Study On The Mineralization Process Of Calcium Phosphate In Giant Vesicles Modified By Ionomycin And Its Influencing Factors

The initial nucleation and growth of bone minerals occurs in matrix vesicles（MVs）.The influx of mineralized ions is regulated by transporters in the membrane components of MVs.Factors such as p H value,supersaturation of mineralized ions,and organic matrix are involved in the regulation of apatite nucleation,growth,and aggregation processes.However,the process of bone mineral mineralization in MVs and the mechanism of related influencing factors need to be further studied.Giant vesicles（GUVs）can be used as a biomimetic model,which can simulate the mineralization environment of MVs and locally regulate the nucleation growth of calcium phosphate.Ionomycin is an ionophore with Ca²⁺ion selective transport function.GUVs were modified with ionomycin to simulate Ca²⁺transmembrane transport in MVs,which can be used to study the formation and phase transformation process of calcium phosphate mineral crystals in vesicles.This is of great significance for fully understanding the formation process of bone minerals in vivo.Herein,GUVs were synthesised by dioleoyl phosphatidyl choline（DOPC）and acid phospholipid dioleoyl phosphatidyl serine（DOPS）as membrane materials to study the influence of ionic phospholipid content and p H value of the solution on the formation and stability of GUVs.GUVs was used as an in vitro mineralization model to investigate the effect of p H value on the phase transformation of calcium phosphate.GUVs were modified with ionomycin to construct GUVs with the function of carrying Ca²⁺ions,and to study the transmembrane transport of Ca²⁺ions and the formation of calcium phosphate in vesicles.GUVs are constructed with DOPC and DOPS as membrane materials.And the influence of ionic phospholipid DOPS content,solution p H,salt（Na Cl）ion concentration,agarose cross-linking degree and other factors on the formation of GUVs has been investigated.Experiments have found that the addition of 20 mol%DOPS accelerates the separation of lipid membranes,and the particle size distribution of vesicles was the most uniform and stable.However,if the content of DOPS exceeds 30 mol%,the fusion of small vesicles will be inhibited by electrostatic repulsion.When solution p H was 7.4,the GUVs produced were the fastest and the particle size distribution was more uniform.Within a certain salt concentration range,as the salt ion concentration increased,it was beneficial to the exfoliation of the lipid layer and promoted lipid fusion to form GUVs.When the salt ion concentration was 50 mmol/L,the lipid layer formed GUVs rapidly and the particle size distribution was more uniform.Evenly.The lower the degree of cross-linking between agarose molecules,the faster the swelling rate of the agarose molecules hybridized with lipids during hydration,and the higher the osmotic pressure generated,the more conducive to the formation of GUVs.The effect of p H on the formation of calcium phosphate and the phase transformation process was investigated in the closed system of GUVs.It was found that in the early stage of mineralization,the p H in the vesicle was in the range of 4-6.And calcium hydrogen phosphate dihydrate（DCPD）was formed as the precursor phase in the vesicles.With the extension of mineralization time,OH^-continued to permeate across the membrane,and the p H value in the bubble increased.At p H 10,the DCPD phase in the vesicles was transformed into the hydroxyapatite（HAp）phase.The change of p H value mainly changes the relative concentration of different proton forms of phosphoric acid,thereby changing the phase composition of calcium phosphate formed by direct precipitation.GUVs with Ca²⁺ion carrying function were constructed by modifying GUVs with ionomycin.The process of ionomycin-mediated Ca²⁺ion transmembrane transport and calcium phosphate mineralization in vesicles were investigated.The results showed that ionomycin could rapidly transport Ca²⁺ions at p H 7.4-9.GUVS combined with 4μmol/L ionomycin could perceive the concentration of Ca²⁺ions from 0.05 to 1 mmol/L faster.The mineralization process mediated by GUVs was a heterogeneous nucleation process.The dissolution rate of amorphous calcium phosphate（ACP）was slowed down and the time in the vesicles was prolonged Under the restriction of vesicles.As Ca²⁺ions continue to be transported across the membrane,the calcium phosphate underwent the phase conversion process of ACP-DCPD-octalcium phosphate（OCP）.