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Application Of Nucleic Acid Aptamer Modified Nanoparticles In Tumor Targeted Immunotherapy And Targeted Hyperthermia

Posted on:2016-03-29Degree:MasterType:Thesis
Country:ChinaCandidate:L Y YuFull Text:PDF
GTID:2134330461476918Subject:Pathology and pathophysiology
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Objective:MUC1 was glycoprotein overexpressed in many types of cancers including breast, ovarian, lung, colon and prostatic cancers, making it a promising target in cancer therapy. Several attempts of MUC1-targeting drug delivery had reported and developed. However, the strategy of using natural killer cells (NK) to eliminate MUC1-positive cancer cells remains to be explored. Natural killer cell is an important type of immune cells within human body. It is associated with anti-tumor reaction, anti-viral infection, and immune regulation. In this study, we developed a novel amphipathie nanoparticle that was modified by CD16 and MUC1 aptamers. The amphipathic nanoparticle could combine with both the CD16-positive natural killer cells and the MUC1-positive tumor cells, and pull the two types of cells together to enhance NK’s cytotoxicity to the tumorcells.Method:The CD16 and MUC1 aptamers were implanted onto nanoparticle via biotin-streptavidin reaction. The aptamers’binding with target cells was verified by flow cytometry. The aptamers’combination with nanoparticles was confirmed by DNA hybridization experiments. The size distribution of the amphipathic nanoparticle was evaluated by DLS assay. The binding of aptamer modified nanoparticle with target cells was tested by competition blocking assay. Dark-field microscopy showed that the amphipathic nanoparticle could recruit NK to the neighbor of MUC1-positive tumor cells. MTS was used to evaluate the immune reaction ofNK to tumor cells.Result:The amphipathic nanoparticle (NP) was constructed by implanted CD16 and MUC1 aptamers onto the NP via biotin-streptavidine reaction. Flow cytometry analysis indicated that the aptamers could bind with the targeted cells. DNA hybridization experiments showed that the aptamers were indeed implanted onto the nanoparticle. The average size of the nanoparticle was 574 nm. Competition blocking assay indicated that the aptamer-modified-nanoparticle could bind to the target cells. Moreover, dark-filed microscopy indicated that the amphipathic nanoparticle could recruit NK to the vicinity of MUC1-positive tumor cells. MTS assay showed that the amphipathic nanoparticle could enhance NK’s immune reaction to MUC1-positive tumor cells but not to MUC1-negative tumor cells.Conclusion:The results suggest that NK cells may be mobilized to target against MUC 1-positive tumor cells.Objective:Glioblastomas is the most aggresive primary intracranial tumor in adults. The survival time of glioblastomas patients is shorter and the mortality is relatively high. Traditional cancer therapies such as chemotherapy, radiotherapy and surgery can’t completely eliminate the tumor cells due to their powerful invasion into normal cells. Therefore, the development of a new method for treating glioblastoma is imperative. Thermal therapy is a new type of cancer therapy methods. Its cytotoxic effect on cancer cells is performed by inducing apoptosis in high temperature. The lack of targeting performance of thermal therapy tends to damage normal cells. Consequently, a more effective thermal therapy method with targeting performance should be explored. Aptamer is a kind of molecular which has been widely studied in recent years. It has high affinity and specificity when binding with target molecular. In this study, we developed a novel aptamer-modified magnetic nanoparticle (Apt-NP) and for the first time detected its ability in improving the efficacy of the targeted thermal therapy in glioblastomas cancer cells in vitro.Method The aptamer was implanted onto nanoparticles via biotin-streptavidin reaction. Apt-NP was characterized by dynamic light scattering (DLS) assay. The binding of aptamer to target cell was evaluated by flow cytometry. The aptamers’ combination with nanoparticles was confirmed by DNA hybridization experiments. The attachment of Apt-NP to target cell was evaluated by Prussian blue staining. Thermal damage under alternative magnetic field was measured by detecting the release of LDH.Results Flow cytometry analysis indicated that the aptamers could bind with the targeted cells. DNA hybridization experiments showed that the aptamers were indeed implanted onto the nanoparticle. The average size of Apt-NP was 574 nm. Prussian blue staining suggested that Apt-NP could bind with the glioblastomas U251 tumor cells but not the MDA-MB-231 cell. Importantly, Apt-NP significantly enhanced the thermal damage to glioblastomas U251 tumor cells but not that to MDA-MB-231 cells.Conclusions Aptamer-modified nanoparticles may have potential utility in glioblastomas targeted thermal therapies.
Keywords/Search Tags:MUC1, CD16, nanoparticle, aptamer, immune therapy, target therapy, thermal therapy, glioblastomas
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