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

Mimi M Yang,William R Wilson,Mingtan Tang,Stephen M F Jamieson,Sasi Bhushan Yarragudi,Zimei Wu

doi:10.1007/s11095-022-03206-0

Mimi M Yang, William R Wilson + Show 4 more

Open Access

https://doi.org/10.1007/s11095-022-03206-0

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Abstract

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.Graphical abstract

Highlights

Cytoplasmic delivery of chemotherapeutic agents to cancer cells using nanoparticles can maximize therapeutic efficacy and safety [1]
Both high EE and DL were achieved (Table 1) by further optimization of previous drug loading conditions19, including increasing the concentration of calcium acetate to 500 mM, and of SN25860 in the loading medium to 4.5 mg/ml with addition of 2.5% (w/v) HP-β-CD and 2% ethanol with pH maintained at 7.0 using PBS
Reducing HP-β-CD concentration to 2% decreased DL to 25.4 ± 2.8% while a further increase of HP-β-CD to > 3% reduced liposome pellet size, as it can disrupt the integrity of liposomal bilayer[39]

Summary

Introduction

Cytoplasmic delivery of chemotherapeutic agents to cancer cells using nanoparticles can maximize therapeutic efficacy and safety [1]. The ability to enter cancer cells followed by rapid release of the payload to circumvent “endosomal escape” are highly sought to enable effective delivery of chemotherapeutics to their targets [9, 11]. PH-sensitive nanoparticles promote endosomal escape by three hypothetical pathways: i) liposomal destabilization in the acidic lumen of endosomes, ii) fusion of liposomes with endosomal membranes, and iii) rupture of endosomal membranes [18]. Our previous studies demonstrated that the DOPE-CHEMS-based PSL enter cells mainly through clathrin-mediated endocytosis [19], followed by endosomal escape primarily by destabilization in the acidic lumen and fusion with the endosomal membrane [19, 20]. Even endowed with pH-sensitivity, as well as fusogenic and membrane destabilizing properties, it seems only a small fraction of PSL are capable of crossing the endosomal membrane, making endosomal entrapment still a bottle-neck for the PSL mediated cytosolic delivery of chemotherapeutics [20, 22, 23]

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