Sequential-targeting nanocarriers with pH-controlled charge reversal for enhanced mitochondria-located photodynamic-immunotherapy of cancer

Targeting delivery of photosensitizers to mitochondria as the most sensitive cellular organelles to reactive oxygen species (ROS) by positively charged polymeric nanocarriers (NCs) is one of the useful methods for efficient photodynamic therapy (PDT). However, the NCs with positively charged mitochondria-targeting moieties are easily cleaned during circulation, restricting their in vivo applications. Herein, to address this issue and enhance in vivo PDT efficacy, we developed a sequential-targeting delivery system consisting of mitochondria-targeting micelles as the core prepared from the cationic amphiphilic copolymer for loading chlorin e6 (Ce6) and a tumor-targeting pH-dependent charge transformational layer as the shell obtained from 2,3-dimethylmaleic anhydride modified Biotin-PEG4000-NH2 (BioPEGDMA) via electrostatic interaction. Concealed by the anionic shell, the as-prepared NCs showed longer retention within the first stage of tumor-targeting. Then, the accumulated NCs conversed to positive charge in tumor extracellular microenvironment (pH ∼ 6.5), which could be more effectively internalized by tumor cells, and the re-exposed triphenylphosphonium (TPP) groups endowed their second-stage targetability to the mitochondria. In vivo experiments revealed that the Ce6-loaded NCs exhibited remarkable tumor inhibition rates of 84.1% and 93.2% on BALB/c nude mice and Kunming mice, respectively, under 660 nm NIR irradiation, and stimulated immune responses with upregulated expression of IFN-γ, TNF-α and CD3+ in tumor tissues, and enhanced activation of CD3+/CD4+, CD3+/CD8+ T lymphocytes and DCs in both tumor tissues and lymph glands. This work provided a new pathway for the development of smart drug delivery system with advanced PDT efficacy. Although the existing targeting delivery of photosensitizers to mitochondria by positively charged nanocarriers (NCs) have efficiently enhanced photodynamic therapy (PDT), their positive charges caused rapid clearance during circulation, which has restricted their in vivo applications. Therefore, we fabricated a novel sequential-targeting NC to solve the problem. The tumor accumulated NCs conversed to positive charge in tumor extracellular microenvironment, and the re-exposed triphenylphosphonium groups initiated second-stage targetability to mitochondria. This system exhibited remarkable tumor inhibition efficiency both in vitro and in vivo. Moreover, as we hypothesized, mitochondria-located PDT could promote immune response, resulting in improvement of PDT. The strategy of sequential targeting-based PDT in combination with augmented immune response showed a novel pathway for the development of smart drug delivery system with advanced PDT.