A Membrane Fluidization Strategy Significantly Boosts Photodynamic Therapy and Antitumor Immunity of Porphyrin-Lipid Nanoparticles

Porphysomes (PS) are self-assembled porphyrin-lipid nanoparticles with intrinsic multifunctionality for cancer theranostics. We recently discovered that incorporating EDTA-conjugated lipids (EDTA-lipids) into lipid nanoparticle formulations significantly enhances intracellular uptake by modulating the cell membrane fluidity. To retain the favorable <i>in vivo</i> properties of PS while improving intracellular porphyrin delivery for effective photodynamic therapy (PDT), we developed a dual cellular- and vascular-enriched porphysome (DE-PS) containing 30 mol % cholesterol, 5 mol % PEG2000-DSPE, 27 mol % pyro-lipid, and 38 mol % EDTA-lipid. DE-PS demonstrated favorable pharmacokinetics (<i>t</i> _₁/₂β = 7.5 h), robust photostability (>99% fluorescence quenching), and strong serum stability over 24 h (>85% fluorescence quenching retained). Compared to PS, DE-PS resulted in an 11-fold increase in KB cancer cell uptake and achieved >95% PDT-induced cell death, in contrast to <5% with PS. Intravital imaging showed enhanced intratumoral porphyrin activation in DE-PS-treated mice, with stronger fluorescence signals observed in tumor vasculature and necrotic regions, leading to superior <i>in vivo</i> PDT efficacy across all drug-light-intervals. Notably, while low-dose PDT (25 J/cm²) with DE-PS had minimal impact on CT-26 tumor growth in immunodeficient NSG mice, it resulted in marked tumor suppression and prolonged survival in immunocompetent BALB/c mice, highlighting the importance of PDT-induced immune activation in the therapeutic response. A single DE-PS PDT treatment led to durable tumor-free survival exceeding 90 days. Upon CT-26 tumor rechallenge, previously cured mice rejected tumor regrowth, demonstrating development of long-lasting immunity without utilizing additional immunostimulants. This study highlights DE-PS as a promising next-generation porphysome-based photosensitizer platform for effective cancer phototherapy and immune modulation.