Construction Of A Nanoparticle Delivery System For The Treatment Of Ulcerative Colitis Based On The Codonopsis Radix Polysaccharides A And The Underlying Mechanism

Objective:Ulcerative colitis（UC）is an inflammatory disease confined to the mucosal surface and submucosa of the intestine.It was believed that UC was caused by weakness of the spleen and stomach and deficiency of qi and blood.Codonopsis pilosula（Franch）Nannf.is the dried root of Codonopsis pilosula,which has the traditional effects of tonifying the qi,benefiting the spleen and lung.Clinically,it is mainly used for the treatment of spleen and stomach disorders,cough and body heat.Using Codonopsis as the monarch drug in the prescription has been recommended by the expert consensus on TCM diagnosis and treatment of UC.Polysaccharide is one of the main pharmacologically active substances of Lu Codonopsis,and its content is an important quality indicator reflecting the authentic quality.An inulin-type fructan Codonopsis Radix polysaccharides A（CPA）with the highest content was isolated from Lu Codonopsis Radix polysaccharides.This compound has significant effect on gastric ulcer and UC,but its low bioavailability has seriously limited its development and clinical application.Nanocarrier delivery system can effectively improve the bioavailability of CPA and take the advantages of nanotechnology to achieve the purpose of prolonging retention time in vivo.Therefore,in this study,the structure of CPA was hydrophobically modified,nanoparticles were prepared by ultrasonic method to improve the bioavailability of CPA.We also investigate the intervention effects of CNSU NPs on UC at the cellular and animal studies and the undelying mechanisms.The article is beneficial to promote the development of health products based on Codonopsis Radix polysaccharides and to promote the healthy and sustainable development of Lu Codonopsis.Methods:1.Construction and characterization of CNSU NPsThe CPA-NH₂（CN）was completed by using oxidizing and ammoniating agents in turn.Covalently combined with the hydrophobic group succinic anhydride（SA）and ursodeoxycholic acid（UDCA）,we obtained the amphiphilic macromolecular compound CPA-NH-NSA-UDCA（CNSU）.The structure of CNSU was characterized by Fourier infrared spectroscopy（FTIR）and nuclear magnetic resonance hydrogen spectroscopy（¹H-NMR）.Furthermore,self-assembled nanoparticles（CNSU NPs）were prepared under sonication.The particle size and potential of CNSU NPs were investigated by transmission electron microscopy（TEM）and dynamic light particle size scattering（DLS）.We also examined the stability of CNSU NPs in artificial gastric fluid（SGF）and artificial intestinal fluid（SIF）.2.In vitro anti-inflammatory activity study of CNSU NPsLipopolysaccharide（LPS）-induced human colonic epithelial cells（NCM 460）and mouse macrophages（RAW 264.7）were used as inflammation models in vitro.The effects of CNSU NPs on the gene expression levels of inflammatory factors IL-6,IL-1β,TNF-α,MCP-1,IFN-γ,i NOS and classical signaling pathway TLR4/NF-κB in cells were determined by real-time fluorescence quantitative polymerase chain reaction（RT-PCR）.3.Therapeutic effect and mechanism of CNSU NPs on Dextran Sulfate Sodium（DSS）-induced inflammatory response in UC miceSeventy male C57BL/6 healthy mice were randomly divided into seven groups:blank control group（Control）,model group（DSS）,positive control group（SASP）,mixture group（CPA,SA,UDCA）,different dose groups of CNSU NPs（CNSU NPs L,CNSU NPs M,CNSU NPs H）.Except for the control group,the UC mouse model was established by drinking 2.5%（w/v）DSS aqueous solution freshly for 8 days,and the treatment group was given drugs by gavage with the volume of 0.1 m L/10 g administration,while the control and model groups were given equal amounts of distilled water by gavage.The therapeutic effects of CNSU NPs on UC mice were examined by disease activity index（DAI）,colon length,colon histopathology score and other inflammatory indexes.The gene expression of inflammatory factors（TNF-α,IL-1β,IL-6）in colon were measured and the mechanism of CNSU NPs was elucidated at the molecular level.In vivo pharmacokinetics of orally administered drugs in mice for 24 h using CNSU NPs wrapped with fluorescent dye Fluorescein isothiocyanate isomer（FITC）.Results:1.Construction and characterization of CNSU NPsThe structure of CNSU analysis by FTIR and ¹H-NMR indicated that the compound CNSU was successfully synthesized.The nanoparticles were prepared by intermolecular forces with a particle size of 350 nm and a surface charge of-30 m V as determined by TEM and DLS.The stability results showed that the particle size decreased after 6 h in SGF and increased after 6 h in SIF,while the surface charge was relatively stable.2.Anti-inflammatory activity study of CNSU NPs in vitroCNSU NPs were able to inhibit the gene expression of inflammatory factors,such as IL-6,IL-1β,TNF-α,MCP-1,IFN-γ,and i NOS in LPS induced NCM 460 and RAW264.7 cells.It suggest that CNSU NPs have a protective effect on LPS-induced cellular inflammatory response.Furthermore,CNSU NPs can significantly reduce the gene expression of TLR4,PI3K,AKT,NF-κB in cells as mentioned above.Therefore,we guess that the protective effect of CNSU NPs on cellular inflammatory response may be related to the inhibition of TLR4/NF-κB signaling pathway activation and expression of other related genes.3.Therapeutic effect and mechanism of CNSU NPs on DSS-induced UC miceCNSU NPs significantly reduced inflammatory manifestations such as weight loss,colonic shortening,elevation of disease activity index（DAI）and colonic pathological damage in UC mice.Compared to SASP and CNSU Mix,the therapeutic effect of CNSU NPs was more pronounced at the same dose.Notably,compared to normal mice,CNSU NPs were metabolized for significantly longer in UC mice and tended to have a longer resident time at sites of colonic inflammation in vivo.Otherwise,CNSU NPs down-regulated the expression of IL-6,IL-1βand TNF-αgenes and inhibited the expression of TLR4,PI3K,AKT and NF-κB in the colon of UC mice.It was further confirmed that the efficacy of CNSU NPs on UC was closely related to inhibit the activation of TLR4/NF-κB signaling pathway.CNSU NPs with long elimination half-life and mean retention time,low clearance and large area under the drug-time curve.Conclusion:In this study,we designed and synthesized the amphiphilic compound CNSU.It was further formed nanoparticles CNSU NPs by prophilic hydrophobic interactions.The nanoparticles have a spherical structure with a particle size of 350 nm and a surface charge of-30 m V.They are stable in the physiological environment.The results showed that CNSU NPs could effectively protect the cellular inflammation stimulated by LPS.Moreover,CNSU NPs have good anti-colitis effects in vivo,which can prolong the retention time of CPA,prevent colon shortening,reverse DAI index.Significantly,compared with the same dose of CPA and CNSU Mix,the therapeutic effect of CNSU NPs was remarkable.Meanwhile,TLR4/NF-κB signaling pathway was involved in the therapeutic effect.In addition,CNSU NPs had no significant toxic side effects on other tissues during the treatment and high oral bioavailability.