Physical-chemical measurement method development for self-assembled, core-shell nanoparticles.

Improvements in dimensional metrology and innovations in physical-chemical characterization of functionalized nanoparticles are critically important for the realization of enhanced performance and benefits of nanomaterials. Toward this goal, we propose a multi-technique measurement approach, in which correlated atomic force microscopy, dynamic light scattering, high performance liquid chromatography and mass spectroscopy measurements are used to assess molecular and structural properties of self-assembled polyplex nanoparticles with a core-shell structure. In this approach, measurement methods are first validated with a model system consisting of gold nanoparticles functionalized with synthetic polycationic branched polyethylenimine macromolecules. Shell thickness is measured by atomic force microscopy and dynamic light scattering, and the polyelectrolyte uptake determined by chromatographic separation and mass spectrometric analysis. Statistical correlation between size, structure and stability provide a basis for extending the methods to more complex self-assembly of nucleic acids and macromolecules via a condensation reaction. From these size and analytical chemical measurements, we obtain a comprehensive spatial description of these assemblies, obtain a detailed interpretation of the core-shell evolution, and identify regions of the parameter space where stable, discrete particle formation occurs.

Interlaboratory comparison of nanoparticle size measurements between NMIJ and NIST using two different types of dynamic light scattering instruments.

The question of how to relate particle sizes measured using a fixed-angle dynamic light scattering (DLS) instrument with those measured using a multi-angle DLS instrument is addressed. A series of nearly monodisperse polystyrene latex (PSL) particles with nominal diameters of 100 nm, 70 nm, 50 nm, and 30 nm were measured using two different types of DLS instruments: one owned by the National Metrology Institute of Japan (NMIJ) of the multi-angle type and the other owned by the National Institute of Standards and Technology (NIST) of the fixed-angle type. The mean particle size of the PSL particles was measured using the multi-angle-type instrument at various scattering angles and at various concentrations of particle suspension. These data were used to establish the functional dependence of the measured particle size on the scattering angle and particle concentration through the least-squares fitting method. The established function was then used to predict the mean particle sizes that would have been obtained if the same scattering angle and particle concentrations as those used at NIST had been selected at NMIJ. The mean particle sizes obtained at NIST and at NMIJ agreed quite well for all four PSL particle samples after compensating for the angle and concentration differences. The result of this study clearly demonstrates that consideration for the dependence of measured particle sizes on the scattering angle and particle concentration is crucial in intra-method comparisons of mean particle sizes obtained using DLS.

Validation of Single Particle ICP-MS for Routine Measurements of Nanoparticle Size and Number Size Distribution.

Single particle inductively coupled plasma-mass spectrometry (spICP-MS) is an emerging technique capable of simultaneously measuring nanoparticle size and number concentration of metal-containing nanoparticles (NPs) at environmental levels. single particle ICP-MS will become an established measurement method once the metrological quality of the measurement results it produces have been proven incontrovertibly. This Article presents the first validation of spICP-MS capabilities for measuring mean NP size and number size distribution of gold nanoparticles (AuNPs). The validation is achieved by (i) calibration based on the consensus value for particle size derived from six different sizing techniques applied to National Institute of Standards and Technology (NIST) Reference Material (RM) 8013; (ii) comparison with high-resolution scanning electron microscopy (HR-SEM) used as a reference method, which is linked to the International System of Units (SI) through a calibration standard characterized by the NIST metrological atomic force microscope; and (iii) evaluation of the uncertainty associated with the measurement of the mean particle size to enable comparison of the spICP-MS and HR-SEM methods. After establishing HR-SEM and spICP-MS measurement protocols, both methods were used to characterize commercial AuNP suspensions of three different sizes (30, 60, and 100 nm) with four different coatings and surface charge at pH 7. Single particle ICP-MS measurements (corroborated by HR-SEM) revealed the existence of two distinct subpopulations of particles in the number size distributions for four of the 60 nm commercial suspensions, a fact that was not apparent in the measurement results supplied by the vendor using transmission electron microscopy. This finding illustrates the utility of spICP-MS for routine characterization of commercial AuNP suspensions regardless of size or coating.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Intranasal delivery of obidoxime to the brain prevents mortality and CNS damage from organophosphate poisoning.

Intranasal delivery is an emerging method for bypassing the blood brain barrier (BBB) and targeting therapeutics to the CNS. Oximes are used to counteract the effects of organophosphate poisoning, but they do not readily cross the BBB. Therefore, they cannot effectively counteract the central neuropathologies caused by cholinergic over-activation when administered peripherally. For these reasons we examined intranasal administration of oximes in an animal model of severe organophosphate poisoning to determine their effectiveness in reducing mortality and seizure-induced neuronal degeneration. Using the paraoxon model of organophosphate poisoning, we administered the standard treatment (intramuscular pralidoxime plus atropine sulphate) to all animals and then compared the effectiveness of intranasal application of obidoxime (OBD) to saline in the control groups. Intranasally administered OBD was effective in partially reducing paraoxon-induced acetylcholinesterase inhibition in the brain and substantially reduced seizure severity and duration. Further, intranasal OBD completely prevented mortality, which was 41% in the animals given standard treatment plus intranasal saline. Fluoro-Jade-B staining revealed extensive neuronal degeneration in the surviving saline-treated animals 24h after paraoxon administration, whereas no detectable degenerating neurons were observed in any of the animals given intranasal OBD 30min before or 5min after paraoxon administration. These findings demonstrate that intranasally administered oximes bypass the BBB more effectively than those administered peripherally and provide an effective method for protecting the brain from organophosphates. The addition of intranasally administered oximes to the current treatment regimen for organophosphate poisoning would improve efficacy, reducing both brain damage and mortality.

A nanoparticle carrying the p53 gene targets tumors including cancer stem cells, sensitizes glioblastoma to chemotherapy and improves survival.

Temozolomide (TMZ)-resistance in glioblastoma multiforme (GBM) has been linked to upregulation of O(6)-methylguanine-DNA methyltransferase (MGMT). Wild-type (wt) p53 was previously shown to down-modulate MGMT. However, p53 therapy for GBM is limited by lack of efficient delivery across the blood brain barrier (BBB). We have developed a systemic nanodelivery platform (scL) for tumor-specific targeting (primary and metastatic), which is currently in multiple clinical trials. This self-assembling nanocomplex is formed by simple mixing of the components in a defined order and a specific ratio. Here, we demonstrate that scL crosses the BBB and efficiently targets GBM, as well as cancer stem cells (CSCs), which have been implicated in recurrence and treatment resistance in many human cancers. Moreover, systemic delivery of scL-p53 down-modulates MGMT and induces apoptosis in intracranial GBM xenografts. The combination of scL-p53 and TMZ increased the antitumor efficacy of TMZ with enhanced survival benefit in a mouse model of highly TMZ-resistant GBM. scL-p53 also sensitized both CSCs and bulk tumor cells to TMZ, increasing apoptosis. These results suggest that combining scL-p53 with standard TMZ treatment could be a more effective therapy for GBM.

Nanoimmunoliposome delivery of superparamagnetic iron oxide markedly enhances targeting and uptake in human cancer cells in vitro and in vivo.

To circumvent the problem of reduction of the supermagnetic properties of superparamagnetic iron oxide (SPIO) nanoparticles after chemical modification to conjugate targeting molecules, we have adapted a tumor-targeting nanoimmunoliposome platform technology (scL) to encapsulate and deliver SPIO (scL-SPIO) in vitro and in vivo without chemical modification. Scanning probe microscopy, confocal microscopy, and Prussian blue staining were used to analyze the scL-SPIO and assess intracellular uptake and distribution of SPIO in vitro. In vivo targeting and tumor-specific uptake of scL-SPIO was examined using fluorescent-labeled SPIO. We demonstrated that SPIO encapsulation in the scL complex results in an approximately 11-fold increase in SPIO uptake in human cancer cells in vitro, with distribution to cytoplasm and nucleus. Moreover, the scL nanocomplex specifically and efficiently delivered SPIO into tumor cells after systemic administration, demonstrating the potential of this approach to enhance local tumor concentration and the utility of SPIO for clinical applications.