Oxygen-Generating Liposomes Boost Dual Light Therapy Against Hypoxic Glioma Cells in Lab Tests

Glioblastoma remains one of the most formidable challenges in clinical oncology, and a new laboratory study now reports a nanoscale strategy designed to strike at one of its defining weaknesses: the oxygen-starved environment inside the tumor. Writing in Discover Nano, researchers describe a lipid nanoplatform called DL-86@CAT@Lip, fabricated with microfluidic technology, that co-delivers a copper phthalocyanine photosensitizer named DL-86 together with the oxygen-generating enzyme catalase. In experiments with C6 rat glioma cells grown under artificially hypoxic conditions, the combined photodynamic and photothermal treatment reduced cell viability to approximately 20.3 percent after irradiation with a 660 nm near-infrared laser. The work is explicitly an in vitro proof of concept, and the authors caution that blood-brain barrier penetration, in vivo biodistribution and long-term safety remain to be established.

The rationale behind the design rests on a well-documented biological obstacle. Glioblastoma is the most prevalent and lethal primary malignant tumor of the adult central nervous system, accounting for more than 45 percent of primary intracranial malignancies, with a stable global annual incidence of 3 to 5 cases per 100,000 population. Even under the current standard of maximum safe surgical resection followed by six weeks of fractionated radiotherapy with concurrent temozolomide chemotherapy and adjuvant maintenance temozolomide, patients achieve a median overall survival of only 12 to 15 months, and the five-year survival rate falls below 5 percent. Two physiological barriers restrict therapy: the intact blood-brain barrier, which impedes the penetration of most macromolecules and nanoscale agents into brain parenchyma, and the characteristic hypoxic tumor microenvironment, which drives malignant phenotypes and multi-drug resistance.

Hypoxia is particularly damaging for photodynamic therapy, or PDT, a light-based treatment that depends on molecular oxygen. The core cytotoxic cascade of PDT relies on oxygen-dependent Type II photochemical reactions to generate singlet oxygen, and hypoxia directly interrupts this reaction chain, causing a precipitous decline in singlet oxygen production and a substantial attenuation of tumoricidal activity. Within glioblastoma lesions, intratumoral partial oxygen pressure persistently falls below 10 mmHg, far lower than the physiological range of 20 to 60 mmHg in normal brain tissue. Singlet oxygen also has an extremely short half-life and an effective diffusion radius of less than 20 nanometers, meaning monomodal PDT can only eliminate superficial tumor cells within the laser irradiation coverage and fails to eradicate deep hypoxic lesions. Photothermal therapy, by contrast, is oxygen-independent: near-infrared photothermal materials convert absorbed light into localized heat through non-radiative relaxation, killing tumor cells through compromised membrane integrity, irreversible protein denaturation and heat-triggered necrosis. Combining the two modalities in a single agent is therefore an attractive strategy for hypoxic tumors.

The photosensitizer at the heart of the new platform is DL-86, a novel water-soluble copper phthalocyanine derivative. Compared with inorganic photothermal materials such as gold nanorods and carbon nanotubes, copper phthalocyanine derivatives offer metabolic degradability, low dark cytotoxicity and high molar extinction coefficients in the near-infrared region. Within the 650 to 700 nanometer biological window, such molecules simultaneously undergo intersystem crossing to generate reactive oxygen species and non-radiative relaxation to release heat, enabling activation of both PDT and PTT cascades without composite functional fillers. However, free DL-86 small molecules suffer from two translational drawbacks identified by the authors: insufficient oxygen supply under hypoxia suppresses the PDT cascade, and free small molecules are rapidly cleared by glomerular filtration with low tumor cellular uptake efficiency.

To overcome these limitations, the team adopted a Dolomite 3200 T-type microfluidic mixer to fabricate the liposomes in a single step. The lipid phase, composed of DSPC, cholesterol and DSPE-PEG2000 at a molar ratio of 7.5:2.5:1 dissolved in ethanol, was mixed with an aqueous phase containing DL-86 and catalase at a fixed flow rate ratio of 1:3 and a total flow rate of 6 milliliters per minute. The layered architecture embeds the hydrophobic DL-86 within the hydrophobic domain of the phospholipid bilayers while encapsulating the water-soluble catalase in the aqueous core, spatially isolating the two components to avoid mutual fluorescence quenching and loss of functional activity. After dialysis purification and lyophilization with trehalose as a lyoprotectant, the resulting nanoparticles were characterized by dynamic light scattering and transmission electron microscopy.

The physicochemical characterization confirmed a well-formed, monodisperse product. DL-86@CAT@Lip exhibited a hydrodynamic diameter of 114.70 ± 0.40 nanometers, with polydispersity index values below 0.3 for all three formulations tested, including blank liposomes at 75.97 ± 0.43 nanometers and DL-86-only liposomes at 97.15 ± 3.35 nanometers. Zeta potential measurements traced the sequential loading: blank liposomes displayed a surface potential of −13.30 ± 1.13 millivolts, shifting to −19.25 ± 0.35 millivolts after DL-86 embedding and rising to −5.99 ± 0.65 millivolts with catalase encapsulation. Transmission electron microscopy showed uniform, regular spherical particles with intact contours. The formulation maintained a stable hydrodynamic diameter of 110 to 120 nanometers over six days both in storage at 4 degrees Celsius in phosphate-buffered saline and in serum stability tests at 37 degrees Celsius in PBS supplemented with 10 percent fetal bovine serum. Quantification against a standard curve yielded an encapsulation efficiency of 23 ± 1 percent and a drug loading content of 10.0 ± 0.2 percent for DL-86, while the phospholipid bilayer preserved more than 85 percent of the native catalytic activity of catalase, which the authors note is markedly superior to the 60 to 80 percent residual activity reported for conventional thin-film hydration liposomes.

Optical testing demonstrated that the platform retained the dual photoresponsive behavior of DL-86. The UV-Vis-NIR absorption spectrum kept the characteristic 670 nanometer absorption peak, confirming that microfluidic mixing, dialysis and lyophilization did not disrupt the conjugated chromophore system. Using methylene blue as a reference, free DL-86 showed a singlet oxygen quantum yield of 24.6 percent, while lipid-formulated DL-86@Lip and DL-86@CAT@Lip both yielded approximately 16.7 percent, a slight reduction the authors attribute to the lipid bilayer restricting collisions between photosensitizer molecules and dissolved oxygen. Under continuous 660 nanometer irradiation at 1 watt per square centimeter for 10 minutes, the 1.0 milligram per milliliter DL-86@Lip group reached a maximum temperature of 40.19 degrees Celsius, and five consecutive laser on-off cycles showed no obvious attenuation in heating, indicating strong anti-photobleaching performance. The cooling-curve calculation yielded a photothermal conversion efficiency of 46.15 percent for the lipid-formulated DL-86. The authors note that cellular phototoxicity experiments used a lower power density of 0.1 watts per square centimeter, and temperature increments between the two power settings cannot be cross-compared.

The functional tests under hypoxia showed the intended oxygen-supply mechanism at work. Using a 1 percent oxygen hypoxic incubator with oxygen monitoring confirming partial oxygen pressure below 10 mmHg, the researchers compared singlet oxygen production among the three formulations. Solution-phase DPBF degradation assays revealed that DL-86@CAT@Lip induced substantially faster degradation of the singlet oxygen probe than DL-86@Lip and free DL-86 under identical irradiation, directly indicating that liposome-encapsulated catalase catalyzes hydrogen peroxide decomposition to generate oxygen and replenish the photochemical substrate. Singlet oxygen production also increased in a dose-dependent manner across DL-86 equivalent concentrations of 0.5 to 15 micrograms per milliliter. At the cellular level, DCFH-DA fluorescence imaging showed that intracellular reactive oxygen species increased with irradiation duration in all groups, with cells treated with DL-86@CAT@Lip generally exhibiting stronger fluorescence than free DL-86, the catalase-mediated enhancement being most evident at the 10 minute irradiation timepoint. Cellular uptake experiments with Nile Red-labeled nanocarriers showed that fluorescence rose progressively over 9 hours and plateaued thereafter, and both liposomal formulations were taken up significantly more efficiently than free DL-86, with DL-86@CAT@Lip showing the strongest intracellular signal.

The cytotoxicity results underpinned the central claim of the study. In dark toxicity screening across the full dosage range of 0.5 to 15 micrograms per milliliter equivalent DL-86, C6 cell viability exceeded 85 percent in all groups after 24 hours without laser exposure, indicating minimal dark cytotoxicity. Under 1 percent oxygen and 660 nanometer laser irradiation delivering a total energy density of 100 joules per square centimeter at 0.1 watts per square centimeter for 17 minutes, the DL-86@CAT@Lip group showed prominent concentration-dependent phototoxicity with significantly lower viability than the DL-86@Lip and free DL-86 control groups, reaching statistical significance at p < 0.001, and reducing viability to approximately 20.3 percent. Calcein-AM/propidium iodide live-dead staining visually confirmed that the vast majority of cells in the DL-86@CAT@Lip field were labeled with propidium iodide after irradiation, while the other groups retained abundant viable cells. Hemolysis testing with rat erythrocytes showed hemolysis rates below 5 percent across all tested concentrations, well under the 10 percent clinical safety threshold for intravenous formulations.

The authors are careful to frame the scope of their findings. They acknowledge that the work constitutes an in vitro proof of concept: detailed dissection of the molecular cell-death pathways, such as distinguishing apoptosis from ferroptosis or necroptosis, and validation of hypoxia-related signaling molecules such as HIF-1α at the protein and gene levels were not performed, and blood-brain barrier penetration, in vivo biodistribution, brain accumulation and long-term biosafety remain to be established. The study also used a rat glioma cell line rather than human glioblastoma cells. These aspects, the team states, will be investigated in future orthotopic intracranial glioma models. Within those limits, the study reports that a microfluidically fabricated, self-oxygen-supplied lipid nanoplatform can simultaneously deliver a dual-responsive photosensitizer and an oxygen-generating enzyme to hypoxic glioma cells in culture, achieving synergistic photodynamic and photothermal killing while maintaining favorable dark biocompatibility and hemocompatibility, and providing a blueprint for further preclinical development of brain tumor phototherapeutic nanomedicines.

Subject of Research: An in vitro study of a self-oxygen-supplied lipid nanoplatform for combined photodynamic and photothermal therapy against hypoxic glioma cells.

Article Title: Microfluidically fabricated self-oxygen-supplied DL-86 lipid nanoplatform for in vitro synergistic photodynamic/photothermal dual phototherapy against hypoxia-tolerant glioblastoma

Article References: Gong, J., Pan, X., Wang, S., Liu, Y., Wang, G., Yu, M., Ke, P. C., & Xu, M. (2026). Microfluidically fabricated self-oxygen-supplied DL-86 lipid nanoplatform for in vitro synergistic photodynamic/photothermal dual phototherapy against hypoxia-tolerant glioblastoma. Discover Nano, 21(1), Article 564. https://doi.org/10.1186/s11671-026-05081-9

Image Credits: AI Generated

DOI: 10.1186/s11671-026-05081-9

Keywords: glioblastoma, photodynamic therapy, photothermal therapy, liposomes, catalase, copper phthalocyanine, microfluidics, hypoxia, nanomedicine, reactive oxygen species, in vitro study, C6 glioma cells