Silica nano-carrier as a sustained delivery system of GDF5 for intervertebral disc regenerative medicine

Abstract

Event Abstract Back to Event Silica nano-carrier as a sustained delivery system of GDF5 for intervertebral disc regenerative medicine Nina Henry1, 2, 3, Johann Clouet1, 3, 4, 5, Pauline Colombier1, 3, Eric Gautron2, Bernard Humbert2, Jean Le Bideau2, Catherine Le Visage1, 3 and Jérôme Guicheux1, 3, 6 1 INSERM, UMRS 791, Center for OsteoArticular and Dental Tissue Engineering (LIOAD), France 2 CNRS, UMR 6502, Institute of Materials Jean Rouxel (IMN), France 3 Université de Nantes, UFR Odontologie, France 4 CHU Nantes, PHU 11 Pharmacie, Pharmacie Centrale, France 5 Université de Nantes, UFR Sciences Biologiques et Pharmaceutiques, France 6 CHU Nantes, PHU 4 OTONN, France Introduction: Intervertebral disc (IVD) degeneration is one of the major causes of low back pain[1]. The sustained delivery of therapeutic factors able to promote IVD regenerative processes in situ is contemplated and biomaterials-based delivery systems are widely studied[2]. Among all biomaterials, mesoporous silicas are of interest with respect to their properties and lack of cytotoxicity[3],[4]. In this context, we studied mesoporous silica nanofibers (MSNFs)[5] as a drug delivery system. The loading and release capacity of MSNFs and the protein-silica interactions were explored using a model protein (lysozyme). MNSFs were then characterized as a bioactive carrier for Growth Differentiation Factor 5 (GDF5), a nucleopulpogenic factor[6]. Methods: Lysozyme was incubated with MSNFs (50x500nm) in different conditions of pH, time and concentrations. Analyses of protein-silica interactions were performed with transmission electron microscopy (TEM), attenuated total reflectance infrared (ATR/IR) and zeta potential (ZP). Release was performed in PBS, pH 7.2 at 37°C for 20 days. Lysozyme concentration and biological activity were measured with BCA method and enzymatic assay, respectively.GDF5 was incubated with MSNFs at pH 7 for 48h at concentrations from 1-4µg/mL. Release was performed in PBS, pH 7.2 at 37°C for 48h. GDF5 concentration and bioactivity were measured with ELISA and by testing the Smad 1/5/8 pathway activation in human adipose stromal cell (hASC) by western blot, respectively. Nucleopulpogenic commitment of hASC by released GDF5 was finally assessed using RT qPCR (aggrecan, type II collagen, OVOS2, PAX1, CD24) and immunohistology (alcian blue, type II collagen, aggrecan, OVOS2, PAX1, CD24). Results and Discussion: Lysozyme was successfully adsorbed on MSNFs and the highest amount of adsorbed lysozyme was obtained at pH 10, concentration of 200 mg/mL and 48h of incubation. Different lysozyme morphologies were observed by TEM. The ATR/IR study, which compared amides, Si-O-Si, Si-OH and water bonds between lysozyme, MSNFs and MSNFs/lysozyme, combined with ZP results, suggest that protein-silica interactions must be mostly driven by hydrogen bonds involving the amide II of the protein. Release experiments showed a burst within the 1st hour followed by a slower release rate, leading to a sustained release for up to 20 days. The biological activity of the released protein was maintained at all time points. We also demonstrated that GDF5 could be adsorbed and released onto MSNFs. GDF5 bioactivity, evaluated by Smad 1/5/8 phosphorylation, was evidenced for higher GDF5-loaded concentrations onto MSNFs and cells commitment was assessed. Conclusion: MSNFs are promising nano-carriers for therapeutic factors delivery. Moreover, protein-silica interactions were extensively described and revealed hydrogen bond-mediated interactions. Further experiments will focus on the association of GDF5-loaded MSNFs with hASC with an adapted scaffold for in vivo proof of concept. The authors would like to thank Nicolas Stephant, Paul Pilet and Françoise Lary for their contributions.; Financial support : FARMA "Etude ET3-683", Région Pays de la Loire "Projet LMA", ANR "REMEDIV" and FRM Projet DBS20131128442.