(562b) Graduate Student Award Session: Cysteine-Conjugated Thermoresponsive Hydrogels As Mucoadhesive Intestinal Scaffolds

Introduction: Inflammatory Bowel Disease (IBD) ­­is a condition that affects 3% of US adults [1]. Conventional treatments are aimed at symptom management and often involve invasive surgery leading to only marginal improvements in post-operational quality of life. Mesenchymal stem cell therapy has shown promise in tissue regeneration through the modulation of immune response from inflammatory to regenerative. However, systemic delivery of stem cells has poor viability and therefore limits the efficacy of this approach [2]. Localized cell delivery could help overcome this limitation, but stem cells often require a niche microenvironment for survival and function. Scaffolds constructed out of biocompatible materials can be engineered to provide a specialized microenvironment for cells, enabling localized delivery. Furthermore, in situ-forming scaffolds, made from stimuli-responsive materials, can facilitate minimally-invasive delivery.

Poly(N-isopropylacrylamide) (pNiPAAm) is a thermoresponsive polymer whose aqueous solution undergoes a phase transformation from sol to gel phase above a Lower Critical Solution Temperature (LCST) of ~32 °C. This proximity of the LCST to human body temperature of 37 °C has led to pNiPAAm copolymers being investigated as in situ-forming scaffolds. Moreover, use of different co-monomers can lend additional functionality to the thermoresponsive polymer, such as introducing responsiveness to pH, changing of the LCST or providing anchor points to enable further functionalization. Previous research has shown p(NiPAAm–co­–Glycidyl Methacrylate (GMA)) polymers can be further functionalized by opening the epoxide rings on the GMA units using a nucleophile, like an amine[3]. These functionalizable thermoresponsive polymers can also be delivered to the intestine via an airbrush spray and can sustain encapsulated cell culture [4]. In this work, sulfhydryl groups were introduced onto the pNIPAAm–co­–GMA thermogelling macromer (TGM) by conjugation of cysteine. These sulfhydryl groups covalently crosslink the scaffold with the intestinal mucus layer, providing mucoadhesion.

Intestinal mucus is a hydrogel, consisting of a hydrated network of cysteine-rich proteins called mucins. Cysteine, a trifunctional amino acid with amine, carboxylic acid and sulfhydryl groups, crosslinks the mucins with disulfide bonds. Mucoadhesive CysTGM polymers were synthesized by conjugating cysteine with TGM via epoxy-amine reaction. Sulfhydryl groups on cysteine in CysTGM form disulfide crosslinks with other sulfhydryl groups, such as those abundant in intestinal mucins and other CysTGM chains. This work covers synthesis and characterization of CysTGM and investigates the kinetics of cysteine conjugation and crosslink formation, its impact on mucoadhesion, and encapsulated cell viability.

Materials and Methods: TGM was synthesized by copolymerization of NiPAAm and GMA in a 10:1 mole ratio as previously described in literature and characterized using DSC, GPC and ¹H NMR [3,4]. CysTGM was synthesized in a room temperature reaction by adding cysteine to a 10 wt % TGM solution and agitating for 12 hours. The material was characterized using DSC, GPC, ¹H and ¹³C NMR. Crosslinking behavior was studied using rheometry and sol-gel fractionation. Cellular response to the material was evaluated by LIVE/DEAD-staining encapsulated fibroblasts at various timepoints up to 14 days of culture and observing using fluorescence microscopy. Mucoadhesion was quantified by measuring the pull-off force required to separate two pieces of porcine intestine bound with the polymer.

Results: Cysteine conjugation was confirmed from the appearance of additional peaks on a ¹³C NMR spectrum, corresponding to opened epoxide rings (Figure 1a). Quantification of conjugated cysteine was done using ¹H NMR. Kinetics of Cysteine conjugation were determined by following the reaction using DSC and tracking the change in the LCST (Figure 1b). It was observed that when the polymer is incubated in a thermogelled state, the gel becomes insoluble in water after removal of the thermal stimulus, indicating crosslinking. Rheological measurements of thermogelled Cys₅₀TGM showed a sharp increase in the storage modulus of the gel by 2 orders of magnitude between 2 and 3 hours confirming the formation of a crosslinked network (Figure 1c). Secondary confirmation for crosslinking was obtained by measuring the polymer content in the sol and gel phases at different times of incubation, which showed a distinct change from sol to gel phase at longer incubation times. Adhesion strength was determined by measuring the force per unit area required to pull apart two pieces of porcine intestine stuck with Cys₅₀TGM and was found to increase compared to the control group (Figure 1d). LIVE/DEAD staining at 0- and 14-day timepoints demonstrated the viability of encapsulated fibroblasts (Figure 1f).

Conclusions: Cysteine-conjugated thermogelling polymers were synthesized and characterized. Applicability as a mucoadhesive intestinal scaffold was confirmed by measuring adhesion strength and the ability of the biomaterial to sustain a cell culture. Hence, cysteine-mediated disulfide bond formation can be a promising pathway to tune scaffold mucoadhesion.

References:

[1] J.M. Dahlhamer, E.P. Zammitti, B.W. Ward, A.G. Wheaton, J.B. Croft, MMWR. Morb. Mortal. Wkly. Rep. 65 (2016) 1166–1169.

[2] D.J. Mooney, H. Vandenburgh, Cell Stem Cell 2 (2008) 205–213.

[3] A.K. Ekenseair, K.W.M. Boere, S.N. Tzouanas, T.N. Vo, F.K. Kasper, A.G. Mikos, Biomacromolecules 13 (2012) 1908–1915.

[4] M.O. Pehlivaner Kara, A.K. Ekenseair, J. Biomed. Mater. Res. - Part A 104 (2016) 2383–2393.