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

Abstract

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.

Statement of significance

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.

Introduction

Photodynamic therapy (PDT) involves the exploitation of a tumor-localized photosensitizer (PS), which can be light-activated by irradiation of the tumor with a specific wavelength of light, to generate cytotoxic reactive oxygen species (ROS), especially singlet oxygen (SO), thus causing cell apoptosis and tissue damage [1], [2]. The key challenges for PDT include tumor nonspecificity, rapid cellular clearance of PSs, and low diffusion radius of SO [3]. Therefore, nano-sized polymeric drug delivery systems designed according to the chemical, structural and biological environment of tumor cells have been extensively developed to improve PDT [4], [5], [6], [7], [8], [9]. Particularly, SO has a short half-life of <40 ns, and can only be effective within a limited distance of <20 nm after generation [10], so it could be superior to deliver PSs to critical subcellular organelles with higher susceptibility to ROS and not just into tumor cells [11,12]. Mitochondrion that acts as the powerhouse of cells regulates the vital actions, such as ATP production, ROS generation, programmed cell death (apoptosis), and oxidative phosphorylation [13,14]. Moreover, mitochondria dysfunction can lead to a series of diseases including Alzheimer's disease, diabetes and cancer [15], [16], [17]. Because the death of cancer cells is tightly linked to their mitochondria, delivering PSs into mitochondria is emerging as a preferential approach for improving the efficiency of PDT. Many delivery systems such as inorganic nanoparticles [18], liposomes [19], and polymeric micelles that are chemically modified with cationic mitochondrial targeting moieties including lipophilic ligand triphenylphosphonium (TPP) and zwitterionic oligopeptides [20], [21], [22], [23] have been constructed to deliver PSs into mitochondria of cancer cells. Lee et al. reported water-soluble globular poly(ethylene glycol) nanoparticles chemically linked with iodomethyltriphenylphosphonium for mitochondria-targeting delivery of Ce6. The in vitro evaluation indicated that these nanoparticles could significantly improve SO generation and increase photodynamic tumor ablation [24]. Furthermore, the mitochondria-targeting supramolecular photosensitizer system was also reported, where SO could be in situ generated in mitochondria under light illumination, leading to enhanced PDT efficacy [25]. Despite nanocarriers (NCs) with positive charges are beneficial to enhance cellular uptake because of the high negative potential of cell membrane and inner mitochondrial membrane, they also cause severe aggregation and rapid clearance during circulation for in vivo drug delivery [26], [27], [28].

To resolve this contradiction, the strategy of pH-controlled negative to positive charge reversal that was triggered by tumor extracellular microenvironment (pH ∼ 6.5) and protective shell of hyaluronic acid that could be digested by hyaluronidase has been verified to be distinctly effective ways for advancing drug delivery [29], [30], [31]. For example, the introduction of β-carboxylic acid groups and chemical modification with anionic 2, 3-dimethylmaleic anhydride (DMA) onto the surface of NCs have been demonstrated to result in the transformed surface charge of the nanoparticles depending on the tumoral environment acidity. Dong et al. have reported a polycaprolactone bearing acid-labile β-carboxylic amide segments conjugated to mPEG with pH-controlled charge conversion for intracellular delivery of doxorubicin, which significantly enhanced cell internalization [29]. Huang et al. have fabricated a nanohybrid consisting of pH-responsive N-(2-hydroxypropyl) methacrylamide (HPMA) co-polymer shells and positive mesoporous silica nanoparticle cores via electrostatic interaction, which achieved prolonged blood circulation and acquired a good tumor inhibition rate of 72.6% on nude mice [30]. The pH-dependent charge-conversional feature of NCs provides a chance for the in vivo applications of mitochondria-targeting drug delivery systems, however, their poor tumor targetability is not good for further mitochondrial uptake only by cancerous cells. Endowing NCs with sequential tumor and mitochondria targeting could greatly improve the therapeutic efficiency of anticancer. Furthermore, early studies have discovered certain levels of immune responses after PDT [32], [33], [34], [35], [36], [37]; thus, we hypothesized that the mitochondria-located PDT induced immune response might be augmented. To our best knowledge, the strategy of sequential targeting-based PDT in combination with enhanced immune response has not yet been reported.

Herein, we attempted to fabricate a sequential-targeting delivery system consisting of the positively charged micelles as core prepared from amphiphilic copolymer for loading Ce6, and a pH-triggered charge transformational layer as shell obtained from anionic 2, 3-dimethylmaleic anhydride (DMA) modified Biotin-PEG₄₀₀₀-NH₂ with tumor-targeting property via electrostatic interaction for advancing PDT efficacy. As shown in Scheme 1, the NCs were firstly tumor-targeted and effectively accumulated in the tumor tissue, and sequentially conversed to positive charge in tumor extracellular microenvironment of pH ∼ 6.5 to accelerate cellular uptake by cancerous cells. The exposed TPP moieties endowed NCs mitochondrial targetability, so that the incorporated Ce6 could generate cytotoxic SO in mitochondria under the 660 nm NIR irradiation to kill cancerous cells for enhanced PDT efficacy. Furthermore, the Ce6 loading content, sequential-targeting capability, charge reversal behaviors of the NCs, the in vitro photodynamic cytotoxicity and in vivo anti-tumor activity of Ce6-loaded NCs, as well as the simultaneously immerged immune responses were comprehensively investigated.