Acriflavine-empowered IR780-PTX albumin nanoparticles for reinforced synergistic photochemotherapy

TL;DR

Researchers developed a new treatment combining chemotherapy and phototherapy to overcome challenges in treating glioma tumors.

Contribution

A novel combination of IR780-PTX nanoparticles and acriflavine hydrogels is introduced to enhance photochemotherapy in hypoxic tumors.

Findings

The IR780-PTX nanoparticles and ACF hydrogels work together to provide an extraordinary antitumor effect.

ACF suppresses HIF-1 activity and HSP70 upregulation, improving phototherapy efficacy in hypoxic conditions.

This approach shows promise for treating tumors with severe hypoxia or therapy-induced resistance.

Abstract

Traditional chemotherapy and radiotherapy for glioma are challenging due to the hypoxia in tumor microenvironment and the inability of chemotherapeutic agents to enter into tumor cells. Phototherapy is a novel therapeutic approach against various tumors in recent years. When combined with chemotherapy, the antitumor efficacy of phototherapy is superior than each alone. However, the combination of chemotherapy and phototherapy is still hampered by the hypoxic tumor microenvironment which upregulates the expression of hypoxia-inducible factor 1 (HIF-1) and its downstream pathways, as well as the thermoresistance caused by the overexpression of heat shock proteins (HSPs). To solve this, the biocompatible albumin-based nanoparticles (NPs) are developed to co-deliver IR780 iodine (IR780) and paclitaxel (PTX) simultaneously at an optimized ratio (IR780-PTX NPs) for synergistic photochemotherapy. Moreover, acriflavine (ACF), a chemical inhibitor of HIF-1, is formulated into intratumorally formed hydrogels to reinforce synergistic photochemotherapy. The continuously released ACF from hydrogel not only relieves the impact of photodynamic therapy-exacerbated tumor hypoxia by suppressing HIF-1 activity, but also efficiently attenuates HSP70 upregulation. The collaboration between IR780-PTX NPs and ACF hydrogels leads to an extraordinary antitumor effect in vitro and in vivo. The reinforced synergistic photochemotherapy via a single molecule by overcoming HIF-1 activity and HSP overexpression provides an effective therapeutic example to treat tumors, especially in those undergone severe hypoxia and/or therapy-induced thermoresistance.

The online version contains supplementary material available at 10.1007/s44307-026-00097-9.

Figures

Schematic illustration of synergistic GBM treatment via IR780-PTX NPs and intratumorally formed ACF hydrogels. (Created in [https://BioRender.com](https://BioRender.com))

Characterizations of IR780-PTX NPs. a CI of IR780 and PTX at different ratios. Data were represented as means ± standard deviation (SD) (*n* = 3). One-way analysis of variance (ANOVA) was used to compare the means of multiple groups. p* < 0.05, *p* < 0.01, p* < 0.001, ns: no significance. b Size distribution of PTX NPs, IR780 NPs and IR780-PTX NPs. c TEM image of IR780-PTX NPs (Scale bar: 200 nm). d Colloidal stability of IR780-PTX NPs. Data were represented as means ± SD (*n* = 3). e In vitro release of PTX from IR780-PTX NPs at pH 7.4 or pH 5.0. Data were represented as means ± SD (*n* = 3).

Cellular uptake and PTT/PDT effects of IR780-PTX NPs. a Cellular uptake of free IR780 and IR780-PTX NPs at 3 h and 6 h of incubation in LN229 cells (Scale bar: 50 μm). b Statistical analysis of IR780 cellular uptake in (a). Data were represented as means ± SEM (*n* = 3). Student’s t-test was used for comparison between two groups. * *p* < 0.05, *p* < 0.01. c Cellular uptake of IR780-PTX NPs under BSA pretreatment (Scale bar: 50 μm). d Subcellular localization of PTX-IR780 NPs in LN229 cells (Scale bar: 20 μm). e AO staining of lysosomes in LN229 cells with or without NIR irradiation (Scale bar

In vitro antitumor effects of IR780-PTX NPs and ACF. a PDT-induced hypoxia in LN229 cells. b HIF-1α expression in LN229 cells after different treatments. c Statistical analysis of HIF-1α expression in (b). Data were represented as means ± SD (*n* = 3). Student’s t-test was used for comparison between two groups. * *p* < 0.05, ns: no significance. d Cytotoxicity of LN229 cells under different treatments at hypoxic condition. e IC_50_ of PTX and IR780 for various formulations at hypoxic condition. One-way ANOVA was used to compare the means of multiple groups. * *p* < 0.05, * *p* < 0.001. f Casp

In vitro suppression of HIF-1 pathway and HSP70 by ACF. a Western blot analysis and (b) statistical analysis of HIF-1α, GLUT-1, MMP9, VEGF, and HSP70 expressions in LN229 cells receiving various treatments. c Cell migration assay (Scale bar: 50 μm). d Statistical analysis of cell migration assay. Data were represented as means ± SD (*n* = 3). e Invasion assay of LN229 cells after different treatments (Scale bar: 20 μm). f Statistical analysis of cell invasion assay. Data were represented as means ± SD (*n* = 3). One-way ANOVA was used to compare the means of multiple groups. * *p* < 0.05, *p*

In vivo antitumor efficacy of IR780-PTX NPs and ACF hydrogels combination. a Dynamic in vivo fluorescence imaging of nanoparticle biodistribution in mice after intravenous injection of free IR780 or IR780-PTX NPs. b Ex vivo NIR fluorescence imaging of excised tumors and major organs from tumor-bearing mice treated with free IR780 or IR780-PTX NPs. c Experimental scheme for in vivo antitumor study. d Tumor growth in mice under different treatment conditions. e Image and (f) weights of excised tumors from mice after different treatments. g H&E staining and TUNEL staining of tumor sections from m

In vivo antitumor effect of IR780-PTX NPs combined with intratumorally formed ACF hydrogels. a Thermal imaging of tumor-bearing mice treated with PBS or IR780-PTX NPs + ACF at indicated time points. b Temperature rise curves of tumors in (a). Data were represented as means ± SD (*n* = 3). c Western blot analysis and (d) statistical analysis of HSP70 expression in tumors receiving various treatments. e ROS generation indicated by DCFH-DA fluorescence and (f) statistical analysis in the slices of tumors receiving various treatments (Scale bar: 200 μm). Data were represented as means ± SEM (*n* =

Full text

Introduction

Glioma is a malignant primary brain tumor, while glioblastoma (GBM) is the most aggressive phenotype, constituting approximately 57% of all gliomas and 48% of all malignant brain tumors (Schaff et al. 2023). Surgical resection, postoperative adjuvant chemotherapy and radiotherapy are clinically employed to treat glioma. Despite recent advancements in therapeutic modalities for glioma, the overall prognosis remains dismal, with a notably low long-term survival rate below 9.8% (Schaff et al. 2023). The glioma therapeutic effectiveness is significantly limited by the complex tumor microenvironment (TME), such as hypoxia in tumor interior and aberrant angiogenesis (Sun et al. 2025). Thus, it is highly necessary to explore new potential treatments for glioma.

Phototherapy is a treatment approach based on light and a photosensitizer (PS). It operates through two primary mechanisms: in photothermal therapy (PTT), the process generates hyperthermia to directly ablate tumors; in photodynamic therapy (PDT), it produces reactive oxygen species (ROS). These ROS oxidize critical biomacromolecules—including nucleic acids, proteins, and lipids—thereby inducing cell apoptosis and necrosis (Wang et al. 2023a; Cai et al. 2025). Phototherapy outcompetes conventional treatments in terms of high spatiotemporal selectivity and less adverse effects (Overchuk et al. 2023). On the other side, the oxygen-dependent ROS production severely hampers the PDT potency due to the hypoxic TME (Yang et al. 2020a). Additionally, the ROS can cause acute damage to the microvessels at the tumor site, leading to vascular blockage and exacerbated tumor hypoxia (Li et al. 2018). However, the apoptosis induced by PTT in tumor cells may be alleviated by heat shock proteins (HSPs), leading to thermoresistance and reduced PTT efficiency (Chen et al. 2023). These largely give rise to glioma relapse and metastasis upon phototherapy. To this end, phototherapy has been extensively combined with other treatment modalities, particularly chemotherapy for enhanced synergistic antitumor effects. For instance, camptothecin-modified polyethylene glycol self-assembled into micelles with IR780 iodine (IR780), which showed significantly boosted antitumor effect of glioma by combining PDT and chemotherapy (Lu et al. 2020). In addition, gold nanoroses or hollow mesoporous copper sulfide nanoparticles (NPs) were committed to PTT with the co-delivery of chemotherapeutic drugs or other therapeutic agents for improved glioma therapy (Dube et al. 2022; Cao et al. 2023).

However, the above-mentioned combinational phototherapies are still frustrated by the complicated TME of glioma. It’s been widely recognized that glioma especially the GBM is featured by tumor hypoxia owing to the underdeveloped vascular system, which efficiently upregulates the expression of hypoxia-inducible factor 1 (HIF-1) (Su et al. 2022). ROS generated in PDT activates extracellular signal-regulated kinase and mitogen-activated protein kinase pathways, causing HIF-1 phosphorylation and increasing its activity (Yun et al. 2023). In addition, it’s also reported that elevating tumor temperature in PTT causes increased HIF-1 expression independent of oxygen level (Moon et al. 2010). As a nuclear transcription factor, HIF-1 upregulates vascular endothelial growth factor (VEGF) and 3’-phosphoinositide-dependent kinase-1 expression, promotes angiogenesis and metabolic reprogramming, and increases tumor resistance against diverse treatments such as chemotherapy due to the overexpression of efflux pumps (Domènech et al. 2021; Li et al. 2025). Therefore, HIF-1 inhibition is pivotal for improving the effectiveness of tumor phototherapy and chemotherapy. Currently, many effective strategies targeting HIF-1 have been developed, including silencing HIF-1α gene via siRNA and antisense oligonucleotides, degrading HIF-1α via hypoxia relief, and suppressing HIF-1 transcriptional activity via a range of inhibitors (Zhang et al. 2022). In particular, acriflavine (ACF) has been widely utilized as a HIF-1 inhibitor in enhancing PDT of various cancers including glioma (Ma et al. 2022). Guo et.al, demonstrated a photo-controlled, platelet-mediated co-delivery strategy for verteporfin and ACF to enhance GBM PDT by simultaneously blocking HIF-1α and YAP signaling, thereby impairing DNA damage repair following PDT (Guo et al. 2025). However, the extraordinary water solubility of ACF and the short half-life in vivo largely restricts its efficacy of HIF-1 suppression in these studies as hypoxia modulation is a long-lasting progress during therapy course (Zhang et al. 2020). In addition, whether ACF could enhance the antitumor effect of PTT is still not fully explored.

Herein, we developed a novel synergistic therapy by combining phototherapy and chemotherapy mediated by albumin-based NPs and simultaneous HIF-1 and HSP70 modulation via automatically formed intratumoral hydrogels delivering ACF (Fig. 1). The near-infrared dye IR780 and paclitaxel (PTX) were simultaneously incorporated into albumin to form the IR780-PTX albumin NPs. However, ACF is a hydrophilic small molecule, whereas PTX and IR780 are hydrophobic, making their efficient co-encapsulation within a single albumin nanocarrier technically challenging. ACF has several limitations, including potential off-target effects, DNA intercalation, phototoxicity, and limited brain penetrability when administered systemically. Therefore, we employed locally administered ACF-loaded hydrogels to achieve sustained intratumoral retention and high local drug concentrations, enabling continuous inhibition of HIF-1 while minimizing systemic exposure. Firstly, based on drug ratio screen to get a better synergistic antitumor effect, IR780 and PTX are self-assembled into bovine serum albumin (BSA) NPs (IR780-PTX NPs), which could accumulate in the tumor for combined phototherapy and chemotherapy. In addition, the albumin-based NPs could also further enhance drug accumulation in GBM through its interaction with specific proteins such as by glycoprotein 60 (gp60) and secreted protein acidic and rich in cysteine (SPARC), both of which are reported to be overexpressed in GBM (Ruan et al. 2018; Yang et al. 2020b; Wei et al. 2025). Meanwhile, sustained release of ACF from the intratumorally formed alginate hydrogels not only inhibits HIF-1 and its downstream proteins, but also suppresses the expression of HSP70 to overcome the thermoresistance against PTT, thus enhancing the synergistic effect of phototherapy and chemotherapy. This study provides a new way to reinforce the synergistic photochemotherapy of glioma and other hypoxic tumors with ACF as a potent enhancer.Fig. 1. Schematic illustration of synergistic GBM treatment via IR780-PTX NPs and intratumorally formed ACF hydrogels. (Created in https://BioRender.com)

Materials and methods

Materials

PTX was procured from Xinhou Biotechnology (Nanjing, China). IR780 and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) were achieved from Sigma Aldrich (MO, USA). ACF was obtained from Aladdin Biochemical Technology (Shanghai, China). Hoechst 33342, 4′,6-diamidino-2-phenylindole (DAPI), Lyso-Tracker Green, and Enhanced BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit was purchased from Vazyme Biotech (Nanjing, China). The Cell Counting Kit-8 (CCK-8) was acquired from APExBIO (Houston, USA). HIF-1α antibody was purchased from Bioss Biotech (Beijing, China). Caspase-3, VEGF, glucose transporter 1 (GLUT-1), matrix metalloproteinase-9 (MMP-9), HSP70 rabbit polyclonal antibody, β-Actin polyclonal antibody, and horseradish peroxidase goat anti-rabbit IgG were procured from ABclonal (Wuhan, China). Image-iT™ Hypoxia Reagents was purchased from Thermo Fisher Scientific (MA, USA), Human LN229 cell line was purchased from Procell Life Science & Technology (Wuhan, China). Dialysis bag was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China).

Fabrication of IR780-PTX NPs

PTX and IR780 were incorporated into albumin nanoparticles at an optimized synergistic ratio based on the screening of free drugs by cell viability assay. In the following, 0.8 mL PTX (5 mg/mL) and IR780 (2.5 mg/mL) ethanol solution was added to 20 mL BSA aqueous solution (25 mg/mL) drop by drop, stirred continuously for 30 min to self-assemble into IR780-PTX NPs, and filtered through a 0.22 μm sterile filter membrane for later use.

Characterization of IR780-PTX NPs

The hydrodynamic diameter IR780-PTX NPs were determined using Dynamic Light Scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK). To assess morphological characteristics, the diluted nanoparticle solution was added onto a copper grid, dehydrated under ambient conditions, and examined under HT7700 transmission electron microscopy (TEM) (HITACHI, Japan). The IR780 content in the NPs was measured by the UV–vis absorbance at 780 nm. PTX content was quantified by the high-performance liquid chromatography (HPLC). IR780-PTX NPs were diluted with acetonitrile to extract the drug, vortexed for 60 s, and centrifuged (16,000 rpm) for 5 min, then the supernatant was analyzed by HPLC with a HC-C18 column (250 × 4.6 mm, 5 μm, HC-C18, Agilent, USA). To evaluate photostability, the relative content of IR780 in NPs was calculated. In detail, the samples were diluted to 5 μg/mL, irradiated with an 808 nm laser (0.5 W/cm^2^, 5 min) in a quartz cuvette, and the relative absorbance of IR780 was measured and compared to non-irradiated samples. Free IR780 dissolved in N, N-Dimethylformamide/water was used for comparison. PTX-IR780 NPs size stability was evaluated through DLS. The NPs were preserved at ambient conditions and their average size was evaluated at 0, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h respectively.

Photothermal effect

To evaluate photothermal performance, PBS, BSA, PTX NPs, Free IR780, IR780 NPs, and IR780-PTX NPs were diluted to the concentration of IR780 (C_IR780_) at 80 μg/mL. 0.8 mL of each sample was transferred to an EP tube containing a thermal probe and irradiated with 808 nm laser (0.5 W/cm^2^) for 10 min. The temperature rise was recorded every 30 s to obtain heating curves. The photothermal effect of various IR780-PTX NPs concentrations (C_IR780_: 25, 50, 75, 100, 150 μg/mL) was assessed as above. The thermal cycling capability of IR780-PTX NPs was explored. IR780-PTX NPs (C_IR780_: 50 μg/mL) and free IR780 (C_IR780_: 50 μg/mL) as a control were irradiated with a laser (808 nm, 0.5 W/cm^2^) up to 46 °C, followed by cooling to room temperature. A digital thermometer monitored the solution real-time temperature at 30 s intervals until the solution could no longer achieve the maximum temperature of the initial irradiation.

Detection of ROS

Singlet oxygen generated by NPs was assessed using 1, 3-Diphenylisobenzofuran (DPBF) as a chemical probe. Free IR780 (C_IR780_: 3 μg/mL) or IR780-PTX NPs (C_IR780_: 3 μg/mL) were mixed with 20 μL DPBF (50 mM) followed by irradiation with 808 nm Near-Infrared laser (NIR) (0.5 W/cm^2^, 10 min). The absorbance at 425 nm was recorded every 5 s.

Drug release in vitro

Dialysis method was used to examine drug release of IR780-PTX NPs in vitro. In detail, IR780-PTX NPs (C_PTX_: 60 μg/mL) was loaded into a dialysis bag (MWCO: 8000 Da) with triplicate and dialyzed in two different buffers (pH = 7.4 and 5.0, respectively) containing 0.05% Tween 80. All dialysis medium was taken and replenished with fresh one at 0.5, 1, 2, 3, 6, 9, 12, 24, and 48 h, while PTX were quantified with HPLC using the protocol mentioned above.

Cellular uptake and drug release

LN229 cells (5 × 10^5^) were seeded into confocal dishes and incubated with 2 mL free IR780 or IR780-PTX NPs (C_IR780_: 5 μg/mL) for 3 or 6 h under dark conditions. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde and counterstained with DAPI for 15 min. The photographs were undertaken by confocal laser scanning microscopy (CLSM) (LSM 710, Carl Zeiss, Germany). BSA was used as a competitive inhibitor to study nanoparticle uptake mechanisms. The BSA was incubated with cells for 4 h before the addition of NPs. The IR780-PTX NPs, IR780-PTX NPs + BSA (C_BSA_: 0.3 mg/mL), and IR780-PTX NPs + BSA (C_BSA_: 3 mg/mL) with triplicates were assessed as above. Moreover, the LN229 cells were incubated with PTX-IR780 NPs for 2 h followed by DAPI and LysoTracker Green staining and were imaged with CLSM. Furthermore, to investigate nanoparticle intracellular distribution after NIR treatment, the LN229 cells (5 × 10^5^ cells) were incubated with 2 mL IR780-PTX NPs (C_IR780_: 5 μg/mL) in dark for 6 h, washed with fresh PBS twice, stained with acridine orange hydrochloride (AO) (20 mg/mL, 15 min), and were irradiated with 808 nm NIR (0.5 W/cm^2^, 5 min) prior to CLSM observation.

Cellular cytotoxicity and apoptosis assay

For IR780/PTX ratio screening, LN229 cells were seeded into a 96-well plate (1 × 10^4^ cells/well) for 12 h and were treated with PTX and IR780 at different ratios. For the cytotoxicity analysis of IR780-PTX NPs and enhancing role of ACF, cells were treated with different concentrations of ACF, PTX NPs, IR780 NPs (L), ACF + IR780-PTX NPs, and ACF + IR780-PTX NPs (L). In cell experiments, the "L" group was defined as laser irradiation treatment (808 nm NIR, 0.5 W/cm^2^, 5 min). The anoxic chamber was employed to simulate the tumor's hypoxic microenvironment in the anoxic group. The cell viability was finally assessed with CCK-8 kits according to the manufacturer’s instructions. For cellular apoptosis assay, LN229 cells were seeded in 6-well plates (5 × 10^5^ cells/well) and treated with PBS, PTX NPs, IR780 NPs (L), IR780-PTX NPs, or IR780-PTX NPs (L) respectively with C_PTX_: 4 μg/mL and C_IR780_: 2 μg/mL. After 12 h, the cells were washed with fresh PBS twice and irradiated. Finally, the cells were washed with PBS, stained with Annexin V-FITC/PI Apoptosis Detection Kit and analyzed by flow cytometry (BD FACSAria III, Becton, Dickinson and Company, USA).

Cellular photothermal effect

LN229 cells were treated with PBS, Free IR780, and IR780-PTX NPs with C_IR780_: 4 μg/mL. After 12 h, the cells were washed with fresh PBS twice and were irradiated with an 808 nm laser (0.5 W/cm^2^). The cell temperature was recorded at a 30 s interval for 5 min.

Intracellular ROS generation

ROS generation in LN229 cells were meticulously detected utilizing a ROS probe DCFH-DA. The cells were treated with PBS, Free IR780 (L), PTX NPs, IR780-PTX NPs, IR780-PTX NPs (L), and IR780-PTX NPs (L) at C_IR780_: 3 μg/mL. After 6 h, the cells were washed, labeled (10 μM DCFH-DA, 30 min), and imaged by CLSM with excitation wavelength at 345 and 488 nm, respectively.

Cellular hypoxia and HIF-1α expression

LN229 cells (5 × 10^5^ cells) were added into the bottom of the confocal dish and incubated with IR780-PTX NPs (C_IR780_: 3 μg/mL) followed by normoxia or hypoxia for 12 h. The cells were washed with PBS, stained with DAPI for 15 min, and stained with Image-iT™ Hypoxia Reagents (10 μM, 30 min) with or without irradiation (808 nm NIR, 0.5 W/cm^2^, 5 min). Eventually, the photographs were undertaken with CLSM at excitation wavelength 345 nm or 488 nm. To detect the expression of HIF-1α, LN229 cells were cultured in 6-well plates at 3 × 10^5^ cells per well for 12 h and treated with PBS, IR780 NPs, and IR780-PTX NPs at a final concentration of 3 μg/mL of IR780 before incubating in an anoxic chamber which mimics the hypoxic condition. Another 12 h later, the medium was removed, while the cells were irradiated with 808 nm laser (0.5 W/cm^2^) for 5 min. The cells were further incubated in the anoxic chamber for 6 h. The total proteins were extracted and HIF-1α was analyzed by western blot.

For western blot, total protein was extracted either from cell lysates or homogenized tumor tissues. The total protein concentration of supernatants from centrifuged lysates was determined by BCA Protein Assay Kit and equilibrated by lysis buffer. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and blocked in TBST (5% skim milk) for 1.5 h. Afterwards, the membrane was incubated with the primary antibody at 4 °C overnight and then incubated with HRP-conjugated secondary antibody at room temperature for 2 h. The protein bands were detected using the chemiluminescence imaging system (CHEMIDOC, Bio-Rad, USA).

Cell migration and invasion

To analyze the lateral migration, LN229 cells were seeded into 24-well plates and cultured until the full confluency. Then a straight line was drawn in the middle of well using a tip and photographic documentation was recorded using a microscope. The cells were treated with PBS, ACF, IR780 NPs (L), IR780-PTX NPs (L), IR780-PTX NPs + ACF, and IR780-PTX NPs + ACF (L) respectively (PTX: 6 μg/mL, IR780: 3 μg/mL, ACF: 6 μg/mL). The drug-containing medium was replaced with fresh one at 12 h. The photographs were taken to record the width and area of the scratches and calculate the migration rate.

The matrigel (10 mg/mL) was diluted to 200 μg/mL using DMEM at 4 °C and added to Transwell chambers (8 μm polyethylene terephthalate membrane, 100 μL), incubated at 37 °C for 1 h to solidify for the invasion assay. LN229 cells were cultured in 6-well plates (3 × 10^5^ cells) and treated with PBS, ACF, IR780 NPs (L), IR780-PTX NPs (L), IR780-PTX NPs + ACF, and IR780-PTX NPs + ACF (L) respectively (PTX: 6 μg/mL, IR780: 3 μg/mL, ACF: 6 μg/mL). The drug-containing medium was replaced with fresh one at 12 h. After different treatments, the cells were collected and resuspended in serum-free medium (5 × 10^5^ cells/mL). Then, 150 μL of the cell suspension was added onto upper chamber, with 800 μL 15% FBS medium in the lower chamber as a chemoattractant. After 24 h, the cells in the lower chamber were fixed (4% paraformaldehyde, 20 min), stained with crystal violet (10 min), and photographed using a microscope.

HIF-1α suppression

LN229 cells were cultured in 6-well plates (3 × 10^5^ cells/well) and treated with PBS, ACF, IR780 NPs (L), IR780-PTX NPs (L), IR780-PTX NPs + ACF, IR780-PTX NPs + ACF (L) (PTX: 6 μg/mL, IR780: 3 μg/mL, ACF: 6 μg/mL). After irradiation, the anoxic treatment was continued for 12 h. The proteins were extracted, and the protein expression levels of HIF-1α, VEGF, GLUT-1, MMP9, and HSP70 were analyzed by western blot.

In vivo biodistribution

The animal experiments were performed under approval from the Animal Ethics Committee of Soochow University (No. 202303A0918). A subcutaneous model of glioma was established by injecting 100 μL LN229 cell suspension (1 × 10^8^ cells/mL) into the right hind limb of nude mice (3–5 weeks, female). The volume of the tumor (length × width^2^/2) was measured using caliper gauges.

Mice were randomly grouped and administered free IR780 or IR780-PTX NPs via intravenous injection (IR780: 4 mg/kg). In vivo fluorescence imaging was performed using an IVIS Lumina III system (Ex 760 nm/Em 800 nm) at different time points after injection. Afterwards, major organs and tumors were collected for ex vivo imaging.

Preparation and characterization of ACF-loaded alginate hydrogel

Sodium alginate hydrogels at varying concentrations (1, 5, 10, 15, 20, 30 mg/mL) were prepared by dissolving different quantities of sodium alginate in an ACF aqueous solution. The injectability of these ACF hydrogels was assessed by extruding them through a syringe into physiological saline containing 1.8 mM CaCl₂. The gelation capability of hydrogels at different concentrations was evaluated by visual inspection after standing. 150 μL of hydrogel was immersed in 3 mL physiological saline containing 1.8 μM CaCl₂ and incubated at 37 °C with shaking at 200 rpm. At selected time points, 0.5 mL aliquots of the supernatant were collected and replenished with equal volume of fresh buffer. The concentration of ACF in the samples was determined by measuring the absorbance at 422 nm, and the cumulative release was calculated accordingly.

Antitumor efficacy in vivo

Once the tumor volume had reached approximately 150 mm^3^, the mice were allocated to seven groups (n = 5). Each group was treated with PBS, ACF hydrogel, PTX NPs, IR780 NPs (L), IR780-PTX NPs (L), IR780-PTX NPs + ACF hydrogel, and IR780-PTX NPs + ACF hydrogel (L) (PTX: 8 mg/kg; IR780: 4 mg/kg). The ACF hydrogel (300 μg/mL, 50 μL) was intratumourally formed via injection of sodium alginate solution containing ACF, while the NPs were injected intravenously on day 0 and 3, respectively. In animal experiments, the “L” means that the groups requiring light exposure were irradiated with laser light (808 nm at 0.5 W/cm^2^, 5 min) 24 h after the intravenous injection. The tumor size and body weight were monitored daily over a 28-day treatment period. The mice were euthanized, and major organs together with tumor tissues were collected for histological evaluation, including Hematoxylin and Eosin staining (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Additionally, blood samples were collected for biochemical analysis of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea (UREA) and creatinine (CREA) levels using an automated clinical analyzer (Architect C8000, Abbott, USA).

Intratumoral ROS generation and photothermal effect in vivo

The mice were divided into four groups (n = 3) and were treated with PBS, IR780 NPs (L), IR780-PTX NPs, and IR780-PTX NPs (L) (IR780: 4 mg/kg). After 24 h, 50 μL DCFA-DA was injected (50 μg/mL) into tumors, which were then subjected to laser irradiation (808 nm, 0.5 W/cm^2^) for 5 min except the PBS group. Afterwards, the mice were sacrificed and the tumors were collected and fixed in 4% paraformaldehyde for 2 h, dehydrated in 30% sucrose solution, and subsequently frozen and embedded for sectioning. The slices were washed, stained with Hoechst 33342, and observed under CLSM. In addition, two groups of tumor-bearing nude mice (n = 3) were treated with either PBS or IR780-PTX NPs + ACF hydrogels as above. Twenty-four hours after administration, the mice were anesthetized and irradiated with 808 nm laser (0.5 W/cm^2^) for 5 min. The tumor temperature was recorded using a thermal imager at 0, 1, 2, 3, 4, and 5 min, respectively.

HSP70 and HIF-1α pathway suppression in vivo

The mice were randomly divided into seven groups (*n *= 3) and treated with the same formulations as the antitumor efficacy study in vivo. Twenty-four hours post the NIR irradiation, the mice were euthanized and the tumor tissues were collected and homogenized to extract total proteins. The protein expression levels of HIF-1α, GLUT-1, MMP9, VEGF, and HSP70 were examined by western blot.

Results

Preparation and characterization of IR780-PTX NPs

The optimized ratio between IR780 and PTX for the co-delivery by albumin-based NPs was firstly examined. The Combination Index (CI) is a tool used to study the synergistic effects of drug combinations, quantitatively describing synergism (CI < 1), additive effects (CI = 1), and antagonism (CI > 1) (Yadav et al. 2015). The CI gradually decreased as the proportion of PTX increased, reaching 0.39 when the ratio of IR780 to PTX was 1:2 (Fig. 2a). At an IR780:PTX:ACF ratio of 1:2:2, the CI value was approximately 0.7, whereas increasing the ACF proportion to 1:2:4 further reduced the CI to ~ 0.5, indicating stronger synergistic efficacy. Although the higher ACF ratio produced a more pronounced synergistic effect, we deliberately selected the lower ratio of 1:2:2 for subsequent experiments to limit the intrinsic cytotoxicity of ACF and to minimize potential interference with downstream mechanistic analyses. Therefore, this ratio was chosen for IR780-PTX NPs preparation using the method in our previous work (He et al. 2024; Xu et al. 2025). DLS revealed average particle sizes of 158 ± 0.7 nm, 167 ± 1.9 nm, and 168 ± 2.3 nm for IR780 NPs, PTX NPs, and IR780-PTX NPs respectively, with the polydispersity indices (PDI) around 0.1, indicating uniform size distribution (Fig. 2b). IR780-PTX NPs displayed a spherical shape in the TEM image (Fig. 2c) with an average particle size of approximate 120 nm (Figure S1), which was smaller than the hydrated particle detected by DLS. The encapsulation efficiency and loading capacity of IR780 in IR780-PTX NPs were 89.8 ± 2.5% and 2.7 ± 0.1%, while those for PTX were 84.4 ± 2.9% and 5.0 ± 0.1%, indicating high encapsulation rate and drug loading capability. These two parameters for IR780-PTX NPs are close to that in our previous study utilizing albumin for multiple drug delivery (He et al. 2024), demonstrating the consistency and robustness of self-assembly technique in fabricating albumin-based nanomedicines. In addition, the particle size of IR780-PTX NPs remained stable over 72 h at room temperature (Fig. 2d), demonstrating an excellent colloidal stability. Finally, a dialysis method was used to investigate the PTX release from IR780-PTX NPs. At two pH conditions (7.4 and 5.0) which mimicked the physiological and lysosomal environments, PTX was gradually released in a similar speed in the first 12 h, which turned much slower afterwards (Fig. 2e). The overall release in 48 h was slightly higher at lower pH (92% vs 79%), which would be desirable for intracellular drug release considering the acidic condition in the lysosome.Fig. 2. Characterizations of IR780-PTX NPs. a CI of IR780 and PTX at different ratios. Data were represented as means ± standard deviation (SD) (n = 3). One-way analysis of variance (ANOVA) was used to compare the means of multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance. b Size distribution of PTX NPs, IR780 NPs and IR780-PTX NPs. c TEM image of IR780-PTX NPs (Scale bar: 200 nm). d Colloidal stability of IR780-PTX NPs. Data were represented as means ± SD (n = 3). e In vitro release of PTX from IR780-PTX NPs at pH 7.4 or pH 5.0. Data were represented as means ± SD (n = 3). f Temperature changes of PBS, BSA, PTX NPs, Free IR780 and IR780-PTX NPs under NIR irradiation (808 nm laser, 0.5 W/cm^2^, 5 min). g UV–vis spectral changes of free IR780 and IR780-PTX NPs before and after NIR irradiation (808 nm laser, 0.5 W/cm^2^, 1 min). h Photothermal cycle curves of free IR780 and IR780-PTX NPs. i ROS generation of PBS, Free IR780, PTX NPs, IR780 NPs, and IR780-PTX NPs. Data were represented as means ± SD (n = 3)

Photothermal effect and ROS generation of PTX-IR780 NPs

The photothermal effect was assessed by measuring temperature changes of NPs under NIR irradiation. PBS, BSA, and PTX NPs showed negligible temperature elevation, while free IR780 caused an approximately 18 °C temperature rise within 5 min and then decreased sharply (Fig. 2f), which may be attributed to dye photobleaching (Wang et al. 2023b). In contrast, IR780-PTX NPs enabled continuous heat generation, which increased the solution temperature to approximate 25 °C upon NIR irradiation without any drop within at least 10 min. Moreover, the heat production of IR780-PTX NPs was positively correlated with the concentration of IR780, reaching a plateau beyond 75 μg/mL (Figure S2). The IR780-PTX NPs showed a noticeable red shift from 770 nm to approximately 790 nm (Fig. 2g), likely due to the molecular aggregation of IR780 within the nanoparticles, leading to enhanced excitation efficiency at 808 nm (Zhang et al. 2019). Notably, under the same NIR exposure conditions, IR780-PTX NPs showed markedly higher absorbance than free IR780 (Fig. 2g and Figure S3), indicating a higher photostability which could reasonably explain the better hyperthermia generation (Fig. 2f). To further investigate the photothermal performance, IR780-PTX NPs were subjected to cyclic NIR irradiation. Compared with free IR780 which withstood only one heating–cooling cycle, IR780-PTX NPs experienced at least 4 heating–cooling cycles without any drop of peak temperature (Fig. 2h). These data suggested that the photostability of IR780 was significantly enhanced when encapsulated into BSA NPs.

Singlet oxygen generation of IR780-PTX NPs was detected and quantified by measuring DPBF absorbance. Either PBS or PTX NPs showed no significant change in DPBF absorbance under NIR irradiation. Free IR780 quickly decreased the fluorescence, while IR780 NPs further reduced DPBF fluorescence, indicating that IR780 NPs could generate a significant amount of ROS more than free drugs (Fig. 2i). The improved photostability of IR780 may be attributed to the stabilized microenvironment provided by BSA NPs (Fig. 2g), similar to our previous study (Wang et al. 2023). Importantly, ROS generation by IR780-PTX NPs was nearly identical to that of IR780 NPs, implying that PTX loading did not influence IR780 ROS production ability. This was in agreement with the phase-change material-based NPs in our previous study (Wang et al. 2023b), and suggested a high flexibility of co-encapsulating other drugs in the BSA NPs to potentiate the phototherapy.

Cellular uptake of IR780-PTX NPs

The cellular uptake of IR780-PTX NPs was investigated in LN229 cells under CLSM. After 3 h of incubation, IR780-PTX NPs were evident in the cytoplasm, showing a faster uptake rate than free IR780 (Fig. 3a). The cellular uptake of NPs was positively dependent on the incubation time. The mean fluorescence intensity of IR780-PTX NPs was constantly superior than that of free IR780, though the gap was narrowed down until 6 h (Fig. 3b). This phenomenon was similar to our previous albumin-based nanomedicines (Wang et al. 2023), which might be owing to the energy consumption for the uptake of albumin NPs, making it slow down compared to the free diffusion of single IR780 molecules. It was reported that some highly expressed proteins on tumor cells such as SPARC are known to bind albumin with high affinity and facilitate the uptake of albumin-based NPs (He et al. 2024). To validate this, free albumin was used as a competitive inhibitor prior to IR780-PTX NPs treatment. As expected, the pretreatment with free albumin markedly reduced the internalization of IR780-PTX NPs in a concentration-dependent manner (Fig. 3c). This finding suggested that both IR780-PTX NPs and free albumin were taken up by LN229 cells via the same pathway. Upon the cellular uptake, IR780-PTX NPs were distributed in the lysosome, as shown by the co-localization of IR780 (red fluorescence) with lysosome (green fluorescence) (Fig. 3d). Lysosome integrity was evaluated using AO staining. The red fluorescence diminished after NIR irradiation indicating the disruption of lysosomes (Fig. 3e). This verified ROS-enhanced lysosomal escape which was beneficial for PTX release and its chemotherapy. The photothermal effect of internalized NPs was assessed by measuring the temperature changes in cell suspensions upon NIR irradiation. Compared with PBS showing negligible temperature variation, IR780-PTX NPs exhibited a rapid temperature increase of approximately 15 °C within 4 min while free IR780 induced a substantially lower temperature elevation of about 10 °C (Fig. 3f), confirming the superior photothermal conversion efficiency of IR780-PTX NPs as previously shown in the solution (Fig. 2g). Next, we examined their ROS generation in tumor cells via a fluorescence reporter DCFH-DA (Fig. 3g). Cells treated with PBS, PTX NPs, and IR780-PTX NPs without NIR irradiation showed minimal green fluorescence induced by ROS caused by their own oxidative metabolism. In contrast, cells treated with free IR780 and IR780-PTX NPs generated a large amount of ROS upon NIR irradiation. As expected, the fluorescence of IR780-PTX NPs-treated cells was stronger than that of free IR780, indicating a higher ROS productivity. Taken together, these results indicate stronger photothermal and photodynamic effects of IR780-PTX NPs in tumor cells, possibly due to increased cellular uptake of nanoparticles and enhanced photostability of IR780 demonstrated above.Fig. 3. Cellular uptake and PTT/PDT effects of IR780-PTX NPs. a Cellular uptake of free IR780 and IR780-PTX NPs at 3 h and 6 h of incubation in LN229 cells (Scale bar: 50 μm). b Statistical analysis of IR780 cellular uptake in (a). Data were represented as means ± SEM (n = 3). Student’s t-test was used for comparison between two groups. * p < 0.05, ** p < 0.01. c Cellular uptake of IR780-PTX NPs under BSA pretreatment (Scale bar: 50 μm). d Subcellular localization of PTX-IR780 NPs in LN229 cells (Scale bar: 20 μm). e AO staining of lysosomes in LN229 cells with or without NIR irradiation (Scale bar: 50 μm). f Temperature changes in cells after different treatments under NIR irradiation. Data were represented as means ± SD (n = 3). g ROS generation in LN229 cells after different treatments with or without NIR irradiation (scale bar: 100 μm)

In the following, a water-soluble hypoxia probe which emits green fluorescence under hypoxic conditions was used to detect the hypoxia level in tumor cells upon PDT. LN229 cells treated with IR780-PTX NPs and laser irradiation (L) showed significantly enhanced green fluorescence compared to non-irradiated NPs (Fig. 4a), indicating a higher hypoxia level in the cells. This caused an obvious HIF-1α overexpression at the same condition (Fig. 4b and c). The HIF-1α expression in IR780-PTX NPs (L) was significantly increased by 56% (p < 0.05) when compared with IR780-PTX NPs without irradiation, though the HIF-1α expression in IR780-PTX NPs was comparable to PBS group (Fig. 4c). Collectively, these data indicated a boosted tumor hypoxia and the consequential HIF-1α upregulation in tumor cells after IR780-PTX NPs treatment and NIR irradiation. Considering the negative role of HIF-1α in tumor phototherapy and chemotherapy, this highly necessitates the discovery of an efficient way to suppress HIF-1α overexpression and enhance the therapeutic outcomes.Fig. 4. In vitro antitumor effects of IR780-PTX NPs and ACF. a PDT-induced hypoxia in LN229 cells. b HIF-1α expression in LN229 cells after different treatments. c Statistical analysis of HIF-1α expression in (b). Data were represented as means ± SD (n = 3). Student’s t-test was used for comparison between two groups. * p < 0.05, ns: no significance. d Cytotoxicity of LN229 cells under different treatments at hypoxic condition. e IC_50_ of PTX and IR780 for various formulations at hypoxic condition. One-way ANOVA was used to compare the means of multiple groups. * p < 0.05, *** p < 0.001. f Caspase-3 and cleaved Caspase-3 protein expression levels under hypoxia upon different treatments. g Relative expression levels of Caspase-3 and cleaved Caspase-3. Data were represented as means ± SD (n = 3). One-way ANOVA was used to compare the means of multiple groups. * p < 0.05, ** p < 0.01, ns: no significance

ACF enhances therapeutic efficacy of IR780-PTX NPs via HIF-1 and HSP70 suppression

In order to fully potentiate the antitumor effect of IR780-PTX NPs, ACF was added and its impact on the cytotoxicity was evaluated. At both hypoxic and normoxic conditions, IR780 NPs and PTX NPs displayed concentration-dependent cytotoxicity (Fig. 4d and S4a). Their combination (IR780-PTX NPs, 1:2 ratio between the two drugs) under NIR irradiation revealed largely improved cytotoxicity and decreased IC_50_ of IR780 compared to single drugs (Fig. 4e and S4a), suggesting the synergy between phototherapy and chemotherapy. Cellular apoptosis under hypoxia was detected to verify this through Annexin V/PI staining followed by flow cytometry analysis (Figure S5). PTX-IR780 NPs (L) treatment increased cell apoptosis to 79.4%, much higher than individual PTX NPs or IR780 NPs (L), reconfirming a synergistic effect by combining PTX and IR780. Free PTX and IR780 drugs used at the same ratio also exhibited a good synergistic therapeutic effect (70.2%), but its effect was inferior compared to PTX-IR780 NPs. Additionally, it’s worthy to note that cell viability under normoxic conditions was lower than hypoxic conditions at the same drug concentration. Quantitatively, the IC_50_ of IR780 NPs (L), PTX NPs, IR780-PTX NPs (L) was 1.33 μg/mL (for IR780), 1.56 μg/mL (for PTX), and 0.08 μg/mL (for IR780) respectively at normoxic conditions, which was boosted to 2.18 μg/mL (for IR780), 3.69 μg/mL (for PTX), 0.48 μg/mL (for IR780) at hypoxic conditions (p < 0.05) (Figure S4b), proving that hypoxia reduces the effectiveness of phototherapy and chemotherapy. To this end, ACF which showed limited cytotoxicity alone in consistence with the literature (Guo et al. 2025) was added into the treatment. The cytotoxicity was further enhanced, as endorsed by the IC_50_ (Fig. 4e). The IC_50_ of PTX and IR780 for IR780-PTX NPs at hypoxic condition was further decreased to 0.63 and 0.32 μg/mL after the addition of ACF (Fig. 4e). These data demonstrated an intriguing enhancement by ACF for the photochemotherapy.

Next, Caspase-3 assessment was conducted to further characterize the apoptosis of LN229 cells under hypoxia receiving various treatments (Fig. 4f and g). ACF caused no clear cleavage of Caspase-3, staying in agreement with the cytotoxicity study and suggesting negligible induction of cellular apoptosis. IR780 NPs led to an obvious apoptosis as shown by cleaved Caspase-3, which was further enhanced by IR780-PTX NPs due to combined phototherapy and chemotherapy. A significant elevation of cleaved Caspase-3 was observed in the IR780-PTX NPs + ACF group following NIR irradiation, whereas the formulation without irradiation showed limited Caspase-3 activation. This was reasonable since IR780 remains inactive in the absence of NIR. These results indicated that the combined treatment by IR780-PTX NPs and ACF under NIR showed the strongest apoptosis-inducing capability.

In the following, the impact of ACF on HIF-1 pathway suppression was investigated. HIF-1α expression was significantly improved in LN229 cells after treatment with IR780 NPs (L) and IR780-PTX NPs (L), probably caused by the hypoxia exacerbation by oxygen consumption in PDT (Fig. 5a and b). The overexpression of HIF-1α led to a significant upregulation of its downstream proteins such as GLUT-1, MMP9, and VEGF. Interestingly, ACF showed almost no impact on HIF-1α expression, either applied alone or combined with IR780-PTX NPs. This is probably due to that instead of tuning HIF-1 expression, ACF binds to HIF-1α and restrains its dimerization with HIF-1β, thus suppressing the transcriptional activity of HIF-1 (Guo et al. 2025; Tedeschi et al. 2025). Furthermore, we found that ACF-mediated HIF inhibition significantly reduced the expression of GLUT-1, MMP9, and VEGF upregulated by IR780-PTX NPs, even regardless of NIR irradiation (Fig. 5a and b). Therefore, combining ACF with IR780-PTX NPs holds great promise for enhancing photochemotherapy in glioma via restricting HIF-1 associated downstream proteins.Fig. 5. In vitro suppression of HIF-1 pathway and HSP70 by ACF. a Western blot analysis and (b) statistical analysis of HIF-1α, GLUT-1, MMP9, VEGF, and HSP70 expressions in LN229 cells receiving various treatments. c Cell migration assay (Scale bar: 50 μm). d Statistical analysis of cell migration assay. Data were represented as means ± SD (n = 3). e Invasion assay of LN229 cells after different treatments (Scale bar: 20 μm). f Statistical analysis of cell invasion assay. Data were represented as means ± SD (n = 3). One-way ANOVA was used to compare the means of multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: no significance

To further confirm the role of IR780-PTX NPs and ACF in glioma therapy and HIF-1 pathway modulation, especially invasion and metastasis, we then evaluated the lateral and longitudinal migration capacity of LN229 cells subjected to various treatments under hypoxic conditions. Cells treated by PBS displayed ~ 50% gap healing due to lateral cell migration, which was suppressed to about 20%−30% by IR780 NPs or IR780-PTX NPs under NIR irradiation. IR780-PTX NPs + ACF (L) exhibited the highest migration inhibition ratio (~ 100%) (Fig. 5c and d). Consistent with the lateral migration, the longitudinal migration assay showed that LN229 cells in the PBS group easily passed through the basement membrane barrier of the Transwell, while relatively only about 35% cells were seen at the bottom layer upon ACF treatment (Figure S6). When combined with IR780-PTX NPs, the longitudinal migration was further suppressed, with only a few penetrating cells observed on the bottom layer. The same trend was observed in the invasion assay, where a layer of Matrigel was coated on the barrier membrane of the Transwell to simulate the tumor extracellular matrix and basement membrane, while tumor cells need to secrete matrix enzymes such as MMPs to digest the basement membrane for metastasis. IR780-PTX NPs + ACF (L) resulted in the least number of cells invading and penetrating through the matrigel for metastasis (Fig. 5e and f). Taken together, these data indicated that IR780-PTX NPs + ACF (L) treatment had the best suppression on GBM migration and metastasis.

The effect of enhancing PTT by ACF was also investigated. It has been well established that tumor cells exposed to hyperthermia could activate HSPs upregulation to develop thermotolerance and reduce the therapeutic efficiency of PTT (Liu et al. 2023; Wang et al. 2023b; Wu et al. 2024). Thus, HSP70 protein expression levels were evaluated in LN229 cells under various treatments. As expected, PTT via IR780 NPs or IR780-PTX NPs and NIR irradiation led to increased HSP70 expression compared to the PBS control. ACF alone markedly downregulated HSP70 by about 80% compared with the control, confirming its strong suppressive function on HSP70 (Fig. 5a and b). More importantly, the combination of ACF with IR780-PTX NPs could significantly decrease such expression even to an extent similar to ACF treatment alone. To the best of our knowledge, this is the first report of ACF as a powerful HSP70 expression inhibitor, providing a novel strategy to enhance tumor phototherapy via tackling the HSP-mediated thermoresistance.

IR780-PTX NPs biodistribution in vivo

Motivated by the encouraging in vitro performance, we subsequently investigated the antitumor effects of IR780-PTX NPs in combination with ACF in vivo. The biodistribution was firstly investigated by NIR imaging in LN229 tumor-bearing mice. After intravenous injection, the fluorescence of IR780-PTX NPs was detectable in tumor localization as early as 2 h and progressively increased until reaching a maximum at approximately 24 h, followed by a slight decline at 48 h (Fig. 6a), suggesting progressive clearance from the tumor tissue. In contrast, there was an obviously lower tumor accumulation of free IR780 within the same duration, although more free dyes were found in tumor regions compared to other parts of the body, as reported in our previous studies (Wang et al. 2023b; He et al. 2024). Ex vivo fluorescence imaging further confirmed the preferential accumulation of IR780-PTX NPs in tumor tissues relative to other organs (heart, liver, spleen, and kidney) (Fig. 6b), demonstrating desirable targeting ability of IR780-PTX NPs to the tumor site. It was worthy to note that IR780-PTX NPs also displayed a considerable distribution in the lung, probably due to the arresting of larger NPs by the capillary vessels over there. Although intratumoral retention and clearance was not quantified using standard pharmacokinetic protocols, these results still provide meaningful insights into the biodistribution, accumulation, and retention of the nanoparticles, which are sufficient to support the interpretation of the observed therapeutic efficacy in our study. Collectively, these findings demonstrated effective tumor-targeting capability of IR780-PTX NPs, which is critical for therapeutic applications.Fig. 6. In vivo antitumor efficacy of IR780-PTX NPs and ACF hydrogels combination. a Dynamic in vivo fluorescence imaging of nanoparticle biodistribution in mice after intravenous injection of free IR780 or IR780-PTX NPs. b Ex vivo NIR fluorescence imaging of excised tumors and major organs from tumor-bearing mice treated with free IR780 or IR780-PTX NPs. c Experimental scheme for in vivo antitumor study. d Tumor growth in mice under different treatment conditions. e Image and (f) weights of excised tumors from mice after different treatments. g H&E staining and TUNEL staining of tumor sections from mice after different treatments (scale bars: 200 μm). h Body weight changes in mice during the experiment period. Data were represented as means ± SD (n = 5). For statistical analysis, One-way ANOVA was used to compare the means of multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001

Antitumor efficacy and safety evaluation in vivo

The antitumor effect of IR780-PTX NPs combined with ACF was then examined using a subcutaneous model of GBM via 2 arounds of formulation injection followed by NIR irradiation 24 h post the injection (Fig. 6c). ACF was incorporated into alginate hydrogel by intratumoral injection of 30 mg/mL alginate on day 0, which would automatically form the hydrogel with intratumor Ca^2+^ of physiological concentration (1.8 mM) (Figure S7a) (Chao et al. 2018). In vitro release studies demonstrated that alginate hydrogels revealed a sustained drug release of ACF for at least 72 h (Figure S7b). In addition, using hydrophilic indocyanine green (ICG) as a fluorescence probe, upon the intratumoral injection and formation of these hydrogels, the fluorescence started to spread in the tumor owing to drug release, which eventually covered the whole tumor region at 48 h post the injection (Figure S8a). After the dissection, there was only fluorescence in the tumor compared to other major organs, suggesting an outstanding intratumoral retaining of drugs by alginate hydrogel (Figure S8b). Collectively, these data suggested that alginate hydrogel would allow effective ACF encapsulation and release for continuous modulation of HIF-1 pathways and HSP70 expression in vivo.

In terms of antitumor effect, PTX NPs or ACF hydrogel led to considerable but insufficient tumor growth suppression, as shown by the fast increase of tumor volume starting from day 10 (Fig. 6d). The phototherapy by IR780 NPs under NIR irradiation led to stronger tumor suppression, while its combination with chemotherapy (IR780-PTX NPs) further enhanced the therapeutic outcome at the same condition. But the tumor suppression ratio was only around 80%, necessitating the combination with other methods to synergistically suppress GBM. Strikingly, the combination of IR780-PTX NPs with ACF hydrogel greatly enhanced the tumor suppression capability to around 100% from day 6 on. Consistent with the tumor volume changes, the weights of the excised tumors also demonstrated a remarkable therapeutic advantage for the IR780-PTX NPs + ACF (L) group, in which complete tumor elimination occurred in 3 of 5 mice (Fig. 6e and f). Furthermore, H&E staining revealed that IR780-PTX NPs + ACF (L) induced the most extensive cellular destruction characterized by pronounced nuclear shrinkage (Fig. 6g). Likewise, TUNEL analysis showed the highest proportion of apoptotic cells in IR780-PTX NPs + ACF (L) group as evidenced by intense brown/yellow staining (Fig. 6g). These results validated the superior antitumor efficacy of the combination of IR780-PTX NPs and ACF hydrogel under NIR irradiation. Next, the preliminary in vivo biosafety of IR780-PTX NPs and ACF hydrogel was evaluated. Compared with the PBS group, all NPs groups maintained steady body weights during the experiment period (Fig. 6h). Histological examination of major organs (heart, liver, spleen, lung, and kidney) showed no obvious lesions or structural abnormalities (Figure S9). In addition, the hemolysis experiments indicated that IR780-PTX NPs did not cause noticeable hemolysis (< 5%) at a concentration up to 1 mg/mL (Figure S10). Moreover, the blood biochemistry analysis was conducted to evaluate liver and kidney functions in mice, whereas no significant differences were found in the levels of AST, ALT, CREA, and UREA between various treatments including PBS, ACF, IR780-PTX NPs (L), and IR780-PTX NPs + ACF (L) (Figure S11). Taken together, these results demonstrated that the combination of IR780-PTX NPs and ACF hydrogel exhibited favorable biosafety characteristics with no detectable systemic toxicity or adverse effects.

Intratumoral ROS generation and photothermal effect in vivo

The in vivo photothermal behavior of the combined therapy was assessed by thermal imaging during NIR irradiation (Fig. 7a and b). As expected, tumors treated with PBS exhibited negligible temperature increase (less than 5 °C increase in 5 min), which was insufficient for effective photothermal treatment (Wang et al. 2023b). In contrast, IR780-PTX NPs + ACF hydrogels produced an obvious heating of tumors by 17 °C within 5 min of NIR irradiation, which could be maintained for at least 4 min. This suggested that IR780 encapsulation by albumin NPs induced an effective hyperthemia for PTT in vivo, which was not affected by the co-delivered PTX and ACF hydrogels. As depicted by the in vitro study, PTT enabled by IR780 NPs and IR780-PTX NPs under NIR irradiation caused clearly upregulated HSP70 expression (Fig. 7c and d), serving as an important mechanism of thermoresistance. But such upregulation by IR780-PTX NPs could be reversed by the combination of ACF hydrogels with IR780-PTX NPs, which revealed an HSP70 expression even lower than PBS control, either with or without NIR irradiation (Fig. 7c and d). Collectively, these data confirmed the important role of ACF as a powerful HSP70 expression inhibitor, which potentiates PTT via overcoming the HSP-mediated thermoresistance.Fig. 7. In vivo antitumor effect of IR780-PTX NPs combined with intratumorally formed ACF hydrogels. a Thermal imaging of tumor-bearing mice treated with PBS or IR780-PTX NPs + ACF at indicated time points. b Temperature rise curves of tumors in (a). Data were represented as means ± SD (n = 3). c Western blot analysis and (d) statistical analysis of HSP70 expression in tumors receiving various treatments. e ROS generation indicated by DCFH-DA fluorescence and (f) statistical analysis in the slices of tumors receiving various treatments (Scale bar: 200 μm). Data were represented as means ± SEM (n = 3). g Western blot analysis and (h) statistical analysis of HIF-1, GLUT-1, MMP9, and VEGF protein expression levels in tumors receiving various treatments. One-way ANOVA was used to compare the means of multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significance

Next, we studied the ROS production within tumors after different treatments using DCFH-DA as a fluorescence probe. Both IR780 NPs and IR780-PTX NPs under NIR irradiation produced massive green fluorescence in the tumor compared to the counterparts without irradiation or PBS control (Fig. 7e). Interestingly, IR780-PTX NPs (L) treatment gave rise to a higher mean fluorescence intensity which was about 20% higher than that for IR780 NPs (L) (p < 0.05) (Fig. 7f). This may be due to the cellular damage caused by PTX, which led to disruption of intracellular redox balance (e.g., regulating the activity of superoxide dismutase) and thus the accumulation of ROS (Ren et al. 2018). Additionally, cell nuclei treated with IR780 NPs and NIR irradiation clearly shrank, while the tumor tissue become loose, as shown by the reduced number of cell nuclei. This indicated typical cellular apoptosis and tumor destruction. In one word, these data indicated efficient ROS generation in tumors by IR780-PTX NPs. It is worthy to note that upon light activation, localized heat generation (Fig. 7a) and ROS production (Fig. 7e) were observed across the tumor region, which may disrupt tumor vasculature and transiently modulate the tumor microenvironment, potentially enhancing drug penetration. Such multimodal therapeutic approaches offer a promising way to partially overcome transport limitations in EPR-poor tumor regions and enhance overall antitumor efficacy. The ROS generation in tumors and PTT also contributed to the regulation of HIF-1α expression and its downstream pathways. Similar to the in vitro study, IR780 NPs and IR780-PTX NPs improved the HIF-1α expression in tumors under NIR irradiation compared to the non-irradiated counterparts and PBS control (Fig. 7g), suggesting the hypoxia deterioration after the phototherapy treatment. The overexpressed HIF-1α lead to the upregulation of its downstream proteins including GLUT-1, MMP9, and VEGF (Fig. 7g). Although ACF hydrogel was impotent to regulate HIF-1α expression, it significantly prohibited the expression of GLUT-1, MMP9, and VEGF that were upregulated by IR780-PTX NPs under NIR irradiation (Fig. 7g). The quantification analysis revealed ~ 50% decreases in the expression of these proteins which were even lower than PBS group (Fig. 7h). This would inhibit tumor glucose uptake, angiogenesis and metastasis, serving as an explanation for the best antitumor efficacy of IR780-PTX NPs together with ACF hydrogels. In short, ACF endowed IR780-PTX NPs with the best antitumor efficacy in vivo via simultaneously regulating both HIF-1 pathways and HSP70 expression.

Discussion

Conventional anti-glioma treatment efficacy is severely hindered by the complex TME such as hypoxia interference, ROS clearance, and drug resistance, thus novel strategy is urgent to be developed (Li et al. 2022). Combined photochemotherapy has its unique advantages in terms of spatio-temporal control and relevant improved therapy outcome, but it is still limited due to the activation of HIF-1 and HSPs in tumor cells (Zhang et al. 2024; Cai et al. 2025). In the present study, we developed an albumin-based nanoplatform to co-deliver IR780 and PTX for synergistic photochemotherapy in combination with sustained release of ACF in alginate hydrogel to further reinforce synergistic photochemotherapy. The combinational therapy exhibited superior antitumor effects in vitro and in vivo, offering a potential smart strategy to treat glioma.

Over the past years, phototherapy, including PDT and PTT, has been demonstrated as a promising approach for cancer treatment due to its high efficiency, controllable spatiality, and minimal invasiveness (Zhang et al. 2019; Cai et al. 2025). However, phototherapy is still limited in clinical application due to the poor water solubility and tumor targeting capability of PSs, the insufficient penetration of light, and the complex microenvironment within tumor (Zhang et al. 2024). In addition, traditional chemotherapy also faces severe adverse effects, poor targeting capability, and increased dose-limiting toxicity in tumor treatment (Karschnia et al. 2025). Thus, the combination of phototherapy and chemotherapy into a single system may offer an outstanding choice to treat malignant tissues. One novel drug delivery system based on liposome which was compose of tirapazamine to destroy tumor cell under hypoxia, BMS-202 to inhibit Programmed Death-1 (PD-1)/Programmed Death Ligand-1 (PD-L1) interaction, and IR820 to generate ROS yields promising antitumor effects in breast cancer treatment (Zeng et al. 2024). The HA/H-I NPs containing IR780 for PTT and PDT, ivermectin for chemotherapy, and hydroxychloroquine for autophagy modulation also exhibit excellent tumor inhibition in vivo and in vitro in colorectal cancer therapy (Ding et al. 2024). In our work, PTX and IR780 were encapsulated into the biocompatible albumin-based NPs for simultaneous phototherapy and chemotherapy upon NIR irradiation, providing another example for synergistic photochemotherapy. Moreover, phototherapy can also be combined with other novel therapeutic approaches. The PD-L1 antibody fragment and indocyanine green are encapsulated into lipid-based nanoparticles for synergistic immunotherapy and phototherapy to treat breast cancer (Park et al. 2024). The disulfide-bridged borylbenzyl carbonate is designed to consume glutathione (GSH) and to encapsulate IR780 to form NPs. The NPs could effectively suppress colon cancer (Jung et al. 2023). These studies demonstrate that the combination of phototherapy with other therapeutics could produce synergistic antitumor effects.

Albumin is an ideal drug delivery platform due to its excellent biocompatibility, lower immunogenicity, and minimal cytotoxicity (Iqbal et al. 2021; Liu et al. 2025). Abraxane, containing PTX, is the first albumin based-NPs approved by FDA in 2005 (Murphy et al. 2025). Since then, albumin-based formulations are extensively developed in various therapeutics, especially in targeted tumor treatment. Through the self-assembly technique driven by the hydrophobic interactions, we created one kind of albumin based-NPs to co-deliver IR780 for PTT/PDT, NLG919 for IDO-1 suppression, and tirapazamine for chemotherapy into cancer cells for synergistic therapy, achieving optimistic antitumor effects in vitro and in vivo (Wang et al. 2023). Another albumin based-NPs were also developed by our group to treat triple-negative breast cancer by co-delivery of IR780 for PTT/PDT and diclofenac for cyclooxygenase-2 inhibition to activate tumor immunogenicity by enhancing T cell infiltration and inducing immunogenic cell death (Xu et al. 2025). As in glioma, ibrutinib and hydroxychloroquine were encapsulated into albumin-based NPs for combined chemotherapy, resulting dramatically glioma eradiation (Yang et al. 2023). Moreover, the albumin-based NPs modified with ^D^CDX co-delivering LY2157299 and celastrol showed enhanced glioma targeting therapy efficacy by remodulating the immunosuppression of TME (Zhu et al. 2022b). In the present study, albumin-based NPs co-delivering IR780 and PTX were also excellent in treating glioma, further proving the benefits of albumin as the drug delivery carrier.

Though synergistic antitumor effects are obtained by photochemotherapy, it still faces several challenges including tumor hypoxia exacerbated by PDT, HIF-1 activated by hypoxia, increased GSH by glycolysis intermediates, and upregulated HSPs during PTT (Cao et al. 2022b; Wang et al. 2023b; Cai et al. 2025). The hypoxia activated HIF-1 could promote the expression of its downstream proteins such as VEGF to stimulate angiogenesis (Lim et al. 2022), GLUT-1 to meet the high glucose consumption via glycolysis (Blum et al. 2005), and MMP9 to promote the synthesis and secretion of matrix metalloproteinases for tumor metastasis (Ke et al. 2025). HSPs could rapidly introduce thermoresistance and inhibit PTT induced cellular damage (Jin et al. 2018; Liu et al. 2023; Wang et al. 2023b; Wu et al. 2024). Both HIF-1 activation and HSPs upregulation induced by PDT and PTT will lead to reduced antitumor efficacy. ACF has been identified as a HIF-1 inhibitor by binding to HIF-1α subunit and restrain its dimerization with HIF-1β, thus suppressing HIF-1 transcriptional activity (Guo et al. 2025; Tedeschi et al. 2025). In the present study, ACF was formulated into alginate hydrogels which could continuously release ACF at the tumor site. With the aid of ACF, the synergistic antitumor effect of IR780-PTX NPs was remarkably reinforced by suppressing HIF-1 activity and its downstream proteins. At the same time, we found that ACF could also inhibit HSP70 expression, which was demonstrated as one of the key factors contributing to thermoresistance (Chen et al. 2023). According to our best knowledge, this may be the first research showing that ACF could inhibit HIF-1 activity and HSP70 expression simultaneously, providing a promising prospect for ACF in future clinical use. In addition, ACF is an FDA-approved drug for local clinical use, providing an important safety and translational reference point. From a clinical perspective, glioma resection typically leaves a postoperative cavity which has been widely recognized as a feasible anatomical space for local drug delivery. Numerous preclinical studies (Cao et al. 2022a; Zhu et al. 2022a) and clinical applications have demonstrated that this resection cavity can be exploited for the implantation of therapeutic agents. A well-established clinical example is the implantation of Gliadel (carmustine wafers) which are biodegradable polymer wafers containing the chemotherapeutic drug carmustine into the brain cavity after glioma surgery, enabling sustained local chemotherapy with minimized systemic toxicity. Although our proof-of-concept experiments were conducted using a subcutaneous tumor model with systemic administration of IR780-PTX nanoparticles and local hydrogel delivery of ACF, the underlying therapeutic principles show strong relevance to real-world clinical scenarios. Both NPs and hydrogels are feasible formulations in clinical settings of tumor treatment. For instance, the administration of albumin-based nanoparticles via intravenous injection is a widely used and clinically validated strategy for drug delivery, as exemplified by Abraxane, which employs albumin nanoparticles to improve the solubility and tumor-targeted delivery of PTX. Given the high hydrophilicity and short pharmacokinetic half life of ACF, we employed a local ACF-loaded hydrogel to achieve sustained intratumoral retention and high local drug concentrations while minimizing systemic exposure. To formulate IR780, PTX and ACF into a single injectable formulation, albumin-based NPs would be a desirable option, which could be realized by conjugating ACF on albumin molecules via linking the active amide groups of both entities by NHS/EDC-based chemistry (Wang et al. 2023). Furthermore, stimulus-responsive linkers such as thioketal (sensitive to ROS), azobenzine (vulnerable to hypoxia) and hydrazone bond (responsive to low pH), could be incorporated in the linkers to allow triggered drug release and attenuated side effects of ACF.

On the other hand, our study also had some limitations. First, the therapeutic performance was not evaluated in an orthotopic glioma model, which represents a more clinically relevant setting. Subcutaneous tumor models differ from orthotopic glioblastoma in several key aspects. Their subcutaneous vasculature has higher permeability and a stronger EPR effect, potentially overestimating nanocarrier delivery. Additionally, they lack the brain-specific microenvironment and invasive growth patterns seen in real glioma (Patrizii et al. 2018). Therefore, while subcutaneous models are useful for proof-of-concept evaluation, they have inherent limitations in predicting therapeutic efficacy in clinical glioma, and future studies using orthotopic intracranial models will be required to further validate the translational potential of the proposed strategy. To achieve this, the nanoparticle platform and hydrogel system will need to be redesigned. The present work resolves only a portion of the challenges in glioma treatment, and considerable efforts will be required in future studies. Second, the IR780-PTX NPs and ACF hydrogels are two independent drug delivery systems though they could work in a synergistic manner. Though separating systemic albumin nanoparticle delivery of IR780-PTX from local hydrogel-based delivery of ACF represents a rational and necessary strategy that aligns with the physicochemical properties and therapeutic objectives of each agent, it will be more convenient for drug administration if the two system could be united together. Third, the long-term biosafety and pharmacokinetics of IR780-PTX NPs and ACF hydrogels have not been systematically evaluated. Although the materials used in this study are biocompatible, rigorously designed experiments are required.

Taken together, our findings provide a novel strategy for reinforcing synergistic photochemotherapy via a single regulatory agent ACF to overcome photochemotherapy induced HIF-1 and HSP-related stress responses for hypoxic tumors.

Supplementary Information

Supplementary Material 1.