In Vivo Targeted Delivery of Antibodies into Cancer Cells with pH-Responsive Cell-Penetrating Poly(disulfide)s

Cell-penetrating poly(disulfide)s (CPDs) are promising vehicles for cytosolic delivery of proteins. However, currently available arginine-rich CPD has rarely been reported for systemic delivery due to its “always” positive charge. Herein,...

Pharmacological Activation of cGAS for Cancer Immunotherapy

When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.

Intracellular Delivery of Virus-Like Particles Using a Sheddable Linker

Intracellular targeting is an important aspect of the efficient delivery of drugs and nanotherapeutics. Cytosolic transport of nanomaterials is often an essential requirement for therapeutic delivery into cells but remains a challenge owing to the endosomal trap and eventual lysosomal degradation of cargo. To address this, we designed a functional carrier that escapes the endosome and delivers biological materials into the cell's cytoplasm. For this purpose, we synthesized a glutathione-sensitive linker that connects the well-known mitochondria targeting lipophilic triphenylphosphonium cation (TPP) to the surface of a proteinaceous nanoparticle based on the engineered virus-like particle (VLP) Qβ. Once in the cytosol, the thiol sensitive linker severs the TPP from the nanoparticle, halting its trafficking to the mitochondria, and marooning it in the cytosol. We demonstrate the successful in vitro cytosolic delivery of a VLP loaded with Green Fluorescent Protein, where evenly distributed fluorescence is observed in A549 lung cancer cells after four hours. We further demonstrate successful cytosolic delivery by showing that encapsulating siRNA inside the VLP promotes luminescence silencing in luciferase expressing HeLa cells more efficiently than VLPs that lack our sheddable TPP linker.

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

Efficacy and Selectivity of FGF2-Saporin Cytosolically Delivered by PCI in Cells Overexpressing FGFR1

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs.