Author Affiliations: Department of Digestive Endoscopy Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China (Wen W, Ma LM, Tang XW, Wang X, Liu L and Fan ZN); Nanjing Medical University, Nanjing 210029, China (Wen W); Department of Gastroenterology, The 81th Hospital of PLA, Nanjing 210002, China (Wen W); Department of Gastroenterology, Changzhou First People’s Hospital, Third Affiliated Hospital of Suzhou University, Changzhou 213003, China (He W and Zhang Y)

Corresponding Author: Zhi-Ning Fan, MD, PhD, Department of Digestive Endoscopy Center, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China (Tel/Fax: +86-25-58509931; Email: fanzhining@njmu.edu.cn)

 

The abstract of this study was presented as a poster at the Asia Pacific Digestive Week (APDW 2013), which was held in Shanghai, China on September 21-24, 2013.

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60410-6

Published online September 17, 2015.

 

 

Contributors: FZN proposed the study. WW, MLM and HW performed research and wrote the first draft. TXW, ZY, WX and LL collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. FZN is the guarantor.

Funding: The study was partially supported by grants from the Jiangsu Province Social Development Program (BL2012031), the National Natural Science Foundation of China (81172266), the Natural Science Foundation of Jiangsu Province (BK2011859) and Jiangsu Innovation of Medical Team and Leading Talents Cultivation (LJ201127).

Ethical approval: This study was approved by the Animal Use and Care Committee of Nanjing Medical University, China.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: One of the major limitations of biliary stents is the stent occlusion, which is closely related to the over-growth of bacteria. This study aimed to evaluate the feasibility of a novel silver-nanoparticle-coated polyurethane (Ag/PU) stent in bacterial cholangitis model in swine.

 

METHODS: Ag/PU was designed by coating silver nanoparticles on polyurethane (PU) stent. Twenty-four healthy pigs with bacterial cholangitis using Ag/PU and PU stents were randomly divided into an Ag/PU stent group (n=12) and a PU stent group (n=12), respectively. The stents were inserted by standard endoscopic retrograde cholangiopancreatography. Laboratory assay was performed for white blood cell (WBC) count, alanine aminotransferase (ALT), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) at baseline time, 8 hours, 1, 2, 3, and 7 days after stent placements. The segment of bile duct containing the stent was examined histologically ex vivo. Implanted biliary stents were examined by a scan electron microscope. The amount of silver release was also measured in vitro.

 

RESULTS: The number of inflammatory cells and level of ALT, IL-1β and TNF-α were significantly lower in the Ag/PU stent group than in the PU stent group. Hyperplasia of the mucosa was more severe in the PU stent group than in the Ag/PU stent group. In contrast to the biofilm of bacteria on the PU stent, fewer bacteria adhered to the Ag/PU stent.

 

CONCLUSIONS: PU biliary stents modified with silver nanoparticles are able to alleviate the inflammation of pigs with bacterial cholangitis. Silver-nanoparticle-coated stents are resistant to bacterial adhesion.

 

KEY WORDS: biliary stent; silver nanoparticles; endoscopic retrograde cholangiopancreatography; bacterial cholangitis

It has been more than 30 years since endoscopic biliary stenting was as a standard palliative treatment for obstructive jaundice due to malignant or benign obstruction of bile tracts.^[1] Many studies^[2-7] have shown that metal biliary stents may offer better palliation and longer patency than plastic stents in patients with malignant biliary obstruction. However, compared to plastic stent, the major disadvantages of metal stents are higher cost and difficult to reposit once deployed. Therefore, the metal stents may be not suitable for benign strictures.^[8]

 

The occlusion of plastic stents remains a major problem in this treatment.^[9] It is believed that microbial biofilms resulted from bacterial colonization and sludge deposition are closely associated with biliary stent clogging.^[10-12] Antibiotic prophylaxis has been employed to reduce the risk of biliary bacterial colonization.^[13] But the antibiotic treatment sometimes leads to allergic reactions (including anaphylaxis) and development of bacterial resistance, and increases costs of medical care. Hence, it is essential to design a new plastic stent to prevent bacterial colonization and biofilm formation.

 

Silver (Ag) nanoparticles have antimicrobial properties and have been seen as a candidate for antibacterial coating of stents.^[14] Coatings using silver salts implantation of metallic silver have been devised.^{[15, 16]} However, they have shown disappointing antibacterial ability because of obliteration or inactivation of the silver coating by blood plasma.

 

In an attempt to address these shortcomings while taking advantage of the broad antimicrobial spectrum of silver, we designed an Ag nanoparticle-impregnated polymer coating on plastic stents. The present study reports the antimicrobial activity of Ag nanoparticles in pig models and their release in vitro.

 

 

Biliary stents modified with Ag nanoparticles (Ag/PU stents) were prepared by coating Ag/PU composite suspension on polyurethane (PU) biliary stents. The Ag/PU composite suspensions were prepared by in situ reduction method. Firstly, 0.5 mL AgNO₃ absolute ethanol solution (1 mmol/L) was mixed with 50 mL tetrahydrofuran (THF) solution containing 5 g PU to obtain a PU/AgNO₃ solution in THF/ethanol. Then 0.5 mL freshly prepared NaBH₄ ethanol solution (0.02 mol/L) was added to the above PU/AgNO₃ solution with vigorous stirring to reduce Ag⁺ to Ag nanoparticles. The bright-brown suspension was obtained after the reaction was completed. This in situ reduction method is able to obtain Ag/PU composite without aggregation of nanoparticles.

 

Then the Ag/PU composite suspension was cast on PU stent with outer diameter of about 8 mm, wall thickness of 0.3 mm, and length of 40 mm. The PU stent was immersed into the Ag/PU suspension for 10 seconds and slowly pulled out. After the fast evaporation of THF solvent, Ag/PU coating was formed on the PU stent. Because THF can partially dissolve the surface of the PU stent, the Ag/PU coating can be firmly attached to the surface of the PU stent. The control PU biliary stent and Ag/PU biliary stent are shown in Fig. 1. Brown color indicates the presence of Ag nanoparticles about 20 nm in diameter.

 

Healthy pigs weighing from 19 to 24 kg (mean 22 kg) were used in the experiments. The experimental procedures were approved by the Animal Use and Care Committee of Nanjing Medical University, China. All the pigs were fasted for two days before the experiments. Anesthesia was induced with 3% pentobarbital by peritoneal injection, followed by an intramuscular injection of 250 mg of ketamine hydrochloride. Cardiac and respiratory parameters were recorded at baseline and 8 hours, 1, 2, 3, and 7 days after the placement of the stent.

 

After the pigs were anesthetized in the left lateral position, e-duodenoscopy was inserted through the esophagus, stomach into the duodenum. A small plastic tube was passed through the duodenoscope into the ampulla. Then 1.0 mL (15-18×10⁸/mL) of E. coli (model ATCC 25922) solution was injected into the bile duct. The temporary obstruction of the nipple was solved with saline injection (Fig. 2).

 

After the success of modeling (about 8 hours later), either PU stents or Ag/PU stents were intervened in 12 pigs with bacterial cholangitis respectively through an endoscope. The guide wire was sent to the duodenal papilla within the conveyor, then a stent was sent to the bile duct by the wire. Two pigs of each group were sacrificed at days 1 and 7, respectively, to observe bacterial adhesion on the surface of the stent. All the animals were killed by euthanasia.

 

Blood samples of the thoracic vein of each pig were taken for analysis of white blood cell (WBC) count at 8, 24, 48, 72 hours, and 7 days after modeling. The samples were partly centrifuged for analyzing aspartate aminotransferase (ALT), inflammatory cells, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) at different time points.

 

At the end of each follow-up, the pigs were euthanized by an intravenous injection of pentobarbital followed by 20 mEq of potassium chloride. All animals underwent a postmortem examination to exclude intra-abdominal hemorrhage, seroperitoneum, or peritonitis. The portion of the common bile duct (CBD) containing the stent was dissected out and immediately fixed in a 4% formaldehyde solution. The specimens were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histological evaluation was performed under a light microscope. The extent of necrosis and the intensity of the inflammation of the CBD wall were assessed by a single pathologist masked to the type of the inserted stent.

 

Bacteria and biofilm adhered on PU or Ag/PU stents at days 1 and 7 were evaluated with propidium iodide (PI) staining and observed under a fluorescence inverted microscope (ZEISS Axiovert 200). The morphology of implanted stents at days 1 and 7 was observed with a scan electron microscope (JSM-6300, JEOL, Tokyo, Japan) after fixation and dehydration and gold coating of the bacteria.

 

To study the kinetics of silver released from the prepared Ag/poly (L-lactic acid) (PLLA) films, the flat or honeycomb-structured Ag/PLLA film was cut into a square shape of 2.0×2.0 cm. After washing with deionized water for ten times and dried, the samples were immersed in 10 mL of the deionized water at 37 �� without stirring. At the predetermined time (0, 1, 10, 20, 30, 60 and 90 days), the samples were taken out and the silver ion content of suspending fluids obtained from each time point was analyzed by an atomic absorption spectrometer (AA, Hitachi 180-80).

 

Data were expressed as mean±SD. The data were analyzed using the SPSS v11.5 software (SPSS Inc., Chicago, IL, USA). A P value of <0.05 was considered statistically significant.

 

 

The temperature and serological test results are shown in Fig. 3. There were no significant differences in body temperature, WBC count, ALT, IL-1β and TNF-α in the two groups before the experiment (P>0.05). The number of inflammatory cells and level of ALT, IL-1β and TNF-α were significantly lower in the Ag/PU stent group than those in the PU stent group (P<0.05).

 

Seven days after stent implantation, the intumescent bile duct was filled with pus (the yellow mucus flowing out from the CBD) in the PU stent group (Fig. 4A), whereas there was mild inflammation in the CBD of the Ag/PU stent group (Fig. 4B).

Examination of tissue sections of the CBD after the implantation of stents in the PU stent group and Ag/PU stent group under a light microscope is shown in Fig. 5. At the 7-day follow-up, severe hyperplasia of the mucosa was seen in the PU stent group and there was extensive necrotic tissue with increased infiltration of inflammatory cells and neutrophils (Fig. 5A). There was a slight inflammation in the Ag/PU stent group and the number of inflammatory cells and neutrophils was less than that in the PU stent group (Fig. 5B).

 

The degree of bacterial adhesion on stents in pig models at days 1 and 7 was examined by a fluorescence microscope (Fig. 6) and a scan electron microscope (Fig. 7). Bacterial biofilm was observed on the surface of PU stent at day 1 (Figs. 6A and 7A). And at day 7, PU stent was fully covered with bacteria (Figs. 6B and 7B). In comparison, few bacteria adhere to the Ag/PU stent (Figs. 6C, D and 7C, D).

 

The amount of Ag⁺ released from Ag/PU stent in deionized water at different periods (0, 1, 10, 20, 30, 60 and 90 days) is illustrated in Fig. 8. This composite coating on PU stent can continuously release Ag⁺ and the Ag⁺ release rate decreases with time. The results suggested that Ag nanoparticles coated on the stent are able to release Ag⁺ with results in bacterial inhibition.

 

 

According to the United States Center for Disease Control and Prevention statistics in 2011, the number of patients died from infection by the built-in medical device ranked No. 5 in the cause of death of hospital patients in the United States. And nosocomial infection in each case leads to an additional cost of 10 000-20 000 US dollors.^[17] Therefore, preventing nosocomial catheter-related infections can greatly reduce the cost of hospitalization and mortality. However, there is currently no artificial device that has the antibacterial features.

 

With broad spectrum antimicrobial activity and excellent antibacterial efficiency,^{[18, 19]} Ag nanoparticles are considered a good candidate for surface coating in medical devices.^[20] Gupta et al^[21] reported that silver nanoparticles might be an answer to the drug-resistant microorganisms. The Ag nanoparticles interact with bacteria and confer antimicrobial activity via Ag⁺.^[22] Hence, the antimicrobial activities of Ag-containing materials are often studied in terms of their Ag⁺ release rate. Our in vitro Ag⁺ release test of PU stents coating with Ag nanoparticles revealed that Ag coating is able to achieve a long-term antibacterial effect with the continuous release of Ag⁺.

 

Choosing pigs as our experimental animals mainly based on two reasons: 1) the diameter of the bile duct in humans (0.5-10 mm) is similar to that in pigs (0.5-8.0 mm);^[23] 2) animals are suitable for noninvasive endoscopic procedure.^[24]

 

We chose bacterial infection-related parameters to monitor bacterial cholangitis, and these parameters included body temperature, WBC count, ALT, IL-1β and TNF-α. The baseline of the parameters was not significantly different in the two groups. After stent placement, body temperature, WBC count and IL-1β increased quickly. They began decreasing in 8 hours and continued to decrease slowly afterward. The three parameters in the Ag/PU stent group decreased faster than those in the PU stent group. The changes of ALT and TNF-α were different from those mentioned above. They increased gradually after stent placement. However, compared with the PU stent group, the intervention of Ag/PU stent could slow down the increase rate in the levels of ALT and TNF-α. The recovery of animals in the PU stent group was slower than that in the Ag/PU stent group.

 

To measure the effectiveness of stents, we did anatomy on the bodies of pigs immediately after they died. Compared with the PU stent group, histological examination showed minor inflammation in the Ag/PU stent group. The PU stent group showed a serious infection and abundant neutrophils infiltration in tissues. However, the Ag/PU stent group showed only mild inflammation in the CBD and the abdominal cavity surrounding it.

 

Bacterial biofilm is easily formed on PU stent in short time with accelerated deposition of biliary sludge. According to the images of a scan electron microscope, the biofilm is composed of highly packed E. coli. This complex 3-D structure of biofilm is strongly resist to antibiotics and difficult to be treated. In contrast, only a small amount of bacteria, shaped as a single colony structure, were attached to the surface of Ag/PU stent. On day 7 after implantation, the number of bacteria increased but there was no sign of biofilm formation. The adhesion of bacteria to PLLA surfaces is the first step to biofilm formation. With the accumulation of bacteria on the film surface, adhesion proteins expressed on bacterial surfaces can bind bacteria to the PU surface or with other nearby bacteria. Then, a 3-D community of bacteria and peptidoglycan envelope can form and lead to the development of biofilm.^[25] Ag/PU stent reduced bacterial adhesion and inhibited biofilm formation at the initial stage. From the long-term antibacterial efficiency of Ag nanoparticles and the Ag⁺ release results, we predicted that Ag/PU stent effectively prevents biofilm formation and growth in a long period of time.

 

In conclusion, we developed a novel biliary stent modified with Ag nanoparticles using a facile preparation method. Ag/PU stent is able to efficiently release Ag⁺ for a long period to enable a long-term antibacterial efficiency. The in vivo experiments in pig models confirmed a milder inflammation and greatly reduced bacterial adhesion on Ag/PU stent. This stent is expected to prolong stent patency time and improve the therapeutic effect in future study.

 

 

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