In vitro evaluation of a targeted and sustained release system for retinoblastoma cells using Doxorubicin as a model drug

Abstract

Purpose: The objective of this study was to develop a novel folate receptor-targeted drug delivery system for retinoblastoma cells using doxorubicin (DOX) as a model drug.

Methods: Biodegradable DOX-loaded poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) micelles (DOXM) were prepared with various solvents (dimethylsulfoxide, acetone, and dimethylformamide). The effects of solvents on entrapment efficiency, particle size, and polydispersity were examined. The effects of thermosensitive gel structure on the release of DOX from the DOXM were also studied. Qualitative and quantitative uptake studies of DOX and DOXM were carried out in Y-79 cell line. Cytotoxicity studies of DOXM were performed on Y-79 cells.

Results: Based on size, polydispersity, and entrapment efficiency, dimethylformamide was found to be the most suitable solvent for the preparation of DOXM. Dispersion of DOXM in PLGA-PEG-PLGA gel sustained drug release for a period of 2 weeks. Uptake of DOX was ∼4 times higher with DOXM than DOX in Y-79 cells overexpressing folate receptors. This was further confirmed from the quantitative uptake studies. DOXM exhibited higher cytotoxicity in Y-79 cells when compared with pure DOX.

Conclusion: These polymeric micellar systems suspended in thermosensitive gels may provide sustained and targeted delivery of anticancer agents to retinoblastoma cells following intravitreal administration.

FIG. 1.

¹H NMR spectrum of poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) conjugate. The corresponding proton peak numbers marked in the NMR are denoted in the inset structure of PLGA-PEG-FOL.

FIG. 2.

Transmission electron microscopy image of doxorubicin-loaded PLGA-PEG-FOL micelles (DOXM). (A) DOXM prepared by dimethylsulfoxide; (B) DOXM prepared by acetone; (C) DOXM prepared by dimethylformamide.

FIG. 3.

In vitro release profile of DOX from (♦) DOXM, (▪) DOX suspended in PLGA-PEG-PLGA thermosensitive gel, and (▴) DOXM suspended in PLGA-PEG-PLGA thermosensitive gel. Each data point is the average of 3 samples. Error bars represent the standard error of mean.

FIG. 4.

Gelation and uniform dispersion of polymeric micelles in PLGA-PEG-PLGA thermosensitive gel. (A) DOXM suspended in PLGA-PEG-PLGA thermosensitive gel at 25°C; (B) DOXM suspended in PLGA-PEG-PLGA thermosensitive gel at 34°C–37°C; (C) scanning electron microscopy images of DOXM suspended in PLGA-PEG-PLGA thermosensitive gel; (D) reverse transition of gel to solution at 25°C.

FIG. 5.

Confocal images of Y-79 cells following treatment with DOX and DOXM. (A) Pure DOX; (B) DOXM; (C) DOXM in the presence of folic acid; (D–K) Z-stack images of DOXM to show that micelles are inside Y-79 cells. Scale bar = 50 μm.

FIG. 6.

Quantitative uptake of DOX in Y-79 cells, using pure DOX, DOX-loaded in PLGA-PEG micelles/nontargeted micelles (DOXMC), DOXM, and DOXM in the presence of folic acid. **P < 0.05.

FIG. 7.

Cell viability studies of DOX in Y-79 cells following treatment with DOX and DOXM.

FIG. 8.

Release mechanism of DOXM from PLGA-PEG-PLGA thermosensitive gel.