Enhancing Photothermal Therapy Efficacy by In Situ Self-Assembly in Glioma

Abstract

The residence time of some small molecular imaging and therapeutic agents in tumor tissue is short and the molecules can be easily dispersed, which decreases treatment efficacy. Therefore, methods that enhance oncotherapy performance are of significant importance. Here, we report an in situ self-assembly strategy aimed at enhancing the photothermal therapy of glioblastomas. The probe, ICG-PEP-c(RGD)fk, consisted of a glutathione-reactive self-assembling polypeptide as the skeleton, indocyanine green (ICG) as a theranostic agent, and cyclic Arg-Gly-Asp [c(RGD)fk] peptides as the targeting group. ICG-PEP-c(RGD)fk was synthesized and found to be assembled in the glutathione environment at 9.446 μM in vitro. Human glioblastoma cell line U87MG-luc with high integrin α_vβ₃ expression was applied to invivo experiments. ICG-PEP-c(RGD)fk provided clearer tumor imaging and had a tumor retention time of 6.12 times longer than that of ICG-c(RGD)fk. In therapeutic experiments, ICG-PEP-c(RGD)fk significantly suppressed glioblastoma growth and the tumor volume was 2.61 times smaller than in the ICG-c(RGD)fk group at the end of the observation period. Moreover, the median survival time of ICG-PEP-c(RGD)fk group was significantly improved by 2.78 times compared with that of the control group. In conclusion, glutathione-reactive self-assembling peptides are capable of increasing the tumor retention time and improving the photothermal therapeutic effect. The in situ self-assembly strategy is a potential and feasible method to enhance oncotherapy.

Graphical Abstract

INTRODUCTION

Brain tumors are life-threatening diseases that affect the central nervous system.¹ A glioma is a common type of brain tumor, which accounts for the majority of malignant brain tumors.² Although the blood-brain barrier is significantly weakened in the presence of tumors, the high interstitial pressure within the tumor prevents anticancer drugs from remaining in the brain for extended periods of time.^3,4 Small molecules such as indocyanine green (ICG) have the advantages of strong tissue penetration and rapid metabolism in vivo, playing an important role in the therapy of malignant tumors.^5,6 However, these molecules can easily disperse, which results in a short duration of the effective concentration in situ.⁷ When modified to nanoparticles, they can achieve longer tumor retention times. However, nanoparticles are limited to reaching ideal therapeutic concentrations, resulting in weak tumor penetration.⁸ Additionally, retention in the reticuloendothelial system may lead to potential toxicity, which can further hinder clinical applications.^9,10 Thus, new strategies, modifications, and additional exploration are strongly desired.

Recently, self-assembled peptides have played an increasingly important role in the fields of bioengineering, drug delivery, and disease treatment.^11,12 Compared with in vitro self-assembly of nanofractures trapped in macrophages, in situ self-assembly strategies are related to the peptide self-assembly response to specific endogenous stimuli (e.g., redox, enzymes, and changes in pH),^13,14 which remain in the corresponding microenvironment. A structure containing a small portion of phenylalanine (Phe) within the peptide is inclined to form a nanomicelle through enzymes or a pH switch.^15,16 Glutathione (GSH) redox-responsive linkers such as disulfide linkages are gaining attention as smart probes because of the high GSH concentrations in tumor cells.^17–19 Therefore, when studying therapeutic strategies for tumors, the combination of a Phe-Phe peptide and the use of disulfide bonds warrants investigation. When disulfide bonds are reduced by GSH, the amphiphilic progelator divides into hydrophilic and hydrophobic parts and the hydrophobic part containing drugs self-assembles into nanostructures.^20,21

In this study, we designed a GSH-responsive Phe-Phe peptide named PEP as a self-assembled platform to enhance photothermal therapy (PTT) of glioma (Scheme. 1). In addition to a cyclic Arg-Gly-Asp peptide named c(RGD)fk, which targeted glioma, and ICG, a theranostic probe was bound as the functional group. We hypothesized that the self-assembled probe would rapidly reach tumor tissue with disruption of the blood-brain barrier and the targeting group. A nanomicelle was further assembled in response to the tumor microenvironment and retained in the tumor tissue. A modified probe, ICG-PEP-c(RGD)fk, named IPR, is desired to prolong the retention time in tumors and improve PTT efficacy.

Scheme 1.

Schematic Diagram of Tumor Microenvironment-Responsive Smart Molecular Probes Entering Tumor Target Sites for Self-Assembly^a

^aThis scheme was created using materials from Biorender.com.

RESULTS AND DISCUSSION

Preparation and Characterization of ICG-PEP-c(RGD)-fk.

ICG-PEP (IP), ICG-c(RGD)fk (IR), and IPR were synthesized (Figure S1) and characterized by mass spectrometry (Figure S2). IPR chemical intermediate compounds in GSH were also identified (Figure S3). The purity of all products was checked by high-performance liquid chromatography (HPLC) (Figure S4). The maximum absorption and emission wavelengths of IP and IPR were 794 and 826 nm, respectively, in dimethyl sulfoxide (DMSO) (Figure 1a, b), which were consistent with those of ICG. Before and after initiating the GSH reaction, there was no change in the absorption wavelengths of IPR in phosphate-buffered saline (PBS) (Figure 1c). After adding GSH, the critical aggregation concentration (CAC) of IPR was determined to be 9.446 μM by examining the excitation spectra of pyrene (Figure 1d). In transmission electron microscopy (TEM) observation of IPR with GSH incubation, micellar structures with an average diameter of approximately 20.74 ± 6.70 nm were observed (Figure 1e). Without incubation, they were not observed by TEM (Figure 1f). Additionally, the CAC of IP was 9.070 μM (Figure S5a), and micellar structures were also found in the TEM image of IP (Figure S5b). The zeta-potential of IPR and IP were −9.23 ± 0.40 mV and −31.83 ± 2.91 mV, respectively. In physiological conditions, we found that nanomicelles could still be formed stably in the serum environment containing GSH (Figure S7).

Figure 1.

Characterization of IPR. (a) UV–vis absorption spectra of ICG, IP, and IPR in DMSO. (b) Emission spectrum of IP and IPR in DMSO. (c) UV–vis absorption spectra of IPR with/without GSH in PBS. (d) Critical assembly concentration of IPR (CAC = 9.446 μM). (e) TEM image of IPR after reaction with GSH (10 mM). (f) TEM image of IPR.

Photothermal Performance of IPR.

The photothermal conversion effect of IPR was investigated at various concentrations and laser power densities. As shown in Figure 2a, e, the temperature increased with the concentration of IPR (2, 5, 10, 15, and 25 μM). When the concentration reached 25 μM, the temperature increased to 73.20 °C after 808 nm laser irradiation for 10 min. As a control, the temperature of water only increased to 30.97 °C. Various power densities of 808 nm laser (0.5, 1, 1.5, and 2 W/cm²) were further to investigate (Figure 2b). The temperature increased from 48.17 to 94.90 °C when the power density increased to 2 W/cm², indicating that IPR efficiently converted light into thermal energy and the heat generation could be finely tuned. The photothermal heating curves of the various probes are shown in Figure 2c. When the concentration was 15 μM, the temperature of IPR with GSH was increased to 59.70 °C after 190 s of continuous laser irradiation and remained at 56.55 °C after 10 min of irradiation. As a control, the temperature of ICG only increased to 52.75 °C at 180 s and IPR reached 52.74 °C at 140 s. The monitored end point temperatures were 38.12 and 43.13 °C, respectively. Next, we examined the photothermal stability of IPR with GSH (Figure 2d) and a cyclic monitoring experiment was conducted to restore the probe to room temperature after the irradiation. After three cycles, the photothermal efficiency of the continuously irradiated probe remained stable (>90% of initial temperature). The photothermal conversion efficiency (η value)^22–24 of IPR with GSH was 37.68%.

Figure 2.

Photothermal properties of IPR. (a) Photothermal heating curves of IPR with GSH solutions at different concentrations (2, 5, 10, 15, and 25 μM) under 808 nm laser irradiation at a power density of 1 W/cm². (b) Photothermal heating curves of IPR (15 μM) with GSH solution under 808 nm laser irradiation at different power densities (0.5, 1, 1.5, and 2 W/cm²). (c) Photothermal heating curves of ICG, IPR, and IPR with GSH solutions under 808 nm laser irradiation at a power density of 1 W/cm². (d) Photothermal stability of IPR with GSH aqueous solution. After 808 nm (1 W/cm²) laser irradiation, the laser is turned off. This cycle is repeated three times. (e) Photothermal heating thermal images of IPR with GSH solutions at different concentrations (2, 5, 10, 15, and 25 μM) under 808 nm laser irradiation at a power density of 1 W/cm².

Characterization of IPR In Vitro.

High and low integrin α_vβ₃-expressing cell lines U87MG-luc (human glioma cell line) and MCF-7 (human breast cancer cell line), respectively, were selected and identified by immunohistochemistry (Figure 3a). For fluorescence imaging of IPR in vitro, IPR or ICG was applied to the cells. In the IPR group, intense fluorescence signals were detected in the U87MG-luc cytoplasm, which were confirmed by fluorescein isothiocyanate isomer (FITC)-phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining (Figure 3b). The fluorescent signal area of ICG per cell in the IPR group (70.37 ± 1.06%) was significantly higher than that in the U87MG-ICG (2.06 ± 0.46%) and MCF-7-IPR (2.57 ± 0.77%) groups, as measured by Image J semiquantitatively.

Figure 3.

Characterization of ICG-PEP-c(RGD)fk in vitro. (a) Integrin α_vβ₃ immunohistochemical staining images of U87MG-luc and MCF-7 cells. Scale bar: 50 μm. (b) Confocal laser scanning microscopy images of U87MG-luc and MCF-7 cells after incubation of ICG and IPR. The cell F-actin were dyed green by FITC-phalloidin, and the nuclei were stained by DAPI. Scale bar: 20 μm. (c) Viability rate of U87MG-luc cells treated with different concentrations of IPR. (d) Cell viability of U87MG-luc cells after incubation with ICG, IP, IR, and IPR (1, 2, 5, 10, 20, 50, and 100 μM) under 808 nm laser irradiation (1 W/cm²) for 3 min.

Without laser irradiation, IPR showed no apparent cytotoxicity in U87MG-luc cells even at concentrations of up to 100 μM, demonstrating its in vitro biocompatibility (Figure 3c). Cell viability was analyzed after photothermal treatment. The results of various groups treated with various concentrations under laser irradiation (1 W/cm²) are shown in Figure 3d. After laser irradiation, U87MG-luc cells treated with IPR (20 μM) displayed a lower viability of 11.84 ± 1.21% (P = 0.046) compared with the PBS group after irradiation for 3 min, and it was also lower than that of ICG (79.37 ± 5.35%), IP (70.36 ± 2.48%), and IR (53.46 ± 2.99%) groups. IPR treatment resulted in significantly stronger inhibition of the viability and proliferation of tumor cells after laser treatment, whereas no obvious cytotoxicity occurred without laser treatment. Owing to the good therapeutic effect and biocompatibility in vitro, this GSH-responsive platform had potential for further application in vivo.

In Vivo Fluorescence Imaging and Biodistribution.

Integrin α_vβ₃-targeted peptide c(RGD)fk and highly integrin α_vβ₃-expressing cell line U87MG-luc were chosen to investigate the in vivo performance of IPR. ICG, IR, and IPR were all detected in orthotopic gliomas by fluorescent imaging at 2 h after injection (Figure 4a). The orthotopic glioma was clearly seen with increased tumor-to-background ratios (2.40 ± 0.03, n = 3) over 12 h in the IPR group, and the tumor contour was also present until 192 h. Comparatively, tumor lesions were undetectable at 72 h in the IR group. The IPR group improved the half-life of ICG in tumor site compared with the IR group (59.46 vs. 9.72 h) by 6.12 times, demonstrating a feasible strategy to prolong tumor retention. Similar imaging patterns were seen in the subcutaneous tumor model (Figure S8a). Quantitative analysis of fluorescence signals in the tumor site was performed. Compared with IR and ICG, IPR significantly prolonged the tumor imaging time through self-assembly within the tumor tissue compared (Figure S8c).

Figure 4.

Fluorescence imaging of U87MG-luc glioma mice in vivo and in vitro. (a) In vivo bioluminescence imaging and fluorescence imaging of three probes in different time. (b) Fluorescence signals of ICG, IR, and IPR in brains harvested at 72 h postinjection. (c) Ex vivo fluorescent imaging of nanoparticles in significant organs (heart, lung, liver, spleen, and kidney) harvested at 72 h postinjection.

In ex vivo fluorescence imaging, intense fluorescent signals in tumors were observed in the IPR group at 72 h postinjection, but were undetectable in IR and ICG groups (Figure 4b). The significant ICG signal from IPR can be detected at the tumor site by tissue fluorescence imaging, while slightly detectable in ICG-injected tissue (Figure S9).

Photothermal Therapeutic Efficacy of IPR in Mouse Models.

The growth of U87MG-luc xenografted tumors was assessed to determine PTT efficacy in vivo. In contrast to IR groups, IPR treatment significantly suppressed the tumor growth (Figure 5a, 38.23 ± 3.80 mm³ vs. 99.68 ± 28.74 mm³, P = 0.01) at the end of the observation period. The tumor volume of the IPR group was 2.61 times smaller than that of the IR group. No obvious behavioral abnormalities were observed in the treated mice. The body weights of the various groups showed no statistical difference (Figure 5b). At the end of the treatments, tumor tissues were excised and weighed. The tumor weight of the IPR group was much smaller compared with that of IR and ICG group (P = 0.028, P = 0.002, respectively, Figure 5c, d). Moreover, the median survival time of the IPR group was 2.78 times longer than that of the control group (Figure 5e). To investigate the systemic toxicity of the various treatments, we examined vital organs of the mice, including the heart, liver, spleen, lung, and kidney, by hematoxylin and eosin (H&E) staining (Figure 5f). The results showed no obvious damage to these organs, indicating that the treatments were well tolerated and did not lead to death or acute side effects at the applied dose.

Figure 5.

Photothermal therapeutic efficacy in U87MG-luc xenograft mouse models. (a, b) Tumor volume curves and weight curves of different groups. (c) After treatment, tumors were harvested and weighted at 16 days. (d) Representative images of tumor form in each group. (e) Survival curves of mice with U87MG-luc tumors after treatment. (f) H&E staining of heart, liver, spleen, lung, and kidney at the end of treatment were presented. +I indicates with laser irradiation. CON refer to the group treated with an equal volume of saline. * P < 0.05, ** P < 0.01, **** P < 0.0001, compared with IPR + I group. Scale bar: 250 μm.

Our results strongly showed that IPR inhibited tumor growth and prolonged the median survival time of U87MG-luc glioma mice. Moreover, IPR treatment was well tolerated, resulting in no body weight loss or obvious hepatic and renal damage, suggesting the safety and efficacy of IPR. Accordingly, it is reasonable to assume that IPR may be an alternative approach to enhancing tumor therapy.

Self-assembly strategies are promising for diagnostic imaging, biosensing, drug delivery, and therapeutic applications.²⁵ The self-assembly of phenylalanine peptide cores in the tumor GSH environment prolongs the retention time of therapeutic molecules in a tumor.^26,27 The aim of this study was to investigate the effects of combining the self-assembly properties of Phe-Phe peptide with c(RGD)fk²⁸ as a targeting group and ICG as a photothermal therapeutic molecule.²⁹ IPR effectively assembled under the GSH condition, while maintaining binding specificity and excellent fluorescence performance. Importantly, IPR notably improved PTT efficacy in glioma via mediated selective retention in tumors.

In situ self-assembly is an appropriate strategy because of the reaction to the specific microenvironment.³⁰ There are several common types of in vivo self-assembly: physical methods (pH),³¹ enzyme reactions (alkaline phosphatase),³² chemical reactions (GSH),³³ and biosurface induction (vancomycin).³⁴ In this study, we used a GSH-responsive group (disulfide bond) and embedded it in Phe-Phe peptides. It provided a suitable method with a high yield and excellent modifiability to develop a GSH-responsive self-assembly strategy. ICG is an ideal fluorescence agent that has been approved by the FDA for 63 years.³⁵ IPR was synthesized successfully and had good water solubility and high fluorescence photostability. In vitro, IPR self-assembled into nanofibers when 9.446 μM was reached, which was in accordance with other studies using the Phe-Phe peptide.^36–38 It formed regular nanomicelles that may deposit in cancer cells. In vitro cytological fluorescence imaging verified IPR retention in U87MG-luc cells.

Photothermal therapy is a minimally invasive treatment modality with minimal toxicity and triggers photosensitization through electromagnetic radiation, such as radio frequencies, microwaves, near-infrared light, and visible light, which converts this energy into heat.^39,40 By exploiting the ability of ICG to generate both heat and reactive oxygen species in response to near-infrared light, ICG can be used for PTT to elicit an antitumor response.^41,42 IPR demonstrated significantly stronger inhibition of the viability and proliferation of tumor cells after laser treatment, while no obvious cytotoxicity had occurred without laser treatment. Owing to the good therapeutic effect and biocompatibility in vitro, this GSH-responsive platform has potential for further application in vivo.

Integrin α_vβ₃-targeted peptide c(RGD)fk and highly integrin α_vβ₃-expressing cell line U87 were chosen to investigate invivo performance of IPR. In situ and subcutaneous tumors of U87 cells were visualized at 2 h after IPR injection and clearly delineated until 192 h postinjection, suggesting a feasible strategy to prolong tumor intention. Furthermore, our results strongly indicated that IPR inhibited tumor growth and prolonged the median survival time of U87 glioma mice. The tumor volume of the IPR group was 2.61 times smaller than that of the IR group at the end of the observation period. Additionally, the median survival time of the IPR group was 2.78 times higher than that of the control group. Accordingly, the IPR treatment was efficient and well tolerated in vivo. It is reasonable to assume that IPR may be an alternative approach to enhancing tumor therapy and may be applied in patients with gliomas in the future.

CONCLUSION

This study provides an effective method of building a GSH-reactive self-assembling probe. It incorporates the advantages of strong tissue penetration and a long functional time in tumors, which improves PTT efficiency in gliomas. Other targets may also be applied in this in situ self-assembly strategy to enhance oncotherapy.

MATERIALS AND METHODS

Preparation of the Self-Assembling Probe.

The synthesis of the small molecule peptide named PEP was carried out using a solidphase peptide synthesis technique with purity of >95%. ICG-N-hydroxysuccinimide ester (ICG-NHS) (10 μmol), PEP (12 μmol), and N-diisopropylethylamine (DIPEA, 200 μmol) were dissolved in 500 μL of DMSO and stirred overnight at room temperature. IP was obtained after purification through a C4 semipreparative HPLC column. The solvent system was H₂O (0.1% TFA, A) and acetonitrile (0.1% TFA, B). IP (10 μmol), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (20 μmol), and N-hydroxysuccinimide (NHS) (12 μmol) were added to anhydrous DMSO (200 μL), followed by stirring in a 1.5 mL centrifuge tube for 4 h at room temperature. The c(RGD)fk (15 μmol) and DIPEA (200 μmol) were then added to the mixture and reacted at room temperature for 8 h. Finally, IPR was collected after purification by HPLC using water/acetonitrile containing 0.1% TFA as an eluent. ICG-NHS (10 μmol), c(RGD)fk (15 μmol), and DIPEA (200 μmol) were also dissolved in 500 μL of DMSO and stirred overnight at room temperature. IR was collected after purification as described above. The synthesized IP, IR, and IPR were characterized by high-resolution mass spectrometry (Orbitrap LC/MS, Q Exactive, Thermo Fisher, Germany). High-resolution mass spectrometry (HRMS, ESI) m/z: [M-H]⁻: C₈₄H₉₇N₈O₁₂S₃⁻: m/z = 1505.6394; found 1505.6429; [M + H]⁺: C₇₂H₉₀N₁₁O₁₁S⁺: m/z = 1316.6536; found 1316.6550; [M + H]⁺ C₁₁₁H₁₃₈N₁₇O₁₈S₃⁺: m/z = 2092.9562; found 2092.9622.

Transmission Electron Microscopy and Critical Assembly Concentration.

After incubation with GSH (10 mM, 20 min) and purification with dialysis membranes (3500), IPR was loaded onto 230-mesh copper grids that had been coated with a continuous thick carbon film to cover the grid surface. Then, the grid was rinsed three times with ddH₂O. Water was carefully allowed to move away from the grid and subsequently touch the edge of the grid to filter paper slivers. After rinsing, the copper grids covered in sample were dried in air. TEM (Tecnai G2 20, FEI, Netherlands) was used for observation.

CAC of IPR and IP were assessed by a fluorescence assay of pyrene. At the beginning, an acetone solution of pyrene (6 × 10⁻⁵ M) was prepared. Then, 20 μL was added to a vial and the acetone was removed by natural evaporation. Afterward, 2 mL aqueous polymer dispersion was added to the solid pyrene residue. The IPR concentration was in the range of 0.39–100 μM. All of them were incubated with GSH (10 mM) for 20 min. The mixture was sonicated for 4 h, followed by incubation at a constant temperature of 40 °C in a water bath for 24 h to equilibrate the pyrene and micelles. The excitation and emission spectra of pyrene were recorded at 25 °C with an excitation wavelength of 334 nm and an emission wavelength of 376 nm.

Photothermal Characteristics of IPR.

To evaluate the photothermal performance of IPR, 808 nm (1.0 W/cm²) laser was used to irradiate different concentrations of IPR (2, 5, 10, 15, and 25 μM) by measuring the solution temperature. Then, 808 nm laser experiments were performed at 15 μM of IPR with different power densities (0.5, 1, 1.5, and 2 W/cm²). We also added solutions of ICG, IP, and IPR (15 μM in 1 mL PBS), followed by and irradiation with an 808 nm (1.0 W/cm²) laser. Temperature images at various times were recorded and measured by an infrared camera (FLIR Systems, Inc.). To evaluate photothermal stability, we irradiated the IPR solution at 808 nm (1 W/cm²) until the temperature no longer increased, and then the laser was switched off to allow the solution to cool naturally to room temperature. The same solution cycle was repeated three times. Then, we calculated the photothermal conversion efficiency using the formula^43,44

$$\eta =\frac{hA\left(\Delta T_{\text{max},\text{mix}}-\Delta T_{\text{max},{\text{H}}_2\text{O}}\right)}{I\left(1-{10}^{-A_{\lambda }}\right)}$$

Where η is the heat transfer coefficient, A is the surface area of the container, ΔT_max,mix and $\Delta T_{\text{max},{\text{H}}_2\text{O}}$ are the temperature change in the IPR dispersion and solvent (water) at the maximum steady-state temperature, respectively, I is the laser power, and A_λ is the absorbance of IPR at 808 nm.

Cell Culture and Identification.

Human glioma cell line U87MG-luc and human breast cancer cell line MCF-7 were maintained in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. Integrin α_vβ₃ expression in U87MG-luc and MCF-7 cells was identified by immunohistochemical staining. The cells were incubated for 4 h with ICG, IR, or IPR (20 μM). The cytoskeleton was stained with FITC-phalloidin and nuclei were stained with DAPI. Confocal laser scanning microscopy was conducted using a Zeiss laser scanning microscope 710 (Carl Zeiss Oberkochen, Germany).

Cell Counting Kit-8 (CCK-8, Solarbio, China) was used to evaluate the cytotoxicity of ICG, IR and IPR in U87MG-luc cells after photothermal treatment. U87MG-luc cells were seeded at 1 × 10⁴ cells/well on 96-well plates and cultured for 48 h. Then, different concentrations (1, 2, 5, 10, 20, 50, and 100 μM) free PBS, ICG, IP, IR, or IPR were added to the cells, followed by incubation for 24 h. After rinsing with PBS, photothermal treatment was performed at an excitation wavelength of 808 nm for 3 min, followed by treatment with CCK-8 reagent (10 μL each well) for an additional 1 h. Absorbance was measured in a microplate reader (absorbance: 450 nm). Corresponding cells without any treatment were used as controls for three replicates.

Animals and Tumor Model.

BALB/c nude male mice (4–5 weeks old) were purchased from Beijing HuaFuKang Animal Technology Co., Ltd. (Beijing, China) and maintained under specific pathogen-free conditions. Animals received care under the Guidance Suggestions for the Care and Use of Laboratory Animals. A 5 μL suspension containing 5 × 10⁵ U87MG-luc cells was slowly implanted into the right corpus striatum of the mice using a stereotactic fixation device. After 1 week, d-luciferin potassium salt (150 mg/kg) was intraperitoneally injected and the mice were imaged using an in vivo imaging system (IVIS Lumina Series III, PerkinElmer) after 10 min.

A total of 50 nude mice were subcutaneously injected with 100 μL serum-free PBS containing 5 × 10⁶ U87MG-luc cells into their upper limbs. Approximately 2 weeks after injection when the tumor volume had reached 60 mm³, the tumor-bearing mice were used for further experiments.

In Vivo Fluorescence Imaging.

Tumor-bearing mice were intravenously injected with ICG, IR, or IPR (30 nmol each, 100 μL, n = 3 per group) under anesthetization with 2% isoflurane in oxygen. Fluorescence images of the mice at various time points (2, 12, 24, 48, 72, and 192 h) were obtained by the imaging system using an 845 nm band-pass filter under 780 nm excitation (excitation/emission, Ex/Em = 780/845 nm). The mice were then sacrificed to obtain major organs, including the heart, lung, liver, spleen, and kidney, and tumors to conduct ex vivo fluorescence imaging.

In Vivo PTT and Toxicity Evaluation.

To measure tumor growth, we used subcutaneous U87MG-luc xenografted tumor models, which were divided into five groups (n = 5 per group): (1) IPR with laser irradiation treatment, (2) IR with laser irradiation treatment, (3) ICG with laser irradiation treatment, (4) equal volume of saline with laser irradiation treatment, and (5) equal volume of saline without laser irradiation treatment. Photothermal treatment was performed twice at 24 and 72 h after intravenous injection of 50 nmol probe. The laser power density of each group was 1.0 W/cm² (808 nm) with an irradiation duration of 5 min. Tumor volume and body weight were measured every 2 days. The tumor volume was calculated by the following formula: tumor volume = length × width²/2. Animals were euthanized when the tumor volume exceeded 1500 mm³. Additionally, the tumor and other vital organs (heart, lung, liver, spleen, and kidney) were sectioned and stained with H&E for histological analysis at 16 days. An additional 25 mice were treated as described above for analyzing survival time.

Statistical Analysis.

All data are presented as the mean ± standard deviation. Data analyses were conducted using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Differences among groups were analyzed by the paired-samples t test, independent-samples t test, and one-way ANOVA. A P value less than 0.05 indicated statistical significance.

Data Availability Statement

All the data that support the findings of this study are available from the authors upon reasonable request.