Water-soluble fullerene derivatives represent emerging molecular nanomaterials for antiviral drug discovery, yet their target selectivity against SARS-CoV-2 proteins remains insufficiently defined. Here, four functionalized [60]fullerenes, C60(OH)24, C60ser, C60ALA, and C60Trp, were evaluated as binders of key SARS-CoV-2 targets involved in viral entry and replication: spike glycoprotein, papain-like protease, 3CL main protease, and RNA-dependent RNA polymerase. Microscale thermophoresis revealed distinct target-dependent binding profiles governed by fullerene functionalization. The C60Trp fullerene was the most active derivative, binding Spike with nanomolar affinity (Kd = 94.8 nM) and showing micromolar affinity toward papain-like protease and 3CL main protease. Docking and molecular dynamics simulations supported the preferential interaction of C60Trp with the spike protein cavity through combined fullerene cage complementarity and polar interactions with lysine and arginine residues. The tested derivatives showed low cytotoxicity in A549 cells, and C60Trp was efficiently internalized. These findings identify tryptophan-functionalized [60]fullerene as a promising multivalent scaffold for SARS-CoV-2 protein targeting.

The rapid development of nanomedicine in recent years has been driven by the exploitation of unique structural properties of nanomaterials for biomedical applications, particularly in cancer therapy. One of the most promising therapeutic approaches is photodynamic therapy (PDT), a minimally invasive treatment modality used in oncology as well as in the management of bacterial infections. PDT is based on the administration of a non-toxic photosensitizer (PS), which upon irradiation with electromagnetic radiation in the presence of molecular oxygen generates reactive oxygen species (ROS), ultimately leading to cancer cell death. Among various nanomaterials, fullerenes have attracted considerable attention due to their exceptional potential for chemical functionalization. The carbon–carbon double bonds present in fullerene cages are highly susceptible to nucleophilic addition, enabling their modification through relatively simple organic reactions, including Bingel–Hirsch and Prato reactions, [4+2] cycloadditions, as well as direct amination or hydroxylation. These synthetic strategies allow for the preparation of water-soluble fullerene derivatives (e.g., C₆₀ and C₇₀), which can be efficiently purified using standard chromatographic or dialysis techniques. As a result, functionalized fullerenes can be applied in a wide range of biological and biomedical applications, including drug delivery systems, antioxidants, photosensitizers, and nanotherapeutics. In the context of photodynamic therapy, fullerene-based nanomaterials have demonstrated the ability to damage various types of cancer cells, including head and neck, skin, breast, and esophageal cancers, which often respond poorly to conventional anticancer treatments. Another important class of compounds used as photosensitizers in PDT are phthalocyanines (PCs), which belong to the porphyrinoid family. Phthalocyanines exhibit strong absorption in the far-red region of the visible spectrum (640–700 nm), corresponding to the therapeutic optical window (600–800 nm). Additionally, they are characterized by high chemical, photochemical, and thermal stability, as well as a high quantum yield of singlet oxygen (¹O₂) generation. The use of nanocarriers, such as fullerene nanoparticles, represents an effective strategy to overcome the limitations of conventional photosensitizers. These nanomaterials can selectively accumulate in tumor tissue either passively, via the enhanced permeability and retention (EPR) effect, or actively, through surface functionalization with ligands that are selectively recognized by specific cancer cell markers. Chemical modification of fullerenes with a phthalocyanine core may further enhance their ability to generate singlet oxygen, hydroxyl radicals, and superoxide anions upon irradiation. Consequently, such hybrid systems have been proposed as effective near-infrared photosensitizers for photodynamic cancer therapy, with broad applicability in oncological nanotechnology. The main objective of this research is the design and synthesis of hybrid fullerene–phthalocyanine nanomaterials for application in the photodynamic therapy of pancreatic cancer. The dedicated hybrid fullerene-based nanomaterial (1) was synthesized by coupling an aldehyde-functionalized zinc phthalocyanine derivative (2) with an aminofullerene (3) via reductive amination using NaBH₄. The resulting C 60 -phtalocyanine hybrid was subsequently reacted with low-molecular polyethyleneimine, yielding the final, water-soluble fullerene nanomaterial (4). The obtained derivative was characterized by MALDI-TOF mass spectrometry and NMR, UV–Vis, FT-IR spectroscopies. In addition, its ability to generate singlet oxygen as well as the superoxide anion radical was evaluated using EPR-spin trapping methodology and time-resolved singlet oxygen phosphorescence measurements. This research is funded by the National Science Centre (Poland) under the PRELUDIUM 23 program (grant no. 2024/53/N/NZ7/01266).

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors, owing to its aggressive physiology, late diagnosis, dense stromal barriers, and limited responsiveness to currently available therapies. In this setting, RNA interference offers an attractive strategy to suppress oncogenic drivers at the post-transcriptional level, including targets that are difficult to modulate with conventional small molecules. Fullerenes are especially compelling in this context because their well-defined carbon cages can be extensively functionalized. Here, we report the development of two water-soluble cationic fullerene nanomaterials based on C60 and C70 scaffolds, each functionalized with branched polyethyleneimine and polyethylene glycol, as nonviral carriers for siRNA delivery to pancreatic cancer cells. We further examine how scaffold identity influences physicochemical properties, gene-silencing performance, and protein-corona composition. Using PANC-1 model, we show that both nanomaterials efficiently complex siRNA and mediate silencing, whereas the C60-based platform displays superior functional performance. We then extend this system to therapeutically relevant cargoes targeting EGFR and KRAS-associated signaling and evaluate its safety in a zebrafish embryo model. Together, these studies identify cationic fullerenes as a promising class of siRNA delivery agents for PDAC and underscore the importance of protein-corona composition as a mechanistic variable in the design of carbon-based nanomedicines.

Understanding the biological activity of hydroxylated fullerene nanomaterials in human fluids is essential for translating these platforms toward biomedical use. Here, we report an integrated experimental and computational characterization of the human serum protein corona formed on hydroxyfullerene C60(OH)24 nanoparticles (HFNPs). The HFNP was prepared via bromination of pristine C60 followed by base-promoted hydrolysis and ion-exchange/dialysis purification, and they were validated by elemental analysis and spectroscopic techniques. In aqueous buffer, C60(OH)24 exhibited a bimodal aggregation state (ca. 23 and 164 nm) with a strongly negative zeta potential (about -57 mV); incubation with human serum increased hydrodynamic diameters (ca. 35 and 242 nm), consistent with corona formation observed by TEM. A tightly bound hard corona was isolated by iterative centrifugation and washing, with BCA analysis indicating that only a small fraction of total serum protein remained associated after corona isolation. Label-free LC-MS/MS quantified 424 proteins across serum controls and HFNP samples; although most identifications overlapped, relative abundances shifted markedly, defining a distinct HFNP corona enriched in complement/coagulation factors, apolipoproteins, and immunoglobulin-associated proteins, with human serum albumin strongly depleted. To rationalize adsorption selectivity, molecular docking and binding affinity calculations were performed for corona-enriched proteins (complement C1s, complement C5, complement factor H, plasminogen, and serum amyloid P-component). The molecular modeling studies indicated that binding is dominated by electrostatic and hydrogen-bonding contacts to the hydroxyl shell of C60(OH)24, with arginine and lysine residues overrepresented among interaction hotspots, whereas aromatic π-π contributions are attenuated by surface hydroxylation. Collectively, these results provide a molecular-level framework for predicting and engineering the blood-exposed interactions of hydroxylated fullerene nanomaterials.

Hexavalent chromium contamination remains a serious challenge for industrial water and wastewater treatment due to its high toxicity, mobility and resistance to conventional remediation methods. Here we report an adsorption-based detoxification approach using a regenerable hybrid nanoadsorbent (GO-C60-PEI) that integrates graphene oxide, [60]fullerene and branched polyethyleneimine. Microscopy and spectroscopic analyses (SEM/TEM, FT-IR, XPS, CHNS) together with N2-sorption confirm successful construction of the hybrid architecture. Cr(VI) uptake is rapid (equilibrium within 45 min) and best described by a Langmuir monolayer model, giving a maximum capacity of 380 mg g- 1 at pH 1.5. The GO-C60-PEI adsorbs quantitative Cr(VI) even under an unfavorable aqueous environment of high ionic strength of the solution (up to 3M of NaNO3), and in the presence of million-fold co-existing ions in relation to Cr(VI). The material can be regenerated within 5 min using 1.5 mL of 2M NH3(aq) assisted by ultrasonication, allowing efficient Cr(VI) recovery. Finally, in vivo toxicological studies using the Drosophila melanogaster model confirmed the absence of toxicity for both the pristine nanomaterial and its Cr(VI)-loaded form, indicating effective detoxification of the treated water. Therefore, the obtained GO-C60-PEI represents a promising and eco-friendly platform for the purification and detoxification of Cr(VI) in water treatment systems.

Pancreatic ductal adenocarcinoma (PDAC) is an exceedingly aggressive malignancy characterized by poor prognosis and a five-year survival rate below 5%. Conventional gemcitabine-based chemotherapy remains largely ineffective, and despite intensive efforts to develop novel therapeutics—including nanomedicines, immunotherapies, and gene-based approaches—no truly efficient treatment strategy has yet emerged. In this work, we developed a series of fullerene-based nanoplatforms designed for both the treatment and diagnosis of pancreatic tumors. As a first approach, we focused on modifying standard chemotherapeutic agents, particularly gemcitabine and the EGFR inhibitor erlotinib. Our synthetic strategy involved direct amination of fullerene cores (C 60 , C 70 , and Gd@C 82 ) with aliphatic amines, followed by conjugation to a modified form of erlotinib. This protocol reliably yielded a highly water-soluble [60]fullerene–erlotinib conjugate in good yields. Spectroscopic analyses (NMR, FT-IR, UV-Vis, and XPS) confirmed the formation of two covalent amide bonds per [60]fullerene scaffold and verified the presence of erlotinib moieties. Dynamic light scattering and TEM analyses revealed that these fullerene derivatives form stable nanoscale aggregates. For the Gd@C82 variant, measurements of T1 and T2 relaxation times underscored its potential as an MRI contrast agent. All synthesized fullerene nanomaterials were evaluated against several pancreatic cancer cell lines. Notably, C 60 BUT demonstrated the strongest cytotoxicity, with IC 50 values ranging from 16.88 to 45.46 µM across PANC-1, PAN02, and AsPC-1 cells. Detailed mechanistic studies showed that these nanoconjugates induced cell cycle arrest at the G0/G1 phase, as evidenced by reduced cyclin E1 and increased p27 levels. Intriguingly, C 60 BUT and C 70 BUT-ERL induced autophagic cell death via mTOR inhibition, while Gd@C 82 EDA-ERL primarily triggered apoptosis in PAN02 and AsPC-1 cells. Additionally, both C 60 BUT and C 70 BUT-ERL effectively suppressed EGFR signaling by downregulating p-EGFR, p-Akt, and PI3K. Importantly, no ultrastructural changes were observed in the midgut epithelium of Drosophila melanogaster, indicating a favorable biocompatibility profile. Finally, we characterized the protein corona that adsorbed onto the Gd@C82EDA-ERL and C 70 BUT-ERL nanomaterials and employed computational modeling to elucidate binding interactions. Together, these findings highlight fullerene-based nanoplatforms as promising candidates for both pancreatic cancer therapy and imaging applications.

Nanotechnology offers promising tools for disease diagnosis through the unique biomolecular corona (BC) that surrounds nanoparticles (NPs) when exposed to biological fluids, creating personalized profiles. In this study, we investigated the capability of BC of polystyrene and silica NPs with diverse surface functionalization in distinguishing eight types of cancer (breast, ovarian, kidney, lung, colon, bladder, uterus, and thyroid) using a straightforward method based on one-dimensional gel electrophoresis. A total of six NPs, silica and polystyrene NPs with various functional groups (plain, NH2, and COOH), were exposed to a total of 43 plasmas, 5 for each of the 8 investigated cancers plus 3 controls, to create personalized BCs. By analyzing the densitometric profiles of proteins' molecular weights, we demonstrated that the BC signatures of NPs can distinguish healthy individuals from those affected by cancer. We revealed that certain BCs could discriminate against specific types of cancer, i.e. ovarian and kidney cancer, and that amination could improve the diagnostic power of the platform. Being fast, cheap, and requiring only a blood sample, if validated on a more comprehensive cohort of patients, this method could serve as a first-line diagnostic tool, guiding decisions on further invasive testing and/or patient monitoring post-surgery or during treatment.

Chronic diabetic wounds affect a significant number of individuals and present considerable challenges in clinical treatment. Ideal diabetic wound dressings should possess antibacterial properties, the ability to regulate redox balance, and the capacity to promote tissue regeneration. Herein, we developed an injectable mixed-valence copper nanocluster-crosslinked hydrogel (HS-Cu) through reductive metal-ligand coordination assembly of Cu2+ and thiourea-grafted hyaluronic acid (HA-NCSN) to achieve the above-mentioned requirements in one-step. Density functional theory (DFT) calculations indicated that the bioactive mixed-valence copper nanoclusters exhibited enhanced photothermal properties and conductivity, enabling HS-Cu hydrogel to effectively combat bacteria and accelerate tissue regeneration through combined with photothermal therapy (PTT) and electrical stimulation (ES). Notably, the abundant thiourea groups inside the hydrogel acted as reactive oxygen species (ROS) scavengers to regulate the redox balance at the wound site. Additionally, in vivo experiments indicated that HS-Cu hydrogel promoted hemostasis in wounds. Overall, the HS-Cu hydrogel accelerated the three phases of diabetic wound healing: hemostasis, inflammation, and proliferation, with specific mechanisms including rapid hemostasis, bacterial eradication by PTT, scavenging of ROS, and wound healing acceleration through ES. All the results indicated that mixed-valence copper nanocluster-crosslinked hydrogel demonstrated great potential in the treatment of diabetic wounds. STATEMENT OF SIGNIFICANCE: This study developed an injectable hydrogel through metal-ligand coordination assembly, which showed great potential for accelerating diabetic wound repair in clinic. The hydrogel demonstrated superior antioxidant capacity, photothermal properties, and conductivity due to the internal bioactive mixed-valence copper nanoclusters formed through the reductive coordination between Cu2+ ions and thiourea groups. By combining photothermal therapy (PTT) and electrical stimulation (ES), this hydrogel effectively addressed critical challenges in diabetic wound healing, including bacterial infection inhibition, oxidative stress reduction, and tissue regeneration acceleration, thereby significantly enhancing wound healing. The study also provides perspectives for the design and clinical application of ion chelation based functional biomaterials.

AIM:Pancreatic ductal adenocarcinoma (PDAC) is recognized as one of the most formidable cancers, largely due to its distinct microenvironment characterized predominantly by extensive desmoplastic stroma. In this study, we synthesized three novel water-soluble fullerene-based nanomaterials targeting EGFR protein. METHODS:The direct amination of fullerene carbon atoms, was followed by conjugation with a modified derivative of the EGFR inhibitor-erlotinib, resulting in the formation of novel water-soluble fullerene derivatives. RESULTS:Further investigation into PAN02 and AsPC-1 cell lines revealed that these fullerene nanomaterials could induce cell cycle arrest in the G0/G1 phase, corroborated by alterations in the expression levels of the p27 and cyclin E1 proteins. Additionally, mechanisms of cell death were identified as autophagy for C60BUT and C70BUT-ERL, and apoptosis for Gd@C82EDA-ERL nanomaterials. CONCLUSIONS:Crucially, the study uncovered the efficacy of synthesized aminofullerenes in inhibiting the EGFR signaling pathway. The further toxicological studies of Gd@C82EDA-ERL fullerene on Drosophila melanogaster, underscored its potential for theranostic applications.

In the present study, we introduce thiosemicarbazone derivatives with high antiproliferative activity against glioblastoma cells and high selectivity against healthy cells. The cellular studies were performed on a 3D spheroid model, which more closely reflects the conditions of an actual tumor. The tested thiosemicarbazone derivatives form complexes with iron ions, triggering the generation of reactive oxygen species, leading to cell death. The multitarget mechanism of action manifests through inhibition of the insulin receptor and the impact on iron metabolism and the disruption in redox homeostasis inducing oxidative stress in the cell. An additional advantage of the tested thiosemicarbazone derivatives is their ability to pass through the blood-brain barrier, which is crucial in the case of brain tumors. The effectiveness of the therapy was confirmed in a fish in vivo model, in which a reduction in tumor size in xerographs was recorded while maintaining minimal toxicity on a Zebrafish model. These results render the studied compounds attractive as potential drugs for therapy against glioblastoma.

The persistent water contamination by toxic metal ions such as Cr(III), Cu(II), Hg(II), and Pb(II) presents significant public health challenges worldwide. This study introduces a novel carbon nanomaterial for efficient multi-elemental sorption, focusing on L-tryptophan functionalized fullerene C60 core stabilized on graphene oxide (GO-C60-Trp). The material structure, characterized by SEM, TEM, ATR-FTIR, and XPS, confirms the successful attachment of C60-Trp onto the graphene oxide surface, improving its physicochemical properties and enhancing durability in a water environment. The adsorption kinetics experiment using GO-C60-Trp indicates predominant chemisorption mechanisms with equilibrium obtained within 15-20 min and a very low adsorbent dose of 50 mg/L. The adsorption nature was also confirmed by adsorption isotherms, which revealed the adsorption capacities of 26, 95, 321, and 278 mg/g for Cr(III), Cu(II), Hg(II), and Pb(II), respectively. The material demonstrates excellent potential for water purification, given its high metal affinity, even in the presence of co-existing ions at high excess. Furthermore, its stability and functional versatility offer enormous possibilities for multiple sorbent regeneration and reuse at least 20 times, aligning with Green Chemistry principles. This innovative approach addresses metal ion contamination in water, offering a sustainable solution for environmental remediation and potential applications in analytical chemistry.

Fullerenes (C60, C70) as carbon nanomaterials can enter the environment through natural processes and anthropogenic activities, while synthetic fullerenes are commonly used in medicine in targeted therapies in association with antibodies, or anticancer and antimicrobial drugs. As the nanoparticles, they can pass through cell membranes and organelles and accumulate in the entire cytoplasm. The red-fluorescent, water-soluble [70]fullerene derivative C70-OMe-ser, which produces reactive oxygen species upon illumination with an appropriate wavelength, passed into the cytoplasm of the middle region in the Drosophila melanogaster digestive system. To determine whether [70]fullerene nanomaterials that produce fluorescence after entering the cell cytoplasm will hurt its homeostasis, it is necessary to investigate the activation of degenerative and possibly regenerative processes. In vivo, studies on the model species D. melanogaster may help to elucidate whether the water-soluble [70]fullerene derivative that produces fluorescence can still be considered among the most promising nanomaterials. The experiment involved feeding insects ad libitum with yeast paste supplemented with 40 µg of fullerenes/mL for 1 week and 1 month. Thus, adult females and males of D. melanogaster were divided into control (CWM, CWF, CMM, and CMF) and experimental groups (FWM, FWF, FMM, and FMF). The quantitative and qualitative analysis enabled the presentation of the effects of the water-soluble [70]fullerene derivatives on cell proliferation and degeneration. Our study presented that [70]fullerene derivative showed a cytoprotective effect and activated cell proliferation. Therefore, we could conclude that analyzed carbon nanomaterials seemed to be safe for the cells into which they have penetrated.

Iron plays a crucial role in various metabolic processes. However, the impact of 5-aminolevulinic acid (ALA) in combination with iron chelators on iron metabolism and the efficacy of ALA-photodynamic therapy (PDT) remain inadequately understood. This study aimed to examine the effect of thiosemicarbazone derivatives during ALA treatment on specific genes related to iron metabolism, with a particular emphasis on mitochondrial iron metabolism genes. In our study, we observed differences depending on the cell line studied. For the HCT116 and MCF-7 cell lines, in most cases, the decrease in the expression of selected targets correlated with the increase in protoporphyrin IX (PPIX) concentration and the observed photodynamic effect, aligning with existing literature data. The Hs683 cell line showed a different gene expression pattern, previously not described in the literature. In this study, we collected an extensive analysis of the gene variation occurring after the application of novel thiosemicarbazone derivatives and presented versatile and effective compounds with great potential for use in ALA-PDT.

Li et al. reported the development and application of a novel hypoxia-sensitive fullerene-based nanotherapeutic system for photodynamic therapy in cancer treatment. This system integrates a [70]fullerene scaffold with amino-modified cyclodextrin and the hypoxia-activatable anticancer prodrug tirapazamine, enhanced with tumor-targeting peptides and disulfide bond donors. The created fullerene nanomaterial exhibits selective accumulation in tumor tissues, facilitated by its enhanced water solubility and targeted specificity.

Microneedles are a novel drug delivery system that offers advantages such as safety, painlessness, minimally invasive administration, simplicity of use, and controllable drug delivery. As a type of polymer microneedle with a three-dimensional network structure, hydrogel microneedles (HMNs) possess excellent biocompatibility and biodegradability and encapsulate various therapeutic drugs while maintaining drug activity, thus attracting significant attention. Recently, they have been widely employed to promote wound healing and have demonstrated favorable therapeutic effects. Although there are reviews about HMNs, few of them focus on wound management. Herein, we present a comprehensive overview of the design and preparation methods of HMNs, with a particular emphasis on their application status in wound healing, including acute wound healing, infected wound healing, diabetic wound healing, and scarless wound healing. Finally, we examine the advantages and limitations of HMNs in wound management and provide suggestions for future research directions.

Hexavalent chromium is much more toxic than trivalent chromium and is severe environmental pollution caused by human activity. The presence of Cr(VI) ions in waters comes from anthropogenic sources, mainly from industries, and poses an enormous danger. Because of the health effects of Cr(VI) ions on humans, even at very low concentrations, it is necessary to control its levels in the water. However, the determination and speciation of Cr (VI) in water samples remains a sophisticated subject, and according to the WHO recommendation, further studies on reliable and validated methods should be continued. In this study, graphene oxide (GO) was modified with tetraethylenepentamine (TEPA) for the highly effective adsorption and determination of Cr(VI) ions by energy-dispersive X-ray fluorescence (EDXRF) and total-reflection X-ray fluorescence spectrometry (TXRF). The experiment shows that Cr(VI) ions can be adsorbed from aqueous solutions at pH 3.5 with a maximum adsorption capacity of 102 mg g-1 using minimal adsorbent doses, 10-50 mu g mL-1, much lower than those of the currently reported adsorbents. These adsorptive properties of GO-TEPA and selectivity toward Cr(VI) in the presence of Cr (III) indicate its potential use as a micro-adsorbent in the determination and speciation of chromium. Due to the high preconcentration factors of 865 and 100, for EDXRF and TXRF, respectively, and high recovery of 98.5-100%, the method based on dispersive micro-solid phase extraction allows obtaining extremely low detection limits of 53 and 3.5 pg mL-1 for EDXRF and TXRF. The exceptional adsorptive properties of GO-TEPA, including the possibility of application in micro-quantities, allow for the development of the ultra-trace method according to the fundamental principles of green analytical chemistry. It significantly expands the possibilities of using the EDXRF, as well as the TXRF technique in water analysis.

Intervertebral disc degeneration (IVDD) is characterized by fibrosis of nucleus pulposus (NP) cells and accelerated surrounding extracellular matrix catabolism. Bioactive hydrogels have shown significant potential in regulating cellular functions and tissue homeostasis. In this work, a dynamic hydrogel (HA-NCSN/Cu) is designed via the reductive chelation of hyaluronic acid grafted with thiourea (HA-NCSN) and Cu 2+ . The reductivity of the grafted thiourea groups of HA-NCSN can quickly reduce part of the chelated Cu 2+ to Cu + . Therefore, during the gelation process, the color of hydrogel become dark immediately, which endowed hydrogel with remarkable photothermal effect. The abundant thiourea groups inside hydrogel can effectively scavenge reactive oxygen species to mitigate the inflammatory stress of NP cells. RNA sequencing analysis further reveals that glutathione signaling pathway is significantly altered. Meanwhile, mild photothermal therapy could activate the TGF-β/Smad pathway in NP cells, promoting the expression and secretion of Aggrecan and Collagen II. Ultimately, the combined modulation of inflammation alleviation and matrix regeneration achieves the restoration of the structure and function of the damaged intervertebral disc, which is also strongly demonstrated by the in vivo animal experiments. All of these results demonstrate the great potential of the dynamic HA-NCSN/Cu hydrogel in IVDD treatment.

Engineered fullerene materials have attracted the attention of researchers in the biomedical sciences, especially when their synthetic methodology is developed to endow them with significant levels of water-solubility and bioavailability. In this study, we synthesized and characterized a water-soluble and red-fluorescent [70]fullerene nanomaterial, which fluoresced at 693 nm with a quantum yield of 0.065 and a large Stokes shift (around 300 nm). The fullerene nanomaterial generated mainly singlet oxygen after illumination with blue LED light, while superoxide anion radical production was minimal. The transmission electron microscopy as well as fluorescent studies of Drosophila melanogaster revealed that prepared [70]fullerene nanoparticles had better bioavailability than pristine [70]fullerene nanoparticles. The designed nanomaterials were observed in the apical, perinuclear, and basal regions of digestive cells, as well as the basal lamina of the digestive system's epithelium, with no damage to cell organelles and no activation of degenerative processes and cell death. Our findings provide a new perspective for understanding the in vivo behavior of fullerene nanomaterials and their future application in bioimaging and light-activated nanotherapeutics.

Introduction:Thanks to recent advances in synthetic methodology, water-soluble fullerene nanomaterials that interfere with biomolecules, especially DNA/RNA and selected proteins, have been found with tremendous potential for applications in nanomedicine. Herein, we describe the synthesis and evaluation of a water-soluble glycine-derived [60]fullerene hexakisadduct (HDGF) with T h symmetry, which is a first-in-class BTK protein inhibitor.Methods:We synthesized and characterized glycine derived [60]fullerene using NMR, ESI-MS, and ATR-FT-IR. DLS and zeta potential were measured and high-resolution transmission electron microscopy (HRTEM) observations were performed. The chemical composition of the water-soluble fullerene nanomaterial was examined by X-ray photoelectron spectrometry. To observe aggregate formation, the cryo-TEM analysis was carried out. The docking studies and molecular dynamic simulations were performed to determine interactions between HDGF and BTK. The in vitro cytotoxicity was evaluated on RAJI and K562 blood cancer cell lines. Subsequently, we examined the induction of cell death by autophagy and apoptosis by determining the expression levels of crucial genes and caspases. We investigated the direct association of HDGF on inhibition of the BTK signalling pathway by examining changes in the calcium levels in RAJI cells after treatment. The inhibitory potential of HDGF against non-receptor tyrosine kinases was evaluated. Finally, we assessed the effects of HDGF and ibrutinib on the expression of the BTK protein and downstream signal transduction in RAJI cells following anti-IgM stimulation.Results:Computational studies revealed that the inhibitory activity of the obtained [60]fullerene derivative is multifaceted: it hampers the BTK active site, interacting directly with the catalytic residues, rendering it inaccessible to phosphorylation, and binds to residues that form the ATP binding pocket. The anticancer activity of produced carbon nanomaterial revealed that it inhibited the BTK protein and its downstream pathways, including PLC and Akt proteins, at the cellular level. The mechanistic studies suggested the formation of autophagosomes (increased gene expression of LC3 and p62) and two caspases (caspase-3 and -9) were responsible for the activation and progression of apoptosis.Conclusion:These data illustrate the potential of fullerene-based BTK protein inhibitors as nanotherapeutics for blood cancer and provide helpful information to support the future development of fullerene nanomaterials as a novel class of enzyme inhibitors.