Peptide-based supramolecular nanomaterials with super-large red-shifted absorption for antitumor therapy

Abstract

Indocyanine green (ICG) is a near-infrared dye with photothermal therapy (PTT) and photodynamic therapy (PDT) effects, widely used in cancer treatment. In this study, a multifunctional nanoplatform (ICG-TP-5) was successfully constructed through the supramolecular co-assembly of ICG and TP-5, enabling multimodal synergistic therapy for tumors. The nanomaterial demonstrated remarkable stability in aqueous solution at 4 °C for at least three months, effectively addressing the inherent limitations of ICG while enhancing its bioavailability. Driven by molecular self-assembly-induced J-aggregation effects, the near-infrared (NIR) absorption peak of the material exhibited super-large red-shift absorption from 785 nm to 947 nm, significantly enhancing tissue penetration depth and photoconversion efficiency. This optical optimization synergistically amplified both PTT and PDT efficacy, offering a promising strategy for treating deep-seated malignancies. Innovatively, this platform integrated PDT/PTT with TP-5-mediated immune activation, establishing a trimodal therapeutic mechanism. In vitro experiments confirmed its selective cytotoxicity against diverse cancer cells, while in vivo studies revealed that the combined therapy markedly suppressed tumor growth and activated systemic antitumor immunity. These findings provide a robust nanomedicine candidate with enhanced stability, deep-tissue penetrability, and multimodal therapeutic synergy, paving the way for precision treatment of aggressive and deep-seated tumors.

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1. Introduction

According to the World Health Organization's International Agency for Research on Cancer (IARC), cancer caused 9.96 million deaths globally in 2020, making it one of the most dreadful diseases affecting human life and health.¹ It also highlights limitations in current cancer treatment strategies. Therefore, seeking an effective treatment means is an important task in the current oncology field.^2–4 In recent years, phototherapeutics, namely photodynamic therapy (PDT) and photothermal therapy (PTT), has gained increasing attention in cancer research as a promising approach.^3–7 Phototherapeutics is a non-invasive method for treating cancers and other diseases.⁸ It has been clinically approved as a minimally invasive treatment that selectively and irreversibly damages malignant tissues or cells while sparing adjacent healthy tissues.^9–12 This makes it a unique and compelling choice for cancer treatment. PDT involves three key components: a photosensitizer (PS), a light source, and oxygen.^13–16 When exposed to light in the presence of oxygen, the photosensitizer produces reactive oxygen species (ROS) that cause the death of tumor cells.^17–20 PTT transforms light energy into thermal energy, effectively exterminating tumor cells via heat damage.^21–25 These innovative cancer treatments have been shown to treat various types of cancer. However, PDT and PTT may not eradicate solid tumors by themselves due to their own defects.^26,27 Combining light therapy with other treatment modalities can yield additional and even synergistic therapeutic effects by leveraging the advantages of each modality and counteracting its disadvantages.^28–30 This synergistic effect may lead to improved antitumor efficacy at lower doses of photosensitizers or at lower power light exposure, thereby substantially reducing the potential toxicity to normal tissues.^31–33

In addition, the therapeutic outcome for PDT and PTT relies on the depth of tissue penetration.^34–36 To achieve successful in vivo implementation of PTT and PDT, the excitation light source used as an external initiator should correspond consistently with the wavelength absorption band of the photosensitizer employed as well as exhibit sufficient tissue penetration depth while minimizing power loss. Most photosensitizers utilized in PDT/PTT exhibit maximum absorption within the visible spectrum.^37,38 However, visible light can also be absorbed by numerous endogenous chromophores present in biological tissues. Additionally, biological tissue induces scattering, which restricts light propagation and directionality.^39,40 Typically, visible light can only penetrate a few millimeters into tissue, posing challenges for PDT/PTT of deeper tumors.⁴¹ Near-infrared light, with its longer wavelength, reduces tissue scattering and achieves deeper penetration than visible light. Therefore, the development of photosensitizers with long wavelength absorption is crucial.

ICG is a typical photosensitizer, which has been widely utilized in clinics with the 785 nm absorption peak and poor stability. The supramolecular assembly of organic dye molecules can result in J-aggregation states with super-large red-shift absorption and enhanced stability.⁴² Inspired by the advantages of supramolecular strategies, herein, we reported a convenient supramolecular strategy to prepare a peptide-based nanomaterial with super-large red-shift absorption and enhanced stability.^43–45 TP-5 plays a critical role in regulating immune responses by promoting T-cell differentiation, enhancing T-cell function, and modulating the activity of other immune cells such as macrophages and dendritic cells. TP-5 has been widely explored in immunotherapy and as an adjuvant in various therapeutic strategies. Therefore, in this study, TP-5 was utilized to co-assemble with ICG, a near-infrared (NIR) fluorescent dye with photothermal and photodynamic properties, to form a multifunctional nanocomplex (ICG-TP-5). This co-assembly not only leverages the inherent immunostimulatory effects of TP-5 but also enhances the PDT and PTT capabilities of ICG. The ICG-TP-5 nanoparticles have a uniform size of 280 nm. Furthermore, the maximum absorption peak of ICG shifted from 785 to 947 nm in ICG-TP-5, demonstrating its potential in treating deep solid tumors (Scheme 1). In addition, this supramolecular nanomedicine addresses the inherent limitations of ICG, such as its susceptibility to decomposition and poor stability in physiological environments. Through co-assembly with TP-5, ICG is effectively encapsulated within the supramolecular architecture, which significantly enhances its stability and resistance to degradation. Experimental results demonstrate that the supramolecular nanomedicine retains its original structural integrity and functional properties even after three months of storage in aqueous solution, exhibiting remarkable stability. This improvement not only extends the shelf life of the nanomedicine but also ensures its consistent performance during in vivo delivery, thereby enhancing its therapeutic efficacy and clinical applicability. In vitro and in vivo studies demonstrated that ICG-TP-5 had a favorable therapeutic impact on several malignancies. Crucially, this ICG-TP-5 nanomedicine not only obliterates tumors directly but also serves as an immune modulator, enhancing the immunological response of mice with cancer and contributing to enduring anticancer effects. These observations underscore the outstanding potential of ICG-TP-5 for propelling advancements in cancer treatment.

Scheme 1 Schematic diagram of a supramolecular nanomaterial of ICG and TP-5 self-assembly and the mechanism diagram of generating super-large red-shifted absorption. This supramolecular nanomaterial can be used for synergistic treatment through PTT, PDT, and immunotherapy.

2. Experimental section

2.1. Preparation of ICG-TP-5 nanomaterials

We created a mixed solution with 15 mL of deionized water, ICG (0.0024 g), and TP-5 (0.0021 g). The pH of the solution was adjusted to 4.5 and then immersed in an oil bath at 70 °C for 5 h. After the reaction, the product was washed three times at a speed of 8000 rpm using a high-speed centrifuge to collect the final nanomaterials.

2.2. Photodynamic performance test

2.2.1. Ultraviolet spectrophotometry. The reactive oxygen species (ROS) detection reagent was 1,3-diphenylisobenzofuran (DPBF, 0.077 mM L⁻¹). The ROS generation yield of ICG-TP-5 (0.001 mM L⁻¹, 808 nm) was determined with methylene blue (MB, 0.001 mM L⁻¹, 635 nm) as a reference compound. The absorption spectra at different time points were recorded using an ultraviolet spectrophotometer. ROS generation yield can be calculated as follows:
wherein Φ_ICG-TP-5 is the ROS generation yield of ICG-TP-5, Φ_MB is the ROS generation yield of methylene blue (Φ_MB = 57%), and K_ICG-TP-5 and K_MB are the decomposition rate constants of ICG-TP-5 and reference compounds for DPBF. F_ICG-TP-5 and F_MB are the absorption correction factors of the sample and methylene blue at the excitation wavelength (F = 1–10^−A). A is the UV absorbance value of each compound at the excitation wavelength.

2.2.2. Fluorescence imaging method. HepG2 cells (1 × 10⁵) were cultured in confocal dishes for 24 h. Then, the culture medium containing different doses of ICG-TP-5 (0, 1/16, 1/32 and 1/64 mg mL⁻¹, 2 mL) was replaced with the original medium of the dish for another 24 h. Next, each confocal dish was irradiated with an 808 nm laser for 8 min (60 mW cm⁻²). After 24 h, the reactive oxygen species produced were stained. First, 2,7-dichlorofluorescein diacetate (DCFH-DA) was diluted with a serum-free medium at 1 : 1000 to a final concentration of 10 mM L⁻¹. The cell culture medium was removed, and 1 mL of diluted DCFH-DA was added. The cells were incubated in the cell incubator for 20 min at 37 °C. The cells were washed three times with a serum-free cell culture medium to remove DCFH-DA that did not enter the cells adequately.

2.3. Photothermal performance test

The aqueous solution of ICG-TP-5 (1/16 mg mL⁻¹, 1 mL) was put into a 1 cm quartz cuvette, and small magnetons were added to stir at a constant speed of 200 rpm. Then, the thermocouple detector was inserted into the solution, the laser-irradiated (808 nm, 60 mW cm⁻²) solution was tested, and the temperature was recorded every 30 s. When the temperature rose to the maximum, the laser was turned off to cool down, and the temperature rise curve was drawn after five cycles. An infrared thermal imager was used to photograph the solution to be tested after laser irradiation every 1 min. In addition, different concentrations of ICG-TP-5 (1/2, 1/8, 1/16 mg mL⁻¹) were tested by irradiation.

2.4. Cytotoxicity assay

2.4.1. CCK-8 cytotoxicity assay. The human cervical cancer cell line HeLa and the human hepatocellular carcinoma cell line HepG2 were purchased from Guangzhou Cellcook Biotech Co., Ltd (Guangzhou, China). Cells were grown in 96-well plates with 1 × 10⁴ cells per well for a total of 24 h after reaching the logarithmic stage of development in the complete DMEM. Next, the original medium was discarded, and a fresh culture medium was added for 24 h with various doses of ICG-TP-5 (0, 0.0625, 0.0313, 0.0156, and 0.0078 mg mL⁻¹, 100 μL). Subsequently, 808 nm laser irradiation (60 mW cm⁻²) was applied for 8 min. After 24 h, 10 μL of the CCK-8 solution was added and incubated in a CO₂ incubator for 2 h. A full-wavelength microplate reader detected the absorbance value at a wavelength of 450 nm. Cell viability was calculated using the following formula:

2.4.2. Cytotoxicity assay of double staining of live and dead cells. HepG2 cells were cultured in confocal dishes at a rate of 2 × 10⁵ per dish for 24 h. Then, according to the following experimental method in 2.4.1, after 24 h of laser irradiation, the medium was removed and washed twice with 1× assay buffer (2 min each time). After immersion, 1 mL of calcein-AM solution (1 μM mL⁻¹) was added and incubated at 37 °C for 25 min in the dark. Next, the samples were stained with PI solution (5 μM mL⁻¹) at room temperature and shielded from light. After 5 min, PI was removed by suction, washed twice with PBS, covered with 500 μL PBS, and sent for inspection.

2.5. Cell uptake assay

Human liver cancer cells HepG2 (1 × 10⁵ cells per dish) and human cervical cancer cells HeLa (5 × 10⁴ cells per dish) were cultured in confocal words for 24 h. Then, the original medium was discarded, and a fresh medium containing different concentrations of ICG-TP-5 (0, 1/16, 1/32, 1/64 and 1/128 mg mL⁻¹, 2 mL) was added. After culturing for 24 h, the medium in the dish was discarded and washed three times with PBS. Then, the cells in the dish were fixed with 4% paraformaldehyde. After 20 min, the fixation was completed and washed three times with PBS. Finally, nuclei were stained with DAPI. After 5 min of staining, the cells were washed three times with PBS and covered with 500 μL of PBS. A laser confocal microscope was used for detection.

2.6. Apoptosis assay

HeLa cells were cultured in 6-well plates with 2 × 10⁵ cells per well. After 24 h, the medium in the original 6-well plate was replaced with a medium with ICG-TP-5 concentrations of 0, 1/16, 1/32, and 1/64 mg mL⁻¹. After 24 h, 808 nm lasers with 60 mW cm⁻² power density were irradiated for 8 min. After illumination, the cells were cultured for 24 h in a 37 °C cell incubator. Finally, apoptosis kit staining was performed. The cell culture medium was aspirated into a suitable centrifuge tube, washed with PBS, and 1 mL of EDTA-free trypsin digestion solution was added to digest the cells. The cells were incubated at room temperature until the HeLa cells were blown off by gentle blowing. The collected cell suspension was transferred to a centrifuge tube, centrifuged at 1000 RPM for 5 min, then the supernatant was discarded, and the cells were collected. The cells were resuspended twice with PBS, centrifuged at 1000 RPM for 5 min, then the supernatant was discarded, and finally, the cells were collected. The cells were gently resuspended by adding a 300 μL Annexin V-FITC binding solution. 5 μL of Annexin V was added, gently mixed, and incubated for 25 min at room temperature. Then, 10 μL of propidium iodide staining solution was added, gently mixed, and incubated for 5 min at room temperature. The samples were immediately analysed on the machine.

2.7. In vivo fluorescence imaging

ICG-TP-5 and ICG solution (0.37 mg mL⁻¹, 40 μL) were injected into tumor bearing mice. After 0, 1, 2, 4, 8, 12, and 24 h, imaging was performed in the NIR-II in vivo fluorescence imaging system (MARS, Artemis Intelligent Imaging, Shanghai, China).

2.8. Body distribution

To explore the enrichment of ICG-TP-5 in various tissues and organs after injecting ICG-TP-5 into the mouse tail vein and allowing it to flow through the blood circulation, mice were injected with ICG-TP-5 at different concentrations. 24 h later, mice's tumors and organs including the heart, liver, spleen, lungs, and kidneys were collected, and specific fluorescence detection was performed on the collected organs using the small animal imaging instrument. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Wannan Medical College and approved by the Institutional Animal Care and Use Committee (IACUC Protocol No. WNMC-AWE-2024430).

2.9. Therapeutic effect in vivo

The mice were divided into six groups (n = 5): the control group (Control), the light group only (light), the drug group only (Medium, 0.37 mg), the low dose group (Low + Light, 0.185 mg), the medium dose group (Medium + Light, 0.37 mg) and the high dose group (High + Light, 0.74 mg). The light was given 6 h after administration, once every two days. Solid tumor samples were collected after seven doses.

2.10. Validation of PTT in vivo

The mice were injected with ICG-TP-5 (0.37 mg, 100 μL) and PBS (100 μL) through the caudal vein, respectively. 6 h later, the tumor site was irradiated using a laser (808 nm, 60 mW cm⁻²). The mice were imaged using an infrared imager.

2.11. H&E staining

The tumors and organs (heart, liver, spleen, lungs, and kidneys) from mice in each group were harvested after 7 days of treatment and fixed in 4% paraformaldehyde for over 24 hours. After fixation, the tissues were trimmed, leveled, and transferred to a dehydration box. Dehydration was performed using a graded ethanol series: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, and anhydrous ethanol for 30 minutes (repeated twice). The tissues were then treated with alcohol–benzene for 10 minutes, followed by two changes of xylene for 10 minutes each. Paraffin infiltration was carried out in three steps, with each step involving immersion in paraffin melted at 65 °C for 1 hour. Following dehydration and paraffin infiltration, the tissues were embedded using an embedding machine. The paraffin-embedded tissue blocks were trimmed and cooled on a freezing stage at −20 °C. Sections of 4 μm thickness were cut using a microtome, floated on warm water to flatten the tissue, and mounted onto slides. The slides were then baked to dry the tissue sections. For staining, the sections were deparaffinized in two changes of xylene for 10 minutes each, followed by rehydration in a descending ethanol series: anhydrous ethanol for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. The sections were subsequently rinsed with distilled water. The sections were stained with hematoxylin dye solution for 5 minutes, rinsed with distilled water, and differentiated in 1% hydrochloric acid alcohol for several seconds. After another rinse with distilled water, the sections were blued in 0.6% ammonia water and rinsed again with distilled water. Counterstaining was performed using eosin dye solution for 3 minutes. Finally, the sections were dehydrated through an ascending ethanol series (95% ethanol for 5 minutes, repeated once, followed by anhydrous ethanol for 5 minutes, repeated twice) and cleared in two changes of xylene for 5 minutes each. The slides were removed from xylene, air-dried briefly, and sealed with neutral gum. The stained tissue sections were imaged and analyzed using a light microscope to evaluate histological features.

2.12. Immunoassay

In order to test whether ICG-TP-5 still had peptide activity after self-assembly, different doses of ICG-TP-5 were administered intravenously according to the above-mentioned groups (n = 3). After 14 days of treatment, mouse serum was collected and the levels of interleukin-2 (IL-2), interferon γ (IFN-γ), and T lymphocyte subsets CD3⁺CD4⁺ and CD3⁺CD8⁺ in serum were detected.

3. Results and discussion

3.1. Characterization of ICG-TP5

Using peptide-based self-assembly technology, ICG and TP-5 were co-assembled at a molar ratio of 1 : 1. The supramolecular nanomaterial ICG-TP-5 was successfully prepared in an aqueous solution at pH 4.5 and 70 °C for 5 hours. Studies revealed that the size of the nanomaterial is closely related to the pH of the solution. When the pH decreases, the size of the nanomaterial significantly increases with the reduction in pH value. However, when the pH increases, the system fails to form stable nanomaterials through self-assembly, and the solution retains its original state without forming an ordered supramolecular structure. This phenomenon indicates that pH plays a critical regulatory role in the self-assembly behavior of ICG-TP-5, further elucidating the pH-dependent mechanism in the formation of supramolecular nanomaterials. SEM and TEM images showed that ICG-TP-5 had a relatively uniform spheroid shape of 280 nm (Fig. 1a and b). The EDX elemental diagram of ICG-TP-5 showed that C, N, O, and S are uniformly distributed in a single sphere (Fig. S1), indicating that the whole sphere had a globular, solid structure.

Fig. 1 (a) SEM and (b) TEM images of ICG-TP-5. (c) Full-wavelength absorption spectra of ICG, TP-5, and ICG-TP-5 in aqueous solution, and ICG-TP-5 in DMSO. (d) Full-wavelength absorption spectra of freshly prepared ICG-TP-5 and ICG-TP-5 stored in water for 1 and 3 months. Emission spectra of ICG-TP-5 excited at (e) 808 nm and (f) 940 nm.

The spectroscopic properties of ICG-TP-5 were investigated in different solutions. The maximum absorption peak of ICG-TP-5 in aqueous solution is at 947 nm, while the absorption peaks of ICG and TP-5 involved in the assembly are at 785 nm and 275 nm, respectively (Fig. 1c). The absorption spectrum of ICG-TP-5 in DMSO solution is the same as that of ICG. These indicate that ICG and TP-5 are self-assembled to form an aggregation structure in ICG-TP-5, which leads to the super-large red-shift of the absorption peak. This super-large red-shift in the absorption spectrum can be attributed to the J-aggregation of ICG dyes in supramolecular assembled ICG-TP-5. J-aggregates usually cause a bathochromic shift.¹⁹ Different from previous reports,⁴⁶ a novel supramolecular nanomedicine ICG-TP-5 was prepared utilizing the optimized self-assembly method. The ICG-TP-5 has a super-large red-shift in its absorption of up to 162 nm compared with ICG.

It is interesting to note that fresh ICG-TP-5 has strong absorption in the region of 600–1100 nm with the peak at 947 nm of ICG aggregation (Fig. 1d). After storing in aqueous solution for one month, obvious absorption peaks were observed at 600–850 nm, typical ICG molecular absorption peaks. These peaks diminished after storing for another 2 months, while the absorption intensity at 947 nm exhibited negligible changes. These results indicate that the unassembled ICG might exist on the surface of ICG-TP-5 nanoparticles, which were degraded in aqueous solution when stored for a long time. In contrast, the assembled ICG in ICG-TP-5 nanoparticles was isolated from water, and they are relatively stable and can be maintained for 3 months without significant decomposition. Crucially, this exceptional stability extends beyond pure water to physiologically relevant environments. Dynamic light scattering (DLS) measurements revealed minimal changes in the hydrodynamic diameter of ICG-TP-5 nanoparticles after incubation in phosphate buffered saline (PBS) and fetal bovine serum (FBS) for 24 hours, with no visible precipitation or aggregation. Furthermore (Fig. S2 and S3), UV-vis-NIR spectroscopy confirmed the persistence of the characteristic J-aggregate absorption peak at 947 nm in normal saline (0.9% NaCl) after 24 hours, with negligible loss in intensity compared to its initial state in water (Fig. S4). These combined results demonstrate the robust structural integrity of the ICG-TP-5 supramolecular assembly across diverse biological buffers and complex media. The fluorescence spectra of ICG-TP-5 were performed using 808 nm and 940 nm lasers as excitation light (Fig. 1e and f). The maximum emission wavelength of ICG-TP-5 was 920 nm when excited at 808 nm, while it was 1162 nm when excited at 940 nm. In none of the cases did the peak of ICG appear in the fluorescence spectra, indicating the formation of ICG aggregation. The largely bathochromic-shifted emission wavelength suggests that ICG-TP-5 has excellent fluorescence imaging ability in near-infrared regions, which is superior to ICG.

3.2. The PDT/PTT performance of the ICG-TP-5

An 808 nm laser was used as the excitation light source to evaluate the potential of ICG-TP-5 as a phototherapy reagent. The power density of the laser decreases with increasing distance from the center position following the beam expander's action (Fig. 2a). To avoid the interference of this phenomenon, we used the central region of the laser spot to perform the experiments (power density of 50–60 mW cm⁻²). ICG-TP-5 was expected to have good phototherapy properties since its component, ICG, has remarkable PDT and PTT activity. The PDT effect of ICG-TP-5 was first evaluated using 1,3-diphenylisobenzofuran (DPBF) as a ROS generation probe under light irradiation (808 nm, 60 mW cm⁻²). The absorption peak of DPBF decreases gradually with increasing time of light irradiation (Fig. 2b), which indicates that ICG-TP-5 is capable of generating ROS. According to the formula, the ROS generation rate of ICG-TP-5 is about 4.9 times that of ICG (Fig. S5). In addition, the intracellular ROS generation ability of ICG-TP-5 was evaluated by taking advantage of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as an indicator in HepG2 cells. The green fluorescence, which indicates ROS levels in cells, became stronger with increasing ICG-TP-5 concentrations (Fig. 2c). These results demonstrate the potential of ICG-TP-5 as a PDT reagent.

Fig. 2 (a) The power density profile of the laser used. (b) Ultraviolet–visible absorption spectra of DPBF at different illumination times (60 mW cm⁻²) in the presence of ICG-TP-5. (c) CLSM images of reactive oxygen species produced by HepG2 cells co-incubated with different concentrations of ICG-TP-5 stained with DCFH-DA. (d) Temperature rising curves of ICG-TP-5 solution with different concentrations (808 nm laser, 60 mW cm⁻²). (e) Temperature elevation of 1/16 mg ml⁻¹ICG-TP-5 solution under 5 irradiation/cooling cycles (808 nm laser, 60 mW cm⁻²). (f) Thermal infrared images of 1/16 mg ml⁻¹ICG-TP-5 solution at different times (808 nm laser, 60 mW cm⁻²).

The PTT effect of ICG-TP-5 was then investigated under irradiation (808 nm, 60 mW cm⁻²). The temperature of the ICG-TP-5 solution was effectively elevated in a concentration-dependent manner (Fig. 2d). Infrared thermal images at a given irradiation time were recorded at a concentration of 1/16 mg mL⁻¹ (Fig. 2e). The results indicated that the temperature of the solution rose rapidly to 42 °C (ΔT = 15 °C) after irradiation for 10 min. Then, five successive irradiation–cooling cycles for ICG-TP-5 were performed to check the stability of its photothermal effect. It can be observed that the highest temperature was above 40 °C for all 5 circles, and there were almost no differences between the 5 circles. These results verify that ICG-TP-5 maintained stable PTT activity over five cycles. In addition, the time for the photothermal effect was set as 10 min, indicating the rapid warming capacity of ICG-TP-5, which was further recorded in the thermal imager images (Fig. 2f).

3.3. Antitumor effect of ICG-TP-5in vitro

In vitro cytotoxicity of ICG-TP-5 was evaluated using human hepatocellular carcinoma HepG2 cells and human cervical cancer HeLa cells by the method of CCK-8 (Fig. 3). No dark toxicity was observed in the experiments since the cell viability is larger than 95% in all cases without irradiation (Fig. 3a and b). The cytotoxicity was also negligible under irradiation when there was an absence of ICG-TP-5. When ICG-TP-5 and 808 nm irradiation (60 mW cm⁻²) coexisted, the obvious cytotoxicity could be detected. The phototoxicity of ICG-TP-5 increases with its concentration in both HepG2 and HeLa cells, but the phototoxicity is higher for HeLa cells than for HepG2 cells at the same concentration (survival rate at 1/32 mg mL⁻¹, in HepG2 cells: 24.9%, HeLa cells: 5.9%).

Fig. 3 Cytotoxicity of ICG-TP-5in vitro. (a) HepG2 cells and (b) HeLa cells were incubated with different concentrations of ICG-TP-5 for 24 h, and the survival status of the cells was detected using the CCK-8 kit. (c) HepG2 cells were incubated with different concentrations of ICG-TP-5 for 24 h, and the cell survival status was detected using the Calcein/PI kit. The scale bar is 50 μm. (*p < 0.05, ****p < 0.0001 vs. the control).

Results of CCK-8 experiments proved that ICG-TP-5 exhibits an obvious killing activity towards cancer cells. In addition, another experiment was performed with different concentrations of ICG-TP-5 to further confirm its phototoxicity to cancer cells. Calcein AM (green fluorescence, staining live cells) and propyl iodide (red fluorescence, staining dead cells) were used to evaluate the survival rate of cancer cells after irradiation. As shown in Fig. 3c, with increasing dose of ICG-TP-5, the number of living cells stained by calcein AM significantly decreased, while the number of dead cells stained by PI significantly increased. When the concentration reached 1/16 mg mL⁻¹, most of the cancer cells in the illuminated area died. This indicates that ICG-TP-5 has an excellent killing effect on HepG2 cells, which is in agreement with the CCK-8 results.

We evaluated the interaction between ICG-TP-5 and the cancer cells by observing the biodistribution using a confocal laser scanning microscope (CLSM). The CLSM images of cancer cells co-incubated with ICG-TP-5 showed clear red fluorescence in a concentration-dependent manner (Fig. 4a and b), and the intensity of red fluorescence gradually enhanced with increasing ICG-TP-5 dose, which suggests that ICG-TP-5 can be efficiently taken up by cancer cells. In addition, a co-staining experiment with ICG-TP-5 and DAPI indicates that the red fluorescence from the ICG-TP-5 is mainly distributed in the cytoplasm.

Fig. 4 CLSM images of (a) HepG2 cells and (b) HeLa cells incubated with different concentrations of ICG-TP-5 and stained with DAPI. ICG-TP-5 was excited at 561 nm to show red fluorescence, and DAPI was excited at 405 nm to show blue fluorescence. (c) Flow cytometry analysis of grid and necrosis in HepG2 and HeLa cells treated with different concentrations of ICG-TP-5.

Phototherapy can kill tumor cells by inducing apoptosis through immunogenic cell death to a certain extent. In order to determine whether the phototoxicity of ICG-TP-5 on cancer cells was associated with apoptosis, we performed double staining analysis at the cellular level using the FITC/PI method using HepG2 and HeLa cells (Fig. 4c). Compared with the control group, the apoptosis rate of ICG-TP-5 co-incubating cancer cells significantly increased after phototherapy. The apoptosis rate was positively correlated with the concentration of ICG-TP-5. The HepG2 and HeLa cells are mainly killed by inducing apoptosis under irradiation, which is consistent with the mechanism of phototherapy.

3.4. In vivo experiment

Based on the fluorescence imaging properties of ICG-TP-5, we further studied its fluorescence imaging effect and biodistribution in the tumor-bearing mice model with ICG as a control. The ICG-TP-5 and ICG were i.v. injected into mice through the tail vein and imaged using IVIS. As shown in Fig. 5a, fluorescence within the tumor can be clearly seen, and after 24 h, the tumor still maintains a certain degree of fluorescence. In addition, the fluorescence intensity in the tumors of mice injected with ICG-TP-5 was several times that of mice injected with ICG (Fig. 5b). The high fluorescence was probably due to the EPR effect and the high stability of ICG-TP-5, indicating high fluorescence imaging capability and a high phototherapy effect.

Fig. 5 In vivo experiment. After intratumoral injection of ICG-TP-5 and ICG, (a) in vivo NIR-II fluorescence images and (b) intratumoral fluorescence intensity of tumor-bearing mice at different time points. (c) In vitro fluorescence images and (d) fluorescence intensity of tumors and major organs (heart, liver, spleen, lungs, and kidneys) in different groups of mice 24 h after the administration of ICG-TP-5 through the tail vein. (e) Tumor images obtained from different groups of mice after 7 treatments. (f) Curves of body weight changes in different groups of mice during treatment. (g) Six hours after caudal intravenous injection of ICG-TP-5 (upper) and PBS (lower), thermal infrared images were obtained by laser irradiation (808 nm, 60 mW cm⁻²) at different times.

To further study the tumor accumulation in tumor-bearing mice induced by ICG-TP-5, we examined the distribution and accumulation of ICG-TP-5 in major organs after caudal vein injection. The mice were sacrificed after being injected with ICG-TP-5 for 24 h. Subsequently, we collected major organs (heart, spleen, lungs, liver and kidneys) and tumor tissue for ex vivo fluorescence imaging (Fig. 5c). Strong fluorescence was observed in the tumor tissues and lungs, while weak fluorescence was observed in the liver. Moreover, the fluorescence intensity data of the organs exhibited in Fig. 5d also showed that ICG-TP-5 could be specifically enriched in tumors and lungs, and the fluorescence intensity was much higher than that of other organs.

Since ICG-TP-5 has a remarkable killing effect in vitro and good tumor accumulation, we conducted a comprehensive evaluation of its antitumor effect in vivo. Tumor-bearing mice were randomly divided into six groups (n = 5): the control group (Control), the light-only group (Light), the drug-only group (Medium), the low-dose group (Low + light), the medium-dose group (Medium + light), and the high-dose group (High + light). Laser irradiation was performed 6 hours after each drug administration, and the drug was administered every two days. After 7 laser treatments, the tumors of the mice were stripped (Fig. 5e). The tumor volumes of the two groups that only added ICG-TP-5 and only received laser irradiation showed a negligible difference from the control group. This excludes the inhibitory effect by adding ICG-TP-5 or laser irradiation independently. Compared with the control group, the phototherapy groups showed significant tumor growth inhibition, indicating that ICG-TP-5 has obvious phototoxicity and an anti-cancer effect after laser irradiation. There was no significant difference in body weight between the experimental groups and the control groups, as shown in Fig. 5f, and no discomfort symptoms were observed in the mice during the whole experiment, indicating that ICG-TP-5 had few side effects on mice up to the high dose.

To explore whether the anti-cancer effects of ICG-TP-5 are related to the effects of PTT, we performed thermal infrared imaging in mice 6 hours after the drug was administered intravenously (Fig. 5g). The administration group (0.37 mg ICG-TP-5) had a higher temperature of 44.0 °C compared with the PBS group, showing the effect of PTT.

Furthermore, the evaluation of serum levels of IL-2, IFN-γ, CD3⁺CD4⁺, and CD3⁺CD8⁺ in mice post-treatment demonstrated that ICG-TP-5 exerts immunomodulatory effects. Given that TP-5 can enhance IL-2 and IFN-γ levels, we assessed these cytokines in the serum following treatment. As illustrated in Fig. 6a and b, the concentrations of IL-2 and IFN-γ across all treatment groups were significantly elevated compared to those in the control group, indicating that TP-5 may retain its activity post self-assembly. In tumor immunoregulation, T lymphocytes are pivotal; specifically, CD3⁺CD8⁺ T cells, also known as cytotoxic T lymphocytes (CTL), and CD3⁺CD4⁺ T cells, referred to as helper T lymphocytes (Th), play crucial yet interrelated roles in anti-tumor immunity. The T cell receptor (TCR) on CTLs is capable of specifically recognizing tumor cell surfaces to elicit cytotoxic responses, while Th cells engage with dendritic cells and B cells to orchestrate a coordinated anti-tumor immune response. To further substantiate the immune effects of ICG-TP-5, we measured serum levels of CD3⁺CD4⁺ and CD3⁺CD8⁺. As depicted in Fig. 6c and d, both populations were significantly increased across all treatment groups relative to controls. Thus, ICG-TP-5 demonstrates capabilities for PDT, PTT, and immunotherapy in inhibiting cancer cell proliferation.

Fig. 6 Histograms of serum (a) IL-2, (b) IFN-γ, (c) CD3⁺CD4⁺ and (d) CD3⁺CD8⁺ contents of mice in different groups after 7 treatments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the control).

In order to further explore the inhibitory effect of ICG-TP-5 on tumor tissues and the inherent potential of ICG-TP-5 as an anti-cancer drug, H&E staining (Fig. 7) was used for morphological analysis of tumor tissues and organs (heart, liver, spleen, lungs and kidneys). No killing effect on tumor cells was observed under light or drug administration alone, while drug + light groups showed a satisfactory anti-cancer effect.

Fig. 7 H&E staining images of tumors and tissues and organs of mice in different groups after 7 treatments. The scale is 50 μm.

In the tumor tissues treated with the Low + Light group and Medium + Light group, the nuclei of some cancer cells atrophied, ruptured or even disappeared, and the complete cell state was lost. In the tumor tissues treated with the High + Light group, the nuclei almost completely disappeared. In addition, the liver slices of the mice shown here exhibited cancer cell metastasis. Compared with the normal hepatocytes, the hepatocytes in the Control, Light and Medium groups showed large hepatocyte nuclei, occasional double nuclei, mitotic images, and an empty bright cytoplasm, accompanied by the hepatocyte cells with obvious atypia. This suggests that the cancer cells have metastasized extensively in the liver. However, in the Low + Light and Medium + Light groups, hepatocytes showed a small amount of cancer metastasis. The morphology of hepatocytes in the High + Light group showed no difference from that in the normal group, and no obvious hepatoma cells were observed (Fig. S6). Furthermore, no light was targeted at the liver site, which excludes direct phototherapy to the liver region, suggesting that ICG-TP-5 plays an immune role in stimulating an immune response to kill metastatic cancer cells in mice. In addition, no obvious pathological abnormalities were observed in other organs. This indicates that ICG-TP-5 has good biocompatibility.

4. Conclusion

Based on peptide self-assembly technology, we have successfully developed the supramolecular nanomedicine ICG-TP-5, which possesses three therapeutic strategies including PDT, PTT and immunotherapy from two classic components with enhanced phototherapy effects. The therapeutic strategies are supported by the following experiments: ROS trapping experiment utilizing DPBF confirms the ROS generation ability and PDT therapeutic effect of ICG-TP-5. The ROS generation yield of ICG-TP-5 is determined to be 13.5%. Temperature rising and in vivo thermal imaging experiments confirm the PTT effect of ICG-TP-5. IL-2 and IFN-γ level measurement experiments confirm the immunotherapeutic effects of ICG-TP-5. It is worth noting that ICG-TP-5 produces a super-large red-shifted absorption of 162 nm, which will undoubtedly increase the anticancer effect and the fluorescence imaging capability of ICG-TP-5. The clinically accessible components ICG and TP-5 demonstrate promising translational potential through this formulation. The enhanced phototherapy efficacy including super-large red-shifted absorption and high stability encourages us for further study.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

The data supporting this study are available from the corresponding author on reasonable request. Animal histopathology datasets require prior ethics approval (Wannan Medical College Ethics Committee, No. WNMC-AWE-2024430).

Supplementary information is available. The Supplementary Information file contains: Figure S1: EDX elemental mapping images of ICG-TP-5.Figures S2–S4: Particle size distribution and stability data of ICG-TP-5 under various conditions.Figure S5: Determination of reactive oxygen production rate. Figure S6: H&E staining images of liver tissues from treated mice. See DOI: https://doi.org/10.1039/d5bm00945f.

Acknowledgements

This research was supported by the Anhui Province Engineering Research Center for Dental Materials and Application and supported by the Program for Excellent Sci-tech Innovation Teams of Universities in Anhui Province (Grant No.: 2023AH010073).

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