Intelligent Nanocarriers Based On Poly(?-Amino Ester)for Tumor Combined Treatment

Background and Objective:The mortality of cancer is increasing by year.Chemotherapy is still a main means for clinical cancer treatment.However,chemotherapeutic drugs are usually lack of targeting ability,thus causing some toxic and side effects on normal tissues or normal cells.Drug resistance caused by long-term chemotherapy is also one of the main reasons for the death of cancer patients.A large number of studies have shown that chemotherapy combined with other treatment methods(such as gene therapy,cyclooxygenase-2(COX-2)inhibition or immunotherapy)has significant synergistic anticancer effects and can effectively overcome cancer multidrug resistance.Nanocarrier systems exhibit unique advantages in tumor-targeted delivery of chemotherapy drugs and combination treatment of chemotherapy with other therapies.In addition,inteligent nanocarriers can controlledly release anticancer drugs by responding the endogenous stimuli of tumor(weak acid condition and redox environment,etc.),which is conducive to enhancing drug efficacy and reducing drug side effects.Poly(?-amino ester)(PBAE)is a kind of biodegradable cationic polymers,which have significant pH-responsability.PBAE being used as a carrier material can realize the controlled release of drugs or genes in tumor cells through the"proton sponge"effect.In this study,we synthesized ssPBAE that possesses pH-and redox-dual responsability through introducing disulfide bond(S-S),and used it as a carrier material to construct two tumor-targeted nanocarrier systems for efficiently co-loading,targeted delivery and controlled release of chemotherapy drug and gene(or COX-2 inhibitor),thus to achieve the reversal of cancer drug resistance and the synergistic antitumor effects.Methods:Section 1:1.First,disulfide-containing poly(?-amino ester)(ssPBAE)was synthesized by Michael addition method.The redox-responsive capability of ssPBAE was investigated by agarose gel electrophoresis.The end-alkene groups of ssPBAE were reacted with diethylenetriamine to form amino-modified ssPBAE(NH-ssPBAE).MTX,a chemotherapy agent,was then conjugated to NH-ssPBAE via amide bond to obtain a polymeric prodrug ssPBAE-MTX.The structure of ssPBAE-MTX were characterized by Fourier transform infrared spectroscopy(FT-IR)and ~1H NMR spectra.The loading content of MTX in ssPBAE-MTX was determined by UV-Vis spectroscopic method.p DNA was complexed with ssPBAE-MTX via electrostatic interaction to form ssPBAE-MTX/p DNA nanocomplexes.A pH-sensitive charge-reversal pullulan derivative,CAPL,was synthesized and then coated on the surfaces of ssPBAE-MTX/p DNA nanocomplexes by electrostatic interaction to obtain CAPL/ssPBAE-MTX/p DNA nanoparticles.CAPL/ssPBAE-MTX/p DNA nanoparticles were morphologically characterized using a transmission electron microscope(TEM),and their sizes,polymer dispersity indexes(PDIs)and Zeta potentials were detected by Zetasizer nano ZS90.pH-and redox-responsive drug release behaviors of CAPL/ssPBAE-MTX/p DNA nanoparticles were evaluated using the dynamic dialysis method.2.After incubation with hepatoma Hep G2 cells,the cellular internalization and the endo/lysosomal escape ability of CAPL/ssPBAE-MTX/TAMRA-DNA nanoparticles were observed using the confocal microscope.Moreover,the cytotoxicity of CAPL/ssPBAE-MTX/p DNA nanoparticles in Hep G2 cells was measured by the CCK-8 assay.Section 2:1.ssPBAE/PLGA/DOX/CXB(PPDC)nanocores were firstly prepared using the emulsion-solvent evaporation method.Hyaluronic acid(HA),a natural CD44-targeting polysaccharide,was then coated on the surface of PPDC nanocores by electrostatic adsorption,thus to obtain HPPDC nanoparticles.The morphology of HPPDC nanoparticles were characterized using TEM,and their sizes,PDI and Zeta potentials were detected by Zetasizer nano ZS90.The loading contents and encapsulation efficiencies of DOX and CXB in PPDC and HPPDC nanoparticles were determined by the high performance liquid chromatography(HPLC)method.The pH-and redox-responsive drug release behaviors of HPPDC nanoparticles were evaluated using the dynamic dialysis method.2.The MDR-reversal activities of HPPDC nanoparticles were investigated in human breast cancer MCF-7 and MCF-7/ADR cell lines,which are sensitive and resistant to DOX respectively.After incubation,the cellular location,intracellular delivery,cytotoxicity and apoptosis-inducing activity of HPPDC nanoparticles in MCF-7 and MCF-7/ADR cells were measured by the confocal microscope,CCK8assay and apoptosis detection kit.We also detected the influence of HPPDC nanoparticles on the m RNA expression of MDR1 and the protein expressions of COX-2 and P-gp in MCF-7/ADR cells,thus to clarify the mechanisms of HPPDC nanoparticles for overcoming cancer multidrug resistance.3.The animal breast cancer model was constructed through inoculating MCF-7/ADR cells into BALB/c nude mice.HPPDC nanoparticles were labeled with a near infrared dye Cy5.5,and their tissue distribution and tumor accumulation in MCF-7/ADR tumor-bearing mice were then investigated using the in vivo fluorescence imaging technique after injection via tail vein.The antitumor effects of HPPDC nanoparticles were also investigated in MCF-7/ADR tumor-bearing mice.These effects included the inhibitory activity against tumor growth and the toxic and side effects on normal organs.At the end of treatment,the mice were sacrificed and their tumor tissues were removed for further detecting the m RNA levels and protein expressions of COX-2 and P-gp protein.Results:Section 1:1.ssPBAE was successfully synthesized by Michael addition.The results of AGE showed that ssPBAE had strong redox responsiveness.ssPBAE was reacted with diethylenetriamine at the end-alkene groups to form NH-ssPBAE.Afterwards,ssPBAE-MTX was synthesized via the formation of amide bond between?-carboxyl group of MTX and amino groups of NH-ssPBAE.The FT-IR and ~1H NMR spectra showed that MTX was successfully conjugated to NH-ssPBAE.In addition,the MTX content in ssPBAE-MTX detected by the UV-Vis spectroscopic was 6.24±0.45%.ssPBAE-MTX was mixed with p DNA to form ssPBAE-MTX/p DNA nanocomplexes via electrostatic interaction.Then CAPL was coated on the surfaces of ssPBAE-MTX/p DNA nanocomplexes via electrical interaction to obtain CAPL/ssPBAE-MTX/p DNA nanoparticles.These nanoparticles displayed a classic“core-shell”structure and the size was about 120 nm with a relatively narrow distribution.Furthermore,CAPL/ssPBAE-MTX/p DNA nanoparticles significantly accelerated the in vitro releases of p DNA and MTX under the acidic and redox conditions mimicking tumor environment.2.In hepatoma Hep G2 cells,CAPL/ssPBAE-MTX/p DNA nanoparticles were effectively uptaken via the asialoglyco protein receptor(ASGPR)-mediated endocytosis,thus realized the intracellular co-delivery of p DNA and MTX.With the endo/lysosomal pH decreasing,these nanoparticles were successfully escaped from the endo/lysosomes into the cytoplasm owing to the charge reversal of CAPL and the“proton-sponge”effect of ssPBAE.In the reductive circumstance of cytoplasm,the rupture of disulfide bond further triggered the degradation of ssPBAE,thus released p DNA and MTX to exert their therapeutic effects.Section 2:1.ssPBAE was mixed with PLGA to prepare PPDC nanocores for co-loading chemotherapeutic agent doxorubicin(DOX)and selective COX-2 inhibitor celecoxib(CXB)using the emulsion-solvent evaporation method.PPDC nanocores had a regularly spherical shape and a compact structure,and their size determined by the dynamic light scattering method was approximately 114 nm with a relatively narrow distribution.Due to the cationic polymer nature of ssPBAE,PPDC nanocores exhibited a highly positively charged surface and their Zeta potential was about+38m V.Then we prepared HPPDC nanoparticles using a simple adsorption method.This nanocarrier system had a classic“core-shell”structure,and the size was approximately 212.6 nm with a relatively narrow distribution.The loading contents of DOX and CXB were 3.92%and 7.98%respectively for PPDC nanocores,and 2.15%and 4.02%respectively for HPPDC nanoparticles.The encapsulation efficiencies of both DOX and CXB were more than 70%.HPPDC nanoparticles were stable in vitro and significantly accelerated the releases of DOX and CXB under the acidic and redox conditions mimicking the endosomal pH and glutathione-rich microenvironment in cancer cells.2.It has been reported that CXB can efficiently overcome cancer multidrug resistance by down-regulating the expression of P-glycoprotein(P-gp),a multidrug resistance(MDR)-associated protein,and inhibited its drug-efflux activity in cancer cells.Our previous report confirmed that the over-expression of P-gp is a main mechanism of drug resistance in huam drug resistant breast cancer MCF-7/ADR cells,and this cell line is also characterized by the high expression of CD44.As HA is a natural ligand for CD44,HPPDC nanoparticles greatly improved the cellular uptake of DOX through the endocytosis mediated by HA/CD44 specific binding.After cell entry,CXB and DOX were effectively released from HPPDC nanoparticles by responding to the low pH of endo/lysosomes and the redox intracellular environment orderly,and thus to exert their COX-2 inhibitory and cytotoxic effects,respectively.In MCF-7/ADR cells,HPPDC nanoparticles showed notably enhanced cell-killing and apoptosis-inducing activities,and also significantly down-regulated the expression of P-gp at both m RNA and protein levels.3.A mouse breast cancer model was successfully constructed through inoculating MCF-7/ADR cells into BALB/c nude mice.After intravenous injection,HPPDC-Cy5.5 nanoparticles showed excellent tumor-targeting ability,which would be helpful to deliver CXB and DOX to the tumor and promote their tumor-accumulations through the EPR and HA/CD44 mediated-active targeting effects.HPPDC nanoparticles significantly inhibited the growth of MCF-7/ADR tumors in the mice,improved the antitumor effects of DOX,and furthermore reduced the protein expressions of COX-2 and P-gp in the tumor tissues.In addition,HPPDC nanoparticles caused no influence on the body weights and the normal organs of MCF-7/ADR tumor-bearing mice.Conclusion:In this study,we synthesized a pH and redox dual-responsive polymer ssPBAE and used it as a carrier material to prepare two kinds of intelligent nanoparticle drug-loaded systems named as CAPL/ssPBAE-MTX/p DNA and HPPDC.CAPL/ssPBAE-MTX/p DNA nanoparticles efficiently promoted the cell entry and controlled release of DOX and gene in hepatoma cells,and also exhibited remarkable chemosensitization effect against hepatoma.HPPDC nanoparticles had a good tumor-targeting performance and a high inhibitory effect on the growth of MCF-7/ADR resistant tumor both in vitro and in vivo.Through down-regulating the expression of COX-2 and P-gp,HPPDC nanoparticles significantly reversed breast cancer drug resistance.Thus it can be seen that nanocarrier systems based on ssPBAE have significant pH and redox dual-responsability,which can be used for efficient co-loading,targeted delivery and controlled release of anti-tumor drugs with different action mechanisms.These nanocarrier systems show many unique advantages and huge application potential for combination cancer treatment.