Photodynamic cancer therapy is still limited in its efficiency because of a lack of targeted methods avoiding non-specific toxicity. To overcome this we developed a system that is solely effective upon cellular uptake and intracellular activation by incorporating redox-sensitive chemistry. We used a nanoprecipitation method to obtain human serum albumin nanoparticles (HSA NP) with a diameter of 295 ± 5 nm and decorated them with the photosensitizer (PS) chlorin e6 (Ce6). The NP were stabilized using a redox-sensitive cross-linker to create a smart drug delivery system that is activated only upon NP disintegration in the reducing intracellular environment. Indeed, our drug delivery NP broke down in an environment emulating the reducing intracellular environment with 10 mM glutathione, but not under extracellular conditions. In contrast, the control cross-linked with glutaraldehyde did not break down in the reducing environment. Upon NP disintegration Ce6 fluorescence doubled as the result of diminished self-quenching. While the Ce6-HSA NP did not produce a significant amount of singlet oxygen upon irradiation, NP disintegration restored singlet oxygen production to about half of the value generated by the free Ce6. In vitro experiments with HeLa cells showed that the smart system was able to kill up to 81% of the cells while the glutaraldehyde cross-linked control only killed 56% of them at a drug concentration of 10 ng/ml. Also, Ce6 immobilization in HSA NP prevented dark toxicity in three different cell lines. For the first time, we demonstrate that it is possible to design a smart NP drug delivery system delivering a PS drug to cancer cells while avoiding toxicity prior to the uptake and irradiation. This finding may provide a means of designing more efficient PDT in cancer treatment.
Photodynamic therapy is a treatment that uses photosensitizer drugs to produce reactive oxidative species that kill cells when exposed to light of a specific wavelength. Our research project focuses on the development of protein nanoparticles as delivery systems for photosensitizers in cancer treatments, with the objective to overcome the lack of target specificity resulting in inappropriate drug retention by non-target tissues. The main goal is to produce nanoparticles that selectively accumulate in the target tissue due to their size, are effectively internalized into the cells via receptor-mediated endocytosis and disintegrate in the reducing environment of the cell, resulting in the activation of the drug. The synthesis method has been optimized to obtain nanoparticles of the desired size that disintegrate rapidly in the intracellular space. The photosensitizer is covalently bound to prevent non-specific release, and folate has been immobilized on the nanoparticle surface. The reversible activation of the drug was confirmed by fluorescence experiments and the therapeutic efficiency of the system was evaluated in HeLa cells. The system dark toxicity and phototoxicity was evaluated in vitro, as well as the internalization, the intracellular localization, and the mechanism of cell death. Note: This abstract was not presented at the meeting. Citation Format: Marimar Benitez, Anna M. Molina, Kai Griebenow. Protein-photosensitizer nanoparticles for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2014-4474
Photodynamic therapy (PDT) is a promising treatment for cancer. It relies on the accumulation of a photosensitizer (PS) drug in the target area and the subsequent irradiation with a light source to induce the formation of reactive oxygen species that cause cell death. The incorporation of these drugs in drug delivery systems (DDS) has proven to increase selectivity and overall efficiency of the therapy in vivo. We have synthesized a new DDS using human serum albumin nanoparticles (HSA NPs) of a discrete size as carriers. The PS drug Chlorin e6, which has proven to be effective for PDT, was covalently linked onto the NPs to prevent non‐selective release. Our NPs were stabilized with a redox‐sensitive crosslinker for targeted activation of the drug in a reducing environment, such as that of the cell cytoplasm. Absorbance and fluorescence spectroscopy experiments showed that the NPs disintegrated and that the PS photoquenching decreased in a 10 mM glutathione solution, resulting in the activation of the drug. The system was physically characterized in terms of size, shape and drug loading. The size obtained (280‐300 nm) was appropriate for accumulation in cancer tissue. Cell internalization and phototoxicity of the system were tested in HeLa cells by cell viability assays and confocal microscopy, showing efficient accumulation of the drug and dose‐dependent cytotoxicity. Our system demonstrated to be redox‐activated, with efficiency comparable to the free drug in vitro. Grant Funding Source: Supported by NIH Research Initiative for Scientific Enhancement (RISE) Program with grant number: 2R25GM061151‐12.
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