Targeted delivery of EV peptide to tumor cell cytoplasm using lipid coated calcium carbonate nanoparticles

Abstract

Intracellular-acting peptide drugs are effective for inhibiting cytoplasmic protein targets, yet face challenges with penetrating the cancer cell membrane. We have developed a lipid nanoparticle formulation that utilizes a pH-sensitive calcium carbonate complexation mechanism to enable the targeted delivery of the intracellular-acting therapeutic peptide EEEEpYFELV (EV) into lung cancer cells. Lipid-calcium-carbonate (LCC) nanoparticles were conjugated with anisamide, a targeting ligand for the sigma receptor which is expressed on lung cancer cells. LCC EV nanoparticle treatment provoked severe apoptotic effects in H460 non-small cell lung cancer cells in vitro. LCC NPs also mediated the specific delivery of Alexa-488-EV peptide to tumor tissue in vivo, provoking a high tumor growth retardation effect with minimal uptake by external organs and no toxic effects.

Fig. 1

Schematic illustration of LCC nanoparticle preparation (a) and TEM imaging of unstained LCC-PEG-AA NPs (b).

Fig. 2

Stability of the pH sensitive calcium carbonate core studied at 5, 10, 15, 20, 30 and 45 min exposure to different pH conditions (pH 5.5, 6.5 or 7.4). The disruption of the formation of calcium cores was measured using dynamic light scattering (average mean value of detected particle intensity per second (%)) (a). Release profiles of Alexa-488-EV peptide formulated in LCC-PEG-AA NPs under different pH conditions were observed by SDS-PAGE analysis followed by imaging with a KODAK imaging system (b).

Fig. 3

EV Peptide uptake by H460 cells. Uptake of Alexa-488-EV peptide formulated in LCC-PEG-AA NPs was observed by confocal microscopy, 40X (b). Free Alexa-488-EV peptide treated cells were used as the comparative control (a).

Fig. 4

MTT assay of H460 cell viability after treatment with 1 μM of EV or EE formulated in LCC-PEG-AA NPs for different incubation time intervals (12, 24 and 36 h). Viabilities were calculated as compared to an untreated control at each time point. P < 0.05 *.

Fig. 5

Cellular apoptosis of H460 lung cancer cells evaluated by flow cytometry. Cells were treated for 36 h with 2 μM of EE or EV peptide formulated in either LCC-PEG NPs or LCC-PEG-AA NPs. A control sample was treated with PBS buffer (pH 7.4). Apoptotic cells (early and late apoptosis) were quantified (right lower panel). P < 0.05 *.

Fig. 6

Tissue distribution of EV peptide. Major organs were taken from H460 tumor bearing mice 4 h after i.v. injection with Alexa-488-EV peptide formulated in LCC-PEG or LCC-PEG-AA NPs (a). Imaging was performed with use of an IVIS 100 imaging system. Quantification of the total average fluorescence intensity (tumor area x fluorescence intensity) of the tumors from each mouse group was estimated using Image J software (b).

Fig. 7

Tumor growth retardation effect of EV peptide. Nude mice bearing human H460 tumor were i.v. injected (0.36 mg/kg) every other day with either PBS, free EV peptide, or EE or EV peptide formulated in LCC-PEG-AA NPs. *: P < 0.05, Mean ± SEM (n=4–5).