Nuclear Targeting And Responsive Drug Release Mediated Breast Cancer Therapy By Glycolipid-like Drug Delivery System

As an important structure of the genetic information base and the crucial regulator of cell activity,the nucleus plays an important role in tumor cell proliferation.It has been proven to be the main interaction site for most therapeutic agents such as anti cancer drugs,gene therapy,free radicals,and heat.The delivery efficiency of therapeutic drugs to their final targets can maximize the therapeutic efficacy and significantly eliminate the off-target and side effects.In this study,we focus on developing the nuclear-targeting micelles through the glycolipid structure specific nuclear transport pathway and the nanoparticles with reactive oxygen species(ROS)sensitive release by structural deformation to achieve high-efficiency targeting and rapid release in tumor nucleus.Chitosan oligosaccharide grafted stearic acid(CSOSA)was synthesized by an amidation reaction with the basic framework material of chitosan oligosaccharide(CSO).In the aqueous solution,CSOSA can self-assemble into micelles.CSOSA with different amino substitution degrees(SD%)were prepared to investigate the capability of the micelles to be transported into the nucleus.The result showed that 9.8%(SD%)was optimal.Confocal laser scanning microscopy(CLSM)results illustrated that CSOSA can be efficiently delivered to the nucleus by interacting with nuclear pore complex(NPC).Folate receptor(FR),which is overexpressed in a variety of cancer cells,is a cell surface receptor that could bind and transcribe folate and its derivatives.In this study,the carboxyl group of folic acid(FA)was linked to the amino group on one side of PEG2000 with the help of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)and N-Hydroxysuccinimide(NHS).Di(N-succinimidyl)3,3'-Dithiodipropionate(DSP)was selected as the bridging agent,and the amino group on the other side of PEG2000 was further linked to the amino group of CSOSA to obtain a tumor-targeted folate modified chitosan oligosaccharide grafted stearic acid(FA-CSOSA).The SD%of FA in FA-CSOSA was 0.8%.The distribution of FA-CSOSA micelles in tumor cells was investigated,and it was found that the FA-CSOSA micelles uptake in 4T1 cells was increased and accumulated in the nucleus.Loaded with chemotherapeutics doxorubicin(DOX)and Pinl inhibitor all trans retinoic acid(ATRA),FA-CSOSA/DOX and FA-CSOSA/ATRA were prepared by dialysis method.The particle sizes of FA-CSOSA/DOX and FA-CSOSA/ATRA were 71.0 ± 1.84 nm and 73.3 ± 0.98 nm,respectively,and the zeta potentials were 21.2 ± 1.0 mV and 25.4 ± 0.8 mV,respectively.To exert the best antitumor effect for 4T1 cells,the optimal synergistic ratio of DOX and ATRA determined by combination index(CI)was 1:1(w/w).The apoptosis rate with the treatment of FA-CSOSA/DOX&FA-CSOSA/ATRA was 83.7%,which was 1.42-fold and 4.68-fold of that treated by CSOSA/DOX&CSOSA/ATRA and DOX&ATRA groups,respectively.DOX can inhibit the synthesis of nucleic acid by inserting DNA into the nucleus.ATRA can be trapped in the Pinl active site and inhibits its catalytic activity,ultimately leading to Pinl degradation.The results of CLSM showed that Pinl protein was mainly distributed in the nucleus.With the aid of nuclear targeting micelles,drug showed better antitumor efficacy because of the higher concentration at the targets.Although traditional chemotherapy has been an effective approach for treating breast cancer in clinics,it was reported that metastasis and enhanced stemness were observed with chemotherapy and lead to neoplastic recurrence and death for cancer patients.To explore the mechanism of the combination therapy of DOX and ATRA,we found that ATRA could reduce DOX-induced stemness and metastasis by inhibiting Pinl protein.The results showed that FA-CSOSA/DOX&FA-CSOSA/ATRA could improve the anti-tumor efficacy significantly with an inhibition rate of 85.5%,which was much higher than that of DOX·HCl(33.9%),DOX&ATRA(45.9%)and CSOSA/DOX&CSOSA/ATRA(72.8%).The mechanism of anti-tumor effect in vivo was investigated by immunohistochemical.The precisely targeted delivery of the FA-CSOSA to action sites significantly enhanced the antitumor efficacy of DOX.The combined therapy could inhibit the expression of Pinl protein,downregulate the expression of stemness-related protein,inhibit tumor metastasis,and reduce the proliferation of tumor cells.Chitosan oligosaccharide thioacetal stearamine conjugate(CSOTKSAm)was synthesized for achieving rapid drug release to maximize efficacy.Thioacetal(TK)was chosen as the crosslinking agent in CSOTKSAm for its responsiveness to ROS.As a photosensitizer,Chlorin e6(Ce6)can produce ROS and be used to control drug release in the nucleus.With the help of NHS/EDC,Ce6 was linked to CSOTKSAm by amide reaction to construct Ce6 modified chitosan oligosaccharide thioketal stearylamine conjugate(Ce6-CSOTKSAm).The particle sizes and morphology of Ce6-CSOTKSAm micelles changed significantly under the condition of H2O2 incubation.Transmission electron microscope(TEM)observation showed that the Ce6-CSOTKSAm micelles expanded from spherical structure with uniform size and clear edge to particles with different sizes and irregular shapes after H2O2 incubation.Loaded with DOX,Ce6-CSOTKSAm/DOX micelles were prepared by dialysis method.The result of in vitro drug release showed that the drug release rate of Ce6-CSOTKSAm/DOX with 10 mM H2O2 was significantly faster than that of the control groups without H2O2.The accumulative DOX release of Ce6-CSOTKSAm/DOX under laser irradiation for 4 h was 2.97 times of that treated with CSOTKSAm/DOX under laser irradiation,and was 3.04 times of Ce6-CSOTKSAm/DOX group without laser irradiation.After laser irradiation,the fluorescence signals of the micelles and the drugs were separated,indicating that the drug was already released.The MTT assay and apoptosis test showed that Ce6-CSOTKSAm significantly increased the cytotoxicity on tumor cells and the proportion of apoptotic cells.It was found that most of Ce6-CSOTKSAm/DOX micelles were distributed in the nucleus after 18 h i.v.injection in 4T1 breast cancer models.Moreover,the tumor inhibition rate of Ce6-CSOTKSAm/DOX group under laser irradiation was 77.1%,which was significantly higher than that of Ce6-CSOTKSAm/DOX without laser irradiation group and non-nuclear targeting Ce6-HCSOTKSAm/DOX group under laser irradiation.The tumor inhibition rate with the same quantity DOX·HCl was 15.3%.Compared to other groups,the Ki67 expression of Ce6-CSOTKSAm/DOX with laser was significantly decreased,while the expression of c-caspase-3 was significantly increased,which means the proliferation of tumor was reduced and the percentage of apoptosis cells was enhanced.The results showed that the ROS sensitive release glycolipid-like nanoparticles could significantly improve the drug distribution to target sites,so as to maximize the anti-tumor efficacy.In this study,the nuclear targeting glycolipid-like drug delivery system CSOSA was synthesized,and the mechanism of nuclear targeting was explored.FA-CSOSA/DOX&FA-CSOSA/ATRA micelles were obtained,which can significantly improve the anti-tumor efficacy by reducing chemotherapy drugs-induced stemness characteristics and metastasis.Futhermore,a ROS sensitive release drug delivery system(Ce6-CSOTKSAm)was constructed,which can further improve the anti-tumor efficacy through the rapid drug release after reaching the target sites.