Bioactive Synthetic Copolymer, Bioactive Macromolecule and Related Methods Thereof
20230399457 · 2023-12-14
Inventors
- Peili Teo (Singapore, SG)
- Jiayi Guo (Singapore, SG)
- Yew Chin Teo (Singapore, SG)
- Eun Ju Park (Singapore, SG)
Cpc classification
C08G2261/3342
CHEMISTRY; METALLURGY
C08G61/08
CHEMISTRY; METALLURGY
C08G2261/1426
CHEMISTRY; METALLURGY
International classification
Abstract
There is provided a bioactive synthetic 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 bioactive macromolecule, a material comprising said bioactive synthetic copolymer, a method of preparing said bioactive synthetic copolymer and a method of preparing said bioactive macromolecule.
##STR00001##
Claims
1. A bioactive synthetic 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): ##STR00043## 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.sup.1 comprises a synthetic polymer 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 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).
3. The copolymer of claim 1, wherein L is heteroalkylene having from 20 carbon atoms to 300 carbon atoms.
4. 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.
5. (canceled)
6. 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.
7. (canceled)
8. 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.
9. 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 mimics, collagen fragments, heparin sulfate, glycosaminoglycans (GAGs) and derivatives thereof.
10. 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), heparin oligosaccharide DP8, DP10, DP12, DP14, DP16, DGEA (SEQ ID NO: 5), (PHypG).sub.n type 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 and hyaluronic acid.
11. The copolymer of claim 1, wherein X comprises an antibiotic, antimicrobial, antibacterial moiety, blood thinning agents or anti-inflammatory agents, optionally wherein X is selected from the group consisting of penicillin, amoxicillin, amphotericin, ciprofloxacin (CIF), atorvastatin, aspirin, streptomycin, ribostamycin and gentamycin.
12. (canceled)
13. The copolymer of claim 1, wherein Y.sup.1 is represented by general formula (III): ##STR00044## wherein 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.sup.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.sup.2 is selected from the group consisting of polypropylene (PP), polyesters, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylates, poly(meth)acrylates, polyamides (PA), and parts thereof, and T is a terminal 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. The copolymer of claim 1, wherein Y.sup.1 is selected from the following general formulae (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IIIf): ##STR00045##
15. A method of preparing a bioactive synthetic copolymer of claim 1, the method comprising: polymerising one or more bioactive macromolecules represented by general formula (IV) with one or more synthetic macromolecules represented by general formula (V) in the presence of a catalyst to obtain the bioactive synthetic copolymer: ##STR00046## 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.sup.1 comprises a synthetic polymer 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.
16. The method according to claim 15, wherein the catalyst comprises a ruthenium complex.
17. The method according to claim 15, wherein the method comprises ring opening metathesis polymerisation (ROMP).
18. (canceled)
19. The method according to claim 15, wherein the method further comprises, prior to polymerizing preparing a bioactive macromolecule by: (i) providing a dicarboxylic anhydride having general formula (VI): ##STR00047## wherein Z.sup.1 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; (ii) reacting said dicarboxylic anhydride having general formula (VI) with a diamine R.sup.4R.sup.3N-L-R.sup.1—NH.sub.2 to obtain an amine having general formula (VII): ##STR00048## wherein R.sup.1 is optionally substituted alkyl; R.sup.3 and R.sup.4 are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, wherein at least one of R.sup.3 and R.sup.4 is H; L is heteroalkylene; and Z.sup.1 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 (iii) reacting said amine having general formula (VII) with an acid-containing bioactive moiety X—C(═O)OH to obtain the bioactive macromolecule, wherein X comprises a bioactive moiety selected from the group consisting of proteins, peptides, carbohydrates, therapeutic/drug molecules and derivatives thereof.
20. The method according to claim 19, wherein the method further comprises, prior to reacting amine having general formula (VII) with X—C(═O)OH, purifying the amine having general formula (VII) to remove impurities.
21. The method according to claim 20, wherein purifying comprises double neutralisation.
22. The method according to claim 21, wherein the double neutralisation comprises a first washing with acid and a second washing with base.
23. A material comprising a copolymer of claim 1 for use in medicine.
24. The material according to claim 23, wherein the material is part of an apparatus selected from the group consisting of wound dressing, skin scaffold, bone scaffold, organoid scaffold, implants, and medical device.
Description
BRIEF DESCRIPTION OF FIGURES
[0232]
[0233]
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
EXAMPLES
[0241] 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 Synthetic Polymer
[0242] 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 build up of a wide library of bioactive macromonomers of various chain length, allowing for quick development of synthetic polymers 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, is made possible using this library. Rapid polymer customization can thus be achieved.
[0243] 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 synthetic material which not only possess therapeutic effects but also, necessary mechanical properties for easy storage and handling.
[0244] 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 to create bioactive synthetic polymers 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 or biodegradable, depending on the needs. The modular synthesis therefore makes application matching to polymer properties much simpler and effective.
Example 2: Method of Preparing Bioactive Synthetic Copolymer
[0245] The method of preparing a bioactive synthetic copolymer in accordance with various embodiments disclosed herein involve creating macromonomers of the bioactive molecules and synthetic polymers 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 synthetic polymer 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.
[0246] In the following examples, brush polymers containing pendant arms of synthetic polymers and bioactive molecules tethered on polyethylene glycol (PEG) moieties, have been created. Synthetic polymers may include poly(caprolactone) (PCL), polyesters such as 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). 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 appropriate synthetic polymer and biomolecule with the bioactivity of interest can be chosen, copolymerized together using the brush polymer technology as disclosed herein by ring opening metathesis polymerization.
[0247] The resultant polymer shows bioactivity of the biomolecule involved while having much better physical and mechanical properties for good material handling and processability. For example, improvement in cell viability or cell proliferation in both the PA-collagen copolymers and PLA-RGD copolymers over controls, were observed.
[0248] The polymers can be subsequently blended with polymers similar to that on the pendant arms 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.
[0249] The synthetic route for preparing a bioactive synthetic polymer in accordance with various embodiments disclosed herein is illustrated in Scheme 2.
##STR00020##
[0250] In the following examples, six types of synthetic polymers were selected for the synthetic polymer side chains on the brush polymers. The exact polymer to be selected is dependent on the nature of the biomedical device to be manufactured, for example whether properties such as biodegradability, flexibility, impact-resistance etc, are required in the device material.
Example 3: Bioactive Macromonomers and Method of Synthesis
[0251] 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., Mw=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.
[0252] 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 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; COL or RGD, where COL may be DGEA, (GPHyp).sub.n or (PHypG).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. 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.
##STR00021##
##STR00022##
Example 4: Synthetic Macromonomers and Method of Synthesis
[0253] Schemes 4.1 to 4.5 show synthetic macromonomers of PCL, PLA, PLGA, PS, PMMA and PA.
[0254] PLA, PLGA, PCL are created using ring opening polymerization on a norbornene dicarboximide linker with a terminal hydroxy group. Briefly, cis-norbornene-exo-2,3-dicarboxylic anhydride is reacted with 3-amino-1-propanol to create 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 Sn(Oct).sub.2 catalyst to form PCL chains on the norbornene dicarboximide linker, N-[3-hydroxylpropyl]-cis-5-norbornene-exo-2,3-dicarboximide (NPH), to give the PCL macromonomer (NB-PCL) (Scheme 4.1) (or NB-PLA macromonomer).
##STR00023##
[0255] 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 Sn(Oct).sub.2 catalyst to provide NB-PLA macromonomer (Scheme 4.2).
##STR00024##
[0256] Poly(lactic-co-glycolic acid) (PLGA) macromonomer is synthesized in 2 steps via a cis-norbornene-exo-2,3-dicarboximide aminopropanol initiator molecule (Scheme 4.3).
##STR00025##
##STR00026## ##STR00027##
[0257] PS is prepared by atom transfer radical polymerization (ATRP) where an azide terminal is formed at the polymer chain end after the polymerization reaction so that the norbornene dicarboximide linker can be “clicked” onto the polymer to create PS (NB-PS) macromonomer (Schemes 4.4a to 4.4c).
[0258] PMMA (NB-PMMA) macromonomer is 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 provide the norbornenyl-functionalized ATRP initiator. NB-PMMA macromonomer is then synthesized by directly growing the polymer from a norbornenyl-functionalized ATRP initiator using CuBr/TMEDA catalytic system (Scheme 4.4d).
[0259] Polyamide (PA) macromonomers can be created by ring opening polymerisation of ε-caprolactam on N-(carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) under reflux conditions using H.sub.2O and H.sub.3PO.sub.3 as catalysts (Scheme 4.5). NCP served as the initiator for ε-caprolactam ROP.
##STR00028##
Example 5: Ring Opening Metathesis Polymerisation Catalysts
[0260] With both the bioactive macromonomer and synthetic polymer (PCL, PLA, PLGA, PS, PMMA or PA) macromonomer, the final bioactive copolymer is prepared by ROMP using Grubbs type catalysts 1 or 2 (Scheme 5).
##STR00029##
Example 6: Bioactive Synthetic Copolymers Examples
[0261]
[0262] Examples of synthetic polymers include poly(caprolactone) (PCL), polyesters such as 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 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, carbohydrates such as glycosaminoglycans or drug molecules containing a carboxylic acid terminal such as certain antibiotics.
[0263] Depending on the targeted application, the bioactive macromonomer can be matched with different types of synthetic polymers to create materials with different physical properties.
[0264] An example would be a bioactive macromonomer comprising RGD. RGD is a peptide sequence that is capable of binding integrins for cell attachment, migration and proliferation. Hence, macromonomer of RGD is created (Scheme 6).
##STR00030##
[0265] The RGD macromonomer can be paired with a biodegradable macromonomer to create skin scaffolds that would degrade in the human body after the patient's own skin has taken over. This macromonomer can also be copolymerized with heparin sulfate bearing macromonomers and polycaprolactone bearing macromonomers to create triblock copolymers that allow bone tissue regeneration, for use as bioresorbable bone scaffolds. 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 patient's needs. The dosage of the therapeutic agent (bioactive macromonomer) can also be tuned to suit a patient's needs by adjusting macromonomer ratios during polymerization.
[0266] 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.
[0267] 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. In vitro experiments of BMP-2 complexed with DP12 showed greater osteogenic differentiation in cells whereas in vivo experiments using rat models showed that DP12 enhanced bone tissue regeneration relative to controls of collagen sponge in polycaprolactone (PCL) tubes. Therefore, PCL copolymers with DP12 macromonomers are created which can then be added to base polymer PCL and fabricated into whole bone implants. By chemically linking DP12 to PCL itself before blending the polymers into base polymer PCL, the present disclosure has advantageously shown that it is possible to localize the GAG on the implant to prevent undesirable side effects such as bone tissue regeneration at any other locations of the body except the implant site. Apart from being used as bone scaffolds, the DP14-PCL/PCL blend can also be used to create skin scaffolds since GAGs are also known to enhance keratinocyte regeneration.
[0268] Apart from GAGs, peptides such as integrin binders or collagen fragments, which are useful towards skin and bone tissue regeneration may also be used. Extracellular peptides such as RGD are able to function as integrin binders to encourage cell attachment, migration and proliferation.
[0269] Apart from RGD peptides, 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 tissues makes it an ideal scaffold material for bones. Without being bound by theory, it is believed that the use of collagen fragments or collagen mimics (COL) in the PCL scaffolds helps to increase the biocompatibility and biomimetic properties of the overall PCL-based scaffold material. 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.
[0270] 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.
[0271] 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.
[0272] In summary, a general strategy has been developed to create bioactive macromonomers for rapid building of bioactive synthetic polymers 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.
[0273] In summary, a series of polymers that bear synthetic polymers such as polystyrene, polyacrylate, poly(meth)acrylate, poly(lactide), poly(lactic-co-glycolic acid), poly(ε-caprolactone) and polyamide with pegylated biomolecules as side chains on a poly(norbornene dicarboximide) backbone have been developed, via ROMP technologies. The biocompatibility, bone growth factor 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 synthetic polymers 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 synthetic polymer 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.
Experimental Procedures
General Procedure
[0274] Ring opening metathesis polymerization (ROMP) reactions, PS (NB-PS) and PCL (NPH-PCL), macromonomer synthesis, bioactive macromonomer syntheses and catalyst 2 synthesis were carried out 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 carried out in a fumehood under atmospheric conditions. All solvents used in the glovebox are anhydrous and used 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). HOBT, HBTU, .sup.iPr.sub.2EtN were purchased from Sigma Aldrich and cis-norbornene-exo-2,3-dicarboxylic anhydride was purchased from Alfa Aesar. DP12 was purchased from Iduron. All purchased reagents were used without further purification.
[0275] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as solvent for all biomolecule-based macromonomers. CDCl.sub.3 is used as solvent for PCL macromonomer. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used. Polystyrene was used as calibration standard. TGA/DSC is measured using TA Instruments SDT2960 simultaneous DSC-TGA.
Synthesis of (H.SUB.2.IMes)(pyr).SUB.2.(Cl).SUB.2.RuCHPh (Catalyst 2)
[0276] 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 min during which a colour change from red to green was observed. Hexanes (16 mL) was added to the green solution and a green solid began to precipitate. The green precipitate was vacuum-filtered, washed with hexanes (4×10 mL), and dried under vacuum to afford catalyst 2 as a green powder.
Synthesis of N-(Hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH)
[0277] 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). To the flask was added 30 mL toluene, followed by triethylamine (84 μL, 0.60 mmol). A Dean-Stark trap was attached to the flask, and the reaction mixture was heated at reflux (135° C.) for 4 h. The reaction mixture was then cooled and concentrated in vacuo to yield a pale yellow oil. This residue was diluted with 30 mL of dichloromethane and washed with 0.2 M HCl (20 mL) and sat. NaCl (20 mL). The organic layer was dried over Na.sub.2SO.sub.4, concentrated in vacuo and dried overnight in a vacuum oven to yield 1.22 g of white solid. .sup.1H NMR (500 MHz, CDCl.sub.3): δ 6.27 (t, J=2.0 Hz, 2H), 3.64 (t, J=6.4 Hz, 2H), 3.53 (q, J=6.1 Hz, 2H), 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).
Synthesis of NPH-PCL Macromonomer by ROP
[0278] NPH-PCL macromonomers with different degree of polymerization (DP) were prepared by ROP. As an 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).sub.2 (0.0037 g, 9.1 μmol) was added to the mixture and the resultant solution was stirred at 110° C. for 90 min and precipitated into methanol. The methanolic solution was then placed in the freezer overnight to result in white precipitate which was filtered and washed with methanol. The residue is then dried under vacuum overnight. GPC analysis (THF): M.sub.n=5,613, PDI=1.08, yield 0.4478 g.
[0279] A standard solution of Sn(Oct).sub.2 of concentration 91 μmol/ml, was prepared and used for ROP reactions.
Synthesis of NPH-PLA Macromonomer by ROP
[0280] NPH-PLA macromonomers with different degrees of polymerization (DP) were prepared by ROP. As an 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).sub.2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times, and was 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 into cold methanol twice. The macromonomer was isolated by decanting the supernatant and dried under vacuum overnight. GPC analysis (THF): M.sub.n=2,471, PDI=1.20, yield 0.600 g. .sup.1H NMR (CDCl.sub.3): δ 6.28 (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).
Synthesis of N-(Hydroxydecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (NDH)
[0281] 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). To the flask was added 20 mL of toluene, followed by triethylamine (80 μL, 0.58 mmol). A homogeneous solution was obtained upon heating. A Dean-Stark trap was attached to the flask, and the reaction mixture was heated at reflux (135° C.) for 4 h. The reaction mixture was then cooled and concentrated in vacuo to yield an off-white solid. This residue was dissolved in 20 mL of CH.sub.2Cl.sub.2 and washed with 0.1 N HCl (10 mL) and sat. NaCl (10 mL). The organic layer was dried over MgSO.sub.4 and concentrated in vacuo to yield 1.96 g of colorless, viscous oil. .sup.1H NMR (500 MHz, CDCl.sub.3): δ 1.20-1.28 (m, 13H), 1.49-1.56 (m, 5H), 2.65 (d, J=1.5 Hz, 2H), 3.26 (t, J=1.5 Hz, 2H), 3.44 (t, J=7.5 Hz, 2H), 3.62 (t, J=6.5 Hz, 2H), 6.27 (t, J=2.0 Hz, 2H).
Synthesis of N-(Pentynoyldecanyl)-cis-5-norbornene-exo-2,3-dicarboximide
[0282] 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 CH.sub.2Cl.sub.2. Pentynoic acid (0.25 g, 2.5 mmol) was added as a solution in 5 mL of CH.sub.2Cl.sub.2 via syringe. The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was washed with water (2×20 mL) and sat. NaCl (20 mL) and dried over MgSO.sub.4. The solvent was evaporated, and the remaining residual was purified by silica gel chromatography (ethyl acetate/hexanes, 1:9 v/v) to give 0.88 g product as a colorless oil (88% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 1.21-1.33 (m, 13H), 1.49-1.54 (m, 3H), 1.62 (t, J=7.5 Hz, 2H), 1.97 (t, J=2.5 Hz, 1H), 2.48-2.57 (m, 4H), 2.67 (d, J=1.5 Hz, 2H), 3.27 (t, J=1.5 Hz, 2H), 3.45 (t, J=7.5 Hz, 2H), 4.09 (t, J=7 Hz, 2H), 6.28 (t, J=2.0 Hz, 2H).
Synthesis of NB-PS Macromonomer by ATRP-Click
[0283] NB-PS macromonomers with different degree of polymerization (DP) were prepared using ATRP, click-reactions. As an example, CuBr (0.1435 g, 1 mmol) was weighed into a 20 ml scintillation vial in the glovebox. Styrene (pre-filtered through basic Al.sub.2O.sub.3, 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 stirring MeOH (400 ml) to give a white precipitate (ppt) in deep blue solution. The ppt was filtered to obtain a blueish white solid that is redissolved in minimal CH.sub.2Cl.sub.2, reprecipitated in MeOH and filtered. This redissolving, precipitation and filtration process is repeated until a pure white solid of PS-Br is obtained. The solid is then dried in a vacuum oven overnight. GPC analysis (THF): M.sub.n=2,523, PDI=1.18.
[0284] PS-Br (0.5 mmol) and NaN.sub.3 (2.5 mmol) were added to a 20 ml scintillation vial in the glovebox, followed by DMF (10 ml) and the mixture was stirred for 48 h to result in a colourless solution with white precipitate of NaBr. The mixture was added to a beaker of stirring MeOH in the fumehood. The white precipitate was filtered, washed with MeOH and dried in a vacuum oven to give PS-N.sub.3 prepolymer.
[0285] In a 20 ml scintillation vial was added PS-N.sub.3 prepolymer (0.1 mmol) and N-(Pentynoyldecanyl)-cis-5-norbornene-exo-2,3-dicarboximide (0.15 mmol) and CuBr (0.01 mmol). THF (2 ml) and PMDETA (0.01 mmol) were added and the mixture stirred at 50° C. overnight. MeOH was added to the cooled reaction mixture to yield a white ppt which was filtered and washed with MeOH, followed by drying in a vacuum oven, to yield NB-PS macromonomer. .sup.1H NMR (CDCl.sub.3): 7.10-6.46 (m), 6.28 (s, 2H), 5.04-4.94 (m, 1H), 4.13-4.0 (m, 2H), 3.51-3.40 (m, 5H), 3.27 (s, 2H), 2.91-2.86 (m, 2H), 2.67-2.56 (m, 2H), 0.92 (br s, 3H).
Synthesis of Norbornenyl-Functionalized ATRP Initiator
[0286] A round-bottom flask was charged with N-(Hydroxypropyl)-cis-5-norbornene-exo-2,3-dicarboximide (NPH) (0.66 g, 3.0 mmol). To the flask was added dichloromethane (12 mL), 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. When the addition was completed, the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was washed with 0.1 M HCl (15 mL), saturated NaHCO.sub.3 solution (15 mL) and sat. NaCl (2×15 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by silica gel chromatography (dichloromethane) to yield the product as a pale yellow solid (0.80 g, 72%). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 6.28 (t, J=1.8 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 3.61 (t, J=7.1 Hz, 2H), 3.28 (s, 2H), 2.69 (d, J=1.8 Hz, 2H), 1.99-1.96 (m, 8H), 1.52 (m, 1H), 1.21 (d, J=9.9 Hz, 1H).
Synthesis of NB-PMMA Macromonomer by ATRP
[0287] NB-PMMA macromonomers with different degrees of polymerization (DP) were prepared using ATRP. As an 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 the polymerization was conducted in a 70° C. oil bath for 3 h. The mixture was filtered through neutral alumina, precipitated into MeOH and filtered. The solid is then dried in a vacuum oven overnight. GPC analysis (THF): M.sub.n=5,158, PDI=1.13. .sup.1H NMR (CDCl.sub.3): δ 6.30 (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).
Synthesis of N-(Carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP)
[0288] 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 Et.sub.3N (410 μL, 2.92 mmol). The flask was fitted with a Dean-Stark trap and heated to reflux for 4h. The mixture was then allowed to cool to room temperature and diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with 1 M aqueous HCl (2×20 mL). The organic layer was washed with saturated aqueous NaCl (20 mL), dried with Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to provide NCP as a pale yellow solid. .sup.1H NMR (500 MHz, CD.sub.3OD, 25° C.) δ 6.26 (t, 2H, J=2.0 Hz), 3.44 (m, 2H), 3.25 (m, 2H), 2.66 (d, 2H, J=1.0 Hz), 2.32 (t, 2H, J=7.2 Hz), 1.63 (m, 2H), 1.55 (m, 2H), 1.46-1.51 (m, 1H), 1.33 (m, 2H), 1.19 (d, 1H).
Synthesis of NCP-PA 6 Macromonomer by ROP
[0289] NCP-PA 6 macromonomers with different degree of polymerization (DP) were prepared by ROP. As an example, ε-caprolactam (2.56 g, 12 mmol) was weighed into a 50 ml round bottom flask (rbf) containing NCP initiator (0.2 g, 0.6 mmol) with nitrogen inlet. Deionized H.sub.2O (5 ml) with H.sub.3PO.sub.3 (0.081 g) were added to the mixture and the resultant mixture was heated at 170° C. for 30 min and maintained at 240° C. for 4 hrs. H.sub.2O was removed by distillation and the reaction was heated at 240° C. under vacuum for another 2 hrs. Beige solid was precipitated from MeOH and washed repeatedly by it. NCP-PA 6 was obtained upon drying in a vacuum oven overnight. .sup.1H NMR [500 MHz, DCO.sub.2D/CD.sub.2Cl.sub.2 (1:4),]: δ 6.42 (br, PA 6), 6.28 (s, 2H, NCP), 3.42 (s, 6H, NCP), 3.14-3.12 (m, PA 6), 2.67 (s, 2H, NCP), 2.14-2.12 (m, PA 6), 1.56-1.53 (m, PA 6), 1.46-1.44 (m, PA 6), 1.29-1.25 (m, PA 6).
Synthesis of NBPEG macromonomer body (for H.SUB.2.N-PEG-NH.SUB.2 .1000, 3,400 and 6000)
[0290] 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): δ=6.36 (t, 2H, NB), 3.67 (s, PEG), 3.21 (s, 2H, NB), 2.74 (s, 2H, NB), 1.92 (s, 2H).
Synthesis of NBPEG.SUB.1000.RGD as Representative Prep for PEG 1000, 3,400 and 6,000
[0291] RGD (with 1 carboxylic acid end 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 μL, 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 is then evaporated to dryness and the oil is added to diethylether (50 ml). The diethylether solution is chilled in a freezer for 48 h and decanted. MeOH (5 ml) is added to the residue to give an orange solution with white ppt. The mixture is passed through a syringe filter and the clear filtrate is evaporated to dryness to give an orange oil of RGDPEGNB at 95% yield. .sup.1H NMR (MeOD): δ=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 ([M−NB]+2H.sup.+).
Synthesis of NBPEG.SUB.1000.DGEA as Representative Prep for PEG 1000, 3,400 and 6,000
[0292] DGEA (with carboxylic acid on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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.
[0293] .sup.1H NMR (500 MHz, CD.sub.3OD, 25° C.): δ 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).
Synthesis of NBPEG.SUB.1000.(GPHyp).SUB.3 .as Representative Prep for Collagen Fragments of Glycine, Proline and Hydroxyproline in Varying Sequence and Chain Length Up to n=6, PEG 1000, 3,400 and 6,000
[0294] (GPHyp).sub.3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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 freeze 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 (0.22 g, yield 50%) was obtained upon filtration and solvent evaporation.
[0295] .sup.1H NMR (500 MHz, CD.sub.3OD, 25° C.): δ 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).
Synthesis of NBPEG.SUB.3400.DP12
[0296] DP12 (0.0211 g, 8.5 μmol) was dissolved in MeOH (1.5 ml) in an 8 ml scintillation vial. .sup.iPr.sub.2EtN (3 μl, 17 μmol) was added and mixture 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., followed by addition of the solution A to give suspension B. Suspension B was then added to NBPEG.sub.3400NH.sub.2 (0.025 g, 7.05 μmol) and stirred at r.t. for 24 hr. The resulting pale yellow mixture was then concentrated with solvent evaporation to give beige mixture. The mixture was dispersed into Et.sub.2O and the mixture placed in freezer for 84 h. The Et.sub.2O was decanted and MeOH was added to the residue to give a pale yellow solution which resulted in an orange oil on filtration of solution followed by solvent evaporation.
[0297] .sup.1H NMR (500 MHz, CD.sub.3OD, 25° C.): 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).
Typical Procedure for ROMP of NPH-PCL Macromonomer and NBPEG.SUB.3400.RGD Macromonomer as Representative Prep for PCL-Peptide Type Copolymers
[0298] NBPEG.sub.3400RGD macromonomer (0.2 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PCL (0.05 g). THF (0.021 M wrt. NPH-PCL) is added and the mixture stirred at r.t. till a clear solution is obtained. A solution of catalyst 1 or 2 in THF (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 2 h at 30° C. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a white ppt. The mixture was centrifuged and mother liquor was decanted. The residue was resuspended in methanol, centrifuge followed by decanting mother liquor again, to wash the residue. The washing with MeOH was carried out 3 times before the final residue was dried overnight in a vacuum oven.
[0299] For PCL-RGD copolymer, GPC analysis (THF): M.sub.n=140,000, PDI=1.21.
Typical Procedure for ROMP of NPH-PLA Macromonomer and NBPEG.SUB.1000.RGD Macromonomer as Representative Prep for PLA-Peptide Type Copolymers
[0300] NBPEG.sub.1000 RGD macromonomer (0.1 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PLA (0.05 g). THF (0.05 M wrt. NPH-PCL) is added and the mixture stirred at r.t. till 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 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a sticky solid. The mother liquor was decanted and the residue washed repeatedly with MeOH followed by drying in vacuum oven. For PLA-RGD copolymer, GPC analyses (THF): M.sub.n=76,681, PDI=1.44.
Typical Procedure for ROMP of NB-PS Macromonomer and NBPEG.SUB.1000.RGD Macromonomer as Representative Prep for PS-Peptide Type Copolymers
[0301] NBPEG.sub.1000 RGD (0.1 eq.) was weighed into a 4 ml glass vial followed by addition of NB-PS (0.05 g). THF (0.6 ml) is added and the mixture stirred at 25° C. till a clear solution is obtained. A solution of catalyst 1 or 2 in THF (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 1 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a white precipitate. The mixture was filtered and the residue washed repeatedly with MeOH followed by drying in vacuum oven.
[0302] For PS-RGD copolymer, GPC analysis (THF): M.sub.n=33,484, PDI=1.41.
Typical Procedure for ROMP of NB-PMMA Macromonomer and NBPEG.SUB.1000.RGD Macromonomer as Representative Prep for PMMA-Peptide Type Copolymers
[0303] NBPEG.sub.1000 RGD macromonomer (0.1 eq.) is weighed into a 4 ml scintillation vial followed by addition of NB-PMMA (0.05 g). THF (0.05 M wrt. NB-PMMA) is added and the mixture stirred at r.t. till 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 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a white precipitate. The residue was resuspended in methanol, centrifuged followed by decanting mother liquor again, to wash the residue. The washing with MeOH was carried out 3 times before the final residue was dried overnight in a vacuum oven.
[0304] For PMMA-RGD copolymer, GPC analysis (THF): M.sub.n=68,512, PDI=1.72.
Typical Procedure for ROMP of NCP-PA6 Macromonomer and NBPEG.SUB.3400.DGEA Macromonomer as Representative Prep for PA-Peptide Type Copolymers
[0305] NBPEG.sub.3400DGEA macromonomer (0.2 eq.) is weighed into a 4 ml glass vial followed by addition of NCP-PA6 (0.12 g). CH.sub.3CO.sub.2H (0.021 M wrt. NCP-PA6) is added and the mixture stirred at 80° C. till a clear solution is obtained. Catalyst 2 (1.25 mol %, 0.05 M in CH.sub.2Cl.sub.2) is added to the solution and the reaction is stirred for 24 h at 80° C. Ethyl vinyl ether is added to the reaction followed by MeOH. The mixture was placed in the freezer for 1d to give beige ppt. The suspension was centrifuged and mother liquor was decanted. The residue was washed repeatedly with MeOH followed by drying in vacuum oven to give a beige solid product. .sup.1H NMR [500 MHz, DCO.sub.2D/CD.sub.2Cl.sub.2 (1:4)]: δ 6.42 (br, PA 6), 3.60 (s, PEG), 3.19-3.13 (m, PA 6), 2.15-2.12 (m, PA 6), 1.62-1.56 (m, PA 6), 1.48-1.42 (m, PA 6), 1.29-1.23 (m, PA 6).
Example 7: Bioactive Synthetic Copolymers Examples—Poly(ε-Caprolactone)-Biomolecule Copolymers as Bioadditives for Human Skin and Bone Tissue Regeneration
[0306] A series of poly(s-caprolactone) (PCL) copolymers with various pegylated biomolecules such as collagen mimics (COL), integrin binding peptides and glycosaminoglycans (GAGs), have been synthesized and characterized. Such copolymers can be used to create tissue regenerating scaffolds in human for either bone or skin regeneration.
[0307] PCL is the synthetic polymer of choice in this example due to its ability to biodegrade in human body without causing local acidity like poly(lactic acid) (PLA) and the material is biocompatible. The incorporation of biomolecules such as heparin oligosaccharide DP12 into PCL would be desired for bone scaffold materials that enable bone tissue regeneration while the material itself biodegrades in the body eventually.
[0308] However, the problem with DP12 is that it is extremely hygroscopic and coating it on PCL tubes before implantation is not ideal as there is no means of controlling the homogeneity of the coating prior to use. Furthermore, as the GAG is highly water soluble, it has a high tendency to leach into the body upon implantation and not remain on the implant itself, for BMP binding where bone tissue regeneration is required. Hence, in this example, PCL copolymers with DP12 macromonomers are created which can then be added to base polymer PCL and fabricated into whole bone implants. By chemically linking DP12 to PCL itself before blending the polymers into base polymer PCL, the present disclosure has advantageously shown that it is possible to localize the GAG on the implant to prevent undesirable side effects such as bone tissue regeneration at any other locations of the body except the implant site.
[0309] Apart from GAGs, peptides such as integrin binders or collagen fragments, are also useful toward bone tissue regeneration. In fact, these biomimetic molecules (GAGs, integrin binders, collagen fragments) are not only useful for bone tissue regeneration but also skin tissue regeneration. Hence, the polymers synthesized in accordance with various embodiments disclosed herein not only serve as bone scaffolds, they can also be employed in skin scaffolds to allow for skin tissue regeneration in patients with large area wounds such as burns patients.
[0310] Extracellular peptides such as RGD are able to function as integrin binders to encourage cell attachment, migration and proliferation. RGD sequence is mostly found in native collagen but is often inaccessible for integrin binding until the collagen is denatured. Hence, it would be useful to isolate RGD sequence from collagen and apply it to the tissue regeneration products directly. RGD can be advantageously used in skin and bone tissue regeneration products as it is able to induce cell growth and angiogenesis through its integrin binding ability. However, as with many biomolecules, it is extremely hygroscopic. In fact, it is more hygroscopic than DP12 where exposure to humid air for 5-10 min turns it from a crystalline solid to liquid immediately. Without anchoring RGD to a synthetic polymer to increase its ease of handling, it is extremely difficult to apply the peptide to the site of repair, especially in a bone defect.
[0311] 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 tissues makes it a desirable scaffold material for bones. In this example, collagen fragments or collagen mimics (COL) were also used in the PCL scaffolds to increase the biocompatibility and biomimetic properties of the overall PCL-based scaffold material. Some collagen mimics such as DGEA and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences have been used as the bioactive moieties (see Scheme 7). Without being bound by theory, it is believed that DGEA supports mesenchymal stem cell adhesion and differentiation to osteoblasts. Apart from being good bone scaffold materials, collagen also make excellent skin scaffold materials since the extracellular matrix (ECM) is largely collagenous material. Hence, the same PCL-COL polymers can also be used as bioadditives to the PCL scaffold matrix for application in skin scaffolds.
##STR00031## ##STR00032##
Thermal Stability
[0312] Thermal stability of the biomacromonomer NBPEGRGD and the NBPCL-PEGRGD ROMP polymer has been measured and compared. Pure RGD shows thermal degradation at 181° C. whereas the macromonomer NBPEG.sub.3400RGD shows a 2-phase mass loss at 220° C. (RGD loss) and 398° C. (PEG loss). However, on copolymerization with NBPCL macromonomer, the overall bioactive synthetic polymer only shows 1 significant mass loss at 393° C., indicating overall improvement in stability of RGD moiety on the synthetic polymer. In fact, thermal stability of NBPCL macromonomer is also improved with the inclusion of NBPEG.sub.3400RGD macromonomer in the overall bioactive synthetic polymer product (
Biocompatibility
[0313] Copolymers were created using PCL as the synthetic polymer and a range of peptides of different properties as the bioactive macromonomer, namely collagen fragment (GPHyp).sub.3: GPHP; collagen mimic: DGEA and integrin binding peptides: SRGDS and RGD. The copolymers were subsequently blended with medical grade PCL and 3D-printed into sheets, before tested for cell viability and biocompatibility against commercially available wound dressing namely Allevyn, which is most commonly used in hospitals.
[0314] As shown in
[0315] Alkaline phosphatase (ALP) assays to check for osteoblast activity was conducted on the materials to determine the materials' compatibility with BMP-2, a bone growth factor necessary for bone tissue growth relative 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 a pluripotent myoblast C.sub.2Cl.sub.2 cell line. PCL-RGD showed excellent ALP activity compared to PCL. At 20% blending in pure PCL, PCL-RGD showed 4 times higher activity after 72 h of incubation. PCL-(GPHyp) also showed improved activity over PCL (
[0316] In summary, a series of polymers that bear bioresorbable PCL side chains and bioactive molecules such as heparin sulfate DP12, collagen mimics or fragments and integrin binders such as RGD have been developed. These copolymers enhance both bone and skin tissue regeneration and would serve as useful bioactive ingredients for tissue regenerating scaffold materials. A general strategy for creating a scaffold material would be through blending of such bioactive ingredients with a base material of similar nature, that is, medical grade PCL itself.
Experimental Procedures
General Procedure
[0317] Ring opening metathesis polymerization (ROMP) reactions, PCL macromonomer (NPH-PCL) synthesis, bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. NBPEG and NPH synthesis was carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used in the glovebox are 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, .sup.iPr.sub.2EtN were 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.
[0318] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as solvent for all biomolecule-based macromonomers. CDCl.sub.3 is used as solvent for PCL macromonomer. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used.
[0319] Synthesis of NBPEG and NBPEGRGD are described in Example 6.
[0320] For BMP-2 binding study, scaffolds were sterilized using 100% ethanol, then rinsed in sterile water before being 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 for 20 minutes at room temperature. BMP-2 alone was added directly to empty wells. Cells were seeded at 2×104 cells/cm.sup.2 in 1 mL of 5% FCS media, directly onto the scaffolds and into the surrounding well. Cells were incubated for 72h (37° C., 5% CO.sub.2) prior to ALP assay.
Synthesis of NPH-PCL Macromonomer by ROP
[0321] NPH-PCL macromonomers with different degree of polymerization (DP) were prepared by ROP. As an 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).sub.2 (0.0037 g, 9.1 μmol) was added to the mixture and the resultant solution was stirred at 110° C. for 90 min and precipitated into methanol. The methanolic solution was then placed in the freezer overnight to result in white precipitate which was filtered and washed with methanol. The residue is then dried under vacuum overnight. GPC analysis (THF): M.sub.n=5,613, PDI=1.08, yield 0.4478 g.
[0322] A standard solution of Sn(Oct).sub.2 of concentration 91 μmol/ml, was prepared and used for ROP reactions.
Synthesis of NBPEG.SUB.1000.DGEA as Representative Prep for PEG 1000, 3,400 and 6,000
[0323] DGEA (with carboxylic acid on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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 a 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.
[0324] .sup.1H NMR (500 MHz, CD.sub.3OD): δ 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).
Synthesis of NBPEG.SUB.1000.(GPHyp).SUB.3 .as Representative Prep for Collagen Fragments of Glycine, Proline and Hydroxyproline in Varying Sequence and Chain Length Up to n=6, PEG 1000, 3,400 and 6,000
[0325] (GPHyp).sub.3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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 freeze 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 (0.22 g, yield 50%) was obtained upon filtration and solvent evaporation.
[0326] .sup.1H NMR (500 MHz, CD.sub.3OD): δ 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).
Synthesis of NBPEG.SUB.3400.DP12
[0327] DP12 (0.0302 g, 8.5 μmol) was dissolved in MeOH (1.5 ml) in an 8 ml scintillation vial. .sup.iPr.sub.2EtN (3 μl, 17 μmol) was added and mixture 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., followed by addition of the solution A to give suspension B. Suspension B was then added to NBPEG.sub.3400NH.sub.2 (0.025 g, 7.05 μmol) and stirred at r.t. for 24 hr. The resulting pale yellow mixture was then concentrated with solvent evaporation to give beige mixture. The mixture was dispersed into Et.sub.2O and the mixture placed in freezer for 84 h. The Et.sub.2O was decanted and MeOH was added to the residue to give a pale yellow solution which resulted in an orange oil on filtration of solution followed by solvent evaporation.
[0328] .sup.1H NMR (500 MHz, CD.sub.3OD): δ 7.81 (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).
Typical Procedure for ROMP of NPH-PCL Macromonomer and NBPEGz.SUB.3400.RGD Macromonomer as Representative Prep for PCL-Peptide Type Copolymers
[0329] NBPEG.sub.3400RGD macromonomer (0.2 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PCL (0.05 g). THF (0.021 M wrt. NPH-PCL) is added and the mixture stirred at r.t. till a clear solution is obtained. A solution of catalyst 1 or 2 in THF (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 2 h at 30° C. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a white ppt. The mixture was centrifuged and mother liquor was decanted. The residue was resuspended in methanol, centrifuge followed by decanting mother liquor again, to wash the residue. The washing with MeOH was carried out 3 times before the final residue was dried overnight in a vacuum oven.
[0330] For PCL-RGD copolymer, GPC analysis (THF): M.sub.n=140,000, PDI=1.21.
Typical Procedure for ROMP of NPH-PCL Macromonomer as Representative Prep for PCL Homopolymer
[0331] NPH-PCL macromonomer (0.63 g) is weighed into a 10 ml scintillation vial followed by addition of THF (0.021 M wrt. NPH-PCL) and the mixture stirred at 27° C. till a clear solution is obtained. A solution of catalyst 1 or 2 in THF (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 2 h at 27° C. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (5 ml) and the mixture placed in the freezer for 1 d to give a white ppt. The mixture was filtered and washed with MeOH repeatedly before the final product was dried overnight in a vacuum oven.
[0332] .sup.1H NMR (500 MHz, CDCl.sub.3): 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.sub.n=225,000, PDI=1.09. TGA: 315.7° C.
Typical Procedure for ROMP of NPH-PCL Macromonomer and NB-mPEG.SUB.5000 .Macromonomer as Representative Prep for PCL-mPEG Type Copolymers
[0333] NB-mPEG.sub.5000 macromonomer (0.1 eq.) is weighed into a 10 ml scintillation vial followed by addition of NPH-PCL (0.5 g). THF (0.021 M wrt. NPH-PCL) is added and the mixture stirred at 45° C. till a clear solution is obtained. A solution of catalyst 1 or 2 in THF (1.25 mol %, 0.05 M) is added to the solution and the reaction is stirred for 2 h at 45° C. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (5 ml) and the mixture placed in the freezer for 1 d to give a white ppt. The mixture was filtered and washed with MeOH repeatedly before the final product was dried overnight in a vacuum oven.
[0334] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 4.07-4.04 (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˜=171,000, PDI=1.21. TGA: 316.4° C.
Example 8: Bioactive Synthetic Copolymers Examples—Polyamide-Peptide Brush Polymers for Use as Bioadditives in Biomedical Devices
[0335] A series of polyamide (PA) copolymers with various pegylated biomolecules such as collagen mimics and integrin binding peptides have been synthesized and characterized using ring opening metathesis polymerization. The brush polymers can be blended with polymers similar to that on the pendant arms to create bioactive materials for use in biomedical devices such as catheters, plastic surgery implants, prosthetic parts, cartilage joint implants etc.
[0336] This example reports another type of bioactive brush polymer using polyamide (PA) and collagen mimics, for use in biomedical devices that are polyamide-based.
[0337] Polyamides (PA) is the synthetic polymer of choice in this example. Polyamides (PA) such as PA 6, PA 12, PA 6,6 are silky thermoplastics that have found significant biomedical applications such as in tubings, surgical guides, prosthetics, sutures and ligament, tendon repair. It is believed that PA has the lowest microbial contamination compared to other materials. PA type polymers can be mixed with a wide variety of additives to achieve many different property variations, allowing the devices to be fabricated using a wide variety of material processing methods such as melt extrusion, 3D-printing and injection molding. However, polyamide chain is polymerized under harsh conditions of high temperature with reduced pressure. This polymer is also insoluble in most solvents, adding to the difficulty of this material preparation.
[0338] PA-based materials with collagen fragments and mimics have been created using ring opening metathesis polymerization (ROMP) techniques. The biocompatibility of collagen and its similarity to human tissues makes it an ideal material for biomedical device. However, as with many biomolecules, it is extremely hygroscopic. Without anchoring the collagen fragments or mimics to a synthetic polymer to increase its ease of handling, it is nearly impossible to create an implant or biomedical device for insertion into human body. Although cross-linked collagen is often used in wound care products, they are also very hygroscopic, existing as gels upon absorption of moisture, rendering them too weak for use as implantable devices on their own. Furthermore, full-length human collagen requires complex synthesis and often show poor solubility in buffers. Short collagen-mimic peptide sequences or fragments which include crucial peptide sequences at a fraction of the length has been used to elicit similar biological response to their full-length collagen counterparts. Collagen mimics such as DGEA (Asp-Gly-Glu-Ala) and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences are incorporated into the synthetic polymer. DGEA is capable of promoting cell adhesion, spreading and osteogenic differentiation which will be advantageous for applications in both skin and cartilaginous bone regeneration.
[0339] On the other hand, polyamide being an FDA-approved polymer for biomedical device usage, still triggers inflammatory responses in the host body as it is after all, a foreign material. Foreign Body Reaction (FBR) may be induced by polyamide, resulting in inflammation around implantation site. Without being bound by theory, it is believed that the use of collagen fragments or collagen mimics (COL) in the polyamide polymeric material can help to increase the biocompatibility and biomimetic properties of the overall polyamide based implant or device. Some possible collagen mimics used include DGEA and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences, in any order. In fact, collagen fragments make excellent skin and bone regeneration materials since the extracellular matrix (ECM) and bone is largely collagenous material. The bone especially, is mineralized collagen and cartilage joints are mostly collagen fibers, glycosaminoglycans and proteoglycans. Hence, the use of collagen-modified polyamide in joint implants may be particularly useful in helping the joints heal by stimulating collagen regeneration at the implantation site. This exact property also makes it suitable for use in plastic surgery implants where cartilaginous bones are required such as in rhinoplasty implants.
[0340] With both the biomacromonomer and synthetic PA macromonomer, the final bioactive polymer is prepared by ROMP using Grubbs type catalysts (Scheme 8).
##STR00033## ##STR00034##
[0341] Upon synthesis, metal catalyst removal and characterization, the bioactive polymers are blended with medical grade PA of choice, depending on application, and processed by either fused filament fabrication (fff) or fused deposition modelling (FDM) type 3D printing, melt extrusion, melt blowing or electrospinning into relevant shapes and tested for biocompatibility. TGA-DSC analyses on the synthesized copolymers are typically carried out before material processing to ascertain thermal properties such as T.sub.g and degradation temperature of material, prior to processing.
[0342] Thermal Stability
[0343] Thermal stability of the biomacromonomer NB-PEG-(GPHyp) and the PA 6 ROMP polymers have been measured and compared (
Biocompatibility
[0344] Biocompatibility tests using human fibroblasts Hs27, were carried out on 3 of the PA-collagen materials, PA6-(GPHyp).sub.3, PA6-(PHypG).sub.3 and PA6-DGEA, where both (GPHyp).sub.3 and (PHypG).sub.3 are collagen fragments and DGEA is a collagen mimic. The bioactive polymers were blended with medical grade PA12, electrospun into sheets of fibers, sterilized with 70% EtOH, dried and incubated for 72 h with human skin fibroblasts (Hs27) before being checked for cell viability using Celltitre-Glo assays. From the cell viability data (
[0345] In summary, a series of brush polymers with polyamide 6 and pegylated biomolecules as side chains, on a poly(norbornene dicarboximide) backbone have been developed, via ROMP technologies. The general strategy presented here forms a method to create bioactive synthetic polymers 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. In this case, the base polymer is polyamide. The synthetic PA6 side chain helps make the biomolecule more compatible with the base polymer PA12, 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. Preliminary cell viability tests showed improvements in cell viability from PA6-collagen materials, over PA6 polymers without the biomolecules and significantly greater improvement in cell viability over pure medical grade PA12. Excellent human skin fibroblast cell growth was observed for PA-collagen material, relative to pure PA12.
Experimental Procedures
General Procedure
[0346] Ring opening metathesis polymerization (ROMP) reactions and bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. PA 6 macromonomer synthesis was carried out under positive N.sub.2 flow on bench. Reactions to obtain NBPEG and N-(Carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP) were carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used are anhydrous and used as purchased. Grubbs catalyst was purchased from Sigma Aldrich and peptides were purchased from Biomatik Inc. PEG diamine were purchased from Alfa Aesar (1,000 and 3,400) or Sigma Aldrich (6,000). HOBT, HBTU, .sup.iPr.sub.2EtN 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.
[0347] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using CD.sub.3OD as solvent for all biomolecule-based macromonomers. DCO.sub.2D/CD.sub.2Cl.sub.2 (1:4) is used as solvent for polyamide peptide macromonomer.
Synthesis of NBPEG macromonomer body (for H.SUB.2.N-PEG-NH.SUB.2 .1000, 3,400 and 6000)
[0348] 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 is 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 (500 MHz, 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).
Synthesis of NBPEG.SUB.1000.DGEA as Representative Prep for PEG 1000-6,000
[0349] DGEA (with carboxylic acid on E and A protected with OMe) (0.109 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 0.52 mmol) was added and the 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 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 a yellow oily mixture. The mixture was dispersed into Et.sub.2O and the mixture 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 (500 MHz, CD.sub.3OD): δ 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).
Synthesis of NBPEG.SUB.1000 .(GPHyp).SUB.n .(n=3) as Representative Prep for Collagen Fragments of Glycine, Proline and Hydroxyproline in Varying Sequence and Chain Length Up to n=6, PEG 1000-6,000
[0350] (GPHyp).sub.3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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 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 a beige mixture. The mixture was dispersed into Et.sub.2O and placed in freezer 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 (0.22 g, yield 50%) was obtained upon filtration and solvent evaporation. .sup.1H NMR (500 MHz, CD.sub.3OD): δ 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).
Synthesis of N-(Carboxypentyl)-cis-5-norbornene-exo-2,3-dicarboximide (NCP)
[0351] 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 Et.sub.3N (410 μL, 2.92 mmol). The flask was fitted with a Dean-Stark trap and heated to reflux for 4h. The mixture was then allowed to cool to room temperature and diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with 1 M aqueous HCl (2×20 mL). The organic layer was washed with saturated aqueous NaCl (20 mL), dried (Na.sub.2SO.sub.4), filtered, and concentrated under reduced pressure to provide NCP as a pale yellow solid. .sup.1H NMR (500 MHz, CD.sub.3OD, 25° C.) δ 6.26 (t, 2H, J=2.0 Hz), 3.44 (m, 2H), 3.25 (m, 2H), 2.66 (d, 2H, J=1.0 Hz), 2.32 (t, 2H, J=7.2 Hz), 1.63 (m, 2H), 1.55 (m, 2H), 1.46-1.51 (m, 1H), 1.33 (m, 2H), 1.19 (d, 1H).
Synthesis of NCP-PA6 Macromonomer by ROP
[0352] NCP-PA6 macromonomers with different degree 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.sub.2 pressure. Deionized H.sub.2O (5 ml) with H.sub.3PO.sub.3 (0.081 g) were added to the mixture and the resultant mixture was heated at 170° C. for 30 min and maintained at 240° C. for 4 h. H.sub.2O was removed by distillation and the reaction was heated at 240° C. under vacuum for another 2 h. A beige solid was precipitated out from MeOH, filtered and the residue washed repeatedly with MeOH to yield NCP-PA6 upon drying overnight in a vacuum oven. .sup.1H NMR [500 MHz, DCO.sub.2D/CD.sub.2Cl.sub.2 (1:4)]: δ 6.42 (br, PA 6), 6.28 (s, 2H, NCP), 3.42 (s, 2H, NCP), 3.14-3.12 (m, PA 6), 2.67 (s, 2H, NCP), 2.14-2.12 (m, PA 6), 1.56-1.53 (m, PA 6), 1.46-1.44 (m, PA 6), 1.29-1.25 (m, PA 6).
Typical Procedure for ROMP of NCP-PA6 Macromonomer and NBPEG.SUB.3400.DGEA Macromonomer as Representative Prep for PA-Peptide Type Copolymers
[0353] NBPEG.sub.3400DGEA macromonomer (0.2 eq.) was weighed into a 4 ml glass vial followed by addition of NCP-PA6 (0.12 g). CH.sub.3CO.sub.2H (0.021 M wrt. NCP-PA6) was added and the mixture stirred at 80° C. till a clear solution is obtained. A solution of catalyst 2 in CH.sub.2Cl.sub.2 (1.25 mol %, 0.05 M) was added to the solution and the mixture is stirred for 24 h at 80° C. Ethyl vinyl ether is added to the reaction followed by MeOH. The mixture was placed in the freezer for 1 day to give a beige ppt. The suspension was centrifuged and mother liquor was decanted. The residue was washed repeatedly with MeOH followed by drying in vacuum oven to give a beige solid product of PA6-DGEA copolymer. .sup.1H NMR [DCO.sub.2D/CD.sub.2Cl.sub.2(1:4), 500 MHz, 25° C.]: δ 6.42 (br, PA6), 3.60 (s, PEG), 3.19-3.13 (m, PA6), 2.15-2.12 (m, PA 6), 1.62-1.56 (m, PA6), 1.48-1.42 (m, PA6), 1.29-1.23 (m, PA6).
Example 9: Bioactive Synthetic Copolymers Examples—Antibiotic-Containing Polystyrene for Use in Tissue and Serum Handling Devices
[0354] Brush polymers containing pegylated antibiotics and polystyrene were created for use as antimicrobial additives in medical use polystyrene to create non-leachable 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 where the tissue or serum is held in, or coated on the device, which tends to be a costlier approach.
[0355] 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.
[0356] 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 especially, is very stable towards gamma and e-beam irradiation, amongst other common medical device polymers, making it a very popular material for tissue handling devices since these two sterilization techniques are the most effective methods for sterilization prior to use. Furthermore, the high clarity in the polymer allows its use in tissue and serum handling devices to enable visual inspection of contents from exterior of device.
[0357] In this strategy, antibiotics is tethered on a polyethylene glycol (PEG) chain that bears a norbornene-exo-dicarboximide (NB) moiety to create a biomacromonomer, followed by ROMP with a polystyrene-bearing norbornene-exo-dicarboximide synthetic macromonomer, to create that eventual antibiotic-containing polystyrene bioadditive. This bioadditive can then be blended into base medical grade polystyrene for device fabrication.
[0358] In penicillin class of antibiotics, the mechanism of action is in the β-lactam ring where the ring binds to the enzyme transpeptidase, preventing the bacteria from forming crosslinks in its cell walls. Crosslinking in peptidoglycans is required for cell wall formation in bacteria cells. By inhibiting cell wall production, bacteria cells die rapidly. Hence, the β-lactam ring of penicillin should be left exposed to bacteria cells for an anti-bacterial effect. Penicillin was chosen to tether via its carboxylic acid terminal, which is fairly distant from the β-lactam ring, thus allowing its reach to bacteria cells. On contact of bacteria cells with the penicillin containing polystyrene, the cells bind to penicillin and its cell walls break as a result of this contact, thus killing the bacteria cells.
[0359] Ciprofloxacin (CIF) is a fluoroquinolone-based broad spectrum antibiotic, especially active against gram negative bacteria such as P. aeruginosa. The fluoroquinolone ring binds to DNA gyrase, an essential bacteria enzyme, preventing bacteria cells from replicating. By tethering CIF via its carboxylic terminal and exposing its fluoroquinolone ring in the side arms of the brush polymer (Scheme 3.2), CIF binding sites are allowed to be available for bacteria cell binding, when the cells comes into contact with the polymer surface, thereby killing the bacteria cells present in the sample containers.
[0360] Aminoglycosides are broad-spectrum antibiotics that are commonly used as anti-infectives in clinical settings. Such antibiotics are bactericidal and contain hydrophilic saccharide units bearing multiple hydroxy and amino functionalities. Antibiotics that are aminoglycoside-based include streptomycin, ribostamycin and gentamicin. These can be connected to the NBPEG moiety via the —CH.sub.2OH (strep), —CH.sub.2NH.sub.2 (rib) or —CH(CH.sub.3)NH.sub.2 (gen) group on the antibiotic molecule, leaving the binding sites on the molecule exposed to bacteria cell binding. Such antibiotics bind to bacteria ribosomal subunit, preventing them from synthesizing essential proteins for growth. Polymers bearing these antibiotics are useful for tissue culture devices apart from penicillin, since they are part of the standard antibiotic recipe for cell culture media. Once the antibiotic-bearing macromonomer is synthesized, they can be copolymerized using ROMP techniques, with a polystyrene-bearing macromonomer, to create the desired brush polymer 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 for blending in base medical grade polystyrene for medical device fabrication.
##STR00035## ##STR00036##
[0361] In summary, antibiotic bonded polystyrene copolymer is developed for the creation of antibiotic containing polystyrene tissue handling devices where the antibiotics are covalently bonded to polystyrene and cannot leach out from the material. This is achieved by using ring opening polymerization metathesis technologies on pegylated antibiotics on norbornene dicarboximide linkers and polystyrene on norbornene dicarboximide linkers. The result is a brush polymer with pendant pegylated antibiotics and polystyrene where the active group on the antibiotic molecule is exposed to bacteria cell binding, for bactericidal effects.
Experimental Procedures
General procedure
[0362] Ring opening metathesis polymerization (ROMP) reactions and bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. NBPEG and NBPS syntheses were carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used in the glovebox are 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). Amoxicillin, Ciprofloxacin, Ribostamycin, HOBT, HBTU, .sup.iPr.sub.2EtN were 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.
[0363] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as solvent for all biomolecule-based macromonomers. CDCl.sub.3 is used as solvent for PS macromonomer. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used.
[0364] Synthesis of NBPEG and NB—PS are described in Example 6.
Synthesis of NBPEG.SUB.1000.CIF as representative prep for PEG 1,000-6,000
[0365] Ciprofloxacin (0.0866 g, 0.26 mmol), was suspended in MeOH (2.5 ml) in a 4 ml vial, in the glovebox. .sup.iPr.sub.2EtN (91 μL, 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 CIF solution from (A), to give suspension (B). Suspension B is then added to NBPEG.sub.1000 (0.25 g, 0.218 mmol) in a 40 ml vial and the mixture stirred at room temperature overnight to give a pale yellow solution with white suspension. The 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 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 freezer for another 48 h to give a yellow oil at the bottom of flask. The ether was decanted and MeOH (3 ml) was added to give a yellow solution with white ppt. The mixture was passed through a syringe filter and the clear filtrate was evaporated to dryness to give a yellow oil of NBPEGCIF. .sup.1H NMR (500 MHz, 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).
Representative Synthesis for PS-Antibiotic Polymer by ROMP Using NBPEGCIF as Example
[0366] NBPEG.sub.1000CIF macromonomer (0.1 eq) was weighed into a 4 ml glass vial followed by addition of NB-PS (0.050 g, 0.030 mmol). THF (0.05 M wrt. NB-PS) was added and the mixture was stirred at r.t. till a clear solution is obtained. A solution of catalyst 2 (referred to in Example 5) in THF (1.25 mol %, 0.05 M) was added to the solution and the mixture was stirred for 2 h at r.t. before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in Methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was washed repeatedly with MeOH followed by drying under vacuum to give a white powdered polymer. .sup.1H NMR (500 MHz, CDCl.sub.3): δ 6.99-7.15 (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.sub.n=30,056, PDI=1.36
Example 10: Bioactive Synthetic Copolymers Examples—Polylactide-Biomolecule Copolymers as Bioadditives for Human Skin and Bone Tissue Regeneration
[0367] A series of brush copolymers containing polylactide (PLA) side chains and pegylated biomolecules including integrin binding peptides, collagen mimics or fragments (COL) and glycosaminoglycans (GAGs), were synthesized via ring-opening metathesis polymerization (ROMP). These copolymers can be utilized as bioadditives in scaffold materials for either human skin or bone tissue regeneration.
[0368] Biomolecules used in this example include heparin oligosaccharide (HS) DP12, DP14, integrin binding peptide such as RGD, collagen fragments with repeating units of glycine, proline and hydroxyproline (G, P, Hyp) in varying sequences and length, and collagen mimic DGEA.
[0369] Polylactide (PLA) is the synthetic polymer of choice in this example as it degrades under physiological conditions to form non-toxic lactic acid which is also present in the human body, i.e. bioresorbable polymer. Due to its biocompatibility and good processability, PLA and its copolymers are commonly being used in medical implants and tissue engineering. Bioactive molecules which can promote skin or bone tissue regeneration are then incorporated into the final polymer via copolymerization in our approach.
[0370] Integrin-binding peptides such as RGD (Arg-Gly-Asp) which are found in several extracellular matrix proteins have been identified as an important motif for cell recognition and cell adhesion. The 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, by covalently attaching the peptide onto the synthetic PLA polymer, it will increase its stability and the ease of handling. These RGD-containing polymers can be subsequently blended with base material to create scaffolds for skin or bone regeneration.
[0371] In addition to RGD peptides, polymers containing collagen mimics or fragments have also been synthesized. Collagen is the most abundant protein in the extracellular matrix and has been widely used in biomaterials to increase biocompatibility and encourage tissue regeneration. However, full-length human collagen requires complex synthesis and often show poor solubility in buffers. Short collagen-mimic peptide sequences or fragments which include crucial peptide sequences at a fraction of the length has been used to elicit similar biological response to their full-length collagen counterparts. Collagen mimics such as DGEA (Asp-Gly-Glu-Ala) and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences are incorporated into the synthetic polymer. Without being bound by theory, it is believed that DGEA promotes cell adhesion, spreading and osteogenic differentiation which will be advantageous for applications in both skin and bone regeneration.
[0372] Heparin sulfate (HS) is a GAG having repeating disaccharide units that have been heavily modified with sulfate groups. Without being bound by theory, it is believed that HS chain of between 5 to 10 disaccharide units is the most active towards binding of bone morphogenetic proteins (BMP). Without being bound by theory, it is believed that HS directly regulates BMP-2-mediated differentiation of myoblasts onto osteoblasts. In particular, the HS fragment with hexa-disaccharide unites (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 rat models revealed improved bone tissue regeneration using DP12 as compared to controls of collagen sponge in polycaprolactone (PCL) tubes. HS interacts with angiogenic factors and induce vascularization. Vascularization is critical in tissue scaffolds to deliver oxygen and nutrients throughout the engineered tissue. In addition, HS can interact with growth factors that stimulates epithelial repair and encourage wound healing. PLA with HS molecules such as DP12 or DP14 incorporated would be desired for use in scaffold materials to allow for skin or bone tissue regeneration. However, HS molecules are highly hygroscopic and cannot be simply coated onto the polymer. The high aqueous solubility of HS also means that it may leach into the body and not remain on the desired site where tissue regeneration is required. In the present approach, macromonomers containing HS molecules are copolymerized with PLA macromonomer to prepare the bioactive copolymer which will be blended with base polymer PLA for skin scaffold or bone implant fabrication. This will ensure the HS molecules will be localized on the implant site and not induce undesirable effects in other parts of the body.
[0373] The final brush copolymers are prepared by ROMP (Scheme 10) using Grubbs type catalyst via copolymerization of PLA macromonomer with bioactive macromonomer.
##STR00037## ##STR00038##
Biocompatibility
[0374] To demonstrate the biocompatibility of the polymers, the materials were tested on human fibroblast cells in vitro. The bioactive synthetic polymer (PLA-RGD) was blended with commercial PLA as base material and electrospun into thin sheets. Commercial base polymer PLA (PLA-bulk) was used as control for this study. The sheets were then tested on human fibroblasts Hs27 and all tested materials showed good biocompatibility with high cell viability after 72 h (
[0375] In summary, a series of brush copolymers that have biodegradable PLA side chains and bioactive molecules such as integrin binding peptides, collagen mimics or fragments (COL) and heparin sulfate (HS) were synthesized. These bioactive polymers can be blended with base material, such as medical grade PLA, to create scaffold materials for use in skin or bone regeneration.
Experimental Procedures
General Procedure
[0376] Ring opening metathesis polymerization (ROMP) reactions and bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. PLA macromonomer (NPH-PLA) synthesis were carried out using standard Schlenk line techniques under nitrogen atmosphere. NBPEG and NPH synthesis was carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used in the glovebox are anhydrous and used as purchased. Grubbs second generation catalyst was purchased from Sigma Aldrich and peptides were purchased from Biomatik Inc. Catalyst 2 ((H.sub.2IMes)(pyr).sub.2(Cl).sub.2RuCHPh) was synthesized according to procedure provided in Example 6. PEG diamine were purchased from Alfa Aesar (1,000 and 3,400) or Sigma Aldrich (6,000). HOBT, HBTU, .sup.iPr.sub.2EtN were 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.
[0377] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD or D.sub.2O as solvent for all biomolecule-based macromonomers. CDCl.sub.3 was used as solvent for PLA macromonomer and ROMP polymers. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used.
[0378] Synthesis of NBPEG, NBPEGRGD, NBPEG(DGEA), NBPEG(GPHyp).sub.3 and NBPEGDP12 are described in Example 6.
Synthesis of NPH-PLA Macromonomer by ROP
[0379] NPH-PLA macromonomers with different degree of polymerization (DP) were prepared by ROP. As an 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).sub.2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times, and was 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 into cold MeOH twice. The mother liquor was decanted and the residue washed with MeOH, followed by drying in vacuum oven.
[0380] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 6.28 (br t, 2H), 5.27-5.08 (m, PLA), 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).
[0381] GPC analysis (THF): M.sub.n=2,471, PDI=1.20, yield 0.600 g.
[0382] Synthesis of NBPEG.sub.3400DP14
[0383] DP14 (0.0285 g, 8.4 μmol) was dissolved in MeOH/DMF (0.5 ml/1.0 ml) in an 8 ml scintillation vial. .sup.iPr.sub.2EtN (2.9 μl, 16.8 μmol) was added and mixture 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., followed by addition of the solution A to give suspension B. Suspension B was then added to NBPEG.sub.3400NH.sub.2 (0.025 g, 7.03 μmol) and stirred at r.t. for 24 h. The resulting pale yellow mixture was centrifuged to remove insoluble impurities. Supernatant was concentrated with solvent evaporation and then precipitated into cold Et.sub.2O and the mixture was placed in freezer for 24 h. Orange powder product (43.5 mg, yield 90%) was obtained upon filtration.
[0384] .sup.1H NMR (500 MHz, D.sub.2O): 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).
Typical Procedure for ROMP of NPH-PLA Macromonomer and NBPEG.SUB.1000.RGD Macromonomer as Representative Preparation for PLA-Peptide Type Copolymers
[0385] NBPEG.sub.1000RGD macromonomer (0.1 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PLA (0.05 g). THF (0.05 M wrt. NPH-PLA) is added and the mixture stirred at r.t. till 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 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a sticky solid. The mother liquor was decanted and the residue washed repeatedly with MeOH followed by drying in vacuum oven.
[0386] For PLA-RGD copolymer, .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA). GPC analysis (THF): M.sub.n=76,700, PDI=1.44.
Typical Procedure for ROMP of NPH-PLA Macromonomer and NBPEG.SUB.1000.(GPHyp).SUB.3 .Macromonomeras Representative Preparation for PLA-COL Type Copolymers
[0387] NBPEG.sub.1000(GPHyp).sub.3 macromonomer (0.1 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PLA (0.05 g). THF (0.05 M wrt. NPH-PLA) is added and the mixture stirred at 45° C. A solution of catalyst 2 in THF (1.25 mol %) is added to the solution and the reaction is stirred at 45° C. for 2 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give sticky solid. The mother liquor was decanted and the residue washed repeatedly with MeOH followed by drying in vacuum oven. .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA). GPC analysis (THF): M.sub.n=97,262, PDI=1.14.
Typical Procedure for ROMP of NPH-PLA Macromonomer and NBPEG.SUB.3400.DP14 Macromonomer as Representative Preparation for PLA-HS Type Copolymers
[0388] NBPEG.sub.3400DP14 macromonomer (0.1 eq.) is weighed into a 4 ml scintillation vial followed by addition of NPH-PLA (0.03 g). THF (0.02 M wrt. NPH-PLA) is added and the mixture stirred at 45° C. A solution of catalyst 2 in THF (1.25 mol %) is added to the solution and the reaction is stirred at 45° C. for 2 h. Ethyl vinyl ether is added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a sticky solid. The mother liquor was decanted and the residue washed repeatedly with MeOH followed by drying in vacuum oven.
[0389] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.27-5.08 (m, PLA), 3.60 (s, PEG), 1.97-1.47 (m, PLA).
Example 11: Bioactive Synthetic Copolymers Examples—PLGA-Peptide and -Oligosaccharides Polymers for Cartilage Tissue Regeneration
[0390] 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, have been pegylated and linked to PLGA, as side chains, on a poly(norbornene-exo-2,3-dicarboximide) backbone, via ring opening metathesis polymerization reactions. The resultant brush polymers can be used as bioadditives for cartilage tissue regeneration materials that are PLGA-based. Preliminary in vitro tests on the bioactive PLGA demonstrated excellent cell viability with some degree of cell proliferation at 72 h.
[0391] Biomolecules used in this example include ECM peptides such RGD, collagen fragments and collagen mimics that are known to regenerate cartilage tissues. The synthetic polymer of choice in this example is PLGA, a bioresorbable polymer that has properties between that of PLA and poly(glycolic acid) (PGA). The overall polymer created is shown to be thermally stable and bioactive polymer that is osteoinductive for use in cartilage implants.
[0392] Using the bioactive synthetic polymer technology in accordance with various embodiments disclosed herein, collagen-bearing synthetic polymers are created that allow collagen to be introduced to synthetic materials without loss in functionality of these collagen fragments. Furthermore, the PLGA side chains in these bioactive synthetic polymer helps increase the thermal stability of collagen and allows efficient blending of an otherwise hygroscopic collagen into base polymer PLGA, which is hydrophobic. The overall PLGA material is not only bioactive but thermally stable and mechanically strong, for material processing and use in meniscal cartilage implants.
[0393] PLGA is chosen as the synthetic polymer due to its better control of polymer crystallinity, melting point and load-bearing capabilities, over its homopolymer counterpart, PGA and PLA where PGA is more crystalline and higher melting than PLA. However, PLGA is non-osteoinductive despite its apparent biocompatibility. Hence, there is a need to introduce a stimulus on 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 by-products of the polymer are lactic acid and glycolic acid, both of which are non-toxic to human.
[0394] To enhance the softness of the material and its hydrophilicity, polyethylene glycol (PEG) is introduced into the bioactive synthetic polymer chain. This same strategy is used in tuning the softness/hardness of material. By adjusting PEG to PLGA side chain content in the bioactive synthetic polymer, as well as the PLGA-COL bioactive synthetic polymer ratio to PLGA base material, overall hardness of the material can be adjusted. This is especially important in articular cartilage implant. To further enhance the strength of the articular cartilage implant, lattice designs in additive manufacturing (AM) of the scaffold may be used/created. Material strength can be greatly enhanced through lattice designs using AM, whilst retaining porosity of material for enhanced osseointegration in scaffold, and lightweight of entire scaffold.
[0395] Apart from ECM peptides, heparin sulfate mimics such as highly sulfated glycosaminoglycans, can also be used to provide necessary stimulus required for articular cartilage regeneration. Glycosaminoglycans (GAGs) are heterogeneous polysaccharides ubiquitously found in mammalian tissues, and heparin sulfate (HS) is a highly sulfated GAG with enormous structural diversity that can interact with a plethora of proteins to regulate many physiological processes. Proteins that interact with HS include growth factors (GF), chemokines, enzyme inhibitors, extracellular matrix proteins and membrane-bound receptors. HS potentiates key GFs responsible for cell proliferation and differentiation, including bone morphogenetic protein BMP-2, which is important in bone growth, as well as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), which is important for blood vessel formation. By incorporating such oligosaccharides into PLGA, BMP binding properties are introduced to the polymer and the material can be used in cartilage implants. Through the combination of material design by advance chemical syntheses and scaffold design fabrication by additive manufacturing, different types of bone scaffolds are created for regeneration of different parts of the bone, depending on need. Alternatively, the scaffolds can also be fabricated by other material processing methods such as melt extrusion, injection molding and electrospinning.
[0396] With both the biomacromonomer and synthetic macromonomer, the final bioactive synthetic polymer is prepared by ROMP using Grubbs type catalysts (Scheme 11).
##STR00039## ##STR00040##
[0397] Upon characterization of bioactive polymers, TG-DSC analysis to ascertain melting point and degradation temperature of polymer is carried out. ICP-MS to ensure metal residues from ruthenium catalysts have been reduced to a minimal, below ISO10993 guidelines for metal catalysts in biomedical devices, is also carried out before material processing. Once these parameters have been ascertained, the materials can be processed into prototypes for in vitro testing to ascertain biocompatibility of material and cell viability.
Biocompatibility
[0398] To demonstrate the biocompatibility of the polymers, the materials on human fibroblast cells were tested in vitro. The bioactive synthetic polymer (PLGA-RGD) was blended with commercial PLGA as base material and electrospun into thin sheets. Commercial base polymer PLGA (PLGA-Bulk), PLGA ROMP homopolymer (PLGA-homo) and PLGA-mPEG.sub.5000 were used as controls for this study. The sheets were then tested on human fibroblasts Hs27 and all tested materials showed good biocompatibility with high cell viability after 72 h (
[0399] In summary, a series of biodegradable polymers that bear biodegradable synthetic polymer side chains of PLGA and bioactive side chains of extracellular matrix peptides or sulfated glycosaminoglycans were developed, for cartilage tissue regeneration. The materials can be processed by a wide variety of material processing methods such as melt extrusion, FFF or FDM type 3D-printing and electrospinning. Preliminary in vitro tests have demonstrated good cell viability and proliferation without the introduction of stem cells or growth factors.
Experimental Procedures
General Procedure
[0400] Ring opening metathesis polymerization (ROMP) reaction and bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. PLGA macromonomer (NPH-PLGA) synthesis were carried out using standard Schlenk line techniques under nitrogen atmosphere. NBPEG and NPH synthesis was carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used in the glovebox are 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, .sup.iPr.sub.2EtN were 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.
[0401] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD as solvent for all biomolecule-based macromonomers. CDCl.sub.3 is used as solvent for PLGA macromonomer. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used.
[0402] Synthesis of NBPEG and NBPEG(peptide) are described in Example 6.
Synthesis of NPH(PLGA) Macromonomer
[0403] NPH-PLGA macromonomers with different degree of polymerization (DP) were prepared by ROP. As an 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).sub.2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times, and was 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 washed with MeOH, followed by drying in vacuum oven.
[0404] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 6.28 (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.sub.n=4,336, PDI−1.27.
Synthesis of NBPEG.SUB.1000.(GPHyp).SUB.3 .as Representative Prep for Collagen Fragments of Glycine, Proline and Hydroxyproline in Varying Sequence and Chain Length Up to n=6, PEG 1000, 3,400 and 6,000
[0405] (GPHyp).sub.3 (0.213 g, 0.26 mmol) was dissolved in MeOH (2.5 ml) in glovebox. .sup.iPr.sub.2EtN (91 μl, 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 freeze 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 (0.22 g, yield 50%) was obtained upon filtration and solvent evaporation.
[0406] .sup.1H NMR (500 MHz, CD.sub.3OD): δ 6.33 (s, 2H), 4.73-.4.44 (br, 4H), 3.65 (s, 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).
Representative Synthesis for PLGA-PEG.SUB.3.4K.(GPHyp).SUB.3 .Polymer by ROMP Using NPH(PLGA) and NB-PEG.SUB.3.4k.(GPHyp).SUB.3 .as Example
[0407] NBPEG.sub.3.4K(GPHyp).sub.3 macromonomer (0.1 eq) was weighed into a 4 ml glass vial followed by addition of NPH(PLGA) (0.050, 0.012 mmol). THF (0.05 M wrt. NPH(PLGA) was added and the mixture was stirred at 40° C. till a clear solution is obtained. A solution of catalyst 2 in THF (1.25 mol %, 0.05 M) was added to the solution and the mixture was stirred for 2 h at 40° C. before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was washed repeatedly with MeOH followed by drying under vacuum. The obtained polymer is white powder.
[0408] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 5.20 (m, 1H, PLA), δ 4.8 (m, 2H, PGA), δ 3.63 (s, 4H, PEG), δ 1.56 (d, 3H, PLA). GPC analysis (THF): M.sub.n=65,166, PDI=1.23.
Example 12: Bioactive Synthetic Copolymers Examples—PMMA-Peptide Copolymers for Use as Bioadditives in Medical Implants
[0409] A series of brush copolymers containing poly(methyl methacrylate) (PMMA) side chains and biomolecules tethered on PEG moieties were synthesized via ring-opening metathesis polymerization (ROMP). Biomolecules may include collagen fragment or 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. These brush polymers can be blended with base polymer PMMA to create bioactive materials for use in biomedical implants such as bone cements, bone implants and craniofacial implants.
[0410] This example reports the facile synthesis of brush copolymers containing PMMA side chains and collagen mimics tethered on PEG moieties, for use as bioactive polymers in PMMA-based biomedical implants.
[0411] PMMA is the synthetic polymer of choice in this example as it is biocompatible, non-degradable and lightweight thermoplastic with good mechanical strength. It is the first synthetic polymer used in biomedical applications and has now been used in various medical implants such as in intraocular lens, rhinoplasty, dentistry and orthopedics. PMMA is also currently the most widely used alloplastic implant material for craniomaxillofacial reconstructions. PMMA polymers are often modified with varying amounts of additives or fillers to achieve the desired properties in the final materials. PMMA-based implant materials can be fabricated using traditional molding methods such as injection molding or extrusion and also 3D-printing. With the rapid progress in 3D printing technology, PMMA has been increasingly utilized in patient-specific biomedical applications for the fabrication of customized medical implant structures.
[0412] In this example, PMMA brush copolymers with collagen fragments or mimics have been synthesized using ROMP. Collagen is the most abundant protein in the extracellular matrix and has been widely used in biomaterials to increase biocompatibility and encourage tissue regeneration. However, full-length human collagen requires complex synthesis and often show poor solubility in buffers. Short collagen-mimic peptide sequences or fragments which include crucial peptide sequences at a fraction of the length may be used to elicit similar biological response to their full-length collagen counterparts. However, as with many biomolecules, these peptides are extremely hygroscopic. Without anchoring the collagen fragments or mimics to a synthetic polymer to increase its ease of handling, it is challenging to create an implant for insertion into human body. Furthermore, PMMA medical implants are foreign materials to the body and can trigger host immune response, leading to inflammation of tissues. PMMA itself also does not support osseointegration of the structure with other structures that it comes in contact with. Therefore, without being bound by theory, it is believed that by incorporating collagen fragments or collagen mimics (COL) in the PMMA polymer, it would help to increase the biocompatibility and biomimetic properties of the material. Some possible collagen mimics used include DGEA (Asp-Gly-Glu-Ala) and collagen fragments bearing varying lengths of glycine, proline and hydroxyproline sequences, in any order. Without being bound by theory, it is believed that DGEA promotes cell adhesion, osteogenic differentiation and osseointegration which will be advantageous for applications in bone or craniofacial implants.
[0413] The final brush copolymers are prepared by ROMP using Grubbs type catalyst (Scheme 12).
##STR00041## ##STR00042##
[0414] The bioactive PMMA polymers can be blended with medical grade PMMA, and processed by either extrusion, 3D-printing or electrospinning into relevant shapes and tested for biocompatibility.
Biocompatibility
[0415] To demonstrate the biocompatibility of the polymers, the materials were tested on human fibroblast cells in vitro. The bioactive synthetic polymer (PMMA-GPHyp) was blended with commercial PMMA as base material and electrospun into thin sheets. Commercial base polymer PMMA (PMMA-bulk), PMMA ROMP homopolymer (PMMA-homo) and PMMA-mPEG.sub.5000 were used as controls for this study. The sheets were then tested on human fibroblasts Hs27 and all tested materials showed good biocompatibility with good cell viability after 72 h (
[0416] In summary, a series of brush copolymers that have PMMA side chains and peptide molecules such as collagen mimics or fragments (COL) have been synthesized. These bioactive polymers can be used as bioadditives to create implant materials for use in orthopedic or cranioplasty.
Experimental Procedures
General Procedure
[0417] Ring opening metathesis polymerization (ROMP) reactions and bioactive macromonomer syntheses were carried out in a Vacuum Atmosphere glovebox under nitrogen atmosphere. PMMA macromonomer (NB-PMMA) synthesis were carried out using standard Schlenk techniques under nitrogen atmosphere. NBPEG and norbornenyl-functionalized ATRP initiator synthesis were carried out in a fumehood under atmospheric conditions, following procedures provided in Example 6. All solvents used in the glovebox are anhydrous and used as purchased. Grubbs second generation catalyst was purchased from Sigma Aldrich and peptides were purchased from Biomatik Inc. Catalyst 2 is synthesized according to the procedure provided in Example 6. PEG diamine was purchased from Alfa Aesar (1,000 and 3,400) or Sigma Aldrich (6,000). HOBT, HBTU, .sup.iPr.sub.2EtN were 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.
[0418] .sup.1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using MeOD or D.sub.2O as solvent for all biomolecule-based macromonomers. CDCl.sub.3 was used as solvent for PMMA macromonomer and ROMP polymers. Gel Permeation Chromatography was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity RI detector. THF was used in sample preparation and a flow rate of 1.0 ml/min at 40° C. was used.
[0419] Synthesis of NBPEG, NBPEG(DGEA) and NBPEG(GPHyp).sub.3 are described in Example 6.
Synthesis of NB-PMMA Macromonomer by ATRP
[0420] NB-PMMA macromonomers with different degree 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 the polymerization was conducted in a 70° C. oil bath for 3 h. The mixture was filtered through neutral alumina, precipitated into MeOH and filtered. The white powder was washed with MeOH followed by drying in vacuum oven overnight. .sup.1H NMR (500 MHz, CDCl.sub.3): δ 6.30 (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.sub.n=5,158, PDI=1.13.
Typical Procedure for ROMP of NB-PMMA Macromonomer and NBPEG.SUB.3400.(GPHyp).SUB.3 .Macromonomer as Representative Prep for PMMA-Peptide Type Copolymers for PEG of MW 1,000-6,000
[0421] NBPEG.sub.3400(GPHyp).sub.3 macromonomer (0.1 eq.) was weighed into a 4 ml scintillation vial followed by addition of NB-PMMA (0.05 g). THF (0.02 M wrt. NB-PMMA) was added and the mixture stirred at 45° C. till 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 at r.t. for 2 h. Ethyl vinyl ether was added to the reaction mixture followed by MeOH (3 ml) and the mixture placed in the freezer for 1 h to give a white precipitate. The mother liquor was decanted and the residue washed repeatedly with MeOH followed by drying in vacuum oven.
[0422] .sup.1H NMR (500 MHz, CDCl.sub.3): 3.65-3.59 (m, PMMA and PEG), 2.00-1.69 (m, PMMA), 1.07-0.75 (m, PMMA). GPC analysis (THF): M.sub.n=65,600, PDI=1.37.
Applications
[0423] The present disclosure provides a new modular synthesis method to create bioactive macromonomers rapidly for construction of bioactive copolymers with synthetic polymer of choice. Bioactive macromonomers may be easily copolymerized with another synthetic copolymer to form bioactive polymers 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 synthetic polymer 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 synthetic polymer 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 synthetic polymer 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 synthetic polymers 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 synthetic polymers disclosed herein may be used as bioadditives for skin or bone scaffold to create stimulus required for skin or bone tissue regeneration.
[0430] Embodiments of the bioactive synthetic polymer disclosed herein may be used in bone scaffolds to make PCL more “bone-like” and more biocompatible as a result. Studies have shown that osteocytes do not bind to PCL and only start binding to PCL after collagen is coated on PCL.
[0431] The present disclosure also provides a bioactive polyamide-peptide brush polymer that possesses both bioactivity to enhance biocompatibility and wound healing, together with structural integrity and mechanical strength. Embodiments of the polyamide-peptide brush polymers can be blended into polymers similar to the synthetic side chains as bioadditives, to create materials for use in medical devices such as catheters, plastic surgery implants, prosthetic parts, cartilage joint implants. Advantageously, embodiments of the bioactive polyamide-peptide brush polymer disclosed herein can be sterilized by heat before implantation and is long lived. Embodiments of the bioactive polyamide-peptide brush polymer disclosed herein allow product customization by 3DP as it is thermally stable. Embodiments of the bioactive polyamide-peptide brush polymer disclosed herein improve biocompatibility of polyamide which can trigger inflammatory response in body.
[0432] The present disclosure also provides a bioactive polystyrene made to be bactericidal whilst still possessing structural integrity and mechanical strength, like a polymer. In various embodiments, antibiotics are attached on the polymer by covalent bonding, therefore preventing leaching of antibiotics into media which can escape into environment if disposal is improperly managed. In various embodiments, there are active sites on the antibiotic molecule left exposed on polymer chain to allow bacterial cell penetration or bacteria RNA binding. Embodiments of the bioactive synthetic polymer disclosed herein allow for antibiotics to be blended into base material of synthetic polymer similar to the synthetic polymer side arms of copolymer, without phase separation. Embodiments of the antibiotic-polystyrene copolymers may be used as bioadditives for biomedical devices to provide bactericidal effect on device without additional drugs added.
[0433] The present disclosure also provides a bioactive poly(lactic-co-glycolic acid) that may be used as bioadditives in cartilage implant material fabrication. For example, the bioactive poly(lactic-co-glycolic acid) may be an acellular biodegradable cartilage scaffold with chondrocyte binding capability for cartilage regeneration. Embodiments of the polymer disclosed herein incorporate acellular implant material, therefore providing a lower regulatory handle and a faster path to market. In various embodiments, bioactivity is localized as biomolecules are covalently bonded to synthetic polymer and cannot leach out. In various embodiments therefore, premature metabolism of sulfated saccharides or unintended BMP binding elsewhere in the body is prevented. In various embodiments, biomolecules bound on polymers are able to bind BMP while staying immobilized on scaffold instead of leaching to other parts of body for undesirable side effects or being metabolized prematurely. Embodiments of the bioactive poly(lactic-co-glycolic acid) may be made to be more like polymer used in base material for device fabrication to allow effective blending of biomolecules into main polymer matrix. In various embodiments, phase separation of the bioactive poly(lactic-co-glycolic acid) is unlikely. Advantageously, in various embodiments, biomolecules show improved thermal stability on binding to polymer, allowing for material processing. For example, the polymer is 3D-printable by fused filament fabrication, fused deposition modelling and/or customized into a scaffold. Embodiments of the bioactive synthetic polymer disclosed herein allow for peptides and oligosaccharides to be blended into base material of synthetic polymer similar to the synthetic polymer side arms of copolymer, without phase separation.
[0434] The present disclosure also provides PMMA-peptide brush polymers that may be blended with base polymer PMMA as bioadditives, to create materials for use in medical devices such as orthopedic or cranial implants. Embodiments of the PMMA-peptide brush polymers disclosed herein allow implant customization and pre-operative fabrication by 3D printing, therefore improving “fit” and reducing surgical time. Embodiments of the PMMA-peptide brush polymers disclosed herein also improve biocompatibility of PMMA and reduce inflammatory response in body. In various embodiments, the biomolecules are covalently bonded to synthetic polymer and cannot leach out.
[0435] 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.