Abstract
We report the design, synthesis, characterization and in vitro testing of a novel nanodrug based on a covalent linking model that allows intracellular controlled release of the pharmaceutical payload. A new synthetic strategy is implemented by direct coupling of as-synthesized (pyridin-2-yldisulfanyl)alkyl carbonate derivatives of camptothecin (CPT) with thiol groups of silica hybrid nanoparticles containing a non-porous core and a mesoporous shell. Upon reaction with thiols in physiological conditions, disulfide bridge cleavage occurs, releasing the naked drug after an intramolecular cyclization mechanism. Additional incorporation of a fluorophore into particles core facilitates imaging at the subcellular level for the monitoring of uptake and delivery. Confocal microscopy experiments in HeLa cervix cancer cells confirms that nanoparticles enter the cells by endocytosis but are able to escape from endo-lysosomes and enter the cytosolic compartment to release their cargo. The incorporation to cells of L-buthionine-sulfoximine, a glutathione inhibitor allows concluding that the intracellular releasing mechanism is mainly driven by the reducing activity of this tripeptide. This camptothecin nanoplatform shows the same cytotoxic activity than the free drug and is clearly superior to those release systems depending on enzymatic hydrolysis (as determined by calculation of the IC50 ratios).
Highlights
The administration of cytotoxic drugs, like antitumorals, constitutes a major challenge for the clinical practice
A new synthetic strategy is implemented by direct coupling of as-synthesizedalkyl carbonate derivatives of camptothecin (CPT) with thiol groups of silica hybrid nanoparticles containing a non-porous core and a mesoporous shell
We have developed a novel nanoparticulated core-shell system combining therapeutic activity and imaging at the subcellular level, based in an external covalent coupling model with a Scheme 2 Artistic representation of cell uptake process and drug release mechanism of RhB-SiO2@mesoporous silica nanoparticles (MSN)-SS-CPT in HeLa cells. (a) Cell internalization by endocytic pathway of the multifunctional nanoparticles and co-localization in endo-lysosomes. (b) Escape of nanoparticles from endo-lysosomal compartments to the cytosol and activation of the redox-responsive linking system by GSH-driven disulfide reduction and further intermolecular cyclization, resulting in the release of free CPT and the corresponding thiolactone. (c) Entrance of CPT into the cell nucleus and inhibition of topoisomerase I during DNA replication
Summary
The administration of cytotoxic drugs, like antitumorals, constitutes a major challenge for the clinical practice Very frequently, their poor pharmaceutical profile and serious undesired effects precludes their practical use in humans.[1,2,3] In this sense, the delivery strategy in chemotherapy has been mostly focused on stimuli-responsive systems that promote controlled release of their payload inside the target cells.[4,5,6,7] Here, redox-sensitive, disulfide-based mechanisms have shown significant efficiency for selective intracellular release, based mainly in glutathione-driven cleavage.[8,9] Otherwise, mesoporous silica nanoparticles (MSN) have been proposed for the delivery and controlled release of small therapeutic molecules and for theranostic systems, due to their large surface area, tunable pore size, multifunctionality and good biocompatibility,[10,11,12,13] and they look like good candidates as vehicles for the delivery of covalently linked anticancer compounds such as camptothecin and paclitaxel.[14,15] the direct coupling of these molecules to an inorganic nanoparticle is circumvented by the reduced therapeutic activity of the structure-modified derivatives.[16] It would be desirable to develop covalent linking models that allow for the controllable release of the free drug after cell entry.[17] Secondly, it is necessary to establish precisely when, where and how the pharmaceutical payload is released into the cells. This allowed us to attach the drug over the pores of the external coating by a cleavable disulfide linker, sensitive to thiols such as glutathione (GSH) or dithiothreitol (DTT), which are able to
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