(T1130-06-34) Mitochondria-Targeted Delivery of Metabolic Modulators to Tumor-Associated Macrophages for Immunotherapy

Purpose: In the tumor microenvironment (TME), tumor associated macrophages (TAMs) are a major type of tumor-supporting cells, contributing to the tumor growth, metastasis, and immune escape. TAMs mainly polarize toward the M2-like pro-tumor phenotype, which is highly influenced by their mitochondrial metabolism. Thus, targeting TAMs and modulating their mitochondria to reprogram their polarization toward the M1-like anti-tumor phenotype is a promising cancer treatment strategy. However, such studies are currently limited and yet remains under investigated. The goal of this work is to design a novel targeted nanoparticle (NP) for targeted delivery of metabolic modulators to TAMs, to reprogram TAMs’ mitochondrial metabolism for cancer immunotherapy.

Methods: The TAM and mitochondria dual-targeted NPs contain a mitochondrial targeting fluoroamphiphile, PEG-F7, a matrix metalloproteinase 2 (MMP2)-sensitive polymer, PEG-pp-PE, and a macrophage targeting ligand, phosphatidylserine (PS). In this design, PEG-pp-PE responds to overexpressed MMP2 for tumor targeting; PS, an “eat-me” signal, enables phagocytosis by macrophages, allowing TAM targeting; and PEG-F7 facilitates mitochondrial targeting (Figure 1). The multifunctional PEG-pp-PE/PS/PEG-F7 NPs were characterized by physicochemical properties including particle size, ζ-potential, drug loading, and drug release. The macrophage and mitochondrial targetability were evaluated in individual cell lines, co-cultured cells, and 3D cell spheroids by confocal microscopy and flow cytometry. The NIR dye-labeled NPs’ biodistribution in breast tumor-bearing mice was studied by animal imaging. The PEG-pp-PE/PS/PEG-F7 NPs’ drug delivery capability was evaluated by perhexiline, an antianginal drug that inhibits mitochondrial carnitine palmitoyltransferase-1/2 (CPT1/2). The perhexiline-induced altered mitochondrial metabolism and TAM re-polarization were examined by ELISA, cell surface marker, PCR, and glycolysis analysis. The immunomodulated anticancer activity were confirmed by MTT, CellTiter-Blue® Cell Viability Assay, and bioluminescence assay.

Results: The PEG-pp-PE/PS/PEG-F7 NPs possessed the conducive physicochemical property with particle size of ≤ 200 nm, ζ-potential of ~-40 mV, drug loading of around 15%, and sustained drug release. The fluorescent dye-labeled targeted NPs showed a doubled phagocytosis and macrophage selectivity compared with the non-targeted NPs. The internalized NPs were ultimately accumulated in mitochondria (Figure 2). Perhexiline-loaded NPs could effectively re-polarize M2 TAMs to antitumor M1 phenotype, as evidenced by the upregulated TNF-α, IL-β, CD80, CD86, MHC2, iNOS, NF-kB, and glycolysis as well as the downregulated IL-10, TGF-β, and CD-206. As a result, the perhexiline-loaded NPs demonstrated immunomodulatory anticancer activity. In vivo, the NPs exhibited excellent tumor targetability compared to the free dye and control NPs (Figure 3).

Conclusion: The approach combines cellular (TAMs) and subcellular (mitochondria) targeting strategies to improve metabolic modulators’ specificity and TAM-centered anti-cancer immune response, which might overcome off-target side effects, immune escape, and immunosuppression. The dual-targeted NPs might be a promising drug delivery platform for developing future precision nanomedicines.

Acknowledgements: There is no conflict of interest disclosed by all authors.


Figure 1. Drug delivery strategy. The Duel Targeting Nano Particles (DTNPs) composed of three components: PEG-pp-PE, for MMP2-sensitive tumor targeting; PS for macrophage targeting; and PEG-F7 for mitochondrial targeting. In the tumor microenvironment, the PS is exposed upon the MMP2 induced PEG deshielding. Then, macrophages engulf the nanoparticles via phagocytosis, allowing them to reach the mitochondria. The metabolic modulator, perhexiline, blocks CPT1/2 to inhibit fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS), resulting in TAMs’ M2 to M1 repolarization.


Figure 2. Macrophage & mitochondria targeting. Cell-based assays to evaluate NPs’ targetability and selectivity of TAMs and mitochondrial accumulation. The MMP-dependent cellular uptake of Rh-PE labeled dual targeting NPs (DTNPs) was evaluated in RAW 264.7 cells by fluorescence microscopy (A.1) & quantified by flow cytometry (A.2). The cell selectivity of Rh-PE labeled DTNPs was assessed in RAW 264.7/4T1 cell co-cultures by confocal microscopy (B.1) and quantified by flow cytometry (B.2). The RAW 264.7 cells were sorted by FITC-F4/80. The mitochondrial accumulation of DTNPs was investigated in RAW 264.7 cells by confocal microscopy upon mitochondrial staining by MitoView™ Green (C). NPs were pretreated with MMP2 for 4h at 37℃.


Figure 3. Tumor targeting. In-vivo biodistribution of DTNPs was conducted on 4T1 tumor-bearing BALB/c mice by a NIR live animal imager. DTNPs exhibited high tumor accumulation and prolonged retention at the tumor site (A). At 48-hour post-injection, the mice were euthanized, and the major organs (heart, lung, liver, tumor, spleen, and kidney) and tumors were collected for ex vivo imaging (B). The tumor-to-organ fluorescence ratio normalized by the tissue weight were assessed (C). * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) indicated significant differences.