Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect.

While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.

pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells.

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6-2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.