Fluorinated Polyamidoamine-mediated The MiR-23b Delivery For The Treatment Of Periodontitis

Periodontitis is one of the main oral diseases.Its clinical manifestations include bleeding gums,formation of periodontal pockets,resorption of alveolar bone and even loosening and shedding of teeth.The pathogenesis of periodontitis is very complex,mainly due to tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),IL-6 and other inflammatory mediators can bind to their corresponding receptors and activate NF-?B signaling pathway.The receptor activator of nuclear factor-?B on the osteoclast membrane was activated by the receptor activator of nuclear factor-?B(RANK).combined with receptor activator of nuclear factor-?B ligand(RANKL)successfully activated osteoclasts and induced alveolar bone resorption.Clinically commonly used treatment methods include periodontal basic therapy,drug therapy and periodontal surgery,but these methods still have limitations,such as the inability to effectively reduce the expression level of inflammatory factors in the periodontal tissue,drug side effects,etc.Therefore,it is of great significance to design a treatment scheme for periodontitis.In recent years,with the deepening of people's understanding of miRNA,more and more scholars have found that abnormal expression of miRNA can appear in the occurrence and development of many diseases.Therefore,miRNA-based gene therapy has become a kind of biomedical therapy with wide application prospect.MiRNA is a non-coding RNA composed of 18-25 nucleotides in length,which can inhibit mRNA translation or degrade mRNA by targeting target mRNAs with partial complementary sequences in the 3'-UTR region.In diseases caused by the down-regulation or deletion of miRNAs,miRNAs can be delivered to target tissues or target cells to restore the expression level of miRNAs and play the same biological functions as endogenous miRNAs,so as to achieve the purpose of alleviating the occurrence and development of the disease.In rheumatoid arthritis,the expression of miR-23b is decreased,and the delivery of miR-23b targeting TAB2/3 can regulate the downstream NF-?B signaling pathway,and ultimately reduce the expression of inflammatory factors such as IL-17,thus achieving the purpose of treating rheumatoid arthritis.The pathogenesis of periodontitis is similar to that of rheumatoid arthritis,so we speculate that miR-23b may have the same effect in periodontitis.Because miRNA is extremely unstable,has negative charge and is easy to be cleared by the kidney and other shortcomings,it is not easy to enter into cells and play its function.Therefore,miRNA must rely on efficient gene delivery system to realize the efficacy as gene drugs and play its biological function.At present,the commonly used gene delivery vectors are mainly divided into viral vectors and non-viral vectors.Compared with viral vectors,non-viral vectors have the advantages of better biosafety and simpler production.Among them,polyamidoamine(PAMAM)is an ideal carrier,mainly because the surface of PAMAM has a high density positive charge,which can effectively assemble nucleic acid molecules,and at the same time,the tertiary amine structure in PAMAM can quickly escape from the intosome/lysosome through the"proton sponge effect".However,the transfection process still has the disadvantages of high cytotoxicity and low transfection efficiency.In the previous study,the group modified the surface of PAMAM by means of fluorination,which not only reduced the positive charge density on the surface of PAMAM,but also increased the affinity between the carrier and the cell membrane,greatly improving the gene transfection efficiency.In this study,we used the previously constructed fluorinated modified PAMAM as the carrier to achieve efficient and accurate delivery of miR-23b,aiming to build a precise gene therapy technology route for periodontitis.Specific research contents are as follows:1.In the second chapter,we aimed to investigate the changes of miR-23b expression level in periodontitis and its role in bone remodeling in bone defects caused by periodontitis.Gingival tissues were extracted from clinical periodontitis patients and rat periodontitis models.The qPCR detection showed that the level of miR-23b in the gingival tissues of patients with periodontitis and rat periodontitis models was significantly reduced,indicating that miR-23b plays a key role in the process of periodontitis.Using fluorinated modified PAMAM as the vector,miR-23b was mediated to deliver to osteoblasts.ALP activity detection kit,ALP staining,ARS staining,qPCR and Western blotting were used to detect the expression levels of ALP,mineralized nodules and osteogenic related factors(Runx2,ALP,OCN,OPN)in osteoblasts.The results showed that miR-23b could significantly increase the expression levels of osteogenic related factors(Runx2,ALP,OCN,OPN)in osteoblasts,and had a certain promoting effect in both early osteogenesis(ALP staining)and late osteogenesis(ARS staining),indicating that miR-23b could improve bone remodeling.It provides a good technical support for periodontal tissue regeneration.At the same time,the fluorinated modified vector can effectively improve the transfection efficiency and promote the function of small nucleic acids,which has a broad application prospect.2.In the third chapter,we aim to explore the anti-inflammatory effect of miR-23b and the relevant mechanisms under in vitro conditions.After delivering miR-23b to the inflammatory cell model of RAW264.7 cells stimulated by LPS using fluorinated modified PAMAM as the vector.The expressions of TNF-?,IL-6 and IL-1? and the expression levels of p-p65 and p65 were detected by qPCR,ELISA and Western blotting.The results showed that miR-23b could inhibit p65 phosphorylation,regulate NF-?B signaling pathway and effectively inhibit the production of inflammatory cytokines(TNF-?,IL-6 and IL-1?)in vitro by targeting Tab2,Tab3 and IKK-?.3.In the fourth chapter,we aim to explore the anti-inflammatory effect of miR-23b and the relevant mechanisms under in vivo conditions.The rat model of periodontitis was established by the ligation wire around the first maxillary molar of the rats.The fluorinated modified PAMAM was used as the carrier to deliver miR-23b to the periodontal tissues of the rats.Clinical indicators,micro-CT,qPCR and immunohistochemistry were used to detect the absorption degree of alveolar bone and the expression changes of related inflammatory factors TNF-?,IL-6 and IL-1?,and H&E staining was used to evaluate the biosafety of the nanocomposites.The results showed that miR-23b delivery could effectively alleviate the expression of inflammatory cytokines TNF-?,IL-6 and IL-1? in periodontitis,inhibit the infiltration degree of inflammatory cells,and effectively reduce the resorption of alveolar bone caused by periodontitis,thus achieving the effective treatment of periodontitis.In conclusion,this study found abnormal expression of miR-23b in periodontitis gingival tissue,and systematically explored the regulatory mechanism of miR-23b in the occurrence and development of periodontitis,thus providing a theoretical basis for miRNA-based precise gene therapy strategy for periodontitis.At the same time,we used fluorinated modified PAMAM as the carrier to realize the efficient and accurate delivery of miR-23b,and explored the effect and mechanism of miR-23b delivery on osteogenesis and inflammation inhibition.The development of this study provides a new idea for further revealing the mechanism of miR-23b in periodontitis,and also provides important theoretical support and technical guidance for the development of efficient miRNA drug delivery system.