Targeted Delivery of Nanoparticulate Cytochrome C into Glioma Cells Through the Proton-Coupled Folate Transporter

Abstract

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.

Optimization and Characterization of Protein Nanoparticles for the Targeted and Smart Delivery of Cytochrome c to Non-Small Cell Lung Carcinoma

“…Fluorescence studies confirmed that both types of formulations (NP-free and NPcontaining) are internalized by LLC cells, but the NPs are ~3 times more effective inducing cell death. Studies from our lab have shown that this internalization is mediated by the FR [12,15]. In these studies, it was confirmed that cells treated with the optimized Cyt c-PLGA-PEG-FA NPs were undergoing an apoptotic cell death [42].…”

“…This system offers higher loading capacity in comparison to other nanocarriers because the core of the protein is made 100% of Cyt c, which is the drug itself. Our group has proved in previous studies the selectivity and effectivity of the decorated NPs in vitro, in HeLa cells, and in vivo, in glioma tumors [12,15]. In this study we demonstrate the superiority of the system when compared to an individual, FA-tagged Cyt c system.…”

“…Several studies have shown that the direct external delivery of Cyt c to the cell cytosol can activate the caspase cascade and induce apoptosis in a dose-dependent manner [9,10]. Our group and others have developed several drug delivery systems for Cyt c which can induce apoptosis selectively in different cancer types by targeting the mitochondrial apoptotic pathway [11][12][13][14][15][16][17]. These systems specifically release the drugs under reducing conditions like the ones found in the cellular cytosol, but not outside the cell, increasing their effective dose and lowering off-targets and premature drug release [18].…”

“…Taking advantage of these systems and their potential for nanotechnology applications in cancer treatment [20], we developed a drug delivery system for Cyt c. The system consists of Cyt c-based NPs decorated on their surface with the amphiphilic co-polymer folate-poly(ethylene glycol)-poly (lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH). The FA moiety of the NPs targets specifically cells overexpressing the FR, and after cellular uptake, the disulfide bond that covalently attaches the co-polymer to the Cyt c NPs is cleaved, Cyt c is released and induces apoptosis [15]. This system offers higher loading capacity in comparison to other nanocarriers because the core of the protein is made 100% of Cyt c, which is the drug itself.…”

Ferritin Nanocages for Protein Delivery to Tumor Cells

“…A tumor-targeting tissue was faced linking to cyt C AS1411 aptamer, which targets and binds to nucleolin, a nucleolar phosphoprotein which is overexpressed on the surface of HepG2 cells [84]. In another study, cyt C was specifically targeted to glioma cells, exploiting the proton-coupled folate transporter, which is overexpressed in these cells [85]. A folate-receptor targeting amphiphilic copolymer (folic acid-PEG-poly(lactic-co-glycolic acid)) This protein, just like transferrin, is efficiently uptaken by HeLa cells and can be detected in the cytoplasm and in the perinuclear space, suggesting a typical clathrin-coated endocytosis pathway mediated by TfR1.…”