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

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.

Keywords: Calcium acetate; Co-localization analysis; Cytosolic delivery; Endosomal entrapment; Proton sponge effect; Remote drug loading; pH-sensitive liposomes.

Fig. 1

Physicochemical characterization and cytotoxicity to EMT6-nfsB breast cancer cells of PSL-SN25860 and NPSL-SN25860 using calcium acetate as a remote loading agent. A) Mechanism for drug loading. B) Representative cryo-TEM micrographs. The red arrows indicate drug precipitates or ‘bundles’ in the liposome cores. C) In vitro pH responsive drug release profiles (mean ± SD; n = 3); and D) Growth curves of EMT6-nfsB cells treated with free drug, PSL and NPSL for 18 h (means ± SEM; n = 3 individual experiments). Blank liposomes were non-toxic at the corresponding concentration range

Fig. 2

The anti-tumor and tumor targeting abilities in CD-1 nude mice following a single intravenous treatment with SN25860 formulations: drug solution, NPSL and PSL with PBS as control. A Change of tumor volume over 2 weeks after inoculation (n = 6–7). Box highlights the change in 48 h following a single treatment with SN25860 formulations at a dose of 1 mmol/kg (512 mg/kg) compared with control. B Intra-tumoral SN25860 concentrations (n = 3), and proportion of intra-tumoral drug to the administered dose after 48 h (ND: not detectable, below 15 ng/g). C Representative pictures of clonogenic plates showing median tumor cell colonies grown from mice following different treatment. D Anti-tumor activity of SN25860 formulations against subcutaneous EMT6-nfsB tumors by ex vivo clonogenic assay 48 h after a single IV dose (n = 3 for control group; n = 6–7 for drug treated groups). Line = geometric mean; bars = SEM. Panel D shows p-values from one way ANOVA with Tukey’s post hoc test for multiple comparison

Fig. 3

Live cell imaging of EMT6-nfsB cells co-existed with PSL (A and B) or NPSL (C and D) containing 500 mM calcium acetate (A and C) or PBS of pH 7.4 (B and D), DIC (grey images) show cell morphology. Rh-PE (red counterstain) was used to label liposomes. Hoechst 33,342 (blue) was used as the nuclear stain. LysoTracker (green counterstain) indicates late endo/lysosomes. The scatterplots were generated by correlation of liposome (Rh-PE) with LysoTracker channel. It was clear that at the dose studied, PSL-Ca treatment caused depletion of endo/lysosomes, which became evident from 60 min onward. Scale bars = 20 µm

Fig. 3

Live cell imaging of EMT6-nfsB cells co-existed with PSL (A and B) or NPSL (C and D) containing 500 mM calcium acetate (A and C) or PBS of pH 7.4 (B and D), DIC (grey images) show cell morphology. Rh-PE (red counterstain) was used to label liposomes. Hoechst 33,342 (blue) was used as the nuclear stain. LysoTracker (green counterstain) indicates late endo/lysosomes. The scatterplots were generated by correlation of liposome (Rh-PE) with LysoTracker channel. It was clear that at the dose studied, PSL-Ca treatment caused depletion of endo/lysosomes, which became evident from 60 min onward. Scale bars = 20 µm

Fig. 4

Fluorescence intensities (FI) of Rh-PE and LysoTracker in EMT6-nfsB cells co-existed with various liposomes. A-D) normalized FI to the respective peak value; E and F) Pearson’s correlation for co-localization of liposomes and endo/lysosomes

Fig. 5

The representative images showing PSL containing 500 mM calcium acetate induces rupture of endo/lysosomes from three different representative cells. (A) The endo/lysosomes of cells were pre-stained with LysoTracker (green counterstain) and treated with PSL dyed with Rh-PE (red counterstain) and the interaction was monitored by live cell imaging. Pictures of cells at 5 min show few endo/lysosomes contain liposomes (red signal). (B) At around 45–55 min, the endo/lysosomes lose fluorescence signals and DIC images (bottom images) indicate loss of endo/lysosome morphology. Representative signals from individual fluorescence channels of the boxed area are shown in upper panels. The endo/lysosomes were observed to have strong presence of liposomes (red fluorescence) just before the rupture

Fig. 6

Images of EMT6-nfsB cells co-existed with PSL (A) or NPSL (B) containing a 250 mM or 500 mM calcium acetate solution or PBS (0.1 M, pH 7.4). Rh-PE (red counterstain) was used to label liposomes. Hoechst 33,342 (blue) was used as the nuclear stain. LysoTracker (green) indicates late endo/lysosomes. PSL-CA treatment caused depletion of late endo/lysosomes, which became evident from 60 min or 120 min depending on the concentration of calcium acetate. Scale bars = 20 µm