In this study, we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to some gliomas. Using the techniques of confocal imaging, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and small interfering (siRNA) knockdown against the PCFT, we demonstrated that Gl261 and A172 glioma cells, but not U87 and primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by cell death. The FA-Cyt c NPs (100 µg/mL), had no cytotoxic effects in astrocytes but caused death in glioma cells, according to their level of expression of PCFT. Whole-cell patch clamp recording revealed FA-induced membrane currents in FA-Cyt c NPs-sensitive gliomas, that were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a route for internalization of FA-conjugated NPs in these glioma cells. Analysis of human glioblastoma specimens revealed that at least 25% of glioblastomas express elevated level of either PCFT or folate receptor (FOLR1). We conclude that the PCFT provides a mechanism for targeted delivery of drugs to some gliomas as a starting point for the development of efficient methods for treating gliomas with high expression of PCFT and/or FOLR1.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells where its release from mitochondria and apoptotic induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and tested their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. The internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) in LLC cells was confirmed by confocal microscopy. NP caspase activation was more efficient than the NP-free formulation. Caspase activity assays showed NPs retained 88–96% Cyt c activity. The NP formulations were more effective in decreasing LLC cell viability than NP-free formulation, with IC50 49.2 to 70.1 μg/mL versus 129.5 μg/mL, respectively. Our NP system proved to be thrice as selective towards cancerous than normal cells. In vivo studies using near infrared-tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.

rugosa lipase nanoparticles as robust catalyst for biodiesel production in organic solvents.Inexpensive but resourceful sources of lipids, for example, used cooking oil (UCO) and brown grease (BG), which often contain large amounts of free fatty acids (FFA), are difficult to convert into biodiesel economically and in good yield. Candida rugosa lipase nanoparticles (cNP) were formed first and subsequently cross-linked nanoparticles (CLNP) were obtained by crosslinking of them. Alternatively, cNP were conjugated to magnetic nanoparticles (mNP) to achieve a cNP-mNP conjugate. All three formulations were employed in three different organic solvents (n-heptane, 1,4-dioxane, and t-butanol) to produce biodiesel using BG and UCO in the transesterification reaction with ethanol and methanol. The radii of nanoparticles (NP) were 5.5, 75, 100, 85 nm for mNP, cNP, CLNP, and cNP-mNP, respectively, as measured by scanning/transmission electron microscopy and dynamic light scattering. The catalytic efficiency (Kcat/KM) of cNP, CLNP, and cNP-mNP was increased ca. -25, -68, -176 folds in n-heptane and -35, -131, -262 folds in 1,4-dioxane compared to the lyophilized lipase in the model transesterification reaction of p-nitrophenyl palmitate (PNPP) with ethanol. In biodiesel formation, the best performance with 100% conversion of BG was achieved under optimum conditions with cNP-mNP, ethanol at a 1:3 molar ratio of lipid-to-alcohol, NP at a 1:0.1 weight ratio of lipid-to-enzyme, and water at a 1:0.04 weight ratio of enzyme-to-water at 30 o C for 35 h. The operational stability of the CLNP and cNP-mNP was sustained even after five consequent biodiesel batch conversions while 50% and 82% residual activity (storage stability) were retained after 40 d.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells were its release from mitochondria and apoptosis induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and test their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA, and compared to a nanoparticle-free formulation. Overexpression of FA in LLC cells and internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) was confirmed by confocal microscopy. Caspase activation assays show NPs retain 88-96% Cyt c activity. The NP formulations were more efficient in decreasing LLC cell viability than the NP-free formulation, with IC50: 49.2 to 70.1 μg/ml versus 129.5 μg/ml, respectively. Our NP system is thrice as selective towards cancerous than normal cells. In-vivo studies using tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.