Purpose: To evaluate the biocompatibility and 6-month in vivo release of bevacizumab from a hyaluronic acid/dextran-based in situ hydrogel after intravitreal injection in rabbit eye. Methods:The in situ hydrogel was formed by the catalyst-free chemical crosslinking between vinylsulfone functionalized hyaluronic acid (HA-VS) and thiolated dextran (Dex-SH) at physiological condition. The pH 7.4 buffered mixture containing HA-VS, Dex-SH, and bevacizumab were injected into the vitreous of rabbit eyes by a 30-G needle. The biocompatibility was evaluated by intraocular pressure measurement, binocular indirect ophthalmoscope (BIO), full-field electroretinogram (ERG), and histology. The concentrations of both total and active bevacizumab in rabbit vitreous were determined by enzyme-linked immunosorbent assay. The concentration of bevacizumab in rabbit vitreous after bolus injection was simulated by onecompartment first order elimination model. Results:A transparent gel was seen in the vitreous after injection. BIO images, ERG, and histology showed that the gel does not induce hemorrhage, retinal detachment, inflammation, or other gross pathological changes in rabbit eyes after injection. While the bolus intravitreal injected bevacizumab follows the first order elimination kinetics in rabbit eye, the in situ gel formation was able to prolong the retention of bevacizumab in rabbit eye at therapeutic relevant concentration for at least 6 months. The concentration of bevacizumab 6 months after injection was about 10 7 times higher than bolus injection. Conclusions:The new in situ hydrogel formulation of bevacizumab was biocompatible and able to prolong the retention of drug in rabbit eyes in vivo at therapeutic relevant concentration for at least 6 months.Translational Relevance: Although proven to be effective, monthly intravitreal injection of bevacizumab or other protein drugs may cause various complications. Extending the residence time of protein therapeutics in the eye can reduce the injection frequency, its associated complications, and treatment cost, which will be beneficial to both the patients and doctors. In this study, we showed that the in situ hydrogel-based controlled release system is a feasible option to tackle this problem.

The fast release rate and the undesirable covalent binding are two major problems often encountered in formulating in situ chemically cross-linked hydrogel as protein release depot, particularly when prolonged release over months is desirable. In this study, we applied the De Gennes' blob theory to analyze and tackle these two problems using a vinylsulfone-thiol (VS-SH) reaction based in situ hydrogel system. We showed that the simple scaling relation ξb ≈ Rg(c/c*)(-v/(3v-1)) is applicable to the in situ hydrogel and the mesh size estimated from the precursor polymer parameters is a reasonable match to experimental results. On the other hand, as predicted by the theory and confirmed by experiments, the drug diffusion within hydrogel depends mainly on polymer concentration but not the degree of modification (DM). The covalent binding was found to be caused by the mismatch of location between the reactive groups and the entanglement points. The mismatch and, thus, the protein binding were minimized by increasing the DM and concentration of the SH polymer relative to the VS polymer, as predicted by theory. Using these principles, an in situ hydrogel system for the controlled release of an antiangiogenic antibody therapeutics bevacizumab for 3 months was developed.

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Low-frequency ultrasound significantly enhanced the penetration of macromolecules via transscleral route. No undesirable side effects have been found for up to 2 weeks after ultrasound application. The study supports that sonication is a potentially safe and effective method to modulate transscleral barriers for delivering macromolecular therapeutics to posterior segment of the eye.

Wet age‐related macular degeneration (wet‐AMD) is a leading cause of irreversible blindness. Current treatment of AMD requires monthly intravitreal injection, which is difficult to be implemented in many parts of the world. In recent years, controlled release of anti‐vascular endothelial growth factor (VEGF) therapeutics has attracted intense research interest aiming to reduce the injection frequency to one or two times per year. In this study, we evaluated the in vivo pharmacokinetics and the long‐term therapeutic efficacy of an in situ hydrogel encapsulating an anti‐VEGF antibody in nonhuman primates. We show that after a single injection of anti‐VEGF controlled release hydrogel, a relatively constant concentration of drug can be maintained in the monkey eye for at least 5 months and the dose was sufficient for the treatment of recurrent choroidal neovascularization induced by repeat laser photocoagulation in monkeys. Our result suggested that when formulated into a controlled release formulation, a single dose of anti‐VEGF may be sufficient for a half‐year treatment and controlled release may be a suitable strategy to reduce the injection frequency in the treatment of AMD in human.

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