Development of Glycopolymer Resin on Desktop Digital Shell Printer

Even though the mechanical properties of the cured polymer were not promising, they were sufficient to allow the exploration/functionalization of these resins by investigating the addition of a carbohydrate groups to the polymer system for biomedical application of hydrogels.

Hydrogels are hydrophilic, which explains their swelling properties and their capability of maintaining encapsulated cell viability. Unfortunately, they are also a poor environment for cell attachment and proliferation, which is unfavourable in en vivo applications. Currently, glycopolymers have attracted a lot of attention from the materials science community due to their biocompatibility and their bioactivity for applications in tissue engineering and targeted drug delivery systems.

Glycopolymers are synthetic polymers with pendant carbohydrates, which if the carbohydrate displays on the cell surface, it can play critical a role in cell-cell recognition, adhesion and signalling between cells [226].For instance; saccharide units in glycopolymers can be capable of molecular recognition and interact with specific carbohydrate receptors. The participation of saccharides in biological recognition has led to the development of glycosylated drug and gene delivery carriers in which the carbohydrate receptors are used to direct the drugs to specific organ or cell targets and of carbohydrate-sensitive biosensors to monitor saccharide molecular recognition processes. Due to their excellent biocompatibility, glycopolymers have been proposed for use as scaffold materials in tissue engineering, while sugar-based hydrogels, are attractive as biocompatible water-absorbent materials [227]. In addition they also play an important role in cell growth regulation, differentiation, adhesion, cancer cell

responses [228].

There are two ways of synthesising glycopolymers; protected and unprotected carbohydrates. There are two major linkages by which carbohydrate chains can be attached to proteins: either N-linked through an asparagines residue or O-linked through a threonine or serine. A given glycoprotein may contain only N-linked, only O-linked, or both types of oligosaccharide chains [229]. The choice of employing protected or unprotected sugars is dependent on the ease of stereospecific functionalization of the sugar, the solubility of the monomer and polymer, the potential incompleteness of the removal of the protective group, and the ease of purification [228]. In this study, the synthesis of glycopolymers synthesis with unprotected carbohydrate and was done in collaboration with a researcher from Department of Chemistry, Warwick University. The synthesis was done according to Ohno et. al. method (1990) and explained as following section.

5.4.1 Preparation of PEGDA/HDPA-GlcAc

5.4.1.1 Synthesis of 1,2,3,4,6-Penta-O-acetyl-beta-D-glucopyranose; Acetyl 2,3,4,6-tetra-o-acetyl-beta-D-glucopyranoside (GlcAc)

Figure 5.10 Molecular structure of 1, 2, 3, 4, 6-Penta-O-acetyl-beta- D- glucopyranose; Acetyl 2, 3, 4, 6-tetra-o-acetyl-beta-D-

glucopyranoside (GlcAc) (http://www.chemblink.com/products/604- 69-3.htm).

119

The synthesis was made by a slight modification of the method proposed by Ouchi et al. [230]. Dry acetone, triethylamine, and acryloyl chloride was added drop wise at 0 °C to cold solution of 1,2,3,4,6-Penta-O-acetyl-beta-D-glucopyranose. The mixture was magnetically stirred for 1 hour at 0 °C and then for another 2 hours at room temperature. The system was diluted with cold water and extracted three times with chloroform. The combined extracts were dried over anhydrous sodium sulfate. After the solvent was evacuated, the crude product was purified by flash silica gel chromatography with a 2:3 (v/v) hexane/ ethyl acetate mixture as an eluent to yield pale yellow syrup. Crystallization of the syrup was induced in cold hexane to give a slightly yellow powder.

5.4.1.2 Deprotection of GlcAc

The protected polymer was dissolved in 80% formic acid and stirred for 48 hours at room temperature, to which water was added and stirred for another 3 hours. The solution was dialyzed against distilled water for 2 days, concentrated in vacuo, and finally lyophilized to give GlcAc as a white powder in quantitative yield. Table 5.6 shows the final formulation of the glycopolymers as a photopolymer resin.

Table 5.6 Formulation of glycopolymers photopolymer resin

Sample Ratio (%)

PEGDA HDPA GlcAc

Control 2.5Glc 5.0Glc 70.0 72.5 70.0 30.0 25.0 25.0 - 2.5 5.0

To study the effect of introducing carbohydrate onto the acrylate branches, the glycopolymers were subjected to swelling ratio and microtensile measurements. The

respectively.

5.4.2 Results and Discussion

Table 5.7 Swelling ratios measurement for each glycopolymers. ± is the standard deviation of three five data obtained at each formulation.

Sample Swelling ratio,Q Elastic Modulus,E(kPa) Control 2.5Glc 5.0Glc 9.9 ± 0.1 11.8 ± 0.2 13.2 ± 0.2 9.84 ± 0.5 8.70 ± 0.3 5.62 ± 0.2

Table 5.7 represents the swelling ratios measurement for each formulation. Swelling studies indicated that the addition of the glycopolymer has a significant effect on the swelling ratio after 24 hours. As a comparison, the swelling ratio has increased 20 and 33% after 2.5 and 5.0 % of GlcAc was added to the PEGDA/HDPA. The modification and increases of GlcAc has increased the molecular weight of the polymer mixture and as a result increases the swelling ratio of the cured polymer. The ability to absorb water and coupled with optimum rate of degradation are amongst the main characteristics required in a drug delivery system. Sol fraction of the material explained the degradation rate of the cured glycopolymers. Figure 5.11 represents the degradation behaviour of glycopolymers after immersion in PBS for 1, 7, 14, 21 and 28 days.

121

Figure 5.11 Sol fractions of glycopolymers after immersion in PBS for up to 28 days. The error bars is the standard deviation of five data obtained at each soaking time.

The engineered carbohydrate based materials has significant effect on the degradation rate of the cured glycopolymers. As a comparison, the rate of degradation has decreased 33% after 5.0% of GlcAc was branched onto the acrylate backbone and immersed for 28 days in the SBF solution. This results show the role of polysaccharide in promoting the hydrolytic scissioning of the polymer backbone hence increased the degradation rate.

However, sugar-based glycopolymers has no significant effect on the tensile strength of the cured polymers as shown in Figure 5.12. These results were related to the shape of fabricated dumb-bell from the gycopolymers resin; which was still not optimal. The sample showed delamination and warping, a sign of stress after post- curing. 0 10 20 30 40 50 60 70 80 90 Control 2.5Glc 5.0 Glc So l f ra ct io n (% )

Figure 5.12 Tensile strength of glycopolymers with different ratio of GlcAc. The error bars is the standard deviation of five data obtained at each soaking time.

Figure 5.13 shows the surface morphology analysis on sample 5.0 Glc and the control sample after fabrication. The morphology clearly shows the differences between the two specimens. The control sample has a smooth and less contaminated surface (Figure 5.13 (a)) whereas the sample with 5.0% GlcAc has induced more stress post-curing, which promotes delamination and uneven surface (Figure 5.13(b)).

Further magnification on the SEM (Figure 5.13 (c)), shows the gap of delamination was about 50µm and the surface roughness shows brittleness of the sample which explained the low tensile strength. Bartolo et. al indicated that as the differences between the temperature within the irradiation volume and the surrounding resin that can produce stress gradients [231]. These suggest that the fabrication needs to be in a controlled environment, which is very interesting to explore in the future

0 2 4 6 8 10 12 14 16 18 Control 2.5 Glc 5.0Glc Ten si le st re ng th (k Pa )

123

.

(a) (b)

(c)

Figure 5.13 Surface morphology of (a) control sample, (b) 5.0% Glc with 100x magnification and (c) 5.0 Glc with 500 x magnifications.

5.5 Conclusion

In this study it was shown that it was possible to synthesize a new photopolymer resin with different type of multifunctional acrylate polymer that compatible with a microstereolithography technique. However, the hydrogels suffered with stress post- curing, which promote delamination and warping of the sample.

For further material development, carbohydrate-based polymers were also synthesized, in order to explore their feasibility with microstereolithography technique to further enhance the diversity of the technique. The glycopolymers was

fabrication is needed in order to tackle the stress problem of the cured sample.

In summary, the highly hydrophilicity of PEG and an active site of PEG chain for easily modification through a variety of synthetic reactions has make PEG an interesting and robust biomaterials for hydrogel. With more knowledge and understanding in the tailoring the polymer properties, structure and mechanism during polymerisation, the development of novel materials that are able to fabricate on the microstereolithography technique will be very exciting. This will open up more possibility of new application of hydrogel in tissue engineering field and offer an alternative option to patients.

125