Construction And Antitumor Activity Of Functional Aptamer Modified Magnetic Nanoparticle Drug-loading System

In recent years,human health is still greatly threatened by cancer,and the incidence of malignant tumors remains high and tends to be younger.Due to the rapid proliferation of cancer cells,it is easy to spread in the body.Early accurate diagnosis and effective treatment can improve the survival rate of cancer patients and reduce the suffering of patients.In clinical,surgery,chemotherapy and radiation therapy were widely used.However,early untransferred solid tumors are usually resected with surgical treatment,but metastatic tumors and non-solid tumors are not applicable.In addition,systemic toxicity,drug resistance,and low selectivity of chemotherapeutic drugs often result in poor therapeutic effect.In order to solve these problems,it is urgent to develop new therapy options to increase the cure rate and reduce the side effects and drug resistance caused by cancer treatment.Recent studies have shown that nucleic acid aptamers can successfully carry small molecules.It recognizes and binds to proteins overexpressed on the surface of tumor cells and can be used as a target for drug delivery.Simultaneously,superparamagnetic nanoparticles' small size and the surface easily modified,so they can play varieties of roles including drug delivery,treatment and imaging in multi-functional magnetic resonance imaging,targeted drug delivery and magnetic hyperthermia.In this study,functional aptamers modified superparamagnetic nanoparticles were used as carriers,the chemotherapeutic drug daunorubicin(DNM)and photosensitizer tetramethylpyridinoline(TMPyP)were immobilized.We successfully builded a drug delivery system with chemical/photodynamic therapy and dual magnetic/aptamer targeting functions.We evaluated the stability,targeting,and sensitive delivery of the complex at in vitro and cytological levels.Efficiency,photosensitivity,migration inhibition and cytotoxicity,the specific research contents are as follows:First,a novel targeted delivery system was constructed using COOH@SPION and aminated Apt-S8/Apt-gc34 nucleic acid aptamers,as follows:(1)Construction of DNM&TMPyP&Apt-S8/Apt-gc34@SPION delivery system and detection of in vitro antitumor activity.COOH@SPION was combined with aminated Apt-S8/Apt-gc34 by EDC/NHS reaction,and the drug TMPyP and DNM were treated with the G-quadruplex structure of the AS1411 aptamer and the hairpin structure of the GC-rich structure at the tail end.The results of studies on aptamer-modified magnetic nanoparticles showed that the drug-loaded system was highly uptaken by tumor cells,and the pH-dependent releaseof DNM in tumor cells was present;nucleolar aptamers(AS1411).The blocking experiment demonstrated that the drug-loading system specifically enters into the tumor cells through the specific binding of the aptamer AS1411 to the shuttle protein nucleolin.The results of the light-protection experiment confirmed that the ROS produced by TMPyP illumination inhibited tumor proliferation,further apoptosis and protein.Western blotting showed that apoptosis was induced under light conditions,and the expression of bcl-2 and c-myc in related proteins was down-regulated during apoptosis.(2)Construction of DNM&TMPYP&Apt-PSA&PEI@SPION delivery system and detection of in vitro anti-tumor activity.Based on the above studies,the aptamer AS1411 and the siRNA were self-assembled into a DNA-RNA hybrid strand(Apt-PSA)by GCG base complementation.Using the PEI-modified SPION with positive charge characteristics,the above-mentioned hybrid chain apartment-psa was composited onto the SPION surface by electrostatic force,and a new targeted delivery system of DNM&TMPyP&Apt-PSA&PEI@SPION was further constructed.Subsequent RT-PCR experiments and Western blotting experiments showed that the expression of p-gp was significantly decreased in MCF-7/ADR cells,demonstrating the effective silencing of MDR1 gene by p-gp siRNA.(3)Construction of DNM&TMPyP&PND&Bio-gc34&PSA&PEI@SPION delivery systems for in vitro antitumor activity assays.The biotin-modified single-stranded oligonucleotide gc34(Bio-gc34),p-gp siRNA and PEI-modified SPION were respectively electrostatically combined to the SPION surface.The sensitizing Chinese medicine pyronaridine,DNM,and TMPyP were loaded onto the annealed Bio-gc34 to further construct a novel targeted delivery system of DNM&TMPyP&PND&Bio-gc34&PSA&PEI@SPION.The cytotoxicity test confirmed that the IC50 of MCF-7/ADR was significantly different between DNM&TMPyP&PND&Bio-gc34&PSA&PEI@SPION compared with DNM alone.The results of RT-PCR and Western blotting showed that after systemic action,the expression of p-gp was significantly decreased in MCF-7/ADR cells,demonstrating that p-gp siRNA can effectively silence the MDR1 gene.The results of this experiment demonstrate the synergistic effect of DNM&TMPyP&PND&Bio-gc34&PSA&PEI@SPION system multi-therapeutics,reducing the amount of DNM in the chemotherapeutic agent;the sensitizer,pyronaridine,can competitively bind to p-gp protein,reducing the transport of DNM from cells.The probability of ensuring the effective concentration of DNM in the cells;combined with p-gp siRNA down-regulated the expression of MDR1 gene,achieving the triple inhibition of p-gp protein,providing a new idea and new treatment for drug-resistant breast cancer.