Preparation Of Folate Functionalized Nanodiamonds As Drug Delivery System And Study Of Its Cell Targeting In Vitro

Chemotherapy is one method of treating tumor disease, but its biggest drawback is without selective recognition to tumor cells and normal cells, which leads to serious side effects to patients in the process of tumor treatment. Based on the fact that receptors overexpressed on the surface of tumor cells have high affinity with its ligand, drugs conjugated or adsorbed the ligand will be transported to the lesion site by receptor-mediated pathway, which can achieve targeted drugs delivery, improve the therapeutic effect and reduce side effects. "With the development of nanomedicine, nano-materials modificated with the appropriate ligand for targeted delivery of drugs has attracted widespread interesting. These ligands include folate, lipoprotein, transferrin, polypeptide, and so on. Folate is a small molecule possessing a low immunogenicity and easy covalently coupled to other substances. Hence folate-modified nanoparticles loaded drugs enable targeted therapy to tumor.In this article, the ND-PEG-FA/DOX was prepared using nanodiamonds (NDs) as a carrier, chemotherapy drug doxorubicin (DOX) as the model drug, folate(FA) for the targeting ligand molecule. The interaction between the ND-PEG-FA/DOX and cells with different amount receptors expression has been conducted mainly by laser scanning confocal microscopy imaging, flow cytometry and MTT assay. The works below were carried out:1. The nanodiamond complexes (ND-PEG-FA) was prepared, where nanodiamond conjugated with PEG-diamine (H2N-PEG-NH2) and folic acid (FA) via covalent bond on the carboxylated NDs, This coupling of prepared compounds was confirmed by FTIR analysis. The coupled amount of PEG is 200μg/mg on the surface of NDs and the coupled amount of FA is 44μg/mg on the surface of ND-PEG-NH2 by UV-visible spectrophotometry and fluorescence spectrophotometry, respectively.2. Doxorubicin hydrochloride (DOX), a chemotherapy drug, was physically adsorbed onto the ND-PEG-FA nanoparticles to obtain system of ND-PEG-FA/DOX. Firstly, saturation equilibrium conditions of the interaction between ND-PEG-FA and DOX were investigated. Secondly, releasing behavior DOX from ND-PEG-FA/DOX in different pH buffer in vitro were studied. The results displaied that optimal time of ND-PEG-FA interacting with DOX is 6h and the ND-PEG-FA nanoparticles showed the maximum adsorption capacity for DOX was (47±1.26)μg/mg. The rate and amount of DOX released from ND-PEG-FA/DOX were dependent on the pH of the medium. ND-PEG-FA/DOX showed a slightly rapid release of DOX at pH 5.5(a simulated intracellular endosomes environmen) and pH 6.5(a simulated tumor environment,) than at pH 7.4(a simulated the physiological environment of the bloodstream). It implied that the less drug release from ND-PEG-FA/DOX in the blood circulation and reduce the damage to normal tissue.3. HeLa, C6, HepG2 and HEK293 cells with different amounts of folate receptors on the cell surface as in vitro models, using MTT assay showed that NDs, ND-PEG-NH2 and ND-PEG-FA material almost no cytotoxicity. The cellular uptake of the ND-PEG-FA/DOX was time, concentration, temperature and energy-dependent by Flow cytometry analysis and is the clathrin-mediated endocytic pathway into the cells. Moreover, ND-PEG-FA/DOX system has a sustained drug release capability and a good active targeting tumor ablity. Using a laser scanning confocal microscope imaging further proves that ND-PEG-FA/DOX has a good active targeting tumor ablity and showed that ND-PEG-FA/DOX nanomedicine was distributed in the cytoplasm and small molecule drug doxorubicin could enter the nucleolus playing a role in inhibiting tumor growth.