| BackgroundSymptomatic biliary strictures can lead to a series of adverse outcomes, such as chronic cholestasis, jaundice,recurrent cholangitis and secondary biliary cirrhosis, and these complications can be serious, irreversible, andlife threatening. Biliary strictures can be caused by a variety of factors, including post-operation biliary fistula,primary sclerosing cholangitis, pancreatitis and biliary complications after liver transplantation, as well asmalignant biliary obstructions. In total,10-30% of patients with advanced chronic pancreatitis experience asymptomatic biliary stricture,4-9% of patients following orthotopic liver transplantation (OLT) develop anastomoticstrictures, and 0.3-0.7%of patients after laparoscopic cholecystectomy may suffer from a major bile ductinjury, leading to post-operative strictures6. Thus, it is essential to provide prompt, effective and durable treatmentof strictures.Fully covered self-expanding metal stents (FCSEMSs) have been recognized as an optimal therapy for benignbiliary strictures and palliative management of malignant biliary obstructions. However, the long-termunobstructed rate of FCSEMSs in clinical usage remains limited. In benign biliary strictures, the average unobstructedperiod is approximately 6-9 months, whereas the average period for malignant biliary obstructions is4-8 months. The formation of bacterial biofilm is the main cause for the re-stricturing of biliary stents in clinicalapplications, and bacterial infection is the leading factor for bacterial biofilm formation. Therefore, development of a novel biliary stent with anti-microbial functionality is urgently needed for clinical applications.Recent applications of nanotechnology in translational medicine require materials and devices designedto interact with the body on subcellular (i.e., molecular) scales with a high degree of specificity that may bepotentially translated into targeted cellular and tissue-specific clinical applications to achieve maximal therapeuticefficacy with minimal side effects. Because of a high surface-to-volume ratio, nanoparticles have becomea well-recognized, effective antimicrobial substance.Objective:In this study, we developed a novel biliary stent coated with silver nanoparticles (AgNPs) and investigated its function in vitro and in vivo.Materials and MethodsA total of 0.0849 g silver nitrate was gently added to a50 ml chitosan solution (pH =3.8), and then both were well mixed as a 10 mmol/1 Ag+chitosan solution. Sixtymilligrams of sodium borohydride was dissolved in 20 ml of distilled deionized water (ddH2O), and then the5 ml mixture was added to the above Ag+chitosan solution at a speed of 0.01 ml per second and 30 min stirring.The above solution was treated with a microfiltration membrane to discard larger particles and prevent particle aggregation. The obtained chitosan nanosilver solution was stored in a 4℃ refrigerator; the quality of the AgNPswas detected with ultraviolet detection at different time points, as previously described.To coat a multilayer film onboth the external and inner layers of the polyester used in a plastic biliary stent. the polyester was treated withsmall-molecule hexamethylenediamineto obtain positive electricity. Multilayer film coats can be formedaccording to two methods:dip assembly and spin assembly. The dip-assembly method was performed with thefollowing steps. The polyester with positive electricity was dipped into a 1 mg/ml heparin solution for 15 min andwas washed three times in ddH2O. Then, the polyester was dipped into a 5 mg/ml chitosan nanosilver solutionfor 15 min and washed three times in ddH2O. The above process was cycled 18 times, and 18 layers of film coatswere obtained. The spin-assembly method was performed with the following steps. One milligram per milliliterof heparin solution (pH= 3.8) with a negative charge was dropped into the polyester with positive electricity andspin coated once. Then, the ddH2O was dropped into the polyester and spin coated once; this step was repeated.Finally,5 mg/ml of chitosan nanosilver solution (pH= 3.8) with a positive charge was dropped into the polyesterand spin coated once; the spin was at 4000 revolutions per second for 30 seconds. The above process was cycled 18 times, and 18 layers of film coats were obtained.All multilayerfilm coat samples were vacuum dried overnight. The contact angles were measured with the circle fitting methodon a JY-82-type contact-angle metre to track the assembly process of the multilayer film coats. A total of 18 layersof film coats were created with the spin-assembly method; the odd-numbered layers were dropped with heparin,and the even number layers were dropped with Ag-chitosan. The ultraviolet-visible spectrum was obtained on theultraviolet and visible spectrophotometer to characterize the AgNPs and track the assemblyprocess. The Ag-loaded multilayer film coats were dipped into hydrofluoric acid for 3-5 seconds and thendropped onto the copper wire mesh. The surface topography of the multilayer film coats was observed with anatomic force microscope and scanning electron microscopy.One millilitre of human plasma (preheated at 37℃ to discardCa2+) was dropped onto the surface of the polyester, and then 1 ml of 0.025 mol/1 CaC12 solution was added. Simultaneously, a small stainless steel hook was stretched into the solution and mixed gently. The formation offibrin was evaluated, and the time that the filament was emerged was recorded. The time is the re-calcificationclotting time. Each sample was measured six times. In addition, Ag-loaded heparin/chitosan multilayer film coatswere stored in a 4℃ refrigerator for 14 days and then used for measurement of the re-calcification clotting time.A Ag+release experiment was conducted. The AgNP biliary stent was placed into 30 ml of PBS, and the new PBS solutionwas changed every 24 hours. At different time points, Ag+ concentrations in the PBS solution were measuredand recorded using inductively coupled plasma mass spectrometry (ICP-MS). Each experiment was repeated atleast three times.Anti-bacteria experiment of Ag heparin/chitosan multilayer film coats in vitro. The sixty-four pigs were randomly divided into two groups:the AgNP group (n=32)and the control (n=32) group. In the AgNP group, the novel AgNP biliary stents were inserted into the commonbile ducts in all of the pigs. Teflon biliary plastic stents served as the controlgroup. For each group, eight pigs were randomly selected to observe the percentage of biliary obstruction andsurvival function, and B ultrasound was used to monitor the position and obstruction of the biliary stent onceeach month. The other 24 pigs were randomly divided into four time points for sample collection and bacterialinfection detection (n=6 at each time point).For 24 pigs in each group, blood samples were collected serially via an ear vein to monitorblood, liver function and cytokine alterations 4,12,24 and 48 weeks after biliary stent implantation. At eachtime point for each group, six pigs were managed with general anaesthesia. The common bile duct with a biliarystent was gently dissociated, and the biliary stent was obtained. Additionally, the bile was collectedto conduct bacterial culture and identification. Anastomosis of the bile duct was performed to performhaematoxylin and eosin (H&E) staining and Masson’s trichrome staining.Statistical analysisThe data are presented as the mean ± standard error (SEM). For parametric data, statisticalsignificance between groups was compared using Students’T-Test. The survival distribution function was evaluated with the Kaplan-Meier survival curve. Statistical analyses were performed with the software packageSPSS for Windows (version 19.0; SPSS, Inc., Chicago, IL, USA). A p-value of less than 0.05 was considered statisticallysignificant.ResultsThe quality ofAgNPs was characterized using ultraviolet detection at different time points. The peak position of the wavelengthof AgNPs was stable at 396 nm during the initial 8 days, which suggested that the obtained Ag-loadedchitosan complex was stable without AgNPagglomeration.The contact angles were measured to track the assembly process of multilayer film coats. The result indicatedthat heparin and chitosan almost presented a strict alternate layer-upon-layer arrangement. The ultraviolet-visible spectrum was detected to characterize AgNPs and track the assembly process. Becauseof the effective ultraviolet absorption of AgNPs at 433 nm, all multilayer film coats at different bilayers presented a maximum absorption at the 433 nm wavelength. The surface topography of the AgNPs biliary stent wasobserved using AFM and SEM.We used the tapping mode of AFM to observe the surface topography of multilayerfilm coats from the dip-assembly and the spin-assembly methods.Different assemblymethods exerted a significant influence on the roughness of the multilayer film. The surface of the multilayer filmfrom the spin-assembly method presented a low roughness (root mean square (RMS)= 14.73 nm) versus thedip-assembly method (RMS = 22.37 nm). Because of less crossover between layers during the spin-assembly process, a flatter-surfaced multilayer film was obtained with the spin-assembly method.Furthermore, we observed the distributionof AgNPs in multilayer film coats produced with the spin-assembly method under SEM and noted that almostall AgNPs presented a uniform distribution.The influence of the AgNP biliary stent on coagulation functionality was evaluated based on the detectionof re-calcification clotting time. The blank polyester served as the control. We found that the polyesterwith AgNPs-heparin (heparin as the outermost layer) presented a significantly increased re-calcification time(108.8±3.1)versus the blank polyester (19.0±1.5, p< 0.001). The polyester with AgNPs-chitosan (chitosanas the outermost layer) also displayed a remarkably prolonged re-calcification time (72.7±3.9) versus thecontrol (p< 0.001). Importantly, after storage of the AgNP biliary stent for 14 days, it still presented a longerre-calcification time (50.2±2.7) than the control (p<0.001), suggesting that the AgNP biliary stent exerted along-term anti-coagulation effect.A Ag+release experiment was conductedthrough measurements of Ag+concentration using ICP-MS. We found that the concentration of Ag+releaseachieved the maximum (8.52±0.81 u g/1) on the first day, was significantly decreased during the initial 10days (2.86v0.58μg/1), and then gradually reduced after 20,30,45 and 60 days, corresponding to 2.53±0.46,2.39±0.29,2.07±0.23 and 1.81±0.17 μg/1, respectively. These data indicated that the Ag+release inthe AgNP biliary stent was an effective and slow process.Because of a high incidence of bacterialinfection after biliary injury, bacterial adherence and biliary sludge are prone to occur, inducing biliary stentocclusion; thus, we investigated the anti-bacterial activity of the AgNP biliary stent. We first detected the anti-E. coli effect of the AgNP biliary stent. After 1 hour of the co-culture of AgNPs andE. coli, the colony number of E. coli was significantly decreased versus the control, and the survival ratioof E. coli was also remarkably reduced in the AgNP group versus the control (p< 0.001). After 2,4 and8 hours of the co-culture of AgNPs and E. coli, the survival ratio of E. coli presented a continuous decrease versusthe control (all p< 0.001). Notably, after storage of the AgNP biliary stent for 1 month, the survival ratio of E.coli continuously declined versus the control under the co-culture of AgNPs and E. coli,which suggested that the AgNP biliary stent exhibited a high-efficiency anti-bacterial activity for both short-andLong-term periods.After biliary injury, the infected bacteria usually include Staph. aureus, Enterococcus and P. aeruginosa, inaddition to E. coli. Staph.aureus is representative of gram-positive bacteria and can be identified using a commercially available Staph. aureus chromogenic medium. We found that the colony number of Staph. aureus wassignificantly decreased in the AgNP group versus the control (p< 0.001). In addition, the colony numbersof Enterococcus and P. aeruginosa were remarkably reduced in the AgNP group versus the control (both p< 0.001,). These data suggested that the AgNPs biliary stent possessed a strong, broad-spectrum anti-bacterialfunction.To further investigatethe effect of AgNPs biliary stents, we performed a preclinical study in sixty-four pigs. For each group, eight pigs were randomly selected in which to observe the percentage of biliary obstruction and survival function. Onemonth after the operation, there were no biliary dilatations in either the intrahepatic or extrahepatic bile ductand the biliary stent was in a proper position in the common bile duct, as shown by B ultrasound. The results indicated that the average time of biliary obstruction wassignificantly prolonged in the AgNP group (72.37±4.23 weeks) versus the control group (40.13±3.30 weeks,p<0.0001), which suggested that the application of the AgNP biliary stent significantly delayed the occurrenceof biliary obstruction versus the control.The average survival time was 73.88±4.06 weeks in eight pigs from the AgNP group, whereas the average survival time was only 41.38±3.24 weeks in the control group. The Kaplan-Meiersurvival curve demonstrated that the survival function was remarkably increased in the AgNP group versus thecontrol (p< 0.0001). These data indicated that the application of the AgNP biliary stent significantly prolongedthe unobstructed period of the bile duct and improved survival time in the preclinical study.To monitor the biliary status in realtime, blood samples were collected serially via an ear vein at 4,12,24 and 48 weeks after biliary stent implantationto test liver function in the other 24 pigs in each group.We found that plasma total bilirubin (TBIL) and direct bilirubin(DBIL) persistently increased from 12 to 24 to 48 weeks post-operation, and plasma γ-L-glutamyl dipeptide(γ-GT) presented a continuous increase from 4 to 48 weeks post-operation in the control group.Importantly, the application of the AgNP biliary stent significantly decreased the levels of plasma TBIL and DBILversus the control from 12 to 24 to 48 weeks post-operation (all p<0.01 or 0.001). Theplasma y-GT level increased from 4 to 48 weeks post-operation (all p< 0.001).To further investigate bacterial infection in bile ducts, six pigs in each group at each time point were sampledand their bile was collected for bacterial culture. We found that the positive rate of bacterial culture in the bilewas persistently elevated in the control group from 4 to 12 to 24 to 48 weeks post-operation, corresponding to 0%, 33.33%,50% and 100%, respectively. Furthermore, the selected bacterial culture media were usedto identify positive bacteria in the bile. The results indicated that the positive bacteria in the bile were primarilyEscherichia coli, Staphylococcus aureus, Quail chicken enterococcus D, Enterobacter cloacae, Klebsiella pneumoniaeand Enterococcus faecalis, of which Escherichia coli was the dominant bacteria, accounting for 6/11 (54.55%).The anastomosis of the bile duct was evaluated to perform H&E and Masson’s trichrome stainingat 48 weeks post-operation. The scar tissue was significantly formed, and substantial fibrocytes could be observedin the control group, whereas the degree and scope of fibrosis were attenuated in the AgNP group, as shownhistologically. Notably, the collagen fibre (staining green) was significantly more abundant, whereas themyofibre was remarkably decreased, in the control group versus the AgNP group.Theseresults suggested that the application of the AgNPs biliary stent significantly decreased the formation of collagenfibres and scar tissue on the anastomosis of bile ducts in vivo.ConclusionsWe developed a novel biliary stent coated with silver nanoparticles (AgNPs) and investigated its efficacyboth in vitro and in vivo. We first identified properties of the AgNP complex using ultraviolet detection.The AgNP complex was stable without AgNP agglomeration, and Ag abundance was correspondinglyincreased with an increased bilayer number. The AgNP biliary stent demonstrated good performance inthe spin-assembly method based on topographic observation. The AgNP biliary stent also exhibited along-term anti-coagulation effect and a slow process of Ag+release. In vitro, anti-bacteria experimentsindicated that the AgNP biliary stent exhibited high- efficiency anti-bacterial activity for both shortandlong-term periods. Importantly, application of the AgNP biliary stent significantly prolonged theunobstructed period of the biliary system and improved survival in preclinical studies as a result ofits anti-microbial activity and decreased granular tissue formation on the surface of the anastomoticbiliary, providing a novel and effective treatment strategy for symptomatic biliary strictures. |
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