Application Of Silk Fibroin-Based Multifunctional Nanomedicine In The Treatment Of Ulcerative Colitis

Ulcerative colitis(UC)is a chronic and relapsed modern refractory disease of unknown etiology.With the improvement of Chinese people’s living standards,the incidence of UC has increased rapidly in recent years,which has seriously threatened people’s healthy life.However,due to the shortcomings of traditional medicines,such as poor efficacy and large toxic,it is imperative to develop new strategy of UC treatment.The refined and personalized strategy for UC diagnosis and treatment are urgently needed in UC clinical treatment.The ideal UC treatment should be include anti-inflammatory and anti-oxidation,mucosal repair,and domestication of intestinal microorganisms.Therefore,the new strategy was required to be able to deliver the bio-active drug to a reasonable cell in a reasonable way with a reasonable mount at a reasonable time in order to achieve maximum efficacy and minimal toxic side effects.Based on this,with the benefit from multi-system fusion and multi-dimensional design,nanomedicine which relied on mature nanotechnology can provide a strong platform support for achieving the above goal.In recent years,researchers have prepared a variety of Nano-drug carriers using various materials,and have been used in the diagnosis and treatment of various diseases.With the rapid development in the recent ten years,nanomedicine have entered the era of precision nanomedicine.Of course,with comparison of synthetic polymers,biological macromolecules such as natural proteins and polysaccharides have good biocompatibility,biodegradability,and non-immunogenicity.Thus the natural materials have received increasing attention in the field of drug delivery.In addition,the special properties of natural biomaterials are worthy of further research and exploration.Among them,silk fibroin not only is a kind of natural biological material with abundant sources,but has the characteristics of pliable molecular chain,good biocompatibility,biodegradability,special biological activity and easy processing and chemical modification,exhibiting great potential as a drug delivery material.In this study,we successfully prepared a silk fibroin-based multifunctional nanoparticle with targeted property(CS-CUR-NPs).And its therapeutic effect on UC was evaluated through reasonable in vivo and in vitro experiments.The optimized desolvation methods was used to prepare various silk fibroin-based nanoparticles(SFNPs),and then the surface of SFNPs was modified with targeting reagent chondroitin sulfate(CS).By in vitro characterization of control release,we discovered for the first time that SFNPs with secondary structure exhibited excellent multiple stimulus response properties.Meanwhile,through a series of cell and animal model experiments,it was verified that the multifunctional CS-CUR-NPs has excellent anti-inflammatory properties and biosafety properties.In detail,after oral or intravenous administration,CS-CUR-NPs could aggregate to the site of inflammatory lesions with programmed fashion,and then were specifically engulfed by target cells to achieve anti-inflammatory,anti-oxidation and mucosal repair.In the other hand,SFNPs can remodel and the intestinal microorganisms well and thus achieving complete reversal of inflammatory stress and alleviating UC symptoms.The main research contents and conclusions of this paper are divided into the following five parts:Part 1: Preparation and characterization of multifunctional targeted SFNPs.Regenerated silk fibroin(RSF)was obtained from silk cocoon through the steps of degumming,dissolving,dialysis,and lyophilization.The drug-loaded SFNPs was prepared by an improved desolvation method,and the classic carboxyl-amino reaction was used to modify CS to the surface of SFNPs.The particle size and Zeta potential of CS-CUR-NPs were characterized by dynamic light scattering(DLS)。 The morphology of SFNPs was observed by FESEM.The secondary structure was analyzed by CD,FTIR and XRD.The dynamic of the controlled release of CS-CUR-NPs was characterized by classical dialysis methods.Results: Using optimized method,we have successfully fabricated CS-CUR-NPs with a uniform diameter distribution(174.5 nm,PDI 0.101)and a negative surface charge(-35.5 mV).And it has good stability in the gastrointestinal simulant.Meanwhile,SFNPs have a compact and stable structure to encapsulate CUR,which attributed to the transformation of β-sheet domain conformation.More importantly,through drug controlled release experiments,we found for the first time that SFNPs with β-sheet microcrystalline structure exhibit excellent multiple stimuli(pH,ROS and GSH)bio-responsiveness.Conclusion: The nanoparticle possessing β-sheet microcrystalline exhibited excellent physical and chemical properties,as well as it can be easy to surface functional modification,while showing excellent multiple stimulus response performance.Part 2: In vitro cell experiment of CS-CUR-NPs.Firstly,MTT was used to detect the toxicity and biocompatibility of various SFNPs to colitis-related cell lines,including colon epithelial cells CT-26 and macrophages Raw 264.7.Laser confocal microscopy(CLSM)and flow cytometry(FCM)were used to qualitatively and quantitatively detect the phagocytosis of various SFNPs by cells.ELISA method was used to detect the secretion of inflammatory factors of macrophages after various NPs treatments.Results: It was excellent biocompatibility of SFNPs that was found by MTT study.There were no obvious cytotoxicity appeared,even when SFNPs were incubated with cells for 48 h.Besides we found that CS surface-modification could effectively promote the phagocytosis of macrophages and thus it showed excellent ability of cell targeting.Importantly,when the macrophages were incubated with CS-CUR-NPs,the secretion of pro-inflammatory cytokines was efficiently inhibited,while the secretion of anti-inflammatory cytokines was opposite.Meanwhile time and dose-dependent characteristics was also detected in this effort.Conclusion: All of those indicated that CS-CUR-NPs can play an effective role for a long-term therapeutic to fight inflammatory diseases.Part 3: In vivo animal model test of CS-CUR-NPs.Dextran sulfate sodium(DSS)was used to induce the UC model of mice.In vivo or ex vivo imaging technology and immunofluorescence test were applied to verify the programmed aggregation of CS-CUR-NPs to the colitis lesions.When the mice with UC were orally or intravenously administrated with various SFNPs,the therapeutic effects of all kinds of SNPs and different administration methods were evaluated.Results: No matter what methods of administration,CS-CUR-NPs could effectively aggregate to colonic lesions and be specifically phagocytized by macrophages in colonic inflammation sites,suggesting the targeting with programmed fashion.The animal model experiment exhibited that the treatment results in the intravenous administration group is better than that in the oral administration group.Besides,the CS targeted modification of NPs could effectively improve the therapeutic outcomes.Conclusion: All of the in vivo study suggested CS-CUR-NPs possessed the capacity of alleviating UC with programmed fashion.Part 4: Evaluation of intestinal microorganism remodeling Domestication by SFNPs.The feces of mice in each experimental group were collected,and then the total DNA of the flora was extracted.The next-generation sequencing technology was used to sequence 16 S r DNA and followed with OTU clustering.Then the intestinal microbial remodeling was analyzed through α diversity analysis,β diversity analysis and other methods.Results: There were significant differences in microbial abundance,diversity,and flora composition between the healthy group and the UC control group.After the intervention of various SFNPs,a unique flora structure was formed,whic is different from the healthy group and the UC control group.Conclusion: in a word,the better the treatment effect of SFNPs were,the closer the abundance,diversity and flora structure of microorganism were to the healthy group.Correspondingly,the intestinal flora of mice treated with CS-CUR-NPs by intravenous injection was closest to that of the healthy group.Part 5: Evaluation of biosafety of SFNPs.Hemolytic performance of CS-CUR-NPs was evaluated by an in vitro blood compatibility test.The biosafety of SFNPs was evaluated by testing blood routine,liver and kidney toxicity,and tissue staining of major organs.Results: the hemolytic properties of CS-CUR-NPs were not observed,even if the concentration of NPs reaches 10 times higher than that in vivo treatment.In addition,no matter what kind of administration of SFNPs,the detected parameters of mice in each experimental group were normal,and there was no significant difference with the healthy group.Conclusion:All of the study persuasively suggested that SFNPs possessed the excellent biocompatibility and biosafety.In summary,in this study we successfully fabricated CS-CUR-NPs which showed excellent physicochemical properties and multi-bioresponsiveness.All of the experiments exhaustively revealed the UC alleviation of CS-CUR-NPs with programmed style as well as the excellent biosafety of SFNPs.This provides a natural stimulus-responsive drug delivery system with great application potential for the treatment of UC diseases.