Synthesis Of Targeted Angents Based On Nanodiamond Coupling Folate Acid And Their Studies In Vitro

In the past few decades,with the increase of cancer incidence,malignant tumor has become the leading killer threatening the human health.However,at present the therapeutic effect of the clinically traditional anti-cancer drugs widely used is poor,and the side effects are larger,so it is a worldwide problem to produce a kind of targeted drugs to treat cancer,which are high efficiency and low toxicity.Nanodiamond(ND),a novel carbon material,due to its small size,large surface,easy modification,high adsorption capacity and good biocompatibility,is widely used as drug carrier.In this paper,we selected ND as the fundamental material,Methotrexate(MTX)and Doxorubicin(DOX)as model drugs,and folate acid(FA)as a targeted ligand to prepare two novel targeted drug delivery systems,including ND-PEG-FA/GLY-MTX and ND-PEG-FA/DOX.Besides,their biological effects with cells were studied.The main works can be divided into the following three parts:1.Using ND as the fundamental material,PEG-diamine(NH2-PEG-NH2),FA and glycidol(GLY)were conjunct with the nanoparticles step by step through a series of chemical reactions to prepare the nanocarrier ND-PEG-FA/GLY,then the drug MTX was loaded on the nanocarrier by ester linkage to synthesis ND-PEG-FA/GLY-MTX nanodrug system.The coupled amount of MTX on the ND-PEG-FA/GLY was 123?g/mg using ultraviolet spectrophotometer(UV).The complexes were characterized by the means of Fourier infrared spectrum(FTIR)and transmission electron microscopy(TEM),which confirmed that the target product was prepared successfully.Moreover,the result of the drug release test showed that ND-PEG-FA/GLY-MTX nanomedicine system could maintain a good stability in the normal tissue,and released a mass of free MTX in the tumor environment.2.The biological effects between ND-PEG-FA/GLY-MTX nanomedicne system and human breast cancer cells(MCF-7)or hippocampal neurons in mice(HT22)were studied by the laser confocal and flow cytometry.The results of flow cytometry demonstrated that the cellular uptake of ND-PEG-FA/GLY-MTX was energy-dependent,clathrin-dependent,caveolin-dependent and folate receptor mediated endocytosis,the amount of nanoparticle into cells was concerned with the receptor expression on the cell surface.In addition to that,it also proved that ND-PEG-GLY nanocarrier and ND-PEG-FA/GLY nanocarrier have good biocompatibility.Compared with ND-PEG-GLY-MTX and free MTX,ND-PEG-FA/GLY-MTX nanoparticles had greater lethality,and worked on the G2 phase of cell cycle.Using the laser confocal,it was found that ND-PEG-FA/GLY-MTX nanoparticles were located in the cytoplasm,which was time-dependent.3.DOX was loaded on the surface of ND-PEG-FA nanocarrier to prepare ND-PEG-FA/DOX nanomedicine,and the amount of the adsorbed DOX was 47?g/mg,which was detected by fluorescence photometer.It was proved that ND-PEG-FA/DOX nanomedicine was synthesized successfully by means of FTIR,TEM and scanning transmission electron microscopy(STEM).The flow cytometry showed that the nanomedicine was uptaken into the cells through folate receptor mediated method.The MTT assay indicated that ND and its intermediates had good biocompatibility,the cytotoxicity of ND-PEG-FA/DOX nanomedicine was concentration-dependent and time-dependent,which showed that this nanomedicine had a slow release behavior.