Study On Preparation,characterization,and Properties Of Self-assembling Nanopeptides Against OP

Osteoporosis(OP)is the most common systemic bone disease in the elderly,with a high incidence in the current aging society.Osteoporosis occurs due to the disruption of the balance between bone resorption and bone formation during bone remodeling.Osteoclasts are migratory multinucleated giant cells,which are formed by the fusion and differentiation of multiple monocytes,and play the role of osteoclast resorption.However,the binding of RANKL protein on osteoblasts to RANK protein on osteoclast precursor cells can cause osteoclast differentiation,and the binding of OPG secreted by osteoblasts to their own RANKL can hinder this binding process.Based on the above physiological regulation mechanism,the RANKL-targeting monoclonal antibody denosumab,which mimics the OPG structure secreted by human osteoblasts,has been launched in many countries.The principle is to competitively inhibit the binding of RANKL to RANK on osteoclast precursor cells,thereby inhibiting osteoclast differentiation.However,the production cost of monoclonal antibodies is high,and the side effects of drugs are large.In this paper,two peptides,BIFP and BIFY,were designed and prepared.Their targeting sequences can specifically bind to RANK and RANKL proteins,respectively,and we modified the peptide molecules to enhance their targeting binding ability.The molecular structures of the two polypeptides are divided into three parts.Hydrophobic carbon chains at the ends can provide the hydrophobic driving force for molecular self-assembly to form nanoparticles.The KLVFF structure in the middle enables the molecules to form a β-sheet structure.The targeting sequence can specifically bind to the target.Due to the structure of the hydrophilic head and hydrophobic tail,the molecules disperse in aqueous solutions in the form of nanoparticles(NPs).When the molecular targeting sequence binds to the target,the molecules self-assemble to form nanofibrous structures(NFs).Among them,the BIFP peptide target sequence(NVLKLCSGE)can specifically bind to the RANK protein on the surface of osteoclast precursor cells,and self-assemble into a nanofiber structure to form a fiber network,preventing RANKL from approaching and binding to RANK,thereby inhibiting differentiation.At the same time,the target sequence of BIFY peptide(YLEIEFSLKHR)can specifically bind to the RANKL protein secreted on the surface of osteoblasts,competitively block the binding of RANK-RANKL,and inhibit the differentiation of osteoclasts.Compared with traditional ligand-receptor binding,the nanofiber network produces multivalent bond effects that enhance the binding of ligands and receptors.In order to verify the function of these two peptides,we carried out in vitro molecular characterization and cell experiments,and observed the transformation process of self-assembly morphology after ligand-receptor interaction in cells by transmission electron microscopy and circular dichroism detection.Under induction,the process of molecular self-assembly into nanofibers was significantly accelerated.In cell experiments,the targeting of peptide molecules was verified by scanning electron microscopy and confocal laser scanning.In the osteoclast differentiation inhibition experiment,tartrate-resistant acid phosphatase(TRAP)staining and detection verified the inhibitory effect of polypeptide molecules on osteoclast differentiation under RANKL-induced state.BIFP and BIFY reduced the number of TRAP-positive cells to 68.07%and 61.21%,respectively.Since the bone resorption function of cells depends on the structure of the actin ring,we also stained the cells with phalloidin-actin ring to observe the effect of polypeptide molecules on it.In the control group induced to differentiate by RANKL protein,multinucleated giant cells were formed in the overall shape,and a large and complete circular actin structure could be observed;in the experimental group,there was no similar structure or only small rings could be seen.The experimental results showed that both peptides had obvious inhibitory effects on the formation of actin rings regardless of the high-concentration group or the low-concentration group.The bone resorption effect of osteoclasts was actually observed by planting the above co-culture system on cowhide bone slices.Among them,BIFP significantly reduced the area of bone resorption pits by 75%compared with the negative control group.The classic mouse model of ovariectomized osteoporosis was selected in the in vivo experiments,and established the polypeptide experimental group,the OVX group(PBS group)and the sham operation group(Sham group).Administered 1 week after castration surgery.The experimental group was injected intraperitoneally every day.After 4 weeks,the mice were sacrificed and their tibias were taken for Micro CT scanning.After reconstruction,the BMD of the BIFP group was 2.55 times higher than that of the Sham group,and the BV/TV was up to 3.99 times.Experiments show that our self-assembly targeting peptide can inhibit the differentiation of osteoclasts and has great potential in the prevention and treatment of osteoporosis.The good biological safety of the polypeptide molecule was proved by observing the tissue sections of various organs and counting the changes in the body weight of mice.No lesions were found in any organ slices of the mice,and the body weight was stable.