Journal of Nanobiotechnology - Latest Articles

QDs versus Alexa: reality of promising tools for immunocytochemistry

Helena Monton — 2009-05-27T00:00:00Z

Background: The unique photonic properties of the recently developed fluorescent semiconductor nanocrystals (QDs) have made them a potential tool in biological research. However, QDs are not yet a part of routine laboratory techniques. Double and triple immunocytochemistries were performed in HeLa cell cultures with commercial CdSe QDs conjugated to antibodies. The optical characteristics, due to which QDs can be used as immunolabels, were evaluated in terms of emission spectra, photostability and specificity. Results: QDs were used as secondary and tertiary antibodies to detect β-tubulin (microtubule network), GM130 (Golgi complex) and EEA1 (endosomal system). The data obtained were compared to homologous Alexa Fluor 594 organic dyes. It was found that QDs are excellent fluorochromes with higher intensity, narrower bandwidth values and higher photostability than Alexa dyes in an immunocytochemical process. In terms of specificity, QDs showed high specificity against GM130 and EEA1 primary antibodies, but poor specificity against β-tubulin. Alexa dyes showed good specificity for all the targets tested. Conclusion: This study demonstrates the great potential of QDs, as they are shown to have superior properties to Alexa dyes. Although their specificity still needs to be improved in some cases, QDs conjugated to antibodies can be used instead of organic molecules in routine immunocytochemistry.

Two-stimuli manipulation of a biological motor

Zorica Ristic — 2009-05-15T00:00:00Z

F1-ATPase is an enzyme acting as a rotary nano-motor. During catalysis subunits of this enzyme complex rotate relative to other parts of the enzyme. Here we demonstrate that the combination of two input stimuli causes stop of motor rotation. Application of either individual stimulus did not significantly influence motor motion. These findings may contribute to the development of logic gates using single biological motor molecules.

Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway

Jinshun Zhao — 2009-04-20T00:00:00Z

Background: Carcinogenicity of nickel compounds has been well documented. However, the carcinogenic effect of metallic nickel is still unclear. The present study investigates metallic nickel nano- and fine particle-induced apoptosis and the signal pathways involved in this process in JB6 cells. The data obtained from this study will be of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles. Results: Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that metallic nickel nanoparticles exhibited higher cytotoxicity than fine particles. Both metallic nickel nano- and fine particles induced JB6 cell apoptosis. Metallic nickel nanoparticles produced higher apoptotic induction than fine particles. Western-blot analysis showed an activation of proapoptotic factors including Fas (CD95), Fas-associated protein with death domain (FADD), caspase-8, death receptor 3 (DR3) and BID in apoptotic cells induced by metallic nickel particles. Immunoprecipitation (IP) western blot analysis demonstrated the formation of the Fas-related death-inducing signaling complex (DISC) in the apoptotic process. Furthermore, lamin A and beta-actin were cleaved. Moreover, we found that apoptosis-inducing factor (AIF) was up-regulated and released from mitochondria to cytoplasm. Interestingly, although an up-regulation of cytochrome c was detected in the mitochondria of metallic nickel particle-treated cells, no cytochrome c release from mitochondria to cytoplasm was found. In addition, activation of antiapoptotic factors including phospho-Akt (protein kinase B) and Bcl-2 was detected. Further studies demonstrated that metallic nickel particles caused no significant changes in the mitochondrial membrane permeability after 24 h treatment. Conclusion: In this study, metallic nickel nanoparticles caused higher cytotoxicity and apoptotic induction than fine particles in JB6 cells. Apoptotic cell death induced by metallic nickel particles in JB6 cells is through a caspase-8/AIF mediated cytochrome c-independent pathway. Lamin A and beta-actin are involved in the process of apoptosis. Activation of Akt and Bcl-2 may play an important role in preventing cytochrome c release from mitochondria to the cytoplasm and may also be important in the carcinogenicity of metallic nickel particles. In addition, the results may be useful as an important reference when comparing the toxicities of different nickel compounds.

Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides

Shashank Sirsi — 2009-04-07T00:00:00Z

Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD.

Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture

Emanuela Jacchetti — 2008-11-17T00:00:00Z

Background: Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. Results: A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. Conclusion: The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.

Hydrophobic silver nanoparticles trapped in lipid bilayers: Size distribution, bilayer phase behavior, and optical properties

Geoffrey Bothun — 2008-11-12T00:00:00Z

Background: Lipid-based dispersion of nanoparticles provides a biologically inspired route to designing therapeutic agents and a means of reducing nanoparticle toxicity. Little is currently known on how the presence of nanoparticles influences lipid vesicle stability and bilayer phase behavior. In this work, the formation of aqueous lipid/nanoparticle assemblies (LNAs) consisting of hydrophobic silver-decanethiol particles (5.7 ± 1.8 nm) embedded within 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers is demonstrated as a function of the DPPC/Ag nanoparticle (AgNP) ratio. The effect of nanoparticle loading on the size distribution, bilayer phase behavior, and bilayer fluidity is determined. Concomitantly, the effect of bilayer incorporation on the optical properties of the AgNPs is also examined. Results: The dispersions were stable at 50°C where the bilayers existed in a liquid crystalline state, but phase separated at 25°C where the bilayers were in a gel state, consistent with vesicle aggregation below the lipid melting temperature. Formation of bilayer-embedded nanoparticles was confirmed by differential scanning calorimetry and fluorescence anisotropy, where increasing nanoparticle concentration suppressed the lipid pretransition temperature, reduced the melting temperature, and disrupted gel phase bilayers. The characteristic surface plasmon resonance (SPR) wavelength of the embedded nanoparticles was independent of the bilayer phase; however, the SPR absorbance was dependent on vesicle aggregation. Conclusion: These results suggest that lipid bilayers can distort to accommodate large hydrophobic nanoparticles, relative to the thickness of the bilayer, and may provide insight into nanoparticle/biomembrane interactions and the design of multifunctional liposomal carriers.

Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

Athena Klutz — 2008-10-23T00:00:00Z

Background: An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced. Results: A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM) and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor. Conclusion: This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR dimers.

Toxicity of CdSe nanoparticles in Caco-2 cell cultures

Lin Wang — 2008-10-23T00:00:00Z

Background: Potential routes of nanomaterial exposure include inhalation, dermal contact, and ingestion. Toxicology of inhalation of ultra-fine particles has been extensively studied; however, risks of nanomaterial exposure via ingestion are currently almost unknown. Using enterocyte-like Caco-2 cells as a small intestine epithelial model, the possible toxicity of CdSe quantum dot (QD) exposure via ingestion was investigated. Effect of simulated gastric fluid treatment on CdSe QD cytotoxicity was also studied. Results: Commercially available CdSe QDs, which have a ZnS shell and poly-ethylene glycol (PEG) coating, and in-house prepared surfactant coated CdSe QDs were dosed to Caco-2 cells. Cell viability and attachment were studied after 24 hours of incubation. It was found that cytotoxicity of CdSe QDs was modulated by surface coating, as PEG coated CdSe QDs had less of an effect on Caco-2 cell viability and attachment. Acid treatment increased the toxicity of PEG coated QDs, most likely due to damage or removal of the surface coating and exposure of CdSe core material. Incubation with un-dialyzed in-house prepared CdSe QD preparations, which contained an excess amount of free Cd2+, resulted in dramatically reduced cell viability. Conclusion: Exposure to CdSe QDs resulted in cultured intestinal cell detachment and death; cytotoxicity depended largely, however, on the QD coating and treatment (e.g. acid treatment, dialysis). Experimental results generally indicated that Caco-2 cell viability correlated with concentration of free Cd2+ ions present in cell culture medium. Exposure to low (gastric) pH affected cytotoxicity of CdSe QDs, indicating that route of exposure may be an important factor in QD cytotoxicity.

Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay

Mark Pereira — 2008-10-01T00:00:00Z

Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 μL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

The margination propensity of spherical particles for vascular targeting in the microcirculation

Francesco Gentile — 2008-08-15T00:00:00Z

The propensity of circulating particles to drift laterally towards the vessel walls (margination) in the microcirculation has been experimentally studied using a parallel plate flow chamber. Fluorescent polystyrene particles, with a relative density to water of just 50 g/cm3comparable with that of liposomal or polymeric nanoparticles used in drug delivery and bio-imaging, have been used with a diameter spanning over three order of magnitudes from 50 nm up to 10 μm. The number n∼s MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmOvayLbaGaadaWgaaWcbaGaem4Camhabeaaaaa@2EB4@ of particles marginating per unit surface have been measured through confocal fluorescent microscopy for a horizontal chamber, and the corresponding total volume V∼s MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmOvayLbaGaadaWgaaWcbaGaem4Camhabeaaaaa@2EB4@ of particles has been calculated. Scaling laws have been derived as a function of the particle diameter d. In horizontal capillaries, margination is mainly due to the gravitational force for particles with d > 200 nm and V∼s MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmOvayLbaGaadaWgaaWcbaGaem4Camhabeaaaaa@2EB4@ increases with d4; whereas for smaller particles V∼s MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmOvayLbaGaadaWgaaWcbaGaem4Camhabeaaaaa@2EB4@ increases with d3. In vertical capillaries, since the particles are heavier than the fluid they would tend to marginate towards the walls in downward flows and towards the center in upward flows, with V∼s MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xH8viVGI8Gi=hEeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGafmOvayLbaGaadaWgaaWcbaGaem4Camhabeaaaaa@2EB4@ increasing with d9/2. However, the margination in vertical capillaries is predicted to be much smaller than in horizontal capillaries. These results suggest that, for particles circulating in an external field of volume forces (gravitation or magnetic), the strategy of using larger particles designed to marginate and adhere firmly to the vascular walls under flow could be more effective than that of using particles sufficiently small (d < 200 nm) to hopefully cross a discontinuous endothelium.