ASTAXANTHIN BASED POLYMER AND USES THEREOF

Abstract

Provided are polymers having a repeating unit including at least one astaxanthin moiety. Also provided are processes for preparing the polymers and various uses thereof.

Claims

1.-30. (canceled)

31. A polymer having a repeating unit comprising at least one astaxanthin moiety.

32. A polymer having the following repeating unit —[ATX]n— wherein ATX is an astaxanthin moiety and n is at least 2.

33. The polymer according to claim 31, wherein the repeating unit further comprises at least one other moiety selected from a bioactive moiety, a biocompatible moiety, hydrophobic moiety, hydrophilic moiety, or any combinations thereof.

34. The polymer according to claim 31, wherein the repeating unit further comprises at least one other moiety selected from a carboxylic acid moiety, an anhydride moiety, an epoxide moiety, an acyl moiety, a phosgene derivative, an isocyanate moiety or any combinations thereof.

35. The polymer according to claim 31, having a MW of at least 15 kD.

36. The polymer according to claim 31, having a MW of between about 15 kDa to about 50 kDa.

37. The polymer according to claim 31, capable of controllably releasing ATX.

38. The polymer according to claim 31, capable of adsorbing at least one protein.

39. The polymer according to claim 31, having an erosion rate of between 24 h to 24 month.

40. The polymer according to claim 31, being at least one of biocompatible, antibacterial, antioxidant, anti-infectious, anti-inflammatory, antithrombotic or any combination thereof.

41. The polymer according to claim 31, being at least one of thermally and mechanically stable.

42. The medical device comprising at least one polymer according to claim 31.

43. The medical device according to claim 42, being an implantable medical device.

44. The medical device according to claim 42, selected from at least one of catheter, valve, stent, scaffold, tissue filler, wound healing patch.

45. The medical device according to claim 42, wherein said polymer forms a coating on the surface of said device.

46. The medical device according to claim 42, being resistant to infection.

47. The method of preventing or inhibiting infections associated with use of an implant, said method comprising treating a surface region of said implant with a polymer according to claim 31.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0100] FIG. 1 presents a scheme of a process for preparing ATX homopolymer, referred herein as pATX, according to an embodiment of the invention.

[0101] FIG. 2 presents a scheme for the preparation of a hydrophobic ATX co-polymer (showing the polymerization of co-polymer poly(astaxanthin-co-suberic acid) referred herein as poly(ATX-co-SA)), according to an embodiment of the invention.

[0102] FIG. 3 presents a scheme for the preparation of a hydrophilic ATX co-polymer (showing the polymerization of co-polymer poly(astaxanthin-co-polyethylene glycol) referred herein as poly(ATX-co-PEG)), according to an embodiment of the invention.

[0103] FIG. 4 provides FTIR-ATR analysis of ATX powder and ATX homopolymer, pATX, according to Example 1 of the present invention.

[0104] FIG. 5 provides gel permeation chromatography (GPC) results of the ATX homopolymer, pATX, according to Example 1 of the present invention.

[0105] FIG. 6 provides NMR analysis of ATX powder and the ATX polymer, poly(ATX-co-SA), according to Example 2 of the present invention.

[0106] FIG. 7 provides gel permeation chromatography (GPC) results of the ATX polymer, poly(ATX-co-SA), according to Example 2 of the present invention.

[0107] FIG. 8 provides DMA results of the ATX polymer, poly(ATX-co-SA), according to Example 2 of the present invention.

[0108] FIG. 9 provides FTIR-ATR analysis of the ATX polymer, poly(ATX-co-PEG), according to Example 3 of the present invention.

[0109] FIG. 10 provides gel permeation chromatography (GPC) results of the ATX polymer, poly(ATX-co-PEG), according to Example 3 of the present invention.

[0110] FIG. 11 provides DMA results of the ATX polymer, poly(ATX-co-PEG), according to Example 3 of the present invention.

[0111] FIG. 12 provides optical density (OD) measurements over 24 hours of inoculated ATX samples, wherein OD570- refers to the optical density at 570 nm and the error bars indicate standard error of mean.

DETAILED DESCRIPTION OF EMBODIMENTS

EXAMPLE 1

Polymerization of Homopolymer Polyastaxanthin (Referred Herein: pATX)

[0112] 1 mmol ATX, 4 mL DCM, and 2 mmol triethylamine were placed in a 50 mL amber scintillation vial. The vial was placed in ice water (exothermic reaction). 1 mmol oxalyl chloride in 4 mL DCM was added under N.sub.2. The reaction vessel was covered with aluminum foil in order to protect the reactants from light, and stirred in the ice water for two hours. To complete the reaction, the reagents were stirred for an additional for 2 h at room temperature. The solution was then precipitated in isopropyl alcohol (IPA). The precipitated product was then collected and dried under vacuum overnight. The reaction yield was calculated from the dry weight of the precipitate. 10 mg were collected and dissolved in THF (2 mL) for molecular weight analysis using gel permeation chromatography (GPC, VISCOTEK equipped with RI and UV detectors using PS standards.). For further analysis, solvent cast films of the polymer were prepared. The remaining polymer powder was dissolved in THF (10% w/v) and after complete dissolution was cast into a polytetrafluoroethylene (PTFE) mold under N.sub.2 for 8 hours and then placed in a desiccator for drying overnight.

[0113] FTIR-ATR analysis of the solid films (recorded on a BRUKER S ALPHA-P, ATR-FTIR) was performed on the prepared films in comparison to ATX powder is displayed in FIG. 4. The formation of ester groups (1737 cm.sup.−1, 1238 cm.sup.−1), with the disappearance of hydroxyl group (3500 cm.sup.−1) are evident to the formation of an ester group within the reaction.

[0114] The average molecular weight of the pATX polymer of Example 1 was evaluated by gel permeation chromatography (GPC) analysis and displayed in FIG. 5. The formation of a high molecular weight polymer was confirmed, where a polymer with weight average molecular weight of 47 kDa with a polydispersity index (PDI) of 1.4 was obtained.

EXAMPLE 2

Polymerization of Copolymer Poly(astaxanthin-co-suberic Acid) [Referred Herein: poly(ATX-co-SA)]

[0115] 1.0 mmol ATX and 1.0 mmol suberic acid were placed in a dry 50 ml amber scintillation vial. 20 ml of dry dichloromethane were added and stirred under N.sub.2 until complete dissolution. 0.5 mmol of 4-dimethylaminopyridin (DMAP) was added followed by 6 mmol of carbodiimide (DIC) over a 3 hour period (various carbodiimide derivatives can be used). The reaction vessel was covered with aluminum foil and stirred overnight. Precipitation was performed using isopropanol. The precipitated product was then collected and dried under vacuum overnight. The reaction yield was calculated from the dry weight of the precipitate. 10 mg were collected and dissolved in THF (2 mL) for molecular weight analysis using gel permeation chromatography (GPC). For further analysis, solvent cast films of the polymer were prepared. The remaining polymer powder was dissolved in THF (10% w/v) and after complete dissolution, cast into a PTFE mold under N.sub.2 for 8 hours and then placed in a desiccator for drying overnight.

[0116] The results of FTIR-ATR analysis performed on the prepared films of the poly(ATX-co-SA) of Example 2 is displayed in FIG. 6. The formation of ester groups (1735 cm.sup.−1) is evident to the formation of a polyester within the reaction.

[0117] The successful synthesis of a high molecular weight polymer poly(ATX-co-SA) of Example 2 was confirmed through GPC analysis, where a polymer with average molecular weight of 103.5 kDa with a PDI of 1.6 was obtained (shown in FIG. 7).

[0118] A dynamic mechanical analysis (DMA) of the obtained poly(ATX-co-SA) sample, using a TA Q800 DMA operated at a temperature range of −30° C. to 120° C., indicated that the glass transition temperature of the resulting polymer is 60° C., as illustrated in FIG. 8.

[0119] A differential scanning calorimetry (DSC) analysis of the same material resulted in Tg of approximately 50° C.

[0120] The cast poly(ATX-co-SA) films have a green metallic color, possibly due to the presence of many conjugated double bonds in the polymer. A film sample was tested according to the Van der Pauw method (ASTM F76). The resulting surface resistivity was 7.084 MΩ/sq, indicating a possibly inherently electrically dissipative polymer.

[0121] For evaluation of the biodegradation potential, poly(ATX-co-SA) film samples were incubated at 37° C. in phosphate buffer saline with pH of 7.4 for 1,3,7, 14 & 30 days. Control samples were kept under vacuum at 0° C. Within 30 days, a 5% mass loss was observed. GPC analysis indicated a molecular weight loss of 60% indicating a potentially bulk erosion mechanisms, where the loss of polymer Mw precedes mass loss.

[0122] Some experiments were conducted using DPTS (4-(N,N′-Dimethylamino) pyridinium 4-toluenesulfonate) as a catalyst, which was added to the reaction vessel after dissolution, before the addition of the carbodiimide.

EXAMPLE 3

Polymerization of Copolymer poly(astaxanthin-co-polyethylene glycol) [Referred Herein: poly(ATX-co-PEG)]

[0123] 5.75 mmol of DIC were added over 3 hrs, under N.sub.2 to a reaction vessel containing 1 mmol ATX, 1 mmol poly(ethylene glycol) bis(carboxymethyl) ether, and 14 mL DCM, to allow a controlled polymerization rate and to obtain high molecular weight polymers. The reaction vessel was covered with aluminum foil and stirred for additional 3 hrs to reach high molecular weight polymers. The solution was then precipitated in IPA. The precipitated product was then collected and dried under vacuum overnight. The reaction yield was calculated from the dry weight of the precipitate. 10 mg were collected and dissolved in THF (2 mL) for molecular weight analysis using gel permeation chromatography (GPC). For further analysis, solvent cast of the poly(ATX-co-PEG) films were prepared. The remaining poly(ATX-co-PEG) powder was dissolved in THF (10% w/v) and after complete dissolution, was cast into a PTFE mold under N.sub.2 for 8 hours and then placed in a desiccator for drying overnight.

[0124] FTIR-ATR analysis performed on the poly(ATX-co-PEG) films prepared according to Example 3 is shown in FIG. 9. The formation of ester groups (1756 cm.sup.−1) and ether groups derived from the PEG (1240 cm.sup.−1) are evident to the formation of a polyester within the reaction.

[0125] The successful synthesis of a high molecular weight polymer was confirmed through GPC analysis (exhibited in FIG. 10), where a polymer with weight average molecular weight of 45 kDa with a PDI of 1.7 was obtained.

[0126] A dynamic mechanical analysis (DMA) of the obtained poly(ATX-co-PEG) of Example 3 indicated that the glass transition temperature of the resulting polymer was 41° C., as illustrated in FIG. 11.

[0127] A differential scanning calorimetry (DSC) analysis of the same material resulted in Tg data of approximately 45° C.

[0128] The cast poly(ATX-co-PEG) films of Example 3 have a green metallic color, possibly due to the presence of many conjugated double bonds in the polymer. Degradation studies in phosphate buffer saline revealed a molecular weight loss of over 50% in four weeks, and mass loss of 20%.

[0129] Table 1 lists some of the polymers provided according to the present disclosure. The different polymers were prepared by using varying comonomers. A wide range of thermal and mechanical properties was achieved as exhibited in the table. The Mw ranged from 19.4 kDa for the poly(ATX-co-dodecandioic acid), while the poly(ATX-co-suberic acid) exhibited a higher Mw of 103.5 kDa. The structure of the various polymers obtained was determined by NMR. NMR spectra were recorded on a Varian NMRS 300 or 500 MHz instrument. .sup.1H NMR chemical shifts are reported in ppm relative to the solvent's residual .sup.1H signal. .sup.13C NMR spectra were recorded at 125 MHz.

[0130] Without being bound by theory, it is believed that the nature of the comonomer alters the polymer's properties. As shown in Table 1, the mechanical property varies as a function of the different monomeric unit that composes the polymer, for example, the Young's modulus ranges from 200 to 900 MPa for the different polymers comprising different monomeric units.

[0131] Thus, it is believed that the polymers disclosed herein can be fabricated into various shapes and forms using conventional techniques, and can be applied as coating for various medical devices.

EXAMPLE 4

Antibacterial Tests of the Polymers of the Invention

[0132] The potential antibacterial properties of polymer based ATX samples were tested against Staphylococcus aureus MRSA252, S. aureus MSSA476 and S. epidermidis 1457. Using solvent casting method [J. Ghosh et al., Polymer, vol. 52, pp. 2.2011 ,650-2660], 96 well plates were coated with the following samples—ATX only, ATX+sebacic acid (SA), ATX+Hexadecanoic acid (HA) and ATX+sebacic acid +2% polyethylene glycol (PEG). Each sample coated 3 wells per row and the last row was left blank as a control.

[0133] 3 ml Trypticase Soy Broth was inoculated with a colony from an overnight plate. Pre-cultures were grown at 37° C. with shaking to mid-exponential phase (2-3 h), and then used to inoculate fresh TSB to a starting OD.sub.600 of 0.01. 200 μl aliquots of this suspension were then inoculated into wells of one full row of the test plate. The plate was then incubated in a FLUOstar OPTIMA microplate reader at 37° C., and the optical density of each well measured every hour over a 24 h period. After 24 h the suspension in each plate was removed. The plate itself was washed three times with phosphate buffered saline, pH 7.4 (PBS), air dried and stained with 150 μl crystal violet solution. The dye bound to any attached cells was solubilized by addition of 150 μl of 95% ethanol.

[0134] Optical density was measured as absorbance at 570 nm using a FLUOstar OPTIMA microplate reader and biofilm positive defined as OD.sub.570 above 1. All S. aureus and S. epidermidis isolates were tested in triplicate in three independent experiments. Each plate also consisted of negative controls (wells without bacterial inoculation). Data was analyzed using unpaired t-tests or one-way ANOVA, and the level of significance set at p<0.05. The ability of the bacteria to form a biofilm in the presence of the ATX samples was determined after culturing for 24 h using a semi-quantitative biofilm assay.

[0135] FIG. 12A shows the effect of the ATX samples on the growth of S. epidermidis 1457 over the course of 24 h. similar results were obtained with S. aureus MRSA252 and S. aureus MSSA476. The figure shows that ATX alone has a significant effect on inhibiting the growth of the bacteria, and suggests that some ATX polymer samples have a similar potential. FIG. 12B shows that ATX samples inhibited the bacteria from forming biofilm on assayed ATX and ATX polymer surfaces in comparison to the control (p≦0.005).

[0136] Recent preliminary studies performed under JIS Z 2801 standard and using S. epidermidis, have confirmed our reported results of the ATX polymer bacteriostatic nature. In addition, these studies show that the ATX polymer is potentially bacteriocidal (not shown). These results suggest that these ATX polymer samples are bacteriostatic in nature and that further optimized copolymers may have a greater bacteriostatic and bacteriocidal activity. In vitro biodegradation studies indicate that the pATX can become biodegradable polymers, with different erosion mechanism (bulk/surface) as function of hydrophobicity of comonomer. This property may be used as a slow release mechanism of the ATX active constituent. Moreover, Such ATX-based polymers are high Mw plastics that can be molded into various shapes and forms. The ATX polymer can be easily applied as a coating for catheters and other medical device, to provide them with the much needed antimicrobial and hemocompatible properties.

[0137] Thus, ATX polymer has the capacity to be tailored towards the localized controlled release of ATX from their polymer form, and/or as bioactive polymers. These intrinsically bioactive materials have the potential to provide antibacterial activity, and anti-thrombotic activity, in addition to anti-oxidative, anti-inflammatory and anti-cancerous properties; and can be used as coatings for medical device and/or modeled as medical device.