Chlorambucil-Loaded Nanomicelles against Hepatocellular Carcinoma

Abstract

For hepatocellular carcinoma (HCC) therapy, an NQO1-responsive chlorambucil-loaded nanomicelle (CLB-AZO-mPEG5) was designed and synthesized to overcome the poor water solubility of chlorambucil and to explore its mechanism against HCC. CLB-AZO-mPEG5 was constructed by conjugating hydrophobic chlorambucil with hydrophilic monomethoxypolyethylene glycol via an azobenzene linker, enabling specific drug release upon cleavage by NQO1, which is highly expressed in the tumor microenvironment. CLB-AZO-mPEG5 nanomicelles exhibited a filamentous nanostructure with good dispersibility, stability, and biosafety. Cellular assays demonstrated that CLB-AZO-mPEG5 significantly inhibited the proliferation of HepG2 cells, and this effect was markedly attenuated upon NQO1 knockdown, indicating that its antitumor activity is NQO1-dependent. In a HepG2-RAW264.7 coculture system, CLB-AZO-mPEG5 significantly upregulated M1 macrophage markers (CD86, iNOS) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), while downregulating M2 markers (CD206, Arg-1) and anti-inflammatory cytokines (IL-10, TGF-β, Arg-1). In a tumor-bearing mouse model, CLB-AZO-mPEG5 significantly reduced tumor volume and inhibited tumor proliferation, showing superior antitumor efficacy compared to the nonresponsive CLB-mPEG5 group. Mechanistic studies using transcriptome sequencing and qPCR confirmed that CLB-AZO-mPEG5 modulates the tryptophan-AhR-Cyp1 axis-related genes, thereby modulating macrophage phenotype in a coculture system and potentially influencing the tumor immune microenvironment. Collectively, CLB-AZO-mPEG5 exerts anti-HCC effects through NQO1-responsive release of chlorambucil, regulation of the tryptophan-AhR-Cyp1 axis, and induction of M1 macrophage polarization, representing a promising targeted nanodelivery system. These results demonstrate that CLB-AZO-mPEG5 nanomicelles represent a promising nanomedicine platform for targeted HCC chemotherapy.