Ionic, structural, and temperature effects on DNA nanoparticles formed by natural and synthetic polyamines

We synthesized analogues of spermine and studied the effects of chemical structure, ionic strength, and temperature on lambda-DNA nanoparticle formation. Effective concentration of polyamines for DNA condensation (EC50) was lowest for hexamines (0.2 microM) and highest for spermine (tetramine, 4.2 microM). The EC50 value increased with [Na+]. Dynamic light scattering showed nanoparticles with hydrodynamic radii (R(h)) of 40-50 nm. Effect of temperature on R(h) was measured between 20 and 70 degrees C. For spermine, R(h) remained relatively stable until 50 degrees C and increased significantly at >60 degrees C. In contrast, the hexa- and penta-valent analogues exhibited a gradual increase in R(h) between 20 and 70 degrees C. The nanoparticles were mainly toroidal, as revealed by electron microscopy (EM). EM studies showed changes in morphology and size of condensed structures with an increase in temperature. A possible mechanism for the differential effects of temperature on DNA nanoparticles might involve different modes of DNA-polyamine interactions.     

Nanoparticles of compacted DNA transfect postmitotic cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.     

Characterization of silver nanoparticles synthesized by using marine isolate Streptomyces albidoflavus

Silver nanoparticles production by the green chemistry approach was investigated using an isolated marine actinomycetes strain. The isolated strain was identified as Streptomyces albidoflavus based on chemotaxonomic and ribotyping properties. The strain revealed production of silver nanoparticles both extracellular and intracellularly. Surface Plasmon Resonance analysis with the function of time revealed that particle synthesis by this strain is reaction time dependent. The produced particles were spherical shaped and monodispersive in nature and showed a single surface plasmon resonance peak at 410 nm. Size distribution histograms indicated production of 10-40- nm-size nanoparticles with a mean size of 14.5 nm. FT-IR spectra of nanopartilces showed N-H, C-H, and C-N stretching vibrations, denoting the presence of amino acid/ peptide compounds on the surface of silver nanoparticles produced by S. albidoflavus. Synthesized nanoparticles revealed a mean negative zeta potential and electrophoretic mobility of -8.5 mV and -0.000066 cm2/Vs, respectively. The nanoparticles produced were proteinaceous compounds as capping agents with -8.5 mV zeta potential and revealed antimicrobial activity against both Gram-negative and -positive bacterial strains. Owing to their small size, these particles have greater impact on industrial application spectra.     

High cooperativity of the SV40 major capsid protein VP1 in virus assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.     

Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents

Ethanol and a variety of solvents are known to activate basal and Gpp(NH)p- and hormone-stimulated adenylate cyclase. We report here that ethanol and other solvents inhibit the activation of adenylate cyclase by forskolin. In the presence of 10 microM forskolin, 2% ethanol gives about 20% inhibition and 5% ethanol gives 40% inhibition of enzyme activity. Analysis of ethanol inhibition at several forskolin concentrations suggests that inhibition is competitive versus forskolin. Thus the effect of ethanol is greater at low forskolin concentrations and minimal at high concentrations. In addition to ethanol, inhibition of forskolin activation was observed with acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol and n-propanol. Dimethyl sulfoxide was inhibitory only at high concentrations (10%). Since some solvent is needed to prepare forskolin solutions and to maintain solubility at higher concentrations, the inhibitory effects reported here are an important consideration in studies employing forskolin activation. To minimize solvent inhibition we recommend that dimethyl sulfoxide be used to prepare forskolin solutions. At concentrations of 5% and less, dimethyl sulfoxide gives little if any inhibition of forskolin activation and causes only small increases in basal activity.     

Fabrication and growth mechanism of ZnO nanostructures and their cytotoxic effect on human brain tumor U87, cervical cancer HeLa,and normal HEK cells

ZnO nanostructures of diverse shape were grown via a solution process with different precursors and conditions. Morphological investigation of the nanostructures was carried out using field emission scanning electron microscopy and transmission microscopy observations and revealed that the nanostructures exhibit a wurtzite phase with an ideal lattice fringe distance of approximately 0.52 nm. The powder crystallinity was examined via X-ray diffraction spectroscopy. Screening results from anticancer studies of the effects on human brain tumor U87, cervical cancer HeLa, and normal HEK cells of ZnO nanostructures of diverse shape were obtained and indicate promising activity that varies with changes in the structure and the size of the particles. Treatment-induced cell death [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and survival assay], growth inhibition, cytogenetic damage (formation of micronuclei), and apoptosis were studied as parameters for the cellular response. Treatment with nanostructures enhanced growth inhibition and cell death in a concentration-dependent manner in both U87 and HeLa cell lines. At higher concentrations (above 15.6 μg/ml) the cytotoxic effects of the nanoparticles were highly synergistic and mainly mediated through apoptosis, implying the possible interactions of lesions caused by the agents. The enhanced cell death due to nanoparticles was accompanied by a significant increase (2–3 fold at 31.25 μg/ml) in the formation of micronuclei in U87 cells. The increase in the formation of micronuclei observed after treatment indicates that these structures may interfere with the rejoining of DNA strand breaks. Among all the nanostructures, nanoparticles and sheets exhibited potent activity against both HeLa and U87 cells. However, despite potent in vitro activity, all nanostructures exhibited diminished cytotoxicity against normal human HEK cells at all effective concentrations.     

Mechanistic differences in DNA nanoparticle formation in the presence of oligolysines and poly-L-lysine

We studied the effectiveness of trilysine (Lys3), tetralysine (Lys4), pentalysine (Lys5), and poly-l-lysine (PLL) (MW 50000) on lambda-DNA nanoparticle formation and characterized the size, shape, and stability of nanoparticles. Light scattering experiments showed EC50 (lysine concentration at 50% DNA compaction) values of approximately 0.0036, 2, and 20 micromol/L, respectively, for PLL, Lys5, and Lys4 at 10 mM [Na+]. Plots of log EC50 versus log [Na+] showed positive slopes of 1.09 and 1.7, respectively, for Lys4 and Lys5 and a negative slope of -0.1 for PLL. Hydrodynamic radii of oligolysine condensed particles increased (48-173 nm) with increasing [Na+], whereas no significant change occurred to nanoparticles formed with PLL. There was an increase in the size of nanoparticles formed with Lys5 at >40 degrees C, whereas no such change occurred with PLL. The DNA melting temperature increased with oligolysine concentration. These results indicate distinct differences in the mechanism(s) by which oligolysines and PLL provoke DNA condensation to nanoparticles.     

Effects of Cerium Oxide Nanoparticles on the Proliferation, Differentiation, and Mineralization Function of Primary Osteoblasts In Vitro

The effects of cerium oxide nanoparticles on the proliferation, differentiation, and mineralization function of primary osteoblasts in vitro were evaluated. The results showed that the cell biological effects of cerium oxide nanoparticles varied with different diameters. The cytotoxicity of cerium oxide nanoparticles on primary osteoblasts varies with the size and incubation time. Sixty-nanometer cerium oxide nanoparticles show significant cytotoxicity on primary osteoblasts at 48 h exposure. Cerium oxide nanoparticles with diameters of 40 nm promoted the differentiation of osteoblasts and the promotion rate was enhanced with increasing concentration. Cerium oxide nanoparticles with diameters of 60 nm promoted the differentiation of osteoblasts at lower concentrations, but turned to inhibit the differentiation at higher concentrations. Cerium oxide nanoparticles promoted the adipogenic transdifferentiation of osteoblasts at all tested concentrations. Moreover, the effects of 60-nm cerium oxide nanoparticles were stronger than that of 40-nm cerium oxide nanoparticles. Cerium oxide nanoparticles promoted the formation of mineralized matrix nodules of osteoblasts at all tested concentrations in a dose-dependent manner and the promotion rate increased with decreasing size. The results showed that cerium oxide nanoparticles had no acute cytotoxic effects on osteoblasts and could promote the osteogenic differentiation and mineralization of osteoblasts. Moreover, the size, concentration, and culture time of nanoparticles have significant influence on the proliferation, differentiation, and mineralization of osteoblasts.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Colorimetric enzymatic activity assay based on noncrosslinking aggregation of gold nanoparticles induced by adsorption of substrate peptides

The mechanisms of colorimetric assays based on aggregation of gold nanoparticles (GNPs) have been separated into two categories, crosslinking, and noncrosslinking aggregation. The noncrosslinking aggregation has recently been emerging as a simple and rapid mechanism and has been applied to enzymatic activity assays and DNA detection. We report here the detailed study of an enzymatic activity assay for protein kinases based on noncrosslinking aggregation. The principle of the assay is to detect kinase activity by utilizing the difference of coagulating ability of a cationic substrate peptide and its phosphorylated form toward GNPs with anionic surface charge. The critical coagulation concentrations (CCCs) of the peptides were about 10(3) times lower than those of the metal cations with the same cationic charges. The multivalent coordination bonds of the functional groups of the peptides with the GNP surface will strongly support the adsorption of the peptide on the GNP surface. The effect of the GNP size (10, 20, 40, 60 nm) on the dynamic range of OD before and after aggregation was studied. The dynamic range became a maximum for 20 nm GNP among those studied. The difference of CCC between the phosphorylated and nonphosphorylated peptides was governed by (1) the ratio between the peptide concentration and the surface area concentration of GNP and (2) the net charge of the peptides. When the assay system was applied to the activity assessment of protein kinase A, the dynamic range of OD was largest for 20 nm GNPs. However, when the peptide concentration was lowered, the largest 60 nm GNP was advantageous because of its smaller specific surface area.     

Amphiphilic core-shell nanoparticles with poly(ethylenimine) shells as potential gene delivery carriers

Spherical, well-defined core-shell nanoparticles that consist of poly(methyl methacrylate) (PMMA) cores and branched poly(ethylenimine) shells (PEI) were synthesized via a graft copolymerization of methyl methacrylate from branched PEI induced by a small amount of tert-butyl hydroperoxide. The PMMA-PEI core-shell nanoparticles were between 130 to170 nm in diameter and displayed zeta-potentials near +40 mV at pH 7 in 1 mM aqueous NaCl. Plasmid DNA (pDNA) was mixed with nanoparticles and formed complexes of approximately 120 nm in diameter and was highly monodispersed. The complexes were characterized with respect to their particle size, zeta-potential, surface morphology, and DNA integrity. The complexing ability of the nanoparticles was strongly dependent on the molecular weight of the PEI and the thickness of the PEI shells. The stability of the complexes was influenced by the loading ratio of the pDNA and the nanoparticles. The condensed pDNA in the complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxity studies using MTT colorimetric assays suggested that the PMMA-PEI (25 kDa) core-shell nanoparticles were three times less toxic than the branched PEI (25 kDa). Their transfection efficiencies were also significantly higher. Thus, the PEI-based core-shell nanoparticles show considerable potential as carriers for gene delivery.     

Extracellular biosynthesis,characterization, optimization of silver nanoparticles (AgNPs) using Bacillus mojavensis BTCB15 and its antimicrobial activity against multidrug resistant pathogens

Abstract

Biosynthesis of metal nanoparticles is an area of interest among researchers because of its eco-friendly approach. Current study focuses at biosynthesis of silver nanoparticles (AgNPs) and optimization of physico-chemical conditions to obtain mono-dispersed and stable AgNPs having antimicrobial activity. Initially Bacillus mojavensis BTCB15 produced silver nanoparticles (AgNPs) of 105?nm. Silver nanoparticles (AgNPs) were characterized by particle size analyzer, UV-Vis Spectroscopy, Fourier transforms infrared spectroscopy (FTIR), Atomic force microscopy (AFM), and X-ray diffraction (XRD). Whereas, under optimal conditions of temperature 55?°C, pH 8, addition of surfactant Tween 20, and metal ion K₂SO₄, about 104% size reduction was achieved with average size of 2.3nm. Molecular characterization revealed 98% sequence homology with Bacillus mojavensis. AgNPs exhibited antibacterial activity at concentrations ranging from 0.5 to 2.5?µg/µl against Escherichia coli BTCB03, Klebsiella pneumonia BTCB04, Acinetobacter sp. BTCB05, and Pseudomonas aeruginosa BTCB01 but none against Staphylococcus aureus BTCB02. Highest antibacterial activity was observed at 0.27?µg/µl and lowest at 0.05?µg/µl of AgNPs indicated by zone of inhibition. Conclusively, under optimum conditions, Bacillus mojavensis BTCB15 was able to produce AgNPs of 2.3?nm size and had antibacterial activity against multi drug resistant pathogens.     

Innovative approaches to the use of polyamines for DNA nanoparticle preparation for gene therapy

Advances in genomic technologies, such as next generation sequencing and disease specific gene targeting through anti-sense, anti-gene, siRNA and microRNA approaches require the transport of nucleic acid drugs through the cell membrane. Membrane transport of DNA/RNA drugs is an inefficient process, and the mechanism(s) by which this process occurs is not clear. A pre-requisite for effective transport of DNA and RNA in cells is their condensation to nanoparticles of ~100 nm size. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major challenge. Multivalent cations, such as natural polyamines are excellent promoters of DNA/RNA condensation to nanoparticles. During the past 20 years, our laboratory has synthesized and tested several analogs of the natural polyamine, spermine, for their efficacy to provoke DNA condensation to nanoparticles. We determined the thermodynamics of polyamine-mediated DNA condensation, measured the structural specificity effects of polyamine analogs in facilitating the cellular uptake of oligonucleotides, and evaluated the gene silencing activity of DNA nanoparticles in breast cancer cells. Polyamine-complexed oligonucleotides showed a synergistic effect on target gene inhibition at the mRNA level compared to the use of polyamines and oligonucleotides as single agents. Ionic and structural specificity effects were evident in DNA condensation and cellular transportation effects of polyamines. In condensed DNA structures, correlation exists between the attractive and repulsive forces with structurally different polyamines and cobalt hexamine, indicating the existence of a common force in stabilizing the condensed structures. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.     

Early Inhibition of Cellular DNA Synthesis by High Multiplicities of Infectious and UV-Inactivated Reovirus

Inhibition of cellular DNA synthesis began 6 to 8 h after reovirus infection at a multiplicity of infection of 10 PFU per cell. However, as the multiplicity of infection was increased to a maximum of 10³ PFU/cell, inhibition of DNA synthesis began earlier after infection (2-4 h postinfection), and the initial rate of inhibition increased. The enhanced inhibition of DNA replication at high virus multiplicities appeared to be selective since RNA synthesis was not detectably altered as late as 9 h postinfection and inhibition of protein synthesis did not begin until 7 to 9 h after infection. Early inhibition of DNA synthesis did not appear to be related to changes in thymidine pool characteristics, thymidine kinase activity, or detectable degradation of cellular DNA. Even though the particle-to-PFU ratio was increased by ultraviolet light inactivation of virus, the ability to induce early inhibition of DNA synthesis was not diminished.     

Structural variability of DNA-containing Mg-pyrophosphate microparticles: optimized conditions to produce particles with desired size and morphology

Our previous studies demonstrated the formation of structurally diverse DNA-containing microparticles (DNA MPs) in PCR with Mg-pyrophosphate (MgPPi) as the structure-forming component. These DNA MPs were referred to major structural types: microdisks (2D MPs) with nanometer thickness and 3D MPs with sophisticated morphology and constructed from intersecting disks and their segments. Little is known about factors that influence both the morphology and size of DNA MPs, and the present study was aimed at fulfilling this gap. We showed that the addition of Mn²⁺ cations to PCR mixtures caused the profound changes in MPs morphology, depending on DNA polymerase used (KlenTaq or Taq). Asymmetric PCR with 20-fold decrease in the concentration of one of two primers facilitated the predominant formation of microdisks with unusual structure. The addition of 1 mM Na-pyrophosphate to PCR mixtures with synthesized DNA and subsequent thermal cycling (10–15 cycles) were optimal to produce microdisks or nanometer 3D particles. Using electron microscopy, we studied also the structure of inorganic micro- and nanoparticles from MgPPi, formed during multiple heating and cooling cycles of a mixture of Mg²⁺ and Na-pyrophosphate in various regimes. Also, we found the conditions to yield planar (Mg·Mn)PPi nanocrystals (diameter ~100 nm and thickness ~10 nm) which efficiently adsorbed exogenous DNA. These inorganic nanoparticles are promising for DNA delivery in transfection studies. Mechanisms to be involved in structural modifications of MPs and perspectives of their practical application are discussed.     

Synthesis of biologically active copper oxide nanoparticles as promising novel antibacterial-antibiofilm agents

Abstract

In this study, we aimed to synthesize copper oxide nanoparticles (CuONPs) mediated by plant extract in an environmentally friendly way and to reveal their potential biological activities. Here we synthesized CuONPs by using different concentrations of aqueous leaf extract of Thymbra spicata at 80?°C to obtain Ts1CuONPs and Ts2CuONPs. Biosynthesized nanoparticles were characterized by using UV-Vis, AFM, FTIR, SEM-EDS, TEM, DLS and zeta potential analysis. The antibacterial activity of the nanoparticles was determined by calculation of the inhibition zone and minimum inhibitory concentration against selected bacterial strains. Moreover, the antioxidant activity of the as-synthesized nanoparticles was evaluated based on DPPH radical scavenging activity. The results indicate that the as-synthesized NPs have an average size of 26.8 and 21?nm for Ts1CuONPs and Ts2CuONPs, respectively. The formed CuONPs have more antibacterial action on gram-positive bacteria compared to gram-negative bacteria. In addition, CuONPs demonstrated good inhibition activity against biofilm formation of Staphylococcus aureus (S. aureus). Furthermore, the results showed that the smaller size of the CuONPs caused the higher cytotoxicity on L929 mouse fibroblast cells. The as-synthesized CuONPs exhibit antibacterial and antibiofilm potential against S. aureus, indicating that they may be attractive candidates to use in future therapeutic applications.     

Silver Nanoparticles Toxicity and Bactericidal Effect Against Methicillin-Resistant Staphylococcus aureus: Nanoscale Does Matter

Silver nanoparticles, which are being used increasingly as antimicrobial agents, may extend its antibacterial application to methicillin-resistant Staphylococcus aureus (MRSA), the main cause of nosocomial infections worldwide. To explore the antibacterial properties of silver nanoparticles against MRSA, the present work includes an analysis of the relation between nanosilver effect and MRSA’s resistance mechanisms, a study of the size dependence of the bactericidal activity of nanosilver and a toxicity assessment of nanoparticles against epithelial human cells. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and MBC/MIC ratio of silver nanoparticles were quantified by using a luciferase-based assay. The cytotoxic effect (CC₅₀ and CC₉₀) of three different nanosilver sizes (10, 30–40, and 100 nm) were assessed in HeLa cells by a similar method. The therapeutic index was used as an indicator of nanosilver overall efficacy and safety. Silver nanoparticles inhibited bacterial growth of both MRSA and non-MR S. aureus in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio?≤?4). Silver nanoparticle’s therapeutic index varied when nanoparticle’s size diminished. At the same dose range, 10 nm nanoparticles were the most effective since they did not affect HeLa’s cell viability while inhibiting a considerable percentage of MRSA growth. Silver nanoparticles are effective bactericidal agents that are not affected by drug-resistant mechanisms of MRSA. Nanosilver size mediates MRSA inhibition and the cytotoxicity to human cells, being smaller nanoparticles the ones with a better antibacterial activity and nontoxic effect.