Glyconanoparticle-DNA interactions: an atomic force microscopy study.

Glyconanoparticles which present carbohydrate and amino groups motifs at their surface were produced. These particles were highly stable and soluble in aqueous solutions. The presence of the carbohydrate groups also allowed the inclusion of more strongly binding groups, without affecting solubility. The binding of a model DNA, plasmid by these nanoparticles was studied by atomic force microscopy, transmission electron microscopy, and gel electrophoresis. Significant differences between the nanoparticles based on their affinities for the DNA were found, with implications for their potential use as nonviral gene delivery agents.     

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (?M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.     

Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles

Silica nanoparticles with a diameter of 100 nm were covalently modified at their surface by adjustable amounts of amine and carboxyl functional groups. Bioconjugation studies of two proteins, streptavidin and streptactin, with the functional nanoparticles resulted in optimum binding of the proteins to a long-chain carboxyl-terminated linker. The surface functionalization of the nanoparticles was monitored by a variety of independent methods, including zeta-potential measurements, dynamic light scattering (DLS), scanning electron microscopy (SEM), particle charge detection titrations (PCD) and elemental analysis. At the surface of the nanoparticles, a functional surface group density of 1.8 amino groups per nm2 was realized. The amine functions were quantitatively transferred to carboxyl groups coupled with a linker elongation. Streptavidin was immobilized by covalent binding to the carboxyl linkers and resulted in a protein density at the surface of the nanoparticles that was three times higher than the highest binding densities at nanoparticles published to date. The binding capacity of the streptavidin-covered nanoparticles for ligand biotin was quantified by titration with biotin-4-fluorescein to 2.5 biotin binding sites per 100 nm2.     

Chip-based optical detection of DNA hybridization by means of nanobead labeling

A new scheme for the detection of molecular interactions based on optical readout of nanoparticle labels has been developed. Capture DNA probes were arrayed on a glass chip and incubated with nanoparticle-labeled target DNA probes, containing a complementary sequence. Binding events were monitored by optical means, using reflected and transmitted light for the detection of surface-bound nanoparticles. Control experiments exclude significant influence of nonspecific binding on the observed contrast. Scanning force microscopy revealed the distribution of nanoparticles on the chip surface.     

Characterization of attractive interaction-driven carbon nanotube composites with Cd-based nanoparticles

Multiwalled carbon nanotube (MWNT) composites with cadmium telluride (CdTe) or cadmium selenide (CdSe) nanoparticles were prepared via electrostatic interaction. The MWNTs were modified with carboxylic acid groups. Both the CdTe and CdSe nanoparticles were stabilized with 2-(dimethylamino) ethanethiol hydrochloride to develop positive charges on their surfaces in water. They were characterized in detail via UV-visible spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The energy state of the MWNTs was significantly modified by the electrostatic binding between the nanoparticles and carboxylated MWNTs, resulting in absorption at approximately 250 nm. XPS analysis also proved the electronic redistribution of the nanoparticles and the MWNTs. The binding energies of the elements Cd, Se, and Te were definitely changed by the attractive interaction between the nanoparticles and the MWNTs. The distribution of the CdTe or CdSe nanoparticles and the morphologies of the MWNT composites were deliberately investigated from TEM images and XRD.     

Study on the binding of procaine hydrochloride to DNA/DNA bases and the effect of CdS nanoparticles on the binding behavior

The interaction between procaine hydrochloride and DNA/DNA bases in the absence and presence of cadmium sulfide (CdS) nanoparticles has been explored in this study by using differential pulse voltammetry, atomic force microscopy (AFM) and so on, which illustrates the different binding behaviors of procaine hydrochloride with different DNA bases. The results clearly indicate that the binding of purines to procaine hydrochloride is stronger than that of pyrimidines and the binding affinity is in the order of G > A > T > C. In addition, it was observed that the presence of CdS nanoparticles could remarkably enhance the probing sensitivity for the interaction between procaine hydrochloride and DNA/DNA bases. Furthermore, AFM study illustrates that procaine hydrochloride can bind to some specific sites of DNA chains, which indicates that procaine hydrochloride may interact with some special sequences of DNA.     

Synthesis of ordered iron oxide nanoparticle arrays in planar DNA complexes

The formation of iron oxide nanoparticles in planar DNA complexes immobilized on substrates has been studied in reactions involving only biogenic reagents (ferritin and ascorbic acid) in aqueous solutions under normal conditions. Using transmission electron microscopy, we revealed ordered quasi-linear arrays of iron oxide nanoparticles 2–4 nm in diameter, which probably resulted from nanoparticle binding to linear DNA molecules. The electron diffraction patterns of the synthesized nanoparticles are characteristic of polycrystalline magnetic iron oxide (magnetite of maghemite) nanoparticles and point to good crystallinity of the nanoparticles. Our results demonstrate the feasibility of the synthesis of ordered arrays of iron oxide nanoparticles using DNA complexes and may have direct implications for the understanding of biomineralization processes and iron metabolism in living systems.     

The work function of doped polyaniline nanoparticles observed by Kelvin probe force microscopy

The work function of polyaniline nanoparticles in the emeraldine base state was determined by Kelvin probe force microscopy to be ～270 meV higher than that of similar nanoparticles in the emeraldine salt state. Normal tapping mode atomic force microscopy could not be used to distinguish between the particles due to their similar morphologies and sizes. Moreover, other potential measurement systems, such as using zeta potentials, were not suitable for the measurement of surface charges of doped nanoparticles due to their encapsulation by interfering chemical groups. Kelvin probe force microscopy can be used to overcome these limitations and unambiguously distinguish between the bare and doped polyaniline nanoparticles.     

Antibacterial activity and increased freeze-drying stability of sialyllactose-reduced silver nanoparticles using sucrose and trehalose

The resistance to current antibiotics results in the emergence of health-threatening bacteria. Silver nanoparticles are known to exhibit broad-spectrum antibacterial activities without the development of resistance. Herein, we developed a green synthetic method for the preparation of silver nanoparticles with sialyllactose instead of toxic chemicals as a reducing agent, which would improve its therapeutic applicability and increase its biocompatibility. Oven incubation, autoclaving and microwave irradiation methods were applied to prepare the silver nanoparticles. High resolution-transmission electron microscopy and atomic force microscopy images revealed mostly spherical and amorphous silver nanoparticles with an average diameter of 23.64 nm. Fourier Transform-infrared spectra suggest that the N-H amide of sialyllactose might be involved in the binding of silver nanoparticles. Based on thermogravimetric analyses, 2,3-sialyllactose-reduced silver nanoparticles are composed of 54.3 wt% organic components and 45.7 wt% metallic silver. Enhanced antibacterial activities of silver nanoparticles (approximately 8-fold) were observed against Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium (minimum inhibitory concentration 16 microg/mL). Next, we employed the use of carbohydrate stabilizers to increase the stability of silver nanoparticles during a freeze-drying process. It was found that sucrose and trehalose were the most effective stabilizers. In addition, silver nanoparticles possessed excellent salt stability as well as on-the-shelf stability in the presence of these stabilizers. Derivatives of sialic acid are known to be anti-influenza agents; therefore, the newly prepared silver nanoparticles may serve as useful antibacterial and antiviral agents to cope with both pathogenic bacteria and viruses in the near future.     

Influence of anions on the formation and properties of chitosan-DNA nanoparticles

Chitosan-DNA nanoparticles were prepared by using different anions (such as chloride, sulfate, citrate, and tripolyphosphate) as mediation agents. The research suggested that the formation and morphological characteristics of chitosan-DNA nanoparticles largely depended on concentration, molecular size, charge number, and chemical structure of anions, as well as chitosan/DNA ratio. The observation by atom force microscopy showed that chitosan-DNA nanoparticles mediated by four anions (in their appropriate range of concentration) had a spherical shape, narrow size distribution, and good monodispersivity. Especially, nanoparticles mediated by sulfate and TPP had a size distribution of 40-50 nm. Additionally, the nanoparticles presented high encapsulation efficiency and good protection of DNA from DNasel digestion. The zeta-potential of nanoparticles could be adjusted moderately by adding different anions and controlling their concentrations, and DNA encapsulation efficiency was not influenced, which would reduce nonspecific interactions with the cell membrane and nanoparticle toxicity. Smaller size and lower zeta-potential will be beneficial for improving gene therapy. In addition, the anion mediation method has potential for the preparation of cationic polymer nanoparticles as drug or gene vectors.     

Covalent immobilization of DNA onto functionalized mica for atomic force microscopy

The immobilization of DNA on the self-assembled monolayer of 3-aminopropyltrimethoxysilane (APTES) on mica wafer functionalized with glutaraldehyde (GA) by chemical bonding was studied by atomic force microscopy (AFM). The DNA used for our investigation was amplified by polymerase chain reaction, and primers were labeled with a -NH2 group at their 5' terminus. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and AFM. Results from XPS and AFM showed that the mica with the APTES and activated with GA can be formed, and the flatness of the mica can be adapted for AFM images. We found that the modified surface was capable of binding DNA molecules so that it withstood a thorough rinsing with a solution of sodium dodecylsulfate. Covalent binding between the aldehyde-terminated membrane and -NH2 groups at both ends of double-stranded DNA resulted in immobilization and straightening of the DNA.     

Characterization of different sequences of DNA on si substrate by atomic force microscopy and gold nanoparticle labeling

In this paper, different sequences of single-strand DNA modified on Si substrate were studied taking advantages of the high resolution of atomic force microscopy (AFM) and signal enhancement of gold nanoparticles. Two sequences of single-strand DNA, as a model, were immobilized on Si substrate and hybridized with their sequence-complementary DNA molecules modified respectively with two sizes of gold nanoparticles. The surface of Si substrate was characterized through detecting the size and coverage of gold nanoparticles by AFM. Results demonstrated that different sizes of gold nanoparticles represented different sequences of DNA immobilized on the substrate. Density and distribution of DNA on Si substrate can be investigated by AFM imaging using gold nanoparticles as topographic markers. Compared to other sensitive methods such as fluorescence energy transfer, X-ray photoelectron, and radiolabeling experiments, this approach is advantageous in terms of high spatial resolution in sub-micrometer scale. This new method will be beneficial in the characterization of DNA immobilized on chip surfaces.     

Immobilization of albumin on magnetite nanoparticles

The magnetite (Fe₃O₄) nanoparticles were prepared by the co-precipitation of ferrous and ferric salts with NH₄OH, and then modified with 3-aminopropyltriethoxysilane (APTES) by silanization reaction and subsequent reaction with glutaraldehyde (GA) to obtain functional groups on their surface. The influence of different terminated groups on protein binding was studied with bare and modified magnetite nanoparticles. Amine terminated magnetite nanoparticles were shown the highest binding ability for immobilization process compared to Fe₃O₄ NPs and GA bonded NPs. This binding ability was shown by using sodium dodecyl polyacrylamide gel electrophoresis technique (SDS-PAGE). Albumin attached magnetite nanoparticles were also examined by Scanning Electron Microscopy (SEM).     

Characterization of gold nanoparticle binding to microtubule filaments

Microtubule (MT) protein filaments were used as templates for fabricating Au nanowires as a bottom-up approach for fabricating building blocks for future integrated circuits. Photochemical reduction methods were employed to form Au nanoparticles which bind and uniformly cover the MT filaments. Synthesis of the MT-templated Au nanowires was characterized using UV/vis spectroscopy and transmission electron microscopy (TEM). In addition, binding between the MT filaments and Au nanoparticles was investigated using surface enhanced Raman spectroscopy (SERS) and X-ray photoelectron spectroscopy (XPS) to establish the nature of the binding sites. A variety of functional groups were identified by SERS to interact with the Au including imidazole, sulfur, aromatic rings, amine, and carboxylate. The imidazole ring in the histidine is the most prominent functional group for Au binding. The results from these studies provide better understanding of the binding between Au and the biotemplate and give insight concerning methods to improve Au coverage for MT-templated Au nanowires.     

Imaging gold nanoparticles for DNA sequence recognition in biomedical applications

The hybridization of single-stranded oligonucleotide-derivatized gold nanoparticles (Au nanoprobes) with double stranded complementary DNA was directly observed by atomic force microscopy (AFM). This specific interaction is the basis for an Au nanoprobe-based homogeneous assay for specific DNA sequence detection, based on salt-induced particle aggregation that is prevented when a complementary target is present. For long DNA targets (linearized plasmid DNA) complicated hybridized target DNA-Au-nanoprobes structures were formed, that were interpreted as the basis for stability of the Au nanoprobes against salt-induced aggregation. For shorter DNA targets (PCR amplified fragments) hybridization with the Au nanoprobes occurred, in the majority of cases, in the expected location of the DNA target fragment containing the specific sequence. The formation of the observed DNA hybridized structures provides evidence at the molecular level for specific hybridization to the target sequence as the method of binding of the Au nanoprobes.     

Fabrication of capped gold nanoparticles by using various amino acids

The production of gold nanoparticles (GNPs) by amino acid is one of the most attractive and interesting subjects in nanobiotechnology. In this study, amino acids have been utilised as a reducing agent and also an agent for capping GNPs. The GNPs were prepared using a reduction solution containing gold cations with optimum concentration of gold salt (5?mM), and also functionalised by glutamic acid, phenylalanine and tryptophan with optimum concentration of amino acids (25?mM). The optimum condition of gold solution and amino acids were achieved by ultraviolet–visible spectroscopy. The size of nanoparticles was obtained 5–20, 10–20 and 20–30?nm, respectively, by transmission electron microscopy and dynamic light scattering techniques. The results obtained from experimental and quantum calculations confirm that amino acids have strong bond while they have anion binding. Moreover, the free carboxylic groups of capped GNPs are one of the suitable and capable beads for binding biological agents. As a result, the medical applications of amino acids and proteins can be used as a practical method due to the strong interaction of peripheral amine groups with nanoparticles.     

Nanocomposites consisting of titanium dioxide nanoparticles and oligonucleotides

The use of various nanoparticles is a promising way to solve the current problem of drug delivery in medicine and biology. Nanocomposites consisting of titanium dioxide and oligonucleotides noncovalently attached to nanoparticles through the polylysine linker (TiO2 x PL-DNA) have been designed to deliver of DNA fragments into cells. Three forms of TiO2 nanoparticles (amorphous, anatase, and brookite) were used for construction of nanocomposites. The size, morphology, and chemical composition of TiO2 nanoparticles and TiO2 x PL-DNA nanocomposites were characterized. DNA fragments in the proposed nanocomposites were shown to retain their ability to form complementary complexes. TiO2 x PL-DNA nanocomposites independently on the form of nanoparticles were shown by confocal microscopy to penetrate into HeLa cells without any transfection agents and physical impact. The presented type of nanocomposites can be applied in the thriving technology of drug delivery to achieve high therapeutic and biological efficacy.     

Physical characterization and macrophage cell uptake of mannan-coated nanoparticles

Previously, we reported on a cationic nanoparticle-based DNA vaccine delivery system engineered from warm oil-in-water microemulsion precursors. In these present studies, the feasibility of lyophilizing the nanoparticles and their thermal properties were investigated. Also, the binding and uptake of the nanoparticles by a macrophage cell line were studied. The nanoparticles (prior to pDNA coating) were freeze-dried with lactose or sucrose as cryoprotectants. The stability of lyophilized nanoparticles at room temperature was monitored and compared to that of the aqueous nanoparticle suspension. The thermal properties of the nanoparticles were investigated using differential scanning calorimetry (DSC). The nanoparticles, coated or uncoated with mannan as a ligand, were incubated with a mannose receptor positive (MR+) mouse macrophage cell line (J774E), at either 4°C or 37°C to study the binding and uptake of the nanoparticles by the cells. It was found that lactose or sucrose (1-5%, w/v) was required for successful lyophilization of the nanoparticles. After 4 months of storage, the size of lyophilized nanoparticles did not significantly increase while those in aqueous suspension grew by over 900%. Unlike its individual components, emulsifying wax (m.p., ~55°C) and hexadecyltrimethyl ammonium bromide, the nanoparticles showed a melting point of ~90°C. Moreover, the DSC profile of the nanoparticles was different from that of the physical mixture of emulsifying wax and CTAB. After 1 hour incubation at 37°C, the uptake of mannan-coated nanoparticles was 50% higher than that of the uncoated nanoparticles. At 4°C and after one hour, the binding of the mannan-coated nanoparticles by J774E was over 2-fold higher than that of the uncoated nanoparticles. This increase in J774E binding could be abolished by preincubating the cells with free mannan, suggesting that the binding and uptake were receptor-mediated. In conclusion, the nanoparticles were lyophilizable, and lyophilization was shown to enhance the stability of the nanoparticles. DSC provided evidence that the nanoparticles were not a physical mixture of their individual components. Finally, cell binding and uptake studies demonstrated that the nanoparticles have potential application for cell-specific targeting.     

Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles

Nanometer-sized fluorescent particles (latex nanobeads) have been covalently linked to DNA binding proteins to probe specific sequences on stretched single DNA molecules. In comparison with single organic fluorophores, these nanoparticle probes are brighter, are more stable against photobleaching, and do not suffer from intermittent on/off light emission (blinking). Specifically, we demonstrate that the site-specific restriction enzyme EcoRI can be conjugated to 20-nm fluorescent nanoparticles and that the resulting nanoconjugates display DNA binding and cleavage activities of the native enzyme. In the absence of cofactor magnesium ions, the EcoRI conjugates bind to specific sequences on double-stranded DNA but do not initiate enzymatic cutting. For single DNA molecules that are stretched and immobilized on a solid surface, nanoparticles bound at specific sites can be directly visualized by multicolor fluorescence microscopy. Direct observation of site-specific probes on single DNA molecules opens new possibilities in optical gene mapping and in the fundamental study of DNA-protein interactions.     

Physical Characterization and Macrophage Cell Uptake of Mannan-Coated Nanoparticles

Abstract

Previously, we reported on a cationic nanoparticle-based DNA vaccine delivery system engineered from warm oil-in-water microemulsion precursors. In these present studies, the feasibility of lyophilizing the nanoparticles and their thermal properties were investigated. Also, the binding and uptake of the nanoparticles by a macrophage cell line were studied. The nanoparticles (prior to pDNA coating) were freeze-dried with lactose or sucrose as cryoprotectants. The stability of lyophilized nanoparticles at room temperature was monitored and compared to that of the aqueous nanoparticle suspension. The thermal properties of the nanoparticles were investigated using differential scanning calorimetry (DSC). The nanoparticles, coated or uncoated with mannan as a ligand, were incubated with a mannose receptor positive (MR+) mouse macrophage cell line (J774E), at either 4°C or 37°C to study the binding and uptake of the nanoparticles by the cells. It was found that lactose or sucrose (1–5%, w/v) was required for successful lyophilization of the nanoparticles. After 4 months of storage, the size of lyophilized nanoparticles did not significantly increase while those in aqueous suspension grew by over 900%. Unlike its individual components, emulsifying wax (m.p., ?55°C) and hexadecyltrimethyl ammonium bromide, the nanoparticles showed a melting point of ?90°C. Moreover, the DSC profile of the nanoparticles was different from that of the physical mixture of emulsifying wax and CTAB. After 1 hour incubation at 37°C, the uptake of mannan-coated nanoparticles was 50% higher than that of the uncoated nanoparticles. At 4°C and after one hour, the binding of the mannan-coated nanoparticles by J774E was over 2-fold higher than that of the uncoated nanoparticles. This increase in J774E binding could be abolished by preincubating the cells with free mannan, suggesting that the binding and uptake were receptor-mediated. In conclusion, the nanoparticles were lyophilizable, and lyophilization was shown to enhance the stability of the nanoparticles. DSC provided evidence that the nanoparticles were not a physical mixture of their individual components. Finally, cell binding and uptake studies demonstrated that the nanoparticles have potential application for cell-specific targeting.