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Activation of caspase‐dependent apoptosis by intracellular delivery of cytochrome c‐based nanoparticles (LB290)
Author(s) -
Morales Moraima,
Figueroa Cindy,
González Tania,
Delgado Yamixa,
Molina Anna,
Griebenow Kai
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb290
Subject(s) - cytochrome c , apoptosis , chemistry , hela , plga , biophysics , cancer cell , intracellular , cytoplasm , glycolic acid , cytotoxicity , drug delivery , biochemistry , cell , in vitro , lactic acid , biology , cancer , genetics , organic chemistry , bacteria
Cytochrome c (Cyt 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 Cyt c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. Consequently, in this work we designed a nano‐sized targeted Cyt c drug delivery system. First, we precipitated Cyt c with a simple solvent‐displacement method to obtain protein nanoparticles (NPs). Cyt c NPs’ surface was chemically linked to poly(lactic‐co‐glycolic) acid‐ thiol endcap (PLGA‐SH) using succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP) to prevent premature dissolution during delivery. SPDP is a heterobifunctional linker containing a disulfide bond which allows the Cyt c release under intracellular reducing conditions. This construct is further on referred to as PLGA‐S‐S‐Cyt c NPs. The size of the Cyt c NPs 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 (EPR) effect. A cell‐free caspase‐3 assay revealed more than 80% of relative caspase activation by Cyt c after the nanoprecipitation and SPDP modification, when compared to native Cyt c. Incubation of HeLa cells with PLGA‐S‐S‐Cyt c NPs showed significant reduction in cell viability after 6 hours. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with DAPI and propidium iodide (PI) after incubation with the Cyt c‐based NPs. Our results demonstrate that the PLGA coating, mediated by SPDP linker, stabilizes Cyt c NPs allowing their delivery to the cytoplasm of the target cells. After smart release of Cyt c into the cytoplasm it was able to induce programmed cell death. Grant Funding Source : Supported by the NIH‐RISE Program Grant Number 2R25GM061151‐12