DNA Aptamer-oriented Paclitaxel Nanoparticles Targeting Research

Target drug delivery has been hot in the treatment of tumors which can greatly improve the drug efficacy and enhance the therapeutic effect. Aptamer is a synthetic oligo-nucleotide which has high affinity and specificity binding with target molecules. Aptamer has attracted more and more attention especially in the study of cancer diagnosis and treatment, and has achieved many significant results.DNA aptamer AS1411is one of the most successful aptamers. AS1411can form a G-quarter three-dimensional structure. specifically targeting tumor surface which highly express nucleolin receptor. AS1411as an antitumor drug has been used for many types of cancer clinical trials. including the kind of acute myeloid leukemia Phase II clinical stage, Phase II clinical stage of metastatic renal cell carcinoma and Phase I clinical stage of renal cell carcinoma and non-small cell lung cancer. All of them have made encouraging therapeutic effect.In this study, we first prepare PTX nanoparticles (NPs-PTX) according to the laboratory patented method. After cross-linking agent EDC/NHS modifying DNA aptamer AS1411to the prepared nanoparticles. we get the Apt-NPs-PTX nanoparticles. The prepared Apt-NPs-PTX average particle size is179nm. Transmission and scanning electron microscopy images of Apt-NPs-PTX show they are almost spherical with smooth surface and uniform distribution inside the nanoparticles. In addition, by circular dichroism we find albumin in Apt-NPs-PTX still keep its secondary structure. The stability data show that the Apt-NPs-PTX in complete medium at37℃can be stabilized to52hours which provide a strong guarantee for future clinical trials.Then we prepare nanoparticles containing fluorescent dye coumarin-6and investigate their size, size distribution and morphology. The results show that the coumarin-6will not influence nanoparticles’ particle size and morphology. Cellular uptake experiments show that Apt-NPs-PTX could target to nucleolin largely expressed human breast cancer cell line (MCF-7). but there is no detectable targeting to normal cells. In addition, we evaluate the cell toxicity of Apt-NPs-PTX. Apt-NPs-PTX show significant killing effect for tumor cells in vitro but not show killing effect for normal cells. We also evaluate the cytotoxieity of erosslinking agent EDC/NHS. DNA aptamer AS1411, human serum albumin (HSA) on MCF-7cells and MCF-10A cells. The results show they do not have significant killing effect, indicating that the main substance which plays a killing effect in Apt-NPs-PTX is PTX.We also investigate the mechanism of cellular uptake Apt-NPs-PTX. Using endostatin to close nucleolin, we find that untreated tumor cells could uptake more Apt-NPs-PTX than NPs-PTX. When close nucleolin, cells uptake Apt-NPs-PTX almost as many as NPs-PTX. which speculate that nucleolin may play an important role in the cellular uptake of Apt-NPs-PTX.In vivo experiment show that Apt-NPs-PTX accumulates twice in nude mice tumor area, which could make drug stay at the tumor site for more long-term. The data indicate a good prospect in the clinical application.