Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles

Abstract

BackgroundCytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery.ResultsCytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles.ConclusionsOur results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death.

Combining Stimulus-Triggered Release and Active Targeting Strategies Improves Cytotoxicity of Cytochrome c Nanoparticles in Tumor Cells

“…In contrast, when exposing the FA-PEG-PLGA-S-S-Cyt c NPs to 10 mM glutathione, Cyt c was completely released from the micelle-like system in less than 24 h. In contrast, the system initially developed (i.e., PLGA-S-S-Cyt c NP) showed an unwanted release of almost 20% under extracellular conditions and only around 80% of the protein was released under reducing conditions. 8 The stability of the improved Cyt c-based NPs in an aqueous non-reducing media may be attributed to their significantly higher surface charges or zeta potential resulting from the micelle-like structure (see Figure 3B). …”

“…8 In brief, 0.5 mg of FA-PEG-PLGA-S-S-Cyt c NP powder was suspended by sonication in 1 ml of 50 mM PBS with 1 mM EDTA at pH 7.4 and glutathione (GHS) concentrations of 0, 0.001, and 10 mM simulating extra- and intracellular conditions. 5 Incubation was performed for various times at 37 °C, and the NPs were pelleted by centrifugation at 14,000 rpm for 10 min.…”

Recent therapeutic applications of the theranostic principle with dendrimers in oncology

“…Interestingly, the delivery of cytochrome c directly into the cytoplasm of cancer cells selectively initiates apoptosis in many cancer cells [78].…”

Combined Effect of Anticancer Agents and Cytochrome C Decorated Hybrid Nanoparticles for Liver Cancer Therapy

“…However, due to the relative size (12.7 kDa) of the protein, intracellular delivery is not possible without the use of excipients [9]. Over the past decade, multiple nanotechnologies have been developed to act as transfection agents, carrying proteins across the cell membrane or permeating across tissue [9,10]. A relatively new type of nanoparticle that has been reported with potential in this area is hybrid iron oxide-gold nanoparticles (HNPs) [11,12].…”