Abstract
Introduction
Intervertebral disk degeneration (IVD) is associated with changes from gelatinous tissue, rich in proteoglycan, to more fibrous tissue with a low proteoglycan content in the cellular microenvironment of the nucleus pulposus (NP) region. Glycosaminoglycan (sGAG) is abundant in NP cells and has a role in enhancing ECM biosynthesis and maintaining cell phenotype. The goal of this work is to develop single-chain antibody fragments (scFvs) to tether to hyaluronic acid (HA) particles for sGAG gene delivery to NP cells. Here, we present identification of NP cell surface markers, isolation of human scFvs against marker proteins and conjugation to prepared HA particles for characterization and gene transfection studies.
Materials and Methods
Microarray studies and RT-PCR identified neural cell adhesion molecule (NCAM) as a marker for NP cells. The terminal-exposed NCAM domain was expressed in Escherichia coliand used to isolate binders from a human scFv antibody fragment phage library. Eluted scFvs were expressed in E. coli, purified and characterized using direct and competitive ELISA. Two candidate scFvs were labelled with FITC for binding studies with NCAM-expressing astrocytes. Meanwhile, HA particles were prepared using an ionic gelation method and layered with SuperFect transfection reagent. Particle size, shape, and charge were characterized using scanning electron microscopy and zeta potential measurement. Conjugation of the scFvs to HA-SuperFect particles was carried out via carbodiimide chemistry and evaluated using fluorescence activated cell sorting (FACS). The transfection efficiency of functionalized particles was studied in adipose-derived stem cells (ADSCs), AF and NP cells using luciferase activity, FACS analysis, and imaging.
Results
Microarray analysis identified NCAM as more highly expressed on NP than on annulus fibrosus cells, which was confirmed by RT-PCR. The outermost, Ig-like NCAM domain was cloned, expressed in the E. coli periplasm with leader and hexahistidine tags, and purified for use in phage display screening. A number of scFvs were isolated after 4 rounds of panning of the human scFv library against the NCAM domain and these were expressed in soluble format and purified. After confirmation of binding specificity by ELISA, two candidate scFvs were labelled with FITC and used to demonstrate binding to NCAM-expressing astrocyte cells. The scFvs were also conjugated to newly synthesized HA particles for further cell delivery and uptake studies. Studies to incorporate therapeutic genes into the particles for delivery to NP cells are underway.
Conclusion
Human scFvs targeting a surface marker on NP cells were produced and tethered to HA particles for targeted delivery of therapeutics. The potential of these functionalized nanoparticles in increasing the specificity and efficacy of HA particle-mediated gene and drug delivery to NP cells is currently being evaluated in cell studies, with the ultimate goal of their use for targeted regeneration of degenerated IVD.
No
None declared