Stimuli-Responsive Multifunctional Nanocarriers For Tumor Combination Therapy

As medical technology continues to innovate and people become more aware of cancer prevention,the death rate from cancer has decreased,but the younger and generalisation of cancer has been accelerated by factors such as environmental changes,work intensity and psychological stress.At present,the clinical treatment for cancer is based on small molecule anti-cancer drugs,combining with a variety of therapies such as traditional radiotherapy and surgery,as well as the emerging combination of Chinese and Western medicine,gene therapy and immunotherapy,etc.However,it still faces difficulties such as less effective treatment than expected and low drug delivery efficiency.Recently,the application of nano-delivery carriers,especially stimuliresponsive nanocarriers,has received great attention in tumor therapy.Stimuliresponsive nanocarriers can take advantage of unique tumor microenvironments to respond to the release of loaded drugs,increasing the efficacy of drugs while reducing the damage to normal tissues;they can also trigger the release of drugs in a remotely controllable,spatio-temporally selective manner in response to exogenous stimuli(light,heat,sound,etc.).Therefore,the design of novel stimuli-responsive carriers that combine multiple endogenous and exogenous stimuli responses is more innovative and challenging for tumor therapy.Chemotherapy has become one of the most basic cancer treatments,taking advantage of systemic drug delivery.Among these,chemotherapeutic agents,represented by platinum drugs,are the first choice for the suppression of malignant tumors.However,the systemic toxicity and primary and secondary drug resistance of platinum drugs in clinical applications have limited their further development.Compared to chemotherapy,gene therapy is capable of modulating the mutated genome in tumors,eradicating tumorigenesis and development at source,and is expected to become a major treatment in tumor therapy in the future.In addition,it is difficult to address the uncertain performance of tumor multiplicity and heterogeneity with a single therapeutic approach,and combination therapy combining two or more therapeutic modalities is gradually becoming a mainstream research direction.In order to break through the above bottleneck of clinical anti-cancer treatment,the construction of multiple stimuli-responsive nanocarriers applied to the combination of chemotherapy and chemotherapy,chemotherapy and gene therapy,is more valuable for clinical application.Based on a comprehensive investigation of current stimuli-responsive nanocarriers and an overall understanding of traditional and emerging therapies such as chemotherapy and gene therapy,this thesis designs acid-responsive and lightresponsive,reduction-responsive and ultrasound-responsive nanocarriers for cancer therapy to achieve the co-delivery of Chinese and Western chemotherapy drugs,chemotherapy and gene drugs,and sonosensitizers and gene drugs,achieving the dual chemotherapy,chemotherapy and gene therapy and other combined treatments.Firstly,we successfully prepared dual-sensitive dual-prodrug nanoparticles with light-controlled endo/lysosomal escape for synergistic photoactivated chemotherapy.The photoactivated Pt(?)prodrug and demethylcantharidin(DMC),a major component of the Chinese medicine Mylabris,were introduced into the dual-sensitive dual-prodrug nanoparticles(DDNP)in a precise ratio of 1:2 to ensure the best synergistic anti-cancer effect.Compared to single-sensitive single-prodrug nanoparticles(SSNP),DDNP could be activated from inert Pt(?)to toxic Pt(?)in a spatiotemporally controlled manner under irradiation,with the ability to release Pt(?)on-demand in an "on-off" manner.In addition,DDNP produced mild oxidative N3·under photostimulation,which disrupted the lysosomal membrane and accelerated the escape from endo/lysosomes,enhancing the chemotherapeutic effect of DDNP.Meanwhile,DMC utilized acid-sensitive DMC-containing ?-carboxylic amide,in response to its release in intracellular acidic microenvironments.DMC could inhibit the enzymatic activity of protein phosphatase PP2A,block the DNA repair pathway brought about by platinum drugs,enhance the sensitivity to platinum drugs,and achieved the synergistic treatment of photoactivated chemotherapy and herbal chemotherapy.Secondly,due to the short wavelength of exogenous light and poor tissue penetration of the photosensitized Pt(?)prodrug,a reduction-sensitive Pt(?)prodrug that responds to the reductive microenvironment of tumor cells was selected and applied in combination therapy.A reduction-sensitive fluorinated-Pt(?)universal transfection nanoplatform(PtUTP-F)was designed for CT45-targeted CRISPR/dCas9 activation,enabling a synergistic and personalised treatment strategy for ovarian cancer.Induced by reductive microenvironment,PtUTP-F disassembled and released gene and toxic Pt(?).At the same time,PtUTP-F was a versatile gene transfection nanoplatform that could load gene and efficiently transfect into a variety of cell lines such as cancer cells and drug-resistant cancer cells,thanks to the excellent endocytosis,endo/lysosomal escape and gene release capabilities of PtUTP-F under fluorinated modifications and proton sponge effects.In addition,PtUTP-F could deliver the dCas9CT45 plasmids,which increased CT45 expression,thereby inhibiting the protein phosphatase PP4C activity and blocking the DNA repair pathway.Ultimately,PtUTPF/dCas9-CT45 sensitized platinum drugs to the chemotherapeutic effects of A2780 tumors and presented a synergistic and personalized treatment based on the combination of CRISPR/dCas9-based gene therapy and chemotherapy.Finally,in order to break through the limitations of exogenous light in terms of wavelength and penetration and to improve the inadequate and unspecific endogenous reduction response,we explored the ultrasound-sensitive system that has showed the advantages of strong tissue penetration and easier clinical translation in recent years,successfully constructed an ultrasound-sensitive targeted liposome(Us-Ma-Lipo)for a gene delivery system.The mannose-modified Us-Ma-Lipo could better target hepatocellular carcinoma cells,promote endocytosis and provide more efficient siRNA transfection performance.The ROS produced by the sonosensitizer hematoporphyrin monomethyl ether(HMME),triggered remotely by ultrasound,had multiple effects:firstly,it caused oxidation of unsaturated phospholipids leading to liposome cleavage and slow release of HMME and siRNA;secondly,it killed cancer cells;thirdly,it was expected to disrupt endo/lysosomal membranes and promote gene escape,thereby enhancing gene transfection capacity.Further,the loaded siBCL-2 could silence antiapoptotic proteins and serve as a synergistic treatment between gene therapy and sonodynamic therapy.In addition,we expected the pEGFP plasmid,which was simply pretreated with Us-Ma-Lipo-loaded chondroitin sulfate and protamine,to exhibit enhanced DNA transfection performance and be applied in the ultrasound-controlled delivery of CRISPR/Cas systems.