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.

Selective visualization of choroidal neovascular membranes.

PURPOSE: Laser-targeted angiography has unique advantages over conventional angiography of the fundus. Its efficacy in visualizing choroidal neovascular membranes was tested in a rat model and compared to that of fluorescein angiography. METHOD: Laser-targeted angiography was performed in rats with choroidal neovascularization (CNV) by injecting heat-sensitive carboxyfluorescein liposomes intravenously, locally releasing a bolus of dye in the choroid with a weak laser pulse, and recording advancement of the bolus on a video camera. Conventional fluorescein angiography also was performed. RESULTS: Laser-targeted angiography revealed CNV as an abnormal pattern of brightly fluorescent vessels. The flow pattern of the bolus and histology, performed in some cases, confirmed the choroidal nature of the vessels. The angiographic visualization was not dependent on dye leakage through the vessels or staining of their walls. Laser-targeted angiography also provided visualization of new vessels that could not be diagnosed by fluorescein angiography. It demonstrated that blood flow was typically more sluggish in CNV than in normal choriocapillaris. Fluorescein angiography failed to demonstrate flow dynamics in all cases of CNV. CONCLUSIONS: This study, in an animal model of CNV, shows that laser-targeted angiography demonstrates CNV and its flow dynamics in a manner not provided by conventional fluorescein angiography. It holds clinical promise as a method to delineate CNV considered difficult or impossible to detect by fluorescein angiography. The method also may permit selective photocoagulation of feeding vessels in the choroid, thereby limiting damage to the overlying retina.

Noninvasive visualization of the choriocapillaris and its dynamic filling.

PURPOSE: The choroidal microvasculature and its circulation are inadequately assessed by presently available techniques. Laser-targeted delivery was applied to generate local, repetitive angiograms of the choriocapillaris in primates. METHODS: Carboxyfluorescein was encapsulated in heat-sensitive liposomes and injected intravenously in monkeys. The liposome contents were then released locally in the choroid by application of a short heat pulse provided by an infrared laser. The bolus of dye spread rapidly downstream from the underlying arterioles into clusters of lobules. Video angiograms were generated with excitation illumination provided by an argon laser. RESULTS: Laser-targeted delivery choroidal angiography performed on three monkeys indicated that the fluorescence was emitted mainly from the choriocapillaris. Clusters of irregular shape with well-defined margins were observed. Adjacent arteries typically supplied separate clusters that fit together like a jigsaw puzzle. The dynamic filling and emptying patterns, recorded at video rate, revealed that macular lobules were filled by a central arteriole and drained by a venous annulus. The average dye transit time through a lobule (n = 10) was 118 +/- 26 msec (mean +/- SD), and the dye transit velocity was 2.53 +/- 0.55 mm/sec. CONCLUSIONS: This study clearly documents the segmental nature of the primate choroidal microvasculature. It also illustrates that choroidal angiography by laser-targeted dye delivery provides information useful for studying the response of the choriocapillaris to physiological and pathologic changes.

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.

Noninvasive visualization of blood flow in the choriocapillaris of the rat.

PURPOSE: The rat has been used to generate models of various eye diseases. However, methods to study the choriocapillaris noninvasively have been inadequate in this species. Laser-targeted angiography was applied to generate local, repetitive angiograms of the choriocapillaris in the rat and to assess the similarity between the choriocapillaris of the rat and that of the subhuman primate. METHODS: Carboxyfluorescein was encapsulated in heat-sensitive liposomes and injected intravenously in rats. The liposome contents were then released locally in the choroid by the application of a short, noncoagulating heat pulse provided by an argon laser. Videoangiograms of the downstream spread of the bolus of dye were generated with excitation illumination provided by another output from the argon laser. RESULTS: Laser-targeted angiography demonstrated that the bolus of dye perfused the choriocapillaris. Clusters of choriocapillaris lobules were observed and appeared similar to those described in the primate. Dynamic filling and emptying patterns also were similar to those of the primate. Lobules were filled by a central arteriole and drained by a venous annulus. CONCLUSIONS: This study demonstrates the feasibility of noninvasively studying the choriocapillaris of the living rat using the technique of laser-targeted angiography. It demonstrates as well the similarity between the rat and the primate choriocapillaris, thus indicating that the rat is an acceptable and convenient model for the study of physiological and pathologic changes in the choroidal vasculature.

Feasibility of laser-targeted photoocclusion of the choriocapillary layer in rats.

PURPOSE: A new method, laser-targeted photoocclusion, was developed to occlude choroidal neovascularization while minimizing damage to the overlying retina. The ability to occlude normal choriocapillary layer in rats was evaluated as a first test of the feasibility of treating choroidal neovascularization with this method. METHOD: A photosensitive agent, aluminum phthalocyanine tetrasulfonate, encapsulated in heat-sensitive liposomes, was administered intravenously along with carboxyfluorescein liposomes. A low-power argon laser (retinal power density of 5.7 W/cm2) locally released a photosensitizer bolus, monitored by the simultaneous release of carboxyfluorescein. A diode laser (operating at 675 nm with a retinal power density of 0.27 W/cm2) activated the photosensitizer with its release. RESULTS: Vessels in the choriocapillary layer were occluded at day 3 after laser treatment and remained unchanged during the 30-day follow-up. Larger choroidal vessels and retinal capillaries remained perfused. Control experiments excluded possible effects of heat or activation of free photosensitizer. Pilot histologic studies showed no damage to the retinal pigment epithelium. CONCLUSIONS: Laser-targeted photoocclusion caused selective occlusion of normal choriocapillaries while sparing overlying retinal pigment epithelium and retinal vessels. The method has potential as a treatment of choroidal neovascularization that may minimize iatrogenic loss of vision.

Feasibility of laser targeted photo-occlusion of ocular vessels.

AIMS/BACKGROUND: Neovascularisation occurs in many major ocular diseases such as diabetes, age-related macular degeneration, and sickle cell disease. Laser photocoagulation is typically used to obliterate the vessels but it also causes severe damage to adjacent normal tissues. This is a very significant limitation especially in the treatment of choroidal neovascularisation which often covers large areas of the posterior pole and the fovea. A method, laser targeted delivery, has been developed capable of releasing drugs locally and non-invasively in the choroidal or retinal vasculature. This method could be used to target a photo-sensitiser to neovascular membranes and cause their selective occlusion by irradiating them. The targeting properties of the method promise to yield a treatment for neovascularisation that does not damage adjacent tissues and thus preserves vision. The purpose of the present study was to test the feasibility of occluding ocular vessels with this method. METHOD: The iris vessels of the albino rat were chosen because the treatment could be assessed unequivocally and followed with time. Aluminium phthalocyanine tetrasulphonate was encapsulated in heat sensitive liposomes and administered systemically. The iris vessels were irradiated with a yellow laser to raise their temperature to 41 degrees C, cause a phase transition in the liposomes and thereby locally release the photosensitiser. The laser was also used to excite the released photosensitiser and cause occlusion. The effect was monitored immediately and for 8 months thereafter. Controls for the effect of the laser and the unencapsulated drug were conducted. RESULTS: The results demonstrated that occlusion can be achieved and sustained for the period of follow up. The controls showed that the effect was not due to heat or to the activation of the low dose of free drug. CONCLUSION: These preliminary findings indicate that laser targeted photo-occlusion is a promising new method for the treatment of neovascularisation.

Occlusion of retinal vessels using targeted delivery of a platelet aggregating agent.

Local laser targeted delivery of a platelet aggregating agent to occlude retinal and choroidal vessels was evaluated in rabbits and rats. Liposomes containing adenosine diphosphate (ADP) were administered intravenously and an argon laser was used to lyse the liposomes in main retinal arteries. Control vessels were treated with the same energy of laser without administering ADP. Fluorescein angiography performed 2 weeks later showed that all the control vessels were perfused. Ninety percent of the ADP-treated arteries showed complete or partial occlusion. Successful occlusion increased with the laser energy and decreased with increasing vessel diameter. Histopathology showed that occlusion was achieved in retinal as well as choroidal vessels. The inner retina remained relatively unaffected at the treatment site but the outer retina was thermally damaged. These preliminary results suggest that targeted delivery of a platelet aggregating agent holds promise for occluding vessels in the fundus.

Systemic toxicology and laser safety of laser targeted angiography with heat sensitive liposomes.

Angiography is currently limited by its lack of local and tissue specificity. The dye rapidly fills both the retinal and choroidal vessels and leaks out of the vessels thus hampering visualization of small vascular beds such as occult choroidal neovascularization. We have developed a method of laser targeted delivery based on encapsulating the dye in heat sensitive liposomes, administering the liposomes intravenously and causing them to release their content by noninvasively warming the target tissue with a laser pulse delivered through the pupil. The local release yields a bright fluorescent bolus which selectively highlights retinal or choroidal vessels. A preliminary investigation of the potential side effects of the method is presented. In rats the systemic toxicity of carboxyfluorescein-entrapped liposomes was compared with that of the free dye. No significant difference was found between the two. Non-human primates exposed to repeated laser targeted angiography were monitored over time and no significant side effects were observed. The safety of the laser exposures was assessed by conventional fluorescein angiography and histopathology. Choroidal laser targeted angiography was achieved without damage. Retinal laser targeted angiography was accompanied by mild and local damage in an area remote from the fovea. The study indicates that laser targeted choroidal angiography can be performed safely and holds promise for diseases such as age related macular degeneration with occult choroidal neovascularization. Further improvements are needed to ensure that no side effects accompany retinal laser targeted angiography.