Abstract
Therapeutic drug carriers can drive their cargo to their target cells. However, an obstacle is usually the entrapment of the drug inside the endolysosomal compartment, which physically impedes its actuation by the impossibility of reaching its molecular site of action. To overcome this hurdle, photochemical internalization (PCI) has been proposed, but the extent of PCI-induced membrane disruption and its capability to allow the release of microparticles is unknown. The aim of the present study was to determine if PCI allows the release of microparticles from the endolysosomal compartment to the cytosol and to analyze at the ultrastructural level the effect of PCI on the membrane surrounding the particles. Confocal microscope allowed us to detect that endolysosomal membranes suffered some disruption after PCI, evidenced by the diffusion of soluble transferrin from the endolysosomes to the cytosol and by a decrease of LAMP1-microparticles co-localization. Transmission electron microscopy (TEM) showed a decrease in the number of well-defined membranes around microparticles after PCI, and scanning TEM combined with energy dispersive x-ray revealed an increase in the width of endolysosomal membranes after treatment. These results suggest that endolysosomal membranes suffered an ultrastructure alteration after PCI, enough to liberate soluble transferrin but not the entire microparticles.
Highlights
The therapeutic effects of many drugs are limited due to several reasons, being the inefficient targeting to the desired cells and the trapping of the drug inside the endolysosomal compartment two of the most important[1,2]
It has been described that using Photochemical Internalization (PCI) therapeutic drugs[26,27], DNA molecules for gene therapy[28] or even nanoparticles[29] are able to escape from the endolysosomal system[23,25,30]
PCI optimization, cells incubated with 20 μg/ml Tf-A488 showed green discrete fluorescent dots under the fluorescence microscope when illuminated with 470–495 nm wavelength corresponding to endosomes or lysosomes containing Tf-A488, regardless of the AlPc concentration used (Fig. 1a)
Summary
The therapeutic effects of many drugs are limited due to several reasons, being the inefficient targeting to the desired cells and the trapping of the drug inside the endolysosomal compartment two of the most important[1,2]. Photochemical Internalization (PCI), developed by Berg et al, is based on Photodynamic Therapy[23,24] In this approach, endolysosomal membranes are disrupted when exposed to light of a particular wavelength that excites a photosensitizer (PS) previously integrated in the membranes. Due to the lack of systematic studies on the mechanisms of endolysosomal membrane disruption, it is not yet clear how macromolecules can escape from the endolysosomal compartment after PCI In one of these few studies, Ohtsuki et al reported that the photoinduced endosomal disruption started with a ROS-induced membrane destabilization that allowed protons to flow to the cytosol, raising the pH of the endosome. This led to the endosomal membrane disruption, the exact mechanism remains unclear[25]
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