Controlled and Extended In Vitro Release of Bioactive Anti-Vascular Endothelial Growth Factors from a Microsphere-Hydrogel Drug Delivery System

ABSTRACT

Purpose: To demonstrate controlled and extended release of bioactive anti-vascular endothelial growth factor (VEGF) agents (ranibizumab or aflibercept) from an injectable microsphere-hydrogel drug delivery system (DDS).

Methods: Anti-VEGF agents were radiolabeled with iodine-125 and loaded into poly(lactic-co-glycolic acid) (PLGA) 75:25 microspheres using a modified double-emulsion, solvent evaporation technique. Microspheres were then suspended in an injectable poly(N-isopropylacrylamide)-based thermo-responsive hydrogel to create a microsphere-hydrogel DDS. Release profiles were performed in phosphate buffered saline at 37°C and at predetermined intervals, release samples were collected. Microspheres were also made using non-radiolabeled anti-VEGFs to determine the bioactivity of the DDS throughout release. Bioactivity and cytotoxicity of release samples were determined using human umbilical vascular endothelial cells (HUVECs) under VEGF-induced proliferation.

Results: The DDS is capable of releasing either ranibizumab or aflibercept for 196 days with an initial burst (first 24 h) of 22.2 ± 2.2 and 13.1 ± 0.5 μg, respectively, followed by controlled release of 0.153 and 0.065 μg/day, respectively. Release samples showed no toxicity in HUVECs at any time. Both anti-VEGFs remained bioactive throughout release with significant inhibition of HUVEC proliferation compared to the drug-free DDS, which showed no inhibitory effect on HUVEC proliferation.

Conclusions: Controlled, extended, and bioactive release for approximately 200 days was achieved for both ranibizumab and aflibercept in vitro. The use of anti-VEGF-loaded microspheres suspended within an injectable, thermo-responsive hydrogel may be an advantageous ocular DDS with the potential to improve upon current therapies.

KEYWORDS:

• Aflibercept
• choroidal neovascularization
• intravitreal drug delivery
• poly(N-isopropylacrylamide)
• ranibizumab

Acknowledgments

The authors would like to thank Dr. William F. Mieler for generously providing ranibizumab and aflibercept. Additionally, the authors would like to thank Dr. Nancy Karuri for allowing us to use her plate reader for the MTS assays. Thank you to Mr. Yang Zhou for his assistance in acquiring scanning electron micrographs of the microspheres. Finally, the authors would like to thank Dr. Eric M. Brey for generously providing the HUVECs and Ms. Brianna Roux for her assistance in culturing the HUVECs.

Declaration of interest

J. J. Kang-Mieler: Patent application (US 2012/0231072 A1, “Thermo-responsive hydrogel compositions”). C. R. Osswald: none.