, affords the PE macromonomer (Scheme 4.3), which can be used in brush polymer formation with either itself or other macromonomers.

##STR00047##

##STR00048##

##STR00049##

Example 5: Ring Opening Metathesis Polymerisation Catalysts

[0347] With both the bioactive macromonomer and synthetic polymer (i.e. PE) macromonomer, the final bioactive polyethylene copolymer is prepared by ROMP using Grubbs type catalysts 1 (Scheme 5).

##STR00050##

Example 6: Bioactive Polyethylene Copolymers Examples

[0348] FIG. 1 shows a bioactive polyethylene copolymer 100 designed in accordance with various embodiments disclosed herein. The bioactive polyethylene copolymer 100 comprises a poly(norbornene dicarboximide) backbone 102, pendant arms of polyethylene 104a, 104b and 104c, and pendant arms of bioactive molecules 106a, 106b and 106c tethered on 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 may be the same or different types of bioactive moieties.

[0349] Examples of bioactive molecules include biomolecules selected from peptide sequences of 3-20 amino acid residues, formed from 20 natural amino acids, collagen mimic peptides from 3-20 amino acid residues in any sequence such as DGEA, (Gly-Pro-Hyp).sub.3 and (Pro-Hyp-Gly).sub.3, (Hyp-Pro-Gly).sub.3, (Gly-Hyp-Pro).sub.3, (Hyp-Gly-Pro).sub.3 and (Pro-Gly-Hyp).sub.3, carbohydrates such as glycosaminoglycans or drug molecules containing a carboxylic acid terminal such as certain antibiotics.

[0350] Collagen is a popular material in skin care and wound care industry due to their skin compatibility and reported ability to regenerate skin tissues. Yet, they are mild enough to prevent overactive tissue regeneration, potentially leading to cancer. The hydrophilicity of collagen itself makes it an attractive material for skincare products as the ability to preserve hydration in skin prevents dermatitis resulting from overly dry skin. Hence, the use of collagen fragments and collagen mimics may be used in PE to create polymers for blending into polypropylene base materials, for diaper manufacturing.

[0351] Naturally occurring biopolymers such as Hyaluronic Acid (HA) and Collagen (COL) have been shown to support cell growth and proliferation. Several studies have shown that HA improves the healing rate of diabetic foot ulcers (DFU) significantly. HA has been reported to enhance cartilage regeneration. Collagen gels or freeze dried collagen pads are available commercially for wound treatments. Integra, a collagen scaffold made of bovine collagen, is currently the gold standard for burns treatment. The ability of collagen to regenerate skin tissues and being a highly biocompatible material, makes it a very attractive material for wound healing materials.

[0352] Besides HA and COL, ArginylGlycylAspartic acid (RGD) peptides are yet another interesting peptide that has been reported to encourage both bone, cartilage and skin cell proliferation. The RGD sequence is the minimal binding domain for fibronectin, a high molecular weight glycoprotein of the extracellular matrix (ECM) that binds to ECM components such as collagen and fibrin. RGD peptide sequences are known to regulate cellular activity by interacting with cell-surface integrins which contribute to wound healing processes. Materials modified with RGD peptides have been reported to facilitate cell adhesion, spreading and wound healing. RGD also allows integrin binding for Transformational Growth Factor (TGF-) activation which is required for regulation of cartilage development. Hence, RGD is an important peptide to consider in material development for wound healing and cartilage repair.

[0353] An example would be the development of a wound dressing material for treatment of chronic wounds where rapid skin reepithelization is required. RGD is a peptide sequence that is capable of binding integrins for cell attachment, migration and proliferation. Hence, macromonomer of RGD is created (Scheme 6a). As dressings are required to be inert with long shelf life and good mechanical strength, a polyethylene-bearing macromonomer is paired with this RGD-bearing macromonomer to create the eventual copolymer of RGD and PE (Scheme 6b). The material has been tested to be biocompatible and enhances reepithelization using human skin equivalent models.

##STR00051##

[0354] As RGD is a good integrin binder and bone growth factor binder, it can also be paired with PE macromonomer to create bioactive copolymers for use as substrate or scaffold material for bone and bone marrow organoid creation. Such a modular approach in building polymers allow the matching of different bioactive macromonomers with synthetic macromonomers to rapidly create mechanically strong therapeutic materials based on the targeted application.

[0355] RGD can be replaced with any peptide sequence via its acid terminal or any carbohydrate or any drug molecule such as amoxicillin or ciprofloxacin that has a carboxylic acid functional group.

[0356] For example, glycosaminoglycans (GAGs) such as heparin sulfate (HS) chains of between 5 to 10 disaccharide units may be used as the bioactive moiety. Without being bound by theory, it is believed that HS chains is active toward bone morphogenetic proteins (BMP), in particular, BMP-2, which is able to transdifferentiate myoblasts to osteoblasts. Without being bound by theory, it is believed that DP12, the HS fragment with hexa-disaccharide units, possesses the highest binding affinity for BMP-2.

[0357] Bioactive materials may also be created with collagen fragments and mimics. The bone is a mineralized collagenous tissue that remodels itself throughout one's lifecycle to adapt to mechanical stress and maintain the integrity of skeletal tissues. Current bone scaffolds are typically made of collagen sponges, occasionally mineralized with some calcium phosphate ceramics such as tricalcium phosphate or hydroxyapatite. The biocompatibility of collagen and its similarity to bone and cartilage tissues makes it an ideal scaffold material for bones and cartilage. Some possible collagen mimics that may be used include DGEA and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences. Without being bound by theory, it is believed that DGEA supports mesenchymal stem cell adhesion and differentiation to osteoblasts. Furthermore, without being bound by theory, it is believed that collagen also makes excellent skin scaffold materials since the extracellular matrix (ECM) is largely collagenous material. Other ECM peptides studied include laminin-derived peptide A5G81, which has been reported to facilitate wound healing in rats.

[0358] Besides tissue-regenerating biomolecules, cell-penetrating peptides such as (IRIK).sub.2 and (IKKI).sub.3 may also be used as the bioactive moiety, for incorporation into non-biofouling materials. Biofouling is a serious problem in biomedical devices such as catheters, gut stents and even wound dressings. The ability to target biofilm-forming bacteria such as P. Aeruginosa whilst not being toxic to human, makes such peptides attractive candidates for biomedical device materials.

[0359] Drug molecules such as antibiotics may also be incorporated into the brush polymers. Theoretically, any drug molecule with a carboxylic acid terminal would allow the creation of these bioactive synthetic polymers. Some of the drug molecules successfully polymerized include amoxicillin and ciprofloxacin. Brush polymers have been created, also for use in antimicrobial devices.

[0360] In summary, a general strategy has been developed to create bioactive macromonomers for rapid building of bioactive polyethylene copolymers by using a modular approach to polymer design and synthesis. Such a modular approach to therapeutic material synthesis allows for therapy customization to suit each patient's needs, hence moving closer to the ideal situation of personal medicine.

[0361] In summary, a series of polymers that bear polyethylene with pegylated biomolecules as side chains on a poly(norbornene dicarboximide) backbone have been developed, via ROMP technologies. The biocompatibility and skin cell viability of some of these polymers were also tested to demonstrate the materials' ability to withstand harsh material processing temperatures without loss in bioactivity. The general strategy presented here forms a method to create bioactive polyethylene copolymers for use as bioadditives in materials for biomedical devices where the bioadditive can be blended with a base polymer of similar type to the polymer side chain on the poly(norbornene dicarboximide) backbone. The polyethylene side chain helps make the biomolecule more compatible with the base synthetic polymer, allowing them to be blended together without phase separation. The formation of the brush polymer also allows the biomolecule to have better structural integrity as compared to the native biomolecule itself, which tends to be extremely hygroscopic, resulting in their poor handling and low processability, as a material.

Example 7: Succinic Acid-Terminated Polyethylene (SA-t-PE)-Peptide Copolymers

7.1. (SA-t-PE).SUB.p.-[(GPHyp).SUB.3.].SUB.q .Copolymer

[0362] This example shows the development of a new strategy to incorporate collagen fragments or mimics into polyethylene by means of brush polymer synthesis, for use in consumer products such as diapers. The bioactive polyethylene created can be blended into medical grade polypropylene (PP) for non-woven fiber production which typically occurs at 220 C. by melt extrusion. Other peptides such as arginyl-glycyl-aspartic acid (RGD), known to enhance skin biocompatibility, can also be made using similar strategies.

[0363] Succinic acid-terminated polyethylene (SA-t-PE) was obtained according to the literature method (Macromolecules 2009, 42, 4356-4358; following the second example in Supporting Information).

7.1.1. PE Macromonomer

[0364] An amine handle was first created on SA-t-PE using hexamethylenediamine (HMDA), followed by connection of the aminated PE to a norbornene dicarboxylic anhydride linker (cis-norbornene-exo-2,3-dicarboxylic anhydride) to create the PE macromonomer (Scheme 7.1).

7.1.2. Peptide Macromonomer

[0365] Peptide macromonomer was prepared by reacting PEG diamine (MW 1,000-6,000) with cis-norbornene-exo-2,3-dicarboxylic anhydride on one amine end, followed by a second condensation reaction with a suitable peptide on the other amine end of PEG diamine (Scheme 7.2).

[0366] Macromonomers of collagen fragments such as (GPHyp).sub.3, (PGHyp).sub.3, collagen mimics such as DGEA and extracellular matrix peptides such as RGD, SRGDS, have also been prepared using this general strategy.

7.1.3. PE-Peptide Copolymer

[0367] Once both macromonomers have been synthesized, ring opening metathesis polymerization are carried out on the macromonomers using Grubbs' catalyst (Scheme 5, catalyst 1) to obtain the desired PE-peptide copolymer (Scheme 7.3).

[0368] The copolymer synthesized showed an average of 6% peptide incorporation in the polymer, despite a PE:peptide monomer ratio of 5:1. This is likely due to poor solubility of peptide macromonomer in benzene. Other non-polar solvents such as toluene are worst, with no more than 2% RGD incorporation. Polar solvents in general do not dissolve PE.

[0369] Upon synthesis, the polymers are checked for residual metals from Grubbs' catalyst using inductively coupled plasma mass spectrometry (ICP-MS) to ascertain that the metal content falls under 0.1 ppm. The FDA permissible inhalation limit for Ru in a class 2B medical device is 0.1 g/g. For a 5 kg baby, this translates to 0.5 mg of Ru. Assuming each disposable diaper uses 10 g of non-woven PE-COL sheet as its cover and the blending ratio of PE-COL in PP is 3%, there is 0.3 g of PE-COL in the material. At 0.1 ppm Ru, the amount of Ru detected in 0.3 g of material is 0.03 g. This is way below FDA limits for a class 2B device. For an application such as diaper, such a Ru limit is insignificant.

##STR00052##

##STR00053##

##STR00054##

7.2. SA-t-PERGD Copolymer

[0370] The example shows the development of brush polymers with pegylated biomolecules such as RGD, HA and collagen fragments with polyethylene side chains, on a poly(norbornene dicarboximide) backbone, for applications in wound healing and cartilage repair.

[0371] Succinic acid-terminated polyethylene (SA-t-PE) was prepared by the literature method (Macromolecules 2009, 42, 4356-4358; following the second example in Supporting Information). The PE used to make SA-t-PE is in the MW range of 1,400-5,000 just right for use in grafting onto linker units such as norbornene, to create macromonomers that would be further polymerized into brush polymers comprising such PE side chains.

7.2.1. PE Macromonomer

[0372] An amine handle was first created on SA-t-PE using hexamethylenediamine (HMDA), followed by connection of the aminated PE to a norbornene dicarboxylic anhydride linker (cis-norbornene-exo-2,3-dicarboxylic anhydride) to create the PE macromonomer (Scheme 7.1).

7.2.2. Peptide Macromonomer

[0373] Peptide macromonomer was prepared by reacting PEG diamine (MW 1,000-6,000 depending on MW range of PE) with cis-norbornene-exo-2,3-dicarboxylic anhydride on one amine end, followed by a second condensation reaction with a suitable peptide on the other amine end of PEG diamine (Scheme 7.2).

7.2.3. PE-Peptide Copolymer

[0374] Once both macromonomers have been synthesized, ring opening metathesis polymerization are carried out on the macromonomers using Grubbs' catalyst (Scheme 5, catalyst 1) to obtain the desired PE-RGD copolymer (SA-t-PERGD) (Scheme 7.4).

[0375] The copolymer synthesized showed an average of 6% RGD incorporation in the polymer, despite a PE:RGD monomer ratio of 5:1. This is likely due to poor solubility of RGDPEGNB macromonomer in benzene. Other non-polar solvents such as toluene are worst, with no more than 2% RGD incorporation. Polar solvents in general do not dissolve PE.

[0376] Upon synthesis, the polymers are checked for residual metals from Grubbs' catalyst using ICPMS to ascertain that the metal content falls under 0.1 ppm. Assuming each cartilage implant (osteochondral plug) uses 1 g of PE to make, at 10% bioactive PE blending ratio, there is 0.1 g of bioactive PE in the implant material. At 0.1 ppm Ru, the amount of Ru detected in 0.1 g of bioactive RGD is 0.01 g. This is way below FDA daily oral exposure limits of 100 g/day or 1 g/day by inhalation.

##STR00055##

7.3. Thermal Stability

[0377] After ICP-MS, thermogravimetric analysis and differential scanning calorimetry (TGA-DSC) measurements were obtained on pure RGD, NBPEGRGD, NBPE homopolymer and PE-co-PEGRGD copolymer (PE-RGD) to ascertain thermal stability of the polymers. From the DSC curve, it is shown that the material undergoes a single-phase degradation/weight loss at >450 C. (467 C.), a temperature much higher than the typical melt processing temperature of PE or PP for either 3D printing or non-woven fiber production (FIG. 2). Hence, enhanced thermal stability of the material was demonstrated despite the incorporation of a biomolecule.

[0378] It was observed that pure RGD degrades at a temperature (150-250 C.) lower than its macromonomer NBPEG.sub.1000RGD (250-400 C.), and even much lower than its PE-RGD copolymer (400-500 C.). NBPEGRGD showed a two phase degradation (weight loss) where the first degradation was due to loss of RGD group, followed by decomposition of the PEG chain, from around 250 C. The PE-RGD copolymer only suffers from 50% weight loss at 466 C., allowing the use of most material processing methods such as melt extrusion or fused filament fabrication (FFF/FDM) to process the polymers into usable devices. The significant enhancement in thermal stability of RGD upon attachment to PEG followed by copolymerization with PE via ROMP on a norbornene dicarboximide backbone, is therefore evident here.

7.4. Biocompatibility

[0379] The bioactive PE was blended with medical grade polypropylene powder in varying ratios from 0.1-10% to create a formulation. The formulation was then melt extruded at 190 C. using a twin screw filament extruder to create Fused Filament Fabrication (FFF) printer quality filaments, which are then fed to the printer to create 22 cm.sup.2 sheets of materials with 1.51.5 mm.sup.2 pores, for ex vivo testing on human skin models.

[0380] To demonstrate the bioactivity of the polymers designed in accordance with various embodiments disclosed herein, human skin testing was carried out using several of the polymers that had the peptides attached on the biomacromonomer. The bioactive synthetic polymer was blended with medical grade PP as base material and PP has a higher melting point of 179 C. The polymer blend was extruded at 220 C. into filaments using a filament extruder followed by FFF printing to give sheets with built in pores. The sheets were then tested on human skin models and clearly, the materials designed in accordance with various embodiments disclosed herein showed much better skin cell viability compared to commercial dressings such as Allevyn (polyurethane foam) and Acticoat (silver-based non-woven dressing) (FIG. 3A to FIG. 3B). These are two most commonly prescribed wound dressings in hospitals, for chronic wound patients. With these skin data, the efficacy of the bioactive synthetic polymers and their ability to withstand harsh material processing temperatures (220 C.) were demonstrated. Bioactivity of the biomolecule attached is not lost even at such temperatures, demonstrating the present disclosure's ability to create bioactive polymers that have high stability for material processing and bioefficacy for various treatments.

[0381] Biocompatibility tests were carried out in SRIS's human tissue lab using human dermis to reconstruct a skin model by regrowing an epidermis layer using keratinocytes obtained from the skin bank. The tests were conducted following standard reported protocols according to Topping, G. et al. (Primary Intention: The Australian Journal of Wound Management, 2006, 14(1), 14-21), the contents of which are fully incorporated herein by reference. Briefly, dressing materials were applied on the skin models with media provided to the skin to support skin viability. Dressings were removed at 24 h (FIG. 3A) and 48 h (FIG. 3B), and the skin samples stained with alamar blue, incubated for 90 min each, and the stains were analyzed by fluorescence spectroscopy to check for the amount of viable cells.

[0382] Copolymers were created using PE as the synthetic polymer and a range of peptides of different properties as the bioactive macromonomer, namely antimicrobial peptide: IRIK; collagen fragment (GPHyp).sub.3: GPHyp; collagen mimic: DGEA and integrin binding peptides: RGD, SRGDS and RGDS. The copolymers were subsequently blended with polypropylene and 3D-printed into sheets, before tested for cell viability and biocompatibility against commercially available wound dressing such as Allevyn and Acticoat.

[0383] From the ex vivo tests, it can be seen that the collagen-based materials, (GPHyp).sub.3 and DGEA both showed enhanced cell viability compared to controls, at 24 h. SRGDS also outperformed control slightly (FIG. 3A). At 48 h, both SRGDS and GPHyp retained good cell viability, demonstrating their biocompatibility with human skin (FIG. 3B). Even the bioactive PE made with an antimicrobial peptide such as (IRIK).sub.2, showed reasonable biocompatibility, compared to Acticoat, the commercial antimicrobial dressing.

[0384] Cross sectional hematoxylin-stained and eosin-stained (H&E) images of human skin samples obtained from preliminary ex vivo wound closure tests after 3 days application of PE-peptide based materials are provided in FIG. 4 and FIG. 5. Comparative example is commercial dressing Allevyn (i.e. polyurethane-based dressing). Control used include poly(norbornene dicarboximide) with PE and mPEG.sub.1000 as side chains. The PE-peptide based materials are macromonomers of PE copolymerized with macromonomers of RGD and blended with polypropylene at ratios of 0.1%, 1% and 3% PE-RGD copolymer, respectively.

[0385] In short, copolymeric materials of PE with various peptides including collagen fragments, collagen mimics, antimicrobial peptides (IRIK).sub.2 and extracellular matrix peptides such as RGD and SRGDS using PEG and norbornene dicarboximide linkers have been prepared. The materials showed enhanced thermal stability as well as good biocompatibility data. In general, peptides of 3-20 amino acids in length in any sequence and oligosaccharides of up to 14 saccharide units, can be used in general.

7.5. Conclusion

[0386] Through the incorporation of pegylated peptides and collagen fragments into brush polymers containing polyethylene (PE) side chains, bioactive polyethylene was created for use in non-woven fibers for consumer products and polyethylene- or polypropylene-based medical devices. To create prototypes for testing, these polymers were blended into medical grade polypropylene as a powder and melt extruded at 220 C. into sheets using an FDM printer. The polymeric sheets have been tested ex-vivo on human skin models and demonstrated excellent biocompatibility with human skin. Other pegylated peptides have also demonstrated good biocompatibility upon incorporation into PE. As shown in the examples, the thermal stability of the peptides improved dramatically upon incorporation into PE-based brush polymers. Peptides of 3-20 amino acids in length in any sequence and oligosaccharides of up to 14 saccharide units, can be used in general.

[0387] Polymeric materials containing pegylated collagen fragments or peptides have been developed with polyethylene, as brush polymers using polynorbornene dicarboximide backbone. The polymers showed bioactivities of the peptides in terms of biocompatibility and enhanced cell viability, using ex vivo human skin models. At the same time, the polymers also showed improved thermal stability, as evident from the TGA-DSC curves which showed material weight loss of 50% only at 467 C. The polymers were melt extruded at 220 C. and showed no loss in bioactivity when tested on human skin, demonstrating the capabilities in developing bioactive polyethylene materials that have both thermal stability and bioactivity like that of collagen. The materials can be melt processed by conventional material processing methods, making them useful materials for consumer products such as diapers. They also demonstrated good mechanical properties like those of polyethylene.

[0388] Through the incorporation of pegylated biomolecules known to improve wound healing into polyethylene, novel materials were created that possess both bioactivity of the biomolecules used, as well as physical properties of polyethylene. Extracellular matrix peptides such as RGD, collagen and laminin-derived peptides such as A5G81, are known to enhance skin cell proliferation and wound healing. Brush polymers were created with these peptides on pegylated side chains, along with polyethylene side chains, on a polynorbornene dicarboximide backbone. The materials were then 3D printed using FFF printers into sheets and tested on human skin models. The ex vivo tests of the materials showed improved cell viability compared to commercial dressings as controls.

[0389] Brush polymers bearing polyethylene side chains and pegylated peptides have been created for use in tissue regeneration materials such as wound dressings and other medical devices. By incorporating peptides into polyethylene, bioactivity was created in an otherwise inert polyethylene. At the same time, the thermal stability of the peptides such as collagen fragments and RGD, were enhanced dramatically. The peptides also did not phase separate from PE. This allowed the bioactive PE to be blended into base polypropylene (PP), for non-woven fiber production in wound dressing manufacturing. The bioactive PE can also be blended into PE for creation of other biomedical devices such as joint implants and stents to improve recovery rates in patients receiving the implants as a result of more biocompatible PE being used.

7.6. Materials and Methods

7.6.1. General Procedure

[0390] Ring opening metathesis polymerization reactions and RGD macromonomer synthesis were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. SA-t-PE macromonomer was synthesized under ambient conditions. All the solvents usedanhydrous benzene and anhydrous methanol from Alfa Aesar, were used as purchased, in the glovebox. Grubbs second generation catalyst was purchased from Sigma Aldrich and all peptides (including RGD peptides) were purchased from Biomatik Inc. HMDA and PEG diamine were purchased from Alfa Aesar. All purchased reagents were used without further purification. Succinic acid terminated polyethylene (MW 15,000 and below) was prepared according to the literature (Macromolecules 2009, 42, 4356-4358; following the second example in Supporting Information).

[0391] .sup.1H NMR spectra were recorded on a Jeol 500 MHz NMR spectrometer. GPC chromatogram were recorded on an Agilent Infinity II High Temperature GPC system equipped with 2*PLgel Mixed B columns (3007.5 mm, particle size 10 m) and 1*PLgel Mixed B guard column (507.5 mm). Eluent is TCB with 1 ml/min flow rate and oven temperature of 160 C. Polystyrene was used as calibration standard.

7.6.2. Synthesis of SA-t-PE Macromonomer (SA-t-PEHMDANB) for PE MW 1,400-5,000

[0392] SA-t-PE (6 mmol) was weighed into a 250 ml round bottomed flask (rbf) followed by addition of toluene (120 ml). HMDA (18 mmol) and triethylamine (6 mmol) were then added. The mixture was then stirred under reflux overnight with a dean stark trap connected, for water removal. The mixture was then cooled and concentrated, followed by addition of MeOH, to give a beige precipitate. The mixture was filtered and residue was washed with MeOH before drying in a vacuum oven overnight, to give SA-t-PEHMDA quantitatively.

[0393] SA-t-PEHMDA (6 mmol) was added to a 250 ml rbf followed by addition of cis-norbornene-exo-2,3-dicarboxylic anhydride (6.5 mmol), toluene (120 ml) and triethylamine (6 mmol). The mixture was then refluxed overnight with a dean stark trap connected, for water removal. The mixture was then cooled and concentrated, followed by addition of MeOH, to give a beige precipitate. The mixture was filtered and residue was washed with MeOH before drying in a vacuum oven overnight, to give SA-t-PE macromonomer (SA-t-PEHMDANB) quantitatively. .sup.1H NMR (C.sub.7D8) 5=5.88-5.85 (m, 2H), 5.16-5.47 (m, 2H), 3.41-3.36 (m, 5H), 2.97-3.36 (m, 4H), 2.64 (bs, 2H).

7.6.3. Synthesis of NBPEG Macromonomer Body (for H.SUB.2.N-PEG-NH.SUB.2.1,000-6,000)

[0394] PEG diamine (1 g) and cis-norbornene-exo-2,3-dicarboxylic anhydride (1 eq.) were added to a 100 ml rbf, followed by toluene (50 ml). Triethylamine (1 eq.) was added and the mixture stirred under reflux overnight, 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 CH.sub.2Cl.sub.2 (30 ml). The organic layer was extracted and combined, washed with sat. NaCl before drying over Na.sub.2SO.sub.4. The material was evaporated to dryness to give a pale orange oil, NBPEG for PEG diamine 1,000 and beige solid for PEG diamine 3,400 and 6,000. .sup.1H NMR (MeOD): =6.36 (t, 2H, NB), 3.67 (s, PEG), 3.21 (s, 2H, NB), 2.74 (s, 2H, NB), 1.92 (s, 2H).

7.6.4. Synthesis of NBPEG.SUB.1000.RGD as Representative Preparation for PEG 1,000-6,000

[0395] RGD (with 1 carboxylic acid on aspartic acid protected with OMe) (0.0937 g, 0.26 mmol), was dissolved in MeOH (2.5 ml) in a 4 ml vial, in the glovebox. .sup.iPr.sub.2EtN (91 L, 0.52 mmol) was added and the mixture 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., followed by addition of the RGD solution from (A), to give solution (B). Solution B is then added to NBPEG.sub.1000 (0.25 g, 0.218 mmol) in a 40 ml vial and stirred at rt overnight. The resultant mixture was then evaporated to dryness and the oil was added to diethylether (50 ml). The diethylether solution was chilled in a freezer for 48 h and decanted. MeOH (5 ml) was added to the residue to give an orange solution with 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 at 95% yield. .sup.1H NMR (MeOD): =7.74 (dd), 7.35-7.42 (m), 6.32 (t), 4.39 (s), 4.20 (s), 3.63 (br, s), 3.60 (d), 3.17 (t), 2.70 (d). MALDI-MS: 661.3 ([MNB]+2H.sup.+).

7.6.5. Synthesis of NBPEG.SUB.1000.DGEA as Representative Preparation for PEG 1,000-6,000

[0396] DGEA (with 2 carboxylic acid end protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 l, 0.52 mmol) was added and mixture 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 addition of the solution A to give suspension B. Suspension B was then added to NBPEGNH.sub.2 (0.25 g, 0.218 mmol) and stirred at r.t. for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to give yellow oily mixture. The mixture was dispersed into Et.sub.2O and the solution placed in a freezer for 48 h. The Et.sub.2O layer was withdrawn and MeOH was added to the residue to give a yellow suspension. Yellow oily product NB-PEG-DGEA (0.28 g, yield 73%) was obtained upon filtration and solvent evaporation. .sup.1H NMR (CD.sub.3OD, 500 MHz, 25 C.): 7.80 (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).

7.6.6. Synthesis of NBPEG.SUB.1000.(GPHyp).SUB.3 .as Representative Preparation for Collagen Fragments of Glycine, Proline and Hydroxyproline in Varying Sequence and Chain Length Up to n=6, PEG 1,000-6,000

[0397] (GPHyp).sub.3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 l, 0.52 mmol) was added and mixture 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 addition of the solution A to give suspension B. Suspension B was then added to NBPEGNH.sub.2 (0.25 g, 0.218 mmol) and stirred at r.t. for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to give beige mixture. The mixture was dispersed into Et.sub.2O and freezed for 48 h. The Et.sub.2O layer was withdrawn and MeOH was added to the residue to give a beige suspension. Beige oily product NB-PEG-(GPHyp).sub.3 (0.25 g, yield 50%) was obtained upon filtration and solvent evaporation.

[0398] .sup.1H NMR (CD.sub.3OD, 500 MHz, 25 C.): 6.33 (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).

7.6.7. Synthesis of NBPEG.SUB.1000.(IRIK).SUB.2 .as Representative Preparation for PEG 1,000-6,000

[0399] (IRIK).sub.2 (with 1 Boc-protected amine) (0.100 g, 0.0875 mmol) was dissolved in MeOH (1.0 ml) in glovebox. .sup.iPr.sub.2EtN (31 l, 0.175 mmol) was added and mixture stirred as solution A. HOBT (0.012 g, 0.0875 mmol) and HBTU (0.033 g, 0.0875 mmol) were dissolved in MeOH (1.5 ml) at 40 C., followed by addition of the solution A to give suspension B. Suspension B was then added to NBPEGNH.sub.2 (0.0835 g, 0.073 mmol) and stirred at r.t. for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation to give yellow oily mixture. The mixture was dispersed into Et.sub.2O and the solution placed in a freezer for 48 h. The Et.sub.2O layer was withdrawn and MeOH was added to the residue to give a yellow suspension. Yellow sticky solid product (0.14 g, yield 70%) was obtained upon filtration and solvent evaporation. The solid was dissolved in dichloromethane (3.0 ml) and trifluoroacetic acid (0.5 ml) and stirred at r.t. for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation and washed with Et.sub.2O to give pale yellow solid. The solid was re-dissolved in dichloromethane (2.0 ml) and MeOH (1.0 ml). Et.sub.3N (200 l) was added and the mixture stirred at r.t. for 24 h. The resulting pale yellow mixture was then concentrated by solvent evaporation and washed repeatedly with Et.sub.2O to give pale yellow solid NB-PEG-IRIK (0.12 g, 63%). .sup.1H NMR (CD.sub.3OD, 500 MHz, 25 C.): 6.32 (s, 2H), 4.50-4.10 (m, 7H), 3.64 (m, 84H), 3.25-3.10 (m, 4H), 3.00-2.80 (m, 5H), 1.90-1.30 (m, 30H), 1.25-1.15 (m, 4H), 1.05-0.85 (m, 24H).

7.6.8. Procedure for ROMP of SA-t-PE Macromonomer and RGD Peptide Macromonomer as Representative Procedure for SA-t-PE-Peptide Copolymers, for Peptide Length of 3-20 Amino Acids in any Sequence

[0400] RGD peptide macromonomer (0.0342 g, 0.0023 mmol) was weighed into a 20 ml vial followed by addition of SA-t-PE (0.2 g, 0.12 mmol). Benzene (2.3 ml) was added and the mixture stirred at 75 C. till a clear solution was obtained. A solution of catalyst 1 in benzene (1.25 mol %, 0.05 M) was added to the solution and the reaction was stirred for 22 h at 75 C. Ethyl vinyl ether was added to the reaction mixture followed by MeOH (15 ml) to give a beige precipitate. The mixture is filtered and the residue washed 5 times with MeOH before being dried overnight in a vacuum oven.

[0401] For SA-t-PERGD copolymer, .sup.1H NMR: 6.0 (s, unreacted SA-t-PE), 5.62-5.38 (m), 3.65 (PEG), 1.46 (t, CH.sub.2), 1.02 (PE CH.sub.3).

Example 8: Amine-Terminated Polyethylene (PE)-Peptide Copolymers

8.1. Amine-Terminated PE-RGD Copolymer

[0402] This example shows a one-pot, 2-step strategy to create a primary amine terminus on polyethylene and its subsequent use in macromonomer synthesis, followed by copolymerization with a biomacromonomer to form bioactive polyethylene copolymers. In this method, linear primary amines were created using polyethylene via hydroaminomethylation and subsequently used in macromonomer formation with cis-norbornene-exo-2,3-dicarboxylic anhydride. Bioactive polyethylene can be created by copolymerizing this PE macromonomer with biomolecule-containing macromonomers. PE-RGD copolymers are reported here as an example of a bioactive PE that is compatible with human skin fibroblasts.

8.1.1. PE Macromonomer

[0403] To obtain a primary amine terminus on polyethylene (PE) for macromonomer formation, hydroaminomethylation of vinyl terminated PE was carried out in a one pot, two-step process where it was first converted into a linear carbonyl via hydroformylation, followed by reductive amination in the presence of hexamethylenediamine (HMDA) or NH.sub.3 gas (Scheme 4.2). The linear to branch ratio here is excellent where branched carbonyl or amine was not detected. Negligible amounts of linear alcohol (<1%) was detected in the crude product.

[0404] Upon obtainment of the amine terminated PE, condensation with cis-norbornene-exo-2,3-dicarboxylic anhydride, afforded the PE macromonomer (Scheme 4.3), which could be used in brush polymer formation with either itself or other macromonomers.

8.1.2. PE-Peptide Copolymer

[0405] To demonstrate formation of bioactive PE using the PE macromonomer, PE macromonomer was reacted with a pegylated RGD macromonomer to obtain a copolymer of PE and pegylated RGD, using ring opening metathesis polymerization (ROMP) with a Grubbs' catalyst (Scheme 8.1).

##STR00056##

8.2. Thermal Stability

[0406] The PERGD copolymer was checked for thermal stability before undergoing material processing. From TGA analysis of the copolymer, it can be seen that the polymer undergoes 50% weight loss at 465 C., indicating its high thermal stability compared to pure RGD itself which has a degradation temperature of less than 200 C. (FIG. 6).

8.3. Biocompatibility

[0407] Subsequently, PERGD copolymer was blended with medical grade PLA and electrospun into nanofibers for biocompatibility tests with Hs27 human fibroblasts grown in Dulbecco's Modified Eagle Medium (DMEM) w/10% FBS and 1% Pen/Strep. From the 72 h cell viability tests on electrospun samples, it can be seen that PERGD shows slight cell proliferation over negative control or pure PLA at 25% PERGD/PLA blending ratio and much better cell viability than commercial wound dressings Acticoat and Allevyn (FIG. 7). Viable cells are essential for cell proliferation.

[0408] In conclusion, amine terminated PE was successfully created using a one pot, 2-step hydroaminomethylation reaction in the presence of HMDA and PE macromonomers were constructed using it. Bioactive PE copolymer was also created and tested successfully for human skin biocompatibility, using PERGD as example.

8.4. Materials and Methods

8.4.1. General Procedure

[0409] Ring opening metathesis polymerization reactions and RGD macromonomer synthesis were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. PEHMDANB macromonomer was synthesized under ambient conditions. PEHMDA synthesis was carried out in a Hastelloy pressure reactor fitted with PTFE gasket, from Parr Instrument company. All the solvents used-anhydrous benzene and anhydrous methanol from Alfa Aesar, were used as purchased, in the glovebox. Grubbs second generation catalyst was purchased from Sigma Aldrich and all peptides were purchased from Biomatik Inc. [Rh(acac)(CO).sub.2], [Ir(COD)Cl].sub.2, xantphos, HMDA, triethylamine and PEG diamine were purchased from Alfa Aesar. All purchased reagents were used without further purification. Vinyl terminated PE (MW 1,400-5,000) was obtained according to the literature method (Macromolecules 2009, 42, 4356-4358; following the second example in Supporting Information).

[0410] .sup.1H NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer. GPC chromatogram were recorded on an Agilent Infinity II High Temperature GPC system equipped with 2*PLgel Mixed B columns (3007.5 mm, particle size 10 m) and 1*PLgel Mixed B guard column (507.5 mm). Eluent is TCB with 1 ml/min flow rate and oven temperature of 160 C. Polystyrene was used as calibration standard.

[0411] The PERGD copolymers were blended with PLA at 1:4 ratio and electrospun into sheets of fibers, which were then sterilized with 70% ethanol, dried and incubated for 72 h with fibroblasts Hs27 before being checked for cell viability using Celltitre-Glo assays.

8.4.2. Synthesis of PEHMDA

[0412] Vinyl terminated PE (0.35 g, 0.25 mmol) and xantphos (0.0036 g, 6.25 mol) were weighed into a 25 ml pressure reactor followed by addition of toluene (3.5 ml). Rh(acac)(CO).sub.2 was then added (0.5 ml, 0.65 mg/ml, 1.25 mol) to the mixture. The vessel was sealed and flush 5 times with CO/H.sub.2 (g) (1:1) and pressurized to 45 bar with the gas. The mixture was stirred at 100 C. for 12 h then cooled. HMDA (0.0581 g, 0.5 mmol) was dissolved in toluene (1 ml) before being added to the cooled mixture, followed by [Ir(COD)Cl].sub.2 (1 ml, 0.84 mg/ml). The vessel was sealed and flushed 5 times with H.sub.2 (g) and pressurized to 20 bar with the gas. The mixture was stirred at 135 C. for 4h before being cooled, followed by addition of MeOH to result in a white precipitate. The precipitate was filtered and the residue washed repeatedly with MeOH, followed by drying in a vacuum oven to yield a white solid product of PEHMDA at 68% yield. .sup.1H NMR (toluene-d8, 90 C.): =0.91 (t, 3H), 1.36 (br, s, 148H), 1.47-1.44 (m), 2.61-2.54 (m, 6H at 68% conversion).

8.4.3. Synthesis of PECH.SUB.2.NH.SUB.2

[0413] Vinyl terminated PE (0.35 g, 0.25 mmol) and xantphos (0.0036 g, 6.25 mol) were weighed into a 50 ml pressure reactor followed by addition of toluene (3.5 ml). Rh(acac)(CO).sub.2 was then added (0.5 ml, 0.65 mg/ml, 1.25 mol) to the mixture. The vessel was sealed and flush 5 times with CO/H.sub.2 (g) (1:1) and pressurized to 45 bar with the gas. The mixture was stirred at 100 C. for 12 h then cooled. [Ir(COD)Cl].sub.2 (1 ml, 0.84 mg/ml) was added to the cooled mixture and the vessel was sealed, flushed with NH.sub.3 (g) 5 times before being pressurized to 3 bar with NH.sub.3 (g). The vessel was further pressurized with another 20 bar H.sub.2 (g). The mixture was then stirred at 135 C. for 3 h before being cooled, followed by addition of MeOH to result in a white precipitate. The precipitate was filtered and the residue washed repeatedly with MeOH, followed by drying in a vacuum oven to yield a white solid product of PECH.sub.2NH.sub.2 at 61% yield. .sup.1H NMR (toluene-d8, 90 C.): =0.90 (t, 3H), 1.34 (br, s, 194H), 2.58 (t, 2H, at 61% yield).

8.4.4. Synthesis of PEHMDANB

[0414] PEHMDA and PECH.sub.2NH.sub.2 cannot be separated from unreacted PE and is used as a mixture. Quantities of PEHMDA and PECH.sub.2NH.sub.2 used are calculated based on percentage of each in sample mixture containing unreacted PE.

[0415] PEHMDA (0.2 mmol) and cis-norbornene-exo-2,3-dicarboxylic anhydride (0.04 g, 0.25 mmol) were weighed into an rbf, followed by addition of toluene (20 ml) and Et.sub.3N (28 L, 0.2 mmol). The flask was equipped with a dean stark trap and the mixture refluxed for 12 h. The reaction was cooled and MeOH was added to the mixture to give a white precipitate in a pale yellow solution. The mixture was filtered and the residue washed with MeOH repeatedly to yield an off white product. .sup.1H NMR (toluene-d8, 90 C.): 5.89 (s), 5.87 (s), 3.42 (t), 3.01 (t), 2.53-2.60 (m), 2.19 (s), 1.37 (br s), 0.92 (t). Product exists a mixture of PEHMDANB and unreacted PE.

8.4.5. Procedure for ROMP of PEHMDANB Macromonomer and RGD Peptide Macromonomer as Representative Procedure for PE-Peptide Copolymers, for Peptide Length of 3-20 Amino Acids in any Sequence

[0416] RGD peptide macromonomer (0.0342 g, 0.0023 mmol) is weighed into a 20 ml vial followed by addition of PEHMDANB (0.12 mmol). Benzene (2.3 ml) is added and the mixture stirred at 75 C. till a clear solution is obtained. A solution of Grubbs' catalyst (2.sup.nd generation) in benzene (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 22 h at 75 C. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (15 ml) to give a beige precipitate. The mixture is filtered and the residue washed 5 times with MeOH before being dried overnight in a vacuum oven. For PERGD, .sup.1H NMR (toluene d8, 90 C.): =5.88 (s), 5.85 (s) (unreacted PEHMDANB), 5.46-5.42 (m), 3.53 (br s, PEG), 3.4 (t), 2.57-2.53 (m), 1.36 (br s, PE CH.sub.2), 0.91 (br s, PE CH.sub.3). Sample contains unreacted PE.

Example 9: Applications

[0417] The present disclosure provides a new modular synthesis method to create softer and more biocompatible PE/PP blends. Embodiments of the bioactive polyethylene copolymer disclosed herein possess one or more of the following properties: [0418] non-toxic to skin; [0419] increased thermal stability for material processing compared to pure collagen which denatures at 37 C.; and [0420] increased structure integrity compared to pure collagen, oligopeptides or oligosaccharides, which exists in gels or hygroscopic crystals.

[0421] Advantageously, embodiments of the bioactive polyethylene copolymer disclosed herein allow for biomolecule (e.g., collagen) to be blended into base material of synthetic polymer similar to the synthetic polymer side arms of copolymer (e.g., polypropylene) without phase separation, despite the opposing material properties existing between the hydrophobic PP and hydrophilic collagen.

[0422] Embodiments of the bioactive polyethylene copolymer disclosed herein showed good thermal stability and biocompatibility data. Advantageously, embodiments of the bioactive polyethylene copolymer disclosed herein can be used to make consumer care products such as diapers and sanitary products or biomedical devices such as joint implants, gut stents, wound dressings, cartilage implants.

[0423] The present disclosure provides a new modular synthesis method to create bioactive macromonomers rapidly for construction of bioactive copolymers with bioactive molecule of choice, depending on the targeted application or bioactivity required. Bioactive macromonomers may be easily copolymerized with polyethylene to form bioactive polyethylene copolymers with desired physical and mechanical properties. Advantageously, there is increased stability of bioactive molecule upon connection to polymer linker. Embodiments of the strategy disclosed herein allow for any peptide, carbohydrate or drug molecule to be used in polymer synthesis without loss of bioactivity. Embodiments of the strategy disclosed herein also allow rapid build up of bioactive macromonomer library. Any bioactive molecule with a carboxylic acid group may be used. In summary, the present disclosure provides a highly versatile strategy for biomedical material customization.

[0424] Embodiments of the method disclosed herein allow macromonomers to be paired with synthetic polymer of choice to create bioactive polymer that has both mechanical and physical properties of synthetic polymer and biological activity of bioactive molecule.

[0425] Embodiments of the method disclosed herein is an easy strategy to create different types of bioactive polymers that are chemically bonded instead of physical blends of bioactive molecules into synthetic polymers.

[0426] Advantageously, non-cell or growth factor-based bioactivity is/are provided on the polymer disclosed herein. Embodiments of the bioactive polyethylene copolymer disclosed herein possess both bioactivity to enhance therapeutic effects such as tissue regeneration, biofilm eradication etc, and also structural integrity and mechanical strength, like a polymer. Embodiments of the bioactive polyethylene copolymer disclosed herein allow for biomolecule to be blended into base material of synthetic polymer similar to the synthetic polymer side arms of copolymer, without phase separation. Embodiments of the method disclosed herein allow the polyethylene to become biocompatible to human tissues upon modification with biomolecules. Embodiments of the method disclosed herein allow a wide range of biomolecules to be used to achieve any desired therapeutic effect. Embodiments of the method disclosed herein also allow a good range of synthetic polymers to be used to achieve different mechanical, physical properties required in material for targeted biodevice.

[0427] Embodiments of the bioactive polyethylene copolymers disclosed herein may be used as bioadditives for biomedical devices to provide therapeutic effects to device material itself.

[0428] Embodiments of the method disclosed herein use non cell- or growth factor-based therapy, which allow for long shelf life of device or materials such as scaffold and prevent unwanted or uncontrolled bioactivity (for e.g., tissue regeneration).

[0429] Embodiments of the bioactive polyethylene copolymers disclosed herein may be used as bioadditives for wound dressings, cartilage implants or bone scaffold to create stimulus required for skin, cartilage or bone tissue regeneration.

[0430] It will be appreciated by a person 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 disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.