Tarik F. Massoud

Gold Nanoparticles: A Revival in Precious Metal Administration to Patients

Gold has been used as a therapeutic agent to treat a wide variety of rheumatic diseases including psoriatic arthritis, juvenile arthritis, and discoid lupus erythematosus. Although the use of gold has been largely superseded by newer drugs, gold nanoparticles are being used effectively in laboratory based clinical diagnostic methods while concurrently showing great promise in vivo either as a diagnostic imaging agent or a therapeutic agent. For these reasons, gold nanoparticles are therefore well placed to enter mainstream clinical practice in the near future. Hence, the present review summarizes the chemistry, pharmacokinetics, biodistribution, metabolism, and toxicity of bulk gold in humans based on decades of clinical observation and experiments in which gold was used to treat patients with rheumatoid arthritis. The beneficial attributes of gold nanoparticles, such as their ease of synthesis, functionalization, and shape control are also highlighted demonstrating why gold nanoparticles are an attractive target for further development and optimization. The importance of controlling the size and shape of gold nanoparticles to minimize any potential toxic side effects is also discussed.

Correlation of the Angioarchitectural Features of Cerebral Arteriovenous Malformations with Clinical Presentation of Hemorrhage

Superselective angiography is the most accurate technique in the analysis of brain arteriovenous malformation (AVM) angioarchitecture. Therefore, we reviewed the selective and superselective angiograms of 100 consecutive patients with intracerebral AVMs. Our purpose was to determine which parameters of angioarchitecture were significantly correlated with a clinical presentation of hemorrhage. The vascular characteristics evaluated on the angiograms were the size of the AVM, the location of the AVM, the type of nidus, the type of feeders, the characteristics of venous drainage, and the number and location of aneurysms. The parameters found to correlate with hemorrhage were deep venous drainage (P = 0.01), feeding by perforators (P = 0.01), intranidal aneurysm(s) (P = 0.004), multiple aneurysms (P = 0.001), feeding by the vertebrobasilar system (P = 0.002), and location in the basal ganglia (P = 0.04). Six parameters of AVM angioarchitecture were correlated with a clinical presentation of hemorrhage. Among these parameters, three (feeding by perforators, number of aneurysms, and presence of intranidal aneurysms) were well displayed by superselective angiogram.

Bioluminescence resonance energy transfer (BRET) imaging of protein–protein interactions within deep tissues of living subjects

Identifying protein–protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

Ion implantation and protein coating of detachable coils for endovascular treatment of cerebral aneurysms: Concepts and preliminary results in swine models

OBJECTIVE Complete anatomic obliteration remains difficult to achieve with endovascular treatment of wide-necked aneurysms using Guglielmi detachable platinum coils (GDCs). Ion implantation is a physicochemical surface modification process resulting from the impingement of a high-energy ion beam. Ion implantation and protein coating were used to alter the surface properties (thrombogenicity, endothelial cellular migration, and adhesion) of GDCs. These modified coils were compared with standard GDCs in the treatment of experimental swine aneurysms. METHODS In an initial study, straight platinum coils were used to compare the acute thrombogenicity of standard and modified coils. Modified coils were coated with albumin, fibronectin, or collagen and underwent Ne+ ion implantation at a dose of 1 x 10(15) ions/cm2 and an energy of 150 keV. Coils were placed in common iliac arteries of 17 swine for 1 hour, to evaluate their acute interactions with circulating blood. In a second study, GDCs were used to treat 34 aneurysms in an additional 17 swine. GDCs were coated with fibronectin, albumin, collagen, laminin, fibrinogen, or vitronectin and then implanted with ions as described above. Bilateral experimental swine aneurysms were embolized with standard GDCs on one side and with ion-implanted, protein-coated GDCs on the other side. The necks of aneurysms were evaluated macroscopically at autopsy, by using post-treatment Day 14 specimens. The dimensions of the orifice and the white fibrous membrane that covered the orifice were measured as the fibrous membrane to orifice proportion. Histopathological evaluation of the neck region was performed by light microscopy and scanning electron microscopy. RESULTS Fibronectin-coated, ion-implanted coils showed the greatest acute thrombogenicity (average thrombus weight for standard coils, 1.9 +/- 1.5 mg; weight for fibronectin-coated coils, 8.6 +/- 6.2 mg; P < 0.0001). By using scanning electron microscopy, an intensive blood cellular response was observed on ion-implanted coil surfaces, whereas this was rare with standard coils. At Day 14, greater fibrous coverage of the necks of aneurysms was observed in the ion-implanted coil group (mean fibrous membrane to orifice proportion of 69.8 +/- 6.2% for the ion-implanted coil group, compared with 46.8 +/- 15.9% for the standard coil group; P = 0.0143). CONCLUSION The results of this preliminary experimental study indicate that ion implantation combined with protein coating of GDCs improved cellular adhesion and proliferation. Future application of this technology may provide early wound healing at the necks of embolized, wide-necked, cerebral aneurysms.

Molecular Imaging of Drug-Modulated Protein-Protein Interactions in Living Subjects

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor κB promoter/enhancer elements using tumor necrosis factor α, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

Biophysical Mechanisms of Stroke

BACKGROUND Stroke is the third leading cause of death and the leading cause of long-term disability in the United States. Although a host of genetic, biochemical, physiological, anatomic, and histological factors have been implicated, to varying degrees, in the pathogenesis of stroke, biophysical factors are believed to play a significant role in the development, diagnosis, and therapy of stroke. The purpose of this review article is to identify, describe, and illustrate these causes and biophysical and hemodynamic mechanisms predisposing a person to stroke, which often form the basis for novel methods of diagnosis and therapy. SUMMARY OF REVIEW This mini-review begins by describing the physical principles that govern the flow of blood through normal and stenosed carotid artery bifurcations. In addition to the tortuosity, curvature, and tensile forces of the carotid artery bifurcation, the effects of biophysical phenomena from flowing blood such as viscous forces, pressure forces, velocity, kinetic energy, momentum, impulse, shear stress, and vibrational displacements exerted by the flowing blood on the vessel wall are conducive to abnormal flow behavior and patterns, degrading the vessel wall and creating the potential for stroke. CONCLUSIONS Recent advances in the treatment of stroke are based on increasing knowledge of its underlying biophysical mechanisms, as well as on better-publicized advances in imaging instrumentation and procedures for the management and treatment of patients.

The Fate and Toxicity of Raman-Active Silica-Gold Nanoparticles in Mice

Gold-core nanoparticles designed for imaging by Raman spectroscopy in patients are generally nontoxic in mice, causing only temporary liver inflammation when given intravenously. Minimal Toxicity of Nanoparticles for Raman Imaging Nanoparticles are just the right size to interact with molecules and cells. But how best to harness them as tools in the service of medicine? The authors of this paper have pursued one application: nanoparticles created to image specific cells and molecules from inside living animals—and eventually patients—via Raman spectroscopy, a method based on inelastic light scattering. Although the Raman effect is weak, a gold core inside the nanoparticles boosts the Raman signal enough so that it can be detected inside living tissue. To prepare the ground for use of these nanoparticles in imaging colorectal cancer in patients, Thakor et al. thoroughly tested their toxicity in mice. When introduced through the colon, these gold-core nanoparticles did not cross the gut lining into the body of the mice, a result that bodes well for their future as diagnostic and treatment vehicles for gut diseases. The authors first followed the fate of the silica-gold nanoparticles after the intravenous injection of a high dose into mice. Although their intravenous injection did cause temporary inflammation and some apoptosis in the liver 24 hours later, the nanoparticles were taken up by macrophages in the liver and spleen and eventually cleared from the body through the reticulo-endothelial system. A comprehensive survey of the mice revealed no ill effects of the nanoparticles on their general health or behavior. Their ECGs, blood pressure and heart rate were normal, and a panel of measurements of blood cells and chemistry revealed no effect of the particles. When the authors administered the nanoparticles into the colon, through the rectum, there was minimal evidence that the particles even passed into the animals’ circulation. Even the limited reaction in the liver seen after intravenous administration was absent and the particles were cleared within 5 minutes. The silica-gold nanoparticle tested in this paper can be coated with specific targeting molecules; the addition of one of these—a heptapeptide—did not increase the toxicity after treatment via the colon. These results set the stage for Raman spectroscopic imaging of these targeted, gold-core nanoparticles in diagnosis of colorectal cancer or other disease of hollow viscera. Attachment of premalignant cancer specific targeting groups to the particle would allow detection of early lesions with a endoscopic Raman probe, an approach that could be extended to other clinical situations. Raman spectroscopy is an optical imaging method that is based on the Raman effect, the inelastic scattering of a photon when energy is absorbed from light by a surface. Although Raman spectroscopy is widely used for chemical and molecular analysis, its clinical application has been hindered by the inherently weak nature of the Raman effect. Raman-silica-gold-nanoparticles (R-Si-Au-NPs) overcome this limitation by producing larger Raman signals through surface-enhanced Raman scattering. Because we are developing these particles for use as targeted molecular imaging agents, we examined the acute toxicity and biodistribution of core polyethylene glycol (PEG)–ylated R-Si-Au-NPs after different routes of administration in mice. After intravenous administration, PEG-R-Si-Au-NPs were removed from the circulation by macrophages in the liver and spleen (that is, the reticuloendothelial system). At 24 hours, PEG-R-Si-Au-NPs elicited a mild inflammatory response and an increase in oxidative stress in the liver, which subsided by 2 weeks after administration. No evidence of significant toxicity was observed by measuring clinical, histological, biochemical, or cardiovascular parameters for 2 weeks. Because we are designing targeted PEG-R-Si-Au-NPs (for example, PEG-R-Si-Au-NPs labeled with an affibody that binds specifically to the epidermal growth factor receptor) to detect colorectal cancer after administration into the bowel lumen, we tested the toxicity of the core nanoparticle after administration per rectum. We observed no significant bowel or systemic toxicity, and no PEG-R-Si-Au-NPs were detected systemically. Although additional studies are required to investigate the long-term effects of PEG-R-Si-Au-NPs and their toxicity when carrying the targeting moiety, the results presented here support the idea that PEG-R-Si-Au-NPs can be safely used in living subjects, especially when administered rectally.

Anatomical and morphological factors correlating with rupture of intracranial aneurysms in patients referred for endovascular treatment

Abstract The size of intracranial aneurysms is the only characteristic shown to correlate with their rupture. However, the critical size for rupture has varied considerably among previous accounts and remains a point of controversy. Our goal was to identify statistically significant clinical and morphological factors predictive of the occurrence of rupture and aneurysm size in patients referred for endovascular treatment. We retrospectively recorded the following factors from 74 patients who presented with ruptured (40) or unruptured (34) aneurysms: aneurysm morphology (uni/multilobulated), location (anterior/posterior), maximum diameter, diameter of the neck, and the patients age and sex. We performed stepwise discriminant, and stepwise and logistic regression analysis to identify factors predicting rupture and the size of the aneurysm at rupture. The mean diameter of the ruptured aneurysms was 11.9 ± 6.3 mm, range 3.0–33.0 mm, and that of the unruptured aneurysm 13.5 ± 5.8 mm, range 5.0–30 mm. Stepwise discriminant analysis identified aneurysm morphology (P < 0.001) and location in the intracranial circulation (P < 0.001) as statistically significant factors in predicting rupture. Stepwise regression analysis revealed that aneurysm morphology and the size of the neck were predictors of aneurysm size at rupture.

Polymer Nanoparticles Mediated Codelivery of AntimiR-10b and AntimiR-21 for Achieving Triple Negative Breast Cancer Therapy

The current study shows the therapeutic outcome achieved in triple negative breast cancer (TNBC) by simultaneously antagonizing miR-21-induced antiapoptosis and miR-10b-induced metastasis, using antisense-miR-21-PS and antisense-miR-10b-PS delivered by polymer nanoparticles (NPs). We synthesized the antisense-miR-21 and antisense-miR-10b loaded PLGA-b-PEG polymer NPs and evaluated their cellular uptake, serum stability, release profile, and the subsequent synchronous blocking of endogenous miR-21 and miR-10b function in TNBC cells in culture, and tumor xenografts in living animals using molecular imaging. Results show that multitarget antagonization of endogenous miRNAs could be an efficient strategy for targeting metastasis and antiapoptosis in the treatment of metastatic cancer. Targeted delivery of antisense-miR-21 and antisense-miR-10b coloaded urokinase plasminogen activator receptor (uPAR) targeted polymer NPs treated mice showed substantial reduction in tumor growth at very low dose of 0.15 mg/kg, compared to the control NPs treated mice and 40% reduction in tumor growth compared to scramble peptide conjugated NPs treated mice, thus demonstrating a potential new therapeutic option for TNBC.

Molecular imaging of homodimeric protein–protein interactions in living subjects

Homodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation‐based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N‐terminal (N‐hRLUC) or C‐terminal (C‐ hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail‐to‐ head homodimer). This was monitored using luminometry (68‐fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7‐fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant‐TK to generate N‐hRLUC mutant TK and mutant TK‐C‐hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo‐ dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8‐3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N‐hRLUC or C‐hRLUC ‘spacers’) to monitor incremental lack of association of the tail‐to‐head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein–protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects.