The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP؉E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ؎ 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

Natural biopolymers are widely used in the field of drug and gene delivery. In this study, alginate nanoparticles were prepared using water‐in‐oil microemulsion as a template followed by calcium crosslinking of guluronic acid units of alginate polymer. After collected by ultracentrifugation, alginate nanoparticles were analyzed by electron microscopy to obtain the size and morphology which were varied with the ratio of water, oil, and surfactant used. To examine the potency of Ca‐alginate nanoparticles as carriers for gene delivery, GFP‐encoding plasmids were encapsulated in these nanoparticles to investigate the degree of endocytosis by NIH 3T3 cells and ensuing transfection rate. Our results showed that Ca‐alginate nanoparticles with an average size around 80 nm in diameter are very efficient gene carriers, in comparison with plasmid DNA condensed by polyethyleneimine (PEI).

Inhaled nanoparticles have been reported to contribute to deleterious effects on human health. In this study, we investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged; 30 nm) and single wall carbon nanotubes (SWCNT) on alveolar epithelial cell barrier properties. Transmonolayer resistance (Rt) and equivalent shortcircuit current (Ieq) of primary rat alveolar epithelial cell monolayers were measured in the presence and absence of varying concentrations of apical nanoparticles. In some experiments, apical-to-basolateral fluxes of radiolabeled mannitol or inulin were determined with or without apical UAPS exposure and lactate dehydrogenase (LDH) release was analyzed after UAPS or SWCNT exposure. Results revealed that exposure to UAPS decreased Rt and Ieq significantly over 24 hours, although neither mannitol nor inulin fluxes changed. Positively charged QD decreased Rt significantly (with subsequent recovery), while negatively charged QD did not. Rt decreased significantly after SWCNT exposure (with subsequent recovery). On the other hand, PNP exposure had no effects on Rt or Ieq. No significant increases in LDH release were observed after UAPS or SWCNT exposure. These data indicate that disruption of alveolar epithelial barrier properties due to apical nanoparticle exposure likely involves alteration of cellular transport pathways and is dependent on specific nanoparticle composition, shape and/or surface charge.

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Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm(-2) for 10 min which was then maintained at 6 W cm(-2) for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.