PH-Responsive Nano-Drug Delivery System With Size-Switching And Charge-Reversal Properties For Tumor Therapy

The low delivery efficiency of nanomedicines and limited tumor penetration are still huge challenges in treating breast cancer.First,owing to the high interstitial fluid pressure(IFP)and dense extracellular matrix in the tumor microenvironment(TME),traditional nanomedicines are mainly distributed in the vicinity of tumor blood vessels after extravasation from same,and further from the blood vessel blood vessel towards the deep tumor area,the drug concentration is almost zero,hence,the therapeutic effect is seriously affected.Second,owing to the physiological characteristics of solid tumors,particle size is one of the key factors affecting blood circulation time,tumor accumulation,and tumor penetration of nanoparticles.Therefore,to achieve effective curative effects,there is an urgent need for a drug delivery system that can fully penetrate and easily accumulate in the tumor.Based on this,in this study,cationic liposomes(CLS)and polyamidoamine(PAMAM G5.0)dendrimers were used as carriers,and doxorubicin(DOX)was used as a model drug.The p H-responsive nano-drug delivery system CALS/PDMA@DOX with particle size-switching and charge-reversal hierarchical delivery functions in the tumor microenvironment(TME)was constructed by electrostatic adsorption.The research content of this thesis is mainly divided into the following three parts:Part Ⅰ:Preparation and characterization of CALS/PDMA@DOX(1)The preparation method of nanoformulation is as follows:First,DOX-loaded p H-responsive cationic liposomes(CALS)with uniform particle size and good stability were successfully prepared via“film dispersion method”and“ammonium sulfate gradient method”.Subsequently,DOX was successfully loaded into PAMAM(PAMAM@DOX)via physical stirring.To mask PAMAM@DOX surface positive charge,its surface primary amino group was acylated by 2,3-dimethylmaleic anhydride(DMA)to generate acid-sensitiveβ-carboxylate amide bond,the DOX-loaded PDMA@DOX nanocarriers with p H-responsive negative charge were successfully synthesized.Finally,the nanocomposite CALS/PDMA@DOX with p H-responsive with particle size-switching function was obtained by adsorbing PDMA@DOX on the surface of CALS by electrostatic force.A series of methods such as ~1 H NMR,UV-Vis,TEM,and particle size potential,etc.were used to characterize the nanoformulation.(2)In vitro characterization results showed that the particle size and potentials of the successfully prepared CALS was approximately 122 nm and+14.5 m V,respectively.DMA successfully inverted the surface charge of PAMAM@DOX to negative to obtain PDMA@DOX with particle size and potentials of approximately 15 nm and-15.0 m V,respectively.Transmission electron microscopy(TEM)and other methods proved that PDMA@DOX was successfully adsorbed on the surface of CALS by electrostatic force,and the nanocomposite CALS/PDMA@DOX with particle size and potential of 166nm and+1.8 m V were successfully prepared.CALS/PDMA@DOX showed a drug loading of 9.47%,of which CALS accounted for 3.22%and PDMA@DOX accounted for 6.25%.(3)The results of in vitro drug release experiments showed that the p H-responsive drug release behavior in vitro was good.At p H 5.0,the released amount within 72 h was close to 60%,which could achieve high-efficiency release of DOX.Compared with the release curves of CALS and PDMA@DOX alone,there was no significant difference between the release amount and the sum of the corresponding amounts of the two,indicating that the combination of the two does not affect the drug release.(4)The results of p H sensitivity experiments showed that in vitro simulation of TME for p H sensitivity experiments,the results showed that CALS/PDMA@DOX could be decomposed into two positively-charged nanoformulations of CALS and PAMAM@DOX at p H 6.5,therapy achieving the better stratify anti-tumor effect in the superficial and deep area of tumor tissue,resulting in enhancing tumor treatment effect.(5)Hemolysis experiments showed that the nanoformulation had good biocompatibility when the concentration of the nanoformulation was within the range of 0.025-3 mg/ml,and could be used for intravenous injection.Part Ⅱ:In vitro antitumor activity of CALS/PDMA@DOXThe mouse breast cancer cell 4T1 was used as the modal to investigate the antitumor activity of CALS/PDMA@DOX in vitro.(1)In vitro cellular uptake,apoptosis and proliferation inhibition experiments showed that the p H-responsive particle size-switching nano-drug delivery system CALS/PDMA@DOX could be was quickly taken up by 4T1 cells,and the cellular uptake reached 98.7%at 2 h close to saturation.Under the condition of p H 6.5,CALS/PAMAM@DOX could significantly inhibit the proliferation of 4T1 cells,and at the same time,the apoptosis rate of 4T1cells was as high as 89.70%,which could effectively induce the apoptosis of 4T1 cells and exert excellent anti-tumor effect.(2)The results of lysosomal co-localization experiments showed that CALS/PDMA@DOX could effectively release PAMAM@DOX and CALS at p H 6.5,therapy accelerating drug escape from lysosomes and enhancing the lethality to tumor cells.(3)The results of the 3D multicellular spheroid penetration experiment showed that under the condition of p H 6.5,the small particle size PAMAM@DOX decomposed by CALS/PDMA@DOX could achieve deep penetration of tumor tissue and enhance the lethality to tumor cells in the deep area,therapy improving the tumor treatment effect.Part Ⅲ:In vivo antitumor activity of CALS/PDMA@DOXThe 4T1 tumor-bearing BALB/c mouse was used as the animal model to investigate the antitumor activity of CALS/PDMA@DOX in vivo.(1)In vivo tissue distribution of the nanoformulation showed that the unique nanoparticle size of CALS/PDMA@DOX made it easy to pass through the tumor blood vessels and enter the tumor tissue,which could better accumulate in tumor tissue and prolong the residence time.(2)Tumor vascular affinity experiments showed that CALS decomposed by CALS/PDMA@DOX under TEM conditions could preferentially target tumor angiogenesis endothelial cells,making it more beneficial for tumor treatment.(3)In vivo tumor penetration experiments showed that CALS/PDMA@DOX could penetrate into the deep area of tumor tissue and evenly distribute in the entire tumor tissue,which helped to enhance the lethality to the entire tumor and exerted its anti-tumor effect more fully.(4)A series of pharmacodynamics experiments showed that the nano-drug delivery system,CALS/PDMA@DOX,could significantly reduce the toxicity of DOX,reduced the damage of DOX to important organs,and it showed biological safety.In addition,CALS/PDMA@DOX had a significant inhibitory effect on tumor cells in tumor-bearing mice,the tumor inhibition rate was as high as 82.6%,resulting in an excellent tumor treatment effect.In summary,in this study,the p H-responsive nano-drug delivery system CALS/PDMA@DOX with particle size-switching and charge-reversal was successfully constructed.In the TME,CALS/PDMA@DOX undergoes charge inversion and decomposes into two positively-charged CALS and PAMAM@DOX,which exert anti-tumor effect in layers in the superficial and deep area of tumor tissues.Among them,CALS preferentially targets tumor angiogenesis endothelial cells and kills tumor cells in the superficial area surrounding the tumor blood vessels.Owing to its small size and positive charge,PAMAM@DOX is easy to penetrate deep into the tumor tissue and kill tumor cells on deep area far away from the tumor blood vessels.CALS/PDMA@DOX can improve the drug delivery efficiency and enhance the drug penetration ability in tumor tissue by changing the nanoparticle size,increasing the surface charge,and improving the cellular uptake,thereby enhancing the treatment of breast cancer.In conclusion,CALS/PDMA@DOX is feasible to effectively reduce the toxic and side effects of the drug,enhance the drug delivery efficiency and tumor tissue penetration.In the meantime,the features of high bioavailability and significant tumor inhibition effect provide a feasible strategy for nano-drug delivery system.