pH-responsive and folate-coated liposomes encapsulating irinotecan as an alternative to improve efficacy of colorectal cancer treatment

Abstract

Irinotecan (IRN) is a semisynthetic derivative of camptothecin that acts as a topoisomerase I inhibitor. IRN is used worldwide for the treatment of several types of cancer, including colorectal cancer, however its use can lead to serious adverse effects, as diarrhea and myelosuppression. Liposomes are widely used as drug delivery systems that can improve chemotherapeutic activity and decrease side effects. Liposomes can also be pH-sensitive to release its content preferentially in acidic environments, like tumors, and be surface-functionalized for targeting purposes. Herein, we developed a folate-coated pH-sensitive liposome as a drug delivery system for IRN to reach improved tumor therapy without potential adverse events. Liposomes were prepared containing IRN and characterized for particle size, polydispersity index, zeta potential, concentration, encapsulation, cellular uptake, and release profile. Antitumor activity was investigated in a murine model of colorectal cancer, and its toxicity was evaluated by hematological/biochemical tests and histological analysis of main organs. The results showed vesicles smaller than 200 nm with little dispersion, a surface charge close to neutral, and high encapsulation rate of over 90%. The system demonstrated prolonged and sustained release in pH-dependent manner with high intracellular drug delivery capacity. Importantly, the folate-coated pH-sensitive formulation had significantly better antitumor activity than the pH-dependent system only or the free drug. Tumor tissue of IRN-containing groups presented large areas of necrosis. Furthermore, no evidence of systemic toxicity was found for the groups investigated. Thus, our developed nanodrug IRN delivery system can potentially be an alternative to conventional colorectal cancer treatment.

Keywords: Antitumor activity; Folate-coated; Irinotecan, polyethylene glycol; Liposomes.

Fig. 1.. pH dependent release of IRN.

The percent release profile of SpHL-IRN at pH 7.4 (red lines) and pH 5.0 (blue lines) is shown over 24 h. Note: *P < 0.05 as compared with free-IRN (pH 5 and pH 7); +P < 0.05 between the SpHL-IRN pH 7.4. Data are expressed by the mean (n = 3) ± S.D. of the mean. All data were analyzed by one-way ANOVA analysis of variance followed by Tukey’s post-test.

Fig. 2.. Liposomal encapsulated IRN is efficiently taken up in colon cancer cells.

Confocal microscopy images of CT26 murine colon cancer tumor cells after 24 h-incubation with A = free IRN, B = SpHL-IRN or C = SpHLIRN-Fol treatments (IRN concentration 5 μM). Amplification of 63 ×. Diode 370 nm (excitation of IRN).

Fig. 3.. Liposomes containing IRN are more effective than free drug.

(A) Antitumor effect of SpHL (control group), IRN, SpHL-IRN, and SpHL-IRN-Fol on the growth of CT26 colon tumor-bearing female BALB/c mice. Each treatment was intravenously administered 3 times, every 2 days, at dose of 30 mg/kg. (B) Volume tumoral at the end of the experiments, day 8, after treatments. Note: Data are expressed by the mean ± standard deviation of the mean (n = 6). Growth curves were analyzed by regression. a Represents statistical differences (P < 0.05) as compared with control group. b Represents statistical differences (P < 0.05) as compared with free-IRN. All data were analyzed by one-way ANOVA analysis of variance followed by Tukey’s post-test.

Fig. 4.. Evaluation of kidneys and liver toxicity.

Biochemical parameters of control mice treated with 3 doses of IRN formulations were evaluated for kidneys toxicity (A and B), liver toxicity (C and D) and body weight (E). Data expressed as mean ± S.D. of the mean (n = 6). Note: Evaluation of the biochemical parameters was carried out 10 days after treatment of mice with the IRN formulations. Results were expressed as the mean ± standard deviation. Abbreviations: ALT (alanine aminotransferase), and AST (aspartate aminotransferase). All data were analyzed by one-way ANOVA analysis of variance followed by Tukey’s post-test (P > 0.05).

Fig. 5.. Histopathological analysis of tumor necrosis.

Histological sections of primary tumor from CT26 colorectal tumor-bearing female BALB/c mice treated with free IRN, SpHL-IRN or SpHL-IRN-Fol stained by hematoxylin & eosin. (A) control group at amplification of 2x. (B) Free-IRN-treated at amplification of 2x. (C) SpHL-IRN-treated at amplification of 2x. (D) SpHL-IRN-Fol-treated at amplification of 2x. (E) control group at amplification of 20x. (F) Free-IRN-treated at amplification of 20x. (G) SpHL-IRN-treated at amplification of 20x. (H) SpHL-IRN-Fol-treated at amplification of 20x. The black asterisks indicate areas of tumor necrosis.

Fig. 6.. Histopathological analysis of liver and intestinal toxicity.

Histological sections of tissues from CT26 colorectal tumor-bearing female BALB/c mice treated with free IRN, SpHL-IRN or SpHL-IRN-Fol stained by hematoxylin & eosin. (A) control group liver. (B) Fre-IRN-treated liver. (C) SpHL-IRN-treated liver. (D) SpHL-IRN-Fol-treated liver. (E) control group large intestine. (F) Free-IRN-treated large intestine. (G) SpHL-IRN-treated large intestine. (H) SpHL-IRN-Fol-treated large intestine. (I) control group kidney. (J) Free-IRN-treated kidney. (K) SpHL-IRN-treated kidney. (L) SpHL-IRN-Fol-treated kidney. Amplification of 40x.