Intravesical delivery of rapamycin via folate-modified liposomes dispersed in thermo-reversible hydrogel

Abstract

Purpose: To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel.

Methods: Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement.

Results: R-CL and R-FL were successfully prepared, at approximately <160 nm, 42% entrapment efficiency, and 57 μg/mg drug loading. FL cellular uptake was enhanced over 2-fold than that of CL; folate receptor-mediated endocytosis was confirmed using a competitive assay with folic acid pretreatment. In vitro cytotoxic effects increased dose-dependently. Rap-loaded liposomes inhibited mTOR signaling and induced autophagy in urothelial carcinoma cells. With gelation time of <30 seconds and gel duration of >12 hrs, both R-CL/P407 and R-FL/P407 preparations transformed into gel immediately after instillation into the mouse bladder. Drug release from the liposomal gel was erosion controlled. In orthotopic bladder cancer mouse model, statistically significant differences in ROI values were found between R-CL/P407 and R-FL/P407 groups at day 11 (P=0.0273) and day 14 (P=0.0088), indicating the highest tumor growth inhibition by R-FL/P407.

Conclusion: Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.

Keywords: antitumor efficacy; autophagy; bladder cancer; enhanced uptake; mTOR signaling; prolonged retention.

Figure 1

Schematic representation of intravesical instillation of R-FL/P407, followed by gelation in the bladder, erosion-controlled R-FL release, and enhanced absorption via FR-mediated endocytosis. Abbreviations: R-FL, rapamycin-loaded folate-modified liposome; P407, poloxamer 407; DP_2KF, distearoylphosphatidylethanolamine-polyethylene glycol₂₀₀₀-folate; FR, folate receptor.

Figure 2

Characterization of prepared liposomes. Notes: (A) Optimization of ligand density of R-FL evaluated by flow cytometry. Liposomes were loaded with DiI for MFI quantification. (B) Transmission electron microscopy images of liposomes. Scale bar indicates 200 nm. (C) Size distribution of R-FL by frequency. Data represent the means ± SD (n=3). Abbreviations: MFI, mean fluorescence intensity; DP_2KF, distearoylphosphatidylethanolamine-polyethylene glycol₂₀₀₀-folate; R, rapamycin; R-CL, rapamycin-loaded conventional liposome; R-FL, rapamycin-loaded folate-modified liposome.

Figure 3

Stability evaluation of R-FL during storage at 4 °C (●) and 25 °C (△) for 4 weeks. Notes: Statistical analysis was performed using the Student’s t-test (*P<0.05 versus 4 °C). Data represent the means ± SD (n=3).

Figure 4

FR expression and comparison of the liposomal cell uptake in FR-positive cell lines. Notes: (A) Western blot for FR-expression. GAPDH was used as a loading control to ensure the equal loading of gels. (B) Flow cytometry results for treatment effect after 2 h incubation. (C) Competitive assay of FL internalization under folic acid-untreated or folic acid-pretreated condition. (D) Confocal images for the time-dependent intracellular translocation of FL. White scale bar represents 100 µm. Data represent the means ± SD (n=3). Statistical analysis was performed using the Student’s t-test (*P<0.05 versus the paired group). Abbreviations: FR-α, folate receptor α; CL, conventional liposome; FL, folate-modified liposome.

Figure 5

Effects of Rap-loaded formulations on growth inhibition in URCa cells. Notes: (A) MTT assay with various concentration of Rap-loaded formulations. Data represent the means ± SEM (n=6). (B) Colony forming assay with Rap-loaded formulations (1 μg/mL). Abbreviations: Rap, rapamycin; R-CL, rapamycin-loaded conventional liposome; R-FL, rapamycin-loaded folate-modified liposome.

Figure 6

Effects of Rap-loaded formulations on mTOR inhibition and autophagy induction in URCa cells. Notes: (A) Inhibition of phosphorylation of mTOR and mTOR downstream proteins. (B) Induction of AMPKα activation and ULK phosphorylation at Ser757. (C) Accumulation of p62/ATG5 and cleaved LC3B-II. Actin was used as a loading control. Abbreviations: Un, untreated; Rap, rapamycin; R-CL, rapamycin-loaded conventional liposome; R-FL, rapamycin-loaded folate-modified liposome; Baf, bafilomycin A1.

Figure 7

Drug release and gel erosion characteristics of Rap-loaded liposomal gels. Notes: (A) In vitro drug release profile as a function of time. (B) Gel erosion profile as a function of time. (C) Correlation between liposomal Rap release and gel erosion. Data represent the means ± SD (n=3). Abbreviations: Rap, rapamycin.

Figure 8

Comparison of in vivo antitumor efficacy of different samples in the orthotopic bladder cancer model in C3H mice. Notes: (A) In vivo imaging acquired via BLS. For statistical analyses, tumor bioluminescence following treatment was normalized against the initial bioluminescence on day 4. (B) ROI-time plots for quantitative comparison (*P<0.05, **P<0.01 versus R-CL/P407). Arrows indicate instillations. Data represent the means ± SD (n=7). (C) Inhibition of mTOR signaling and induction of cleaved PARP. The blots are representative of three independent experiments. Abbreviations: R, rapamycin; R-CL, rapamycin-loaded conventional liposome; R-FL, rapamycin loaded folate-modified liposome; P407, poloxamer 407; BLS, bioluminescence signal; ROI, region of interest.

Figure S1

Stability evaluation of R-CL during storage at 4 °C (●) and 25 °C (△) for 4 weeks. Notes: Data represent the means ± SD (n=3). Abbreviations: PDI, polydispersity index; ZP, zeta potential.