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

Abstract

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.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC)

“…Our fast infrared NP detection system could be used as a tumor diagnostic agent for folate overexpressing cancers, and further development of the system could have theragnostic potential [ 40 ]. Previously, Cyt c NPs stabilized by the hydrophobic polymer poly (lactic-co-glycolic) acid (PLGA) demonstrated that these NPs were an efficient method to induce cell death in lung carcinoma cell culture, and bound to the tumor site in the LLC murine model [ 29 ]. Recently, we overcame dose limitations previously seen in the PLGA-based NPs for Cyt c by designing smaller Cyt c-based NPs coated with a low-molecular-weight polymer, FA–PEG.…”

“…Crosslinked Cyt c–PEG–FA NPs were synthesized by first obtaining protein NPs using a nanoprecipitation method [ 29 ]. Briefly, 5 mg/mL of Cyt c dissolved in ultrapure water was solvent-precipitated by adding acetonitrile at a 1:4 (water/acetonitrile) volume ratio at a constant rate of 300 mL/h using an automated syringe pump.…”

“…The supernatant was used to determine the concentration of released Cyt c by UV-vis spectroscopy using a NanoDrop 2000c (Thermo Scientific, Waltham, MA, USA). The wavelength used to measure the concentration of non-reduced Cyt c (0 mM and 0.001 mM GSH) was 530 nm, and reduced Cyt c (10 mM GSH) was 550 nm [ 29 ]. The amount of released Cyt c from the NPs to the PBS/GSH dissolvent (observed at 550 nm) was used to construct cumulative Cyt c release profiles at incrementally higher reducing conditions using GSH as the reducing agent.…”

“…Caspase activation by Cyt c was measured in LLC cell lysate following the procedure previously reported in the literature [29]. Briefly, 5 × 10 6 LLC cells were resuspended in 100 µL of lysis buffer, and cells were lysed with four freeze-thaw cycles using liquid nitrogen and a water bath at 37 • C. Then, the cell lysate was centrifuged at 11,000 rpm for 20 min at 4 • C, and the supernatant (lysate) was collected.…”

“…To date, various nano vehicles have been explored to facilitate the intracellular delivery of Cyt c for therapeutic purposes with different degrees of success [ 27 , 28 ]. Recently, our research group overcame biocompatibility and off-target limitations commonly seen in anticancer therapeutics by designing a Cyt c-based drug delivery system (DDS) coated with a biodegradable polymer, PLGA–PEG–FA, which is 253 nm in size [ 29 ]. This DDS showed a tumor-targeting capability and no cytotoxicity after an in vivo injection using a lung carcinoma immune-competent mouse model [ 29 ].…”