Extracellular vesicles for improved intracellular accumulation of photosensitizers

Introduction. Photodynamic therapy (PDT) is based on the use of photosensitizers (PS) that generate cytotoxic reactive oxygen species (ROS) upon light exposure. The efficacy of PDT depends on the efficient PS intracellular accumulation and preservation of their photodynamic activity. However, the limited solubility and poor pharmacokinetics of many PS necessitate the development of efficient delivery systems. Extracellular vesicles (EVs) have been proposed as promising nanocarriers for PS delivery.

Materials and methods. EVs were isolated from human embryonic kidney (HEK293) cells using ultrafiltration. The photosensitizer 5,15-bis(3-methoxy(4-(6-pyridylhexyloxy)phenyl)-10,20-di(ethynylphenyl)porphyrinato zinc (ZnP) was loaded in EVs by sonication, followed by removal of non-encapsulated ZnP by size-exclusion chromatography. Photodynamic activity of ZnP-EVs was assessed using the MTT assay on A549 cells after irradiation with red light (660 nm). ZnP-EV-induced ROS formation in A549 (human lung adenocarcinoma) cells was evaluated by flow cytometry. ZnP release was studied in a model medium (phosphate-buffered saline supplemented with 10% fetal bovine serum, pH 7.4).

Results. After ZnP-loading, EV recovery rate was approximately 40%. ZnP encapsulation resulted in a slight increase in EV size and zeta potential, consistent with membrane incorporation. Both free ZnP and ZnP-loaded EVs showed no dark toxicity in A549 cells at tested concentrations. Photoactivity assay confirmed that ZnP remains functional within EVs, while flow cytometry revealed that ZnP-EVs generate ROS more efficiently than free ZnP. Analysis of ZnP release from EVs demonstrated that only 16% of ZnP was released within the first 24 h, suggesting more selective PS accumulation in target tissues.

Conclusions. The results demonstrate that EVs derived from HEK293 cells preserve the photodynamic properties of the encapsulated PS, enable controlled release under biologically relevant conditions and enhance ROS generation compared to free ZnP.

This work was supported by the Russian Science Foundation research grant No. 25-75-00151, https://rscf.ru/project/25-75-00151/