title: Activation of caspase-dependent apoptosis by intracellular delivery of Cytochrome c-based nanoparticles.
abstract: BACKGROUND
  Cytochrome 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.
  
  
  RESULTS
  Cytochrome 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.
  
  
  CONCLUSIONS
  Our 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.
authors.forename: Moraima
authors.initials: M
authors.lastname: Morales-Cruz
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Figueroa
authors.forename: Tania
authors.initials: T
authors.lastname: González-Robles
authors.forename: Yamixa
authors.initials: Y
authors.lastname: Delgado
authors.forename: Anna
authors.initials: A
authors.lastname: Molina
authors.forename: Jessica
authors.initials: J
authors.lastname: Méndez
authors.forename: Myraida
authors.initials: M
authors.lastname: Morales
authors.affiliation: Departments of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan 00931, PR, USA. kai.griebenow@gmail.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Griebenow
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D019344:Lactic Acid; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer
chemical_list: D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D045304:Cytochromes c; D053148:Caspase 3