BMP2 gene delivery mediated by chitosan-ss-PEI non-viral vector and investigation of BMP2 signaling regulation

Abstract

Osteoporotic fractures are still the major health concerns in many developed

societies especially when the incidence of that tremendously increased with the aging

population. However, the outcomes of osteoporotic fracture treatment have not been

entirely satisfactory due to the poor quality of bone substance. Inspiringly, bone

morphogenetic protein 2 (BMP2) with the ability to accelerate bone formation

showed advantages over the conventional treatment. The only problem needed to

overcome is its short half-life which resulted in the requirement of readministration

and extremely high cost. As a solution to that, gene therapy provides a promising way

to sustainably release this protein at the regeneration site. Since viral vectors have

been hampered by genetic toxicity and immunogenicity, nanoscaled non-viral vectors

offer an attractive means for gene delivery.

Chitosan as non-viral vector has been widely investigated for its excellent

biocompatibility. Most efforts have been given to improve its low transfection

efficiency. In this study, chitosan was first modified with octaarginine, one of cell

membrane penetrating peptides, and showed enhanced transfection activity, but which

was not significant as expected. Following that, low molecular weight

polyethyleminine (PEI) was introduced to modify chitosan through bioreducible

disulfide linkage, denoted as Chitosan-ss-PEI. PEI is an efficient non-viral vector but

hampered by molecular-weight dependent toxicity. The developed Chitosan-ss-PEI

showed good biocompatibility in MTT assay in three different cell lines, during which

cells were maintained 80% of viability when the concentration of this vector was up

to 100 μg/mL. The optimal transfection efficiency of Chitosan-ss-PEI was higher than

that of PEI 25k and comparable to Lipofectamine in delivering luciferase reporter

gene. GFP expression mediated by Chitosan-ss-PEI also showed similar results.

Chitosan-ss-PEI was then applied to deliver BMP2 gene to skeletal system cells

and exhibited the osteogenic ability. For C2C12 myoblast cells, this system inhibited

their myoblast differentiation and induced the osteogenic differentiation. It also

showed stronger effect in promoting the differentiation of immature osteblast-like

MG63 cells and in inducing C3H10T1/2 mesenchymal stem cells osteogenic

differentiation in term of ALP activity and mineralization ability compared with other

commercial available non-viral vectors. Primary MSCs such as bone marrow stromal

cells (BMSC) and human umbilical cord blood mesenchymal stem cells

(hUCB-MSC), are usually more difficult to transfect, but they showed stronger

osteogenic differentiation ability induced by this system comparing with the cell lines.

BMP2 usually requires extremely high concentration to realize its function.

Through the investigation of BMP2 signaling regulation in this study, it was found

that parathyroid hormone (PTH) could increase the access of BMP2 ligands to their

receptors by negatively influencing BMPs antagonist network, resulted in enhanced

BMP2 activity in bone remodeling and in promoting the commitment of MSC to

osteoblast lineage both in vitro and in vivo. This course involved the endocytosis of

PTHR with a complex of LRP6, which organized antagonist network on the cell

surface to shield the BMPs receptors. Novel approaches are expected to be developed

based on this mechanism with the purpose of intensifying the therapeutic effect of

BMPs.