High-throughput cell optoporation system based on Au nanoparticle layers mediated by resonant irradiation for precise and controllable gene delivery.

The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3-10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.

Detection and imaging of bacterial biofilms with glutathione-stabilized gold nanoclusters.

Bacterial biofilms colonize chronic wounds and surfaces of medical devices, thus making the development of reliable methods for imaging and detection of biofilms crucial. Although fluorescent identification of bacteria is sensitive and non-destructive, the lack of biofilm-specific fluorescent dyes limits the application of this technique to biofilm detection. Here, we demonstrate, for the first time, that fluorescent glutathione-stabilized gold nanoclusters (GSH-AuNCs) without targeting ligands can specifically interact with extracellular matrix components of Gram-negative and Gram-positive bacterial biofilms resulting in fluorescent staining of bacterial biofilms. By contrast, fluorescent bovine serum albumin-stabilized gold nanoclusters and 11-mercaptoundecanoic acid - stabilized gold nanoclusters do not stain the extracellular matrix of biofilms. According to molecular docking studies, GSH-AuNCs show affinity to several targets in extracellular matrix, including amyloid-anchoring proteins, matrix proteins and polysaccharides. Some experimental evidence was obtained for the interaction of GSH-AuNCs with the lipopolysaccharide (LPS) that was isolated from the matrix of Azospirillum baldaniorum biofilms. Based on GSH-AuNCs properties, we propose a new fluorescent method for the measurement of biofilm biomass with a limit of detection 1.7 × 105 CFU/mL. The sensitivity of the method is 10-fold higher than the standard biofilm quantification with the crystal violet assay. There is a good linear relationship between the fluorescence intensity from the biofilms and the number of CFU from the biofilms in the range from 2.6 × 105 to 6.7 × 107 CFU/mL. The developed nanocluster-mediated method of biofilm staining was successfully applied for quantitative detection of biofilm formation on urinary catheter surface. The presented data suggest that fluorescent GSH-AuNCs can be used to diagnose medical device-associated infections.

Gold nanoparticles in chemo-, immuno-, and combined therapy: review [Invited].

Functionalized gold nanoparticles (GNPs) with controlled geometrical and optical properties have been the subject of intense research and biomedical applications. This review summarizes recent data and topical problems in nanomedicine that are related to the use of variously sized, shaped, and structured GNPs. We focus on three topical fields in current nanomedicine: (1) use of GNP-based nanoplatforms for the targeted delivery of anticancer and antimicrobial drugs and of genes; (2) GNP-based cancer immunotherapy; and (3) combined chemo-, immuno-, and phototherapy. We present a summary of the available literature data and a short discussion of future work.

Biomedical Applications of Multifunctional Gold-Based Nanocomposites.

Active application of gold nanoparticles for various diagnostic and therapeutic purposes started in recent decades due to the emergence of new data on their unique optical and physicochemical properties. In addition to colloidal gold conjugates, growth in the number of publications devoted to the synthesis and application of multifunctional nanocomposites has occurred in recent years. This review considers the application in biomedicine of multifunctional nanoparticles that can be produced in three different ways. The first method involves design of composite nanostructures with various components intended for either diagnostic or therapeutic functions. The second approach uses new bioconjugation techniques that allow functionalization of gold nanoparticles with various molecules, thus combining diagnostic and therapeutic functions in one medical procedure. Finally, the third method for production of multifunctional nanoparticles combines the first two approaches, in which a composite nanoparticle is additionally functionalized by molecules having different properties.

Penetration of pegylated gold nanoparticles through rat placental barrier.

Penetration of pegylated (enveloped in polyethylene glycol) gold nanoparticles 5 and 30 nm in diameter through the placental barrier was studied in pregnant rats injected intravenously with these particles in a dose of about 0.8 mg Au/kg on day 10 of gestation. The particles were visualized in tissues by silver nitrate autometallography; the total content of gold in the fetuses was evaluated by atomic adsorption spectroscopy. Gold nanoparticles were detected in the fetal liver macrophages and in the spleens; high total content of gold in the fetuses was demonstrated for particles of both sizes. The data suggest that gold nanoparticles penetrate through rat placental barrier in vivo. No morphological changes were detected in the liver, kidneys, spleen, and brain of fetuses.

Colorimetric and dynamic light scattering detection of DNA sequences by using positively charged gold nanospheres: a comparative study with gold nanorods.

We introduce a new genosensing approach employing CTAB (cetyltrimethylammonium bromide)-coated positively charged colloidal gold nanoparticles (GNPs) to detect target DNA sequences by using absorption spectroscopy and dynamic light scattering. The approach is compared with a previously reported method employing unmodified CTAB-coated gold nanorods (GNRs). Both approaches are based on the observation that whereas the addition of probe and target ssDNA to CTAB-coated particles results in particle aggregation, no aggregation is observed after addition of probe and nontarget DNA sequences. Our goal was to compare the feasibility and sensitivity of both methods. A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models. In the case of GNRs, unexpectedly, the colorimetric test failed with perfect cigar-like particles but could be performed with dumbbell and dog-bone rods. By contrast, our approach with cationic CTAB-coated GNPs is easy to implement and possesses excellent feasibility with retention of comparable sensitivity--a 0.1 nM concentration of target cDNA can be detected with the naked eye and 10 pM by dynamic light scattering (DLS) measurements. The specificity of our method is illustrated by successful DLS detection of one-three base mismatches in cDNA sequences for both DNA models. These results suggest that the cationic GNPs and DLS can be used for genosensing under optimal DNA hybridization conditions without any chemical modifications of the particle surface with ssDNA molecules and signal amplification. Finally, we discuss a more than two-three-order difference in the reported estimations of the detection sensitivity of colorimetric methods (0.1 to 10-100 pM) to show that the existing aggregation models are inconsistent with the detection limits of about 0.1-1 pM DNA and that other explanations should be developed.

Gold nanoparticles in biology and medicine: recent advances and prospects.

Functionalized gold nanoparticles with controlled geometrical and optical properties are the subject of intensive studies and biomedical applications, including genomics, biosensorics, immunoassays, clinical chemistry, laser phototherapy of cancer cells and tumors, the targeted delivery of drugs, DNA and antigens, optical bioimaging and the monitoring of cells and tissues with the use of state-of-the-art detection systems. This work will provide an overview of the recent advances and current challenges facing the biomedical application of gold nanoparticles of various sizes, shapes, and structures. The review is focused on the application of gold nanoparticle conjugates in biomedical diagnostics and analytics, photothermal and photodynamic therapies, as a carrier for delivering target molecules, and on the immunological and toxicological properties. Keeping in mind the huge volume and high speed of the data update rate, 2/3 of our reference list (certainly restricted to 250 Refs.) includes publications encompassing the past 5 years.

Mutagenic effect of gold nanoparticles in the micronucleus assay.

Mutagenic activity of gold nanoparticles of different sizes were studied by micronucleus assay. Karyological analysis was performed and the count of micronucleoli in interphase bone marrow polychromatic erythrocytes in white outbred rats was determined. The animals orally received gold nanoparticles 16 and 55 nm in diameter and gold nanoshells 160 nm in diameter once a day for 7 days in a dose of 0.25 mg gold/kg. To ensure stability and biocompatibility, the surface of nanoparticles was functionalized with polyethylene glycol molecules. There were no significant differences in the number of micronucleoli in comparison with the control group, which suggests that gold nanoparticles of the specified size administered orally in the specified doses do not exhibit mutagenic activity.

On the Enhanced Antibacterial Activity of Antibiotics Mixed with Gold Nanoparticles.

The bacterial action of gentamicin and that of a mixture of gentamicin and 15-nm colloidal-gold particles on Escherichia coli K12 was examined by the agar-well-diffusion method, enumeration of colony-forming units, and turbidimetry. Addition of gentamicin to colloidal gold changed the gold color and extinction spectrum. Within the experimental errors, there were no significant differences in antibacterial activity between pure gentamicin and its mixture with gold nanoparticles (NPs). Atomic absorption spectroscopy showed that upon application of the gentamicin-particle mixture, there were no gold NPs in the zone of bacterial-growth suppression in agar. Yet, free NPs diffused into the agar. These facts are in conflict with the earlier findings indicating an enhancement of the bacterial activity of similar gentamicin-gold nanoparticle mixtures. The possible causes for these discrepancies are discussed, and the suggestion is made that a necessary condition for enhancement of antibacterial activity is the preparation of stable conjugates of NPs coated with the antibiotic molecules.

[Dependence of antimicrobial effect of micelle-capsulated therapeutics on the micelle size].

With the method of dynamic light scattering it was shown that the average size of micelles in the series of formulations based on various clindamycin salts, i. e. ClindHCl+Tween-20, ClindBz+Tween-20, ClindHCl+Cremafor-EL and ClindBz+Cremafor-EL increased from 6 to 20 nm. Investigations with the agar diffusion method revealed that the bactericidic action of the micelle-capsulated therapeutics did not depend on the micelle size within 6 to 20 mn. The concentration of the micellar clindamycin or gentamicin equal to 0.05 mcg/ml was bacteriostatic with respect to Micrococcus (Sarsina) luteus.

Advances in high-throughput screening: biomolecular interaction monitoring in real-time with colloidal metal nanoparticles.

The post-genomic era is revolutionizing the drug discovery process. The new challenges in the identification of therapeutic targets require efficient technological tools in order to be properly addressed. Label-free detection systems use proteins or ligands coupled to materials of which the physical properties are measurably modified upon specific interactions. Among the label-free systems currently available, the use of metal nanocolloids offers enhanced throughput and flexibility for real-time biomolecular recognition monitoring at a reasonable cost.

Spectroturbidimetry of fractal clusters: test of density correlation function cutoff.

I have measured the relative increase in turbidity τ/τ(t = 0) as a function of wavelength exponent w = -∂(log τ)/∂(log λ) in the process of the reaction-limited cluster-cluster aggregation of polystyrene latex with a particle diameter of 90 nm. It is shown that this dependence is not sensitive to particular details of sample preparation and is described for independent measurements by one master curve. Theoretical calculations made in the single-scattering and Berry-Percival mean-field theory approximations considering small-angle light-scattering effects and cluster polydispersity also demonstrate that this dependence is sufficiently universal and its shape can be practically determined solely by the choice of the density correlation function cutoff model. Comparing the experimental τ/τ(0) = ƒ(w) curves with calculated theoretical plots I conclude that the Gaussian cutoff model is the best among all the models of the exponential family. This is in accord with a previous finding [Langmuir 8, 2964-2069 (1992)] that was obtained by using angular scattering from diffusion-limited cluster-cluster aggregation soot aggregates.

Orientational averaging of light-scattering observables in the J-matrix approach.

The formalism of the quantum theory of angular momentum is used for orientational averaging of the a matrix, the Hermitian tensorJ(+)J, and the direct product T(*)(vv')T(Tmicromicro'). These results are independent of the nature of waves and scatterers. Equations for ?J? and ?J(+)I? are interpreted as specific forms of the generalized Wigner-Eckart theorem for the matrix elements of operators J and J(+)J which are calculated in terms of symmetrical top eigenfunctions. The averaged values of the above three types of tensor are used for the analytical calculation of a complete set of incoherent light-scattering observables, i.e., the total scattering and extinction cross sections and the Mueller matrix elements.