A novel biliary stent coated with silver nanoparticles prolongs the unobstructed period and survival via anti-bacterial activity

Abstract

Symptomatic biliary stricture causes life-threatening complications, such as jaundice, recurrent cholangitis and secondary biliary cirrhosis. Fully covered self-expanding metal stents (FCSEMSs) are gaining acceptance for treatments of benign biliary stricture and palliative management of malignant biliary obstructions. However, the high rate of FCSEMS obstruction limits their clinic use. In this study, we developed a novel biliary stent coated with silver nanoparticles (AgNPs) and investigated its efficacy both 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 correspondingly increased with an increased bilayer number. The AgNP biliary stent demonstrated good performance in the spin-assembly method based on topographic observation. The AgNP biliary stent also exhibited a long-term anti-coagulation effect and a slow process of Ag(+) release. In vitro anti-bacteria experiments indicated that the AgNP biliary stent exhibited high-efficiency anti-bacterial activity for both short- and long-term periods. Importantly, application of the AgNP biliary stent significantly prolonged the unobstructed period of the biliary system and improved survival in preclinical studies as a result of its anti-microbial activity and decreased granular tissue formation on the surface of the anastomotic biliary, providing a novel and effective treatment strategy for symptomatic biliary strictures.

Figure 1. Surgery process of biliary stent implantation and collection in pigs.

(a) Animals were managed with general anaesthesia and mechanical ventilation. (b) A neuromuscular blockade was administered to ensure complete paralysis. (c) Vital signs were monitored in real time using vital sign monitors. (d) Common bile ducts were gently dissociated and cut out, and approximately 5 cm-long biliary stents were implanted into the biliary ducts. (e) To suture the biliary duct, 5–0 absorbable suture material was used. (f) Animals were fasted from food and water for 12 hours, and appropriate antibiotics were administered to prevent infections. (g) The common bile duct with a biliary stent was gently dissociated. (h) The biliary stent was collected.

Figure 2. Property characterization of Ag-loaded heparin/chitosan multilayer film coats.

(a) The quality of AgNPs was characterized by ultraviolet detection at different time points. The peak position of the wavelength of AgNPs was stable at 396 nm during the initial 8 days. (b) Contact angle of chitosan-Ag0/heparin multilayer films versus the layer number. Heparin and chitosan almost exhibited a strict alternate layer-upon-layer arrangement. (c) UV-visible spectra of chitosan-Ag0/heparin multilayer films versus the bilayer number. All multilayer film coats at different bilayers presented a maximum absorption at 433 nm wavelength. The maximum absorption presented a linear increase with the increased number of bilayers during the assembly process.

Figure 3. Topography and effect of the AgNP biliary stent.

(a) The surface topography of the AgNP multilayer film coats from the dip-assembly and the spin-assembly methods, respectively, was observed with atomic force microscope (AFM). (b) The surface topography of AgNP multilayer film coats from the spin-assembly methods was observed with scanning electron microscopy (SEM). (c) The macroscopic morphology of the AgNP biliary stent is shown. (d) The influence of the AgNP biliary stent on coagulation function was evaluated based on the detection of re-calcification clotting time. The blank polyester served as the control. AgNPs-heparin: heparin as the outermost layer of the polyester; the polyester with AgNPs-chitosan: chitosan as the outermost layer of the polyester. ***p < 0.001. (e) A Ag⁺ release experiment was conducted through measurements of Ag⁺ concentration at different time points using the inductively coupled plasma mass spectrometry (ICP-MS).

Figure 4. Anti-bacterial function of the AgNP biliary stent in vitro.

(a) The colony number of E. coli was significantly decreased after 1 hour of co-culture of AgNPs and E. coli versus the control. (b) The survival ratios of E. coli were compared at different time points of AgNPs and E. coli co-culture versus the control. AgNPs-1M: AgNPs biliary stent storage for 1 month. (c) The colony number of Staph. aureus was compared in AgNPs and E. coli co-culture versus the control. Magnification: 400×. (d) The colony number of Enterococcus was compared in AgNPs and E. coli co-culture versus the control. Magnification: 400×. (e) The colony number of P. aeruginosa was compared in AgNPs and E. coli co-culture versus the control. Magnification: 400×. IOD: integral optical density; HPF: high-power fields. The bacterial number was determined with the spread plate method.

Figure 5. AgNP biliary stent prolonged the unobstructed period and survival in vivo.

(a) Biliary dilatations in both the intrahepatic and extrahepatic bile duct (top) and biliary stent position in the common bile duct (below) at one month after the operation were observed with B ultrasound. (b) The percentage of biliary obstruction occurrence in the AgNP group and the control group were compared using a Kaplan-Meier survival curve. (c) The Kaplan-Meier survival curve demonstrated the survival function in sixteen pigs from the AgNPs group versus the control group.

Figure 6. AgNP biliary stent maintained good biliary status in vivo.

To monitor biliary status in real time, blood samples were collected serially via an ear vein at 4, 12, 24 and 48 weeks after biliary stent implantation to test liver function in the other twenty-four pigs for each group. Plasma levels of (a) total bilirubin (TBIL) and (b) direct bilirubin (DBIL) and (c) γ-L-glutamyl dipeptide (γ-GT) were compared between the AgNP group and the control group at different time points post-operation. **p < 0.01; ***p < 0.001. (d) The histology of bile duct anastomosis in the AgNP group and control group was observed by haematoxylin and eosin (H&E) staining. Magnification: 100×; 400×. (e) The collagen fibre (staining green) and myofibre (staining red) of bile duct anastomosis in the AgNPs group and control group were observed using Masson’s trichrome staining. Magnification: 400×.