Development of indocyanine green loaded long circulating and folate-receptor-targeted PLGA nanoparticles for photodynamic therapy of breast cancer

Purpose. The purpose of this study was to develop a long-circulating and folate-receptor (FR) targeted indocyanine green (ICG) nanoparticles with sustained-release properties, to enhance tumor uptake. We have hypothesized that dual surface modification with mPEG and folic acid (FA) in the nanoparticles may increase the particles' blood circulation time and may result in specific recognition toward tumors with over-expression of FR. Methods. The following co-polymers were synthesized: mPEG-PLGA, FA-PLGA, and FA-mPEG-PLGA. Nanoparticles were prepared with PLGA, mPEG-PLGA, FA-PLGA, FA-mPEG-PLGA or blends of FA-PLGA and mPEG-PLGA with molar ratio 1:3 and 5:3 (Blend 1 and 2 respectively). The specificity of FR-targeting nanoparticles was demonstrated by comparative intracellular uptake of these nanoparticles using human breast cancer cells, MDA-MB-231 (with high FR density) and MCF-7 (with low FR density), and a normal cell line, HEK-293 (with low FR density). The specific targeting was further confirmed by a competitive inhibition assay, in which MDA-MB-231 cells were cultured in a media with or without FA and by evaluating the effect of low temperature (4°C) on ICG uptake. In vitro photodynamic antiproliferation was studied at three activation energy densities, 0.045, 0.9 and 0.135 J/mm². Biodistribution of ICG solution and nanoparticles was studied in MDA-MB-231-bearing athymic mice by measuring the fluorescence intensity of ICG in dorsal and ventral regions using an imaging system. In vivo photodynamic anti-cancer activity of the FR-targeted nanoparticles was investigated on athymic mice bearing MDA-MB-231. Results. Characterization of these nanoparticles showed that Blend 1 and 2 nanoparticles exhibited desired properties: dual surface modification with PEG and FA, sustained release (39.66 +/- 4.91 % and 43.7 +/- 7.96 % release over 24 hr, respectively), suitable particle size (233 nm and 240 nm, respectively), satisfied entrapment efficiency (51.37 +/- 4.28 % and 50.26 +/- 0.72 %, respectively) and stable for 3 months at -20°C (0.36 +/- 0.01 % and 0.94 +/- 0.01 %). FA-PLGA and Blend nanoparticles enhanced the in vitro uptake by the MDA-MB-231 cells than the other nanoparticles by 3--26 times (p<0.05) after 24 h incubation. FA-PLGA and Blend nanoparticles exhibited 30--70% higher (p<0.05) in antiproliferation against the MDA-MB-231 cells than the other two cells while mPEG-PLGA nanoparticles showed similar antiproliferation against the three cells. All the photodynamic antiproliferation activities were increased with the increase of the irradiation energy. There existed a correlation between the cellular uptake of ICG and the antiproliferation effect. The cellular uptake of FA-tagged nanoparticles was suppressed in presence of 12 nM FA in the media by 1.2 to 3 times and at 4°C by 1.2 to 4.5 times supporting the role of folate receptor-mediated endocytosis. The maximum growth inhibition for MDA-MB-231 cells was 64.43 +/- 2.78 % by Blend 2 nanoparticles at 786 nm with 0.135 J/mm² energy density and 0.15 mW/mm² fluency. The uptake of FA-PLGA and Blend nanoparticles by MDA-MB-231 cells was 43--51 times higher (p<0.05) than that by MCF-7 and HEK-293 cells demonstrating the targeting ability toward cancer cells with overexpression of FR through surface modification with FA. After i.v. injection, high accumulation of Blend nanoparticles in tumor region was observed, followed by FA-PLGA nanoparticles. Blend nanoparticles had a steady ICG fluorescent intensity in tumor region till the 7th day of the treatment while the intensity due to FA-PLGA nanoparticles or mPEG-PLGA nanoparticles reduced after 4 days or sooner. The ratios of sum of the ICG intensities in the tumor region by Blend nanoparticles vs. to other nanoparticles were in the range of 1.21 to 2.82 during the 7 days photodynamic treatment. All the nanoparticle formulations and ICG solution had similar fluorescence intensity in the middle region of dorsal side during the 7 days treatment. Compared to the control group without any treatment Blend 2 nanoparticles resulted in 73% reduction in tumor weight (p<0.001), while the treatment of FA-PLGA nanoparticles and m-PEG nanoparticles resulted in 47% and 46% reduction (p>0.05), respectively. The reduction in tumor weight by Blend 2 nanoparticles was also significantly higher (p<0.05) than the FA-PLGA or mPEG-PLGA nanoparticles, demonstrating the higher efficiency against the FR over-expressed tumor and by the dual surface modification of nanoparticles with FA and mPEG than the single surface modification with either FA or mPEG. Conclusion. Thus, an ICG-loaded folate-targeted mPEG-PLGA nanoparticles with high cellular uptake efficiency of ICG and high photodynamic anti-cancer activity against FR over-expressing tumor cells was developed through the dual surface modification of PLGA nanoparticles with both FA and mPEG. (Abstract shortened by UMI.)...