Construction Of Targeted Drug Delivery System Of Nanodiamond And Its Interaction With The Cells

More serious environmental pollution and some bad habits of people lead to a high incidence of cancer, as well as cancer treatment is low efficient nowadays, which induce its high mortality rate. In the conventional methods of cancer treatment, chemotherapy is considered one of the most simple and effective methods. However, it often causes the failure of chemotherapy in particular for solid tumors, due to the more hysteretic diagnosis of cancer, the side effect of small molecule anti-cancer drug and multidrug resistance of cancer cells. The small molecule anti-cancer drug is rapidly diffused the whole body after blood administration. Simultaneously, not only cancer cells are killed, but also normal cells division faster are given great toxicity. With the development of nanotechnology, nanomedicine provides a new way and idea of cancer diagnosis and treatment. Nanotechnology application in medical diagnostic imaging can improve the detection sensitivity, such as magnetic resonance imaging, ultrasound imaging and X-ray computed tomography so on. Recently the development of in vivo imaging technology and multi-photon excitation scanning technology promote the application of fluorescence imaging in cancer diagnosis. The developments of these new technologies become possible for the earlier diagnosis of the tumor. In addition, the incomplete structure of tumor angiogenesis makes nanoparticles aggregate near the tumor through enhanced permeability and retention effect. The anticancer drugs prepared into nanodrugs or loaded on nanocarriers can achieve the passive targeting tumor therapy. Furthermore, some nanoparticles modified on the surface by the ligand can bind to specific angiogenesis or cancer cells to obtain the active targeting tumor therapy. The targeting delivery drug system can reduce the dosage and side effects of anticancer drugs, and also inhibit the multidrug resistance of tumor cells to improve treatment efficiency. The materials as nanocarriers investigated at present mainly include, but are not limited to liposomes, polymers, carbon nanotubes, micelles and nanoparticles so on. Recently, owing to its biocompatibility, chemical stability, optical properties, surface functionalization capabilities and easy synthesis, nanodiamond has been emerged as a new nanomedicine candidate material. The researches of the biological application of nanodiamonds are focused on drug carriers and fluorescence imaging agents and are in their infancy.In this paper, the targeting nanocarrier was constucted from detonation nanodiamond used as a carrier material, polyethylene glycol as a crosslinking agent, and transferrin as a targeting molecule, respectively. The interaction between the loading drug nanocarrier and the cells, the slow-sustained releasing drug from nanocarriers in the cellul and the targeting of nanocarriers are investigated using doxorubicin (DOX) as a model drug, normal liver cells (L-02 cells) and hepatoma cells (HepG2 cells) as cell models, respectively. The main contents are as follows:1. The amount of surface carboxylic acid on oxidized is measured by conductometric backward titration and calculated 3.36×105 carboxylic groups per ND particle (the size～140 nm).2. The results of physical adsorption behavior of ND indicate that Cit3- can enhance the adsorption of DOX onto ND, and the Cl- can regulate the releasing DOX from ND. The adsorption behavior of Tf on ND is coincident with Langmuir model which is a kind of isothermy absorbed behavior. The maximum adsorption amount of DOX on ND is (176.46±2.13) μg/mg in PBS (pH 7.4) and the adsorption capacity is the maximum at the pH (5.5) which is equal to the isoelectric point of Tf, probably because the minimum solubility of Tf at the isoelectric point leads to increasing hydrophobic interactions with the ND. The difference of cellular uptake of ND and ND-Tf are analyzed qualitatively and quantitatively by laser scanning confocal microscope and flow cytometry. The results indicate that ND-Tf is endocytosed easier than ND and more beneficial in intracellular fluorescent imaging.3. ND can be covalently conjugated with polyethlene glycol (PEG) to increase its dispesity and stability, which is conducive to endocytosis. The cellular uptake of ND-PEG-DOX is approximately two times of free DOX, but which can enhance the DOX uptake as compared to DOX alone and decrease the cytotoxity of DOX. The change of fluorescent intensity of intracellular ND determined by flow cytometry is consistent to the side scatter. So the amount of intracellular nanoparticles can be determined by the side scatter. The process of releasing DOX from intracellular ND-PEG-DOX is tracked by laser scanning confocal microscope. The result indicates that DOX released from ND-PEG-DOX composites had a slow and sustained drug release capability. The mechanism of cellular uptake of ND-PEG-DOX is clathrin-mediated endocytosis, which is different from free DOX that is diffused into cell.4. Using Tf as a targeting molecule, the targeting delivery drug nanocarrier, ND-PEG-Tf is constructed. The results of intracellular fluorescent intensity of ND-PEG-Tf determined by flow cytometry indicate that the endocytosis of ND-PEG-Tf is time-and concentration-dependent. The amount of cellular uptake of ND-PEG-Tf is related to the expressing TfR level on cell surface. The inhibition experiment using free Tf and Fe3+ shows that the endocytosis of ND-PEG-Tf is a TfR-mediated pathway. So ND-PEG-Tf can selectively enter the tumor cells and target drug delivery according to the difference of TfR between normal cells (low-expressed) and tumor cells (over-expressed). The cytotoxity is measured by MTT assay. The result show that ND-PEG-Tf-DOX is more toxitic to over-expressed TfR tumor cells. Simultaneously, ND-PEG-Tf-DOX can reduce toxic to normal cells compared with free DOX. So ND-PEG-Tf can potentially be acted as targeting cancer cells and effective drug delivery for cancer therapy.5. ND-PEG-Tf can not affect cell growth which is demonstrated by the cell growth curve after the endocytosis of ND-PEG-Tf. The intracellular ND-PEG-Tf can be transmitted to the daughter cells, which is determined by flow cytometry and laser scanning confocal microscope. The amount of intracelluar ND-PEG-Tf is reduced with the increasing number of cell division. The results indicate that ND-PEG-Tf is biocompatible and can be acted as a targeting drug delivery nanocarrier.