On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase

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

Gel and solid matrix systems for the controlled delivery of drug carrier-associated nucleic acids

In order to achieve a sustained pharmacological activity of oligonucleotides (ODNs) and avoid repeated administrations, we have developed a new concept of delivery system that combine sustained release and improved intracellular penetration. These systems are designed for the intravitreal delivery of antisense ODNs. The first concept consisted in using liposomes dispersed in a thermosensitive gel (poloxamer 407). After intravitreal administration in a rabbit model, liposomes and liposomes-gel formulations provided, 1-day postinjection, significantly higher drug levels than the control solution of the oligothymidilate pdT16. In addition, there was no significant difference in the amounts of pdT16 found in the vitreous humor between the liposomes and liposomes-gel. Nevertheless, because of their better stability in the absence of poloxamer, liposomes alone allowed to a larger extent to control the delivery of ODNs as compared to liposome-gel formulations since 37% of the ODNs were still found in the vitreous 15 days after administration. In addition, the ODNs found in the vitreous humor were protected against degradation by their encapsulation within liposomes. The second approach consisted in designing microspheres allowing to release in a controlled fashion pdT16. The ODN was encapsulated within poly(lactide-co-glycolide) microspheres alone or associated with polyethylenimine (PEI) at different nitrogen/phosphate (N/P) ratios. The introduction of PEI in the internal aqueous phase resulted in a strong increase of the ODN encapsulation efficiency. PEI affected microsphere morphology inducing the formation of very porous particles yielding to an accelerated release of pdT16. Porosity and controlled delivery was prevented by introducing sodium chloride in the external preparation medium. When incubated with HeLa cells, microspheres encapsulating pdT16/PEI complexes allowed an improvement of the intracellular penetration of the released ODN. Both liposomes and microspheres are suitable for local delivery of ODNs

Administration intra-vitréenne de liposomes pour la vectorisation de principes actifs [The use of liposomes as intravitreal drug delivery system].

Liposomes are vesicular lipidic systems allowing encapsulation of drugs. This article reviews the relevant issues in liposome structure (composition and size), and their influence on intravitreal pharmacokinetics. Liposome-mediated drug delivery to the posterior segment of the eye via intravitreal administration has been addressed by several authors and remains experimental. Liposomes have been used for intravitreal delivery of antibiotics, antivirals, antifungal drugs, antimetabolites, and cyclosporin. Encapsulation of these drugs within liposomes markedly increased their intravitreal half-life, and reduced their retinal toxicity. Liposomes have also shown an attractive potential for retinal gene transfer by intravitreal delivery of plasmids or oligonucleotides

Delivery of antisense oligonucleotide to the cornea by iontophoresis.

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea

Design of poly-epsilon-caprolactone nanospheres coated with bioadhesive hyaluronic acid for ocular delivery.

This study was performed to design a new ocular drug delivery system based on poly-ε-caprolactone (PCL) biodegradable nanospheres (NS) coated with a bioadhesive polymer, hyaluronic acid (HA), in order to combine ophthalmic prolonged action with the ease of application. The aim of this work was to investigate three strategies to attach HA on NS surface: (1) coating the core by chain entanglement with HA; (2) coating NS by HA adsorption; (3) coating NS by electrostatic interactions between negatively charged HA and a cationic surfactant (stearylamine, SA, or benzalkonium chloride, BKC). A radioimmunoassay technique, usually used for HA quantification in serum, was transposed to determine the amount of HA on the NS. The results show that HA is strongly attached on NS positively charged by cationic surfactant. This system is stable and not influenced by dilution. These results show the possibility of using cationic surfactants to obtain a HA coating by electrostatic interactions. BKC, approved for ophthalmic administration, was retained because it was more firmly anchored within the PCL matrix and the amount of HA attached was high (41.6 μg HA/mg PCL). Moreover, the yield of fixation reached 50%. Therefore, by using a simple preparation method, it was possible to obtain stable HA and intact HA-coated NS

A new delivery system for antisense therapy: PLGA microspheres encapsulating oligonucleotide/polyethyleneimine solid complexes

Microspheres for the controlled release of an antisense oligonucleotide against the Transforming growth factor β1 were designed. Free oligonucleotide or its solid complexes with polyethylenimine (PEI) at different nitrogen/phosphate (N/P) ratios, were encapsulated within poly(lactide-co-glycolide) (PLGA) microspheres prepared by the multiple emulsion-solvent evaporation technique. The encapsulation of the oligonucleotide in form of solid complexes, the N/P ratio, as well as the PLGA type affected microspheres characteristics in term of loading, morphology, oligonucleotide distribution inside matrix and in vitro release profile. The designed microspheres allow the encapsulation and slow release of oligonucleotide/PEI solid complexes that should be effectively internalized inside cells. © 2002 Elsevier Science B.V. All rights reserved