Anti-cancer activity of doxorubicin-loaded liposomes co-modified with transferrin and folic acid

Abstract

Cancer-specific drug delivery represents an attractive approach to prevent undesirable side-effects and increase the accumulation of the drug in the tumor. Surface modification of nanoparticles such as liposomes with targeting moieties specific to the up-regulated receptors on the surface of tumor cells thus represents an effective strategy. Furthermore, since this receptor expression can be heterogeneous, using a dual-combination of targeting moieties may prove advantageous. With this in mind, the anti-cancer activity of PEGylated doxorubicin-loaded liposomes targeted with folic acid (F), transferrin (Tf) or both (F+Tf) was evaluated. The dual-targeted liposomes showed a 7-fold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cell monolayers. The increased penetration and cell association of the dual-targeted liposomes were also demonstrated using HeLa cell spheroids. The in vitro cytotoxicity of the doxorubicin liposomes (LD) was then evaluated using HeLa and A2780-ADR ovarian carcinoma cell monolayers. In both these cell lines, the (F+Tf) LD showed significantly higher cytotoxic effects than the untargeted, or single-ligand targeted liposomes. In a HeLa xenograft model in nude mice, compared to the untreated group, though the untargeted LD showed 42% tumor growth inhibition, both the (F) LD and (F+Tf) LD showed 75% and 79% tumor growth inhibition respectively. These results thus highlight that though the dual-targeted liposomes represent an effective cytotoxic formulation in the in vitro setting, they were equally effective as the folic acid-targeted liposomes in reducing tumor burden in the more complex in vivo setting in this particular model.

Keywords: A2780-ADR; Cancer; Doxorubicin; Dual-targeting; Folic acid; HeLa; Liposomes; Nanomedicine; Nanoparticles; Receptor targeting; Transferrin; Xenograft.

Figure 1

Cumulative release of Dox from liposomes at pH 5.0 and pH 7.4 at 37°C. Data are represented as mean +/− S.D. (n = 3)

Figure 2

Characterization of (A) transferrin and (B) folate receptor expression. Data are represented as mean +/− S.D. (n =3)

Figure 3

(A) Cell association of liposomes on HeLa cells in media with folic acid and (B) without free folic acid as determined by flow cytometry (n=3) followed by (C) their internalization as determined by confocal microscopy on monolayers (scale bar = 15μm) Data are represented as mean +/− S.D.

Figure 4. Penetration of rhodamine-labeled liposomes into HeLa cell spheroids

(A) Representative images of slices 50–110 μm in depth (scale bar = 50μm). (B) Profile of fluorescence with increasing depth into the spheroid (2 mg/mL liposomes). (C) The cumulative fluorescence of slices (50–110μm) at 2 different liposomal concentrations as quantified using Fiji data analysis software. Data are represented as mean +/− S.D. (n = 4)

Figure 5

In vitro cytotoxicity of the various liposomal formulations on (A) HeLa cells (B) A2780-ADR and (C) summary of IC₅₀ values. Data are represented as mean +/− S.D. (n=3)

Figure 6. Evaluation of formulations in a HeLa xenograft model

(A) Tumor growth inhibition. (B) Normalized body weight (%). (C) Representative images of excised tumors. (D) Excised tumor weights. (E) Excised tumor volumes. Data are represented as mean +/− S.D. (n = 4)