Designing and characterizing the plant-based chitosan-modified poly (lactic-co-glycolic acid) nanoparticles for salmonella and e. coli-induced gastroenteritis

Abstract

A global mortality rate of over 1.6 million deaths was reported in 2017 due to gastroenteritis. The highest mortality rate is reported annually in sub-Saharan Africa and South Asia. In Namibia, the national prevalence of gastroenteritis was 17% in 2014 and to date, gastroenteritis is responsible for 5% of all deaths in children under the age of 5. In the Ohangwena region, 23.8% of children were reported to have gastroenteritis from a total sample size of 530 children in 2020. Salmonella species and Escherichia coli were responsible for most bacterial gastroenteritis cases reported in Windhoek in 2018. Some of the key challenges in the management of gastroenteritis are: 1) Antibiotics and antidiarrheal agents used in the treatment of gastroenteritis have side effects in children and immunocompromised patients. 2) The high treatment doses required for the effective treatment of gastroenteritis are harmful to the beneficial microbiota within the digestive system. 3) Salmonella species and E. coli which cause gastroenteritis, have shown over 60% resistance to the available antibiotics. 4) Many orally administered antibiotics and antidiarrheal agents have reduced bioavailability at the site of infection, within the alimentary canal. Extracts derived from medicinal plants are used in ethnomedicine in African countries including Namibia as alternative treatments for Salmonella and E. coli induced gastroenteritis. However, the lack of information on the presence and quantity of bioactive compounds present, their safety, and efficacy. Moreover, plant-based treatments are faced with numerous challenges within the GI tract which reduce their bioavailability and efficacy limiting their mainstream uses in treating gastroenteritis. The use of stable nano-particles that deliver plant-based medicine has attracted attention in the field of drug development. Such an approach could be an option for effectively delivering plant-based ii medicines, safely to the lower gastro intestinal tract, the site of Salmonella and E. coli with the added benefits of controlled release at this site to increase bioavailability reducing required doses. Benefits would also include reduced side effects and higher efficacy of antidiarrheal compounds from medicinal plant extracts. This study then aimed at designing and characterizing the plant-based chitosan-modified poly (lactic-co-glycolic acid) nanoparticles for Salmonella and E. coli-induced gastroenteritis. Medicinal plants used as an alternative treatment for gastrointestinal infection were collected from Iikokola village in the Omusati region in Namibia. Organic and aqueous extracts were prepared from the collected plants, screened for the present bioactive compounds, and tested for their potential in eliminating multidrug-resistant clinical and reference strains of Salmonella and E. coli that are commonly associated with bacterial gastroenteritis. The antibacterial activity against clinical and reference strains of Salmonella and E. coli and cytotoxicity to human fibroblast cells (NIH/3T3) of prepared extracts were also determined. Plant extracts with potent antibacterial activity against clinical and reference strains of Salmonella and E. coli were encapsulated into poly (lactic-co-glycolic acid) nanocarriers coated with chitosan (CMPLGA) and characterized nanoparticles’ morphology, size, zeta potential, polydispersity index, encapsulation efficacy, and pH based in vitro release in simulated gastrointestinal tract conditions. Furthermore, the stability of the designed nanoparticles was determined by monitoring the physicochemical properties, in-vitro cytotoxicity, and antibacterial activity of lysed and un-lysed nanoparticles stored at 4 and 25 ºC for 24 weeks. As part of this study, an ethnomedicinal survey was conducted and 19 medicinal plants that are used in Iikokola village to treat gastrointestinal conditions and other diseases were iii documented. Crude extracts of five of the frequently used medicinal plants used in this study (Lantana camara, Terminalia sericea, Grewia tenax, Albizia anthelmintica, and Chorcorus trides) were used to validate the phytochemical compositions of these plants and were found to contain multiple bioactive compounds. The collected plants showed potent antibacterial activity against multidrug-resistant clinical Salmonella and E. coli strains. Mild cytotoxicity to the mouse fibroblast cells (NIH/3T3) line was observed for the five plant extracts. All extracts showed an IC50 above 50 µg/ml. Encapsulating the selected plant extracts in CMPLGA enhanced the antibacterial properties of the plant extracts, reduced the cytotoxicity, and improved the in vitro release properties of the encapsulated extracts at pH 1.2 and 7.4. Lastly, factors such as lyophilization showed a change in the physicochemical properties of the formulated CMPLGA nanoparticles. Prolonged storage at 25 ºC tremendously reduces the efficacy of the CMPLGA nanoparticle, while storing at 4 ºC has only shown a minimal decrease in the efficacy of the CMPLGA nanoparticles. Overall, the use of CMPLGA nanoparticles enhanced the antibacterial activity of the plant extracts in this study and improved their in vitro release properties in simulations at a pH of 1.2 (stomach pH) and 7.4 (ileum pH). The use of CMPLGA nanoparticles also reduced the cytotoxicity of the extracts by over 20%. The toxicology and release properties of the formulated CMPLGA nanoparticles should be evaluated in an in vivo model as part of their development for mainstream use in treating Salmonella and E. coli-induced gastroenteritis.