A new method of optically guided controlled release was experimentally evaluated with liposomes containing a molecular load and gold nanoparticles (NPs). NPs were exposed to short laser pulses to induce transient vapor bubbles around the NPs, plasmonic nanobubbles, in order to disrupt the liposome and eject its molecular contents. The release efficacy was tuned by varying the lifetime and size of the nanobubble with the fluence of the laser pulse. Optical scattering by nanobubbles correlated to the molecular release and was used to guide the release. The release of two fluorescent proteins from individual liposomes has been directly monitored by fluorescence microscopy, while the generation of the plasmonic nanobubbles was imaged and measured with optical scattering techniques. Plasmonic nanobubble-induced release was found to be a mechanical, nonthermal process that requires a single laser pulse and ejects of the liposome contents within a millisecond timescale without damage to the molecular cargo and that can be controlled through the fluence of laser pulse.

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1×105 and 2×105 MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 hours after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6>>>AAV2/9>AAV2/8. The transduction efficiency of AAV2/6 was 30-50 fold higher (p <0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4 hrs was considerably low compared to 30 hrs suggesting that the transgene delivery did not reach its peak at 4 hrs. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.

Increased glycolytic flux is not a direct cause for PLT morphology change and spontaneous activation during storage lesion development. Reduction of glucose utilization may increase PLT loss during storage.

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The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrPSc) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions (≥ 3 logs of infectivity) by all techniques used. This data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.

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