(267e) Biomimetic Modification of Thermoresponsive Hydrogel Scaffolds for Enhanced Mucoadhesion

Authors: 
Kanetkar, N., Northeastern University
Ekenseair, A., Northeastern University
Introduction: Inflammatory Bowel Disease (IBD) causes chronic ulcer formation in the intestinal mucosa and affects 1.9 million people in the United States 1. Current treatment options involve invasive surgeries and/or long-term, systemic anti-inflammatory treatments that lead to poor overall quality of life. Cell-based therapies could provide more effective treatment options that focus on regeneration of damaged tissue. Systemic delivery of stem cell suspensions has been shown to help regenerate a variety of damaged tissues, however cell viability and therefore effectiveness remain suboptimal 2. Biomaterial scaffolds can overcome this limitation by sustaining cell populations for a longer duration. In situ forming scaffolds can furthermore deliver cells without invasive surgery.

Poly(N-isopropylacrylamide) (pNiPAAm) is a polymer that forms a gel above a Lower Critical Solution Temperature (LCST) of ~32 °C due to a shift in the balance between hydrophobic and hydrophilic interactions. Polymer chains stabilized by hydrogen bonding at lower temperatures collapse into a globule structure to form a gel at high temperatures due to increased hydrophobicity. pNiPAAm and its copolymers have been investigated as in situ forming scaffolds due to the proximity of its LCST to body temperature. Previous research has established the ability of p(NiPAAm–co­–Glycidyl Methacrylate (GMA)) to form conformal coatings on-demand on intestinal tissue surfaces when delivered via an airbrush spray and to sustain populations of encapsulated cells 3. Here, cysteine pendant groups were introduced on the polymer backbone that react and bind to the intestinal mucus layer.

Intestinal mucus is a hydrogel composed of a network of cysteine rich proteins called mucins. Cysteine, which mediates chemical network formation through disulfide bonds, is a trifunctional amino acid with amine, carboxylic acid and sulfhydryl motifs. GMA contributes epoxide groups to the thermogelling macromer (TGM), which can react with the amine group on cysteine with high specificity through an amine-epoxide reaction. Sulfhydryl groups on cysteine can then form disulfide bonds with other sulfhydryl groups, including those abundant in intestinal mucins. This work investigated the efficacy and kinetics of cysteine conjugation, the kinetics of disulfide bond formation, the viability and activity of encapsulated cells, and the impact of conjugation on mucoadhesion.

Materials and Methods: TGM was obtained by copolymerization of pNiPAAm and GMA in a 10:1 mole ratio 3,4. Cys-TGM was obtained by reacting Cysteine with a 10% TGM solution at room temperature such that 10% of the epoxides have a cysteine pendant group. Differential scanning calorimetry (DSC), gel permeation chromatography (GPC), 1H NMR spectroscopy, and rheometry were used for material characterization. Crosslinking behavior was studied by obtaining sol-gel fractions under varied conditions. Cellular response was evaluated by LIVE/DEAD® assays. Mucoadhesion was determined by the pull-off force required to separate two pieces of porcine intestine bound with the polymer.

Results & Discussion: The kinetics of cysteine conjugation onto the polymer was followed over time using DSC. The peak LCST of Cys-TGM increased from 30.24 ± 0.12 to 32.05 ± 0.18, indicating progress of the reaction. This increase in the LCST can be attributed to increased potential for hydrogen bonding in Cys-TGM chains, which then requires higher energy to undergo a coil-globule transition to form a gel. Whereas TGM exhibited completely reversible thermogelation due to the absence of network-forming chemical crosslinks, Cys-TGM formed crosslinked gels which remained insoluble upon reducing the temperature below LCST. This network formation was accelerated when the gels were kept in a thermogelled state over several hours which was confirmed by measuring the polymer content in the sol and gel fractions. Net movement of the polymer from sol phase to gel phase indicated network formation which reached a critical point at 4 hours of incubation and was complete after 6 hours. Swelling ratio at formation was determined at 37°C for various degrees of cysteine conjugation for a 10 weight % polymer solution after 12 hours of incubation. Results indicated that cysteine conjugation above 30% equivalents was sufficient to prevent syneresis (shrinkage) of the gel upon formation. Adhesion to intestinal mucus was determined by measuring the normal force required to pull apart two CysTGM-coated pieces of porcine intestine incubated at 37°C for 6 hours. Cysteine conjugated TGM showed superior adhesive strength compared to unmodified TGM and a statistically significant increase compared to a mucus-only control. LIVE/DEAD® staining at various timepoints after seeding demonstrated the viability of encapsulated cells in gels with different cysteine content. Cells encapsulated in unmodified TGM initially showed higher death in comparison with the cysteine containing gels. Gels with 30% equivalent cysteine showed similar low death compared to 50% equivalent cysteine gels but showed a higher migratory behavior as opposed to clustered proliferation, indicating that there is an optimum value for the degree of conjugation.

Conclusions: Cysteine modified thermogelling polymers were synthesized, characterized and tested in the context of applicability as an intestinal scaffold. Cysteine-mediated disulfide bond formation was shown to be a promising pathway to enhance scaffold mucoadhesion. Cysteine modification did not hinder the viability of encapsulated cells cultured over a long term.

References:

  1. The National Institute of Diabetes and Digestive and Kidney Diseases. Digestive Diseases Statistics for the United States. Available at: https://www.niddk.nih.gov/health-information/health-statistics/digestive.... (Accessed: 10th February 2018)
  2. Mooney, D. J. & Vandenburgh, H. Cell Delivery Mechanisms for Tissue Repair. Cell Stem Cell2,205–213 (2008).
  3. Pehlivaner Kara, M. O. & Ekenseair, A. K. In situ spray deposition of cell-loaded, thermally and chemically gelling hydrogel coatings for tissue regeneration. J. Biomed. Mater. Res. - Part A104,2383–2393 (2016).
  4. Ekenseair, A. K. et al.Synthesis and Characterization of Thermally and Chemically Gelling Injectable Hydrogels for Tissue Engineering. Biomacromolecules13,1908–1915 (2012).
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