Intrathecal Magnetic Drug Targeting: A New Approach to Treating Diseases of the Central Nervous System

Intrathecal (IT) drug delivery is a standard technique which involves direct injection of drugs into the cerebrospinal fluid (CSF)-filled space within the spinal canal to treat many diseases of the central nervous system. Currently, in order to reach the therapeutic drug concentration at certain locations within the spinal canal, high drug doses are used. With no method to deliver the large drug doses locally, current IT drug delivery treatments are hindered with wide drug distributions throughout the central nervous system (CNS) which cause harmful side effects. In order to overcome the current limitations of IT drug delivery, we have developed the novel method of intrathecal magnetic drug targeting (IT-MDT). Gold-coated magnetite nanoparticles are infused into a physiologically and anatomically relevant in vitro human spine model and then targeted to a specific site using external magnetic fields, resulting in a substantial increase in therapeutic nanoparticle localization at the site of interest. Experiments aiming to determine the effect of key parameters such as magnet strength, duration of magnetic field exposure, location of magnetic field, and ferrous implants on the collection efficiency of our superparamagnetic nanoparticles in the targeting region were performed. Our experiments indicate that intrathecal magnetic drug targeting and implant-assisted IT-MDT are promising techniques for concentrating and localizing drug-functionalized nanoparticles at required target sites within the spinal canal for potential treatment of diseases affecting the central nervous system.

Mechanism of Cell Lysis in Microfluidic Channel With Integrated Nanocomposite Electrodes

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV–Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.

Multiscale Modeling of Flow Induced Thrombogenicity Using Dissipative Particle Dynamics and Molecular Dynamics

The coagulation cascade of blood may be initiated by flow induced platelet activation, which prompts clot formation in prosthetic cardiovascular devices and arterial disease processes. While platelet activation may be induced by biochemical agonists, shear stresses arising from pathological flow patterns enhance the propensity of platelets to activate and initiate the intrinsic pathway of coagulation, leading to thrombosis. Upon activation platelets undergo complex biochemical and morphological changes: organelles are centralized, membrane glycoproteins undergo conformational changes, and adhesive pseudopods are extended. Activated platelets polymerize fibrinogen into a fibrin network that enmeshes red blood cells. Activated platelets also cross-talk and aggregate to form thrombi. Current numerical simulations to model this complex process mostly treat blood as a continuum and solve the Navier-Stokes equations governing blood flow, coupled with diffusion-convection-reaction equations. It requires various complex constitutive relations or simplifying assumptions, and is limited to μm level scales. However, molecular mechanisms governing platelet shape change upon activation and their effect on rheological properties can be in the nm level scales. To address this challenge, a multiscale approach which departs from continuum approaches, may offer an effective means to bridge the gap between macroscopic flow and cellular scales. Molecular dynamics (MD) and dissipative particle dynamics (DPD) methods have been employed in recent years to simulate complex processes at the molecular scales, and various viscous fluids at low-to-high Reynolds numbers at mesoscopic scales. Such particle methods possess important properties at the mesoscopic scale: complex fluids with heterogeneous particles can be modeled, allowing the simulation of processes which are otherwise very difficult to solve by continuum approaches. It is becoming a powerful tool for simulating complex blood flow, red blood cells interactions, and platelet-mediated thrombosis involving platelet activation, aggregation, and adhesion.

Engineering Drug Delivery Vehicles With Multiphase Microfluidics

In our work, we propose the use of multiphase microfluidics to prepare drug delivery vehicles with complex structures, such as core-shell capsules, multicompartment microspheres and nonspherical particles; by tailoring the spatial distribution of drugs, unconventional drug release profiles can be achieved. To avoid the use of harmful organic solvents, we introduce the use of aqueous two-phase systems in microfluidics to generate the emulsion templates for making these novel delivery vehicles. By manipulating the interfacial characteristics of the emulsion templates, complex structures with hydrophilic and hydrophobic compartments can be prepared for separate encapsulation and sequential release of both hydrophilic and hydrophobic drugs. We will discuss the fundamental problems that need to be addressed to generate these drug delivery vehicles and highlight their potential by demonstrating their release characteristics.

Interaction With Nanoscale Topography: The Use of Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces To Modulate Physiological Focal Adhesions in Endothelial Cells

Achieving cell adhesion, growth and homeostasis on an underlying biomaterial surface may be a desirable feature in implant device design and tissue engineering. Insight has been gained from numerous cell patterning strategies where spatial cues and physical constraints have been shown to regulate the structure and function of cells. Despite significant advances in modifying substrates for cellular attachment, migration and proliferation, the achievement of confluent and aligned growth of functional endothelial cells on cardiovascular blood-contacting implants under physiologically significant wall shear stress has proven difficult. Recently we have reported on a method that enhances cellular adhesion under flow conditions on synthetic polymer surfaces, without reliance on pro-adhesive protein biomaterials, which are often thrombogenic. In this method we utilize electron beam lithography and size-dependent self-assembly to fabricate line arrays of nanowells allowing entrapment and retention of charged nanoparticles, covalently conjugated with a RGD adhesive ligand, GRGDSPK. This approach is an additive strategy of combining substrata topographic alteration, electrostatic charge and biochemical ligands, all uniquely incorporated as an ensemble of charged, ligand-bearing nanoparticles entrapped in arrays of nanowells. However, the modulation of endothelial cell physiologic mechanisms as a result of ensemble surface exposure remains to be characterized. In this report, we extend our studies and probe cell physiologic mechanisms altered as a result of nanofeatured surface exposure. We first examined the functional intactness or normalcy of endothelial cells adherent to the nanofeatured ensemble surface utilizing standard immunostaining and flow cytometry methods. We found β1-integrin expression dominated quiescent adherent endothelial cells while αVβ3-integrins expression was more common in migratory cells. Endothelial cells were noted to express high levels of PECAM-1 over time when exposed to nanofeatured surface and RGD peptides. For understanding the contribution of the nanofeatured surface (entrapped RGD conjugated nanoparticles) to cell adhesion, cytochalasin B was used to alter cell spreading. Confocal microscopy illustrated the uptake of nanoparticles in endothelial cells on composite surfaces, as well as the inhibition of this endocytosis by cytochalasin B. After prohibiting the cells from engulfing nanoparticles, we found an 80% reduction in cell adhesion; suggesting that an endocytic mechanism might play a role in maintaining cell adhesion. Nanofeatured ensemble surfaces appear to be good substrates for achieving a high level of EC adhesion, with maintained growth and stability.

Critical Long Bone Defect Treated by Magnetic Scaffolds and Fixed by Permanent Magnets

Diaphyseal bone defect represents a significant problem for orthopaedic surgeons and patients. In order to improve and fasten bone regenerating process we implanted HA biodegradable magnetized scaffolds in a large animal model critical bone defect. A critical long bone defect was created in 6 sheep metatarsus diaphysis; then we implanted a novel porous ceramic composite scaffold (20.0 mm in length; 6.00 mm inner diameter and 17.00 mm outer diameter), made of Hydroxyapatite that incorporates magnetite (HA/Mgn 90/10), proximally fixated by two small cylindrical permanent parylene coated NdFeB magnets (one 6.00 mm diameter magnetic rod firmly incorporated into the scaffold and one 8.00 mm diameter magnetic rods fitted into proximal medullary canal, both 10.00 mm long); to give stability to the complex bone-scaffold-bone, screws and plate was used as a bridge. Scaffolds biocompatibility was previously assessed in vitro using human osteoblast-like cells. Magnetic forces through scaffold were calculated by finite element software (COMSOL Multiphysics, AC/DC Model). One week after surgery, magnetic nanoparticles functionalized with vascular endothelial growth factor (VEGF) were injected at the mid portion of the scaffold using a cutaneous marker positioned during surgery as reference point. After sixteen weeks, sheep were sacrificed to analyze metatarsi. Macroscopical, radiological and microCT examinations were performed. Macroscopical examination shows bone tissue formation inside scaffold pores and with complete coverage of scaffolds, in particular at magnetized bone-scaffold interface. X-rays show a good integration of the scaffold with a good healing process of critical bone defect, and without scaffolds mobilization. These datas were confirmed by the microCT that shown new formation of bone inside the scaffolds, in particular at magnetized bone-scaffold interface. These preliminary results lead our research to exploiting magnetic forces to stimulate bone formation, as attested in both in vitro and in vivo models and to improve fixation at bone scaffold interface, as calculated by finite element software, and moreover to guide targeted drug delivery without functionalized magnetic nanoparticles dissemination in all body. Histological analysis will be performed to confirm and quantify bone tissue regeneration at both interfaces.

Modeling and Simulation of Hydrodynamic Interaction of DNA in a Micro-Fluidic Channel

The motion of DNA (in the bulk solution) and the non-Newtonian effective fluid behavior are considered separately and self-consistently with the fluid motion satisfying the no-slip boundary condition on the surface of the confining geometry in the presence of channel pressure gradients. A different approach has been developed to model DNA in the micro-channel. In this study the DNA is assumed as an elastic chain with its characteristic Young’s modulus, Poisson’s ratio and density. The force which results from the fluid dynamic pressure, viscous forces and electromotive forces is applied to the elastic chain in a coupled manner. The velocity fields in the micro-channel are influenced by the transport properties. Simulations are carried out for the DNAs attached to the micro-fluidic wall. Numerical solutions based on a coupled multiphysics finite element scheme are presented. The modeling scheme is derived based on mass conservation including biomolecular mass, momentum balance including stress due to Coulomb force field and DNA-fluid interaction, and charge transport associated to DNA and other ionic complexes in the fluid. Variation in the velocity field for the non-Newtonian flow and the deformation of the DNA strand which results from the fluid-structure interaction are first studied considering a single DNA strand. Motion of the effective center of mass is analyzed considering various straight and coil geometries. Effects of DNA statistical parameters (geometry and spatial distribution of DNAs along the channel) on the effective flow behavior are analyzed. In particular, the dynamics of different DNA physical properties such as radius of gyration, end-to-end length etc. which are obtained from various different models (Kratky-Porod, Gaussian bead-spring etc.) are correlated to the nature of interaction and physical properties under the same background fluid environment.

Predicting Endothelial Cell Phenotypes in Angiogenesis

Controlling endogenous angiogenesis, or the formation of new capillaries, is a potential therapeutic strategy for numerous diseases including cancer, stroke and cardiovascular disease (1–4). These efforts have been met with mixed success in the clinic, partly due to an inadequate understanding, and thus control, of the mechanisms that influence the endothelial cells that form capillaries (1, 3). In order to control angiogenesis in an effort to improve treatment responses, quantitative information about endothelial cell behavior must be used to build accurate models of vascular network formation. In this paper, we introduce a method to identify and classify endothelial cell responses to angiogenic stimuli through sophisticated image analysis. The presented automated image processing tools and classification framework allow for rapid quantitative investigations of cellular images. Results of our analysis demonstrate that endothelial cells can be grouped into distinct morphological phenotypes as a function of their responses to combinations of angiogenic growth factor stimuli. Information on phenotypic behavior and responses will be applied towards predicting and guiding cell behavior for therapeutic design.

Material Properties of Porcine Corneal Stroma in Unconfined Compression

The tensile properties of the cornea have been extensively studied while there are fewer studies on its compressive stiffness. The mechanical properties and structure of the cornea like many other connective tissues are derived from the function and properties of their extracellular matrix. The corneal extracellular matrix, stroma, is a polyelectrolyte gel composed of collagenous fibers embedded in an aqueous matrix. The cornea has two different functions: optical and mechanical. It is the main refractive component of the visual system and it is an effective barrier resisting the deformation caused by external and internal stresses. A necessary condition for corneal optical properties and transparency is the maintenance of a pseudo hexagonal arrangement of the collagen fibers inside the extracellular matrix. This regular arrangement is attributed to the interaction of collagen fibers with the proteoglycans. Under physiological conditions, the proteoglycans are ionized and form a hydrated gel in the empty space between the collagen fibrils by attracting the water and solutes. The interaction of the negatively fixed charges of the proteoglycans with themselves and with the free ions inside the interstitial fluid contributes to the corneal swelling pressure and subsequently to its compressive properties.