Novel Polymeric Micelles As Enzyme-sensitive And Nuclear-targeted Dualfunctional Drug Delivery Vehicles For Enhanced 9-nitro-20(s)-camptothecin Delivery And Antitumor Efficacy

9-nitro-20（S）-camptothecin（9-NC）is a broad-spectrum antitumor drug in tumor treatment field.It is effective for a variety of malignant solid tumors such as ovarian cancer,rectal cancer and leukemia.But its clinical application and antitumor efficacy was limited by its structural instability,poor solubility and its low oral bioavailability.In order to avoid the disadvantages of the anti-tumor drug 9-NC and give full play to its anti-tumor effect,this study designed a bifunctional polymeric micelle with enzyme sensitivity and nuclear targeting based on the tumor microenvironment（TME）for 9-NC injection administration.Briefly,chrysin（CHR）as aπ-conjugated moiety was immobilized on the terminal of PCL in amphiphiles of TAT-PCL and combined with peptide ALAL as a linker on Hyaluronic acid（HA）chains to yield ultimate amphiphiles of CHR-PCL-TAT-ALAL-HA（HATPC）.In this drug delivery system,HA acts as an active tumor targeting via CD44 receptor-mediated endocytosis and peptide ALAL could be cut off in lysosome in tumor cells due to the high expression of cathepsin B,leading to lysosomal escape while generating secondary polymeric micelles targeted to the tumor cell nucleus via exposed peptide TAT,thereby improving its ability to target antitumors.This designed nanomicelles could protect the anti-tumor drug from transformation,thus specifically kill tumor cells and reduce the toxic and side effects.The structures of polymeric amphiphiles and their intermediate products were identified by nuclear magnetic resonance hydrogen spectroscopy（¹H-NMR）,mass spectrometry（ESI-MS）and gel permeation chromatography（GPC）.The dual-functional amphiphilic polymer HATPC and 9-NC were self-assembled to obtain spherical 9-NC micelles by dialysis,and their encapsulation efficiency and drug loading were measured by ultraviolet spectrophotometer.The laser particle size analyzer and transmission electron microscope were used to characterize the particle size,zeta potential and morphology.The safety of micelles was investigated through the material toxicity experiment,hemolysis experiment and H&E staining section experiment.The stability of polymeric micelles,the release characteristics in vitro,the uptake of SKOV3 cells and the anti-tumor effect in vivo and in vitro were systematically studied.The results of ¹H-NMR and ESI-MS indicated that the intermediate product of the polymeric material had synthesized,and the molecular weight of the grafted PCL block was 2k according to the integral calculation.The GPC spectrum showed that the molecular weight of the final product was 10140,indicating that the enzyme-sensitive and nuclear-targeted bifunctional polymeric material was successfully synthesized.The micelle size was uniform（121.6±5.79 nm）,with well morphology distribution（PDI=0.256）.And it has a negative surface charge（-23.2±0.5 mV）,which was stable in the blood circulation.The encapsulation efficiency and drug loading were 85.09±2.93%and12.92±0.88%,respectively.It was detected that under simulated tumor cell lysosome environment,HATPC micelles showed two particle size peaks,and the particle size was significantly reduced by using dynamic light scattering（DLS）,confirming the enzyme sensitivity of 9-NC/HATPC micelles.The nuclear targeting of micelles was analyzed by confocal laser scanning microscope（CLSM）.The results showed that 9-NC/HATPC micelles were more effective than free 9-NC and traditional polymeric micelles mPEG_2k-PCL_2k.It is concentrated in the nucleus of tumor cells,so these micelles show the highest cytotoxicity to SKOV3 tumor cells in vitro and in vivo.IC₅₀0 of 9-NC/HATPC micelles was measured by cytotoxicity test as 0.03μg/m L,and the apoptosis rate of 9-NC/HATPC micelles detected by flow cytometry was 58.5%.After injection of micelles to tumor-bearing mice,the tumors obviously got controlled,9-NC/HATPC drug-loaded micelles.In addition,the safety of micelles was further investigated.The material toxicity experiment,hemolysis experiment and H&E staining section experiment had confirmed that the drug delivery system has good biological safety,reduced the toxic and side effects on normal tissues and organs,and no hemolysis.When the polymeric material concentration is 0.8mg/m L,the hemolysis rate was only 0.133%.This enzyme-sensitive and nuclear-targeted dual-function drug delivery system（HATPC）successfully delivered 9-NC to the tumor cell nucleus,improved its anti-tumor effect,and provided a basis for the construction of such nuclear targeted delivery systems.