Improved Size Determination by Nanoparticle Tracking Analysis: Influence of Recognition Radius.

Nanoparticle tracking analysis (NTA) is a light scattering technique that measures the size distribution of suspended nanoparticles. A key parameter in NTA measurements is the recognition radius, which distinguishes individual, tracked particles from one another. However, by defining a finite radius, the displacement of tracked particles is effectively restricted, translating into an overestimation of particle size. Herein, we introduce a modified probability model that describes the restricted displacement of a tracked particle and enables more accurate nanoparticle size determination. The analytical performance of the modified displacement probability was validated through computational simulations and experimental measurements. While conventional models typically overestimated nanoparticle size by over 20%, the modified probability model estimated nanoparticle size with an error of less than 6%. The modified probability model is compatible with conventional NTA measurement readouts and thus should find wide application for more accurately determining the size distribution of suspended nanoparticles.

Validation of Size Estimation of Nanoparticle Tracking Analysis on Polydisperse Macromolecule Assembly.

As the physicochemical properties of drug delivery systems are governed not only by the material properties which they are compose of but by their size that they conform, it is crucial to determine the size and distribution of such systems with nanometer-scale precision. The standard technique used to measure the size distribution of nanometer-sized particles in suspension is dynamic light scattering (DLS). Recently, nanoparticle tracking analysis (NTA) has been introduced to measure the diffusion coefficient of particles in a sample to determine their size distribution in relation to DLS results. Because DLS and NTA use identical physical characteristics to determine particle size but differ in the weighting of the distribution, NTA can be a good verification tool for DLS and vice versa. In this study, we evaluated two NTA data analysis methods based on maximum-likelihood estimation, namely finite track length adjustment (FTLA) and an iterative method, on monodisperse polystyrene beads and polydisperse vesicles by comparing the results with DLS. The NTA results from both methods agreed well with the mean size and relative variance values from DLS for monodisperse polystyrene standards. However, for the lipid vesicles prepared in various polydispersity conditions, the iterative method resulted in a better match with DLS than the FTLA method. Further, it was found that it is better to compare the native number-weighted NTA distribution with DLS, rather than its converted distribution weighted by intensity, as the variance of the converted NTA distribution deviates significantly from the DLS results.

Gene delivery to carcinoma cells via novel non-viral vectors: nanoparticle tracking analysis and suicide gene therapy.

Suicide gene therapy of oral squamous cell carcinoma (OSCC) may be a viable approach to the treatment of this cancer. However, human OSCC cells are relatively resistant to efficient transfection by non-viral vectors. To identify an optimal vector for gene delivery, we compared the transfection activities and efficiencies of Glycofect, Metafectene, Metafectene Pro, Metafectene Easy and FuGENE HD, using the OSCC cell line, HSC-3, and the cervical carcinoma cell line, HeLa. The size distribution and ζ-potential of the complexes of these vectors with plasmid DNA were assessed by nanoparticle tracking analysis and electrophoretic mobility measurements, respectively. Metafectene Easy and FuGENE HD mediated the highest transfection activity (measured as luciferase expression) and efficiency (measured as the percentage of cells transfected with ß-galactosidase). These vectors were used to deliver a plasmid encoding herpes simplex virus thymidine kinase, followed by ganciclovir treatment. By day 9, HeLa cell viability was 22±3% of controls with FuGENE HD and 26±3% with Metafectene Easy. The viability of HSC-3 cells was 42±25% with FuGENE HD, and 58±28% with Metafectene Easy. The reduction in viability was statistically significant in both cases (p⩽0.005; average of 3 independent experiments), although there was considerable variability between experiments with the HSC-3 cells.

Adsorption of amelogenin onto self-assembled and fluoroapatite surfaces.

The interactions of proteins at surfaces are of great importance to biomineralizaton processes and to the development and function of biomaterials. Amelogenin is a unique biomineralization protein because it self-assembles to form supramolecular structures called "nanospheres", spherical aggregates of monomers that are 20-60 nm in diameter. Although the nanosphere quaternary structure has been observed in solution, the quaternary structure of amelogenin adsorbed onto surfaces is also of great interest because the surface structure is critical to its function. We report studies of the adsorption of the amelogenin onto self-assembled monolayers (SAMs) with COOH and CH(3) end group functionality and single crystal fluoroapatite (FAP). Dynamic light scattering (DLS) experiments showed that the solutions contained nanospheres and aggregates of nanospheres. Protein adsorption onto the various substrates was evidenced by null ellipsometry, X-ray photoelectron spectroscopy (XPS), and external reflectance Fourier transform infrared spectroscopy (ERFTIR). Although only nanospheres were observed in solution, ellipsometry and atomic force microscopy (AFM) indicated that the protein adsorbates were much smaller structures than the original nanospheres, from monomers to small oligomers in size. Monomer adsorption was promoted onto the CH(3) surfaces, and small oligomer adsorption was promoted onto the COOH and FAP substrates. In some cases, remnants of the original nanospheres adsorbed as multilayers on top of the underlying subnanosphere layers. Although the small structures may be present in solution even though they are not detected by DLS, we also propose that amelogenin may adsorb by the "shedding" or disassembling of substructures from the nanospheres onto the substrates. This work suggests that amelogenin may have a range of possible quaternary structures that interact with surfaces.

Surface properties of Toxoplasma gondii oocysts and surrogate microspheres.

The physical properties that govern the waterborne transmission of Toxoplasma gondii oocysts from land to sea were evaluated and compared to the properties of carboxylated microspheres, which could serve as surrogates for T. gondii oocysts in transport and water treatment studies. The electrophoretic mobilities of T. gondii oocysts, lightly carboxylated Dragon Green microspheres, and heavily carboxylated Glacial Blue microspheres were determined in ultrapure water, artificial freshwater with and without dissolved organic carbon, artificial estuarine water, and artificial seawater. The surface wettabilities of oocysts and microspheres were determined using a water contact angle approach. Toxoplasma gondii oocysts and microspheres were negatively charged in freshwater solutions, but their charges were neutralized in estuarine water and seawater. Oocysts, Glacial Blue microspheres, and unwashed Dragon Green microspheres had low contact angles, indicating that they were hydrophilic; however, once washed, Dragon Green microspheres became markedly hydrophobic. The hydrophilic nature and negative charge of T. gondii oocysts in freshwater could facilitate widespread contamination of waterways. The loss of charge observed in saline waters may lead to flocculation and subsequent accumulation of T. gondii oocysts in locations where freshwater and marine water mix, indicating a high risk of exposure for humans and wildlife in estuarine habitats with this zoonotic pathogen. While microspheres did not have surface properties identical to those of T. gondii, similar properties shared between each microsphere type and oocysts suggest that their joint application in transport and fate studies could provide a range of transport potentials in which oocysts are likely to behave.

Changes in the quaternary structure of amelogenin when adsorbed onto surfaces.

Amelogenin is a unique protein that self-assembles into spherical aggregates called "nanospheres" and is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin onto substrates is of great interest because protein-surface interactions are critical to its function. We report studies of the adsorption of amelogenin onto self-assembled monolayers containing COOH end group functionality as well as single crystal fluoroapatite, a biologically relevant surface. We found that although our solutions contained only nanospheres of narrow size distribution, smaller structures such as dimers or trimers were observed on the hydrophilic surfaces. This suggests that amelogenin can adsorb onto surfaces as small structures that "shed" or disassemble from the nanospheres that are present in solution.

Active site control of myosin cross-bridge zeta potential.

The electrical properties of contractile proteins contribute to muscle structure and perhaps function but have not been characterized adequately. Electrophoretic mobility, mu(e), is sensitive to the net electric charge and hydrodynamic size of a molecule in solution. Zeta potential, zeta, particle charge, Q(e), and particle charge-to-mass ratio are proportional to mu(e). We measured mu(e) for nucleotide complexes of skeletal muscle heavy meromyosin (HMM) and subfragment 1 (S1). The results indicate that mu(e) for HMM changes depending on the ligand bound in the active site. The changes in electric charge appear to occur mainly on the S1 moieties. For HMM(MgATPgammaS)(2) and HMM(MgADP.P(i))(2) the values of mu(e) are -0.077 and -0.17 (microm/s)/(V/cm), respectively. For these complexes, mu(e) is independent of [ATP], [ADP], and [P(i)]. When P(i) dissociates from HMM(MgADP.P(i))(2) to form HMM(MgADP)(2), mu(e) decreases to -0.61 (microm/s)/(V/cm). This large decrease in mu(e) is independent of free [ADP] or [ATP]. Increasing [P(i)], on the other hand, increases mu(e) for HMM(MgADP)(2) to values near those observed for the steady-state intermediate. For HMM, mu(e) = -0.34 and is independent of P(i). MgADP binding to HMM decreases mu(e) to -0.57 (microm/s)/(V/cm), and the dissociation constant is 9 microM. Taken together, these data indicate that mu(e) and, thus, zeta are controlled by ligand binding to the active site. The magnitudes of the particle charge-to-mass ratios for the HMM complexes are all in a range that falls within published values determined for a variety of other proteins. Possible roles that the observed nucleotide-dependent changes in cross-bridge electric charge might have in the contractile cycle in muscle are considered.