(W1230-02-11) Intestinal Delivery of Live Biotherapeutic Products by pH-Sustaining Hydrogels and Their Therapeutic Efficacies on DSS-Induced Colitis

Purpose: The gut microbiome is closely linked to gastrointestinal health and disease status. Oral administration of live microorganisms such as live biotherapeutic products (LBPs) is now considered a promising therapeutic strategy, especially for refractory diseases such as inflammatory bowel disease. In this study, intestinal delivery of encapsulated LBPs was achieved by neutralizing hydrogen ions that penetrate the hydrogel in a stomach without inhibiting intestinal release. Furthermore, therapeutic efficacy of the LBPs-encapsulating hydrogel was assessed in DSS-colitis model.

Methods: Nanostructured hydroxyapatite/alginate (HAp/Alg) composite hydrogel was fabricated in one step gelation/crystallization process. Lactobacillus rhamnosus GG (LGG) was used as a model live biotherapeutic products strain. The hydrogel was characterized by using electron microscopy, Fourier-transform infrared spectroscopy. The gastric pH protective effect and intestinal pH release of the hydrogel was evaluated by plate counting. Therapeutic effect of LGG-encapsulating hydrogel was evaluated in DSS-induced colitis model, in terms of body weight, disease activity index, colon length, spleen weight, and colon section staining.

Results: An ideal probiotics delivery system should minimize loss of viability during fabrication by applying simple manufacturing processes, effectively protecting the encapsulated probiotics in a stomach, and releasing them in the intestine. Here, we fabricated the HAp/Alg composite hydrogel in one step, via extrusion of a diammonium hydrogen phosphate ((NH₄)2HPO₄) and Alg mixture into a calcium chloride solution (Figure. 1). The Alg hydrogel was formed via the interaction between the calcium and the guluronic acid in the Alg polymer; the HAp crystallizes gradually from the source calcium and phosphate. Encapsulated probiotics require pH homeostasis in order to maintain their enzymatic function and to survive in a harsh low-pH environment. Without this homeostasis, the function and survival of the encapsulated probiotics are threatened in a stomach pH condition (Figure 2). We used cFDA, a fluorescent dye, to assess enzymatic activity of HAp/Alg composite hydrogel-encapsulated LGG. Two hours after gastric pH treatment, the fluorescence intensity of the HAp/Alg composite hydrogel was maintained, while that of the Alg hydrogel beads was lost. To quantify the protective effect of the HAp/Alg composite hydrogel beads against gastric pH, we quantified LGG survival via plate counting. After treatment with simulated gastric fluid at pH 2, the survival of the LGG in the Alg hydrogels decreased sharply, to 3.55 log CFU/g. For the HAp/Alg composite hydrogel-encapsulated LGG, survival increased gradually as the concentration of the precursor diammonium hydrogen phosphate increased. In HAp/Alg Group 3, 9.19 log CFU/g of the encapsulated LGG survived, similar to the initial count. To observe their therapeutic effects, we administered a HAp/Alg hydrogel bead orally in a DSS-induced ulcerative colitis mouse model (Figure 3). The DSS model and healthy control groups did not differ in body weight during the adaptation period. Unlike the healthy control group, body weight of mice was reduced in the colitis, Alg, and HAp/Alg groups after colitis induction. After DSS administration ceased, body weight recovered more rapidly in the HAp/Alg group than in the colitis and Alg groups. DAI was constant in the healthy control group, indicating that no colitis was induced. On Day 10 after colitis induction, the colitis and Alg groups both exhibited severe bleeding and diarrhea, reflected in the high DAI scores, although DAI was lower in the HAp/Alg group than in the Alg group. Colon length was significantly shorter following ulcerative colitis induction than in the healthy control group. The average colon lengths were 106.13, 64.56, 76.02, and 96.68 mm, in the healthy control, colitis, Alg, and HAp/Alg groups, respectively. Spleen weight was significantly higher in the colitis and Alg groups than in the HAp/Alg groups. Overall, the LGG-encapsulated HAp/Alg composite hydrogels ameliorated ulcerative colitis, as indicated by the changes in body weight, DAI, colon length, and spleen weight.

Conclusion: With the increasing efforts to utilize live microorganisms for therapeutic purposes, the demand for effective delivery systems has increased. To address this, we developed an HAp/Alg composite hydrogel for the intestinal delivery of LGG. We fabricated the HAp/Alg composite hydrogels via a one-step ionic Alg gelation and HAp crystallization process. The pH-sustaining nanostructured HAp/Alg composite hydrogel achieved gastric protection and intestine-targeted release of LGG. These findings suggest that this composite system can achieve successful intestinal delivery of live microorganisms and ameliorate colitis.

Figure 1. Fabrication of nanostructured hydroxyapatite/alginate (HAp/Alg) composite hydrogel. LGG, Lactobacillus rhamnosus GG.

Figure 2. Survival of encapsulated LGG after gastric pH challenge.

Figure 3. Therapeutic effects on ulcerative colitis of LGG encapsulating HAp/Alg composite hydrogels.