Light-driven chloride transport kinetics of halorhodopsin

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl /protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle

Biocompatibility and Biodegradation Studies of Subconjunctival Implants in Rabbit Eyes

Sustained ocular drug delivery is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Biodegradable subconjunctival implants with controlled drug release may circumvent these two problems. In our study, two microfilms (poly [d,l-lactide-co-glycolide] PLGA and poly[d,l-lactide-co-caprolactone] PLC were developed and evaluated for their degradation behavior in vitro and in vivo. We also evaluated the biocompatibility of both microfilms. Eighteen eyes (9 rabbits) were surgically implanted with one type of microfilm in each eye. Serial anterior-segment optical coherence tomography (AS-OCT) scans together with serial slit-lamp microscopy allowed us to measure thickness and cross-sectional area of the microfilms. In vitro studies revealed bulk degradation kinetics for both microfilms, while in vivo studies demonstrated surface erosion kinetics. Serial slit-lamp microscopy revealed no significant inflammation or vascularization in both types of implants (mean increase in vascularity grade PLGA50/50 12±0.5% vs. PLC70/30 15±0.6%; P = 0.91) over a period of 6 months. Histology, immunohistochemistry and immuno-fluorescence also revealed no significant inflammatory reaction from either of the microfilms, which confirmed that both microfilms are biocompatible. The duration of the drug delivery can be tailored by selecting the materials, which have different degradation kinetics, to suit the desired clinical therapeutic application

Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imaging and Treating Choroidal Neovascularization

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo

We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help with expressing the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at in the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.Yeshttp://www.plosone.org/static/editorial#pee

Drug discovery in ophthalmology: past success, present challenges, and future opportunities

BACKGROUND: Drug discovery has undergone major transformations in the last century, progressing from the recognition and refinement of natural products with therapeutic benefit, to the systematic screening of molecular libraries on whole organisms or cell lines and more recently to a more target-based approach driven by greater knowledge of the physiological and pathological pathways involved. Despite this evolution increasing challenges within the drug discovery industry are causing escalating rates of failure of development pipelines. DISCUSSION: We review the challenges facing the drug discovery industry, and discuss what attempts are being made to increase the productivity of drug development, including a refocusing on the study of the basic biology of the disease, and an embracing of the concept of ‘translational research’. We consider what ophthalmic drug discovery can learn from the sector in general and discuss strategies to overcome the present limitations. This includes advances in the understanding of the pathogenesis of disease; improvements in animal models of human disease; improvements in ophthalmic drug delivery and attempts at patient stratification within clinical trials. SUMMARY: As we look to the future, we argue that investment in ophthalmic drug development must continue to cover the whole translational spectrum (from ‘bench to bedside and back again’) with recognition that both biological discovery and clinical understanding will drive drug discovery, providing safe and effective therapies for ocular disease