(190bk) Suprachoroidal Space Injection of in-Situ Forming Bevacizumab-Hyaluronic Acid Hydrogel Using a Microneedle to Increase Drug Retention Time

Suprachoroidal space (SCS) injection has been developed for better targeting of drug delivery to the posterior segment of the eyes to treat posterior ocular diseases (e.g., age-related macular degeneration (AMD), diabetic macular edema (DME), and posterior uveitis). Injecting drugs into the SCS, a potential space lying between the choroid and the sclera, allows the drugs to flow circumferentially at the choroid-sclera interface toward the posterior pole (e.g., the macula and around the chorioretinal layers) with high bioavailability. The SCS injection using a microneedle, which is less than 1 mm length, can deliver drugs to the posterior in less invasive manner. Since however, SCS is adjacent to the choroid, which are a vascular layer, the injected drugs especially small molecules can be washed away rapidly through the choriocapillaries within several hours. Thus, injecting drug formulation enabling sustainable-release into the SCS would be the best for posterior ocular treatment. Thus, in this study, bevacizumab (Bev), anti-VEGF drug, was formulated to in-situ forming hyaluronic acid (HA) hydrogel to maximize retention time in the SCS. To further increase the retention time in the SCS, Bev was reduced mildly to functionalize, then, the reduced Bev was covalently bound to the HA chains with poly(ethylene glycol) diacrylate (PEGDA), crosslinker. Since HA and PEG are biocompatible, those were used for long-term Bev releasing system. The formulation of the in-situ hydrogel was optimized by the in vitro test, and the released Bev from the hydrogel and the retention time were analyzed. Based on the ex vivo result, the in vivo injection is going to be performed.

Bev was reduced mildly by 2-Mercaptoethylamine. The reduced Bev (0.8 mg) was reacted to 0.8 mg of PEGDA (3 kDa) at 37 °C for 2 day. Then, 2% (w/v) of thiolated HA solution (40 μL) was mixed with the Bev-PEGDA conjugate (20 μL). Once the formulation was liquid form right after the injection, then, around 10 min after the mixing in-situ forming Bev-HA hydrogel was synthesized. The hydrogel was further incubated at 37 °C for 30 min. To measure the Bev releasing from the hydrogel in vitro, 1 mL of Hank’s balanced salt solution (HBSS) buffer was added to the hydrogel and the buffer was changed at each time point. Collected Bev was analyzed by Bradford assay and ELISA test to measure the concentration and the activity of the drugs.

To optimize the synthesis of the in-situ forming Bev-HA hydrogel, the reaction time between the reduced Bev and PEGDA was varied from 20 min, 4 h, 1, to 2 day at 37 °C. The burst release of the Bev in vitro was measured for 2 days. As a result, the hydrogel, which was synthesized with the 2 day reaction of the Bev and PEGDA, released minimum the Bev 16% among the other reaction times (e.g., 20 min, 4h, and 1 day). Since, the functional group of the reduced Bev is not many in the peptide chain, the reaction takes for 2 days and shows minimum release for 2 days.

The PEGDA amount was varied from 0.8, 1.6, to 3.2 mg. Interestingly, when the amount was 0.8 mg, the Bev releasing rate for 2 days was minimum, 16%, among the other PEGDA amounts (e.g., 1.6 and 3.2 mg). As increased PEGDA amount up to 3.2 mg, since unreacted PEGDA is increased simultaneously, it competes with the reduced Bev, thereby decreasing the Bev binding efficiency.

Since HA concentration in the hydrogel is correlated to the mechanical strength, rigidity, and the retention time of the drugs, the concentration was varied from 1 to 2% (w/v). When the HA concentration was 1%, the hydrogel maintained its shape for maximum 75 days, then, dissolved in the buffer. While the 2% HA hydrogel in vitro released the Bev for over 8 months (94.2% released for 241 days) and maintained its hydrogel shape. Activity of the released Bev was confirmed by ELISA, and the Bev activity was ranged from 66 to 124%. The released Bev even after 8 months still had the activity to VEGF.

To verify the effect of covalent bonding between Bev and HA in the hydrogel, non-reduced Bev was utilized to the formulation of in-situ forming HA hydrogel. Since there is no covalent bonding in the hydrogel between the Bev and the HA, 82.5% of Bev was released within 28 days. It indicates that covalent bonding between Bev and HA contributes the sustainable release of the drugs. Based on the in vitro releasing test, we are going to perform in vivo test into the New Zealand White rabbit eye.

In-situ forming Bev-HA hydrogel in vitro released Bev for over 8 month without dissolution in the buffer. The covalent bonding between Bev and HA contributes the rigidity of the hydrogel to further increase retention time. These results suggest a strategy for long-term Bev releasing to treat posterior diseases in the SCS.