Preparation And Cell Biological Properties Of Nano Hydroxyapatite By Template Method Of Citric Acid Source Carbon Dots

Hydroxyapatite（HAp）,the main inorganic component of bone and teeth,is widely used in bone tissue engineering.It has been confirmed that small-size nano HAp can inhibit the proliferation of a variety of tumor cells,but it is difficult to prepare nano HAp with uniform size and controllable size.HAp prepared by conventional methods often has the problem of uncontrollable particle size.Template method,which contains functional groups,has spatial confinement effect.It can control the size,morphology and structure of materials,and is widely used in the preparation of nanomaterials.The preparation of HAp by carbon dots template is a new method developed in recent years.Citric acid source carbon dots is an ideal template because of its small size（3～5 nm）and abundant functional groups on the surface of spherical carbon core.At present,the preparation methods of citric acid source carbon dots need to be improved,and the influence mechanism of the surface structure of carbon dots on the template effect needs to be further studied.Glutamate has the ability to promote biomineralization.In this paper,nano HAp was prepared by using citric acid carbon dots and glutamic acid modified citric acid carbon dots as templates.The effects of carbon dots on the size and structure of HAp were studied,and the cytocompatibility of nano HAp and its effect on osteosarcoma cells were studied.（1）Citric acid carbon dots and glutamic acid modified carbon dots were prepared by direct pyrolysis.The results showed that the citric acid carbon dots and glutamic acid modified carbon dots prepared by pyrolysis had good water solubility and higher yield than those prepared by hydrothermal method;The carbon core of citric acid carbon dots is amorphous,and the average particle size is 4.24±0.57 nm,and there are-COOH,–C=O and-OH groups on the surface;The carbon core of glutamic acid modified carbon dots crystallizes partially,and the particle size is smaller than that of citric acid carbon dots,with an average size of 3.42±0.67 nm.The results showed that with the increase of the ratio of glutamic acid to citric acid,the amount of glutamic acid bonded to the surface of carbon dots increased in a certain range.（2）HAp nanoparticles with different particle sizes were prepared by hydrothermal method using citric acid carbon dots as template.The results showed that the optimal reaction conditions for the hydrothermal preparation of HAp were 150℃and p H 11.5.When the concentration of citric acid carbon dots is less than 2.0 mg/m L,the phase,group and chemical state of HAp are less affected by the template.With the concentration increase of carbon dots,the average particle size of HAp increases from 71.33 nm to 84.48 nm,and then decreases to67.74 nm.The lattice constants a and c have a similar change trend with the increase of citric acid carbon dots;When the carbon dots concentration was more than or equal to 2.0 mg/m L,the size of HAp decreased,the aggregation increased and the crystallinity decreased.The results of cell experiment showed that the nano HAp prepared by citric acid carbon dots did not affect the morphology and proliferation of normal cells,but had inhibitory effect on osteosarcoma cells（143b）.Compared with the control group,the nano HAp prepared by citric acid carbon dots had stronger inhibitory effect on osteosarcoma cells,and there was no obvious size dependent cytotoxicity in this size range,and the maximum inhibition rate was only 20%.（3）HAp nanoparticles with different sizes were prepared by hydrothermal method using glutamic acid modified carbon dots as template.The results show that the amount of glutamic acid has little effect on the size of HAp,but it affects its crystallinity and the agglomeration phenomenon increases.With the increase of glutamic acid content,the crystallinity of the product decreased,and the optimal mass ratio of citric acid to glutamic acid was 4:1;The average particle size of HAp decreased from 71.33 nm to 49.20 nm with the increase of the concentration of glutamic acid modified carbon dots.However,when the concentration of carbon dots was greater than or equal to 1.5 mg/m L,the aggregation of HAp increased and the crystallinity decreased slightly.Glutamic acid modified the surface of carbon dots was beneficial to improve the template effect of carbon dots.The template effect of glutamic acid modified carbon dots is favorable for CO₃^2-to replace PO₄^3-in HAp to form B-type carbonate substituted hydroxyapatite.The lattice constants of HAp are smaller than commen hydroxyapatite.The results of cell experiment showed that the nano HAp prepared by glutamic acid modified carbon dots did not affect the morphology and proliferation of normal cells.Compared with the HAp of the control group,the HAp prepared by glutamic acid modified carbon dots template had stronger inhibitory effect on osteosarcoma cells and showed obvious size dependent cytotoxicity.The sample with an average length of 49.2 nm had the strongest inhibitory effect and the maximum inhibitory rate was over 30%.It showed good antitumor activity.