(B-42) Extended Release of Doxorubicin-Loaded Nanocarriers from In-Situ Forming Hydrogels for Myofibroblast Precursor Depletion as a Treatment for Posterior Capsular Opacification

Introduction:: Cataracts are the second-leading cause of blindness worldwide, and the number of cataract cases is projected to double within ten years. Over 30% of adults and nearly all children develop posterior capsular opacification (PCO) or clouding of the lens capsule following cataract surgery. Current treatment strategies are insufficient and pose severe adverse effects; therefore, a prophylactic treatment strategy delivered during cataract surgery is a considerable unmet need. Injectable stimuli-responsive gels were designed using poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)) (PLGA-PEG-PLGA) triblock copolymers. These self-assembled gels are optically clear at physiological temperatures and were engineered by varying the lactic acid (LA) to glycolic acid (GA) ratio, and the PLGA/PEG ratio. Previous studies showed preliminary specificity and cytotoxicity of gel-released nanospheres to myofibroblast precursors. The purpose of this study is to compare release and therapeutic efficacy of different doses of a targeted nanocarrier loaded with doxorubicin to deplete cells responsible for PCO.

Materials and Methods:: Antibody-targeted nucleic acid nanocarriers loaded with doxorubicin (G8:3DNA®:Dox) were loaded into injectable PLGA-PEG-PLGA triblock copolymer hydrogels. Human rhabdomyosarcoma (RD) cells were cultured under constant flow of Dulbecco's Modified Eagle Medium (10% fetal bovine serum, 1% antibiotic/antimycotic) for 28 days. RD cells were selected due to their expression of a specific marker for the myofibroblasts responsible for PCO, brain-specific angiogenesis inhibitor 1 (BA1). The following 4 treatment groups (n=4) were evaluated, at 50 µL of: 0.3 ng/ µL 3DNA®/Dox/G8 nanoconjugate-loaded nanogel, 3 ng/ µL 3DNA®/Dox/G8 nanoconjugate-loaded nanogel, nanogel alone and a negative control group of Phosphate Buffered Saline (PBS). Cells were stained at predetermined time points through the covalent, dead cell-specific Live-or-Dye NucFix Red Staining Kit (Biotium, Fremont, CA) and then immediately fixed with 2% paraformaldehyde followed by permeabilization with 0.5% Triton X-100. BAI1 was localized with the G8 IgM mAb. The primary
antibody was visualized using affinity-purified, F(ab’)2 goat anti-mouse IgM m-chain conjugated with a fluorophore (Bio-techne,Minneapolis, MN). Nuclei were stained with Hoechst dye (Biotium). Immunofluorescence was analyzed with an inverted fluorescent microscope (Zeiss) equipped with AxionCam ICm1 camera and Multi-Image-04 ZEN 2 lite image analysis software program.


Results, Conclusions, and Discussions:: The formulation transitions into a hydrogel at physiological temperatures and is optically clear. Additionally, we show the release of G8:3DNA:Dox from thermogels at a low (0.3ng/µL) and high (3.0ng/µL) concentration under microfluidic conditions in PBS. Release of the nanocarriers under these conditions was sustained for up to 4 weeks for both loading concentrations. This indicates that transport of the nanocarriers out of the hydrogel is controlled, and no burst release is taking place. The targeted nanocarriers specifically depleted BA1⁺ cells, with 83.6% of BA1⁺ cell depletion by day 7 for the high loading concentration. The lower loading concentration also reached almost full BA1+ cell depletion at 14 days, at 85.3% targeted depletion. After 28 days, both drug loading conditions exhibited over 90% depletion of BAI1+ cells. The percentage of BA1+ cells in cultures also decreases over 28 days, with 1.2% and 2% of cells being BAI1+ for the low and high concentrations, respectively, compared to 15.0% in the control.



Sustained release of G8:3DNA:Dox nanocarriers from optically clear PLGA-PEG-PLGA hydrogels that form at physiological temperature allows for improved delivery of therapeutic agents in a dynamic environment. Here, we showed that our novel hydrogel formulations can sustain controlled release of nanocarriers for up to 4 weeks for both loading conditions. We have also shown specific depletion of BA1⁺ cells in RD cell cultures, as well as a significant decrease in the relative abundance of BAI1+ cells in cultures over time for both loading concentrations. Controlled drug release from our hydrogel system allows for the lower dose of drug to have therapeutically relevant effects, potentially decreasing the side effects associated with delivery of cytotoxic drugs. Given the observed decrease in BAI1+ cells, we hypothesize that targeted depletion over time can help downregulate further myofibroblast differentiation in lens cell cultures, thereby preventing PCO. The healing process of cataract surgery can last for weeks, and thus having a system that can release a therapeutically relevant dose of drug during that time is necessary. This system has the potential to impact every cataract surgery patient, decreasing the incidence of PCO and associated complications.


Acknowledgements (Optional): : This work was funded by the Cooper Foundation (Myo/Nog cell program to M.B. and M.G-W.).


References (Optional): : Osorno, L. L., Mosley, R. J., Poley, P. L., Bowers, J., Gorski, G., Gerhart, J., Getts, R., George-Weinstein, M., & Byrne, M. E. (2022). Sustained Release of Antibody-Conjugated DNA Nanocarriers from a Novel Injectable Hydrogel for Targeted Cell Depletion to Treat Cataract Posterior Capsule Opacification. Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics, 38(6), 404–411. https://doi.org/10.1089/jop.2021.0111

Gerhart, J., Greenbaum, M., Casta, L., Clemente, A., Mathers, K., Getts, R., & George-Weinstein, M. (2017). Antibody-Conjugated, DNA-Based Nanocarriers Intercalated with Doxorubicin Eliminate Myofibroblasts in Explants of Human Lens Tissue. The Journal of pharmacology and experimental therapeutics, 361(1), 60–67. https://doi.org/10.1124/jpet.116.239079