Photodynamic therapy (PDT) induces localized oxidative stress through the light-activated generation of reactive oxygen species (ROS), leading to targeted cell death. While conventional PDT protocols inducing marked photocytotoxicity have been extensively characterized, the cellular responses to low-concentration PDT, here defined as a reduced photosensitizer concentration under standard light fluence and irradiance, remain poorly understood. In this study, the effects of low-concentration PDT (0.33 µM) using pheophorbide a (Pheo), either in free form or encapsulated within poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) micelles, were examined in three cell models: primary human dermal fibroblasts, human HCT-116 carcinoma cells, and canine MDCK-1 epithelial cells. At the reduced Pheo concentration, PDT induced consistent morphological alterations across all models, including cytoskeletal remodeling, cell rounding, and early apoptotic features. These effects appeared earlier and were more pronounced with encapsulated Pheo, underscoring the influence of formulation on PDT efficiency. Antioxidant treatments (Sodium-L-ascorbate or N-Acetyl-L-cysteine) mitigated the morphological changes and cell death, supporting the involvement of oxidative stress in these responses. Compared with conventionally cytotoxic PDT, the low-concentration approach triggered slower cell-death dynamics, with caspase activation preceding plasma membrane permeabilization. Together, these findings show that low-concentration PDT induces oxidative stress responses across diverse cell types, while their magnitude and kinetics remain formulation-dependent. These findings provide a framework for further investigating the temporal and mechanistic dimensions of PDT-induced oxidative stress and their potential relevance to photodynamic priming, immune modulation, and combination therapies.
Lipid-based nanocarriers have established themselves as a cornerstone of modern drug delivery, offering a versatile platform for the encapsulation of diverse therapeutic payloads. Their utility spans from small hydrophobic and hydrophilic molecules to complex, high-molecular-weight biologics, such as proteins and nucleic acids. Although many manufacturing approaches are available, their effectiveness, scalability, and control over particle size distribution can vary widely according to the preparation strategy and process conditions. This study systematically compared bench-scale thin-film hydration (TFH) followed by extrusion with scalable microfluidic mixing, and Impingement Jets Mixing (IJM), under standardized conditions. Liposomes composed of phosphatidylcholine S100 and cholesterol (3:1 mass ratio; final total lipid concentration of 10 mg/mL) were prepared using either Milli-Q water or phosphate-buffered saline (PBS, pH 7.4) as the aqueous phase, targeting particle sizes of approximately 100 and 200 nm. The resulting formulations were characterized in terms of size, polydispersity, ζ-potential, colloidal stability, particle concentration and phospholipid recovery. All manufacturing approaches generated liposomes within the targeted nanoscale ranges. For formulations targeting 100 nm, hydrodynamic diameters ranged approximately 90–125 nm in H2O and 75–130 nm in PBS, whereas formulations targeting 200 nm exhibited diameters of approximately 170–195 nm in H2O and 145–200 nm in PBS. Formulations targeting 100 nm generally exhibited greater colloidal stability and lower polydispersity than their 200 nm counterparts, while particle concentration varied markedly among manufacturing methods. Despite these differences, phospholipid recovery remained relatively high (approximately 55% to 95% in H2O and from 55% to nearly 100% in PBS). Integrated NTA and phospholipid analyses revealed strong inverse correlations between particle concentration and estimated phospholipid content per particle in both H2O (Spearman r = -0.811, p = 0.0007) and PBS (r = -0.902, p < 0.0001). Overall, these findings demonstrate that liposome manufacturing technology should be considered a critical formulation and process-design parameter, as the selection of an appropriate production strategy requires the simultaneous consideration of product quality, material recovery, reproducibility, and scalability.
