Study on the antigenicity of metallofullerenol: antibody production, characterization, and its enzyme immunoassay application.

With their intriguing structures and properties, metallofullerenols have attracted considerable attention in biological and medical applications. Due to the increasing biomedical interest, effective detection methods are important to monitor and control metallofullerenols. However, the detection of metallofullerenols becomes very difficult after polyhydroxylated modification due to the lack of detectable features. Antibody-based immunoassay methods have been important tools for detection and will better meet the needs of analysis of metallofullerenols. Thus, the antigenicity of metallofullerenol has been studied for the first time. In this study, no immune response was detected when metallofullerenol Gd@C82(OH)x was used as immunogen. However, the polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen, indicating that metallofullerenol can act as hapten. The specificity of the obtained antibody was investigated. It has been found that the hydroxyl groups on the surface of the carbon cage, the encapsulated metal, and the size and shape of the carbon cage did not affect the recognition specificity of the antibody. Based on the obtained antibody, an indirect competitive enzyme immunoassay was developed for the determination of metallofullerenol with detection limits of 18 ng/mL in PBS. This enzyme immunoassay method was successfully used to detect metallofullerenol in serum. This work can provide an innovative way to determine metallofullerenols. Graphical abstract The polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen. Based on the obtained antibody, a competitive enzyme immunoassay was developed for the determination of metallofullerenol.

Nanovaccines and their mode of action.

Nanosized particles including nanovaccines are a novel approach to the development of vaccines to combat diseases. Nanovaccines have the promise to utilize the immune system to cure infections and to prevent infections and diseases from spreading. Rational vaccine development requires an understanding of vaccine mediated stimulation of the immune system. We review here immunostimulatory properties of nanovaccines including their immunogenicity, adjuvant properties, inflammatory responses and the mechanisms of uptake and stimulation of immune cells. Examples of various nanoparticles currently being developed as vaccines are also provided.

Production of monoclonal antibodies against fullerene C60 and development of a fullerene enzyme immunoassay.

The aim of the present study was to produce monoclonal anti-fullerene C(60) antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C(60) both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C(60) carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein-conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C(60) a specially selected water-organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C(60) in solution was 2 µg L(-1). Fullerene C(60) was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C(60) from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C(60) in different tissues.

An anti-tumor nanoparticle, [Gd@C82(OH)22]n, induces macrophage activation.

[Gd@C82(OH)22]n, a fullerene-based nanoparticle, exhibits potent anti-tumor effects in mouse tumor-bearing models without detectable toxicity and the pathological studies revealed a massive infiltration of leukocytes in the residual tumors of [Gd@C82(OH)22]n-treated mice. We report here that [Gd@C82(OH)22]n promotes macrophages secreting pro-inflammatory cytokines IL-6 and TNF-alpha, enhances the expression of MHC-II and costimulatory molecules such as CD40 and CD54, increases endocytosis and cell adhesion. Furthermore, [Gd@C82(OH)22]n-treated macrophages became functionally activated as illustrated by their capacity to activate allogeneic T cells. Taken together, our results indicate that [Gd@C82(OH)22]n nanoparticle is a potent activator of macrophages, which may in part account for its potent anti-tumor effect.

Biomedical activities of endohedral metallofullerene optimized for nanopharmaceutics.

Endohedral metallofullerenes, a novel form of carbon-related nanomaterials, currently attract wide attention for their potential applications in biomedical fields such as therapeutic medicine. Most endohedral metallofullerenes are synthesized using C60 or higher molecular weight fullerenes because of the limited interior volume of fullerene. It is known that the encapsulated metal atom has strong electronic interactions with the carbon cage in metallofullerenes. Gd@C82 is one of the most important molecules in the metallofullerene family, known as Magnetic Resonance Imaging (MRI) contrast agent candidate for diagnostic imaging. Gadolinium endohedral metallofullerenol (e.g., Gd@C82(OH)22) is a functionalized fullerene with gadolinium trapped inside carbon cage. Our group previously demonstrated that the distinctive chemical and physical properties of Gd@C82(OH)22 are dependent on the number and position of the hydroxyl groups on the fullerene cage. The present article summarizes our latest findings of biomedical effects of Gd@C82(OH)22 and gives rise to a connected flow of the existing knowledge and information from experts in the field. It briefly narrates the synthesis and physico-chemical properties of Gd@C82(OH)22. The polyhydroxylated nanoparticles exhibit the enhanced water solubility and high purity, and were tested as a MRI contrast agent. Gd@C82(OH)22 treatment inhibited tumor growth in tumor-bearing nude mice. Although the precise mechanisms of this action are not well defined, our in vitro data suggest involvements of improved immunity and antioxidation by Gd@C82(OH)22 and its size-based selective targeting to tumor site. The review critically analyzed the relevant data instead of fact-listing, and explained the potential for developing Gd@C82(OH)22 into a diagnostic or therapeutic agent.

Fullerene (C60) immunoconjugates: interaction of water-soluble C60 derivatives with the murine anti-gp240 melanoma antibody.

The first fullerene (C60) immunoconjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy (FIT).

Morphologically virus-like fullerenol nanoparticles act as the dual-functional nanoadjuvant for HIV-1 vaccine.

Fullerenol, which self-assembles into virus-sized nanoparticles, is designed as a dual-functional nanoadjuvant to generate comparable immune responses to the HIV DNA vaccine. It shows promising adjuvant activity via various immunization routes, decreasing the antigen dosage and immunization frequency while maintaining immunity levels and inducing TEM -biased immunity to combat the infection at early stage. The underlying mechanisms by which fullerenol-based formulation induces above-mentioned polyvalent immune responses are involved in activating multiple TLRs signaling pathways.