The Experimental Study On The Synergistic Treatment Of Drug Resistance In Anaplastic Thyroid Carcinoma With Sustained And Controlled Release Composite Nanoparticles

Anaplastic thyroid carcinoma(ATC)is a rare malignant tumor with a poor prognosis.Surgery,radiotherapy,chemotherapy and other strategies are often used to control the progression of the disease.However,surgery and radiotherapy often extend the therapeutic range and cause damage to the surrounding tissue and organs of the tumor.Although chemotherapy has some effect,it has been mired in the dilemma of multiple drug resistance(MDR).To solve the problem of multi-drug resistance,improve therapeutic efficiency,and solve the problem of integrated clinical application,we developed a spatio-temporal slow-release nanoparticle based on polyamine-amine dendrimer G4(PAMAM-G4)and Low-intensity focused ultrasound(LIFU)(PAMAM-DOX@Lip-PIP-IR).In addition,the anaplastic thyroid carcinoma was treated by the combined synergic killing effect of Doxorubicin(DOX)and Piperine(PIP).The nanoparticles reach the tumor region via the mitochondrial targeting molecule IR780,which enables the accumulation of nanoparticles in the target region.Meanwhile,the drug release is stimulated by LIFU.The persistent release molecule PAMAM gradually releases DOX in the target region and continuously releases DOX.The drug concentration in the target region can continue to reach a therapeutic concentration to achieve the combined synergistic drug therapy effect of ATC.IR780 has the function of fluorescence imaging and photoacoustic imaging,which also provides a development direction for the "visualization" of tumor therapy.Objective:The mitochondria targeted sustained and controlled release composite nanoparticles PAMAM-DOX@L-PIP-IR were prepared to detect their physical and chemical properties,investigate their photoacoustic imaging and fluorescence imaging capabilities,and evaluate the distribution and targeting of the nanoparticles in cells and in vivo,their drug release ability,and their combined synergistic therapeutic effect in vivo and in vitro.Methods:PAMAM-DOX@L-PIP-IR nanoparticles were prepared by thin-film hydration and sonoseismic method.PAMAM-DOX was used as the core and lipid membranes containing IR and PIP were used as the shell.The target nanoparticles were synthesized by sonoseismometry.The encapsulation and loading rates of DOX,PIP and IR780 in nanoparticles were determined and calculated.In vitro study of PAMAM-DOX@L-PIP-IR nanoparticles suitable for treatment concentration,treatment cycle,drug release curve of DOX,drug release conditions of LIFU,and evaluate the mitochondrial targeting of nanoparticles.The phagocytosis and targeting of nanoparticles in cells were detected by LCSM.CCK-8 was used to evaluate the safety of slow and controlled release nanoparticles on C643 cells and the cytotoxic effect and synergistic therapeutic effect of nanoparticles released by LIFU.Laser confocal microscopy was also used to observe staining of live and dead cells to assess the cytotoxic effect and synergistic therapeutic effect of nanoparticles on C643 cells,and flow cytometry was further used to validate this.In terms of imaging,fluorescence imaging of small animals was used to track the distribution and circulation path of nanoparticles in vivo,once again verifying the targeting effect of nanoparticles on tumors.Photoacoustic imaging studies the relationship between photoacoustic signals and nanoparticle concentration and the sustainability of signals in vivo and in vitro.In vivo,the biological safety of nanoparticles was evaluated by taking the blood and viscera of mice at different days after the injection of nanoparticles.To observe and measure the change and corresponding relationship between tumor volume and treatment time and frequency in tumor-bearing mice treated with different conditions,so as to evaluate the synergistic therapeutic effect of nanoparticles on ATC transplanted tumors.Results:PAMAM-DOX@L-PIP-IR nanoparticles were successfully prepared.The nanoparticles have a typical core-shell structure,spherical and uniform distribution.The cell activity experiment indicated that PAMAM-DOX@L-PIP-IR nanoparticles had good biosafety at the treatment concentration,and laser confocal microscope observed that the nanoparticles had obvious mitochondrial targeting.The nanoparticles showed obvious drug release under low power focused ultrasound irradiation.PAMAM-DOX@L-PIP-IR nanoparticles combined with LIFU can induce obvious apoptosis of cells and the effect is better than that of monotherapy group,indicating that it does play a synergistic role in combination therapy.In vivo photoacoustic imaging and fluorescence imaging showed that the nanoparticles have good tumor targeting and can effectively accumulate in the tumor area,which is conducive to in vivo treatment.In vivo treatment experiments showed that PAMAM-DOX@L-PIP-IR nanoparticles combined with LIFU group could cause significant increase in cell apoptosis and decrease in cell proliferation,and the therapeutic effect was significant compared with other groups.In addition,blood tests and H&E staining of major organs showed that there was no significant difference between the PAMAM-DOX@L-PIP-IR nanoparticle group and the control group,indicating the safety of nanoparticles.Conclusion:In this study,the sustained and controlled release nanoparticle PAMAM-DOX@L-PIP-IR was successfully prepared,which has obvious mitochondrial targeting and biosafety.Aiming at the characteristics of high malignant degree,strong drug resistance and high recurrence rate of anaplastic thyroid carcinoma,the nanoparticle used DOX,a chemotherapeutic drug carrying dendrimer polyamide-amine PAMAM,as a sustained-release molecule,and encapsulated Chinese patent medicine extract piperine PIP with anti-MDR effect and iodide IR780 with mitochondrial targeting in lipid shells.It has the function of anti-MDR effect and slow release,which solves the problem of drug resistance of ATC to chemotherapy drugs and achieves the effect of long-term treatment.In addition,the nanoparticle combined with low-intensity focused ultrasound(LIFU)can release the drug at a fixed point inside the tumor,achieving the effect of fixed-point drug release and synergistic therapy.The nanoparticles can also be used to monitor the treatment process through photoacoustic and fluorescence imaging to achieve "visual" treatment.In conclusion,PAMAM-DOX@L-PIP-IR nanoparticles can effectively kill tumor cells in coordination with LIFU,providing a new diagnosis and treatment method for ATC.