Doxorubicin/p53 Gene Co-Delivery System Based On Polyphosphoesters For Lung Cancer Therapy

In recent years,the incidences of cancer in the world remain high.It has become one of the most important diseases threatening human health.At present,chemotherapy is commonly used in clinical treatment.However,there is a severe side effect in the body and it is easy to produce multidrug resistance,which makes the utilization rate of drugs lower and the effect of treatment is not good.In addition,due to the complexity of cancer pathology,a single treatment method is difficult to achieve the desired therapeutic effect.Therefore,combination therapy is used in the treatment of cancer,especially the combination therapy of drugs and genes.The p53 gene is an important tumor suppressor gene in the human body.The p53 protein encoded by the gene plays an important role in the process of regulating cell cycle regulation and inducing cell apoptosis.The mutation and deletion of p53 gene will lead to cell genetic instability and insensitivity to chemotherapy.Clinical studies have shown that the introduction of exogenous p53 gene can inhibit the growth of tumor,promote the apoptosis of tumor cells,enhance the sensitivity of cancer cells to chemotherapeutic drugs(such as DOX,CPT,PTX,etc.),improve the utilization of drugs and improve the therapeutic effect.Based on the combination of DOX and p53 genes,two kinds of nanoparticles co-loaded DOX and p53 genes are designed and synthesized by utilizing biocompatible and biodegradable polymer materials(such as polyphosphate,polyethylene glycol,etc.)and using ring opening polymerization(ROP),"click" chemical reaction and Michael addition polymerization to achieve combined treatment of drugs and genes.The main contents of this thesis include the following two parts:1.Preparation,characterization and properties of DOX and p53 gene co-loaded mixed micelles based on polyphosphoestersThe pH-responsive DOX prodrug and the polycation carrier were prepared via a combination of the ring-opening polymerization(ROP)and "click" chemistry reaction,respectively.First,the block copolymer precursor(abbreviated as mPEG-b-PBYP)containing alkyne on side chain was obtained by ROP between the mPEG and 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane(BYP).Then a pH-sensitive DOX derivative(DOX-hyd-N3)containing azide group was synthesized.Second,the polymeric prodrug(abbreviated as mPEG-b-PBYP-hyd-DOX)was prepared via the CuAAC reaction between the DOX-hyd-N3 and mPEG-b-PBYP.And the polycation carrier(abbreviated as mPEG-b-PBYP-g-DAE)was obtained via the thiol-yne reaction between mPEG-b-PBYP and 2-dimethylaminoethanethiol hydrochloride.The structures of mPEG-b-PBYP-hyd-DOX and mPEG-b-PBYP-g-DAE were characterized by magnetic resonance spectroscopy(NMR),gel permeation chromatography(GPC),ultraviolet-visible spectrophotometer(UV-vis),fourier transform infrared spectroscopy(FT-IR)and high performance liquid chromatography(HPLC)analyses.The two kinds of polymers were self-assembled into mixed micelles in aqueous solution and then combined with the p53 gene by electrostatic action to form nanoparticles that co-loaded the DOX and p53 gene.At the same time,the size and morphology of the mixed micelles were characterized by dynamic light scattering(DLS)and transmission electron microscopy(TEM).And the release of DOX in the nanoparticles was studied by fluorescence spectrophotome;the ability of the mixed micelle condensd gene was tested by zeta potential and gel retardation assay.Furthermore,in vitro cell experiments have proved the biocompatibility of polymer precursors,the effect of nanoparticles on cancer cell inhibition and the process of effective release of DOX and p53 gene after the nanoparticle endocytosis enters the A549 cells.Meanwhile,the gene transfection effect of nanoparticles at different N/P ratios can be observed by confocal laser scanning microscope.The above results prove that the DOX/p53 gene co-loaded nanoparticles have broad application prospects in the treatment of lung cancer.2.Preparation of pH-responsive prodrug used for DOX and p53 gene co-loading via Michael addition polymerization.Michael addition polymerization is a kind of stepwise polymerization,which is now widely used during the polymer synthesis.In this chapter,a pH-sensitive DOX prodrug(DOX-hyd-PEAEP-g-PDMAEMA),which contains a protonated cationic block,was synthesized by the combination of Michael addition polymerization and "click" chemical reaction.The polymeric prodrug could immobilize DNA and form a DOX/p53 gene co-delivery system.First,the Michael addition reaction was used to synthesize the straight chain phosphate polymer(PEAEP)with the alkynyl group in the side chain.And then the copolymers with the azido group(PDMAEMA-N3)and the DOX derivative(DOX-hyd-N3)were synthesized.Finally,two compounds containing azide groups were connected to the main chain of the polyphosphoester by the CuAAC reaction to form the protonated DOX prodrug DOX-hyd-PEAEP-g-PDMAEMA.The structures of the prodrug were characterized by magnetic resonance spectroscopy(NMR),gel permeation chromatography(GPC),ultraviolet-visible spectrophotometer(UV-vis),fourier transform infrared spectroscopy(FT-IR)and high performance liquid chromatography(HPLC)analyses.The polymeric prodrug could self-assemble into micelles.We use dynamic light scattering(DLS)and transmission electron microscopy(TEM)to observe the particle size and morphology.Then we used a fluorescent spectrophotometer to detect the rate of DOX release in vitro.At the same time,we used zeta potential measurement to verify the ability of the prodrug micelles condensd p53 gene.The biocompatibility of PEAEP and the anti-proliferation activity of the DOX/p53 gene co-loaded nanoparticles against tumor cells were investigated by MTT assays and laser scanning confocal microscope.The above results prove that the DOX/p53 gene co-loaded nanoparticles have broad application prospects in the treatment of cancer.