Construction Of Functionalized Curdlan Nanosystem And Applications In Tumor Therapy

Cancer is currently one of the main causes of human death.Although traditional treatments have achieved certain curative effects,the diversity of tumor development and the side effects of traditional therapy still hinder the treatment of cancer.Therefore,it is extremely urgent to develop new drugs and safe and efficient therapy to overcome the defects of traditional treatment.The rapid development of nanotechnology has brought better solutions for tumor precision treatment.Various of nanomaterials have been successfully applied in tumor treatment,and new treated methods have also shown good prospects.In the work described in this thesis,combined with the characteristics of tumor microenvironment,a variety of nano-platforms have been constructed for gene therapy,multi-drug synergistic therapy and immunotherapy,which based on curdlan for the purpose of safe,precise and efficient tumor treatment.The main contents of the research are as follows:(1)RNA interference(RNAi)is a technology to effectively regulate proto-oncogenes for tumor therapy.While naked siRNA enters the body,it is quickly degraded in the plasma and rapidly cleared from the body,and the resulting degradation products may also have immunogenicity.Therefore,the primary limitation of clinical application is how to deliver therapeutic siRNA to target cells safely and effectively.To solve such problems,we constructed a targeted delivery platform(CTL-PEG-FA),which grafted curdlan with lysine tripeptide by click reaction,and then modified with PEG-linked folic acid.The carrier could bind siRNA at low N/P ratio and enter cells through folate-mediated endocytosis and charge interaction,improving the uptake of siRNA by HepG2 cells.After Bcl-2 siRNA entered cells and down-regulated Bcl-2 protein,it caused severe damage to tumor cells through apoptosis mechanism.Click reaction solved the contradiction between functional modification and loading capacity in gene vector design.At the same time,CTL-PEG-FA had good biocompatibility and targeting capacity,which ensured the safety and precision of treatment.It provides a certain research basis for the development of curdlan biomaterials and RNAi therapy.(2)The combined use of multiple drugs is an effective way to balance drug activity and side effects.Meanwhile,the drug delivery systems also enhance the specificity of drugs.Silica nanoparticles have attracted much attention due to their diverse morphologies,large specific surface area,stable structures and excellent biosafety.The surface modification or attachment of different organic/inorganic molecules on silica holds endless prospects for the design and synthesis of multifunctional nanostructures.Therefore,in this part,a curdlan-coated degradable silica hybrid carrier(Ce6-Dox@TS@Erl/Cur-DMMA,abbreviated as CDS@EC)was synthesized to deliver photosensitizer(Ce6),doxorubicin(Dox)and erlotinib(Erl).A p H-responsive amide bond and a singlet oxygen-responsive thione unit were introduced into curdlan and silica respectively,to realize the sequential release of Erl and Dox under dual-stimulated conditions.There were three stages during the treatment process of CDS@EC.First,the negative charge of CDS@EC could ensure stability for the stable circulation;Secondly,the slight acidity in TME could trigger the charge reversal of curdlan to release Erl and activate the caspase8 pathway;Finally,650 nm laser irradiation was introduced to produce ~1O₂ and break the thione for the release of Dox,realizing the synergistic treatment of the two drugs.The strategy can accurately control the release and time interval of drugs by endogenous and exogenous stimulation to maximize the synergistic effect.(3)In tumor immunotherapy,the repolarization of M2-type tumor-associated macrophages(TAMs)to M1-type is significant to recover the anti-tumor immune activity.Considering the special relationship between mitochondria and tumor cells apoptosis,it is a novel strategy to establish the synergistic effect of promoting apoptosis and activating immunity.In this section,a series of p H-responsive curdlan nanoparticles(CP NPs)were synthesized,aiming to induce mitochondrial damage and cell apoptosis consisting with the tumor immune microenvironment remodel.Under acidic conditions,the imine bond in CP NPs was broken and PEG chains were removed.The exposed positive charge made CP NPs to target mitochondria,cause the decrease of mitochondrial membrane potential and the increase of membrane permeability,and finally induce apoptosis of 4T1 cells through mitochondrial damage.CP40 also had the ability to promote repolarization of TAMs to M1 and increased the release of inflammatory factors in TME.Using transwell to simulate TME in vitro,it was found that the killing effect of 4T1 cells,M2 TAMs and CP40 NPs co-culture system was more than 90%.CP40 is simple to synthesize and have dual functions to tumor cell killing and immune cell activation,which verifies the rationality of the integrated design for treatment and prognosis.