Polymeric composition

Abstract

A polymeric composition comprising (i) a plurality of monomers selected from (a) a carboxylic acryloyi monomer; (b) a sulfonic acryloyi monomer; (c) an amine acryloyi monomer; (d) a hydroxyl acryloyi monomer; (e) an alkyl acryloyi monomer; and (f) a polyalkylene hydroxyl acryloyi monomer; (ii) a divalent metallic crosslinking agent; and (c) a stabilizing agent is disclosed herein. Also provided are the use of said polymeric composition as a hydrogel coating material, a method of synthesizing the polymeric composition and the use of the hydrogel material.

Claims

1. A polymeric composition comprising a plurality of monomers in the presence of a stabilizing agent, wherein said monomers are selected from each of: a) an alkaline earth metallic cross-linked carboxylic acryloyl monomer; b) an alkaline earth metallic cross-linked sulfonic acryloyl monomer; c) an amine acryloyl monomer; d) a hydroxyl acryloyl monomer; e) an alkyl acryloyl monomer; and f) a polyalkylene hydroxyl acryloyl monomer, such that monomer a), monomer b), monomer c), monomer d), monomer e) and monomer f) are present in said polymeric composition.

2. The polymeric composition according to claim 1, wherein said carboxylic acryloyl monomer is a compound of formula I: ##STR00014## wherein m is an integer from 1 to 16; and R.sup.1 is independently selected from the group consisting of hydrogen, alkyl and alkenyl.

3. The polymeric composition according to claim 1, wherein said sulfonic acryloyl monomer is a compound of formula II: ##STR00015## wherein q is an integer of 1 to 16; and R.sup.2 is independently selected from the group consisting of hydrogen, alkyl and alkenyl.

4. The polymeric composition according to claim 1, wherein said amine acryloyl monomer is a compound of formula III: ##STR00016## wherein p is an integer of 1 to 12; R.sup.3 is independently selected from the group consisting of hydrogen, alkyl and alkenyl; and R.sup.4 is an alkyl.

5. The polymeric composition according to claim 1, wherein said hydroxyl acryloyl monomer is a compound of formula IV: ##STR00017## wherein r is an integer of 1 to 12; and R.sup.5 is independently selected from the group consisting of hydrogen, alkyl and alkenyl.

6. The polymeric composition according to claim 1, wherein said alkyl acryloyl monomer is a compound of formula V: ##STR00018## wherein s is an integer of 0 to 6; R.sup.6 is independently selected from the group consisting of hydrogen, alkyl and alkenyl; and R.sup.7 is alkyl.

7. The polymeric composition according to claim 1, wherein said polyalkylene hydroxyl acryloyl monomer is a compound of formula VI: ##STR00019## wherein n is an integer of 2 to 100; and R.sup.8 is independently selected from the group consisting of hydrogen, alkyl and alkenyl.

8. The polymeric composition according to claim 1, wherein said stabilizing agent is a zwitterionic monomer.

9. The polymeric composition according to claim 8, wherein said zwitterionic monomer is a protein-based acryloyl monomer.

10. The polymeric composition according to claim 9, wherein said protein-based acryloyl monomer is a peptide-based acryloyl monomer.

11. The polymeric composition according to claim 10, wherein said peptide-based acryloyl monomer is an amino acid-based acryloyl monomer.

12. The polymeric composition according to claim 11, wherein the amino acid of said amino acid-based acryloyl monomer is selected from the group consisting of a L-lysine, a D-lysine, a L/D-lysine, a glycine, a serine, a phenylalanine, a glutamic acid, an ornithine, an aspartic acid, a proline and a hydroxyproline groups.

13. The polymeric composition according to claim 1, wherein said alkaline earth metal is selected from the group consisting of beryllium, magnesium, calcium, strontium, barium and radium.

14. The polymeric composition according to claim 1, wherein said polymeric composition is a hydrogel coating material.

15. A method for forming a polymeric composition comprising a plurality of monomers in the presence of a stabilizing agent, wherein said monomers are selected from each of: a) an alkaline earth metallic cross-linked carboxylic acryloyl monomer; b) an alkaline earth metallic cross-linked sulfonic acryloyl monomer; c) an amine acryloyl monomer; d) a hydroxyl acryloyl monomer; e) an alkyl acryloyl monomer; and f) a polyalkylene hydroxyl acryloyl monomer, such that monomer a), monomer b), monomer c), monomer d), monomer e) and monomer f) are present in said polymeric composition, wherein the method comprises the steps of: i) providing a solution mixture of the alkaline earth metallic cross-linked carboxylic acryloyl monomer, the alkaline earth metallic cross-linked sulfonic acryloyl monomer, the amine acryloyl monomer, the hydroxyl acryloyl monomer, the alkyl acryloyl monomer, the polyalkylene hydroxyl acryloyl monomer, the stabilizing agent and a source of an alkaline earth metallic cross-linker; and ii) polymerizing said solution mixture with an acid initiator to thereby synthesize said polymeric composition.

16. The method according to claim 15, wherein said metallic cross-linker source is an alkaline earth metal salt selected from the group consisting of beryllium chloride, magnesium chloride, calcium chloride, strontium chloride, barium chloride, radium chloride, alkaline earth metal bromide, alkaline earth metal iodide, alkaline earth metal nitrate, alkaline earth metal sulfate, alkaline earth metal citrate and alkaline earth metal oxalate.

17. The method according to claim 15, wherein said acid initiator is a glutaric acid selected from the group consisting of -ketoglutaric acid and -ketoglutaric acid or an inorganic acid.

18. The method according to claim 15, wherein said solution mixture is provided in a solvent selected from an organic solvent or an aqueous solvent.

19. The method according to claim 15, further comprising the step of providing said stabilizing agent at a concentration in the range of 0 to 100 mol % to said solution mixture.

20. The method according to claim 15, wherein said polymerizing step is undertaken for a period of time in the range of 10 minutes to 60 minutes.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

(2) FIG. 1 shows the concentrations of the monomers and methacryloyl L-lysine (MLL) used in the synthesis of hydrogels A-J.

(3) FIG. 2 is a series of Field Emission Scanning Electron Microscopy (FESEM) images of hydrogels A-J. Scale bar: 10 m.

(4) FIG. 3 shows the non-specific protein adsorption on hydrogels A-J and nitrocellulose membrane (positive control).

(5) FIG. 4 demonstrates a S. aureus attachment on hydrogels A-J after 24 hours of culture. The bacteria were cultured on an uncoated petri dish as a control. Scale bar: 50 m.

(6) FIG. 5 shows an E. coli attachment on hydrogels A-J after 24 hours of culture. The bacteria were cultured on an uncoated petri dish as a control. Scale bar: 50 m.

(7) FIG. 6 demonstrates a fibroblasts attachment on hydrogels A-J after 24 hours of culture. The cells were cultured on an uncoated petri dish as a control. Scale bar: 100 m.

(8) FIG. 7 shows a viability of human primary fibroblast cultured for 24 hours in extracting medium used in incubating hydrogels A-J. The cells cultured in the absence of extracting medium were normalized as 100% viable.

(9) FIG. 8 shows a hemolysis of rabbit red blood cells incubated with hydrogels A-J. In this assay, red blood cells treated with 0.2% Triton-X were used as a positive control, and red blood cells in PBS were used as a negative control.

(10) FIG. 9 shows an image of hydrogel G after subcutaneous implantation in mice for two months.

(11) FIG. 10 shows a histological analysis of tissues near hydrogel G that was subcutaneously implanted for two months. The tissues were stained with Hematoxylin and Eosin (H&E).

(12) FIG. 11 shows the collagen formation in tissues near hydrogel G two months after subcutaneous implantation of hydrogel. The tissues were stained with Masson's trichrome.

EXAMPLES

(13) Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

(14) Materials

(15) All chemical reagents were from Polysciences, Inc. (Warrington, Pa., U.S.A), Sigma-Aldrich Corp. (St. Louis, Mo., U.S.A.) and Merck KGaA (Darmstadt, Germany), and were used as received unless otherwise stated. Staphylococcus aureus (S. aureus; ATCC No. 6538) and Escherichia coli (E. coli; ATCC No. 25922) were purchased from American Type Culture Collection (ATCC; Manassas, Va., U.S.A.) and reconstituted according to standard protocols. Mueller-Hinton broth (MHB) was purchased from BD Diagnostics (Sparks, Md., U.S.A.) and used to prepare the microbial growth medium according to the manufacturer's instructions. Phosphate-buffered saline (PBS, 10, pH=7.4) was purchased from 1st BASE (Singapore), and Luria broth containing 1.5% agar used for agar plate preparation was obtained from Media Preparation Unit (Biopolis Shared Facilities, A*STAR, Singapore).

Example 1

(16) Hydrogel Preparation

(17) Briefly, carboxylic monomer (1 mmol) and sulfonic monomer (1 mmol) were dissolved in 1 M CaCl.sub.2 solution in de-ionized water to obtain the Ca-complex of cross-linking monomers. Subsequently, tert-amine monomer (2 mmol), hydroxyl-terminated monomer (1 mmol), isopropyl-terminated monomer 5 (1 mmol) and PEG monomer (1 mmol) were mixed and added to the Ca-complex cross-linker solution. The zwitterionic monomer, MLL, was dissolved in 1 N HCl at various concentrations (0-100 mol %), and added to the monomer solution. The monomer solution was further diluted with 1 M CaCl.sub.2 solution, and used as a stock monomer solution. This monomer solution was further diluted with de-ionized water to the desired level, dependent on the optimal rigidity, hardness and flexibility of the gel achievable and polymerized using oxoglutaric acid initiator (10% solution in deionized water) under ultraviolet light for 30 minutes to obtain transparent hydrogels. The hydrogels obtained with different MLL concentrations were termed A-J (see FIG. 1).

(18) Field Emission Scanning Electron Microscopy (FESEM)

(19) FESEM analyses were performed with a field emission scanning electron microscopy (JEOL JSM-7400F) under an accelerating voltage of 4.0-6.0 keV. All the hydrogel samples were first polymerized using an initiator under ultraviolet light for 30 minutes to obtain transparent hydrogels. The morphologies of hydrogel were then examined with field emission scanning electron microscopy.

(20) FIG. 2 shows the FESEM images of hydrogels A-J that incorporated lysine functionality in the material at various concentrations for systematic studies. FESEM studies showed that the materials have an interconnected porous structure (FIG. 2). Based on FIG. 1, FIG. 2A is hydrogel A with a 0% of MLL concentration. Similarly, FIG. 2B is hydrogel B with a 2.5% of MLL concentration, FIG. 2C is hydrogel C with a 5% of MLL concentration, FIG. 2D is hydrogel D with a 10% of MLL concentration, FIG. 2E is hydrogel E with a 20% of MLL concentration, FIG. 2F is hydrogel F with a 25% of MLL concentration, FIG. 2G is hydrogel G with a 30% of MLL concentration, FIG. 2H is hydrogel H with a 50% of MLL concentration, FIG. 2I is hydrogel I with a 75% of MLL concentration and FIG. 2J is hydrogel J with a 100% of MLL concentration. Based on the images, hydrogel G possessed larger pores, which collapsed after lyophilization.

Example 2

(21) Protein Adsorption

(22) Protein adsorption is commonly employed to determine if there are protein/protein footprints on the surface of a material. Since all proteins have positively and negatively charged residues randomly distributed on their surface, they can easily adhered to positively or negatively charged surfaces. Protein adsorption is associated with a biologically active material and is not desirable for an anti-fouling material.

(23) To examine protein adsorption on the hydrogel surface, a commonly used model protein, bovine serum albumin (BSA), was employed. Typically, the BSA was dissolved separately in phosphate buffered saline (PBS) solution at a concentration of 100 g/ml, and added to hydrogels that were formed in a 24-well plate. Nitrocellulose membrane was used as a positive control as it could adsorb a lot of proteins. The adsorption was allowed to proceed at 37 C. overnight. After incubation, the BSA solution was collected. The bicinhoninic acid (BCA) method was applied to quantify the amount of BSA by using Micro BCA. Protein assay reagent kit (Pierece, U.S.A.). The amount of BSA was calculated by measuring the absorbance at 562 nm. Assuming that the BSA that no longer remained in solution was adsorbed on the surface of the hydrogel, the percentage of BSA adsorption could be calculated using the following formula. Protein adsorption (%)=100[(OD of BSA solution incubated with the hydrogel)/(BSA solution at a concentration of 100 g/ml)100], where OD=optical density at 562 nm. The mean value and standard deviation were calculated from 3 replicates for each sample.

(24) FIG. 3 shows that the positive control, nitrocellulose membrane, adsorbed over 70% of the bovine serum albumin (BSA) on its surface after overnight incubation. In contrast, hydrogels A to J adsorbed less than 35% of the BSA on their surfaces. In particular, hydrogel G displayed excellent resistance to protein adsorption, showing less than 10% BSA adsorption.

Example 3

(25) Bacteria Adhesion

(26) Bacteria tend to adhere to the surfaces of medical devices. This poses a serious concern for infection as it may lead to the need to remove the implanted device.

(27) Staphylococcus aureus (S. aureus) was selected for the bacteria adhesion study since it is a Gram-positive coccal bacterium frequently found on the skin. In addition, Escherichia coli (E. coli) were chosen for the bacteria adhesion study as it is a common Gram-negative bacterium. The bacteria concentration in Mueller Hinton Broth (MHB) was adjusted to an OD reading of 0.1 at a wavelength of 600 nm on a microplate reader (TECAN, Switzerland), which corresponded to 10.sup.8 CFU/mL. 500 l of suspension of E. coli (10.sup.5 CFU/mL) and S. aureus (10.sup.3 CFU/mL) was added to the hydrogels formed in a 24-well plate. After incubation at 37 C. for 24 hours, the hydrogel surfaces were washed 3 times with PBS. To visualize the viable bacterial cells on the hydrogel surfaces, a LIVE/DEAD Baclight bacterial viability kit (Invitrogen) was used. Hydrogel was soaked in a dye solution at room temperature in the dark for 15 minutes. The stained bacteria were observed with fluorescence microscopy (Zeiss, Germany).

(28) FIGS. 4 and 5 show that very few, if any S. aureus and E. coli bacteria, respectively, are adhered to the surfaces of hydrogels A to J after 24 hours of incubation. In contrast, a large number of bacteria were attached onto the petri dish (positive control). This demonstrated that the hydrogel materials were resistant to the adhesion of S. aureus and E. coli, which are among the most prevalent pathogens causing infections in the U.S. hospitals.

Example 4

(29) Cell Adhesion

(30) A good anti-fouling material should prevent cell adhesion, since cell adhesion would lead to foreign body reaction and cause an inflammatory response after implantation.

(31) Human primary dermal fibroblasts (ATCC, PSC-201-010) were used in the cell adhesion study as they tend to adhere to surfaces easily, and can be cultured without substantial difficulty. Fibroblasts were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 2% penicillin streptomycin at 37 C. in a 5% CO.sub.2 incubator. 500 L of fibroblasts at 110.sup.5 cells/ml were seeded on the surface of hydrogels in a 24-well plate. After 24 hours, the culture medium was replaced with the same amount of the extracting medium from the incubated hydrogels, and cultured for another 48 hours. Next, the attachment and morphology of the cells were assessed using Live/Dead Assay kit. The staining solution was prepared by adding 5 L of Calcein stock solution (4 mM solution in dimethyl sulfoxide (DMSO)) and 20 L of ethidium homodimer-1 (EthD-1) stock solution (2 mM solution in 1:4 DMSO/H.sub.2O) to 10 ml of PBS solution in a dark environment. The staining solution was then added to the wells, and incubated at 37 C. in a 5% CO.sub.2 incubator for 45 minutes. The stained cells were analyzed by fluorescence microscopy.

(32) FIG. 6 shows that very few fibroblasts were adhered to hydrogels C, D and F, as compared to petri dish. All the adhered cells have normal morphology and were alive. The other 7 hydrogels did not appear to have any fibroblasts adhered to their surfaces, indicating their excellent resistance against cell adhesion.

Example 5

(33) In Vitro Cytotoxicity

(34) a) Hemolysis Assay

(35) It is important that an anti-fouling coating for implantable biomedical device does not cause hemolysis. To test for hemolysis, fresh rabbit blood was diluted to 4% (by volume) with PBS, and the diluted blood (100 L) was placed on each hydrogel sample in a 96-well plate. 100 L of PBS were then added to each well. To allow hemolysis to occur, the plate was incubated for 1 hour at 37 C. The 96-well plate was then centrifuged at 2200 rpm for 5 minutes. Aliquots of 100 L of supernatant from each well were transferred to a new 96-well plate, and OD readings were recorded at a wavelength of 576 nm to assess hemoglobin release using the microplate reader (TECAN, Sweden). In this assay, red blood cells treated with 0.2% Triton-X were used as a positive control, and red blood cells in hydrogel-free PBS were used as a negative control. Percentage of hemolysis was calculated using the following formula. Hemolysis (%)=[(OD of sampleOD of negative control)/(OD of positive controlOD of negative control)]100. The mean value and standard deviation were calculated from 3 replicates for each sample.

(36) b) Cell Viability

(37) To determine if the hydrogel materials were biocompatible, cytotoxicity of human primary dermal fibroblasts (HDFs) was examined. HDFs were purchased from ATCC (PSC-201-010), and cultivated using Dulbecco's Modified Eagle Medium supplemented with 10% FBS with 5% CO.sub.2 and humidified atmosphere. The medium was changed every 2 days. The hydrogels were incubated in the culture medium at 37 C. for 24 hours to extract any soluble substances in the materials. HDFs were seeded in 96-well culture plates at 110.sup.4 cells/well. After 24 hours, the culture medium was replaced with the same amount of the extracting medium from the incubated hydrogels. The cells were subsequently cultured for another 24 hours. The effect of materials on HDF cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. MTS solution was added 12 h after cells were treated with the hydrogels. After 3 hours of incubation at 37 C. in 5% CO.sub.2, the light absorbance was measured at a wavelength of 490 nm with a microplate reader. The cell viability was obtained as follows: (number of viable cells with hydrogel treatment)/(number of viable cells without hydrogel treatment). Experiments were conducted in triplicates for each sample.

(38) c) Results

(39) The cells proliferated well without showing any reduction in viability (FIG. 7). FIG. 8 shows that rabbit red blood cells remained healthy with <10% hemolysis in the presence of hydrogels. These two studies showed that hydrogels A-J were not toxic to HDFs and rabbit red blood cells.

Example 6

(40) In Vivo Implantation

(41) a) Source of Mice

(42) Adult C57BL/6 mice (8-week-old, 18-22 g) were used for animal studies. All mice eyes were examined for absence of ocular pathology before experiments were initiated. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Biological Resource Centre, Agency for Science, Technology and Research (A*STAR), Singapore.

(43) b) In Vivo Hydrogel Implantation

(44) The hydrogel were implanted subcutaneously in mice for 1 week and 2 months. The mice were anesthetized by ketamine (150 mg/kg) and xylazine (10 mg/mL) via intraperitoneal injection (I.P.). A longitudinal incision (1 cm) was made on the central dorsal surface using surgical scissors to provide access to the subcutaneous space. Next, subcutaneous pockets were created with a blunt forceps for the implantation of hydrogel disks. The incision was closed with suture. The mice were sacrificed at 1 week and 2 months. The hydrogel and surrounding skin tissues were collected immediately, and prepared for histological analysis. The fixed samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) by the standard protocol. To investigate the collagen formation in tissues near the subcutaneously implanted hydrogel, the tissues were stained with Masson's trichrome.

(45) c) Results

(46) The new zwitterionic hydrogel materials demonstrated very good resistance against protein adsorption, bacteria adhesion and cell adhesion. They also show very good biocompatibility in the cell viability and hemolysis studies. In particular, hydrogel G provided excellent resistance against protein adsorption and was selected for further animal studies. It was implanted subcutaneously in C57BL/6 mice. FIG. 9 shows that hydrogel G was clear and transparent after 2 months of implantation. Representative H&E staining images revealed numerous cells found after 2 months of implantation (FIG. 10). These finding suggested that hydrogel G elicited very little inflammatory response after 2 months of implantation. This weak inflammatory reaction could be attributed to the hydrogel's resistance against non-specific protein adsorption, cell adhesion and bacteria adhesion. Masson's trichrome stain, which stains collagen blue, cytoplasm red and nuclei black, was used to examine capsule formation. After implantation of hydrogel G for 2 months, no collagen capsule formation was found surrounding the material (FIG. 11), indicating that hydrogel G did not elicit substantial foreign body reaction.

INDUSTRIAL APPLICABILITY

(47) The polymeric composition may be used as an anti-fouling coating material and may be used in a variety of applications such as biomedical implants and devices, drug delivery systems, contact lenses and tissue scaffolds to protect the implants or devices from bacteria or cell adhesion, foreign body reaction and resist non-specific protein adsorption, thus minimizing biofouling and potential infections.

(48) The polymeric composition may be applied as hydrogel or as an anti-fouling hydrogel coating material on biomedical materials to develop a hemocompatible non-fouling surface that may be flexible to contour according to a variety of biomaterials. The polymeric composition may comprise six acryloyl monomers comprising carboxylic, sulfonic and amine (such as tert-amine) groups, where the hydroxyl-, alkyl-(such as isopropyl-) and polyalkylene hydroxyl (PEG)-terminated monomers facilitate the formation of hydrogel with greater elasticity and transparency, and a stabiling agent (such as amino acid-terminated methacryloyl-L-lysine (MLL)) was used to enhance the zwitterionic property of the hydrogel. The polymeric composition may comprise a metal salt solution to provide a source of a metal as a cross-linker that forms a complex with the carboxylic and sulfonic groups to strengthen the mechanical properties. This polymer may be a porous material with great transparency and soft elastic nature that could be molded into various shapes, and excellent absorbent capability. The polymeric composition may demonstrate very good resistance against protein adsorption, bacteria adhesion and cell adhesion, and may display very good biocompatibility.

(49) It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.