A potential method for local drug and dye delivery in the ocular vasculature.

We propose a new drug and dye delivery system that would allow repeated release of substances in the ocular vasculature by an externally controlled mechanism. The substances are encapsulated in heat-sensitive liposomes, which are lysed by locally applying a heat pulse produced by an argon laser. The system was tested by investigating the release of carboxyfluorescein encapsulated in the liposomes. The liposome suspension was incubated at 37 degrees or 38.5 degrees C and irradiated at different powers and pulse durations. The amount of dye released was monitored by fluorophotometry and compared with the concentration obtained when the liposomes were lysed at their transition temperature of 41 degrees C. The results showed that 85% of the encapsulated substance can be released. Moreover, a dramatic contrast was observed between the fluorescence before and after the lysis. Presently the energy density is higher than but close to the maximal permissible exposure for humans. The release mechanism with the short laser pulse appeared to be similar to that present when liposomes were heated slowly.

Visualization of the retinal microvasculature by targeted dye delivery.

Although fluorescein angiography has proven to be an important tool in the diagnosis and management of retinal vascular diseases, it is subject to certain limitations, namely the presence of the choroidal background, which usually precludes a detailed examination of the retinal microvasculature. Moreover, the inability to repeat the bolus reduces the chance of obtaining high-quality photographs of early phases, and does not allow for a complete binocular examination or for testing the response to induced physiologic changes. We have developed a method of targeted dye delivery that consists of encapsulating the dye in lipid vesicles, injecting them intravenously, and causing them to release their contents locally when a short heat pulse is induced in a retinal artery by a laser. This method was applied in the rhesus monkey in order to visualize the retinal microvasculature. A well-defined bolus and absence of background fluorescence permitted both following of the dye front through the vasculature and clear imaging of the capillary network over the whole posterior pole. The bolus delivery could be repeated as many as 100 times in 45 min without significant loss of contrast. The comparison of these results with conventional fluorescein angiography illustrated the advantage of the new method. The examination of the safety of the delivery system indicates that there is no major obstacle to the eventual application to humans.

Quantitative analysis of retinal hemodynamics using targeted dye delivery.

A new method designed to allow repeated mapping of retinal hemodynamics on a macro- and microcirculatory level was evaluated in the primate eye. The method, called "targeted dye delivery," consists of encapsulating a fluorescent dye in temperature-sensitive liposomes, injecting the liposomes systemically, and using a light pulse from an argon laser to release a bolus of dye in a targeted retinal vessel. The follow-up of the well-defined dye front thus generated allows calculation of the blood flow and capillary transit time. Evaluation of targeted dye delivery in a monkey indicated that centerline blood velocity and the vessel diameter can be measured with a reproducibility of 10% and 4%, respectively, in vessels that are 40 microns and larger. These measurements yielded flow values that had a reproducibility of 10% on the same day and 13% on different days. The normalization of flow rate by the vessel diameter was consistent with theoretic estimates and promises to be a circulation indicator independent of variations between individual and species. The transit time across capillary beds at different locations was found to be similar, thus indicating that the method could be used to evaluate the local viability of the microcirculation.

Feasibility of targeted drug delivery to selective areas of the retina.

A new method was developed to deliver locally a bolus dose of a drug to the retinal vasculature. The targeted delivery system was based on encapsulating the drug in heat-sensitive liposomes, which are injected intravenously and lysed in the retinal vessels by a heat pulse generated by a laser. To test if substances delivered in the vessels could also penetrate into the surrounding tissue, 6-carboxyfluorescein was encapsulated in liposomes and used as a marker for drug penetration. Moderate argon laser pulses were applied to the retinal vessels of Dutch pigmented rabbits to induce breakdown of the blood-retinal barrier (BRB). A suspension of liposomes at a dose of 2 ml/kg body weight, corresponding to a carboxyfluorescein dose of 12 mg/kg, was injected into the ear vein. The dye was released from the liposomes proximal to the damaged portion of the vessel. Fundus fluorescein angiograms were recorded with a video camera and digitized for subsequent image analysis. The penetration of carboxyfluorescein into the retinal tissue was evaluated by comparing the fluorescence intensity of the area around the damaged vessel with that of an adjacent control area. The dye penetration increased with the numbers of laser applications (P less than 0.001). The leakage was localized distally to the released site and was restricted to areas with a disrupted BRB. The mass of carboxyfluorescein that penetrated gradually spread with time. Both veins and arteries could be used for the targeted delivery. These results indicated that this delivery system, which is fully controllable by laser through the pupil, can deliver drugs inside the vasculature and into the retinal tissue wherever the BRB is disrupted.

Feasibility of blood flow measurement by externally controlled dye delivery.

We are developing a new method of delivering substances locally and repeatedly in the retinal vasculature under external control. This delivery system is based on encapsulating the substance in heat-sensitive lipsomes, which are injected intravenously and lysed by a heat pulse delivered by a laser. The feasibility of using this system with dyes and creating a sharp dye front was tested in vitro and in vivo. The results indicate that the background fluorescence of intact liposomes is minimal but in contrast a dramatic increase in fluorescence is achieved where the dye is released. In vivo tests indicated that only the selected vascular branch fluoresced. Moreover, a sharp dye front could be obtained repeatedly and preserved over significant distances. The presence of a sharp dye front allowed measurements, in vitro, of blood velocity which correlated well (r = 0.985, P less than 0.001) with the average blood velocity values calculated from the known flow rate.