Construction Of Porous Organic Framework Nucleic Acid Carriers For Tumor Diagnosis And Treatment

With the development of nanotechnology,the combination of nanotechnology and anti-tumor therapy to develop more effective anti-tumor treatment methods has shown great application prospects.Among them,nucleic acid delivery technology based on nanocarriers occupies an important position in the fields of tumor treatment and tumor diagnosis.Although a variety of nucleic acid delivery vectors have been developed,there are still many factors that plague their further clinical application.A common problem is that it is difficult to control the particle size and morphology of the nanocomposite formed by the carriers and nucleic acid,which will seriously affect the delivery efficiency of the nucleic acid carrier and the process stability of the largescale production of nucleic acid drug preparations.Under normal circumstances,cationic polymer carriers compress nucleic acid through electrostatic action to form nanocomposite particles.This "bottom-up" electrostatic assembly process is poorly controllable,and it is difficult to form uniform and controllable nanocomposite particles.In this thesis,we proposed a "top-down" nucleic acid vector construction strategy.Firstly,positively charged porous organic framework nanoparticles with uniform and stable size were prepared,and then the negatively charged nucleic acid material was compounded to realize the nucleic acid loading and delivery.We further explored the application of the constructed series of nucleic acid vectors in tumor treatment and tumor diagnosis.First,we conducted a preliminary exploration of the "top-down" nucleic acid vector construction strategy.A cationic flexible organic framework nanoparticle PEIPor NPs was prepared by chemical cross-linking method using cationic polymer hyperbranched polyethyleneimine with a molecular weight of 25K(PEI25K)and photosensitizer tetraaldehyde phenylporphyrin.The nano particles were simple to prepare,and had good dispersibility and uniform size.The PEI-Por NPs prepared by us retain the fluorescence and photodynamic properties of the porphyrin monomer,and could be used for fluorescence imaging and photodynamic antitumor therapy.In addition,the positively charged PEI-Por NPs had a strong nucleic acid carrying capacity and a good gene transfection effect.Studies had found that photodynamic therapy could cause the immunogenic death of tumor cells,thereby improving the immunosuppressive microenvironment of the tumor site and enhancing the ability of immune cells to kill tumor cells.Silencing the programmed cell death-ligand 1(PDL1)on the surface of tumor cells could further enhance the killing ability of T cells on tumor cells.Based on this,we used PEI-Por NPs to carry shPD-L1 plasmid DNA and applied it to the anti-tumor treatment of solid tumors.Animal experiment results showed that after loading shPD-Ll plasmid DNA,PEI-Por NPs could achieve efficient photodynamic/gene immune anti-tumor combined therapy under the guidance of photoacoustic imaging and fluorescence imaging.In order to further improve the biodegradability and biosafety of the constructed nucleic acid vector,we used mesitylene,N-Boc-p-phenylenediamine,PEI1.8K and cystamine hydrochloride as raw materials to prepare the covalent organic framework nanoparticles(CLZU NPs(LZU-PEI1.8K,LZU-Cys NPs))by a one-pot method.Furthermore,the preparation process of CLZU NPs was explored in detail,and the controllable adjustment of the size of nanoparticles was realized.The CLZU NPs prepared by us had an inherent porous structure,and the surface was positively charged,and could carry nucleic acid substances through electrostatic,?-? interaction,and hydrophilic and hydrophobic interactions.Among them,LZU-Cys NPs could be slowly degraded under the action of reducing glutathione due to the disulfide bond in the backbone,and had good biodegradability.Cell experiment results showed that CLZU NPs had good biocompatibility and good gene transfection efficiency.Furthermore,we used this vector to carry out anti-tumor gene therapy in vivo.The results of animal experiments showed that the prepared LZU-Cys/DNA complex could effectively inhibit tumor growth after loading shVEGF plasmid DNA.Finally,we used the biodegradable nucleic acid carrier LZU-Cys NPs to construct a nano-nucleic acid diagnosis and treatment platform that integrates gene silencing and gene detection.In addition to gene therapy,nucleic acid materials can also be tested for genes in cells after modification.Cell experiment results showed that the prepared LZU-Cys NPs had a good gene silencing effect,and could effectively distinguish normal cells from tumor cells after carrying nucleic acid probes.The results of gene detection experiments after gene silencing showed that after using our nanocarriers for gene silencing in the same cell,the efficiency of gene silencing could be further observed by the method of gene detection.This integrated nucleic acid delivery platform provides a new idea for the evaluation of gene transfection effects and tumor diagnosis.This paper proposed a "top-down" nucleic acid vector construction strategy,and constructed a series of nucleic acid delivery systems based on porous organic framework nanoparticles.The results showed that the "top-down" nucleic acid delivery strategy was an effective strategy to realize the controllable preparation of nucleic acid nanocarriers.The porous organic framework nucleic acid carrier constructed by this strategy had the advantages of low cytotoxicity and high nucleic acid delivery efficiency.