Synthetic mucus biomaterials for local delivery of monoclonal antibodies in inflammatory bowel disease

Introduction: Inflammatory bowel disease (IBD) is an autoimmune condition affecting millions of people worldwide. IBD is characterized by chronic inflammation of the gastrointestinal tract with unpredictable disease flares that require continuous or multiple rounds of therapy throughout patient’s lifetime. Monoclonal antibodies (mAbs) have emerged as highly effective biologic therapies for IBD and TNF-α neutralizing mAbs have proven successful in maintaining IBD remission. TNF-α mAbs (∼100 mg/dose) are clinically given through intravenous or subcutaneous infusion. As a result of the systemic delivery route, mAbs used to dampen inflammation pose a potential risk of immunosuppression which render the patient susceptible to infections. Further, systemic administration of mAbs only allows a fraction of the therapeutic payload to reach their intended tissue making the therapy very expensive. Motivated by this, we have developed mucin-based hydrogels for local delivery of mAbs in IBD to limit systemic exposure and reduce dose requirements for improved therapeutic outcomes. Mucus is naturally enriched with peptides and antibodies to help control pathogen invasion and to prime the immune system. These natural binding pockets on mucins make it the ideal biomaterials for encapsulation and controlled release of mAbs. To evaluate the potential use of mAb-loaded synthetic mucus biomaterials to treat IBD, we evaluated the anti-inflammatory effect of the formulation against macrophage activation in vitro and in vivo in a mouse model of IBD.

Materials and

Methods: To synthesize mucin-based hydrogels, pig gastric mucin (PGM) was crosslinked with 10kD 4-arm PEG thiol. To evaluate mAb release kinetics, mucin hydrogels were loaded with FITC-labeled IgG to enable fluorescence reading. To assess biocompatibility in vitro, HEK293T cells were treated with mucin gels and viability was assessed using resazurin assay. The anti-inflammatory effect of TNF-a targeting mAb-loaded hydrogels was evaluated in RAW264.7 mouse macrophages stimulated with lipopolysaccharide (LPS). Following treatment, macrophage polarization and cytokine production (e.g., TNF-a, IL-6, IL-10) was assessed via flow cytometry and ELISA, respectively. In vivo biodistribution in the colon was evaluated following local delivery in healthy mice and TNBS-induced mouse model of IBD. Female C57BL/6 mice were given Cy5 labeled IgG loaded mucin gel intrarectally following saline enema. At 0 hour, 2 hours and 4 hours post administration, mice were sacrificed to isolated colons for IVIS imaging. To induce IBD, TNBS was given colorectally on Day 0 following saline enema. Mice were monitored daily for weight loss on Day 1, Day 2 and Day 3 to confirm onset of disease. On Day 3, mice were given Cy5-labeled IgG solution and Cy5 labeled IgG loaded mucin gel intrarectally to evaluate biodistribution. At 2 hours and 4 hours post administration, mice were sacrificed to isolate colons for IVIS imaging. To evaluate efficacy, TNBS induced IBD mice were treated with anti-TNF-α loaded mucin gels for a total of 3 daily treatments. Twenty-four hours following final treatment mice were weighed, and colorectal tissues were obtained for histological analysis.

Results, Conclusions, and Discussions: Results and

Discussion: We successfully formulated synthetic mucus biomaterials via crosslinking with 4-arm PEG thiol and mAb loading up to 1% w/v was achieved via Fc mediated interactions with mucin biopolymers (Fig. 1A). HEK293T cell viability showed no statistical difference between control and synthetic mucus gel treatments indicating biocompatibility of the gels (Fig. 1B). Drug release studied in PBS alone showed initial burst release of 20% and a total of 90% within 24 hours. Drug release in conditions mimicking the protease rich gastrointestinal tract showed increased drug release reaching 100% in 24 hours. The differences in drug release can be attributed to the degradation of hydrogel backbone i.e., mucins by proteases. However, both drug release in both conditions showed korsemeyer peppas diffusion coefficient n < 0.45 indicating diffusion dominant drug release (Fig. 1C). Decrease in CD80 expression (Fig. 1E) and TNF-a production (Fig. 1F), representative of proinflammatory macrophages was observed upon treatment with TNF-α targeting mAb loaded synthetic mucus hydrogels. This anti-inflammatory effect on LPS-treated macrophages shows retention of mAb bioactivity when loaded in the gel. Further, biodistribution studies in healthy mice showed retention less than 2 hours (Fig. 1G). However, gel retention of up to 4 hours in TNBS induced mouse model of IBD was observed (Fig. 1H). This may be attributed, at least in part, to charge-mediated interactions between the negatively charged synthetic mucus gels and positively charged inflamed regions of the colorectal tract in IBD. Studies are underway in this model to determine if gel-mediated retention of mAbs loaded in synthetic mucus biomaterials enhances their therapeutic effect.

Conclusions: We have successfully formulated TNF-α targeting mAb loaded synthetic mucus biomaterials without impacting the bioactivity of mAbs. Synthetic mucus hydrogels exhibit biodegradability and controlled drug release in the presence of proteases present in the gastrointestinal tract. In addition, these gels address disadvantages of traditional mAb delivery and can be used for local delivery of TNF-α targeting mAbs in IBD treatment. In our future work, efficacy will be evaluated in TNBS induced IBD mouse model.

Acknowledgements (Optional): This work was supported by the NIH R21 Award EB030834