| Nanocarriers carrying chemotherapeutic drugs for anti-tumor therapy are currently a research focus of cancer therapy.The nanocarrier-based drug delivery system has many advantages for cancer therapy,which can not only enhance the solubility for the poor water-soluble chemotherapeutic drugs,but also achieve targeting drug delivery and intracellular controlled drug release through grafting some targeting groups and responsive chemical bonds in the carriers.Conventional nanocarriers only have the ability to respond to a single kind of stimuli or the ability to targeting a single kind of targets,whereas the real in vivo microenvironment is relatively complex,and a single stimulus-response may not allow the nanocarriers to respond quickly to intracellular or external stimuli after entering the tumor cells,which make the rapid release of drugs unable to achieve.Meanwhile,with the development of anti-tumor research,research on carrier active targeting abilities are shift gradually from the cell level to the organelle level.The aim of this thesis is to construct polymer nanocarriers with multiple targeting and multi-stimulus-responsive abilities,and characterize and evaluate their relative properties,in vitro drug release,and in vitro and in vivo antitumor effects.In Chapter 2,we have introduced disulfide bond into copolymer chain through ring opening polymerization of caprolactone with single protection cystamine as initiator,avoiding the low grafting rate via polymer chain grafting method.Then the PEG-SS-PCL with targeting groups was successfully synthesized by the condensation reaction,and the antitumor drug DOX was linked to the end of the hydrophobic segment via hydrazone bond which is unstable bond in acidic condition.The in vitro drug release study showed that under the condition of double stimulation(pH and GSH),a rapid release of drugs have occurred,resulting from the changes of micellar structure.Results of confocal laser scanning microscopy(CLSM)showed that the dual responsive active targeting micelles can be internalized by tumor cells easier,and after entering into the cells,drugs can be rapidly released from micelles under the intracellular acidic and redox stimulation.The blank micelles are almost non-toxic for both normal and tumor cells,while,drug loaded the dual responsive active targeting micelles from PBA-PEG-SS-PCL-hyd-DOX has the best anti-tumor effect.Finally,the in vivo antitumor effect of drug-loaded micelles further carried out with H22 tumor-bearing mice.The results indicated that PBA-PEG-SS-PCL-hyd-DOX micelles had a better targeting effect to tumor tissues,achieving the best antitumor effect.The results of histological analyses(H&E staining and TUNEL staining)also showed that the blank micelles were non-toxicity on tumor cells,while PBA-PEG-SS-PCL-hyd-DOX micelles had the best antitumor effect.In Chapter 3,we designed a step-by-step multiple stimuli-responsive nano-micelles combining chemo-photodynamic for enhancing cancer therapy.The results of DLS show that the size of micelles from the prepared copolymers would change in the acidic environment and reducing environment.HPLC results further confirmed that in the acidic environment,the prodrug of small molecules PBA-SS-CPT was firstly break from the copolymer,and then after GSH stimulation,and prodrug would further convert into CPT.In the experiments of cell phagocytosis,the results of flow cytometry showed that protoporphyrin could enhance the endocytosis of micelles through the photochemical internalization.Cytotoxicity study of the drug loaded micelles showed that the Dex-g-PpIX-g-PBA-SS-CPT(DPPSC)micelles would have an enhanced cytotoxicity under the light irradiation due to the existing of photosensitizer PpIX.At the same time,since the micelles would gradually respond to the extracellular and intracellular microenvironment,leading to efficient drug release,which further resulted in increased cell toxicity.It can be seen that the short time light irradiation can promote the whole micelle to escape from the lysosome.Meanwhile,due to the acidic environment of the lysosome,PBA-SS-CPT can be cut off from the micelle and enter the cytoplasm.The CLSM images of the cells cultured with different micelles were analyzed by using a variety of colocation analysis methods,which qualitatively and quantitatively reflected the step-by-step multiple stimuli-responsive drug release of the micelles.The results showed that DPPSC micelles were gradually released in the cell.In addition,the transformation of PBA-SS-CPT to CPT were further verified through smashing cells cultured with different micelles or drugs and determining the mixture by High Performance Liquid Chromatography(HPLC).After the treatment of nude mice bearing PANC-1 tumor with micelles and free drugs,the mice in the DPPSC micelle group had the slowest tumor growth,and the body weight changed little,and they also presented the longest survival time in two different phototherapy strategies.From the results of ex vivo histological analyses(H&E staining and TUNEL staining)of tumor sections at the end of the treatment period,it can be found that the blank materials were safety and tumors of DPPSC micelles groups in two different irradiation strategies have the most obvious apoptosis phenomenon,which indicated the antitumor efficacy of DPPSC micelles in the level of histology.In the last chapter,the nanoparticles with double targeting abilities(magnetic targeting and mitochondria targeting)were synthesized by using Fe3O4 as magnetic materials and TPP as the active targeting functional group,combining chemotherapy(DOX)and photothermal therapy(polydopamine,PDA).By means of near infrared imaging,the temperature of the dopamine coated particles was detected as increasing 18 ? rapidly after 6 minutes.The drug release experiment showed that the release of DOX under acid condition were faster than under neutral conditions,and the NIR irradiation-induced the rapid release of DOX.In cell compatibility experiments,the black material showed no cytotoxicity to the cells,but when under the NIR irradiation,the cytotoxicity to the cells increased.Because of the breakdown of disulfide bonds,TPP is exposed which induced the nanoparticles targeted the tumor cell mitochondria.Furthermore the heat sensitive tumor cell mitochondria were destroyed which accelerated the apoptosis of the tumor cells.Therefore,the cytotoxicity of Fe3O4@PDA-TPP/-SS-mPEG and Fe3O4@PDA-TPP/-mPEG showed significant difference.Meanwhile,from the live/dead cell staining images,it can be observed that only cells in the light irradiation region were killed.CLSM images of the cells cultured with nanoparticles showed that only the disulfide bond-linked PEG can be cleaved from the nanoparticles,thereby exposing the TPP targeting groups and targeting to mitochondria.These results were furtherly verified by the fluorescence colocalization analysis results.In animal experiment,group with double targeting,combining chemotherapy and photothermal therapy,in the light irradiation,showed the slowest tumor growth,almost unchanged mice body weight and the highest survival rate of mice,indicating better inhibition of tumor growth and high safety. |
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