Hydroxyl-modified fullerene C₇₀(OH)₈ induces pyroptosis for cancer therapy

Abstract

Fullerene (C₇₀), a promising new photosensitizer, faces challenges in its biological applications due to its extreme hydrophobicity. In order to enhance the solubility of fullerene (C₇₀) and facilitate its biological applications, we synthesized a novel hydroxyl-modified fullerene compound (C₇₀(OH)₈) with excellent photosensitizing properties. The introduction of hydroxyl groups allows it to self-assemble with DSPE-PEG(2000); therefore, we prepared it as a nanomedicine (C₇₀(OH)₈@NP). Under white light irradiation, C₇₀(OH)₈@NP stimulates the production of reactive oxygen species (ROS). Furthermore, results have demonstrated that a substantial amount of ROS can also be generated within cells, resulting in cell death. We found that C₇₀(OH)₈@NP can induce both apoptosis and pyroptosis in HeLa cells and identified its mechanism of cell death through the activation of caspase 3/gasdermin E pathways. Importantly, C₇₀(OH)₈@NP demonstrates significant anti-tumor activity in a nude mouse tumor-bearing model. These results highlight the potential of novel fullerene compounds as photodynamic therapy agents.

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Introduction

Carbon, a fundamental element in materials science, manifests in diverse allotropes with distinct structural and electronic properties. Among these, fullerene—a closed-cage carbon nanomolecule—has garnered significant scientific interest since the landmark synthesis of C₆₀ by Curl, Kroto, and Smalley in 1985.^1,2 Unlike graphite and diamond, which have simpler structures, fullerenes consist of a combination of pentagonal and hexagonal carbon rings, giving them remarkable curvature-dependent reactivity, photostability, and ability to accept electrons.^3,4 Based on their diverse activities, fullerenes have been extensively studied for their cellular antioxidant properties and other biological effects in plants.^5–8 These properties have driven research into their potential biomedical applications, especially in photodynamic therapy (PDT).^9–11 In PDT, light activates photosensitizers to produce cytotoxic reactive oxygen species (ROS), which can eradicate malignant cells. Despite their promise, pristine fullerenes such as C₆₀ and C₇₀ face translational challenges due to their extreme hydrophobicity and poor biocompatibility. To address these issues, functionalization strategies—such as conjugation with hydrophilic groups such as carboxyl and hydroxyl groups or introducing quaternary ammonium salt groups—have been developed to improve solubility and biological targeting.^12–14 Despite these advancements, the structure-dependent therapeutic mechanisms of fullerene derivatives remain relatively unexplored.

PDT has emerged as a versatile clinical modality, enabled by advances in light-delivery technologies for treating various malignancies, including those of the skin, viscera, and brain.^15,16 The anticancer effects of PDT can manifest directly by inducing cancer cell death or indirectly through damage to the vascular system and activation of the immune response.^17,18 The effectiveness of a photosensitizer in PDT is primarily determined by its ability to sustain ROS generation through type I (electron transfer) or type II (energy transfer) pathways without experiencing photodegradation.¹⁹ C₆₀ and C₇₀ fullerenes offer several advantages over conventional photosensitizers, such as higher photostability and reduced photobleaching.^20,21 However, C₇₀ fullerene presents distinct advantages that position it as a superior candidate for PDT, as its higher quantum yield of ROS generation translates to greater efficiency in converting light energy into cytotoxic oxidative stress, overwhelming cellular antioxidant defenses and triggering apoptosis.²² Although C₆₀ exhibits greater stability, this characteristic also restricts its potential as a drug due to its resistance to decomposition in biological environments. In contrast, C₇₀ possesses moderate stability and can be metabolized and degraded, offering greater potential for development as a biological reagent. However, research on C₇₀ remains limited.²¹ Ikeda et al. found that fullerenes (acting as sensitizers), encapsulated in a hydrophobic γ-cyclodextrin (γ-CD) cavity, displayed an exchange reaction with liposomal and cell membranes only in the case of C₇₀, but not in the case of C₆₀.²³ Therefore, C₇₀'s photophysical robustness and enhanced biocompatibility position it as a next-generation photosensitizer, effectively addressing key limitations of conventional PDT agents like C₆₀.

Inspired by these insights, we present the design of C₇₀(OH)₈@NP, a polyhydroxy-functionalized C₇₀ derivative integrated with a lipid–polymer conjugate, aimed at tumor-targeted PDT. This novel agent addresses solubility challenges and unveils an unconventional cell death mechanism. As shown in Scheme 1, unlike apoptosis, C₇₀(OH)₈@NP triggers pyroptosis—a lytic, inflammatory pathway mediated by caspase 3 and gasdermin E (GSDME)—marking the first instance of fullerene-induced pyroptosis in cancer therapy. In vivo studies demonstrate strong antitumor efficacy, highlighting the untapped potential of C₇₀ as a photosensitizer in PDT. Our findings redefine the understanding of fullerene-mediated cytotoxicity, providing a strategic framework for optimizing carbon-based therapeutics and offering new insights into programmed cell death pathways.

Scheme 1 (a) Preparation process of C₇₀(OH)₈@NP. (b) Schematic representation of the mechanism of C₇₀(OH)₈@NP-induced pyroptosis and apoptosis of tumor cells.

Results and discussion

Synthesis and characterization

Compound C₇₀Cl₈ was synthesized from C₇₀ in a single step using the previously reported procedure.^24,25 To obtain hydroxylated C₇₀, we explored various classical hydrolysis conditions; however, none of these methods produced the desired product. When we treated C₇₀Cl₈ with an excess of acetic acid (CH₃COOH) and AgClO₄ in o-dichlorobenzene (o-DCB), we successfully isolated C₇₀(OH)₈ with a yield of 20%, and the synthesis route is shown in Fig. 1a. We believe that the reaction benefited from the phase transfer effect of acetic acid, which facilitated effective contact between water and the fullerene derivative. The use of o-dichlorobenzene (o-DCB) as a solvent resulted in the system being suspended both at the beginning and the end of the reaction due to the insolubility of C₇₀Cl₈ and the significant polarity of the product. We tested several solvent mixtures, including chloroform/acetone, chloroform/tetrahydrofuran (THF)/water, and trichloroethylene (TCE)/methanol; however, the best separation results were achieved using a tetrachloroethane/methanol (7 : 3) mixture as the eluent. All plots exhibited fluorescence characteristics similar to those of C₇₀(OCH₃)₈, with the final plot representing the target product.²⁴ The proton nuclear magnetic resonance (¹H NMR) spectra corroborated the structures depicted in Fig. 1, with all signals appearing sharp, including those from the hydroxyl groups when dimethyl sulfoxide (DMSO) was employed as the solvent. The protons displayed four peaks in the ¹H NMR spectrum (Fig. 1b), indicating the symmetry of the product, which was also evident in the ¹³C NMR data (Fig. 1c). In the sp³ region of the ¹³C NMR spectrum, there were four groups of peaks corresponding to tertiary carbons bonded to the hydroxyl groups on the carbon cage. Additionally, cross peaks of the hydroxyl hydrogen and the sp³ carbons were detectable in the ¹H-detected heteronuclear multiple-bond correlation (HMBC) spectrum (Fig. 1d).

Fig. 1 Synthesis and characterization. (a) Synthetic route and characterization of C₇₀(OH)₈. (b) ¹H NMR spectra of C₇₀(OH)₈. (c) ¹³C NMR spectra of C₇₀(OH)₈. (d) HMBC spectra of C₇₀(OH)₈.

Preparation and photoactivity of C₇₀(OH)₈@NP

We synthesized C₇₀(OH)₈ and conducted solubility tests, which revealed that its poor water solubility hinders its dissolution in the culture medium and limits cellular uptake. To address this challenge, as shown in Fig. 2a, we encapsulated C₇₀(OH)₈ using DSPE-PEG (2000), resulting in a water-soluble nanomedicine designated as C₇₀(OH)₈@NP. DSPE-PEG (2000), an FDA-approved amphiphilic phospholipid polymer conjugate, enhances the water solubility of compounds and serves as a promising medicinal excipient.²⁶ To estimate the concentration of C₇₀(OH)₈, we measured its absorbance at 405 nm (Fig. S1†). Dynamic light scattering (DLS) experiments indicated that the hydrodynamic diameter of C₇₀(OH)₈@NP is approximately 7 nm (Fig. 2b). Furthermore, transmission electron microscopy (TEM) demonstrated that C₇₀(OH)₈@NP exhibits a spherical morphology with a radius of approximately 5–10 nm (Fig. 2c).

Fig. 2 Characterization and activity of C₇₀(OH)₈ and C₇₀(OH)₈@NP. (a) Preparation strategy for C₇₀(OH)₈@NP. (b) Size distribution of C₇₀(OH)₈@NP obtained via DLS. (c) Morphology of C₇₀(OH)₈@NP observed through TEM. (d–f) UV–vis spectra of DPBF after oxidation using C₇₀(OH)₈@NP, C₇₀(OH)₈, and the solvent (DSPE) under white light irradiation (2.5 mW cm⁻²). (g) Absorption curve of DPBF at 416 nm indicating the ability of C₇₀(OH)₈ and C₇₀(OH)₈@NP to promote singlet oxygen (¹O₂) generation. (h–j) Emission spectra of DHE after oxidation using C₇₀(OH)₈@NP, C₇₀(OH)₈, and the solvent (DSPE) under white light irradiation (2.5 mW cm⁻²). (k) Emission curve of DHE at 635 nm demonstrating the ability of C₇₀(OH)₈ and C₇₀(OH)₈@NP to promote O₂⁻ generation. (l) Decay traces of C₇₀(OH)₈ (20 μg mL⁻¹) in water recorded under nitrogen (N₂) and oxygen(O₂) atmospheres.

To evaluate the reactive oxygen species (ROS) generated by C₇₀(OH)₈@NP, we used the 1,3-diphenylisobenzofuran (DPBF) probe to measure the generation of singlet oxygen (¹O₂) based on the change in absorption intensity (ΔA) at 416 nm.²⁷ We also utilized dihydroethidium (DHE) to detect superoxide anions (O₂⁻) with excitation/emission wavelengths of 518/616 nm.²⁸ As shown in Fig. 2d–g, C₇₀(OH)₈@NP, when irradiated in DPBF (60 μM) under an air atmosphere (DMSO/H₂O = 50%/50%) with white light at an intensity of 2.5 mW cm⁻², produced a high yield of singlet oxygen (ΔA ∼ 0.43, background corrected). In contrast, C₇₀ generated low yields of singlet oxygen (ΔA < 0.08, background corrected), likely due to its poor water solubility, which hinders the yield of ¹O₂. Fig. 2h–k show that the total yield of O₂⁻ is nearly identical; however, the rate of the reaction of C₇₀(OH)₈@NP is significantly faster than that of the reaction of C₇₀(OH)₈, highlighting the importance of solubility. As control, there is almost no production of ROS under dark conditions; the absorption spectrum of DPBF under dark conditions is depicted in Fig. S2† and the excitation spectrum of DHE under dark conditions is shown in Fig. S3.†

Then, we performed nanosecond transient absorption spectroscopy to monitor the triplet excited states of C₇₀(OH)₈ in deaerated water. As illustrated in Fig. 2l, C₇₀(OH)₈ at a concentration of 20 μg mL⁻¹ exhibited negative absorption centered at 390 nm, indicating ground state bleaching (Fig. S4†), with positive absorption peaking at 410 nm. The lifetime of the triplet state was found to be approximately 72.3 μs. However, in the presence of oxygen, there was significant quenching of the triplet state of C₇₀(OH)₈, reducing the lifetime to 6.9 μs, which is much shorter than that observed for C₇₀(OH)₈ under a nitrogen atmosphere. This finding supports the conclusion that C₇₀(OH)₈ acts as a photosensitizer, sensitizing oxygen in its triplet state.

Photocytotoxicity and cell death mechanism

Next, we performed the cytotoxicity assay in HeLa cells. Under white light irradiation (30 mW cm⁻² for 10 or 20 minutes), we observed that C₇₀(OH)₈ significantly inhibited the growth of HeLa cells, with IC₅₀ values of 18.2 μg mL⁻¹ and 9.8 μg mL⁻¹, respectively (Fig. 3a). In contrast, when HeLa cells were treated with C₇₀(OH)₈@NP under the same irradiation conditions, the IC₅₀ values decreased to 5.1 μg mL⁻¹ and 2.9 μg mL⁻¹ (Fig. 3b). This difference in cytotoxicity can be attributed to the enhanced solubility of C₇₀(OH)₈@NP, which facilitates better cell uptake. Furthermore, both C₇₀(OH)₈ and C₇₀(OH)₈@NP exhibited no cytotoxicity towards HeLa cells in the dark, highlighting their safety. These phototoxicity results suggest that C₇₀(OH)₈ may serve as a new PDT photosensitizer (Fig. 3c).

Fig. 3 Cytotoxicity and ROS. Cytotoxicity of C₇₀(OH)₈ (a) and C₇₀(OH)₈@NP (b) towards HeLa cells when treated under white light conditions (30 mW cm⁻², 10 min and 20 min) and dark conditions (c). (d) Live HeLa cells stained with DCFH-DA for intracellular ROS production after treating with C₇₀(OH)₈ and C₇₀(OH)₈@NP under white light conditions (30 mW cm⁻², 10 min and 20 min) and dark conditions. (e) Apoptosis index of HeLa cells treated with C₇₀(OH)₈@NP (control), C₇₀(OH)₈@NP (20 μg mL⁻¹ under dark conditions), and C₇₀(OH)₈@NP (2.5, 10, and 20 μg mL⁻¹ at 30 mW cm⁻², 10 min) for 24 h detected by the AnnexinV/PI flow cytometry assay.

We utilized confocal microscopy with DCFH-DA probes to monitor the production of reactive oxygen species (ROS) following the treatment of HeLa cells with C₇₀(OH)₈@NP under white light conditions (30 mW cm⁻² for 10 minutes). This observation aligns with our in vitro results, indicating that cell death is associated with ROS production. As evidence, we conducted cytotoxicity experiments using N-acetylcysteine (NAC, an ROS scavenger);^29,30 we pre-treated HeLa cells with NAC (10 μM) three hours before administering subsequent treatments. The results indicated that the cell survival rate in the NAC group (with an IC₅₀ value of 7.4 μg mL⁻¹) was significantly higher than that in the no NAC group (with an IC₅₀ value of 5.1 μg mL⁻¹), and within the concentration range of 2.5–50 μg mL⁻¹, the cell survival rate varied between 19% and 3%, demonstrating the protective effect of NAC, likely due to its interaction with ROS (Fig. S5†). As illustrated in Fig. 3d, no ROS were detected in the dark group when cells were treated with C₇₀(OH)₈@NP, confirming low dark cytotoxicity and underscoring the inherent safety of the photosensitizers. To further investigate the type of cell death induced by C₇₀(OH)₈@NP, we conducted Annexin V-FITC/PI staining. The results presented in Fig. 3e demonstrate that exposure to C₇₀(OH)₈@NP under white light conditions (30 mW cm⁻² for 10 minutes) resulted in both early and late apoptosis in HeLa cells in a concentration-dependent manner. This suggests that C₇₀(OH)₈@NP can effectively induce cell apoptosis.

Through flow cytometry analysis, we have determined that the cells have undergone apoptosis. To further investigate the cell death pathway, we evaluated apoptosis-related proteins using western blotting. Caspase 3, a cysteine-aspartate protease, is a crucial member of the caspase family. It plays a vital role in the process of cell apoptosis and is regarded as the most significant terminal executioner enzyme in the apoptotic pathway.³¹ Caspase 3 typically exists in the form of an inactive zymogen. When cells receive apoptotic signals, caspase 3 is activated, resulting in the production of cleaved caspase 3, which induces cell apoptosis through a cascade amplification effect. At the onset of cell apoptosis, the activation of caspase 3 leads to the cleavage and inactivation of PARP, thereby promoting cell death.^32,33 Consequently, cleaved caspase-3 and cleaved PARP serve as markers of apoptosis in HeLa cells. The results presented in Fig. 4a indicate an upregulation of cleaved PARP and cleaved caspase-3 when HeLa cells were treated with C₇₀(OH)₈@NP (20 μg mL⁻¹) under white light conditions (30 mW cm⁻² for 10 minutes). Furthermore, since photosensitizers have been shown to induce cell pyroptosis through caspase-3-mediated activation of GSDME protein, a novel form of cell death distinct from apoptosis,³⁴ we analyzed the downstream GSDME protein. The results demonstrated that GSDME was activated, exposing the N-terminus (GSDME-N), which signifies the occurrence of cell pyroptosis. These findings suggest a mechanism in which both apoptosis and pyroptosis may be involved, as illustrated in Fig. 4b.

Fig. 4 Cell death mechanism. (a) Expression of key apoptosis- and pyroptosis-related proteins in HeLa cells treated with C₇₀(OH)₈@NP under white light conditions (30 mW cm⁻², 10 min) detected using western blotting. (b) The proposed mechanism of cell death induced by C₇₀(OH)₈@NP.

Antitumor effects in vivo

We further investigated the antitumor effects of C₇₀(OH)₈@NP in vivo using a HeLa tumor-bearing model in BALB/c female nude mice. Once the tumor volume reached approximately 100 mm³, we randomly divided the mice into three groups (n = 4 each). On the first and third days of the treatment, the groups received different interventions: one group was treated with PBS, another received C₇₀(OH)₈@NP under dark conditions, and the third group was administered C₇₀(OH)₈@NP under irradiation conditions. The treatment involved an intratumoral dose of 2 mg kg⁻¹ C₇₀(OH)₈@NP with exposure to white light at a power density of 0.2 W cm⁻² for 10 minutes. The treatment process is shown in Fig. 5a; we measured tumor volumes every two days over a period of 15 days using a Vernier caliper (tumor volume = length × width²/2).³⁵ As shown in Fig. 5b and c, the C₇₀(OH)₈@NP-irradiation group exhibited a significant reduction in tumor growth, while the control group and the C₇₀(OH)₈@NP-dark group showed almost no inhibition of tumor growth, with the tumor volume in the C₇₀(OH)₈@NP-irradiation group reduced to approximately 24.0% of the control group (Fig. 5d), and the tumor weight at the endpoint of treatment indicated that the treatment effect of the C₇₀(OH)₈@NP-irradiation group was significant, as the tumor weight was only 35.0% of that observed in the control group. Additionally, we assessed body weight (Fig. 5e) and conducted hematoxylin and eosin (H&E) staining on major organs (heart, liver, spleen, lungs, and kidneys). The results showed no significant tissue damage or apparent histopathological abnormalities in any of the groups (Fig. S6†), suggesting that C₇₀(OH)₈@NP has a reasonable safety profile. Furthermore, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay of tumor sections (Fig. 5f) revealed a marked increase in apoptotic cells within the C₇₀(OH)₈@NP-irradiation group.³⁶ This finding is consistent with the results from extracorporeal flow cytometry, providing supporting evidence for our proposed mechanistic pathway. Therefore, these results further demonstrate that C₇₀(OH)₈@NP has antitumor effects in vivo, indicating its potential as a PDT photosensitizer.

Fig. 5 Antitumor effects in vivo. (a) Schematic illustration of the antitumor process in mice under white light irradiation at a power density of 0.2 W cm⁻² for 10 minutes. (b and c) Tumor volume curves and digital photographs of the mice of different treatment groups (Control, C₇₀(OH)₈@NP-Dark, and C₇₀(OH)₈@NP-Light). (d) Tumor weight after all the mice of the above listed different groups were sacrificed. (e) Body weight over 15 days of all mice of the above listed different groups. (f) Tumor slice of the above listed different groups analyzed by the TUNEL assay for in situ tumor cell apoptosis, scale bar = 100 μm. The results are presented as the mean ± SD; ***p < 0.001 compared with the control group.

Conclusions

In summary, we have synthesized a novel hydroxyl-modified fullerene compound, C₇₀(OH)₈, which exhibits unprecedented photosensitizing properties. Its high hydrophobicity has been improved, and its water solubility can be significantly enhanced through self-assembly with DSPE-PEG (2000). Under white light irradiation, C₇₀(OH)₈@NP stimulates the production of reactive oxygen species, leading to cell death. The western blotting assay revealed that C₇₀(OH)₈@NP activates cleaved PARP, cleaved caspase 3, and GSDME-N, indicating that HeLa cells undergo both apoptosis and pyroptosis. Additionally, C₇₀(OH)₈@NP demonstrates significant in vivo anti-tumor activity in a nude mouse tumor-bearing model. These results highlight the potential of this novel fullerene as a photodynamic therapy agent, promoting its biological application through multi-hydroxyl modification and providing new candidate photosensitizers for future photodynamic therapy, and this advancement also significantly broadens the application scope of fullerenes.

Author contributions

Conceptualization: H. Z., J.-L. Z. and L. G. Data curation: H. Z. and Z. L. Investigation: H. Z., J.-L. Z. and L. G. Project administration: H. Z. and Z. L. Writing – original draft: H. Z. and Z. L. Writing – review & editing: H. Z., Z. L., X.-F. Z. and J.-L. Z. Funding acquisition: X.-F. Z., J.-L. Z. and L. G. Supervision: J.-L. Z. and L. G.

Data availability

The original contributions presented in the study are included in the article and its ESI† and further inquiries can be directed to the corresponding authors.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Jun-Long Zhang acknowledges the financial support from the National Natural Science Foundation of China (22131003). Liangbing Gan acknowledges the financial support from the Beijing National Laboratory for Molecular Sciences (BNLMS-CXXM-201904) and the National Natural Science Foundation of China (22362029). Xiao-fei Zhu acknowledges the financial support from the Science Research Foundation of Jilin Province (YDZJ202301ZYTS478).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qi00548e

‡ These authors contributed equally.

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