Development and in vitro investigation of methylene blue-containing nanoparticle platforms for photodynamic therapy.

摘要

A variety of novel nanoparticle platforms (NPs) have been developed to facilitate the administration of photo sensitizers (PSs) for photodynamic therapy (PDT). With the ultimate goal of a dynamic nano-platform for cancer diagnosis and therapy, the NPs encapsulate methylene blue (MB) as an effective PS and, upon light exposure, deliver sufficient singlet oxygen (1O 2) to induce in vitro cytotoxicity.;Three types of MB-containing sub-200 nm nanoparticles, composed of polyacrylamide, sol-gel silica and organically modified silicate (Ormosil), respectively, were synthesized. It was found that the polyacrylamide NPs exhibited the most efficient delivery of 1O2, per MB molecule, but the lowest loading of MB per NP mass; while the sol-gel silica NPs showed the highest MB loading but the least efficient 1O2 delivery. Furthermore, the protection of MB from plasma enzymes by the NP matrix was successfully demonstrated, i.e. a negligible reduction of MB, encapsulated in the polyacrylamide NP, in the presence of a reducing enzyme.;Preliminary in vitro PDT studies using the MB-containing polyacrylamide NPs, without incubation, were conducted on rat C6 glioma tumor cells. Following light irradiation, efficient cell killing by singlet oxygen, attacking the membrane from outside the cell, was illustrated by fluorescence confocal microscopy. Furthermore, to deliver the NPs into the cell, a scheme to conjugate targeting peptides to the NPs via a cleavable or non-cleavable linkage was introduced. First, amine-functionalized polyacrylamide NPs with a diameter of approximately 30 nm were prepared and characterized. Their surface was sequentially conjugated with a cell-penetrating peptide, Tat and a mitochondrial targeting signal peptide, Mito-8, respectively. As a result, rapid peptide-mediated internalization of the fluorescence-labeled NPs was observed after incubation with C6 glioma cells, and such NPs also induced significant PDT effects on cells when loaded with MB. In addition, 100 nm amine-functionalized silica NPs were prepared with a coating of polyethylene glycol (PEG), to improve their colloidal stability and enhance the circulation time in vivo. When coupled with the Tat peptide, the silica-based NPs with MB encapsulation for PDT and fluorescent labeling for imaging, were taken up by cells in vitro, and revealed promising photodynamic results as well.