Cellular Uptake, Organelle Enrichment, and In Vitro Antioxidation of Fullerene Derivatives, Mediated by Surface Charge.

Hydrophilic fullerene derivatives get notable performance in various biological applications, especially in cancer therapy and antioxidation. The biological behaviors of functional fullerenes are much dependent on their surface physicochemical properties. The excellent reactive oxygen species-scavenging capabilities of functional fullerenes promote their outstanding performances in inhibiting pathological symptoms associated with oxidative stress, including neurodegenerative diseases, cardiovascular diseases, acute and chronic kidney disease, and diabetes. Herein, fullerene derivatives with reversed surface charges in aqueous solutions are prepared: cationic C60-EDA and anionic C60-(EDA-EA). Under the driving force of membrane potential (negative inside) in the cell and mitochondria, C60-EDA is much rapidly taken in by cells and transported into mitochondria compared with C60-(EDA-EA) that is enriched in lysosomes. With high cellular uptake and mitochondrial enrichment, C60-EDA exhibits stronger antioxidation capabilities in vitro than C60-(EDA-EA), indicating its better performance in the therapy of oxidation-induced diseases. It is revealed that the cellular uptake rate, subcellular location, and intracellular antioxidation behavior of fullerene derivatives are primarily mediated by their surface charges, providing new strategies for the design of fullerene drugs and their biological applications.

Gadofullerene Nanocrystals: A Robust, High-Efficiency Myelosuppressive Protector in Radiotherapy.

Radiotherapy is the current frontline method for cancer treatment, while the severe systemic side effects (e.g., myelosuppression) limit its application because it generates excessive reactive oxygen species. Therefore, there is a pressing need to develop effective strategies for radiotherapy protection. Here, we explored a robust myelosuppressive protector using gadofullerene nanocrystals (GFNCs) to protect mice against radiation injury, which was induced by different doses of X-rays (3, 4.5, and 6 Gy). Our data show that the radiotherapy-induced myelosuppression was remarkably reduced by the high radical scavenging abilities of GFNCs. In addition, GFNCs could normalize the oxidative stress-related indexes, such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Of note, GFNCs provided protection of the bone marrow in tumor-bearing mice without interfering with the antitumor properties of radiotherapy. Thus, GFNCs may play a promising role in radioprotection during radiotherapy.

Positron Emission Tomography/Magnetic Resonance Imaging of Glioblastoma Using a Functionalized Gadofullerene Nanoparticle.

Water-soluble gadofullerene nanomaterials have been extensively investigated as magnetic resonance imaging (MRI) contrast agents, radical scavengers, sensitizers for photodynamic therapy, and inherent antineoplastic agents. Most recently, an alanine-modified gadofullerene nanoparticle (Gd@C82-Ala) with excellent anticancer activity has been reported; however, the absolute tumor uptake of Gd@C82-Ala is still far from being satisfactory, and its dynamic pharmacokinetics and long-term metabolic behaviors remain to be elucidated. Herein, Gd@C82-Ala was chemically modified with eight-arm polyethylene glycol amine to improve its biocompatibility and provide the active sites for the attachment of a tumor-homing ligand (cRGD) and positron emission tomography (PET) isotopes (i.e., 64Cu or 89Zr). The physical and chemical properties (e.g., size, surface functionalization condition, radiochemical stability, etc.) of functionalized Gd@C82-Ala were properly characterized. Also, its glioblastoma cell targeting capacity was evaluated in vitro by flow cytometry, confocal fluorescence microscopy, and dynamic cellular interaction assays. Because of the presence of gadolinium ions, the gadofullerene conjugates can act simultaneously as T1* MRI contrast agents and PET probes. Thus, the pharmacokinetic behavior of functionalized Gd@C82-Ala was investigated by PET/MRI, which combines the merits of high resolution and excellent sensitivity. The functionalized Gd@C82-Ala-PEG-cRGD-NOTA-64Cu (NOTA stands for 1,4,7-triazacyclononane-triacetic acid) demonstrated much higher accumulation in U87-MG tumor than its counterpart without cRGD attachment from in vivo PET observation, consistent with observation at the cellular level. In addition, Gd@C82-Ala-PEG-Df-89Zr (Df stands for desferrioxamine) was employed to investigate the metabolic behavior of gadofullerene conjugates in vivo for up to 30 days. It was estimated that nearly 70% of Gd@C82-Ala-PEG-Df-89Zr was excreted from the test subjects primarily through renal pathways within 24 h. With proper surface engineering, functionalized Gd@C82-Ala nanoparticles can show an improved accumulation in glioblastoma. Pharmacokinetic studies also confirmed the safety of this nanoplatform, which can be used as an image-guidable therapeutic agent for glioblastoma.

Photo-triggered gadofullerene: enhanced cancer therapy by combining tumor vascular disruption and stimulation of anti-tumor immune responses.

Efficient treatment of primary tumor and preventing cancer metastasis present intriguing alternatives to cancer therapy. Herein, for the first time, we reported the photo-triggered nano-gadofullerene (Gd@C82-Ala, abbreviated Gd-Ala) induced malignant tumor vascular disruption by shortening the light interval between Gd-Ala administration and light illumination, where oxygen in blood vessels was employed efficiently to produce cytotoxic reactive oxygen species (ROS). The produced ROS could not only destroy the tumor cells but also devastate the vascular endothelial cells corresponding to the loss of intercellular junctions and vessels disruption. Notably, the irradiated Gd-Ala could enhance dendritic cells (DCs) maturation, which further secreted tumor necrosis factor-α (TNF-α) and interleukin-12 (IL)-12, and then activated T lymphocytes by up-regulation of cluster of differentiation CD4+ and CD8+ T lymphocytes. Furthermore, the down-regulation of matrix metalloprotein 2 (MMP2) and MMP9 also reduce the rate of tumor metastasis. This work explored a new biomedical application of gadofullerene, thereby providing a smart carbon nanomaterial candidate for tumor ablation and inhibition of cancer metastasis.

Amino acid modified [70] fullerene derivatives with high radical scavenging activity as promising bodyguards for chemotherapy protection.

Despite the great efforts for tumor therapy in the last decades, currently chemotherapy induced toxicity remains a formidable problem for cancer patients, and it usually prohibits the cancer therapy from successful completion due to severe side effects. In general, the main side effects of chemotherapeutic agents are from the as-produced reactive oxygen species (ROS) that not only harm the tumor cells but also damage the patients' organs. Here we report the application of amino acid derivatives of fullerene (AADF) in the chemotherapy which strongly scavenge the excess ROS to protect the tested mice against the chemotherapy-induced hepatotoxicity and cardiotoxicity. Two amino acids, i.e., L-lysine and ß-alanine were separately employed to chemically modify C70 fullerene, and L-lysine derivative of fullerene (C70-Lys) exhibits superior radical scavenging activity to ß-alanine derivative of C70 (C70-Ala). As expected, C70-Lys show much better protective effect than C70-Ala against the chemotherapy injuries in vivo, which is verified by various histopathological, haematological examinations and antioxidative enzyme studies. Moreover, the L-glutathione level is increased and the cytochrome P-450 2E1 expression is inhibited. They are potentially developed as promising bodyguards for chemotherapy protection.

Biodegradable, Hydrogen Peroxide, and Glutathione Dual Responsive Nanoparticles for Potential Programmable Paclitaxel Release.

Reactive oxygen species (ROS) and glutathione (GSH) dual responsive nanoparticulate drug delivery systems (nano-DDSs) hold great promise to improve the therapeutic efficacy and alleviate the side effects of chemo drugs in cancer theranosis. Herein, hydrogen peroxide (H2O2) and GSH dual responsive thioketal nanoparticle (TKN) was rationally designed for paclitaxel (PTX) delivery. Compared to other stimuli-sensitive nano-DDSs, this dual responsive DDS is not only sensitive to biologically relevant H2O2 and GSH for on-demand drug release but also biodegradable into biocompatible byproducts after fulfilling its delivering task. Considering the heterogeneous redox potential gradient, the PTX loaded TKNs (PTX-TKNs) might first respond to the extracellular ROS and then to the intracellular GSH, achieving a programmable release of PTX at the tumor site. The selective toxicity of PTX-TKNs to tumor cells with high levels of ROS and GSH was verified both in vitro and in vivo.

RF-assisted gadofullerene nanoparticles induces rapid tumor vascular disruption by down-expression of tumor vascular endothelial cadherin.

The tumor vasculature with unique characteristics offers an attractive target for anti-cancer therapy. Herein, we put forward a novel antitumor therapeutic mechanism based on the gadofullerene nanocrystals (GFNCs), the agent we have previously shown to efficiently disrupt tumor vasculature by size-expansion with assistance of radiofrequency (RF). However, the tumor vascular disrupting mechanism of RF-assisted GFNCs treatment was not further studied. In the present work, a rapid tumor blood flow shutdown has been observed by the vascular perfusion imaging in vivo and vascular damages were evident 6 h after the RF-assisted GFNCs treatment. Importantly, a significant down-expression of tumor vascular endothelial cadherin (VE-cadherin) treated by RF-assisted GFNCs was further investigated, which caused vascular collapse, blood flow shut-down and subsequent tumor hemorrhagic necrosis. These findings set forth a systematic mechanism on the superior anti-tumor efficiency by RF-assisted GFNCs treatment.

Biocompatible [60]/[70] Fullerenols: Potent Defense against Oxidative Injury Induced by Reduplicative Chemotherapy.

Chemotherapy as a conventional cancer treatment suffers from critical systemic side effects, which is generally considered as the consequence of reactive oxygen species (ROS). Fullerenes have been widely studied for their excellent performance in radicals scavenging. In the present study, we report a solid-liquid reaction to synthesize fullerenols and their application as ROS scavengers in chemotherapy protection. The solid-liquid reaction is carried out without catalyst and suitable for mass production. The novel [60]/[70] fullerenols show a high stability in water, and the [70] fullerenols (C70-OH) exhibit radical scavenging capability superior to that of [60] fullerenols (C60-OH) in chemotherapy protection. The mouse model for single and reduplicative chemotherapy-induced liver injury demonstrates their protective effects in the chemotherapeutic process, which is confirmed by histopathological examinations and hematological index. The increase of the hepatic l-glutathione (GSH) level and downregulated expression of the cytochrome P-450 2E1 (CYP2E1) give the possible mechanism associated with the impact of fullerenols on the metabolism of doxorubicin. The novel fullerenols may be promising protective agents to satisfy the demand for future clinical chemotherapy.

Amino acid functionalized gadofullerene nanoparticles with superior antitumor activity via destruction of tumor vasculature in vivo.

Researchers have been puzzled of the therapy of malignant tumors and the current therapeutic strategies are always accompanied by toxicity or side effects. Developing efficient nanodrugs could reduce the dosage and greatly improve the therapeutic effects in cancer treatments. Here we initially reported a novel kind of gadofullerene nanoparticles functionalized with amino acid (ß-alanine), which exhibited a superior antitumor activity in hepatoma H22 models via a novel therapeutic mechanism. The involvement of ß-alanine improved the tumor inhibition rate up to 76.85% for a single treatment by strengthening the interaction with radiofrequency (RF) and extending blood circulation time. It realized a highly antivascular treatment to cut off the nutrient supply of tumor cells by physically destroying the abnormal tumor blood vessels assisted by RF. In situ and real-time observation of the vascular change was conducted using the dorsal skin fold chamber model, which corresponded to the erythrocyte diapedesis in histopathological examination. The ultrastructural changes of vascular endothelial cells were further investigated by environmental scanning electron microscopy and transmission electron microscopy. Long-term toxicity evaluation showed that the GF-Ala nanoparticles could be eliminated from the mice after several days and no obvious toxicity was found to the main organs. All these encouraging results suggest GF-Ala nanoparticles are valuable for the significant therapeutic potential with high-efficacy and low-toxicity.

Fullerene/photosensitizer nanovesicles as highly efficient and clearable phototheranostics with enhanced tumor accumulation for cancer therapy.

A novel phototheranostic platform based on tri-malonate derivative of fullerene C70 (TFC70)/photosensitizer (Chlorin e6, Ce6) nanovesicles (FCNVs) has been developed for effective tumor imaging and treatment. The FCNVs were prepared from amphiphilic TFC70-oligo ethylene glycol -Ce6 molecules. The developed FCNVs possessed the following advantages: (i) high loading efficiency of Ce6 (up to ∼57 wt%); (ii) efficient absorption in near-infrared light region; (iii) enhanced cellular uptake efficiency of Ce6 in vitro and in vivo; (iv) good biocompatibility and total clearance out from the body. These unique properties suggest that the as-prepared FCNVs could be applied as an ideal theranostic agent for simultaneous imaging and photodynamic therapy of tumor. This finding may provide a good solution to highly efficient phototheranostic applications based on fullerene derivatives fabricated nanostructures.

A Versatile and Clearable Nanocarbon Theranostic Based on Carbon Dots and Gadolinium Metallofullerene Nanocrystals.

Nanocarbons such as carbon nanotubes, graphene derivatives, and carbon nanohorns have illustrated their potential uses as cancer theranostics owing to their intrinsic fluorescence or NIR absorbance as well as superior cargo loading capacity. However, some problems still need to be addressed, such as the fates and long-term toxicology of different nanocarbons in vivo and the improvement of their performance in various biomedical imaging-guided cancer therapy systems. Herein, a versatile and clearable nanocarbon theranostic based on carbon dots (CDs) and gadolinium metallofullerene nanocrystals (GFNCs) is first developed, in which GFNCs enhance the tumor accumulation of CDs, and CDs enhance the relaxivity of GFNCs, leading to an efficient multimodal imaging-guided photodynamic therapy in vivo without obvious long-term toxicity. Furthermore, biochemical analysis reveals that the novel nanotheranostic can harmlessly eliminate from the body in a reasonable period of time after exerting diagnostic and therapeutic function.

Synergistic Effect of Human Serum Albumin and Fullerene on Gd-DO3A for Tumor-Targeting Imaging.

A macromolecular magnetic resonance imaging (MRI) contrast agent was successfully synthesized by conjugating the gadolinium/1,4,7,10-tetraazacyclododecane-1,4,7-tetracetic acid complex (Gd-DO3A) with 6,6-phenyl-C61 butyric acid (PC61BA) and upon further modification with human serum albumin (HSA). The final product, PC61BA-(Gd-DO3A)/HSA, has a high stability and exhibits a much higher relaxivity (r1 = 89.1 mM(-1) s(-1) at 0.5 T, 300 K) than Gd-DO3A (r1 = 4.7 mM(-1) s(-1)) does under the same condition, producing the synergistic positive effect of HSA and C60 on the relaxivity of Gd-DO3A. The in vivo MR images of PC61BA-(Gd-DO3A)/HSA-treated tumor-bearing mice show strong signal enhancement for the tumor area due to the enhanced permeability and retention effect. The maximum accumulation of PC61BA-(Gd-DO3A)/HSA at the tumor site was achieved at 4 h postinjection, which may guide surgery. The results from the hematology and histological observations indicate that PC61BA-(Gd-DO3A)/HSA has no obvious toxicity in vivo. These unique properties of PC61BA-(Gd-DO3A)/HSA enable them to be highly efficient for tumor-targeting MRI in vivo, possibly providing a good solution for tumor diagnosis.

Radioactive lutetium metallofullerene 177LuxLu(3-x)N@C80-PCBPEG derivative: a potential tumor-targeted theranostic agent.

A radioactive metallofullerene 177LuxLu(3-x)N@C80 was firstly synthesized by means of neutron irradiation on Lu3N@C80. After modification by methoxypolyethylene glycol amine, in vivo investigation on tumor-bearing mice was performed. The results reveal favorable affinity toward tumors, suggesting that the obtained 177LuxLu(3-x)N@C80-PCBPEG could be promising for tumor diagnosis and therapy.

Amphiphilic trismethylpyridylporphyrin-fullerene (C70) dyad: an efficient photosensitizer under hypoxia conditions.

Amphiphilic trismethylpyridylporphyrin-C70 (PC70) dyad with improved photosensitization has been successfully prepared. The PC70 dyad forms a liposomal nanostructure through molecular self-assembling. An increased absorption coefficient in the visible region, good biocompatibility, and high photostability were observed on the self-assembling structure. Surprisingly, in comparison with previously reported photosensitizer porphyrins, PC70 exhibited an enhanced photodynamic therapy (PDT) effect under hypoxia conditions. Further investigations illustrated that PC70 went through an extremely long-life triplet state (211.3 µs) under hypoxia, which enabled the exiguous oxygen to approach and interact with the activated (3P-C70)* more efficiently and produce more singlet oxygen. This would overcome the problem of existing photosensitizers of low PDT efficiency in cancerous tissues under hypoxia. The excellent properties of PC70 dyad make it a promising phototherapeutic agent, especially for the treatment of early- and late-stage cancers under shallow and hypoxia tissues.

G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain.

Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.

In vitro and in vivo photothermally enhanced chemotherapy by single-walled carbon nanohorns as a drug delivery system.

Single-walled carbon nanohorns (SWNHs) have exhibited many special advantages in biomedical applications. Herein, doxorubicin-loaded SWNHs (DOX-SWNHs) are prepared and further modified by amphiphilic deoxycholic acid modified-hydropropyl chitosan (DCA-HPCHS) to improve their biocompatibility. The obtained DOX-SWNH/DCA-HPCHS drug delivery system (DDS) possesses high stability in physiological media and excellent photothermal properties when exposed to laser irradiation in the near-infrared (NIR) region, which dramatically enhances the chemotherapy of DOX. Cell viability assays show that the growth of 4T1 cells are remarkably inhibitory under the conditions of incubation with DOX-SWNH/DCA-HPCHS and subsequent exposure to 808 nm laser irradiation to produce mild photothermal heating to 43 °C. Further investigation reveals that the photothermally enhanced chemotherapy derived from a promotion of DOX-SWNH/DCA-HPCHS uptake by the cancer cells rather than a light-triggered release of DOX. DOX-SWNH/DCA-HPCHS in combination with the use of laser irradiation exhibits a much better anticancer effect than the controls. Hence, the DOX-SWNH/DCA-HPCHS as a multifunctional DDS has been proposed and is hopeful for medicinal use in the future.

Enhanced photodynamic efficiency of an aptamer-guided fullerene photosensitizer toward tumor cells.

Fullerene-based photosensitizers exhibit great potential in photodynamic therapy (PDT). Based on the high photodynamic efficacy of trimalonic acid-modified C70 fullerene (TF70), we constructed an aptamer-guided TF70 photosensitizer and investigated its photodynamic effect. Conjugation of the novel aptamer (named R13) could effectively enhance the PDT efficiency of TF70 against A549 lung cancer cells in the presence of serum. The lysosomal location of the TF70-R13 conjugate inside cells facilitates the production of intracellular reactive oxygen species (ROS), which can efficiently kill cells, under light irradiation. The enhanced photodynamic efficiency, along with its good biocompatibility in the dark, makes TF70-R13 a highly promising photosensitizer for tumor-specific PDT.

Fullerene-induced increase of glycosyl residue on living plant cell wall.

In this work, we have investigated the change of cell wall for the tobacco plant cell (Nicotiana tobacum L. cv. Bright Yellow) under the repression of water-soluble carboxyfullerenes (C70(C(COOH)2)(2-4)). The adsorption of C70(C(COOH)2)(2-4) on cell wall led to the disruption of cell wall and membrane, and consequently, cell growth inhibition. Results from atomic force microscopy (AFM) force measurement and confocal imaging revealed an increase of the glycosyl residue on the cell wall of carboxyfullerene-treated cells, with a time- and dose-dependent manner, and accompanied by the elevated reactive oxygen species (ROS). Moreover, the stimulation-sensitive alteration of glycosyl residue and ROS was demonstrated, which suggested a possible protection strategy for the plant cells under fullerene repression. This study provides the first direct evidence on the change of plant cell wall composition under the repression of fullerene and is the first successful application of AFM ligand-receptor binding force measurement to the living plant cell. The new information present here would help to a better understanding and assessment of the biological effect of fullerenes on plant.

Polystyrene spheres-assisted matrix-assisted laser desorption ionization mass spectrometry for quantitative analysis of plasma lysophosphatidylcholines.

The quantitative analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a challenge due to the poor reproducibility originating from the heterogeneity of the matrix-analyte crystals. Polystyrene (PS) colloidal spheres have superior monodispersed property and can self-assemble to form photonic crystals. With the assistance of PS spheres, a uniform matrix-analyte cocrystal was constructed for the quantitative analysis of plasma lysophosphatidylcholines (LPCs). The matrix and the solvent in MALDI MS analysis were optimized, and the reproducibility and the accuracy were investigated in detail. This is the first report about the very simple method of PS spheres-assisted MALDI MS for quantitative analysis, where it is believed that this approach will greatly expand the applications of MALDI MS.

pH-responsive mechanism of a deoxycholic acid and folate comodified chitosan micelle under cancerous environment.

Smart pH-responsive polymeric micelles have attracted much attention as one of the most promising drug delivery candidates. In this paper, a different substitution of deoxycholic acid (DCA) and folic acid (FA) comodified hydroxypropyl chitosans (HPCHS) were synthesized for doxorubicin (DOX) targeted delivery and controllable release. The results indicate that the DOX-release behavior is pH-responsive and closely related with the grafting proportions of the two hydrophobic ingredients. The pH-responsive mechanism for the optimized (6%DCA)-HPCHS-(0.1%FA) was suggested, resulting from a synergistic effect of gradual hydrolysis of the amido bond and electrostatic repulsion between the subsequently protonated DOX and the amino residue of the chitosan backbone under a cancerous microenvironment. Moreover, the DOX/(6%DCA)-HPCHS-(0.1%FA) micelle as a promising targeted drug delivery system in cancer therapy was evaluated by cell growth inhibition assays and confocal laser microscopy in vitro. The results clearly demonstrate a controlled release of its cargo and promoted curative efficacy of DOX.