Introduction: Half of the world population is infected with Helicobacter pylori (H. pylori)[1]. Currently used triple therapy is inefficient in 20% of the cases and since persistent infection leads to development of gastric cancer, alternative treatments urge[2]. Chitosan microspheres (Mics) have recently been proposed as promising H. pylori-binding system, due to their antimicrobial/mucoadhesive properties[3],[4]. This work aims to evaluate the efficiency of orally administrated Mics with different sizes and degree of acetylation to fight H. pylori gastric infection in C57BL/6 mice.
Materials and Methods: Large (XL) and small (XS) Mics were produced from chitosan with different degrees of acetylation (DA 6% and 16%) using an aerodynamically driven system and named as XL16, XL6, XS16 and XS6. Mics were crosslinked with genipin (10mM, 45min) followed by lyophilization. Particles size and morphology were evaluated by laser diffractometry (Mastersizer), high-throughput optical microscopy (IN Cell Analyzer) and scanning electron microscopy (SEM). Mics stability in simulated gastric fluid (24h; 37°C) and ability to adhere FITC-labelled H. pylori (SS1 and J99 strains) (pH 6.0; 2h; RT; 90rpm) were assessed by inverted fluorescence microscopy. Cytotoxicity of increasing amounts of Mics was evaluated in vitro using AGS gastric cell line through metabolic activity measurement (resazurin assay) and immunocytochemistry (cytoskeletal morphology). Efficiency of Mics was evaluated in H. pylori infected C57BL/6 male mice (n=12/group). Mice were orogastrically inoculated twice with 1010 SS1 H. pylori strain and, after 3 weeks, daily with 4 different Mic treatments (1000 XL Mics and 100000 XS Mics) for 14 days. Control groups with non-infected animals (with and without Mics administration) were performed. Mice physical appearance and weight variations were monitored. Animals were sacrificed and stomachs collected to assess H. pylori infection by plating (CFU counting), PCR and histological analysis (Modified Giemsa). Gastric and liver histopathology (H&E staining) was performed to evaluate Mics toxicity.
Results and Discussion: Porous XL16 and XL6 Mics with ~ 160 µm and XS16 and XS6 Mics with ~ 40 µm were obtained. Mics were stable in simulated gastric fluid and bind different strains of H. pylori. No in vitro cytotoxicity was observed up to the highest amount of Mics tested (5000 XL Mics; 100000 XS Mics), except when 5000 XL16 Mics were used (45% decrease of cell metabolic activity). The presence of H. pylori in the gastric mucosa of infected mice (without treatment) was confirmed by CFU counting, PCR and modified Giemsa staining. Infection levels of ~104 CFU’s/stomach were observed, as well as small atrophy in the distal portion of the stomach and some inflammatory infiltrates. Statistically significant reduction of infection was verified for treatments with XL6 Mics and XS16 Mics (90.3% and 89.7%, respectively) without causing gastric or hepatic toxicity. These results, together with our previous findings, suggest that larger and more mucoadhesive microspheres (XL6 Mics) can better remove H. pylori present in the mucus layer, and smaller and less mucoadhesive microspheres (XS16 Mics) can also penetrate the gastric pits and remove therein adherent bacteria.
Conclusion: Orally administrated chitosan microspheres can be used as treatment to fight H. pylori infection by binding these bacteria in the stomach and removing them through the gastrointestinal tract.
FEDER and Portuguese Foundation for Science and Technology (FCT) for financial support through projects UID/BIM/04293/2013, PTDC/CTM-BPC/121149/2010, EXPL/CTM-BIO/0762/2013 and PTDC/CTM-BIO/4043/2014.
References:
[1] Wroblewski, L. E.; Peek, R. M.; Wilson, K. T., Helicobacter Pylori and Gastric Cancer: Factors That Modulate Disease Risk. Clin Microbiol Rev 2010, 23 (4), 713-739
[2] Goncalves, I. C.; Henriques, P. C.; Seabra, C. L.; Martins, M. C. L., The Potential Utility of Chitosan Micro/Nanoparticles in the Treatment of Gastric Infection. Expert Review of Anti-Infective Therapy 2014, 12 (8), 981-992.
[3] Fernandes, M.; Gonçalves, I. C.; Nardecchia, S.; Amaral, I. F.; Barbosa, M. A.; Martins, M. C. L., Modulation of Stability and Mucoadhesive Properties of Chitosan Microspheres for Therapeutic Gastric Application. International Journal of Pharmaceutics 2013, 454 (1), 116-124
[4] Gonçalves, I. C.; Magalhães, A.; Fernandes, M.; Rodrigues, I. V.; Reis, C. A.; Martins, M. C. L., Bacterial-Binding Chitosan Microspheres for Gastric Infection Treatment and Prevention. Acta Biomaterialia 2013, 9 (12), 9370-9378