Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.

Ultrasound in combination with microbubbles has recently been considered by gene delivery scientists to be an interesting approach to enhance gene transfer into cells. Its low toxicity and simplicity to apply in vivo without major complications make this technology (sonoporation) especially attractive. Sonoporation of DNA has been evaluated in vivo by the injection of free plasmid DNA (pDNA) together with microbubbles (as used in diagnostic imaging) in the bloodstream. However, the in vivo gene-transfer efficiency in these experiments remained rather low. Both the enzymatic degradation of the injected pDNA as well as the low pDNA concentration in the neighborhood of sonoporated cell membranes may explain this low efficiency. Therefore, we developed polymer-coated microbubbles that can bind and protect the pDNA. Coating albumin-shelled microbubbles with poly(allylamine hydrochloride) (PAH) makes the surface charge of the microbubbles positive without drastically affecting the size distribution of the microbubbles, thereby not affecting the ultrasound responsiveness and injectability. The cationic coating allowed both to bind up to 0.1 pg of DNA per microbubble as well as to protect the bound DNA against nucleases. Finally, the PAH coating significantly increased the lifetime of the microbubbles (half-life ≈ 7 h), making them more convenient for in vivo applications because more microbubbles are expected to reach the target organ. Binding and nuclease protection of DNA by polymer-coated diagnostic microbubbles has, to our knowledge, never been demonstrated. We conclude that these LbL-coated microbubbles might be significant in the further development of ultrasound-mediated gene delivery.

Modifying the surfaces of LPXs with hydrophilic PEG chains prevents them from aggregating in vitreous. In this way, LPXs are obtained that can freely move in vitreous, an absolute criterion for reaching the retina after intravitreal injection.

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