Decreasing the aggregation of photosensitizers to facilitate energy transfer for improved photodynamic therapy

Abstract

The mode of energy transfer between photosensitizers and oxygen determines the yield of singlet oxygen (¹O₂), crucial for photodynamic therapy (PDT). However, the aggregation of photosensitizers promotes electron transfer while inhibiting pure energy transfer, resulting in the generation of the hypotoxic superoxide anion (O₂⁻) and consumption of substantial oxygen. Herein, we achieve the reduction of the aggregation of photosensitizers to inhibit electron transfer through classical chemical crosslinking, thereby boosting the production of ¹O₂. Specifically, we constructed a cross-linked hydrogel-like nanophotosensitizer (HA-TPP NHs) via amidation reactions between hyaluronic acid (HA) and tetrakis(4-aminophenyl)porphyrin (TATPP). In HA-TPP NHs, porphyrin is anchored at the crosslinking sites, preventing their close proximity. Simultaneously, HA-TPP NHs swell in a physiological environment due to water absorption, further increasing the distance between porphyrin molecules to avoid their aggregation. Compared to porphyrin–hyaluronic acid assembling nanoparticles (HA-TPP NPs), we find that the ¹O₂ generation efficiency of HA-TPP NHs is elevated by over 80%. Furthermore, leveraging the targeting capabilities of hyaluronic acid, HA-TPP NHs demonstrate a remarkable anticancer effect in in vitro and in vivo experiments. This study offers a novel insight and method for improving the therapeutic efficacy of PDT.

---

1. Introduction

The energy transfer mode can affect the catalytic performances of photosensitizers, dominating the therapeutic effect of photodynamic therapy.^1–3 Pure energy transfer modes such as physical collision change the spin direction or the orbital energy level of oxygen valence electrons to produce singlet oxygen (¹O₂).^4,51O₂ with strong oxidation capacity can destroy almost all cellular components (cell membrane,^6,7 proteins,^8,9 nucleic acid, etc.),¹⁰ showing hypertoxicity towards tumor cells, which is the dominant factor in determining the efficacy of photodynamic therapy (PDT). The electron transfer mode transforms O₂ into the superoxide anion (O₂⁻) with weak oxidation capacity,^11,12 and this process often occurs in mitochondria-related diseases such as cancers due to the leakage of electrons.^13,14 In tumor, O₂⁻ can be eliminated by over-expressed superoxide dismutase (SOD), and forms low-toxicity hydrogen peroxide (H₂O₂).^15–18 The aggregation of porphyrin photosensitizers promotes their electron transfer due to the lower LUMO–HOMO gap,^19,20 leading to the consumption of O₂ but generation of O₂⁻,²¹ which seriously influences the production of ¹O₂. Moreover, the major drawback is that widely used photosensitizers, with strong hydrophobicity and big π-conjugated structures, are prone to aggregate especially under physiological conditions.^22–24 Therefore, addressing the aggregation of photosensitizers will be of significance for further improving the clinical efficacy of PDT.

The essence of aggregation is that the interaction between photosensitizers themselves is stronger than that between photosensitizers and solvents.^25–27 Under physiological aqueous conditions, the most abundant substance is water, so enhancing the hydrophilicity of photosensitizers can decrease the aggregation of photosensitizers, such as the in vitro synthesis of water-soluble photosensitizers (TPPS4,^28,29 tetrakis(4-aminophenyl)porphyrin,^30,31 tetrakis(4-pyridyl)porphyrin,^32,33etc.), or the introduction of hydrophilic components on photosensitizers (glutathione–porphyrin,^34,35 PEG–porphyrin,³⁶etc.). Besides, the physical barrier effect is also used to reduce the aggregation of photosensitizers.^37–41 Zhang et al. reported that the POSS-porphyrin alternating copolymer reduces its aggregation through the barrier effect of POSS with a large size.³⁸ Huang et al. reported that photosensitizers were loaded into pillararenes by host–guest chemistry. Obviously, they have made some progress in decreasing the aggregation of photosensitizers, but there exist some limitations to some degree.^42,43 For example, water-soluble photosensitizers suffer from severe leakage in the process of in vivo delivery,⁴⁴ leading to serious toxicity and side effects, and the physical barrier effect will prevent photosensitizers from coming into contact with O₂,^45,46 unconducive to the production of ¹O₂.

Herein, we provide a chemical crosslinking fixation strategy for reducing the aggregation of photosensitizers and improving PDT. In detail, hydrogel-like nanoparticles (HA-TPP NHs) are constructed by crosslinking biocompatible hyaluronic acid (HA) and water-soluble 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TATPP) (Scheme 1a). TATPP, as the skeleton component, is fixed on the hydrogel-like skeleton so as to separate from each other, thus reducing the aggregation of photosensitizers. Furthermore, the distance between components can become larger in physiological solution due to the swelling of HA-TPP NHs by absorbing water, further lessening the aggregation of photosensitizers (Scheme 1b). Besides, the hydrogel-like structure allows the O₂ molecule to move freely in and out of HA-TPP NHs. Thus, HA-TPP NHs showed more production of ¹O₂. Meanwhile, in combination with the targeting ability of hyaluronic acid for the CD44 receptor,^47,48 HA-TPP NHs showed a remarkable anticancer effect in in vitro and in vivo experiments. Taking into consideration the manufacture, biocompatibility and therapeutic effect, HA-TPP NHs have the potential for clinical application. Notably, this study provides novel insights for improving the efficacy of other clinical photosensitizers (chlorin e6, porphyrin IX, hematoporphyrin, etc.).

Scheme 1 The preparation and anti-cancer mechanism of HA-TPP NHs. (a) The preparation of HA-TPP NHs and HA-TPP NPs. Inset shows the transmission electron microscopy (TEM) images of HA-TPP NHs and HA-TPP NPs respectively. (b) The chemical crosslinking fixation of photosensitizers for reducing their aggregation to produce more singlet oxygen had a better killing effect on tumor cells. Scale bar = 50 nm.

2. Experimental section

2.1 Materials

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC·HCl), dimethyl formamide (DMF), nitro blue tetrazolium (NBT), propionic acid (PA), dimethyl sulfoxide (DMSO), p-nitrobenzaldehyde, pyrrole, SnCl₂, N-hydroxysuccinimide (NHS), and 1,3-diphenylisobenzofuran (DPBF) were all purchased from Adamas. Hyaluronic acid was purchased from Aladdin. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), singlet oxygen sensor green reagent (SOSG) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime. All other reagents were purchased from commercial resources.

2.2 Preparation of the porphyrin–hyaluronic acid crosslinking nanohydrogel (HA-TPP NHs) and porphyrin–hyaluronic acid nanoparticles (HA-TPP NPs)

2.2.1 Synthesis of 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TATPP). p-Nitrobenzaldehyde (7.32 g, 60 mmol) was dissolved in 240 mL of propionic acid. After three cycles of purging with nitrogen for 15 min and vacuumizing for 5 min, pyrrole (4.02 g, 60 mmol) was added slowly to the solution at 135 °C. After reacting for 4 h, the insoluble products were collected and washed with ethanol, DMF and pyridine respectively. After drying under vacuum, the black powder 5,10,15,20-tetrakis(4-nitrophenyl)porphyrin (TNTPP) was obtained in 70% yield. Then, the black powder (7.94 g) was added into 400 mL of concentrated hydrochloric acid, and stirred for 0.5 h. Then stannous chloride (33.88 g, 150 mmol) was added to the above solution, and the system was heated to 80 °C for 1 h. After heating, deionized water (200 mL) was added to the reaction and cooled to room temperature for 24 h. The green precipitate formed was collected by suction filtration, and the precipitate was re-dissolved in deionized water (200 mL). The pH value was adjusted using ammonia until the solution turned purple, and then the purple product was collected by suction filtration. The crude product was purified to obtain a purple powder with 95% yield.

2.2.2 Synthesis of the crosslinking hydrogel of HA and TATPP (HA-TPP HG). Hyaluronic acid (400 mg, 1.0 mmol) and NHS (67.8 mg, 0.59 mmol) were dissolved in 30 mL of deionized water and stirred for 1 h. Then, 20 mL of DMSO containing TATPP (40.2 mg, 0.059 mmol) was added into the above solution. After stirring for 0.5 h, EDC (112.6 mg, 0.59 mmol) in 10 mL of DMSO was added into the above system. After that, the mixture was stirred for 24 h at room temperature. The raw products were then removed by dialysis (molecular weight cut-off = 12 000 Da) against DMSO for 24 h. Fresh DMSO was replaced every 12 h to ensure the complete removal of NHS, EDC and free TATPP. Subsequently, DMSO was removed by dialysis against deionized water for 24 h, with fresh water being replaced every 4 h. Finally, the crosslinking compound HA-TPP HG was obtained as a brown-yellow solid using a vacuum freeze dryer.

2.2.3 Preparation of the porphyrin–hyaluronic acid crosslinking nanohydrogel (HA-TPP NHs). 40 mg HA-TPP HG was dissolved in 20 mL of 1.0 M NaOH solution, and treated with ultrasound for 1 h to obtain HA-TPP NHs. NaOH was then removed by dialysis. The HA-TPP NHs were concentrated using an ultrafiltration device, and the final concentration of porphyrin in HA-TPP NHs was adjusted to 1.0 mg mL⁻¹.

2.2.4 Preparation of the porphyrin–hyaluronic acid nanoparticles (HA-TPP NPs). 10 mg HA-TPP was dissolved in 5 mL of DMSO, and this solution was slowly added into 20 mL of deionized water, and then the system was stirred for 1 h. After that, the system was subjected to dialysis against deionized water for 24 h. HA-TPP NPs were concentrated using an ultrafiltration device, and the final concentration of porphyrin in HA-TPP NPs was adjusted to 1.0 mg mL⁻¹.

2.3 Characterization

¹H NMR spectra were recorded at 400 MHz using a BRUKER AV400 spectrophotometer in DMSO-d₆. The UV-Vis spectra were recorded using a Thermo Scientific Evolution 220 spectrophotometer. The fluorescence spectra were obtained using an F-4500 fluorescence spectrophotometer. Dynamic light scattering measurements were carried out using a BECKMAN COULTER Delsa Nano particle analyser. Transmission electron microscopy analysis was performed on a JEOL 200CX at 100 kV. Confocal laser scanning microscopy (CLSM) images were obtained using a Nikon confocal microscope. MTT assay was performed using a microplate reader 5 (Bio-TekELx800, USA).

2.4 In vitro study of O₂⁻ production

O₂⁻ can specifically reduce nitro blue tetrazolium (NBT) into blue complexes, so its reduction reaction can be used to evaluate the O₂⁻ generation performance of HA-TPP NHs. In detail, 50 μL of an aqueous solution of NBT (2 mg mL⁻¹) was added to 2.0 mL of 10 μg mL⁻¹ HA-TPP NH solution, and then the system was irradiated with a 650 nm laser (1.0 W cm⁻²) for different time durations (0 s, 15 s, 30 s, 45 s, 60 s, 90 s, 120 s, 150 s, 180 s). The absorbance spectra of the solution in the wavelength range of 450–800 nm were recorded using a Thermo Scientific Evolution 220 spectrophotometer.

2.5 In vitro study of ¹O₂ production

¹O₂ can specifically degrade 1,3-diphenylisobenzofuran (DPBF), so its degradation can be used to evaluate the ¹O₂ generation performance of HA-TPP NHs. In detail, 20 μL of DPBF DMF solution (2 mg mL⁻¹) was added to 2.0 mL of 10 μg mL⁻¹ HA-TPP NH solution, and then the system was irradiated with a 650 nm laser (0.2 W cm⁻²) for different time durations (0 s, 10 s, 20 s, 30 s, 40 s, 50 s, 60 s). The absorbance spectra of the solution in the wavelength range of 300–500 nm were recorded.

2.6 Cell culture, cellular uptake and cytotoxicity in vitro

2.6.1 Cellular uptake and localization. Briefly, 4T1 cells or HEK 293t cells were seeded in 2 mL culture dishes (2 × 10⁵ cells per dish), and cultured for 24 h. The culture medium was removed, and the cells were treated with the culture medium containing HA-TPP NHs for another 4 h and 24 h respectively. The cells were stained with DAPI. After that, the cells were washed with PBS, and cellular uptake and localization were evaluated by observing the fluorescence of DAPI and TPP (λ_ex = 405 nm, λ_em = 435 nm and λ_ex = 640 nm, λ_em = 720 nm) with CLSM.

2.6.2 Detection and localization of ROS in 4T1 cells. 4T1 cells were cultured in 2 mL culture dishes for 24 h. The culture medium was then replaced with HA-TPP NHs. After 24 h, the cells were incubated with SOSG for 20 min. Then, the cells were washed with PBS, and irradiated with a 650 nm laser for 1 min (0.3 W cm⁻²). The levels of intracellular ROS and their localization were evaluated by detecting the green fluorescence of SOSG (λ_ex = 488 nm, λ_em = 525 nm).

2.6.3 Dark cytotoxicity of HA-TPP NHs towards 4T1 cells. 4T1 cells were seeded into 96-well microplates (5 × 10³ cells per well) and cultured for 24 h. Then, the culture medium was replaced with fresh medium containing HA-TPP NHs at porphyrin concentrations of 0, 0.31, 0.62, 1.25, 2.5, 5.0, 10, 20, and 40 μg mL⁻¹, respectively. After incubation for 24 h, the culture medium was replaced with FBS-free medium containing MTT. Finally, the MTT solution was replaced with DMSO after co-incubation for 4 h. Cell proliferation was evaluated using a microplate reader by comparing the absorbance at 490 nm with the control.

2.6.4 Light cytotoxicity of HA-TPP NHs. 4T1 cells were seeded into 96-well microplates (5 × 10³ cells per well) and cultured for 24 h. Then the culture medium was replaced with fresh medium containing HA-TPP NHs at porphyrin concentrations of 0, 0.16, 0.31, 0.62, 1.25, 2.5 and 5.0 μg mL⁻¹, respectively. After an additional 24 h of incubation, the cells were irradiated for 5 min (0.2 W cm⁻²) and then cultured for another 24 h. Subsequently, the culture medium was replaced with fresh medium containing 0.6 mg mL⁻¹ MTT. Finally, the MTT solution was replaced with DMSO after co-incubation for 4 h. Cell proliferation was evaluated using the same method as described above.

2.7 In vivo biocompatibility and PDT antitumor effect

2.7.1 In vivo biocompatibility. All animal experiments were conducted in accordance with the protocols and the care regulations approved by the administrative committee of laboratory animals at Tongji University (animal ethics number: SHDSYY-2022-T0047). Seven-week-old female Kunming mice (∼35 g) were sourced from Shanghai Laboratory Animal Center. After intravenous (i.v.) injection of 8 mg porphyrin per kg of HA-TPP NHs (in 200 μL of saline), with an equal volume of PBS as the control, their body weight was measured every three days (n = 3 for each group). Over one month, no significant changes were observed compared to the control group. At 3 and 30 days, the mice were sacrificed to collect their major visceral organs (heart, liver, spleen, lungs and kidneys) and blood. The organs were fixed in 10% formalin solution for histopathological analysis using hematoxylin and eosin (H&E) staining assay. The blood samples were used to analyse the blood biochemical and hematological parameters.

2.7.2 Anticancer effect in 4T1 tumor-xenografted Balb/c mice. Balb/c nude mice (∼18 g) were subcutaneously (s.c.) implanted with 1 × 10⁶ 4T1 cells in the right leg to generate xenografted tumors. When the tumor volume reached approximately 70–100 mm³ (a week post-inoculation), the mice were randomly allocated for different treatments (6 mice per group). To assess the anticancer effect of HA-TPP NHs, 100 μL of PBS, HA-TPP NHs, HA-TPP NPs, and HA-TPP NHs were injected into the 1st, 2nd, 4th and 5th group mice, respectively. After 24 h, the 2nd, 4th and 5th group mice were irradiated with a 650 nm laser for 10 min (0.50 W cm⁻²). After treatment, the tumor size was recorded every two days using a digital caliper, and their volumes (V) were calculated (V = L (mm) × W (mm)²/2), and then normalized to their initial volume (V₀) to obtain the relative tumor volume (VV₀⁻¹). Pathological sections of tumors were collected 48 h post-treatment for hematoxylin and eosin (H&E) staining analysis. Mice were euthanized once the tumor volume reached 1000 mm³.

3. Results and discussion

3.1 Construction and characterization of HA-TPP NHs

HA-TPP NH nanomaterials were prepared by the method of crosslinking reaction between tetrakis(4-aminophenyl)porphyrin and hyaluronic acid, followed by hydrolysis under alkaline conditions. Thus, we synthesized tetrakis(4-nitrophenyl)porphyrin and reduced it to synthesize TATPP using the stannous chloride reduction method,⁴⁹ and the successful synthesis of TATPP was confirmed using mass spectra and ¹H NMR spectra (Fig. S1 and S2†). Next, we crosslinked TATPP with hyaluronic acid via an amidation-mediated crosslinking reaction (Fig. 1a), obtaining crosslinked products. By analyzing the ¹H NMR spectrum and infrared spectra of the crosslinked product (Fig. 1b and c), we confirmed its successful synthesis.

Fig. 1 Preparation and characterization of HA-TPP NHs and HA-TPP NPs. (a) The concrete crosslinking structures. (b) ¹H NMR spectra of HA-TPP NHs. (c) Infrared spectra of HA-TPP NHs, TATPP and HA. (d) TEM image and DLS size of 340.0 nm (PDI: 0.200) of HA-TPP NHs. (e) TEM image and DLS size of 157.7 nm (PDI: 0.121) of HA-TPP NPs. (f) Zeta potential of HA-TPP NPs and HA-TPP NHs. (g) Stability of HA-TPP NPs and HA-TPP NHs.

After that, the crosslinked product was dispersed in a 1.0 M NaOH solution, ultrasonicated for 1 h, and then purified through ultrafiltration, obtaining porphyrin–hyaluronic acid nano-crosslinking products (HA-TPP NHs). Transmission electron microscopy (TEM) images showed that the size of the nanocomposite crosslinked product was around 200 nm (Fig. 1d), using a dynamic light scattering (DLS) size of 340.0 nm (PDI: 0.200), indicating good stability and dispersion. Besides, we also constructed porphyrin–hyaluronic acid nanoparticles (HA-TPP NPs) through amphipathicity-mediated assembly. The size of HA-TPP NPs in the TEM image ranged from 80 to 120 nm (Fig. 1e), with a DLS size of 157.7 nm (PDI: 0.121). The surface potential of HA-TPP NHs was −17.42 mV (Fig. 1f), slightly lower than that of HA-TPP NPs due to the protonation of amino-containing TATPP in HA-TPP NPs. Additionally, the DLS sizes of both HA-TPP NHs and HA-TPP NPs did not show significant changes after 2 weeks, indicating their good stability (Fig. 1g).

3.2 HA-TPP NHs generated ROS highly efficiently

According to the previous literature,⁵⁰ once photosensitizers undergo aggregation, their UV-Vis absorption peaks will redshift, and their emissions will also quench. As shown in Fig. 2a, the UV-Vis absorbance spectra of TATPP redshifted with its concentration especially its strongest absorption peak (420 nm), and the redshift distance increased with increasing concentration. Thus, this result demonstrated that TATPP aggregated in aqueous solution. Additionally, the significant quenching of the fluorescence intensity of TATPP further verified its aggregation in aqueous solution (Fig. 2b). Next, we evaluated the aggregation state of TATPP in HA-TPP NPs and HA-TPP NHs. As shown in Fig. 2c, at the same porphyrin concentration, the UV-Vis absorption peaks of HA-TPP NPs were at 439 nm, 529 nm, 578 nm and 666 nm and the absorption peaks of HA-TPP NHs were at 420 nm, 528 nm, 576 nm and 663 nm. Therefore, TATPP in HA-TPP NPs will aggregate, while the chemical crosslinking fixation strategy can prevent the aggregation of TATPP in HA-TPP NHs. Furthermore, as shown in Fig. 2d, at the same porphyrin concentration, the fluorescence intensity of HA-TPP NHs was over 20 times stronger than that of HA-TPP NPs, which further proved that the strategy can avoid the aggregation of TATPP.

Fig. 2 HA-TPP NHs for avoiding the aggregation of TATPP to generate ROS highly efficiently. (a) UV-Vis absorbance spectra and (b) fluorescence spectra of TATPP with concentrations. (c) UV-Vis absorbance spectra and (d) fluorescence spectra of HA-TPP NHs and HA-TPP NPs. (e) Reduction of NBT with HA-TPP NPs upon 650 nm laser irradiation. (f) Reduction of NBT with HA-TPP NHs upon 650 nm laser irradiation. (g) Degradation of DPBF with HA-TPP NPs upon 650 nm laser irradiation. (h) Degradation of DPBF with HA-TPP NHs upon 650 nm laser irradiation. (i) Relative absorption of DPBF after treating with HA-TPP NHs and HA-TPP NPs.

After that, we evaluated the production of O₂⁻ from HA-TPP NPs and HA-TPP NHs. As shown in Fig. 2e and f, NBT solution containing HA-TPP NPs or HA-TPP NHs showed no absorption in the wavelength range of 450 nm to 800 nm without laser irradiation. Upon laser irradiation, the absorption appeared in the relevant wavelength range and it increased with the irradiation time, suggesting that both HA-TPP NPs and HA-TPP NHs could produce O₂⁻ under laser irradiation and the production of O₂⁻ increased with the irradiation time. It is evident that the production rate of O₂⁻ in the HA-TPP NP-treated group is faster than that in the HA-TPP NH-treated group, which indicated that the aggregation of TATPP can promote electron transfer between photosensitizers and O₂, thereby enhancing the generation of O₂⁻.

Subsequently, the production of ¹O₂ from HA-TPP NPs and HA-TPP NHs was measured. DPBF is a commonly used detection probe for singlet oxygen, and its UV absorption in the range of 350–450 nm decreases after reacting with singlet oxygen.⁵¹ As shown in Fig. S3,† the UV-visible absorption of DPBF did not change under 650 nm laser light irradiation. In contrast, the UV-visible absorption of DPBF in both the HA-TPP NH group and HA-TPP NP group decreased significantly with the irradiation time under laser irradiation (Fig. 2d and e). Meanwhile, DPBF in the HA-TPP NH group degraded faster than that in the HA-TPP NP group, and the degradation rate of DPBF in the HA-TPP NH group is approximately 2.09 times higher than that in the HA-TPP NP group. The results indicated that both HA-TPP NHs and HA-TPP NPs can produce singlet oxygen under laser irradiation, and HA-TPP NHs have a better capability of producing singlet oxygen. Therefore, our proposed crosslinking fixation strategy can reduce the aggregation of the porphyrin photosensitizer and further improve its yield of ¹O₂ under light illumination.

3.3 HA-TPP NHs killed tumor cells highly efficiently

We assessed the cellular uptake of HA-TPP NHs by confocal microscopy taking advantage of the red fluorescence of porphyrin under light irradiation. As shown in Fig. 3a, 4T1 cells and HEK 293t cells were cultured with HA-TPP NHs for different time durations, and obviously the red fluorescence was observed in both 4T1 cells and HEK 293t cells and increased with the co-incubation time, indicating that HA-TPP NHs were time-dependently taken up by the cells. Additionally, due to the existence of HA in HA-TPP NHs and the over-expressed HA receptor (CD44) in cancer cells, 4T1 cells exhibited a better uptake for HA-TPP NHs compared to HEK 293t cells.

Fig. 3 Cytotoxicity evaluation of HA-TPP NHs in vitro. (a) Intracellular uptake of HA-TPP NHs. (b) The fluorescence intensity changes of the singlet oxygen sensor green (SOSG) fluorescent probe in 4T1 cells incubated with HA-TPP NHs or HA-TPP NPs under laser irradiation. (c) Cell viability of 4T1 cells after 24 h of co-incubation with HA-TPP NHs or HA-TPP NPs at porphyrin concentrations of 0, 0.31, 0.62, 1.25, 2.5, 5.0, 10.0, 20.0, and 40.0 μg mL⁻¹ respectively without illumination. (d) Cell viability of 4T1 cells after 24 h of co-incubation with HA-TPP NHs or HA-TPP NPs at porphyrin concentrations of 0, 0.16, 0.31, 0.62, 1.25, 2.5, and 5.0 μg mL⁻¹ respectively with illumination. Scale bar, 20 μm.

After that, the singlet oxygen generation capability of HA-TPP NHs in the cells was evaluated using the singlet oxygen sensor green fluorescent probe. As shown in Fig. 3b, without 650 nm laser irradiation, 4T1 cells cultured with HA-TPP NHs and HA-TPP NPs respectively did not exhibit any significant green fluorescence, meaning that there was no singlet oxygen. Upon 650 nm laser irradiation (0.3 W cm⁻², 3 min), strong green fluorescence was detected in 4T1 cells cultured with HA-TPP NHs and HA-TPP NPs, and the green fluorescence intensity in HA-TPP NHs was stronger than that in HA-TPP NPs. The results suggested that both HA-TPP NHs and HA-TPP NPs can produce singlet oxygen under light illumination while HA-TPP NHs can generate more singlet oxygen. This also validated our proposed strategy, where the immobilization of the porphyrin photosensitizer to the hydrogel backbone reduces the self-aggregation of the photosensitizer and improves the production of singlet oxygen.

Subsequently, an MTT experiment was carried out to evaluate the killing effect of HA-TPP NHs on tumor cells. As shown in Fig. 3c, in the absence of light, the cell survival rate remains above 95% even at a concentration of porphyrin up to 40 μg mL⁻¹, indicating that the material exhibits good cell safety under dark conditions. When exposed to 650 nm laser radiation, both HA-TPP NHs and HA-TPP NPs showed obvious cytotoxicity at a porphyrin concentration of 0.62 μg mL⁻¹ (Fig. 3d). Furthermore, the phototoxicity in the HA-TPP NH group was significantly higher than that in the HA-TPP NP group. These results further proved that the hyaluronic acid–porphyrin cross-linked curing could reduce the aggregation of the photosensitizer and increase the singlet oxygen yield, thus improving the photodynamic killing performance of tumor cells.

3.4 Anticancer effect of HA-TPP NHs on tumor cells in vivo

Before evaluating the antitumor effect of HA-TPP NHs in vivo, the biological safety of HA-TPP NHs in Kunming mice was evaluated. Nine mice were evenly divided into three groups, and HA-TPP NHs were administered through the tail vein. The weight of the mice was measured every 3 days. As shown in Fig. S4,† compared with the control group, it was obvious that HA-TPP NHs did not affect the growth of the mice. Furthermore, the blood biochemical parameters and hematological data of the mice in the HA-TPP NH-treated group (Fig. 4a and b) were nearly identical to those in the control group. Additionally, the pathological hematoxylin and eosin (H&E) stained images of major organs (heart, liver, spleen, lungs and kidneys) did not show obvious damage (Fig. S5†), which was similar to the control group. These findings demonstrate that HA-TPP NHs exhibit excellent biological safety.

Fig. 4 Biosafety of HA-TPP NHs. (a) The blood biochemical parameters and (b) hematological data obtained from the mice after intravenous injection of HA-TPP NHs (10 mg kg⁻¹, 200 μL, n = 3, mean ± s.d.) at 3 and 30 days with injection of 200 μL of PBS as the control.

The photodynamic antitumor effect of HA-TPP NHs was then evaluated against tumor xenografts in 4T1-tumor-bearing mice under 650 nm laser irradiation (0.5 W cm⁻², 5 min). The tumor volume, weight, and mouse body weight after laser exposure were recorded (Fig. 5a–d). As shown in Fig. 5a, the tumor in the control group grew rapidly, but tumor growth was significantly inhibited in both the HA-TPP NH-treated and HA-TPP NP-treated groups. Furthermore, compared to HA-TPP NPs, HA-TPP NHs displayed a better inhibitory effect for tumor growth. The isolated tumor weight and images further proved this (Fig. 5c, d and S6†). The H&E-stained images of tumor tissues in each group after 48 h of treatment are shown in Fig. 5e, and no tissue necrosis was found in the control group, laser group, and HA-TPP NH group without laser irradiation; however, serious tissue necrosis was found in the HA-TPP NPs + laser and HA-TPP NHs + laser groups. These results confirm that HA-TPP NHs under light irradiation have higher photodynamic therapeutic efficacy. Meanwhile, there was almost no side effect on the growth of mice during tumor treatment (Fig. 5b), indicating that HA-TPP NH-mediated PDT exhibit excellent safety. Therefore, the crosslinking fixation strategy can reduce the aggregation of porphyrin photosensitizers to promote the production of singlet oxygen and improve the efficacy of PDT.

Fig. 5 Anti-tumor assessment of HA-TPP NHs in vivo. (a) Tumor growth curves of mice after treatment (V₀ and V stand for the tumor volume before and after treatment, respectively). (b) Body weight of tumor-bearing mice. (c) Weight of isolated tumors. (d) Images of isolated tumors after excision. (e) Pathological H&E stained images of the tumor section at 48 h after treatment (n = 5, mean ± s.d.). Scale bars: 100 μm. P values were obtained with one-way ANOVA followed by post hoc Tukey's test; p values **P < 0.01, ****P < 0.0001.

4. Conclusions

In summary, we successfully prepared porphyrin–hyaluronic acid nano-hydrogel photosensitizers (HA-TPP NHs) based on a chemical crosslinking reaction. In HA-TPP NHs, the porphyrin photosensitizer exhibited minimal aggregation phenomena; thus, HA-TPP NHs generated plenty of highly toxic singlet oxygen upon laser irradiation. Meanwhile, by combining the targeting capability of HA, HA-TPP NHs generated good toxicity towards tumor cells. In both in vitro and in vivo experiments, HA-TPP NHs exhibited an excellent anti-cancer effect on 4T1 tumor-bearing mice. This strategy, which reduces self-aggregation of photosensitizers by fixing photosensitizers in the hydrogel skeleton to enhance photodynamic therapy, is not only applicable for porphyrin photosensitizers but also other photosensitizers with aggregation quenching. This study provides new insights into the development of hydrophobic photosensitizers for PDT and holds great potential for practical applications.

Author contributions

Zhenhua Wang, Jianyang Ao, and Chaochao Wang designed the related experiments and prepared the nanomaterials. Zhenhua Wang, Gang Liu, Jianyang Ao, Yun Meng and Yurong Zhang evaluated the functions of nanomaterials in vitro and in vivo. Jianyang Ao, Jieyun Shi, and Chaochao Wang analysed and arranged the data. Zhenhua Wang, Jianyang Ao, Yelin Wu, and Chaochao Wang wrote the paper. All authors discussed the results and commented on the manuscript.

Data availability

The data supporting this article have been included as part of the ESI† and the digital data of this study are available from the corresponding authors upon reasonable request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation and the Postdoctoral Science Foundation of China. The authors greatly acknowledge the financial support from the National Natural Science Foundation of China (grant no. 82202304), the China Postdoctoral Science Foundation (2021M692434, 2023M742659), and the Postdoctoral Fellowship Program of CPSF under grant number GZC20241236.

References

1. J. Großkopf, T. Kratz, T. Rigotti and T. Bach, Chem. Rev., 2022, 122, 1626–1653.
2. B. Zheng, D. Zhong, T. Xie, J. Zhou, W. Li, A. Ilyas, Y. Lu, M. Zhou and R. Deng, Chem, 2021, 7, 1615–1625.
3. H. Wang, J. Guo, W. Lin, Z. Fu, X. Ji, B. Yu, M. Lu, W. Cui, L. Deng, J. W. Engle, Z. Wu, W. Cai and D. Ni, Adv. Mater., 2022, 34, 2110283.
4. C. Lochenie, S. Duncan, Y. Zhou, L. Fingerhut, A. Kiang, S. Benson, G. Jiang, X. Liu, B. Mills and M. Vendrell, Adv. Mater., 2024, 36, 2404107.
5. X. Zhang, Y. Hou, X. Xiao, X. Chen, M. Hu, X. Geng, Z. Wang and J. Zhao, Coord. Chem. Rev., 2020, 417, 213371.
6. H. Shi, T. Wu, M. Duan, J. Yu, M. Liu, X. Wen, L. Wang and Y. Xu, Nano Lett., 2024, 24, 6939–6947.
7. Y. Jiang, Z. Wang, J. Huang, F. Yan, Y. Du, C. He, Y. Liu, G. Yao and B. Lai, Chem. Eng. J., 2022, 439, 135788.
8. R. L. Jensen, J. Arnbjerg and P. R. Ogilby, J. Am. Chem. Soc., 2012, 134, 9820–9826.
9. R. L. Jensen, J. Arnbjerg, H. Birkedal and P. R. Ogilby, J. Am. Chem. Soc., 2011, 133, 7166–7173.
10. J. Davies, D. Burke, J. R. Olliver, L. J. Hardie, C. P. Wild and M. N. Routledge, Gut, 2007, 56, 155.
11. S.-j. Liu, F.-t. Li, Y.-l. Li, Y.-j. Hao, X.-j. Wang, B. Li and R.-h. Liu, Appl. Catal., B, 2017, 212, 115–128.
12. Y.-Y. Wang, Y.-C. Liu, H. Sun and D.-S. Guo, Coord. Chem. Rev., 2019, 395, 46–62.
13. J. Marin-Corral, J. Minguella, A. L. Ramirez-Sarmiento, S. N. A. Hussain, J. Gea and E. Barreiro, Eur. Respir. J., 2009, 33, 1309–1319.
14. J.-a. Kim, Y. Wei and J. R. Sowers, Circ. Res., 2008, 102, 401–414.
15. G. H. W. Wong and D. V. Goeddel, Science, 1988, 242, 941–944.
16. G. H. W. Wong, J. H. Elwell, L. W. Oberley and D. V. Goeddel, Cell, 1989, 58, 923–931.
17. Y. Du, X. Zhao, F. He, H. Gong, J. Yang, L. Wu, X. Cui, S. Gai, P. Yang and J. Lin, Adv. Mater., 2024, 36, 2403253.
18. S. Zhang, X. Ji, Z. Liu, Z. Xie, Y. Wang, H. Wang and D. Ni, J. Am. Chem. Soc., 2024, 146, 22530–22540.
19. C.-L. Wang, C.-M. Lan, S.-H. Hong, Y.-F. Wang, T.-Y. Pan, C.-W. Chang, H.-H. Kuo, M.-Y. Kuo, E. W.-G. Diau and C.-Y. Lin, Energy Environ. Sci., 2012, 5, 6933–6940.
20. L. Chen, Y. Jiang, H. Nie, P. Lu, H. H. Y. Sung, I. D. Williams, H. S. Kwok, F. Huang, A. Qin, Z. Zhao and B. Z. Tang, Adv. Funct. Mater., 2014, 24, 3621–3630.
21. X. Li, D. Lee, J. D. Huang and J. Yoon, Angew. Chem., Int. Ed., 2018, 57, 9885–9890.
22. H.-J. Schneider, Acc. Chem. Res., 2015, 48, 1815–1822.
23. Y. Xu, Z. Han, P. Du and X. Lu, TrAC, Trends Anal. Chem., 2023, 165, 117136.
24. J. M. Park, K.-I. Hong, H. Lee and W.-D. Jang, Acc. Chem. Res., 2021, 54, 2249–2260.
25. F.-p. Zhai, H.-e. Wei, Y. Liu and F.-y. Hu, J. Mol. Model., 2019, 25, 181.
26. B. S. Li, X. Huang, H. Li, W. Xia, S. Xue, Q. Xia and B. Z. Tang, Langmuir, 2019, 35, 3805–3813.
27. W. Pedrycz and M. Song, IEEE Trans. Fuzzy Syst., 2011, 19, 527–539.
28. N. Tsolekile, V. Ncapayi, G. K. Obiyenwa, M. Matoetoe, S. Songca and O. S. Oluwafemi, Int. J. Nanomed., 2019, 14, 7065–7078.
29. Y. Li, C. Liu, X. Bai, F. Tian, G. Hu and J. Sun, Angew. Chem., Int. Ed., 2020, 59, 3486–3490.
30. C. C. Wamser, R. R. Bard, V. Senthilathipan, V. C. Anderson, J. A. Yates, H. K. Lonsdale, G. W. Rayfield, D. T. Friesen and D. A. Lorenz, J. Am. Chem. Soc., 1989, 111, 8485–8491.
31. S. Belali, A. R. Karimi and M. Hadizadeh, Int. J. Biol. Macromol., 2018, 110, 437–448.
32. D. Wang, L. Niu, Z.-Y. Qiao, D.-B. Cheng, J. Wang, Y. Zhong, F. Bai, H. Wang and H. Fan, ACS Nano, 2018, 12, 3796–3803.
33. W. Zhou, X. Ma, J. Xiao, X. He, C. Liu, X. Xu, T. Viitala, J. Feng and H. Zhang, Small Sci., 2024, 4, 2300192.
34. C. Wang, P. Zhao, D. Jiang, G. Yang, Y. Xue, Z. Tang, M. Zhang, H. Wang, X. Jiang, Y. Wu, Y. Liu, W. Zhang and W. Bu, ACS Appl. Mater. Interfaces, 2020, 12, 5624–5632.
35. C. Deng, M. Zheng, S. Han, Y. Wang, J. Xin, O. Aras, L. Cheng and F. An, Adv. Funct. Mater., 2023, 33, 2300348.
36. M. Sibrian-Vazquez, T. J. Jensen and M. G. H. Vicente, J. Photochem. Photobiol., B, 2007, 86, 9–21.
37. T. Liu, Y. Yang, S. Cao, R. Xiang, L. Zhang and J. Yu, Adv. Mater., 2023, 35, 2210895.
38. J. Jin, Y. Zhu, Z. Zhang and W. Zhang, Angew. Chem., Int. Ed., 2018, 57, 16354–16358.
39. X. Du, C. Zhao, Y. Luan, C. Zhang, M. Jaroniec, H. Huang, X. Zhang and S.-Z. Qiao, J. Mater. Chem. A, 2017, 5, 21560–21569.
40. Q. Wang, W. Wang, B. Yan, W. Shi, F. Cui and C. Wang, Chem. Eng. J., 2017, 326, 182–191.
41. X.-Y. Lou, G. Zhang, M.-H. Li and Y.-W. Yang, Nano Lett., 2023, 23, 1961–1969.
42. Z. Liu, T. Cao, Y. Xue, M. Li, M. Wu, J. W. Engle, Q. He, W. Cai, M. Lan and W. Zhang, Angew. Chem., Int. Ed., 2020, 59, 3711–3717.
43. J. Su, H. Sun, Q. Meng, P. Zhang, Q. Yin and Y. Li, Theranostics, 2017, 7, 523–537.
44. S. S. Lucky, N. M. Idris, Z. Li, K. Huang, K. C. Soo and Y. Zhang, ACS Nano, 2015, 9, 191–205.
45. Z. Jiang, Y. Liu, R. Shi, X. Feng, W. Xu, X. Zhuang, J. Ding and X. Chen, Adv. Mater., 2022, 34, 2110094.
46. L. Boivin and P. D. Harvey, ACS Appl. Mater. Interfaces, 2023, 15, 13844–13859.
47. J. Gao, F. Wang, S. Wang, L. Liu, K. Liu, Y. Ye, Z. Wang, H. Wang, B. Chen, J. Jiang, J. Ou, J. C. M. van Hest, F. Peng and Y. Tu, Adv. Sci., 2020, 7, 1903642.
48. K. Y. Choi, H. S. Han, E. S. Lee, J. M. Shin, B. D. Almquist, D. S. Lee and J. H. Park, Adv. Mater., 2019, 31, 1803549.
49. N. M. Loim, N. V. Abramova and V. I. Sokolov, Mendeleev Commun., 1996, 6, 46–47.
50. V. Punitharasu, M. F. M. Kavungathodi and J. Nithyanandhan, ACS Appl. Mater. Interfaces, 2017, 9, 32698–32712.
51. R. Vankayala, C.-C. Lin, P. Kalluru, C.-S. Chiang and K. C. Hwang, Biomaterials, 2014, 35, 5527–5538.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr04593a

‡ These authors contributed equally to this work.

---