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

Abstract

Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nano-sized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20% while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as anti-tumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.

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

“…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].…”

“…Protein nanoprecipitation is a structure-and activity-preserving technique that permits the synthesis of Cyt c nanoparticles (NPs) with high induction of caspase activation [17]. Around 80-90% of the caspase activity is retained in Cyt c NPs compared to the native protein (100% activity) in cellfree caspase assays [12,13,16,17]. 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).…”

Sequentially Site‐Specific Delivery of Apoptotic Protein and Tumor‐Suppressor Gene for Combination Cancer Therapy

“…CytoC binds apoptotic protease activating factor‐1 in the cytoplasm to activate caspase‐mediated apoptosis pathway . Efficient transport of CytoC to the cytoplasm of cancer cells results in significantly higher level of apoptosis . In addition, p53 gene, a tumor‐suppressor gene, is involved in inhibiting development and growth of tumors, and can induce apoptosis independently of the CytoC release .…”

Effect of the Folate Ligand Density on the Targeting Property of Folated‐Conjugated Polymeric Nanoparticles

“…It has the high affinity with folate receptors (FRs), which are over‐expressed in most tumor cells and low‐expressed in non‐epithelial tumors and normal tissue cells. Furthermore, it has the advantages of small size, stability, easy modification, and low production cost . FA ligands have been conjugated to Pluronic/polylactide and Pluronic/poly(lactide‐ co ‐glycolide) block copolymers previously by our groups.…”