Synthesis and characterization of ureidopyrimidinone substituted gelatin based elastomeric hydrogel

• Main Author: Panwar, Amit
• Other Authors: Tan Lay Poh
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Materials
• Online Access: https://hdl.handle.net/10356/144360
• Institution: Nanyang Technological University

Elastomeric hydrogels have potential applications in implantable stretchable electronics devices including heart sensors/monitors, drug patches etc. Synthetic polymers polyacrylamide and hybrid gels have been used for the fabrication of stretchable electronics device fabrication. Replacement of synthetic polymer elastomeric hydrogels with natural protein based elastomeric hydrogels would provide better biocompatibility, biodegradability and sustainability. Gelatin is a protein-based animal derived biopolymer and has great potential for biomedical applications due to its biocompatibility, biodegradability and presence of cell adhesion RGD domains. As gelatin is produced by acid or base hydrolysis of collagen along with thermal treatments, gelatin loses most of its physical and covalent crosslinking after production. Due to this, it could only form a weak hydrogel below sol-gel temperature (20-25OC) with limited elastomeric properties [1, 2]. To enhance the stretchability of gelatin hydrogels, supramolecular interactions need to be introduced in gelatin hydrogel. In this dissertation, a novel method for hydrogel fabrication was developed and optimized to enhance the stretchability of gelatin via supramolecular interactions. During elastomeric film fabrication, gelatin was exposed to water-tetrahydrofuran (water-THF) co-solvent system which caused structural transformation and has resulted in an elastomeric micro-porous film with a Young’s modulus, ultimate tensile strength and strain at break of 78.71 ± 0.48 kPa, 516.7 ± 106.3 kPa and 1269±80. %, respectively. Structural transformation caused by co-solvent system involved transition from an aggregated state in water to spherical shaped coacervates at 43.8% THF with >90% transmittance in solution state. Further fusion of small spherical coacervates to form larger spherical coacervates took place with further increase in THF to 60%. This could have been caused by presence of multi droplet phase in water-THF co-solvent systems, which has caused fusion of smaller coacervates to form larger coacervates with increase in THF proportion. Further increase in THF proportion to 80% have resulted in formation of an elastomeric micro-porous film. To remove the solvent, the film was dried in vacuum and rehydrated again at room temperature. The film underwent swelling till ~90% water content and started breaking into pieces due to loss of integrity. To enhance the stability elastomeric film, an external supramolecular crosslinking of ureidopyrimidinone (Upy) was introduced in gelatin. Upy is a hydrophobic self-assembled moiety developed by Meijer, which can undergo strong dimerization via quadrupole hydrogen bonding [3]. Upy was synthesized and substituted in gelatin via two step methods. Substitution was carried out in two steps with one step involving substitution of Upy to amino groups and the second step involves the addition of catalyst for Upy substitution at hydroxyl groups. To achieve different degrees of Upy substitution, Upy feed in the reaction was varied with 0.10, 0.30 and 0.50 g/2g gelatin to synthesize GEUPYII (0.10), GEUPYII (0.30) and GEUPYII (0.50). In situ co-solvent optimization studies have shown that the transmittance at 600 nm increases with increase in THF proportion as contrary to the gelatin alone without Upy substitution which had no significant change in transmittance till 43.8% THF. This could be due to decrease in hydrophobic aggregation due to solubility of Upy in THF as per earlier studies [4]. All Upy substituted gelatin derivatives were studied for their structural transformation at 50% THF. At 50% THF with 24 hours of incubation at 37 oC, some dissolved polymer fraction of gelatin, GEUPYII (0.10) and GEUPYII (0.30) sedimented at the bottom and the rest remained in supernatant. However, GEUPYII (0.50) remained as stable solution after incubation. From the SEM analysis of sedimented film and supernatant, it was observed that in case of gelatin the sedimented coacervates formed a continuous porous film due to aggregation of coacervates, as gelatin is not compatible with THF. In case of GEUPYII (0.10), the sedimented film was not continuous, whereas in GEUPYII (0.30), the coacervates present in sedimented film had fibrous and spherical structures and did not fuse to form a film. In case of GEUPUYII (0.50), no significant sedimentation was observed but only spherical coacervates in the solution state. Presence of spherical aggregates in GEUPYII (0.50) could be due to the presence of stable moving droplet phase (MDP)/ coacervates formed during phase inversion at 50% THF. Due to higher Upy substitution in GEUPYII (0.50), the interactions between the solute and the solvent has increased, which may had prevented the coalescence of coacervates at 50% THF [5]. However, further increase in THF proportion had caused fusion of coacervates to form an elastomeric film similar to gelatin. This could be due to coalescence of coacervates during coarsening which took place through non-elastic collision of coacervates in liquid phase [5]. GEUPYII (0.50) was chosen to study the fusion of coacervates and elastomeric film fabrication, since it had the highest Upy substitution required to form a supramolecular network. In GEUPYII (0.50), adjustment of THF proportion from 50% to 52.5%, 60% and 70% THF and instantaneously freezing of samples after 30 seconds mixing followed by freeze-drying for SEM analysis had shown the formation of large micro-sized spherical coacervates, probably caused by fusion of smaller aggregates in case of 60% and 70% THF. Also, the proportion of large micro-sized coacervates was higher in 70% THF in comparison to 60% THF, demonstrated the effect of THF on the fusion of aggregates. GEUPYII (0.50) elastomeric film fabrication was carried out at 80% THF, similar to gelatin. The elastomeric film of GEUPYII (0.50) 80% THF had 10.13 ± 1.3 kPa Young’s modulus with 207.96 ± 4.8 kPa ultimate tensile strength and 1405.9 ± 47.9 % strain at break. The GEUPYII (0.50) 80% THF film had a higher strain at break but lower Young’s modulus (10.13 ± 1.3 kPa vs 78 .71 ± 0.48 kPa) and ultimate tensile strength (207.96 kPa vs 516.7 ± 106.3 kPa) as compared to gelatin elastomeric film. GEUPYII (0.50) 80% THF film was air dried in vacuum and was rehydrated with water. Water adsorption reached to ~70% within 2 minutes and remained constant when observed for 48 hours and was stable. Rehydrated air-dried films of GEUPYII (0.50) had 318.7 ± 44.4 %, strain at break with 27.35 ± 2.7 kPa, Young’s modulus and 88.57 ± 50.3 kPa, ultimate tensile strength. Rehydration of air-dried films had lower strain at break (318.7 ± 44.4 %, vs 1405.9 ± 47.9 %), lower ultimate tensile strength (88.57 ± 50.3 kPa vs 207.96 ± 4.8 kPa) and higher Young’s modulus (27.35 ± 2.7 kPa vs 10.13 ± 1.3 kPa) as compared to GEUPYII (0.50) 80% THF films. This could be due to decrease in dissipative supramolecular networks and increase in hydrophobic interactions due to replacement of water-THF co-solvent with water which had decreased the stretchability and increased the stiffness of the hydrogel, respectively. The abovementioned study has established a novel method of elastomeric film fabrication for gelatin based hydrogels and Upy crosslinking reinforced gelatin has been fabricated into a stretchable hydrogel with higher stretchability in comparison to native gelatin based hydrogel and covalently cross-linked gelatin based hydrogels.