Boris Polyak

High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents

A cell delivery strategy was investigated that was hypothesized to enable magnetic targeting of endothelial cells to the steel surfaces of intraarterial stents because of the following mechanisms: (i) preloading cells with biodegradable polymeric superparamagnetic nanoparticles (MNPs), thereby rendering the cells magnetically responsive; and (ii) the induction of both magnetic field gradients around the wires of a steel stent and magnetic moments within MNPs because of a uniform external magnetic field, thereby targeting MNP-laden cells to the stent wires. In vitro studies demonstrated that MNP-loaded bovine aortic endothelial cells (BAECs) could be magnetically targeted to steel stent wires. In vivo MNP-loaded BAECs transduced with adenoviruses expressing luciferase (Luc) were targeted to stents deployed in rat carotid arteries in the presence of a uniform magnetic field with significantly greater Luc expression, detected by in vivo optical imaging, than nonmagnetic controls.

Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles

Targeting gene therapy remains a challenge. The use of magnetic force to achieve this was investigated in the present study. It was hypothesized that nanoparticles with both controllable particle size and magnetic properties would enable magnetically driven gene delivery. We investigated this hypothesis by creating a family of novel biodegradable poly‐meric superparamagnetic nanoparticle (MNP) formulations. Polylactide MNP were formulated using a modified emulsification‐solvent evaporation methodology with both the incorporation of oleate‐coated iron oxide and a polyethylenimine (PEI) oleate ion‐pair surface modification for DNA binding. MNP size could be controlled by varying the proportion of the tetrahydrofuran cosolvent. Magnetically driven MNP‐mediated gene transfer was studied using a green fluorescent protein reporter plasmid in cultured arterial smooth muscle cells and endothelial cells. MNP‐DNA internalization and trafficking were examined by confocal microscopy. Cell growth inhibition after MNP‐mediated adiponectin plasmid transfec‐tion was studied as an example of a therapeutic end point. MNP‐DNA complexes protected DNA from degradation and efficiently transfected quiescent cells under both low and high serum conditions after a 15 min exposure to a magnetic field (500 G). There was negligible transfection with MNP in the absence of a magnetic field. Larger sized MNP (375 nm diameter) exhibited higher transfection rates compared with 185 nm‐ and 240 nm‐sized MNP. Internalized larger sized MNP escaped lysosomal localization and released DNA in the perinuclear zone. Adiponec‐tin plasmid DNA delivery using MNP resulted in a dose‐dependent growth inhibition of cultured arterial smooth muscle cells. It is concluded that magnetically driven plasmid DNA delivery can be achieved using biodegradable MNP containing oleate‐coated magnetite and surface modified with PEI oleate ion‐pair complexes that enable DNA binding.—Chorny, M., Polyak, B., Alferiev, I. S., Walsh, K., Friedman, G., Levy, R. J. Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles. FASEB J. 21, 2510–2519 (2007)

The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds.

One of the major challenges in engineering thick, complex tissues such as cardiac muscle, is the need to pre-vascularize the engineered tissue in vitro to enable its efficient integration with host tissue upon implantation. Herein, we explored new magnetic alginate composite scaffolds to provide means of physical stimulation to cells. Magnetite-impregnated alginate scaffolds seeded with aortic endothelial cells stimulated during the first 7 days out of a total 14 day experimental course showed significantly elevated metabolic activity during the stimulation period. Expression of proliferating cell nuclear antigen (PCNA) indicated that magnetically stimulated cells had a lower proliferation index as compared to the non-stimulated cells. This suggests that the elevated metabolic activity could instead be related to cell migration and re-organization. Immunostaining and confocal microscopy analyses supported this observation showing that on day 14 in magnetically stimulated scaffolds without supplementation of any growth factors, cellular vessel-like (loop) structures, known as indicators of vasculogenesis and angiogenesis were formed as compared to cell sheets or aggregates observed in the non-stimulated (control) scaffolds. This work is the first step in our understanding of how to accurately control cellular organization to form tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct with potential applicability for transplantation.

Cardiac tissue engineering in magnetically actuated scaffolds

Cardiac tissue engineering offers new possibilities for the functional and structural restoration of damaged or lost heart tissue by applying cardiac patches created in vitro. Engineering such functional cardiac patches is a complex mission, involving material design on the nano- and microscale as well as the application of biological cues and stimulation patterns to promote cell survival and organization into a functional cardiac tissue. Herein, we present a novel strategy for creating a functional cardiac patch by combining the use of a macroporous alginate scaffold impregnated with magnetically responsive nanoparticles (MNPs) and the application of external magnetic stimulation. Neonatal rat cardiac cells seeded within the magnetically responsive scaffolds and stimulated by an alternating magnetic field of 5 Hz developed into matured myocardial tissue characterized by anisotropically organized striated cardiac fibers, which preserved its features for longer times than non-stimulated constructs. A greater activation of AKT phosphorylation in cardiac cell constructs after applying a short-term (20 min) external magnetic field indicated the efficacy of magnetic stimulation to actuate at a distance and provided a possible mechanism for its action. Our results point to a synergistic effect of magnetic field stimulation together with nanoparticulate features of the scaffold surface as providing the regenerating environment for cardiac cells driving their organization into functionally mature tissue.

Particles and microfluidics merged: perspectives of highly sensitive diagnostic detection

AbstractThere is a growing need for diagnostic technologies that provide laboratories with solutions that improve quality, enhance laboratory system productivity, and provide accurate detection of a broad range of infectious diseases and cancers. Recent advances in micro- and nanoscience and engineering, in particular in the areas of particles and microfluidic technologies, have advanced the “lab-on-a-chip” concept towards the development of a new generation of point-of-care diagnostic devices that could significantly enhance test sensitivity and speed. In this review, we will discuss many of the recent advances in microfluidics and particle technologies with an eye towards merging these two technologies for application in medical diagnostics. Although the potential diagnostic applications are virtually unlimited, the most important applications are foreseen in the areas of biomarker research, cancer diagnosis, and detection of infectious microorganisms. FigureThere is a growing need for diagnostic technologies that provide laboratories with solutions that improve quality, enhance laboratory system productivity, and provide accurate detection of a broad range of infectious diseases and cancers. In this review, we will discuss many of the recent advances in microfluidics and particle technologies with an eye towards merging these two technologies for application in medical diagnostics such as microfluidic device to monitor molecular secretions in real-time as demonstrated in this figure.

Force Dependent Internalization of Magnetic Nanoparticles Results in Highly Loaded Endothelial Cells for Use as Potential Therapy Delivery Vectors

PurposeTo investigate the kinetics, mechanism and extent of MNP loading into endothelial cells and the effect of this loading on cell function.MethodsMNP uptake was examined under field on/off conditions, utilizing varying magnetite concentration MNPs. MNP-loaded cell viability and functional integrity was assessed using metabolic respiration, cell proliferation and migration assays.ResultsMNP uptake in endothelial cells significantly increased under the influence of a magnetic field versus non-magnetic conditions. Larger magnetite density of the MNPs led to a higher MNP internalization by cells under application of a magnetic field without compromising cellular respiration activity. Two-dimensional migration assays at no field showed that higher magnetite loading resulted in greater cell migration rates. In a three-dimensional migration assay under magnetic field, the migration rate of MNP-loaded cells was more than twice that of unloaded cells and was comparable to migration stimulated by a serum gradient.ConclusionsOur results suggest that endothelial cell uptake of MNPs is a force dependent process. The in vitro assays determined that cell health is not adversely affected by high MNP loadings, allowing these highly magnetically responsive cells to be potentially beneficial therapy (gene, drug or cell) delivery systems.

Tracking inflammation in the epileptic rat brain by bi-functional fluorescent and magnetic nanoparticles

Correct localization of epileptic foci can improve surgical outcome in patients with drug-resistant seizures. Our aim was to demonstrate that systemically injected nanoparticles identify activated immune cells, which have been reported to accumulate in epileptogenic brain tissue. Fluorescent and magnetite-labeled nanoparticles were injected intravenously to rats with lithium-pilocarpine-induced chronic epilepsy. Cerebral uptake was studied ex vivo by confocal microscopy and MRI. Cellular uptake and biological effects were characterized in vitro in murine monocytes and microglia cell lines. Microscopy confirmed that the nanoparticles selectively accumulate within myeloid cells in the hippocampus, in association with inflammation. The nanoparticle signal was also detectable by MRI. The in vitro studies demonstrate rapid nanoparticle uptake and good cellular tolerability. We show that nanoparticles can target myeloid cells in epileptogenic brain tissue. This system can contribute to pre-surgical and intra-surgical localization of epileptic foci, and assist in detecting immune system involvement in epilepsy.

Magnetic Nanoparticle-Mediated Targeting of Cell Therapy Reduces In-Stent Stenosis in Injured Arteries

Although drug-eluting stents have dramatically reduced the recurrence of restenosis after vascular interventions, the nonselective antiproliferative drugs released from these devices significantly delay reendothelialization and vascular healing, increasing the risk of short- and long-term stent failure. Efficient repopulation of endothelial cells in the vessel wall following injury may limit complications, such as thrombosis, neoatherosclerosis, and restenosis, through reconstitution of a luminal barrier and cellular secretion of paracrine factors. We assessed the potential of magnetically mediated delivery of endothelial cells (ECs) to inhibit in-stent stenosis induced by mechanical injury in a rat carotid artery stent angioplasty model. ECs loaded with biodegradable superparamagnetic nanoparticles (MNPs) were administered at the distal end of the stented artery and localized to the stent using a brief exposure to a uniform magnetic field. After two months, magnetic localization of ECs demonstrated significant protection from stenosis at the distal part of the stent in the cell therapy group compared to both the proximal part of stent in the cell therapy group and the control (stented, nontreated) group: 1.7-fold (p < 0.001) less reduction in lumen diameter as measured by B-mode and color Doppler ultrasound, 2.3-fold (p < 0.001) less reduction in the ratios of peak systolic velocities as measured by pulsed wave Doppler ultrasound, and 2.1-fold (p < 0.001) attenuation of stenosis as determined through end point morphometric analysis. The study thus demonstrates that magnetically assisted delivery of ECs is a promising strategy for prevention of vessel lumen narrowing after stent angioplasty procedure.

How can we predict behavior of nanoparticles in vivo

The emergence of nanotechnology and nanoparticles has brought conceptually new possibilities for administration of therapeutic agents. Sustained release, targeting, altered pharmacokinetic, reduced toxicity and increased drug bioavailability make nanoscale drug delivery systems highly attractive for improving the therapeutic index of many drugs. It is quite striking that nanotechnology not simply represents a miniaturization of larger objects, but rather enables preparation of materials at the nanometric scale with physical and chemical properties which dramatically differ from those of bulk materials. The main reason for this remarkable change in nanomaterial behavior is its enormous surface-to-volume ratio, which provides a very large interfacial surface area as driving force for enhanced interaction of nanomaterial with surrounding it molecules. Therefore, upon intravenous administration, nanoparticles inevitably form layers of adsorbed biomolecules (mainly proteins) known as a ‘protein corona’ [1,2]. The adsorption of proteins on the surface of nanoparticle is regulated by protein-nanoparticle binding affinity and by protein–protein interactions. Proteins that directly adsorb to the nanoparticle surface with high affinity (usually characterized by long desorption rates in the order of several hours [3]) form the first layer of tightly bound proteins known as the ‘hard corona’. There is a second stage, which consists of proteins interacting with this firmly bound proteinnanoparticle complex via low-affinity protein–protein interactions, forming the socalled ‘soft corona’ and consisting of loosely bound proteins. The complex structure of protein corona alters the size and interfacial composition of nanoparticles, conferring them a new biological identity, which is what is ‘seen’ by cells in reality. The biological identity of nanoparticles, which could be significantly different from their original synthetic identity, determines the physiological behavior of nanoparticles influencing their colloidal stability, targeting capability, kinetics of circulation, transport, cellular uptake and organ accumulation, degradation, drug release, signaling and toxicity [4]. Because the relative quantities of the adsorbed proteins on the surface of nanoparticles do not necessarily correlate with their abundance in blood plasma, the composition of protein corona for each particular type of nanomaterial is unique and influenced by multiple factors. The complexity and uniqueness of the protein corona composition in each particular case of nanomaterial does not allow one to reliably predict behavior of nanomaterials in vivo, which remains one of the major challenges in achieving a predictable and safe use of nanoparticlebased drug delivery systems in therapeutic applications. How can we predict behavior of nanoparticles in vivo?

Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention.