Design and Evaluation of Intracellular Machinery Directed Stereocomplexed Nanomicelles as Therapeutic Intervention Against Mycobacterium

Abstract

Mycobacterium has been a major challenge since emergence of extensively resistant strains. Presence of thick waxy envelop, efflux of drug and maintenance of reducive environment via mycothione reductase (Mtr) pathway help Mycobacterium to survive intracellularly and serve as major contributing factors towards development of drug resistance. Resistance to rifampicin has also been associated with decreased intracellular accumulation of drug due to involvement of mentioned mechanisms. The main objective of this study was to investigate polyethylene imine (PEI) based stereocomplexed nanomicelles for delivery of rifampicin against intracellular Mycobacterium using in vitro and in vivo evaluation. Rifampicin (Rif) loaded isotactic micelles (PEI-g-PLLA and PEI-g-PDLA), stereocomplexed nanomicelles (StM), cholesterol conjugated stereo complexed nanomicelles PEI(CH)-StM, and cholesterol conjugated thiolated stereo complexed nanomicelles thPEI(CH)-StM were prepared by process of self-assembly. The nanoformulations were subjected to in vitro analysis followed by in vivo analysis against two strains of Mycobacterium i.e., Mycobacterium bovis and Mycobacterium tuberculosis (H37-Rv). In comparison to isotactic nanomicelles, StM exhibited a higher encapsulation efficiency and drug loading with 84% of drug release in 48hrs. At pH 5 (endosomal pH) with minimal initial burst. MIC of StM was found to be lower, showed better uptake, and minimum cytotoxicity in murine alveolar macrophages as compared to PEI-g-PLLA and PEI-g PDLA nanomicelles. StM showed higher distribution in macrophage rich organs in comparison to free drug along with improved half-life, AUC, AUMC, and MRT in comparison to isotactic nanomicelles indicating maximum bioavailability and efficacy of StM. In comparison to isotactic nanomicelles and StM, PEI(CH)-StM and thPEI(CH)-StM showed better in vitro results. thPEI(CH)-StM) showed improved in vitro drug release, intestinal mucopenetration, macrophage uptake, mycobacterial inhibition potential and biocompatibility as compared to cholesterol conjugated stereocomplexed nanomicelles. The in vivo efficacy of nanomicelles was found to be closely related in both strains of Mycobacterium. MIC of thPEI(CH)-StM against M. bovis and M. tb (H37-Rv) was found to be significantly higher as compared to free drug, PEI(CH)-StM, and StM. thPEI(CH)-StM also exhibited efflux pump inhibition and prolonged retention in alveolar macrophages along with Mtr inhibition with Ki value of 2.74 and IC50 of 4.73 µg/mL. In vivo studies demonstrated significantly DRSML QAU ix higher reduction in both M. bovis and M. tb (H37-Rv) CFU by thPEI(CH)-StM, a preferential uptake in lungs along with improved pharmacokinetics, and reduced dosing frequency in comparison to PEI(CH)-StM and StM. Thus, rifampicin loaded thPEI(CH)-StM can be regarded as a stable, biocompatible, and efficient nanocarrier for improved anti-mycobacterial potential of rifampicin via cholesterol conjugation and inhibition of efflux pump and Mtr