Shear induced carboplatin binding within the cavity of a phospholipid mimic for increased anticancer efficacy

Abstract

Vesicles 107 ± 19 nm in diameter, based on the self-assembly of tetra-para-phosphonomethyl calix[4]- arene bearing n-hexyl moieties attached to the phenolic oxygen centres, are effective in binding carboplatin within the cavity of the macrocycle under shear induced within a dynamic thin film in a continuous flow vortex fluidic device. Post shearing the vesicles maintain similar diameters and retain carboplatin within the cavity of the calixarene in a hierarchical structure, with their size and morphology investigated using DLS, TEM, SEM and AFM. Location of the carboplatin was confirmed using NMR, FTIR, ESI-MS and EFTEM, with molecular modelling favouring the polar groups of carboplatin hydrogen bonded to phosphonic acid moieties and the four member cyclobutane ring directed into the cavity of the calixarene. The loading efficiency and release profile of carboplatin was investigated using LC-TOF/MS, with the high loading of the drug achieved under shear and preferential released at pH 5.5, offering scope for anti-cancer drug delivery. The hierarchical structured vesicles increase the efficacy of carboplatin by 4.5 fold on ovarian cancer cells, lowered the IC50 concentration by 10 fold, and markedly increased the percent of cells in the S-phase (DNA replication) of the cell cycle.

Figure 1

VFD processed P4C6-carboplatin host-guest vesicles: (A) TEM image, scale bar 500 nm (10 nm for inset), (B) SEM image, scale bar 600 nm, (C) AFM image, (D) sectional height profile of a collapsed vesicle and elemental mapping of host-guest complex with energy-filtered transmission electron microscopy (EFTEM) for (E) unfiltered, (F) carbon and (G) platinum.

Figure 2

Schematic of the binding of carboplatin within the cavity of P4C6, MOPAC2012 PM7 geometry optimized model of the P4C6-carboplatin complex and ¹H NMR spectra of (A) carboplatin, (B) carboplatin with 1.0 equivalent of P4C6 after probe sonication, and (C) carboplatin with 1.0 equivalent of P4C6 after VFD processing.

Figure 3

Release profile of carboplatin (CPt) from CPt-P4C6 host-guest vesicles at pH 7.4 and pH 5.5 over 48 h (n = 3) (Mean ± SD). Statistical analysis was performed using a Student’s t-test. Values of *P < 0.05 and **P < 0.01 were considered statistically significant.

Figure 4

Carboplatin-P4C6 induced apoptosis of SKOV-3 cells and caused cell arrest in the S phase. (A) Dose-response curves for P4C6, CPt (=carboplatin) and CPt-P4C6 against SKOV-3 cells after 24 h incubation, and (B) corresponding IC₅₀ for P4C6, CPt and CPt-P4C6. Flow cytometric analysis of SKOV-3 cells treated with (C) 5 μM P4C6, (D) 1 μM CPt, and (E) 1 μM CPt-P4C6 for 24 h followed by treatment with the reagents in the Dead Cell Apoptosis Kit. (F) Cell cycle analysis of SKOV-3 cells treated for 12 h with negative control (cell culture media), 5 μM P4C6, 1 μM CPt, and 1 μM CPt-P4C6. Statistical analysis was performed using a Student’s t-test. Values of *P < 0.05 and ***P < 0.005 were considered statistically significant.