2.2. NanoVP is Quenched in Saline and Partially Unquenched in Serum‐Containing Media

VP is a modified porphyrin derivative that displays a chlorin‐type spectrum in organic solvents. In DMSO, the absorption spectrum of VP is characterized by a distinct Q band in the near‐infrared region at 687 nm and a strong Soret band at 435 nm (Figure  2a). The absorption spectra of NanoVP, free‐form VP, and liposomal VP were identical in DMSO, where VP is mainly in its monomeric form. At an excitation wavelength of 435 nm, the fluorescence emission spectra of NanoVP in DMSO can be recorded at ≈700 nm (Figure 2a). In PBS, liposomes maintain some monomeric VP, showing a minimal decrease in the Soret band without a red‐shift displacement of the Q band (Figure 2b). In contrast, when NanoVP is well‐dispersed in PBS, a broadening of the Soret band and a red‐shifted Q band were observed (Figure 2b), suggesting that monodispersed NanoVP consists of self‐assembled J‐aggregates. NanoVP is highly quenched in PBS with a quenching ratio, defined as the ratio of unquenched fluorescence intensity in DMSO and quenched fluorescence intensity in PBS (FL_DMSO/FL_PBS), of 328 at 5 µM VP (Figure 2c). This highly quenched NanoVP is likely driven by the spatial confinement of a large number of VP molecules (≈700 000) in a 100 nm diameter nanoparticle. In contrast, the quenching ratios of free‐form VP and liposomal VP are only up to 78 and 2, respectively (Figure 2c). VP quenching can decrease the photochemical production of singlet oxygen (¹O₂). Upon light activation (690 nm, 10 J cm⁻², 10 mW cm⁻²), highly quenched NanoVP and free‐form VP in PBS did not produce any significant amount of ¹O₂, as indicated by the minimal singlet oxygen sensor green (SOSG) signal (Figure 2d). On the contrary, light activation of liposomal VP generates an up to 34‐fold higher SOSG signal compared to NanoVP. We also confirmed that light activation of NanoVP, free‐form VP, or liposomal VP results in limited photothermal effects (ΔT = 2–3 °C; Figure S8, Supporting Information).

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Figure 2

Photochemical characterization of NanoVP, free‐form VP, and liposomal VP. a) Representative absorbance and fluorescence (FL) spectra of 5 µM NanoVP, free‐form VP, and liposomal VP in DMSO. b) Representative absorbance spectra of NanoVP, free‐form VP, and liposomal VP in PBS (red) and DMSO (blue). c) Quenching ratio of NanoVP, free‐form VP, and liposomal VP between 0.625–5 µM. Quenching ratio is defined as the ratio of unquenched fluorescence intensity in DMSO and quenched fluorescence intensity in PBS (FL_DMSO/FL_PBS). d) Singlet oxygen production from NanoVP, free‐form VP, and liposomal VP‐mediated PDT (10 J cm⁻², 10 mW cm⁻²). e) Quenching ratio of NanoVP as a function of serum concentration in PBS. f) The time‐dependent de‐quenching (fluorescence signal recovery; Excitation/Emission: 435/700 nm) of 5 µM NanoVP in PBS, DMSO, and complete cell culture medium supplemented with 10% fetal bovine serum at 37 °C. g) TEM images of NanoVP in 0% human serum, h) 1% human serum, and i) 10% human serum. j) The size of NanoVP in 0%–10% human serum as quantified from TEM images using ImageJ. One‐way ANOVA with multiple comparison test was used to calculate significant differences, where * P < 0.05, ** P < 0.01, *** P < 0.001. N ≥ 3. Error bar shows the standard error of the mean.

While NanoVP is quenched and non‐photoactivable in PBS during storage, we hypothesized that NanoVP could be unquenched and photoactivated in serum‐containing media. Figure 2e shows that increasing the serum protein level from 0.15% to 10% v/v decreases the NanoVP quenching ratio in serum from 92 to 3.2. Disaggregation of NanoVP in fetal bovine serum (FBS)‐containing media is a time‐dependent process. After adding NanoVP to 10% v/v FBS‐containing media, a rapid increase in VP fluorescence within 30 minutes is followed by a plateau of the signal, whereupon the signal remains constant at 38% of that fully dissolved in DMSO (Figure 2f). Through TEM, we probed interactions between NanoVP and human serum. Compared to PBS (0% human serum) (Figure 2g), the size of NanoVP as measured from TEM images using ImageJ remains intact after 2 h of 1% human serum incubation (Figure 2h), and a protein corona may form around the NanoVP (Figure 2h). In contrast, NanoVP in 10% human serum has a 49% decrease in diameter in the same time period, but evidence of particles is still visible via TEM (Figure 2i,j).

2.3. NanoVP is Unquenched in Cancer Cells, Improving Photosensitizer Uptake and PDT Efficacy

We sought to understand the unquenching and photochemical activity of NanoVP in cancer cells. U87 cells both in medium (Figure  3a) and in PBS (Figure 3b) were able to unquench NanoVP in a time‐dependent manner, with superior fluorescence recovery compared to free‐form VP and liposomal VP. To check the recovery of photochemical activity of NanoVP in U87 cancer cells, dichlorodihydrofluorescein diacetate (DCFDA) was used as a fluorescent probe for the detection of intracellular ROS generation (Figure 3c). Surprisingly, upon light activation (690 nm, 10 J cm⁻², 50 mW cm⁻²), NanoVP produced significantly higher intracellular ROS (≈2‐fold), compared to that of liposomal VP and the positive control (100 µM H₂O₂), despite identical treatment conditions.

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Figure 3

In vitro PDT efficacy of NanoVP in glioblastoma cells. Unquenching of NanoVP by U87 cells in a) culture media and b) in PBS in a time‐dependent manner. c) Light activation (690 nm, 10 J cm⁻², 50 mW cm⁻²) of NanoVP, free‐form VP, and liposomal VP resulted in reactive oxygen species (ROS) production in U87 cells. d) The VP concentrations in U87 cells were determined via extraction method at 24 h post‐incubation with 0.25 µM NanoVP, free‐form VP, or liposomal VP. e) Quantitative analyses of NanoVP, free‐form VP, and liposomal VP fluorescence signals in U87 cells. Fluorescence signals were normalized to the largest signal for each concentration. f) Representative fluorescence images of NanoVP, free‐form VP, and liposomal VP accumulated within U87 cells. Scale bar = 50 µm. g) U87 cell viability measured via MTT assay at 24 h after PDT using NanoVP, free‐form VP, or liposomal VP (690 nm, 0–5 J cm⁻², 10 mW cm⁻²). h) Dark toxicity of NanoVP, free‐form VP, and liposomal VP in U87 cells. Cell viability was measured via MTT assay 72 h after photosensitizer incubation. j) Representative fluorescence images of the TMRE probe and phase constant images of U87 cells at 30 min after PDT (690 nm, 10 J cm⁻², 50 mW cm⁻²). i) Mitochondrial membrane potential depolarization was quantified via TMRE probe 1 h after PDT (10 J cm⁻², 50 mW cm⁻²) using NanoVP, free‐form VP, or liposomal VP. Mitochondrial membrane potential depolarization was calculated using the formula: ΔΨm = 1‐(Tf/Tc), where Tf is the TMRE fluoresce signal from FCCP or a treatment group, and Tc is the TMRE fluoresce signal from the control group. k) Quantification of total and cleaved caspase 3 expressions in U87 cells at 1 h post‐PDT (690 nm, 10 J cm⁻², 50 mW cm²). N ≥ 3. A two‐tail (total) and one‐tail (cleaved) t‐test was used to calculate significant differences. l) Representative immunoblotting showed changes in total and cleaved caspase 3 expressions in U87 cells at 1 h after PDT. One‐way ANOVA with multiple comparison tests was used to calculate significant differences, where * P < 0.05, ** P < 0.01, and *** P < 0.001. N ≥ 3. Error bar shows the standard error of the mean.

To investigate why NanoVP‐PDT treatment resulted in greater intracellular ROS generation, we first probed intracellular photosensitizer concentration. The therapeutic efficacy of PDT depends on the concentration of photosensitizer in cancer cells. U87 cells treated with NanoVP had twice as much intracellular VP (129.8 ± 29.1 ng mg⁻¹ of protein) compared to liposomal VP (66.2 ± 8.1 ng mg⁻¹ of protein) (Figure 3d). We further confirmed enhanced uptake of NanoVP via fluorescence imaging of U87 cells treated with free VP, NanoVP, and liposomal VP, finding enhanced uptake of free VP and NanoVP over liposomal VP at all concentrations (Figure 3e,f). We found a 49.9% increase in uptake of NanoVP in noncancerous 3T3 mouse fibroblast cells compared to cells treated with liposomal VP (Figure S9a, Supporting Information). Intracellular ROS generation and cellular uptake of NanoVP was similar to that observed from free VP, which is clinically nonviable due to the organic solvent carrier that is needed to solubilize VP.

Increased uptake of NanoVP and increased singlet oxygen production results in a 400% stronger anti‐GBM PDT effect than liposomal VP, as evaluated by MTT (Figure 3g). NanoVP and free‐form VP had a half‐maximal inhibition concentration (IC₅₀) of 0.2 µM × J cm⁻² in U87 cells, while liposomal VP had a 4‐fold higher IC₅₀ of 0.8 µM × J cm⁻². PDT IC₅₀ in noncancerous 3T3 cells was 0.4 µM × J cm⁻² for NanoVP and 0.8 µM × J/cm² for liposomal VP (Figure S9b, Supporting Information). These IC₅₀ values indicate a potentially greater therapeutic index for PDT using NanoVP (0.2 µM × J cm⁻² difference in IC₅₀s between cancerous and noncancerous cells) compared to PDT using liposomal VP (no differences in IC₅₀s). Considering recent efforts using VP in a light‐independent manner to treat glioblastoma (NCT04590664),^{[ 22 , 23 , 24 ]} we treated U87 and 3T3 cells with higher concentrations of NanoVP in complete darkness. NanoVP exhibited an IC₅₀ of 10.5 µM in U87 cells and 3T3 cells; free VP performed similarly (Figure 3h; Figure S9c, Supporting Information). In contrast, liposomal VP exhibited no dark cytotoxicity in the range of concentrations tested, presumably due to the poor intracellular VP accumulation.

We next explored the mechanisms of NanoVP‐mediated PDT cell killing. VP preferentially accumulates in mitochondria and light activation of VP depolarizes the mitochondrial membrane potential (ΔΨm) to trigger apoptosis.^{[ 25 , 26 , 27 ]} By tetramethylrhodamine ethyl ester (TMRE) assay, NanoVP‐PDT (690 nm, 10 J cm⁻², 50 mW/cm⁻²) induced the largest degree of ΔΨm depolarization (Figure 3i,j). P‐triflouromethoxyphenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation uncoupler, was used as a positive control. Relative to untreated controls, NanoVP‐PDT depolarizes ΔΨm by nearly 60%, while liposomal VP‐mediated PDT results in depolarization of ΔΨm by ≈35%. PDT‐induced mitochondria damage triggers the intrinsic apoptosis pathway, leading to caspase‐3 activation.^{[ 28 , 29 , 30 , 31 ]} Immunoblotting results indicate that PDT using NanoVP or free‐form VP induced a ≈2.4‐fold increase in cleaved caspase‐3 one‐hour post‐light activation (Figure 3k,l). In addition, NanoVP and free‐form VP‐mediated PDT significantly reduced total (pro) caspase‐3 levels by ≈50%, resulting in a cleaved‐to‐total caspase‐3 ratio of ≈4.4. Liposomal VP‐PDT resulted in the lowest ratio of ≈2.2. Next, we investigated the interaction of NanoVP with other cellular components. Neutral red uptake (NRU) assay revealed that light‐activation (690 nm, 1 J cm⁻², 50 mW cm⁻²) of NanoVP, free‐form VP, or liposomal VP does not affect lysosomal integrity in U87 cancer cells (Figure S10, Supporting Information). Previously, we have shown that VP can be effluxed by ATP‐binding cassette (ABC) drug transporters (breast cancer resistance protein, ABCG2; and P‐glycoprotein, P‐gp), reducing PDT efficacy.^{[ 18 ]} Using a human breast cancer cell line MCF‐7, and its sub‐lines ABCG2‐overexpressing MCF‐7 MX100 and or P‐gp‐overexpressing MCF‐7 TX400,^{[ 18 , 32 ]} we found that NanoVP remains a substrate of ABCG2 and P‐gp (Figure S11, Supporting Information).

2.4. NanoVP‐PDT Enhances Acute Tumor Control in a Xenograft Mouse Model of GBM

NanoVP pharmacokinetics were evaluated in mice. Animals received 0.5 mg kg⁻¹ NanoVP via tail vein injection. We observed an initial spike in <5 min, followed by a plateau in plasma levels from 15 min post‐injection to 6 h post‐injection (Figure  4a). Complete clearance was observed at 72 h. The pharmacokinetic profile of NanoVP was found to be similar to that of both VP in DMSO and liposomal VP as reported by others.^{[ 33 , 34 ]} Qualitative comparison of biodistribution at 2 and 24 h post‐injection in mice bearing flank U87 tumors indicates that NanoVP and liposomal VP may have similar biodistribution profiles Figure S12, Supporting Information). Quantification of VP levels in mice bearing flank GBM39 tumors receiving 0.5 mg kg⁻¹ NanoVP IV revealed persistently high levels in highly vascularized tissues (e.g., heart, lung, kidneys, liver, spleen), and tumor‐to‐normal tissue (skin) ratios of 0.3–1.8 around the time of intended treatment (1–3 h post‐injection) (Figure 4b; Table S1, Supporting Information). Clearance was observed by 72 h, consistent with pharmacokinetic data.

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Figure 4

The phototoxicity and biodistribution of NanoVP in U87 glioblastoma xenograft mouse model. a) Pharmacokinetics of NanoVP after 0.5 mg kg⁻¹ IV administration. b) NanoVP biodistribution as measured via HPLC‐MS/MS at various time points after 0.5 mg kg⁻¹ IV NanoVP administration to mice bearing flank GBM39 tumors, expressed as percent injected dose per organ. Grey boxes with an “X” indicate no data. c) Kaplan‐Meier plot of tumor diameter greater than 1.5 cm (N = 7–8 animals per group). PDT treatment (100 J cm⁻², 100 mW cm⁻²) was initiated ≈14 days after subcutaneous U87 cancer cell implantation when tumor volumes reached ≈100 mm³. Mice were randomized into groups that received i) no‐treatment, ii) Liposomal VP (0.5 mg kg⁻¹), and iii) NanoVP (0.5 mg kg⁻¹). d) Tumor volume was longitudinally monitored and calculated using the standard estimation formula, V = ½ × length × width², where length equals the maximum tumor diameter in millimeters and width equals the diameter that is perpendicular to the length. Tumor volumes were normalized to the initial volume at the time of treatment. e) The specific growth rate (SGR) of tumors 0–11 days and 11–25 days post‐PDT were determined using the following formula: SGR = (1/V)(dV/dt), where V is tumor volume and t is time. f) Quantification of the dark, necrotic surface area above the tumor that was impacted by PDT treatment. g) Representative digital images of tumors at 6 days post‐PDT. One‐way ANOVA with multiple comparison test was used to calculate significant differences, where *P < 0.05, **P < 0.01, and ***P < 0.001. N ≥ 3. Error bar shows the standard error of the mean.

We next assessed the efficacy of NanoVP‐PDT in a U87 flank tumor PDT mouse model. Once tumors reached ≈100 mm³, mice received 0.5 mg kg⁻¹ NanoVP or liposomal VP via tail vein injection. After a 2 h drug‐light interval, tumors received top‐down illumination for PDT (690 nm, 100 J cm⁻², 100 mW cm⁻²). In this model, a single cycle of NanoVP‐PDT modestly improved animal survival (median survival: 33 days), compared to no‐treatment control (median survival: 25) and liposomal VP‐PDT (median survival: 31 days) (Figure 4c). From 2–11 days after treatment, NanoVP‐PDT reduced tumor volume by up to 35%, while tumors continued to grow in no‐treatment and liposomal VP‐PDT control groups (Figure 4d). Tumors treated with NanoVP‐PDT had a specific growth rate (SGR) of −4.3%/day from day 0–11 post‐PDT. At 11 days post‐treatment, tumor regrowth began, and the SGR increased to 5.3%/day, similar to the SGR observed in control and liposomal VP‐PDT groups (Figure 4e). In addition to changes in tumor volume, noticeable visual differences on tumor surfaces were observed (Figure 4f). At six days post‐treatment, NanoVP‐PDT resulted in a significantly larger dark, necrotic area that covered most of the tumor (30.5 mm²), while liposomal VP‐PDT led to a smaller (13.6 mm²) and more localized necrotic patch (Figure 4f,g). Treatment of flank tumors was well tolerated with steady weight gain in all treatment groups (Figure S13, Supporting Information).

2.5. NanoVP‐PDT Prolongs Survival in an Orthotopic GBM PDX Mouse Model

While U87 is an established GBM model, we further evaluated the efficacy of NanoVP in GBM39 cells, which is a flank‐passaged patient‐derived xenograft (PDX) model.^{[ 35 ]} We first evaluated the efficacy of NanoVP‐PDT in GBM39 cells cultured in vitro. Based on our pharmacokinetics results and the apparent dissociation of NanoVP in the blood, we hypothesized that NanoVP may functionally be a molecularly dispersed formulation of VP. We identified tris‐buffered tween‐20 (TBST) as a solubilizing aqueous solution for VP and added this molecularly dispersed formulation of the drug as a control (Figure S14, Supporting Information). We also compared NanoVP‐PDT, free VP (in DMSO)‐PDT, TBST VP‐PDT, and liposomal VP‐PDT to 5‐ALA‐PDT, which is currently in clinical trials for GBM (NCT04391062, NCT04469699, NCT03897491). In GBM39 cells, we found that NanoVP‐PDT has the lowest IC₂₅ of ≈0.38 µM × J cm⁻², outperforming free VP‐PDT (IC₂₅  0.62 µM × J cm⁻²), TBST VP‐PDT (IC₂₅  0.86 µM × J cm⁻²), liposomal VP‐PDT (IC₂₅  1 µM × J cm⁻²), and 5‐ALA‐PDT (IC₂₅  110 mM × J cm⁻²) (Figure  5a). This may be partially attributable to increased photosensitizer uptake with NanoVP over liposomal VP, TBST VP, and 5‐ALA (PpIX) (Figure 5b).

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Figure 5

PDT management of orthotopic patient‐derived xenograft glioblastoma. a) In vitro GBM39 cell viability measured via CellTiter Glo® 24 h after PDT treatment (VP formulations: 0.25 µM, 690 nm, 20 mW cm⁻², 0–10 J cm⁻²; 5‐ALA: 5 mM, 635 nm, 50 mW cm⁻², 0–50 J cm²). b) The uptake of photosensitizer in GBM39 cells in vitro was measured via extraction after 24 h of incubation with 1 µM free VP, liposomal VP, TBST VP, NanoVP, or 5 mM 5‐ALA. In the case of 5‐ALA‐treated cells, PpIX was quantified. For in vivo experiments, 5 × 10⁵ GBM39 cells were implanted intracranially. After two weeks, interstitial PDT with liposomal VP, NanoVP, or TBST VP (0.5 mg kg⁻¹ IV, 2 h drug‐light interval, 12 J cm⁻¹, 40 mW cm⁻¹) or 5‐ALA (20 mg kg⁻¹ IV, 4 h drug‐light interval, 60 J cm⁻¹, 100 mW cm⁻¹) was performed. c) Kaplan‐Meier plot for survival after treatment with PDT with liposomal‐PDT or NanoVP‐PDT; P < 0.05 by Log‐rang test. d) GBM39 cells express luciferase; total bioluminescence was used as a proxy for tumor size at 1‐week post‐treatment (day 21) and normalized to pretreatment values (day 14) (N = 8 for Control, N = 6 for liposomes, N = 9 for TBST VP, N = 10 for 5‐ALA, N = 14 for NanoVP). Two‐way ANOVA with multiple comparison test was used to calculate significant differences, where * P < 0.05, ** P < 0.01, **** P < 0.0001. Error bar shows the standard error of the mean. e) Representative images showing better tumor control with NanoVP at day 21, 1‐week post‐treatment.

To evaluate the potential of NanoVP to control orthotopic GBM and improve animal survival, we injected 5 × 10⁵ GBM39 PDX cells expressing luciferase into the right striatum of nude mice. We utilized the GBM39 PDX, which is serially passaged in mice rather than cultured in dishes, to better recapitulate the tumor biology observed in patients. Fourteen days post‐injection, when IVIS signal was robust and when mortality was first observed, we performed interstitial PDT (0.5 mg kg⁻¹ NanoVP, liposomal VP, or TBST VP via tail vein injection; 12 J cm⁻¹, 40 mW cm⁻¹, 690 nm, 2 h drug‐light interval) by inserting a laser fiber (MedLight RD10 fiber, Modulight ML6600 laser) through the burr hole initially used to inject cells. Tolerable conditions for VP‐PDT were established via a dose‐escalation study and 5‐ALA‐PDT conditions were established based on current clinical trial parameters. 5‐ALA is dosed at 20 mg kg⁻¹ with a 4 h drug‐light interval in the clinic, with ≈10 min added to surgical time and irradiances and fluences exceeding those explored in this study (Table S2, Supporting Information). Body weight (Figure S15, Supporting Information) as part of a body condition score was used to monitor animal health over the course of the study. We observed no increase in temperature at the site of PDT, as measured by a FLIR infrared camera under conditions used to perform VP‐PDT (690 nm, 40 mW cm⁻¹), but we did observe an increase in temperature at the site of PDT with 5‐ALA‐PDT (635 nm, 100 mW cm⁻¹) treated mice (Figure S16a, Supporting Information). Upon examination of the laser fiber itself, we identified a significant increase in temperature during and immediately after 5‐ALA‐PDT using 635 nm laser (Figure S16b, Supporting Information). Ultimately, we observed a potential zone of necrosis surrounding the laser insertion site in 5‐ALA PDT‐treated mice, while the neuropil recovered after PDT in control and NanoVP‐PDT mice (Figure S16c, Supporting Information).

We observed a 7‐day median survival benefit in mice treated with NanoVP over untreated mice (28 days versus 21 days, P < 0.05, Figure 5c) and a 10‐day median survival benefit over mice treated with liposomal VP‐PDT (28 days versus 18 days, P < 0.05, Figure 5c). This significant survival benefit was likely due to the superior acute control of tumor growth. When normalized to pre‐treatment bioluminescent signal, the bioluminescent signal one week post‐treatment had the smallest increase in signal in the NanoVP‐PDT group (mean 369% increase), while liposomal VP‐PDT (mean 1590% increase), control (mean 1270% increase), TBST VP (mean 2030% increase), and 5‐ALA PDT (mean 1680% increase) groups had significant increases in the bioluminescent signal (Figure 5d,e). While orthotopic interstitial PDT with NanoVP provided a significant median survival benefit and significant reduction of tumor burden, these results point out challenges in achieving long‐term improvements in treatment response for GBM, highlighting the need for developing combination strategies to provide durable tumoricidal control in the future.

Transmission Electron Microscopy (TEM)

The size, morphology, and microstructure of NanoVP were studied using TEM (JEOL JEM‐2100 LaB6, 200 kV). For conventional TEM, NanoVP (10 µL) was pipetted onto a Lacey carbon grid (Ted Pella) and air‐dried overnight before the examination. To exclude the possibility of artifacts due to sample dehydration, ionic liquid (3 µL, Hilem IL‐1000, Hitachi) was used as a pretreatment reagent for TEM examination of wet specimens. Images were taken at high magnifications (10 000–100 000X). Due to small particle sizes and to enhance diffraction intensity, selected area electron diffraction was used to collect electron diffraction patterns. For serum dissociation studies, NanoVP (final concentration 100 µM) was mixed with 1% or 10% v/v human serum (Fisher Scientific) for 2 h at 37 °C before drying on a Lacey carbon grid. Samples were imaged under the same conditions as pure NanoVP samples.

NanoVP Stability, Photoactivity, and Photosensitizer Release Profile

The stability of NanoVP particles in PBS at 4 °C was determined by monitoring their hydrodynamic size and polydispersity index using a particle sizer analyzer for up to one year. NanoVP singlet oxygen (¹O₂) generation, self‐quenching, and drug release were studied in 96‐well plates as described previously.^{[ 25 ]} NanoVP and singlet oxygen sensor green (SOSG) at 5 µM were mixed and irradiated with 690 nm light (10 J cm⁻², 10 mW cm⁻²; ML6600, Modulight). A multi‐mode microplate reader (Synergy Neo2, BioTek) acquired fluorescence signals of VP (Excitation/Emission: 435/650–750 nm) or SOSG (Excitation/Emission: 504/525 nm) before and after light irradiation. Self‐quenching (FL_DMSO/F_PBS) is defined as the fluorescence after disruption of the NanoVP using DMSO (FL_DMSO) divided by the fluorescence of the NanoVP in PBS (FL_PBS). Photosensitizer release from NanoVP (unquenching) was studied by monitoring the gain in VP fluorescence signal in PBS/DMSO buffer (0%–100% v/v) at room temperature or in Eagle's Minimum Essential Medium (EMEM, Cellgro) with 0%–10% v/v fetal bovine serum (FBS, Gibco) at 37 °C.

Cell Cultures

The human glioblastoma U87 cell line and mouse 3T3 fibroblast cells were obtained from ATCC and cultured per the vendor's instructions. The human breast cancer MCF‐7 parental cell line, the P‐gp‐overexpressing MCF‐7 TX400 subline, and the ABCG2‐overexpressing MCF‐7 MX100 subline were cultured in EMEM supplemented with 10% FBS, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin, and 0.01 mg ml⁻¹ insulin (Sigma) as previously described.^{[ 18 ]} Cells were maintained in 5% CO₂ at 37 °C and tested to be free of mycoplasma (MycoAlert, Lonza).

GBM39 cells expressing luciferase were serially passaged in the flank of female J:NU mice (4‐5 weeks old, #0 0 7850, Jackson Laboratory). Tumors were dissociated using a gentleMACS tumor dissociation kit (Miltenyi Biotec) before in vitro culturing. Cells were cultured as recommended by the Mayo Clinic Brain Tumor Patient‐Derived Xenograft National Resource using the FBS cell culture protocol. Briefly, cells were cultured in DMEM containing 2.5% FBS, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin for 48 h, then media was exchanged to DMEM containing 10% FBS and antibiotics for at least 24 h before experimentation. Cells were dissociated and used in experiments, but not passaged serially. Assays were performed within 14 days of tumor dissociation.

Preparation of TBST VP

VP was dissolved in chloroform at a concentration of 250 µM and chloroform was evaporated under reduced pressure with a rotary evaporator. Tris‐buffered tween‐20 (TBST, 0.5%) was prepared by diluting a stock solution (ThermoScientific) with phosphate‐buffered saline (diluted to 1X from Calbiochem OmniPur 10X PBS (Millipore, 6507)). 1 mL of surfactant solution was added to a tube containing VP film and the samples were placed in a shaker at 37 °C at 100 rpm for 24 h. Samples were filtered using sterile syringe filters with 0.22 µm pore size (Millipore‐Sigma).

Evaluation of Photosensitizer Uptake and PDT Responses In Vitro

Cells were cultured overnight in a 35‐mm Petri dish or 96‐well black wall plates (1–3.3 × 10⁴ cells cm⁻²) and then incubated with photosensitizers (i.e., NanoVP, free VP in DMSO, TBST VP, or liposomal VP at 0.25 µM; 5‐ALA at 5 mM) for 24 h for U87 cells or 1.5 h for GBM39 cells. Subsequently, cells were washed twice with PBS and incubated with a photosensitizer‐free complete medium. Photosensitizer uptake in cells was determined using extraction methods followed by VP fluorescence measurements (Excitation/Emission: 435/690 ± 20 nm, Synergy Neo2, BioTek) or visualized using fluorescence imaging (Lionheart, BioTek) as described previously.^{[ 25 ]} PDT was performed by exposing the cells to 690 nm light (0–10 J cm⁻², 10–50 mW cm⁻², bottom illumination; ML6600, Modulight). The generation of intracellular ROS was studied using 2′,7′‐dichlorofluorescin diacetate probe (DCFDA, Thermo Fisher) and the mitochondrial membrane potential was examined via TMRE assay (tetramethylrhodamine ethyl ester, Abcam). Expressions of total and cleaved caspase 3 were examined by immunoblotting (Supplementary Methods). At 24 h after PDT, cell viability was determined using MTT [3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide] assay, neutral red assay (Abcam), or CellTiter Glo (Promega) following the vendor's protocol. For dark toxicity evaluation, cells were incubated with media containing photosensitizers (0–40 µM) for 72 h, followed by the MTT assay. The photosensitizer efflux by ATP‐binding cassette (ABC) transporters was studied in MCF‐7 and its multi‐drug resistant sublines (MCF‐7 TX400 and MCF‐7 MX100) by adapting our protocol (Supporting Methods).

In Vivo Photodynamic Therapy and Photosensitizer Biodistribution

Animal protocols (R‐MAR‐22‐16, R‐MAY‐20‐19, R‐MAY‐23‐27) were approved by the University of Maryland, College Park Institutional Animal Care and Use Committee, which has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 2011 with PHS Assurance Number D16‐00172 and USDA Certificate Number 51‐R‐0095. Xenograft mouse models of glioblastoma were established by subcutaneously injecting U87 cells (1 × 10⁶ cells in PBS/Matrigel) into the flank of a J:NU mouse (4‐5 weeks old, Jackson Laboratory). Tumor volumes were longitudinally monitored using calipers and calculated using the standard estimation formula, V = ½ × length × width², where length equals the maximum tumor diameter in millimeters and width equals the diameter that is perpendicular to the length. Treatments were initiated 2 weeks post‐implantation when tumors reached ≈100 mm³. At 2 h post‐IV injection of photosensitizers (i.e., NanoVP or liposomal VP; 0.5 mg kg⁻¹) or PBS, a vertical 690 nm laser beam (100 J cm⁻², 100 mW cm⁻²; ML6600, Modulight) was focused on the tumor to activate PDT. A cloth was used to protect animal skin from light exposure. Change in tumor volume was monitored for up to 2 months. The specific growth rate (SGR) of tumors was estimated using the equation (1/V)(dV/dt), where V is tumor volume and t is time. To examine photosensitizer biodistribution, tumor and normal tissues were collected at 2‐ and 24 h post‐injection of photosensitizers.

GBM39 cells were passaged and dissociated as described above. 5 × 10⁵ GBM39 cells in 5 µL of PBS were stereotactically injected using a Hamilton syringe positioned at 2 mm right lateral to bregma and 3 mm deep in female J:Nu mice (4‐5 weeks old, #0 0 7850, Jackson Laboratory). Fourteen days post‐implantation, PDT was performed. Mice were randomly divided into NanoVP‐PDT, liposomal VP‐PDT, TBST VP‐PDT, 5‐ALA‐PDT and no treatment groups. Mice received 0.5 mg kg⁻¹ tail vein injections of NanoVP, TBST VP, or liposomal VP and, after 2 h, received 12 J cm⁻¹ of 690 nm light (40 mW cm⁻¹) interstitially through the burr hole used to implant tumor cells (RD10 laser fiber, ML6600 laser, Modulight). Mice treated with 5‐ALA PDT received 20 mg kg⁻¹ 5‐ALA IV 4 h before laser irradiation with 635 nm light (100 mW cm⁻¹, 60 J cm⁻¹). Power level was measured using the Modulight MLACAL external calibration unit. All but 5 mm from the tip of the RD10 fiber, matching the depth that the fiber penetrated the mouse brain, was covered with black masking tape (Thor Labs) before measuring power according to manufacturer instructions.

Photodynamic Opening of the Blood‐Brain Barrier in Rodents

Animal protocols were approved by the University of Maryland, School of Medicine Institutional Animal Care and Use Committee. Sprague‐Dawley rats (4‐5 weeks old, Envigo) received an IV injection of NanoVP (0.25 or 0.5 mg kg⁻¹) or 5‐aminolevulinic acid (5‐ALA, 20 or 125 mg kg⁻¹) at 30 min or 4 h before PDT. PDT parameters, including photosensitizer concentration, drug‐light interval, irradiance, and radiant exposure, were selected based on our experience or others’ clinical work. At 30 min post‐photosensitizer injection, rats were anesthetized with isoflurane, secured within a stereotaxic frame, and a craniotomy was performed to expose the right cerebral hemisphere (Supporting Methods). PDT was performed by light activation of the exposed brain (NanoVP: 690 nm, 80 or 100 J cm⁻²; 5‐ALA: 635 nm, 60 or 80 J cm⁻²; 40 or 85 mW cm⁻²; ML6600, Modulight). At 90 minutes post‐PDT, rats received an IV injection of Evans blue dye (2%, 4 mL kg⁻¹) to determine the blood‐brain barrier integrity using imaging and extraction methods (Supporting Methods). In a small group of PDT animals that did not receive Evans blue injection, brain tissues were collected, sectioned, and processed for i) histological (hematoxylin and eosin stain, H&E) analysis and ii) Luxol Fast Blue staining of myelin/myelinated axons and Nissl bodies. H&E and immunohistochemistry slides were imaged using the whole slide Aperio AT2 scanner system (Leica Biosystems) (Supplementary Methods). All image analysis was accomplished using Aperio and Halo imaging analysis software (v3.3.2541.300; Indica Labs), and image annotations were performed by a pathologist (B.K). Fields were excluded if they contained large areas of artifact such as folds or tears.

Statistical Analyses

Results are presented in mean ± standard error of the mean (SEM). Statistical tests were carried out using GraphPad Prism (GraphPad Software). Specific tests and the number of repeats are indicated in the figure captions. Reported P values are two‐tailed. One‐way or two‐way ANOVA statistical tests with Tukey post‐hoc tests were performed.

IVIS Imaging

IVIS bioluminescent in vivo imaging (PerkinElmer) and LivingImage software were used to measure tumor burden over time in the orthotopic GBM model. Mice received 150 mg kg⁻¹ intraperitoneal injection of luciferin (Promega VivoGlo) ten minutes before imaging, then were anesthetized and imaged. Regions of interest of equal size were used to quantify total flux (photons/second).

National Institutes of Health grant R21EB028508 (HH), National Science Foundation grant NSF 2030253 (HH), NCI‐UMD Partnership for Integrative Cancer Research grant (MMG, HH). We acknowledge the support of the Maryland NanoCenter and its AIMLab. We thank Sijie Hao, PhD and Christian Pick, PhD of the BioWorkshop core facility in the Fischell Department of Bioengineering at the University of Maryland, College Park for training and access to the Waters Xevo TQD Mass Spectrometer and NanoBrook Omni. We thank Divya Muthusamy and Marzyeh Kheradmand‐Hajibashi for assistance in mass spectrometer operation. We thank Steven Jay, PhD and Stephanie Kronstadt, PhD for access to and training on the NanoSight LM10. We thank Beth Bauer, DVM and Betre Legesse of the Department of Laboratory Animal Resources Imaging Core for access to and training on the IVIS Spectrum imaging system. We thank George Leiman for editorial assistance.