Antitumor Effect of Bleomycin Nanoaerosol in Murine Carcinoma Model.

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma.

Light-to-Heat Converting ECM-Mimetic Nanofiber Scaffolds for Neuronal Differentiation and Neurite Outgrowth Guidance.

The topological cues of fibrous scaffolds (in particular extracellular matrix (ECM)-mimetic nanofibers) have already proven to be a powerful tool for influencing neuronal morphology and behavior. Remote photothermal optical treatment provides additional opportunities for neuronal activity regulation. A combination of these approaches can provide "smart" 3D scaffolds for efficient axon guidance and neurite growth. In this study we propose two alternative approaches for obtaining biocompatible photothermal scaffolds: surface coating of nylon nanofibers with light-to-heat converting nanoparticles and nanoparticle incorporation inside the fibers. We have determined photoconversion efficiency of fibrous nanomaterials under near infrared (NIR) irradiation, as well as biocompatible photothermal treatment parameters. We also measured photo-induced intracellular heating upon contact of cells with a plasmonic surface. In the absence of NIR stimulation, our fibrous scaffolds with a fiber diameter of 100 nm induced an increase in the proportion of ß3-tubulin positive cells, while thermal stimulation of neuroblastoma cells on nanoparticles-decorated scaffolds enhanced neurite outgrowth and promoted neuronal maturation. We demonstrate that contact guidance decorated fibers can stimulate directional growth of processes of differentiated neural cells. We studied the impact of nanoparticles on the surface of ECM-mimetic scaffolds on neurite elongation and axonal branching of rat hippocampal neurons, both as topographic cues and as local heat sources. We show that decorating the surface of nanofibers with nanoparticles does not affect the orientation of neurites, but leads to strong branching, an increase in the number of neurites per cell, and neurite elongation, which is independent of NIR stimulation. The effect of photothermal stimulation is most pronounced when cultivating neurons on nanofibers with incorporated nanoparticles, as compared to nanoparticle-coated fibers. The resulting light-to-heat converting 3D materials can be used as tools for controlled photothermal neuromodulation and as "smart" materials for reconstructive neurosurgery.

Rapid Generation of Neurospheres from Hippocampal Neurons Using Extracellular-Matrix-Mimetic Scaffolds.

3D models of brain organoids represent an innovative and promising tool in neuroscience studies. However, the process of neurosphere formation in vitro remains complicated and is not always very effective. This is largely due to the lack of growth factors, guidance cues, and scaffold structures commonly found in tissues. Here we present a new, simple, and efficient method for generating neurospheres using scaffolds composed of electrospun nylon fibers with a diameter of 40-180 nm, which makes them similar to the brain extracellular matrix (ECM) components. Several main advantages of the proposed method should be highlighted. The method is fast, and the biomaterial consumption is low. Also, the resulting neurospheres are attached to the scaffold nanofibers. This not only provides the experimental convenience but also suggests that the resulting organoid models can potentially demonstrate fundamentally new properties, being closer to the nervous tissue in vivo. We demonstrate the influence of the fibrous scaffold structure on the formation, morphology, and composition of neurospheres and confirm adequate functional activity of the cellular components of these spheroids. The proposed approach can be further used for drug screening, modeling of neurodevelopmental, neurodegenerative disorders, and, potentially, therapeutic tissue engineering.

Rapid Ultrasensitive Gel-Free Immunoblotting with Magnetic Labels.

Immunoblotting is widely used for the detection of proteins using specific antibodies. We present here a new immunoblotting method, which is characterized by exceptional sensitivity, rapidness, and low consumption of antibodies. A thin conductive layer between touching hydrophilic cellulose membranes instead of polyacrylamide gel is used for the electrophoretic separation of proteins. Contrary to common Western blotting, the separation occurs in nondenaturing conditions. The membrane surface is smoothed by deposition of the cellulose layer and modified with azidophenyl groups, allowing for the photochemical in situ immobilization of proteins, which are carried out after the electrophoresis. Thus, the additional step of transferring the protein from the gel onto the membrane is eliminated. Specific protein bands are then visualized by decoration with magnetic beads. The limit of detection of interleukin IL-1ß reaches 0.3 fg or ∼104 molecules, whereas the total blotting time is about 5 min. The application of the technique is demonstrated by the detection of IL-1ß, total IgA, and IgA specific to Mycobacterium tuberculosis antigen in the exhaled breath samples, obtained from healthy subjects and tuberculosis patients.

Ballistic Penetration of Highly Charged Nanoaerosol Particles through a Lipid Monolayer.

To be used as a drug, inhaled nanoaerosol particles (NAPs) must first penetrate the lipid layer on top of the lung fluid before they will be able to reach the lung epithelium. We investigated how the penetration of NAPs through a model lipid monolayer (LM) depends upon their charging level and size. It was shown that deposition of NAPs 20-200 nm in diameter and charged to the Rayleigh limit gradually increased the surface tension of a dipalmitoylphosphatidylcholine monolayer (DPPC), indicating a loss of lipid molecules from the monolayer. This phenomenon was reproduced with a variety of NAPs produced from glucose, proteins, and polymers. Transfer of the lipid material into the subphase was documented by direct visualization of lipid nanoparticles in the subphase with atomic force microscopy after deposition of glucose NAPs on a DPPC monolayer, followed by collection of the lipid nanoparticles on a mica surface. Partial restoration of tension upon storage indicates that some of the lipid may return to the monolayer. Experiments with the deposition of highly charged calibrated polystyrene nanoparticles showed that the amount of lipid removed from the surface was roughly proportional to the overall surface area of the deposited NAPs. When the number of charges on the NAPs was reduced from their Rayleigh level of 103-104 units to 1-10 units, no notable changes in monolayer surface tension were observed even with prolonged deposition of such NAPs. It was therefore concluded that only highly charged NAPs of a certain size acquire sufficient speed from their attraction by mirror charges to enable ballistic penetration through a lipid monolayer.

Nanoaerosols reduce required effective dose of liposomal levofloxacin against pulmonary murine Francisella tularensis subsp. novicida infection.

BACKGROUND: The Institute of Theoretical and Experimental Biophysics in Moscow recently developed a new nanoaerosol generator. This study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary Francisella tularensis subsp. novicida (F. novicida) infections in BALB/c mice. RESULTS: First, the analysis of quantum dots distribution in cryosections of murine lungs demonstrated that nanoaerosols penetrate the alveoli and spread more homogenously in the lungs than upon intranasal delivery. Second, the generator was used to aerosolize the antibiotic levofloxacin to determine the effectiveness of nanoaerosolized levofloxacin as treatment against F. novicida. The generator was capable of delivering a sufficient dose of nanoaerosolized liposome-encapsulated levofloxacin to rescue mice against 100LD50 of F. novicida. CONCLUSIONS: The nanoaerosol-delivered dosage of liposome-encapsulated levofloxacin required to rescue mice is approximately 94× lower than the oral required dose and approximately 8× lower than the intraperitoneal dose required for rescue. In addition, treatment with nanoaerosols consumes less total volume of therapeutic solutions and is gentler on sprayed material than the aerosolization by a conventional three-jet Collison nebulizer as seen by the preservation of liposomes. This could represent a significant advance for the use of expensive therapeutics and lung directed therapies.

Knockdown of Fruit Flies by Imidacloprid Nanoaerosol.

This report describes the effects of nanoaerosol particles (NAPs) from imidacloprid (IMI) on fruit flies. NAPs were produced using a newly developed generator which employs electro-hydrodynamic atomization of IMI solution in ethanol. Exposure of Drosophila melanogaster to the IMI NAPs at a concentration of C = 2.7 ± 0.1 ng/cm(3) caused knockdown in half of the flies in T50 = 88 ± 14 min at 22 °C and in T50 = 36 ± 2 min at 33 °C. A number of special experiments precluded IMI volatilization and contact or oral action of IMI upon exposure to the NAPs. It was shown that only the fraction of NAPs in the size range of 7-300 nm is responsible for the knockdown and that dependence of T50 on the NAPs' fraction mass follows Haber's rule, C × T50 = const. Comparison with the oral doses obtained when flies were fed an IMI-sucrose mixture revealed that the inhaled doses that caused knockdown were 2 orders of magnitude lower than the oral ones. This new technology may be used to quickly eliminate insects with nanoaerosols of nonvolatile insecticides in greenhouses and other closed environments.

Dry Lung as a Physical Model in Studies of Aerosol Deposition.

A new physical model was developed to evaluate the deposition of micro- and nanoaerosol particles (NAPs) into the lungs as a function of size and charges. The model was manufactured of a dry, inflated swine lung produced by Nasco company (Fort Atkinson, WI). The dry lung was cut into two lobes and a conductive tube was glued into the bronchial tube. The upper 1-2-mm-thick layer of the lung lobe was removed with a razor blade to expose the alveoli. The lobe was further enclosed into a plastic bag and placed within a metalized plastic box. The probability of aerosol deposition was calculated by comparing the size distribution of NAPs passed through the lung with that of control, where aerosol passed through a box bypassing the lung. Using this new lung model, it was demonstrated that charged NAPs are deposited inside the lung substantially more efficiently than neutral ones. It was also demonstrated that deposition of neutral NAPs well fits prediction of the Multiple-Path Particle Dosimetry (MPPD) model developed by the Applied Research Associates, Inc. (ARA).

Are reactive oxygen species generated in electrospray at low currents?

It was demonstrated that electrospraying (ES) of solvents from a glass capillary proceeds without emission of light provided that the current is kept below a certain critical level (<100 nA at positive potential and <25 nA at negative potential for 96% ethanol; < 40 nA at positive potential for water). Though the onset of corona, as detected by the appearance of light, was always accompanied by a break in the current-voltage slope, such breaks also happened before the onset of corona, so they cannot be used as an adequate indicator of corona ignition. Of four ROS studied (hydrogen peroxide, ozone, hydroxyl radicals, and superoxide anions), only H2O2 and ozone were found to be generated at a current of 150-200 nA in detectable quantities: with a yield of 0.5-1 H2O2 molecules per electron at positive potential and 1.5-3 at negative potential. Despite the low yield of the ROS, jack bean urease was shown to be inactivated when the enzyme solution with a concentration below 20 µg/mL was electrosprayed at a current of 200 nA. Addition of 0.1 mM EDTA totally protected the activity of the electrosprayed urease.

Computer simulation of gas-phase neutralization of electrospray-generated protein macroions.

The process of neutralizing hydrated multicharged gas-phase protein ions with small counterions was simulated using a molecular dynamics (MD) technique. Hen egg white lysozyme (HEWL) molecules with different numbers of positive charges, both dry and solvated by up to 1500 water molecules, were first equilibrated. Simulations revealed that the hydration layer over a highly charged protein surface adapted a spiny structure with water protrusions composed of oriented water dipoles. MD simulations of the neutralization process showed that the impact of a small dehydrated single-charged counterion with a dehydrated HEWL ion bearing eight uncompensated charges resulted in a short local increase in temperature by 600-1000 K, which quickly (in 3-5 ps) dissipated over the whole protein molecule, increasing its average temperature by 20-25 K. When the protein ion was solvated, no drastic local increase in the temperature of the protein atoms was observed, because the impact energy was dissipated among the water molecules near the collision site.