| Gene therapy is designed to introduce the foreign genes into targeted tissues or cells to correct or compensate abnormal genes by gene transfer technology,thus restoring their normal biological functions and achieving disease treatment.In the selction of an appropriate therapeutic gene,microRNAs(miRNAs)with RNA interference(RNAi)play an important role in the occurrence and development of critical diseases due to their precise recognition of intracellular targets,thus making them key targets for various disease gene therapies.MiRNAs are a class of non-coding single-strand small RNA with 21-23 nucleotides,which can post-transcriptionally target the mRNA,and subsequently lead to the degradation mRNA or inhibition its translation process.For diseases caused by down-regulation or deletion of miRNA,the delivery of miRNA mimics can efficiently restore the miRNA level in targeted tissues or cells,realizing the biological function as endogenous miRNAs to prevent the development of diseases.However,the small nucleic acid is vulnerable and easily degraded by nucleases in the blood;Meanwhile,it is not easily taken up by the cells due to its negative charge.Therefore,an efficient gene delivery system is highly required for miRNA to exert their biological function and achieve their therapeutic effects.The gene delivery vectors include viral gene delivery vectors and non-viral gene delivery vectors.However,the viral gene delivery system has been hindered by its high immunogenicity in the clinic.Compared with viral ones,non-viral gene delivery vectors have been widely concerned due to their lower cytotoxicity,immunogenicity and ease of production.Among them,dendrimer polyamidoamine(PAMAM)have been widely studied due to their unique properties.Firstly,the amine groups on the surface of PAMAM provides a high density of positive charge,which can efficiently bind and condense the nucleic acids into nanoparticles.Secondly,the nanoparticles can protect nucleic acid molecules from degradation by nucleases in blood.Thirdly,the dendrimer PAMAM structure contains a large number of tertiary amines,which can accelerate the endosomal escape of the nanoparticles through the "proton sponge" effect.Further,the surface of the dendrimer is suitable for chemical modifications to achieve multifunctionalities.For example,amine groups on the surface of PAMAM can be modified by a targeted ligand,accelerating the endocytosis by the targeted tissues or cells.Therefore,the special physicochemical properties of PAMAM make them superior to other linear polymer materials and have greater potential in the application of nucleic acid drug delivery systems.Nevertheless,the transfection efficiency of PAMAM is still not satisfactory,and its high cytotoxicity limits its application in gene delivery.Therefore,construction of efficient,low toxicitic PAMAM derivatives by nucleobase or fluorinated modification of the amine groups on PAMAM is of great significance for the precise delivery of miRNA.The researches and results are as follows: 1.Construction of nucleobase-modified dendrimer-mediated miR-23 b delivery for lung cancerCancer is a public health concern worldwide,and lung cancer is one of the most common malignancies.Up to now,surgery is still the preferred approach for treating lung cancer,but high recurrence caused by tumor metastasis will lead to treatment failure.miRNA-based gene therapy can inhibit tumor proliferation and migration on the genetic level,providing a new option for cancer treatment.In this part,nucleobase-modified dendrimer AP-PAMAM was successfully constructed by modification of PAMAM with 2-amino-6-chloropurine.The modified AP-PAMAM can tailor the binding and release profile of nucleic acids through hydrogen bond interaction,leading to an improvement of the transfection efficiency.In addition,since the amine groups of PAMAM are shielded,the positive charge of AP-PAMAM decreases,resulting in a reduction of cytotoxicity.Then,the delivery of miR-23 b by AP-PAMAM in lung cancer cell A549 was successfully achieved,and the proliferation of tumor cells was efficiently inhibited by the induction of cell apoptosis and cycle arrest.After the transfection,miR-23 b activated both the mitochondria-dependent apoptotic pathway and the death receptor-dependent apoptotic pathway in tumor cells,triggerring the cell apoptosis.Moreover,intracellular delivery of miR-23 b also reduced the expression of the oncogenic proteins Bcl-2 and Survivin and increased the expression of the tumor suppressor protein PTEN.Besides apoptosis,miR-23 b also down-regulated the expression of Cyclin D1,leading to a cell cycle arrest at S phase.In addition,the expression of protein MMP-9,which is closely related to tumor metastasis,is significantly inhibited after miR-23 b delivery,thus achieving an ability to inhibit tumor migration and invasion.In conclusion,nucleobase-modified PAMAM carrier can efficiently mediate miR-23 b delivery,which is expected to be a new approach for cancer gene therapy.2.Construction of fluorinated dendrimer-mediated miR-23 b delivery for rheumatoid arthritisIn rheumatoid arthritis(RA)pathophysiology,excessive release of inflammatory cytokines and insufficient apoptosis in inflammatory tissues can exacerbate disease progression.Therefore,designing miRNA delivery systems for apoptosis and inflammatory responses is highly required to alleviate and treat this disease.In this chapter,we constructed a fluorinated PAMAM derivate(FP)for efficient delivery of miR-23 b by a fluorination method.FP/miR-23 b can trigger the mitochondrial apoptotic pathway to induce apoptosis,using macrophages RAW264.7 as a model.Meanwhile,miR-23 b also targeted IKK-?,TAB2 and TAB3 to inhibit the NF-?B signaling pathway,leading to inhibition of the pro-inflammatory cytokine expressions such as TNF-?,IL-1? and IL-6.These results demonstrated that the miR-23 b delivery could induce synergistic therapeutic effect between anti-inflammation and anti-proliferation to achieve the RA treatment.Subsequently,an adjuvant-induced arthritis rat model was constructed to evaluate the therapeutic effect of the FP/miR-23 b nanoparticles.After treatment,FP/miR-23 b can passively target inflamed joints through the ELVIS effect and increase the expression of miR-23 b in the arthritic joints.In the joints of treated arthritis rats,the infiltration of synovial tissue was significantly inhibited,and the expression of proinflammatory cytokines in the affected joints and serum were decreased or recovered to normal levels.Thus,these results revealed that FP/miR-23 b nanoparticles achieved excellent therapeutic efficacy through amelioration of inflammation,inhibition of bone tissue erosion,and improvement of the athletic ability.Finally,there is no significant renal and hepatic toxicity after administration of FP/miR-23 b nanoparticles into normal rats,indicating the good biocompatibility of the nanoparticles.The above results indicated that fluorinated PAMAM-mediated small nucleic acid delivery can achieve rheumatoid arthritis treatment by the synergistic effect between apoptosis induction and inflammation inhibition,providing a new enlightenment for the treatment of RA and other autoimmune diseases.In summary,we have constructed a class of highly-efficient and low-toxic small nucleic acid delivery systems,which can efficiently delivery small nucleic acids into different disease models to realize their therapeutic efficacy by targeting intracellular targets.Meanwhile,our studies provided a new insight for the design and evaluation of nanomedicine for the critical diseases. |
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