Construction Of HER3 Targeted Aptamer-drug Conjugate Delivery System And Its Antitumor Effect

Cancer is a predominant threat to human health.Despite the development of multiple cancer therapy strategies for combined therapy,immunotherapy,and even precision medicine,the chemotherapy remains to be the first-line treatment for some cancer patients.How to overcome and avoid the side effects caused by non-targeted distribution of chemotherapy drugs,has been one of the hot spots of clinical concern.Targeting delivery of toxic drugs has huge application in cancer therapy with its high efficacy and safety,which can improve the drug concentration in the targeted tissue and reduce the systemic toxicity.Two antibody-drug conjugates(ADC)approved by the FDA have been confirmed their validity.However,the preparation process of ADC have many problems that are not well controlled,such as the drug-antibody ratio(DAR)and the immunogenicity of naked antibodies.Aptamers(Apt)are one kind of unique single-stranded oligonucleotides wih lots of advantages,such as the high specificity and affinity with the target and low immunogenicity compared to the antibodies.In addition to being used as drug alone(example: Macugen approved by FDA),they can be used as targeting vectors in drug delivery systems.This paper focused on theproof-of-concept study in targeted delivery systems for chemotherapeutic drugs,that is aptamer-drug conjugate(ApDC).The ApDC in the current study demonstrates the enhanced drug therapy efficiency and the reduction of drug side effects.In this paper,we tried to screen the high affinity and high specificity of candidate Apt using extracellular domain(ECD)of human epidermal growth factor receptor 3(HER3)as target by systematic evolution of ligands by exponential enrichment(SELEX)technology.Liposome-doxorubicin(Lip-DOX)was prepared by thin film dispersion method firstly.Then aptamer-liposome-doxorubicin(Apt-Lip-DOX)was constructed with the aptamer by "post-insertion method".The Apt-Lip-DOX preparation process was investigated and optimized by physical and chemical properties such as encapsulation efficiency,particle size,zeta potential and release degree.In vitro experiments,the half maximal inhibitory concentration(IC50)and endocytosis pathway of the ApDC system were investigated in MCF-7HER3+,BT474HER3+cells.In vivo experiments,the MCF-7HER3+ tumor bearing mice model was selected to examine the advantages of the Ap DC in terms of targeting,efficacy and safety compared with other delivery fromats.Part ?: Screen and identification of HER3 ECD aptamer.HER3ECD plasmid was transiently transfected into the 293 E cells,and the protein was purified.The candidate Apts of HER3 ECD was obtained by the optimized "QPCR-magnetic bead sorting" SELEX method.Then the affinity curves were obtained by enzyme linked immunosorbent assay(ELISA).Two candidate aptamers(#7 and #13)were screened with their affinity constant were Kd = 116 ± 23 nM and Kd = 98 ± 9.7 nM,respectively.The results showed that the aptamers had high affinity and specificity for breast cancer cell withhigh expression of HER3 by flow cytometry and the laser confocal microscopy experiments.Part ?: Construction and Optimization of ApDC Delivery System.First,the preparation process of Lip-DOX was optimized and the ApDC was prepared by "post-insertion method" connection with the Apt-DSPE-PEG2000.The physical and chemical properties of Apt-Lip-DOX were determined.The particle size was 170 ± 25 nm and the zeta potential was-45 ± 5 mV.The entrapment efficiency of DOX was 55 ± 5% by ultrafiltration combined with high performance liquid chromatography(HPLC).The results showed that the release rate of free DOX was close to 100% at 1h,and the release rate of Apt-Lip-DOX was 20% after 24 hours under the same conditions,which proved the sustained release capacity and stability of the delivery system.Part ?: The cellular endocytosis and pharmacodynamics of ApDC system in vitro.At the cellular level,the MCF-7HER3+ and 293THER3-were selected to compare the dominant effect of Apt in the delivery system.Different endocytosis inhibitors were used after the addition of quantum dots(QD)encapsulated ApDC system into the cells.Live cell monitoring observation was performed to explore the mechanism of ApDC pathway.The results showed that the entry of ApDC was dependent on the expression of HER3 and the presence of Apt,showing the advantages of HER3 Apt,and the delivery system endocytosis was via the clathrin dependent pathway.The IC50 values of different delivery forms of DOX were measured by real time cell assay(RTCA),including the MCF-7 HER3+ and BT474 HER3+ cells and 293 T HER3-cell as negative control.The results showed that the IC50 values of Lip-DOX was lower than free DOX in the three cells,and there was a statistically significant difference(P <0.05),which showed the advantages of liposome delivery system.The IC50 values of Apt-Lip-DOX was statistically significant compared with free DOX in MCF-7 and BT474 cells(P <0.01),but it did not show the advantage in 293 T cells,which indicated the targeting role of aptamer in increased cytotoxic effect of tumor cells in vitro,laying a foundation for subsequent experiments.Part ?:Tumor targeting,efficacy and toxicity of Ap DC in tumor-bearing mice.MCF-7 tumor-bearing mice were selected to investigate the tumor targeting,efficacy and safety of Ap DC in vivo.1)Tumor targeting: Apt-Lip-QD was prepared by wrapping QD.MCF-7 tumor-bearing mice were injected with ApDC and Lip-QD control group via tail vein by animal live imaging.Fluorescence distribution in vivo was determine 1 h post administration.The results showed that ApDC showed good targeting,and there was a statistically significant difference(P <0.01)compared with the Lip-QD in the anatomical tumors.2)Tumor inhibitory efficacy: Several groups were investigated including blank control group(Blank,equal volume of saline)and drug-treatment groups of DOX,Lip-DOX and Apt-Lip-DOX.The DOX dose of experimental groups were 5 mg / Kg by intravenous injection every three days.The results showed that the anti-tumor effect of Apt-Lip-DOX was significantly different(P <0.01,P<0.001)compared with the blank group after 18 days of administration,and showed a greater advantage than the DOX group(P < 0.05).3)The toxicity study in vivo : The weight of the mice were monitored during the durg-treatment period.Survival rate of 60 days was observed and the histopathological sections were performed upon the sacrifice.The results showed that the state of Apt-Lip-DOX group was maintained at normal level during the administration period.In contrast,the body weight decreased sharplyin DOX group at 20 days.The survival rates within 60 days of different groups(DOX,Lip-DOX,Apt-Lip-DOX)were 0,40% and 100% respectively.In the DOX group,the H&E slices of the heart and liver showed steatosis,scattered in the bleeding point,vasodilating bleeding and the focal liver cell necrosis and so on.There were also mild lesions in the Lip-DOX group,and with the normal ones in the Apt-Lip-DOX group.In conclusion,the targeted chemotherapy using the ApDC format could provide better tolerability and efficacy compared with non-targeted delivery in relatively low-dose toxic drugs.In this study,we established and improved the SELEX technology route to provide basis for other target screening.Targeted HER3 treatment strategy will bring new ideas for tumor targeted therapy or reversal of EGFR inhibitor resistance.Take DOX as an example,through the construction of ApDC delivery system,its targeting,anti-tumor effect,etc.,we achieve the highly efficient and low toxicity of chemotherapy drugs delivery.