title: Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.
abstract: 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 thiol-disulfide 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.
authors.affiliation: Department of Chemistry, University of Puerto Rico , Río Piedras Campus, P.O. Box 23346, San Juan, Puerto Rico 00931-3346, United States.
authors.forename: Jessica
authors.initials: J
authors.lastname: Méndez
authors.forename: Moraima
authors.initials: M
authors.lastname: Morales Cruz
authors.forename: Yamixa
authors.initials: Y
authors.lastname: Delgado
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Figueroa
authors.forename: Elsie A
authors.initials: EA
authors.lastname: Orellano
authors.forename: Myraida
authors.initials: M
authors.lastname: Morales
authors.forename: Alina
authors.initials: A
authors.lastname: Monteagudo
authors.forename: Kai
authors.initials: K
authors.lastname: Griebenow
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000008:administration & dosage / Q000737:chemistry*; D016503:Drug Delivery Systems; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D012822:Silicon Dioxide; D045304:Cytochromes c; D053148:Caspase 3