Evaluation of In Vitro and In Vivo Release of Recombinant Human Nerve Growth Factor From Bioengineered Human Stromal Lenticule

Molly Tzu-Yu Lin¹Yu-Chi Liu^1,2*Isabelle Lee¹Domitilla Mandatori³Nicola Detta⁴Jing Ying Evelina Han¹Letizia Pelusi⁵Leonardo Mastropasqua⁶Mario Nubile⁷Franca Cattani⁸Tiziana Romeo⁸Marcello Allegretti⁸Assunta Pandolfi³Harminder S Dua⁹Jodhbir Mehta^1,2*

• ¹Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
• ²Department of Cornea and External Eye Disease, Singapore National Eye Centre, Singapore, Singapore
• ³Department of Medical, Oral and Biotechnological Sciences, Center for Advanced Studies and Technology (CAST), StemTeCh Group,“G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy
• ⁴Dompé Farmaceutici SpA, Via Tommaso de Amicis, Naples, Italy
• ⁵Department of Medicine and Sciences of Aging, G. d'Annunzio University of Chieti and Pescara, Chieti, Italy
• ⁶Ophthalmology Clinic, Department of Medicine and Aging Science, "G. d'Annunzio" of Chieti-Pescara, Chieti, Italy
• ⁷Ophthalmology Clinic, Department of Medicine and Aging Science, 'G. d'Annunzio' of Chieti-Pescara, Chieti, Italy
• ⁸Dompé farmaceutici SpA, Via Campo di Pile, L'Aquila, Italy
• ⁹Department of Cornea and External Disease, Nottingham Hospital, Nottingham, United Kingdom

Small incision lenticule extraction (SMILE)-derived lenticules have been repurposed as biocompatible scaffolds to incorporate and release therapeutic substances for ocular therapeutics. We aim to investigate the in vitro and in vivo release profiles of recombinant human nerve growth factor (rhNGF) from bioengineered human stromal lenticules prepared with microparticles incorporated with rhNGF (rhNGF-MPs) for up to 1 and 4 weeks, respectively. Upon bioengineered lenticule implantation, slit lamp, Anterior Segment Optical Coherence Tomography, and In Vivo Confocal Microscopy were performed to assess corneal biocompatibility, central corneal thickness (CCT), corneal nerve -fiber density (CNFD), -branch density, and -fiber length. Rabbit cornea, tears and aqueous humour were collected to quantify rhNGF release in vivo. A rapid in vitro release of rhNGF was detected until day 2, and with sustained release over 7 days. The pattern remains comparable even with the presence of antibiotic-antimycotic, trypan blue or fluorescein. Throughout the in vivo follow-up, no signs of corneal haze, edema, infiltration, or pathological increase in CCT wereas observed. A significant increase in CNFD (p=0.001) at week 4 was reported in rhNGF-MPs than in Blank-MPs group. Finally, significantly higher NGF content in rabbit cornea and tear was found in rhNGF-MPs group compared to the endogenous NC group (p=0.035 and p=0.043, respectively). Bioengineered lenticules exhibit sustained rhNGF release for at least 7 days in vitro and up to 1 month in vivo. These results, together with absence of adverse effects, and significant increase in CNFD at 4 weeks after lenticule implantation suggest its promising potential for clinical use.