Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells
| Autor: | Myraida Morales, Kai Griebenow, Yamixa Delgado, Alina Monteagudo, Elsie A. Orellano, Cindy M Figueroa, Moraima Morales Cruz, Jessica Méndez |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Hemeprotein
Glycosylation Pharmaceutical Science Succinimides Apoptosis environment and public health Article HeLa chemistry.chemical_compound Drug Delivery Systems Drug Discovery Humans Viability assay biology Caspase 3 Cytochrome c Circular Dichroism Chemical glycosylation Cytochromes c biology.organism_classification Silicon Dioxide Cytosol enzymes and coenzymes (carbohydrates) Cross-Linking Reagents Biochemistry chemistry embryonic structures biology.protein cardiovascular system Molecular Medicine Nanoparticles HeLa Cells |
| Popis: | Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl- 6-(3'-(2-pyridyldithio)-propionamido) hexanoate (SPDP) and incorporated into SH-functionalized MSN by thioldisulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)�mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24�72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs. |
| Databáze: | OpenAIRE |
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