INTRODUCTION:

Hydrogels are a class of condensed matter crosslinked with chemical bonds or physical nodes containing massive aqueous solvents.¹ These are an emerging kind of soft materials with huge application potentials in various fields including tissue engineering, drug delivery, catalyst supports, adsorbents, cosmetics and so on.^2-5 The most attractive and important aspect of hydrogel is its biocompatibility and biodegradability which ensures its application in biomedical field.^5-9 This is largely promoted by its high water content and a similar physiochemical nature of hydrogels to the native extracellular matrix.¹⁰ The carbohydrate based hydrogels holds significance in the biomedical field because of their glycotargeting ability of carbohydrate pendants present in the polymer network.^11-14

The carbohydrate pendants in the glycopolymeric network can be recognized by the cell surface carbohydrate binding prolectins and this makes them a unique class of materials for targeted drug delivery and controlled release applications.^15-18 One of the best methods for the synthesis of glycopolymeric (carbohydrate containing) hydrogels is chemical grafting of vinylic monomers such as acrylonitrile, acrylamide and acrylic acid onto low cost and biodegradable polysaccharides such as starch, chitosan and cellulose or synthetic glycopolymers like PMAGlc by using various initiating systems followed by cross linking with hydrophilic crosslinkers.^19,20 Recent studies towards the synthesis of biodegradable materials revealed that incorporation of biodegradable cross linkers into a non-biodegradable but biocompatible polymer could transform the latter into a biodegradable material.^21,22 This observation triggered efforts to make biodegradable carbohydrate based crosslinkers. There are a few reports of carbohydrate based crosslinker^23,24 but their comparative study with commercially available crosslinkers is yet to be investigated.

Encouraged by the previous findings, we hereby report synthesis of a novel cross linker starting from D-glucose. The cross linker was used to fabricate hydrogel and its applicability was compared with another cross linker N,N’-Methylenebisacrylamide (MBA) The compounds were characterized by ¹H NMR. Investigation of properties of hydrogels and their characterization was achieved via various analytical techniques such as TGA, DSC, FTIR and SEM.

EXPERIMENTAL DETAILS:

General.

All chemicals and dry solvents (THF, MeOH, and DMF) were obtained from commercial sources and used without any further purification. DCM and pyridine were dried using calcium hydride. Toluene was dried using calcium chloride. Thin layer chromatography (TLC) was performed on silica gel plates precoated with fluorescent indicator with visualization by UV light or by dipping into a solution of 5% (v/v) conc H₂SO₄ in ethanol and heating. Silica gel (100−200 mesh) was used for column chromatography. ¹H NMR were recorded on a 400 MHz instrument (JEOL, Japan). The chemical shifts in parts per million (ppm) are reported downfield from TMS (0 ppm). Multiplicities of ¹H NMR spin couplings are reported as s for singlet, d for doublet, t for triplet, q for quartet, dd for doublet of doublet, or m for multiplet and overlapping spin systems. Values for apparent coupling constants (J) are reported in Hz.

Synthesis

The strategies for synthesis of bis-acrylate and grafting of Polymethacrylated glucose (PMAGlc) by polyacrylamide (PAM) is shown in scheme 1 and 2 respectively.

(6S)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl methacrylate (2)

A stirred solution of 1 (10.0 g, 0.038 mol) and methacrylic anhydride (10.0 ml, 1.8 equiv) in pyridine (50.0 ml) was heated at 65 ⁰C for 5 h under nitrogen atmosphere. Water (25 ml) was added and the reaction mixture was stirred for an additional hour at 65 ⁰C. The reaction temperature was lowered to RT and stirred for 18 h. At this point, the reaction mixture was diluted with petroleum ether (200 ml) and washed with 5% NaOH (3x, pH ~ 7.0) and water (3 X 100ml). The combined organic layers were dried over Na₂SO₄. 4-Methoxy phenol (0.032 g) was added before the evaporation of the organic solvent as a polymerization inhibitor. The crude product was distilled under reduced pressure to afford the desired compound 2 in 80% yield as thick syrup which crystallized upon standing at RT.²⁵

¹H-NMR (400 MHz, Chloroform-D) δ 6.13-6.06 (1H), 5.87 (d, J = 3.8 Hz, 1H), 5.60 (t, J = 1.6 Hz, 1H), 5.26 (d, J = 1.6 Hz, 1H), 4.51 (d, J = 3.3 Hz, 1H), 4.23 (t, J = 3.8 Hz, 2H), 4.04-4.08 (m, 1H), 3.98-4.01 (m, 1H), 1.93 (s, 3H), 1.50 (s, 3H), 1.38 (s, 3H), 1.28 (s, 6H)

(6S)-5-((R)-1,2-dihydroxyethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl methacrylate (3)

12 g of starting material 2 was dissolved in 100 ml of 4:1 mixture of AcOH: H₂O. The reaction mixture was stirred at RT for 12 h. The reaction proceeding was checked with TLC and on reaction completion excess of AcOH was evaporated under reduced pressure to obtain the crude. The crude was neutralized with NaHCO₃ and extracted with EtOAc (3 X 100 ml). The obtained EtOAc layer was evaporated under reduced pressure to get the product as white powder and was taken to next step without purification.²⁶

(6S)-5-formyl-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl methacrylate (4)

Compound 3 (1.2 g, 4.1 mmol) was dissolved in 1:1 mixture of MeOH-H₂O. NaIO₄ (5.4 mmol) was added slowly and stirred vigorously. Mixture was stirred at RT for 3 h. Filtration was done and residue was washed two times with ethyl acetate (2 x 50 ml). Organic layer was washed with brine (60 ml), dried over sodium sulphate and concentrated under reduced pressure to furnish 4. The presence of a singlet at 9.65 ppm in proton NMR indicated the presence of aldehyde functionality.

¹H-NMR (400 MHz, Chloroform-D) δ 9.65 (s, 1H), 6.09 (d, J = 3.4 Hz, 1H), 6.04 (s, 1H), 5.61 (s, 1H), 5.54 (d, J = 3.4 Hz, 1H), 4.77 (d, J = 3.4 Hz, 1H), 4.62 (d, J = 3.4 Hz, 1H), 1.88 (s, 3H), 1.32 (s, 3H), 1.23 (s, 3H)

(6S)-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl methacrylate (5)

The aldehydic compound 4 (1.2 g, 4.6 mmol) was dissolved in 20 ml of DCM and cooled in an ice bath (0-5˚C). Sodium borohydride (3 eq) was added slowly in small portions. The resulting mixture was stirred at RT for 6 h. The obtained white mixture was diluted with DCM, washed with H₂O (3 x 50 ml), brine (50 ml) and dried over Na₂SO₄. The organic phase was concentrated under reduced pressure and further dried under high vacuum to obtain 5.

((6S)-6-(methacryloyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methyl methacrylate (6)

A stirred solution of 5 (2.0 g, 7.7 mmol) and methacrylic anhydride 1.7 ml (11.6 mmol) in pyridine (5.0 ml) was heated at 65 ˚C for 5 h. Water (25 ml) was added and the reaction was stirred for an additional hour at 65 ˚C. The reaction temperature was lowered to 30°C and stirred for 1 h. At this point, the reaction mixture was diluted with DCM (100 ml) and washed with 5% NaOH (3x, pH ~ 7.0) and water (3 X 50 ml). The combined organic layers were dried over Na₂SO₄. 4-Methoxy phenol (0.032 g) was added before the evaporation of the organic solvent as a polymerization inhibitor. The crude product was distilled under reduced pressure to afford the desired compound 6 which was purified on silica-gel column chromatography (0-15 % EtOAc/pet ether) to yield 1.38 g (55%) as thick syrup.

¹H-NMR (400 MHz, Chloroform-D) δ 6.11 (s, 2H), 5.96 (d, J = 3.7 Hz, 1H), 5.62 (s, 1H), 5.57 (s, 1H), 5.33 (d, J = 3.0 Hz, 1H), 4.55-4.61 (m, 2H), 4.32-4.34 (m, 2H), 1.92 (s, 3H), 1.91 (s, 3H), 1.53 (s, 3H), 1.31 (s, 3H)

(3R)-4,5-dihydroxy-2-((methacryloyloxy) methyl)tetrahydrofuran-3-yl methacrylate (7)

Compound 6 (1.2 g, 3.6 mmol) was dissolved in 10 ml of 85% aq. formic acid and reaction mixture was stirred at room temperature for 12 h. TLC indicated completion of reaction. Excess solvent was co-evaporated with toluene and dried under vacuum. The residue was precipitated in EtOAc (30 ml), washed with EtOAc: hexane (1: 5) (3 x 5ml) and was vacuum dried to obtain 7.

Poly-methacrylated glucose (PMAGlc, 9)

PMAGlc was synthesized according to the literature with some modification. Briefly, compound 2, MAIGlc (3-O-methacryloyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose) the monomer was polymerized in DMF in heating condition (80 ⁰C) using AIBN as the free radical iniator.²⁷ The viscose liquid on precipitation in excess hexane yielded solid polymer 8 (PMAIGlc, Scheme 2) in good yield. The deprotection of acetonide groups was achieved by treating with 85% formic acid at room temperature for 12 hours to obtain PMAGlc (Compound 9, Scheme 2). The obtained polymer was soluble in water, methanol and DMSO.

Synthesis of polyacrylamide (PAM)-g-PMAGlc hydrogel (10)

Graft copolymerization of polyacrylamide onto PMAGlc to prepare hydrogels was investigated (in a separate study) by varying the concentration of MBA and acryl amide and their optimized concentration is reported here. The hydrogel preparation was achieved by free-radical crosslinking polymerization using MBA or bis-acrylate as cross linker taking optimized concentration of MBA. Briefly, 500 mg of PMAGlc was dissolved in 30 ml of water to which 4 g of acrylamide was dissolved with stirring. To this solution 0.8 ml (10% aq. solution) of MBA was added. Nitrogen gas was passed through the reaction mixture for 30 min to make the solution oxygen free. The polymerization was initiated at room temperature by addition of 0.4ml (10% aq. solution) of potassium persulphate as chain initiator and tetramethylethylenediamine (TEMED) 0.2 ml (10% aq. solution) as accelerator with vigorous stirring. Subsequently, the solution started to become viscose and finally converted to solid gel in an hour (10, scheme 2). The reaction mixture was left undisturbed for 24 h to ensure completion of reaction. The gels were removed from beaker and were placed in acetone for a day to get rid of acrylamide homopolymer. Further, the gels were kept in distilled water for 2 days to wash out any unreacted compounds. Hydrogels were removed from water and were dried at 50 ⁰C in a vacuum oven till a constant weight was observed. Same procedure was followed to produce another set of hydrogel using bis-methacrylate as the cross linker and the hydrogels were named as Hydrogel1 (HG1) and hydrogel2 (HG2) respectively.

Characterization and property evaluation of hydrogels

The FTIR spectra of samples were recorded to analyze structural changes using IRPRESTIGE21 (Shimadzu corporation, Japan.) at room temperature using KBr palette. The samples were scanned over the range of 4000−400 cm⁻¹. Thermogravimetric analysis and DSC study was carried out using NEZTSCH, Model no- STA 449 F3 JUPITER having Rhodium furnace. About 5 mg of the sample was placed in a crucible and analyzed over the temperature range of 30–700 ⁰C at 10 ⁰C/min with a nitrogen flow rate of 30 ml/min. Morphological analysis of the dried hydrogels were observed by scanning electron microscopy(JEOL Japan, JFM/6390LV).

Swelling behavior

The swelling behavior of the two sets of hydrogels was studied in water in neutral condition at room temperature. Dried samples (W_d) were immersed in deionized water to swell and the swollen samples were removed from the solution, quickly wiped with filter paper and weighed (Ws). The weight change was monitored at different time intervals till the hydrogels showed no weight change. The swelling ratio was calculated using the following empirical relationship. The measurements were made in triplicate and average data was used for calculation.

Ws- W_d

Swelling ratio=------------

W_d

RESULTS AND DISCUSSION:

Synthesis

The synthesis of bis-methacrylate was achieved in six steps starting from glucose diacetonide, 1 as shown in scheme 1. Glucose diacetonide was methacrylated using methacrylic anhydride in nitrogen atmosphere. Formation of 2 was confirmed by its ¹H NMR (Figure 1). Compound 2 on treatment with 80% acetic acid furnished a dihydroxy compound 3 which .was subjected to periodate cleavage to obtain aldehydic compound 4. The ¹H NMR spectrum of 4 showed a peak at 9.65 ppm confirming the cleavage of dihydroxy compound. The aldehydic compound 4 was reduced using NaBH₄ to produce 5 which was again methacrylated to obtain bis-methacrylated sugar.6. The ¹H NMR (Figure 2) of 6 showed presence of two methyl peaks originating from methacrylate functionality. Compound 6 was treated with 85% Formic acid to obtain the desired compound 7 with an overall yield of 24 % over six steps. The synthesis of PMAGlc was achieved from a similar scheme starting from D-Glucose. The starting material D-Glucose on treatment with acetone/H₂SO₄ formed glucose diacetonide which was methacrylated and polymerized to form PMAIGlc 8 which on treatment with 85 % formic acid formed water soluble polymer, polymethacrylated glucose 9 (PMAGlc). The ¹H NMR (Figure 3) of the deprotected polymer showed signal for one methyl group and absence of vinylic protons suggesting complete polymerisation and deprotection of acetonide groups. The signals between 3-5 ppm correspond to –OH, -CH- and –CH₂- groups.

Figure 1 ¹H NMR spectrum of compound 2

Figure 2 ¹H NMR spectrum of compound 6

Figure 3 ¹H NMR spectrum of PMAGlc

FTIR spectrum

The PAM-g-PMAGlc hydrogels were characterized by FTIR spectrum as shown in Fig. 4. The spectrum of both the hydrogels is consistent. As shown in the labeled spectrum the absorption peak at 3305 cm^-1is due to N-H asymmetric stretching while 3190 cm^-1 belongs to a symmetric N-H stretching, The peak around 2940 belongs to –CH and –CH₂ stretching, 1708 cm^-1 attributes to a C=O stretching of acrylate and 1161 cm^-1 belongs to a C-O-C stretching respectively. The absorption peak at 1635 cm^-1 is due to carbonyl stretching of –CONH₂ group. The peak at 1485 belongs to C-N stretching of amide units.

Figure 4 FTIR spectrums of hydrogels

Thermogravimetric and DSC thermogram analysis

The thermal properties of the hydrogels (HG1 and HG2) were studied by TGA and DSC. The TGA curves are shown in Figure 5 while DSC thermograms are shown in figure 6. It is evident from figure 5 that thermodynamic stability of both the hydrogels upto 350 ⁰C is almost same but at higher temperature HG2 shows higher stability than the other. The DSC thermograms suggests that hydrogel1 and 2 both have nearly same glass transition temperature (Tg) but their crystalline nature is very different. HG2 shows an exothermic peak at 177 ⁰Cwhile there is no such peak in HG1. Although both the hydrogels exhibit a melting peak, the area of the melting peak in case of HG2 is much higher (241.4 J/g) than that of HG1 (138.2 J/g) suggesting highly ordered arrangement in HG2 or greater crystalline behavior.

Figure 5 Thermal degradation curves of hydrogels

Figure 6 DSC thermograms of the hydrogels

Swelling behavior

In order to compare the swelling behavior, swelling ratio of the hydrogels was measured which is shown in figure 7. Hydrogel obtained using bis-methacrylate as cross linker (HG2) showed non-linear swelling rate and the equilibrium swelling was achieved at a later time than that of other hydrogel (HG1) and also the equilibrium swelling ratio was slightly lower than that of hydrogel obtained using MBA. This might be due to greater hydrophilicity of bis-methacrylate causing non-uniform cross linking. This observation is also supported by SEM images.

Figure 7 Effect of cross linker on the swelling rate of hydrogels

Morphology

The morphological features of the prepared hydrogels have been studied by recording SEM images of dried hydrogels as shown in Figure 8. Both images confirm formation of layered structure. While Image a shows homogeneity in the hydrogel, there is some heterogeneity seen in image b which indicates non-uniform cross linking in the network which might be due to mismatch in the hydrophilicity of bis-methacrylate and acrylamide.

Figure 8 SEM image of (a) HG1 (b) HG2

CONCLUSION:

In summary, we have described an effective approach for the synthesis of a novel bis-methacrylated sugar as a cross linker. The high yield of the reaction sequence makes it apt for bulk synthesis. The comparative study of MBA and bis-methacrylate as cross linker to prepare hydrogel by graft polymerization of acrylamide onto PMAGlc was studied. The formation of hydrogel was confirmed by Fourier transform infrared spectroscopy and SEM images. Thermogravimetric analysis confirmed the stability of hydrogels. DSC thermograms showed that hydrogel resulting from bis-methacrylate is more crystalline than the other.

REFERENCES:

1 Terech P and Weiss RG. Low molecular mass gelators of organic liquids and the properties of their gels. Chemical Reviews. 97; 1997: 3133-3160 .

2 Elisseeff J, Puleo C, Yang F and Sharma B. Advances in skeletal tissue engineering with hydrogels. Orthodontics and craniofacial research. 8; 2005: 150-161.

3 Christensen L, Breiting V, Janssen M, Vuust J and Hogdall E. Adverse reactions to injectable soft tissue permanent fillers. Aesthetic Plastic Surgery. 29; 2005: 34-48.

4 Drury JL and Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 24; 2003: 4337-4351.

5 Peppas N, Bures P, Leobandung W and Ichikawa H. Hydrogels in pharmaceutical formulations. European journal of Pharmaceutics and Biopharmaceutics. 50; 2000: 27-46.

6 Bhattarai N, Gunn J and Zhang M. Chitosan-based hydrogels for controlled, localized drug delivery. Advanced Drug Delivery Reviews. 62; 2010: 83-99.

7 Wichterle O and Lim D. Hydrophilic gels for biological use. Nature. 185; 1960: 117-118.

8 Yoshida R et al. Comb-type grafted hydrogels with rapid deswelling response to temperature changes. Nature. 374; 1995: 240-242.

9 Wang Q, Dordick JS and Linhardt RJ. Synthesis and application of carbohydrate-containing polymers. Chemistry of Materials. 14; 2002: 3232-3244.

10 Appel EA et al. Ultrahigh-water-content supramolecular hydrogels exhibiting multistimuli responsiveness. Journal of the American Chemical Society. 134; 2012: 11767-11773.

11 Dordick JS, Linhardt RJ and Rethwisch DG. Chemical and biochemical catalysis to make swellable polymers. Chemtech. 24; 1994: 33-39.

12 Chen X, Dordick JS and Rethwisch DG. Chemoenzymic Synthesis and Characterization of Poly (alpha-methyl galactoside 6-acrylate) Hydrogels. Macromolecules. 28; 1995: 6014-6019 .

13 Bahulekar R et al. Polyacrylamide containing sugar residues: synthesis, characterization and cell compatibility studies. Carbohydrate Polymers. 37; 1998: 71-78.

14 Suh JKF and Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 21; 2000: 2589-2598.

15 Kopecek J et al. Polymers for colon-specific drug delivery. Journal of Controlled Release. 19; 1992: 121-130.

16 Dwek RA. Glycobiology: toward understanding the function of sugars. Chemical Reviews. 96; 1996: 683-720.

17 Lee YC and Lee RT. Carbohydrate-protein interactions: basis of glycobiology. Accounts of Chemical Research. 28; 1995: 321-327.

18 Simanek EE, McGarvey GJ, Jablonowski JA. and Wong CH. Selectin-carbohydrate interactions: from natural ligands to designed mimics. Chemical Reviews. 98; 1998: 833-862.

19 Sugahara Y and Ohta T. Synthesis of starch‐graft‐polyacrylonitrile hydrolyzate and its characterization. Journal of Applied Polymer Science. 82; 2001: 1437-1443.

20 Patel G, Patel C and Trivedi H. Ceric-induced grafting of methyl acrylate onto sodium salt of partially carboxymethylated sodium alginate. European Polymer Journal. 35; 1999: 201-208.

21 Khelfallah NS, Decher G and Mésini PJ. Synthesis of a new PHEMA/PEO enzymatically biodegradable hydrogel. Macromolecular Rapid Communications. 27; 2006: 1004-1008.

22 Casadio YS, Brown DH, Chirila TV, Kraatz HB and Baker MV. Biodegradation of Poly (2-hydroxyethyl methacrylate)(PHEMA) and Poly {(2-hydroxyethyl methacrylate)-co-[poly (ethylene glycol) methyl ether methacrylate]} Hydrogels Containing Peptide-Based Cross-Linking Agents. Biomacromolecules. 11; 2010: 2949-2959.

23 Paterson SM, Clark J, Stubbs KA, Chirila TV and Baker MV. Carbohydrate‐based crosslinking agents: Potential use in hydrogels. Journal of Polymer Science Part A: Polymer Chemistry.49; 2011: 4312-4315.

24 Ajish JK, Kumar KA, Subramanian M and Kumar MD. Glucose based bisacrylamide crosslinker: synthesis and study of homogeneous biocompatible glycopolymeric hydrogels. RSC Advances.4; 2014: 59370-59378.

25 Christensen SM, Hansen HF and Koch T. Molar-scale synthesis of 1, 2: 5, 6-di-O-isopropylidene-α-D-allofuranose: DMSO oxidation of 1, 2: 5, 6-di-O-isopropylidene-α-D-glucofuranose and subsequent sodium borohydride reduction. Organic process Research and Development. 8; 2004: 777-780.

26 Tsui HC and Paquette LA. Reversible Charge-Accelerated Oxy-Cope Rearrangements. The Journal of Organic Chemistry. 63; 1998: 9968-9977.

27 Ambrosi M. et al. Influence of preparation procedure on polymer composition: synthesis and characterisation of polymethacrylates bearing β-D-glucopyranoside and β-D-galactopyranoside residues. Journal of the Chemical Society, Perkin Transactions. 1; 2002: 45-52.

Received on 26.06.2016 Modified on 18.07.2016

Asian J. Research Chem. 2016; 9(7): 343-349.

DOI: 10.5958/0974-4150.2016.00052.3