Dendro[C(60)]fullerene DF-1 provides radioprotection to radiosensitive mammalian cells.

In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1's lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.

Antioxidant single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) and ultrashort SWCNTs (US-SWCNTs) were functionalized with derivatives of the phenolic antioxidant, butylated hydroxytoluene (BHT). By using the oxygen radical absorbance capacity (ORAC) assay, the oxygen radical scavenging ability of the SWCNT antioxidants is nearly 40 times greater than that of the radioprotective dendritic fullerene, DF-1. In addition, ORAC results revealed two divergent trends in the antioxidant potential of SWCNTs, depending on the type of functionalization employed. When existing pendant sites on US-SWCNTs were further functionalized by either covalent or noncovalent interactions of the existing pendant sites with a BHT derivative, the amount of BHT-derivative loading proportionately increased the overall antioxidant activity. If, however, functionalization occurred via covalent functionalization of a BHT-derivative directly to the SWCNT sidewall, the amount of BHT-derivative loading was inversely proportional to the overall antioxidant activity. Therefore, increasing the number of pendant sites on the SWCNT sidewalls by covalent functionalization led to a concomitant reduction in ORAC activity, suggesting that the nanotube itself is a better radical scavenger than the BHT-derivatized SWCNT. Cytotoxicity assays showed that both nonfunctionalized and BHT-derivatized SWCNTs have little or no deleterious effect on cell viability. Therefore, SWCNTs may be attractive agents for antioxidant materials and medical therapeutics research.

An iodinated liposomal computed tomographic contrast agent prepared from a diiodophosphatidylcholine lipid.

Herein we report a novel vesicle-forming iodinated contrast agent for applications in computed tomographic (CT) imaging and drug delivery. Specifically, we have chemically modified a phosphatidylcholine lipid that is commonly used in liposome formation to create an iodinated lipid that self-assembles into approximately 50-150 nm iodoliposomes possessing as-prepared imaging contrast functionality. These iodoliposomes are structurally organized such that the iodinated moieties are contained within the vesicle's bilayer, leaving the liposomal interior unoccupied and thus available for encapsulating drugs. The iodoliposomes were characterized using electron microscopy and dynamic light scattering. We also calculated the iodoliposomes' iodine encapsulation efficiency, which was sufficient for use in current CT imaging protocols. These iodinated liposomes could also serve as multifunctional carriers upon the encapsulation of pharmaceutical agents, permitting simultaneous CT imaging and therapeutic treatment. Alternatively, the commercially available iodinated contrast agent iohexol could be encapsulated inside the iodoliposomes' aqueous core to further enchance their imaging contrast.

New insights into metabolic properties of marine bacteria encoding proteorhodopsins.

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Self assembly of amphiphilic C60 fullerene derivatives into nanoscale supramolecular structures.

BACKGROUND: The amphiphilic fullerene monomer (AF-1) consists of a "buckyball" cage to which a Newkome-like dendrimer unit and five lipophilic C12 chains positioned octahedrally to the dendrimer unit are attached. In this study, we report a novel fullerene-based liposome termed 'buckysome' that is water soluble and forms stable spherical nanometer sized vesicles. Cryogenic electron microscopy (Cryo-EM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies were used to characterize the different supra-molecular structures readily formed from the fullerene monomers under varying pH, aqueous solvents, and preparative conditions. RESULTS: Electron microscopy results indicate the formation of bilayer membranes with a width of ~6.5 nm, consistent with previously reported molecular dynamics simulations. Cryo-EM indicates the formation of large (400 nm diameter) multilamellar, liposome-like vesicles and unilamellar vesicles in the size range of 50-150 nm diameter. In addition, complex networks of cylindrical, tube-like aggregates with varying lengths and packing densities were observed. Under controlled experimental conditions, high concentrations of spherical vesicles could be formed. In vitro results suggest that these supra-molecular structures impose little to no toxicity. Cytotoxicity of 10-200 muM buckysomes were assessed in various cell lines. Ongoing studies are aimed at understanding cellular internalization of these nanoparticle aggregates. CONCLUSION: In this current study, we have designed a core platform based on a novel amphiphilic fullerene nanostructure, which readily assembles into supra-molecular structures. This delivery vector might provide promising features such as ease of preparation, long-term stability and controlled release.

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin.

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp(97) occurs with a pK(a) of 8.2+/-0.1. R94C mutation reduces this slightly to 7.0+/-0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK(a) of Asp(85) by approximately 5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg(94) and Asp(97) in the unphotolyzed state of pR, in comparison to Arg(82) and Asp(85) in bR. Therefore, while fast light-driven H(+) release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.

Time-resolved FTIR spectroscopy of the photointermediates involved in fast transient H+ release by proteorhodopsin.

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

BACKGROUND: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. RESULTS: A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent. CONCLUSIONS: The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

Buckysomes: New Nanocarriers for Anticancer Drugs.

Buckysomes, liposome-like vesicles comprised of dendritic C60 subunits that self-assemble into unilamellar vesicles, are unique nanovectors that have utility in drug delivery. We have prepared paclitaxel-embedded buckysomes (PEBs) and examined biodistriubition profiles with commercially available formulations of the drug. As compared to Abraxane, an albumin-bound formulation of paclitaxel, PEBs showed higher tissue accumulation in the liver and the kidney at 45 and 60 minutes and in the lungs at 30 minutes, making them suitable drug-delivery carriers for short-term therapy to the mentioned organs. These buckysomes can be further functionalized to specifically deliver paclitaxel to the tumor site.

Biomedical applications of functionalized fullerene-based nanomaterials.

Since their discovery in 1985, fullerenes have been investigated extensively due to their unique physical and chemical properties. In recent years, studies on functionalized fullerenes for various applications in the field of biomedical sciences have seen a significant increase. The ultimate goal is towards employing these functionalized fullerenes in the diagnosis and therapy of human diseases. Functionalized fullerenes are one of the many different classes of compounds that are currently being investigated in the rapidly emerging field of nanomedicine. In this review, the focus is on the three categories of drug delivery, reactive oxygen species quenching, and targeted imaging for which functionalized fullerenes have been studied in depth. In addition, an exhaustive list of the different classes of functionalized fullerenes along with their applications is provided. We will also discuss and summarize the unique approaches, mechanisms, advantages, and the aspect of toxicity behind utilizing functionalized fullerenes for biomedical applications.

Antibody-labeled liposomes for CT imaging of atherosclerotic plaques: in vitro investigation of an anti-ICAM antibody-labeled liposome containing iohexol for molecular imaging of atherosclerotic plaques via computed tomography.

We evaluated the specific binding of anti-intercellular adhesion molecule 1 (ICAM-1) conjugated liposomes (immunoliposomes, or ILs) to activated human coronary artery endothelial cells (HCAEC) with the purpose of designing a computed tomographic imaging agent for early detection of atherosclerotic plaques. Covalent attachment of anti-ICAM-1 monoclonal antibodies to pre-formed liposomes stabilized with polyethylene glycol yielded ILs, with a coupling efficiency of the ICAM-1 to the liposomes of 10% to 24%. The anti-ICAM-1-labeled ILs had an average diameter of 136 nm as determined by dynamic light-scattering and cryogenic electron microscopy. The ILs' encapsulation of 5-[N-acetyl-(2,3-dihydroxypropyl)-amino)-N, N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-benzene-1,3-dicarboxamide (iohexol) was determined to be 18% to 19% by a dialysis technique coupled with ultraviolet detection of free iohexol. This encapsulation corresponded to 30 to 38 mg iodine per mL IL solution, and the ILs exhibited 91% to 98.5% iohexol retention at room temperature and under physiologic conditions. The specific binding of the ILs to cultured, activated HCAEC was measured using flow cytometry, enzyme-linked immunosorbent assays, and fluorescence microscopy. The immunosorbent assays demonstrated the specificity of binding of anti-ICAM-1 to ICAM-1 compared with control studies using nonspecific immunoglobulin G-labeled ILs. Flow cytometry and fluorescence microscopy experiments demonstrated the expression of ICAM-1 on the surface of activated HCAEC. Therefore, our iohexol-filled ILs demonstrated potential for implementation in computed tomographic angiography to noninvasively detect atherosclerotic plaques that are prone to rupture.

His-75 in proteorhodopsin, a novel component in light-driven proton translocation by primary pumps.

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.

Buckysomes: fullerene-based nanocarriers for hydrophobic molecule delivery.

We report the preparation and preliminary in vitro studies of nanocarriers termed "buckysomes," which are self-assembled, spherical nanostructures composed of the amphiphilic fullerene AF-1. By inducing AF-1 self-assembly at an elevated temperature of 70 degrees C, dense spherical buckysomes with diameters of 100-200 nm were formed, as observed by electron microscopy and dynamic light scattering. The amphiphilic nature of AF-1 results in the formation of many hydrophobic regions within the buckysomes, making them ideal for embedding hydrophobic molecules to be tested in a drug delivery scheme. After confirming the cellular internalization of buckysomes embedded with the hydrophobic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, we embedded paclitaxel, a highly hydrophobic anticancer drug. The in vitro therapeutic efficacy of the paclitaxel-embedded buckysomes toward suppression of MCF-7 breast cancer cell growth was compared to that of Abraxane, a commercially available, nanoparticle-albumin-bound formulation of paclitaxel. Notably, the paclitaxel-embedded buckysomes demonstrated a similar efficacy to that observed with Abraxane in cell viability studies; these results were confirmed microscopically. Moreover, negative control studies of MCF-7 viability using empty buckysomes demonstrated that the buckysomes were not cytotoxic. The results of our studies suggest that buckysomes prepared from self-assembly of AF-1 at 70 degrees C are promising nanomaterials for the delivery of hydrophobic molecules.