Nano LIFE

The goal of this work is to understand the role of nanoconfinement in designing molecular biosensors. We have been investigating microdevices incorporated with nanoporous membranes as molecular biosensor platforms. Detection of ultra low concentration of biomolecules is the key expectation from the new class of molecular biosensors utilizing nanomaterial. In this paper we have evaluated the role of the physical attributes of nanoporous aluminum oxide membranes in nanoconfinement and enhancing sensitivity of detection of protein biomolecules. In this biosensor configuration we have generated a sandwich assay in a high density array of nanoscale confined spaces generated by overlaying the nanoporous alumina membrane over metallic microscale sensing sites. The binding of the biomolecules results in the perturbation of the electrical double layer due to the binding of the test protein (C-reactive protein). Using electrical impedance spectroscopy (EIS), the capacitance changes in the electrical double layer associated with specific protein binding has been evaluated. The sensor performance metrics of sensitivity and dynamic range have been analyzed with changes in the pore diameter.

About 50% of all candidate drugs that make it past Phase-I clinical trials ultimately fail to receive U.S. Food and Drug Administration (FDA) marketing approval due to human toxicity or bioavailability problems that the drug develops. To remedy this state of affairs, in recent years, researchers have attempted to develop improved in vitro assays which can be applied pre-clinically to produce data with a higher degree of correlation to in vivo responses, and which circumvent the allometric limitations inherent in animal models. Such in vitro assays are intended to enable preclinical research and development groups to better predict the pharmacokinetic and pharmacodynamic action of candidate molecules prior to clinical trials, thereby reducing high late-stage failure rates. While most such in vitro systems comprise a static cell culture environment, microfluidic systems have been shown to provide better results. For example, experiments performed with the HμREL^® microfluidics system demonstrated increased clearance rates and better in vitro–in vivo correlation (IVIVC) than that observed with static culture systems. Several hypotheses were suggested to account for these improvements: (1) enhanced mass transport resulting in better functional efficiency; (2) a flow-induced transduction of gene and protein expression and function; and (3) a flow-induced effect resulting in increased cellular uptake. In this paper, we describe a framework, utilizing computational fluid dynamics (CFD), that can be used to study culture systems with a level of scrutiny and spatial precision not offered using standard in vitro assays. Using this approach, we were able to successfully model experimental observations of increases in the clearance rates of high and medium clearance compounds in the microfluidics system. Based on these results we posit that the increase in clearance is most likely due to the addition of convective transport, and a thinning of the boundary layer present in static and mixed plate cultures systems.

MC-1220 is a highly potent and selective non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV. The objective is to develop formulations for the vaginal delivery of MC-1220 and characterize them in vitro and in vivo (drug uptake, pharmacokinetics, toxicokinetics and vaginal irritation/inflammation). Due to the low aqueous solubility of MC-1220, emulsion-type and liposomal formulations of MC-1220 were developed. After rheological property adjustment (by gelling agents), the toxicity of two types of vaginal formulations of MC-1220 (emulsion [E] and liposomal [LIP] formulations) at 0.1% (E and LIP) and 0.5% (LIP) drug concentration, towards the vaginal mucosa as well as the absorption of the drug through the vaginal epithelium were investigated, after single and multiple administrations in New Zealand white NZW rabbits, for 10 days. Vaginal irritation was found to be within the acceptable range and always lower compared to the irritation caused by positive control formulation (nonoxynol-9), for all the formulation types (and concentrations evaluated). Pharmacokinetic values measured showed that the 0.1% LIP formulation was faster and better absorbed compared to the similar concentration E formulation, although most differences were not significant due to high variations. In conclusion, both types of formulations can be considered as safe for prolonged vaginal administration of MC-1220 or other drugs.

Macrophage foam cells are key components of atherosclerotic plaque and play an important role in the progression of atherosclerosis leading to plaque rupture and thrombosis. Foam cells are emerging as attractive targets for therapeutic intervention and imaging the progression of disease. Therefore, designing nanoparticles (NPs) targeted to macrophage foam cells in plaque is of considerable therapeutic significance. Here we report the construction of an oligonucleotide-functionalized NP system with high affinity for foam cells. Nanoparticles functionalized with a 23-mer poly-Guanine (polyG) oligonucleotide are specifically recognized by the scavenger receptors on lipid-laden foam cells in vitro and ex vivo. The enhanced uptake of polyG-functionalized NPs by foam cells is inhibited in the presence of acetylated-LDL, a known ligand of scavenger receptors. Since polyG oligonucleotides are stable in serum and are unlikely to induce an immune response, they are a promising candidate for developing an NP platform for scavenger receptor-mediated targeting of macrophages that can be optimized for targeting foam cells in atherosclerotic lesions.

Injectable oxygen delivery is an emerging technology that presents an opportunity for improved patient care in a number of medical disciplines. Here, we report on the fabrication and characterization of novel protein-encapsulated oxygen microbubbles (OMBs) designed for intravenous injection. The nanothick albumin encapsulation provided OMBs small enough for transcapillary passage: 99% of the microbubbles were less than 3-μm diameter and less than 1% of the oxygen was encapsulated in microbubbles greater than 8-μm diameter. The protein OMBs were remarkably stable, losing less than 40% of the encapsulated gas over 12 days. Upon injection into an oxygen-depleted saline solution, the protein OMBs rapidly equilibrated by releasing their oxygen core. These results indicate that protein microbubbles may serve as a suitable platform for direct injection of bioactive and therapeutic gases.

Gene therapy holds tremendous promise in prevention and treatment of diseases as the approach is based on regulating the expression of genes that are responsible for pathological conditions. The biggest bottleneck for gene delivery has been the development of safe and efficacious delivery systems. Although non-viral vectors are considered as much safer options than their viral counterparts, they suffer from low transfection efficiency. In this review, we highlight the role of non-condensing polymeric delivery systems for oral and systemic gene delivery. Using evidence from contemporary literature, non-condensing polymeric microparticle and nanoparticle systems afford physical encapsulation of the nucleic acid construct and can be engineered for targeted delivery to tissues and cells. Additionally, these systems have shown less toxicity and afford sustained cytoplasmic DNA delivery for efficient nuclear uptake and transfection for both DNA vaccines and therapeutic genes.

Dendritic cells (DCs) play a pivotal role in immune modulation. Therefore, understanding and regulating the mechanism of DC activation is paramount for functional optimization of any immunotherapy strategy. In particular, the paradoxical ability of DCs to secrete the immune suppressive enzyme indoleamine 2, 3-dioxygenase (IDO) and the suppressive cytokine IL-10 during the course of, and in response to, stimulation is of great interest. 1-Methyl-Tryptophan (1 MT) is a known inhibitor of IDO and has thus been administered in numerous in vitro and in vivo systems to block IDO activity. However, the effect 1 MT has on DCs beyond inhibiting IDO, especially in therapeutic models, has rarely been analyzed. In the current study, we have administered 1 MT via a nanopolymer-based delivery system in conjunction with an antigen (ovalbumin, OVA) and an adjuvant (CpG motif DNA) to determine both the effects of 1 MT on DCs and the resulting efficacy of the polymer-based treatments. 1 MT delivery alone, either via the polymer-based delivery vehicle or dissolved in solution, induced no significant change in DC activation as measured by surface expression of CD80, CD86, and MHCII and several secreted products such as IL-12. These same factors were upregulated however, when 1 MT was delivered in conjunction with OVA and CpG. Although soluble delivery of these components increased the levels of expression and secretion of key proteins, a differential effect of DC stimulation was seen as a result of the polymer delivery system. The T cell suppressive IL-10 secretion was lower with the polymer-based treatments and IL-12 immune-enhancing secretion was increased when 1 MT was supplemented into the polymer system. As a result, including 1 MT in the polymers along with OVA and CpG was seen to have additional effects on DC stimulation and was able to shift DCs to a state more indicative of inducing a Th1-type response.

Thermoresponsive nanocomposites were prepared by immobilizing a 2–3 nm thick phospholipid layer on the surface of superparamagnetic Fe₃O₄ nanoparticles via high-affinity avidin/biotin interactions. Morphological and physicochemical surface properties were assessed using transmission electron microscopy, confocal laser scanning microscopy, differential scanning calorimetry, and attenuated total reflectance Fourier transform infrared spectroscopy. The zeta potential of Fe₃O₄ colloids in phosphate buffered saline (PBS) decreased from -23.6 to -5.0 mV as a consequence of phospholipid immobilization. Nevertheless, heating properties of these superparamagnetic nanoparticles within an alternating magnetic field were not significantly affected. Hyperthermia-relevant temperatures > 40°C were achieved within 10–15 min using a 7-mT magnetic field alternating at a frequency of 1 MHz. Loading of the surface-associated phospholipid layer with the hydrophobic dye dansylcadaverine was accomplished at an efficiency of 479 ng/mg Fe₃O₄. Release of this drug surrogate was temperature-dependent, resulting in a 2.5-fold greater release rate when nanoparticles were exposed to a temperature above the experimentally determined melting temperature of 39.7°C. These data underline the feasibility of preparing novel, stimulus-induced drug delivery systems where payload release from a colloid-immobilized phospholipid assembly is triggered by hyperthermia.

Functional imaging is a novel area in radiological sciences and allows for the non-invasive assessment and visualization of specific targets such as gene and protein expression, metabolic rates, and drug delivery in intact living subjects. As such, the field of molecular imaging has been defined as the non-invasive, quantitative, and repetitive imaging of biomolecules and biological processes in living organisms. For example, cancer cells may be genetically altered to attract molecules that alter the magnetic susceptibility, thereby permitting their identification by magnetic resonance imaging. These contrast agents and/or molecular reporters are seen as essential to the task of molecular medicine to increase both sensitivity and specificity of imaging. Therefore, there are five general principles which need to be fulfilled in order to conduct a successful in vivo molecular imaging study: (1) selection of appropriate cellular and subcellular targets; (2) development of suitable in vivo affinity ligands (molecular probes); (3) delivery of these probes to the target organ; (4) amplification strategies able to detect minimal target concentrations; and (5) development of imaging systems with high resolution. Although there has been a wide range of routes taken to incorporate both imaging agents and a disease-targeting moiety into diagnostic devices, arguably the most interesting of these routes employs the use of nanoparticles. Nanoscale diagnostic systems that incorporate molecular targeting agents and diagnostic imaging capabilities are emerging as the next-generation imaging agents and have the potential to dramatically improve the outcome of the imaging, diagnosis, and treatment of a wide range of diseases. The present review addresses chemical aspects in development of molecular probes based upon gadolinium nanoparticles and their potential role in translational clinical imaging and therapy.

Plasmonic nanomaterials are poised to impact the clinical management of cancer through their ability to convert externally applied energy into localized heat at sites of diseased tissue. However, characterization of plasmonic nanomaterials as cancer therapeutics has been limited to xenograft models, creating a need to extend these findings to more clinically relevant models of cancer. Here, we evaluate the method of photothermal ablation therapy in a genetically engineered mouse model (GEMM) of sarcoma, which more accurately recapitulates the human disease in terms of structure and biology than subcutaneous xenograft models. Using polyethylene glycol (PEG)-coated gold nanorods (PEG-NRs), we quantitatively evaluate the ability of nanoparticles to penetrate and accumulate in sarcomas through passive targeting mechanisms. We demonstrate that PEG-NR–mediated photothermal heating results in significant delays in tumor growth with no progression in some instances. Lastly, by evaluating our photothermal ablation protocol in a GEMM, we observe off-target heating effects that are not detectable in xenograft models and which may be of future clinical interest.

The use of nanomedicine in the war on cancer diseases has progressed significantly in the recent past. Liposomal- and albumin-based chemotherapeutic agents as well as tumor contrast agents (e.g. Gd-DTPA, ferumoxides) have received FDA approval for human clinical use, while many other agents are in different phases of pre-clinical investigation and clinical trials. Plasmonic gold nanoparticles hold great promise as potential theranostic devices for detection and ablation of cancer diseases. This review discusses recent progress in the imaging, photothermal therapy, and nucleic acid/drug delivery using gold nanoparticles (spheres, shells, rods, cages) in vitro and in vivo. Issues relating to toxicity, biocompatibility, biodistribution, cellular uptake, and targeting efficiency are also discussed.

In this paper, we investigate the focalization properties of single-element transducers at intermediate frequencies (500 kHz) through primate and human skulls. The study addresses the transcranial targeting involved in ultrasound-induced blood–brain barrier (BBB) opening with clinically relevant targets such as the hippocampus and the basal ganglia, which are typically affected by early Alzheimer's and Parkinson's disease, respectively. The targeted brain structures were extracted from three-dimensional (3D) brain atlases registered with the skulls and used to virtually position and orient the transducers. The frequency dependence is first investigated and the capability of targeting of different structures is explored. Preliminary in vivo feasibility is investigated in mice at this frequency. A simple, affordable and convenient system is found to be feasible for BBB opening in primates and humans capable of successfully targeting the hippocampus, putamen and substantia nigra and could thus allow for its broader impact and applications.