Antimicrobial Mechanism of Nanochitosan Dihydroartemisinin Against Multidrug-Resistant Mycobacterium Tuberculosis

Objective The present study prepared nanochitosan dihydroartemisinin to observe its antimicrobial effects and mechanisms on multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). Method (1) Nanochitosan dihydroartemisinin was prepared using nanochitosan as the carrier and a magnetic stirrer heated at constant temperature. (2) A laser particle size analyzer was used to detect the particle size and dispersion of nanodrugs, and scanning electron microscopy was used to observe the morphology. (3) The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,Thiazolyl Blue Tetrazolium Bromide (MTT) assay was used to detect the inhibitory effect of dihydroartemisinin (DHA) and nanochitosan dihydroartemisinin on the proliferation of MDR-MTB. (4) Scanning electron microscopy (SEM) was used to observe the morphological structure of MDR-MTB after nanochitosan dihydroartemisinin application. (5) Fluorescein isothiocyanate (FITC)-labeled nanochitosan dihydroartemisinin and laser confocal fluorescence microscopy were used to observe the entry of drugs into the bacteria and the site of action. (6) MDR-MTB was incubated with DHA and nanochitosan dihydroartemisinin for 48 hours, and the protein spectrum was detected using nonlabelled quantitative proteomics. (7) GO signaling pathway analysis of differentially identified proteins was used to understand the functions, classification and metabolic pathways. Results (1) The average particle size of nanochitosan dihydroartemisinin was 227.7 nm, and its dispersion was 0.341 DHA was embedded in nanochitosan sol, which meets the requirements of nanomedicine. (2) A low concentration of DHA and nanochitosan dihydroartemisinin inhibited the growth of MDR-MTB to varying degrees compared with the negative control group, and the antimicrobial effect of nanochitosan dihydroartemisinin was better than DHA (P < 0.05). Nanochitosan dihydroartemisinin and DHA produced no inhibitory effect on MDR-MTB at concentrations ≥100 µg/mL. (3) SEM results showed that both drugs weakened the stereoscopic sense of tuberculosis bacterium, and the surface was not smooth. Nanochitosan dihydroartemisinin induced bacterium collapse or fragmentation and partial loss of the cytoplasm, and the morphological damage was more serious than in the negative control group. (4) Laser confocal microscopy showed that FITC-labeled nanochitosan dihydroartemisinin entered the body of MDR-MTB, and the fluorescence intensity increased gradually from the surface to the interior. (5) Nonlabelled quantitative proteomics revealed 1771 different proteins in MDR-MTB after drug action and 141 different proteins with statistical significance (P < 0.05). Thirty-one upregulated proteins and 110 downregulated proteins were detected. (6) GO analysis showed that nanochitosan dihydroartemisinin altered the protein profile of MDR-MTB, which was related to the molecular function, biological process and cell localization of MDR-MTB. Signal pathway analysis showed that the different proteins were involved in 92 signal transduction pathways. A metabolic pathway (ribosomal biosynthesis pathway) was obtained using enrichment analysis. Two key proteins involved in this pathway were ribonuclease III and oligonucleotide ribonuclease. Conclusion Certain concentrations of nanochitosan dihydroartemisinin and DHA exhibited antimicrobial effect on MDR-MTB, destroyed the morphology and structure of bacteria, and inhibited bacterial growth. The antimicrobial effect of nanochiFunding Information: This research was funded by the Key Project of Sichuan Science and Technology Department (Grant No. 2019YFS0090) and the Project of Mianyang Science and Technology Bureau (Grant No. 150170).Conflict of Interests: The authors declare no conflict of interest.