Study On The Targeted Nano-Hydroxyapatite Loading Doxorubicin By Biomimetic Method

Nanao-Hydroxyapatite (nano-HA) is the main inorganic component of human hard tissues. It is applied in clinic as the bone replacement material and drug carrier because of good biological compatibility and activity. However, it is difficult to precisely control the morphology, size and orientation of nano-HA through traditional methods. In addition, it has some difference with natural bone in biocompatibility and bone conductibility. Gelatin which contains eighteen kinds of amino acids is the product of collagen hydrolysate and its chemical composition is similar to collagen. Therefore, it can provide nucleation sites to mineralization of nano-HA as the replacement of collagen. The aim of this study is to prepare nano-HA/gelatin nanoparticles by using gelatin as biomimetic template, which are more similar to inorganic compoment of natural hard tissue through biomimetic method. Nano-HA with targeted effect to special ligand of cancer cells was realized by carbodiimide grafting of cell-membrane receptor ligand folic acid (FA) on nano-HA surface aminized with dendrigraft polymer, polyethyleneimine (PEI). Meanwhile, doxorubicin (DOX) was loaded in nano-HA which targeted to tumor cells. The drug delivery was controlled in local and the targeting nano-HA with DOX can increase the rate of inhibition on tumour cells. Nano-HA was prepared by biomimetic method with difference mass percent of gelatin, 0,20%,40%,60%,80%, and 100%, respectively. The effect of the different content of gelatin on the size, morphology, orientation and crystalline of nano-HA and of gelatin were studied. In addition, the proportion of gelatin with the different secondary structures in the prepared nano-HA was calculated. The results showed that the sizes of nanocomposite decreased from 150 nm to 50 nm and the morphology transformed from irregular ball to a needle stick with the increasing content of gelatin. Preferred orientation along c axis is up to maximum when the mass percent of gelatin is 80. The proportion of gelatin with (3-sheet secondary structure increased with the increase of content of gelatin under the influence of calcium ion and is up to maximum when the mass percent of gelatin is 80%. Compared with other secondary structures, P-sheet coulld explore more residual amino acids that can provide the most nucleation sites. Preferred orientation along c axis becomes the most stronger because the spacing between amino acid and amino acid is close to the distance between Ca2+ and Ca2+ that nano-HA grows along c axis. So the c axis orientation gradually enhance.The surface of nano-HA is aminized with FA in order to target cancer cells and then loaded with doxorubicin. The results show that the grafting percents of FA firstly increase from 31.18% to 43.86% and then decrease to 41.18%. Encapsulationpercentage of doxorubicin increases from 8.62% to 41.18% with the increase of content of gelatin. HA-80%Gel is firstly loaded with doxorubicin during the formation of it and then encapsulationpercentage. In this way, encapsulationpercentage of doxorubicin is up to 57.92% and the grafting percents of FA is 43.12%. The reason is that nano-HA can wrap doxorubicin in the process of nucleation and improve the load quality of doxorubicin. The results of releasedynamics show that the releasing speed of HA-80%Gel-DOX-FA is significantly slower than the HA-80%Gel-FA-DOX and HA-FA-DOX. The releasing rate of HA-80%Gel-DOX-FA and HA-80%Gel-FA-DOX is 83.9% and 50.4%(pH=7.4, t=240h), respectively. HA-FA-DOX release in full during 96 h. The releasing rate of HA-80%Gel-DOX-FA and HA-80%Gel-FA-DOX is 87.36% and 51.32%(pH=5.8, t=240h). The releasing speed is faster in acid condition because nano-HA degrades more quickly.Macrophages and liver cancer cellsare cultured with nano-HA, nano-HA-80%Gel, doxorubicin, HA-80%Gel-FA-DOX and HA-80%Gel-DOX-FA in order to evaluate the cytotoxicity of macrophages and inhibition rate of liver cancer cells. The results of inverted microscope and fluorescenceshow that macrophages cultured with nano-HA and nano-HA-80%Gel in 5 days growadheringto thewalland is in good condition. Doxorubicin can make macrophages apoptosis. HA-80%Gel-FA-DOX and HA-80%Gel-DOX-FA can reduce toxicity of doxorubicin, so as to slow down macrophages apoptosis. Nano-HA and nano-HA-80% Gel has certain inhibitory effect on the liver cancer cells.HA-80%Gel-FA-DOX and HA-80%Gel-DOX-FA can target to liver cancer cells, but the targeting of HA-80%Gel-DOX-FA is stronger. The results of MTT show that nano-HA and nano-HA-80%Gel have good biocompatibility to macrophages. The cytotoxicity of HA-80%Gel-FA-DOX and HA-80%Gel-DOX-FA is up to level 2 and can effectively reduce toxicity of doxorubicin. HA-80%Gel-DOX-FA has the most stronger inhibition on liver cancer cells that demonstrate loading doxorubicin during the prepared process of HA-80%Gel and then grafting FA can improve the effectiveness and target of particles.In this study, targeted nano-HA which loaded DOX was prepared by biomimetic mineralization and the influence of secondary structures of geltin on mineraliazation was discussed. The drug encapsulation efficiency and drug release kinetics in two different ways were studied to provide a novel method for targageting to tumor cells and releasing of drug in long-term. The study has a potential prospect.