pH‐sustaining nanostructured hydroxyapatite/alginate composite hydrogel for gastric protection and intestinal release of Lactobacillus rhamnosus GG

Abstract The gut microbiome is closely linked to gastrointestinal health and disease status. Oral administration of known probiotic strains is now considered a promising therapeutic strategy, especially for refractory diseases such as inflammatory bowel disease. In this study, we developed a nanostructured hydroxyapatite/alginate (HAp/Alg) composite hydrogel that protects its encapsulated probiotic Lactobacillus rhamnosus GG (LGG) by neutralizing hydrogen ions that penetrate the hydrogel in a stomach without inhibiting LGG release in an intestine. Surface and transection analyses of the hydrogel revealed characteristic patterns of crystallization and composite‐layer formation. TEM revealed the dispersal of the nanosized HAp crystals and encapsulated LGG in the Alg hydrogel networks. The HAp/Alg composite hydrogel maintained its internal microenvironmental pH, thereby enabling the LGG to survive for substantially longer. At intestinal pH, the encapsulated LGG was completely released upon disintegration of the composite hydrogel. In a dextran sulfate sodium‐induced colitis mouse model, we then assessed the therapeutic effect of the LGG‐encapsulating hydrogel. This achieved intestinal delivery of LGG with minimal loss of enzymatic function and viability, ameliorating colitis by reducing epithelial damage, submucosal edema, inflammatory cell infiltration, and the number of goblet cells. These findings reveal the HAp/Alg composite hydrogel as a promising intestinal‐delivery platform for live microorganisms including probiotics and live biotherapeutic products.

obtaining desired health benefits or therapeutic effects to the host.
Microbiome-based therapeutics are considered a promising therapeutic alternative for treating refractory diseases such as inflammatory bowel disease that are difficult to cure with traditional small-molecule medicine. 1,2 Certain microorganisms, such as probiotic strains, which were once regarded as food or food supplements, have gained attention as therapeutic agents. 3 The United States Food and Drug Administration (FDA) has created a category for live biotherapeutic products, which includes live microorganisms that can be used to prevent, treat, and cure disease. 4 In 2021, therapy for Clostridium difficile infection, using live firmicutes spores, passed FDA phase-3 approval. 5 Accordingly, there has been a rapid increase in expectations for microbiome therapeutics based on live microorganisms once regarded as probiotic strains in industry and academia.
Encapsulation of probiotics by using alginate (Alg) hydrogels is the most widely used method due to mild gelation process, pHsensitive disintegration, low cost, and versatile application. However, owing to the porous network and hydrophilic nature of Alg hydrogel, it does not sufficiently maintain probiotic viability in the stomach. Furthermore, Alg hydrogels are mechanically unstable in the presence of monovalent ions, presenting another difficulty in their application. This can be addressed by adding surface coatings to Alg hydrogel beads using cationic materials such as chitosan. Reducing the pore size, and consequently maintaining probiotic cytoplasmic pH, enhances probiotic survival. 6 The gastric survival of probiotics in surface-coated hydrogels could potentially be enhanced by increasing the number of coating layers on the hydrogel; however, the coating processes are time-consuming, and probiotic survival declines during storage, possibly due to the effects of cationic coating materials. 7,8 Composite hydrogels, using materials such as carboxymethyl cellulose, 9 xanthan gum, 10 locust bean gum, 11 nanocellulose, 12 and clay, 13 could enhance encapsulated-probiotic survival at gastric pH. However, the increased viscosity of the mixed slurry could present difficulties during manufacturing, especially during the extrusion process. Further, composite hydrogels are subject to strong molecular interactions, potentially delaying their disintegration and release of encapsulated probiotics in the host intestine.
The physical barrier established in Alg-based delivery systems, such as coating and composite hydrogel, may reduce their efficacy in probiotic intestinal delivery by interfering with the fabrication of the probioticloaded hydrogel, protection in the stomach, and release in the intestine.
Physical barriers based on additional coating materials or composite hydrogel networks may passively delay penetration by gastric fluid and thus extend probiotic survival. However, although physical barriers enhance resistance to gastric pH, they can easily interfere with other critical aspects of the probiotic intestinal-delivery system. Few studies have addressed chemical barrier systems comprising pH buffering agents in Alg-based systems. Alg hydrogels are based on ionically crosslinked networks that can be easily disturbed by calcium-chelating agents or monovalent ions. 14,15 Most buffering agents comprise conjugate acid-base pairs that contain monovalent ions; when these monovalent ions dissociate, the hydrogel cannot sufficiently protect the encapsulated probiotics from penetrating gastric fluid due to the disturbed hydrogel network.
Hydroxyapatite (HAp), a bioceramic material, has pH-modulating properties owing to its surface functional groups. 16 Under stomach pH conditions, HAp consumes massive amounts of hydrogen ions as it dissolves, liberating Ca 2+ ions. Ca 2+ ion replenishment via ethylenediaminetetraacetic acid has been reported to enhance the gastric survival of encapsulated probiotics by reinforcing hydrogel networks. 17 HAp, which occurs naturally in bones and teeth, is biocompatible and does not damage live microorganisms. 18 The inclusion of HAp in Alg hydrogels has been considered for the controlled release of drugs and for osteoblast-based bone-tissue recovery. 19

| LGG preparation
For the starter culture, a single LGG colony was inoculated into MRS medium and cultured for 12 h. Then, 5 mL of the starter culture was transferred to 195 mL MRS medium and cultured for 12 h (37 C, with shaking at 220 rpm).
LGG was harvested and washed twice with 0.1% peptone, followed by redispersion in deionized distilled water (ddH 2 O) for encapsulation.

| Encapsulation of LGG in the HAp/Alg composite hydrogel
Hydroxyapatite/alginate (HAp/Alg) composite hydrogels were fabricated using a modified version of a previously reported method. 21 Briefly, 3 g sodium alginate was dissolved in 190 mL ddH 2 O. Different concentrations of (NH 4 ) 2 HPO 4 (50, 100, and 150 mM, for Groups 1, 2, and 3, respectively) were added to the prepared alginate solution, which was then mixed using

| LGG survival at gastric pH
Simulated gastric fluid was prepared as previously described. 22 Briefly, 0.8 g NaCl was dissolved in 395 mL ddH 2 O. When the solution turned clear, the pH was adjusted to 2.0 using 1 M HCl, and ddH 2 O was added to a total volume of 400 mL. Each hydrogel sample (1 g), containing the encapsulated LGG, was added to the simulated gastric fluid. The hydrogels were incubated in the gastric fluid for 2 h (37 C, with shaking at 99 rpm). The hydrogels were degraded using a 10% citrate buffer. Encapsulated-LGG survival was determined by plate counting. To visualize the enzymatic activity of the encapsulated LGG, the hydrogels were incubated with carboxyfluorescein diacetate (cFDA) dye and observed using an in vivo imaging system (IVIS).

| Release of encapsulated LGG at intestinal pH
The intestinal pH solution was prepared by dissolving 2.72 g KH 2 PO 4 in 385 mL ddH 2 O, the pH was adjusted to 7.2 using 1 M NaOH, and ddH 2 O was added to a total volume of 400 mL. Each hydrogel sample (1 g) was incubated in an intestinal pH solution for 4 h. Samples (100 μL) were collected at different time points (0, 1, 2, 3, and 4 h). The release of LGG was evaluated by plate counting. The proportion of LGG (expressed as a percentage) released from the hydrogel was calculated as follows: Released LGG % ð Þ¼ Count of released LGG Initial LGG count Â 100

| Determination of LGG survival
Viable LGG cells were counted using the plate counting method. After treatment, 100 μL of each sample was serially diluted (from 10 0 to 10 À7 ), plated onto MRS agar plates, and incubated at 37 C for 48 h under anaerobic conditions. Finally, the colonies were counted on MRS agar plates.

| Examination of the efficacy of HAp/Alg composite hydrogels in mice
The procedures involving animals were performed according to the

| Statistical analysis
Statistical analyses of all in vitro and in vivo data were performed using one-way and two-way analysis of variance (ANOVA), followed by the Bonferroni test, using GraphPad Prism software (v.5.0; Graph-Pad Software, Inc., LA Jolla, California). Differences were considered statistically significant at p < 0.05.

| Fabrication of the pH-sustaining HAp/Alg composite hydrogel
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. 29 Here with HAp (550-650 cm À1 ). 30 The characteristic peak of mannuronic acid in Alg (1088 cm À1 ) was diminished as the amount of HAp was increased, whereas the peak of guluronic acid (1030 cm À1 ) in Alg remained unaffected. 31 The findings suggest that there is an interaction between the calcium ions in HAp and the mannuronic acids in Alg, whereas the guluronic acids in Alg is involved in the formation of calcium-Alg hydrogel networks.

| Internal pH changes in HAp/Alg composite hydrogel beads under gastric conditions
Gastric pH ranges from 1.0 to 2.5 and threatens the survival of the administered probiotics, disturbing their membrane integrity and causing loss of enzymatic activity due to an excess of hydrogen ions. 32,33 The penetration of acidic gastric fluid into the highly porous hydrogel networks, and the subsequent drop in pH, presents a significant drawback of Alg-based probiotic delivery systems. We sought to avoid this problem via our pH-sustaining HAp/Alg composite hydrogel system.

| Gastric survival of LGG
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 survival of the encapsulated probiotics is threatened in a stomach pH condition (Figure 4a). We observed a sustained pH in the HAp/Alg composite hydrogel beads (Figure 3). We  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 (Figure 7c). Colitis clinical progression was monitored daily by assessing the DAI. 24