Characterization of the functional binding properties of antibody conjugated quantum dots.

Antibody conjugated quantum dots are an emerging technology for high-resolution labeling of biological systems. In this work we determined the number of functional antibodies (i.e., antibodies that are sterically available for functional binding to target proteins) conjugated to semiconductor quantum dots. This is critical for the interpretation of biological data labeled with these methods. We found that the number of available functional antibodies varied significantly for different conjugation methods and are lower than previously estimated. These results may suggest potential strategies for improving quantum dot labeling of biological preparations.

Evaluation of the toxicity of subretinal triamcinolone acetonide in the rabbit.

PURPOSE: Subretinal triamcinolone acetonide (TCA) might be a useful adjuvant in the treatment of subretinal neovascularization. The purpose of this study was to test the toxicity of subretinal TCA in the rabbit eye. METHODS: Sixteen New Zealand rabbits were vitrectomized and injected with either decanted TCA solution or vehicle. The concentration of injected TCA was 2 mg, and the delivered volume was 10 microL. Rabbits were examined on days 1, 3, and 7 and then once a week up to 3 months after surgery. Optical coherence tomography was performed at months 1, 2, and 3. After the last examination, which included electroretinography, the rabbits were killed, and the eyes were enucleated for histopathologic analysis. RESULTS: Subretinal TCA appeared as a white circumscribed deposit. As the drug disappeared, areas of hyperpigmentation were seen adjacent to the drug deposit. Electroretinography tracings were normal, indicating no widespread toxic effect. Histologic analysis revealed areas of absence and/or hyperpigmentation of the retinal pigment epithelium and damage to photoreceptors and the outer nuclear layer of the retina. CONCLUSION: Subretinal injection of TCA crystals can be toxic to the outer retina and retinal pigment epithelium. Whether the potential beneficial effect of such a high-dose steroid in limiting subretinal inflammation, angiogenesis, and proliferation outweighs toxicity can only be determined from a clinical trial.

Intravitreal toxicity of the kenalog vehicle (benzyl alcohol) in rabbits.

PURPOSE: To test the toxicity of intravitreal injections of benzyl alcohol. METHODS: Nine New Zealand rabbits were injected with either a control or a test article at elevating concentrations. The test article was benzyl alcohol calculated to give final injected concentrations of 0.0073%, 0.022%, 0.073%, 0.222%, and 0.733% benzyl alcohol. The 0.022% concentration corresponds to the concentration of benzyl alcohol in human eyes when 0.1 mL of commercial Kenalog (Bristol-Myers Squibb, Princeton, NJ) is used. Baseline examination of the rabbits was performed along with postinjection examinations on days 1, 3, 7, and 14. The eyes were enucleated and examined by light and electron microscopic examinations. RESULTS: Eyes injected with benzyl alcohol concentrations of 0.073%, 0.222%, and 0.733% displayed changes in the outer retina including loss of, and shortening of, outer segments and photoreceptors. CONCLUSIONS: Benzyl alcohol at concentrations modestly higher than what is present in commercial Kenalog is toxic to the rabbit eye. This has been shown in other organ systems. If commercial preserved Kenalog is to be used clinically, decanting the supernatant or using other means to remove the benzyl alcohol may be considered, especially if a volume of >0.1 mL of solution is used. We hypothesize that the noninfectious inflammation seen clinically after Kenalog injection is due to the presence of a toxic preservative at unsafe concentrations.

Intraocular properties of hexadecyloxypropyl-cyclic-cidofovir in Guinea pigs.

OBJECTIVE: We previously reported a long-lasting crystalline lipid pro-drug of cyclic cidofovir, hexadecyloxypropyl-cyclic-cidofovir (HDP-cCDV), to treat experimental retinitis in rabbit eyes. With HDP-cCDV there was a longer intraocular therapeutic effect than with cidofovir (CDV) and no toxicity with 100 microg/eye. It has been known that CDV and related analogues lower intraocular pressure (IOP) after local use, and it is also accepted that the guinea pig is a better model to study this toxicity before human clinical trials. METHODS: HDP-cCDV was intravitreally injected into 10 guinea pig eyes in doses of 4, 9, and 18 microg in 20 microL/eye. An 18-microg quantity is the dose equivalent to 100 microg/eye in the rabbit. Only one eye of each animal received drug and the fellow eye served as the control. After injection, the eyes were monitored with tonometry, ophthalmoscopy, electroretinography (ERG), and histology. RESULTS: Intravitreal injections of doses of 18 microg/eye or lower revealed no toxicity and a high therapeutic index (132,000 to 3300 times higher than the 50% effective concentration for human cytomegalovirus) during 10 weeks of observation. The crystalline drug depot was ophthalmoscopically visible in the inferior vitreous cavity for 5-10 weeks. There was no difference in IOP between the drug-injected and control eyes at any time points (P > 0.05) except for day 3 after drug injection (P = 0.0338). All eyes demonstrated a normal ERG waveform with no differences between the treated and the fellow control eyes (P = 0.85). Histology revealed normal morphology and structures of the retina and ciliary body in all eyes (with or without treatment). CONCLUSION: Crystalline HDP-cCDV may be a long-lasting and safer alternative to cidofovir to treat CMV retinitis without the retinal or ciliary body toxicity observed with CDV.

Efficient gene transfer to retinal pigment epithelium cells with long-term expression.

PURPOSE: To evaluate the safety and efficiency of feline immunodeficiency virus (FIV) vectors for gene delivery into the mammalian retina. METHODS: A first-generation FIV vector was constructed and administered into rabbit eyes at two different concentrations by intravitreal or subretinal routes. A second-generation FIV vector was also constructed and administered subretinally into both rabbit and rat eyes at the same concentration. After vector administration, eyes were monitored using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, and electroretinogram. After the rabbits were killed, eye tissues were processed for light microscopy and immunohistochemical analysis. RESULTS: Administration of both first- and second-generation FIV vectors produced transient vitritis and/or papillitis in rabbits, without other pathologic abnormalities. Retinal pigment epithelium (RPE) cells were the predominant cell type transduced in rabbit eyes, but ganglion cells and Muller cells were also transduced. Transduction was confined to the retinal bleb area. The second-generation FIV vector transduced RPE cells much more efficiently than the first-generation vector (95% vs. 4.5%, respectively; P = 0.0015) in rabbit eyes. In contrast, no toxicity was evident over a 24- to 25-month follow-up period after injection of the second-generation FIV vector into rat eyes. Tropism in the rat eye was similar, including RPE and ganglion cells, and the RPE transduction rate was also high (50%). Transgene expression was persistent in both species over the duration of the experiment. CONCLUSION: Second-generation FIV vectors can efficiently transfer genes into RPE cells with resulting long-term expression, properties potentially valuable to gene therapy approaches to some retinal diseases.

Intraocular properties of urokinase-derived antiangiogenic A6 peptide in rabbits.

To investigate the intraocular properties of an antiangiogenic peptide, A6, a total of 70 New Zealand rabbit eyes were used. For the toxicity study, 0.05 mL of 0.459 M or 0.148 M A6 was injected intravitreally; right eyes received A6, and left eyes received a vehicle. Serial intraocular pressure measurement, slit lamp, and indirect ophthalmoscopy were performed. The rabbit eyes were evaluated by fluorescein angiography, electroretinography, and histology after the scheduled sacrifice. The pharmacokinetics of an intravitreal A6 (0.05 mL of 0.488 M) and a subtenon A6 (0.5 mL of 0.305 M) injection was studied. There was no toxicity observed following the 0.148 M A6 intravitreal injections. In 2 eyes with a 0.459 M A6 intravitreal injection, focal retinal pigmentary change was observed at the injection site, which was contacted by the hyperosmolar drug bolus. Choroidal A6, following the intravitreal injection, remained therapeutic (>or=10 microM) for 72 hours. The vitreous half-life was 19.4 hours. Choroidal concentrations following the subtenon injection were minimal. The low choroidal concentrations observed may relate to the polar nature of A6. More hydrophobic analogs of A6 are likely to cross the retina more efficiently. However, in diseased eyes, in the area of choroidal neovascularization (CNV), the fluid-filled, damaged, edematous retina may permit the drug to enter the choroid in higher concentrations.