Jodie L. Conyers

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.

Protective Effects of Fresh Frozen Plasma on Vascular Endothelial Permeability, Coagulation, and Resuscitation After Hemorrhagic Shock Are Time Dependent and Diminish Between Days 0 and 5 After Thaw

BACKGROUND Clinical studies have shown that resuscitation with fresh frozen plasma (FFP) is associated with improved outcome after severe hemorrhagic shock (HS). We hypothesized that in addition to its effects on hemostasis, FFP has protective and stabilizing effects on the endothelium that translate into diminished endothelial cell (EC) permeability and improved resuscitation in vivo after HS. We further hypothesized that the beneficial effects of FFP would diminish over 5 days of routine storage at 4 degrees C. METHODS EC permeability was induced by hypoxia and assessed by the passage of 70-kDa Dextran between monolayers. Thrombin generation time and coagulation factor levels or activity were assessed in FFP. An in vivo rat model of HS and resuscitation was used to determine the effects of FFP on hemodynamic stability. RESULTS Thawed FFP inhibits EC permeability in vitro by 10.2-fold. Protective effects diminish (to 2.5-fold) by day 5. Thrombin generation time is increased in plasma that has been stored between days 0 and 5. In vivo data show that day 0 FFP is superior to day 5 FFP in maintaining mean arterial pressure in rats undergoing HS with resuscitation. CONCLUSION Both in vitro and in vivo studies show that FFP has beneficial effects on endothelial permeability, vascular stability, and resuscitation in rats after HS. The benefits are independent of hemostasis and diminish between days 0 and 5 of storage.

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.

Cellular Association and Assembly of a Multistage Delivery System

The realization that blood-borne delivery systems must overcome a multiplicity of biological barriers has led to the fabrication of a multistage delivery system (MDS) designed to temporally release successive stages of particles or agents to conquer sequential barriers, with the goal of enhancing delivery of therapeutic and diagnostic agents to the target site. In its simplest form, the MDS comprises stage-one porous silicon microparticles that function as carriers of second-stage nanoparticles. Cellular uptake of nontargeted discoidal silicon microparticles by macrophages is confirmed by electron and atomic force microscopy (AFM). Using superparamagnetic iron oxide nanoparticles (SPIONs) as a model of secondary nanoparticles, successful loading of the porous matrix of silicon microparticles is achieved, and retention of the nanoparticles is enhanced by aminosilylation of the loaded microparticles with 3-aminopropyltriethoxysilane. The impact of silane concentration and reaction time on the nature of the silane polymer on porous silicon is investigated by AFM and X-ray photoelectron microscopy. Tissue samples from mice intravenously administered the MDS support co-localization of silicon microparticles and SPIONs across various tissues with enhanced SPION release in spleen, compared to liver and lungs, and enhanced retention of SPIONs following silane capping of the MDS. Phantom models of the SPION-loaded MDS display negative contrast in magnetic resonance images. In addition to forming a cap over the silicon pores, the silane polymer provides free amines for antibody conjugation to the microparticles, with both VEGFR-2- and PECAM-specific antibodies leading to enhanced endothelial association. This study demonstrates the assembly and cellular association of a multiparticle delivery system that is biomolecularly targeted and has potential for applications in biological imaging.

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

In this study, the ability of the C60 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.

Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy.

TNF-α (tumor necrosis factor-α) is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL) in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1) mechanical properties, employing atomic force microscopy; 2) cytoskeletal organization, through fluorescence microscopy; and 3) membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h), for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells); the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with controls. Collectively, these results demonstrate that sufficiently high levels of circulating TNF-α have similar effects on different endothelial districts, and provide additional information for unraveling the possible correlations between circulating pro-inflammatory cytokines and systemic vascular dysfunction.

Contact- and agonist-regulated microvesiculation of human platelets

After exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets.We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbβ3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.

Frame isotropic multiresolution analysis for micro CT scans of coronary arteries

We analyze localized textural consistencies in high-resolution Micro CT scans of coronary arteries to identify the appearance of diagnostically relevant changes in tissue. For the efficient and accurate processing of CT volume data, we use fast algorithms associated with three-dimensional so-called isotropic multiresolution wavelets that implement a redundant, frame-based image encoding without directional preference. Our algorithm identifies textural consistencies by correlating coefficients in the wavelet representation.

Quantification of roughness of calcific deposits in computed tomography scans of human coronary arteries

Objectives:The incidence of coronary artery disease has been shown to be greater in patients with calcific deposits than in those without. It has been suggested that the pattern of distribution of coronary calcific deposits within coronary arteries is of greater predictive value for acute coronary events than the overall quantity. Whether roughness of calcific deposits is a predictor of acute coronary events is not known. We derived and tested an algorithm, Voxel-Based Bosselation (VBB), for noninvasive quantification of roughness of calcific deposits in human coronary arteries imaged by computed tomography (CT). Methods and Results:VBB was tested on 213 coronary calcific deposits from electron beam CT scans of 27 patients. This algorithm evaluates the 3-dimensional connectedness of surface voxels of each deposit: smooth masses have low VBB and rough masses high VBB. The algorithm was calibrated with artificially generated phantoms as well as background noise mimicking calcific deposits and surrounding heart tissue. The VBB algorithm is applicable to calcific deposits of all scales and gradations. The VBB values of the deposits in this study did not correlate with deposit size further supporting its validity as a measurement of roughness. The VBB index corresponded directly with visual reconstruction using Phong-shaded algorithms. Conclusions:The VBB index, derived here, is a noninvasive method of quantifying the roughness of calcific deposits in CT scan data which can now be used in future clinical studies to determine possible correlations with increased plaque vulnerability and major acute coronary events.