Inhibition of Bacterial Biofilm Formation on Ureteral Stent Surface by Jelleine-1 Hydrogel Coating Doped with Copper Nanoparticles

doi:10.21203/rs.3.rs-3591911/v1

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Inhibition of Bacterial Biofilm Formation on Ureteral Stent Surface by Jelleine-1 Hydrogel Coating Doped with Copper Nanoparticles

https://doi.org/10.21203/rs.3.rs-3591911/v1

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Aims: Ureteral stent is commonly used after urological surgeries. But, pathogenic bacteria can colonize the surface of the stent and gradually build a biofilm. Biofilms can not only promote the growth and reproduction of pathogenic bacteria, but alsoprotect them from adverse factors (e.g., physical or chemical factors), increasing the risk of urinary tract infections.

Material and methods: In this study, the natural antibacterial peptide Jelleine-1 was made into a hydrogel loaded with copper nanoparticles, which was coated with a dip-coating machine to obtain a novel type of antibacterial ureteral-coated stent.

Key Findings: Verified the feasibility of coating antibacterial peptides on in vivo stent, endowing them with specific biological activity.In vitro antibacterial experiments showed that the coated stent has good antibacterial activity and can inhibit the formation of biofilms. Furthermore, in vivo antibacterial experiments were performed in female rabbits. The results showed that the bacterial count was reduced by 83% compared to that in the uncoated group.

Significance: Our antibacterial ureteral coated stent significantly reduced the risk of stent-related urinary tract infections.

ureteral stent

urinary tract infection

biofilm

metal nanoparticles

antimicrobial peptides

A ureteral stent is a commonly used tool in urology, with functions such as drainage, relieving obstruction, controlling infection, and preventing ureteral stenosis^[1]. However, ureteral stent is not ideal for this purpose. Retaining ureteral stent in the body can cause a series of complications such as pain, hematuria, frequent urination, crusting, and urinary tract infections^{[2, 3]}. The incidence of tract infections is as high as 11%^[4]. Moreover, pathogenic bacteria can attach to the surface of the stent, gradually forming a biofilm and enhancing resistance to clinical antibiotics^[5], and poor clinical treatment can lead to urinary sepsis, septic shock, and even death^[6].

Biofilms are usually composed of lipids, polysaccharides, nucleic acids, and proteins, which are complex three-dimensional structures that provide stability and strong adhesion to bacteria, and can maintain the survival and proliferation of bacterial colonies^{[7, 8]}. Protection of pathogenic bacteria from physical and chemical factors^{[9, 10]}. After inserting a ureteral stent, pathogenic bacteria can form biofilms on the surface of the stent within a short period. However, the use of antibiotics can only control infection and delay the formation of biofilms, but it cannot clear the formed biofilms^[11], and the long-term use of antibiotics can also lead to drug resistance.

After implantation of a ureteral stent, a regulating membrane (e.g., albumin, fibrinogen, and uromodulin) forms on the surface of the stent in a short period^[12]. The conditioning film alters the physical and chemical properties of the stent surface, rendering the potential anti-adhesion mechanism ineffective^[13]. Conditioning membranes can promote the initial "weak interaction" (charge-mediated interaction) between pathogens and the stent surface to transform into stronger irreversible adhesion, leading to bacterial adhesion and colonization on the material surface, followed by the formation of biofilms^[14]. Stent crusting is also considered a factor in biofilm formation, and stent surface crusting is a common phenomenon, with up to 22–100% of stent exhibiting a certain degree of crusting after removal^[15]. It provides an uneven surface for bacterial adhesion and biofilm formation on stent ^[16].

The risk factors for colonized bacteriuria are longer indwelling time of the ureteral stent, diabetes, female sex, chronic renal failure, diabetic nephropathy, and cancer, among which the indwelling time of the stent is the only changeable risk factor^[17–19]. Currently, there are many strategies for ureteral stent, including stent coating^[20], surface modification^[21], reduction of stent materials^[22], and biodegradable stent^[23]. These strategies have achieved good results in reducing colonization of pathogenic bacteria on the surface of the ureter.

Antibacterial peptides are short-chain amino acid sequences that exhibit antibacterial activity. Owing to their unique non-specific physical targeting of cell membranes and their mechanism of action in breaking down cell membranes to kill microorganisms, it is not easy to induce bacterial resistance^{[24, 25]}. The natural antibacterial peptide Jelleine-1 (J-1) was extracted from natural royal jelly^[26]. J-1 has a wide antibacterial spectrum, with a significantly higher antibacterial effect on E. coli than on Staphylococcus aureus, and J-1 has satisfactory biocompatibility^[27]. After adding adenosine diphosphate (ADP), J-1 self-assembled to form a hydrogel. Nanocopper has broad-spectrum antibacterial activity, which can inhibit the growth of bacteria, fungi, and viruses by producing reactive oxygen species (ROS), leading to lipid peroxidation, protein oxidation, and DNA degradation^[28]. The antibacterial activity of Cu nanoparticles can be further enhanced by coating them with an antibacterial peptide hydrogel. Here, we developed a new type of ureteral stent coating using this excellent antibacterial material and evaluated its antibacterial and biofilm properties to reduce pathogen colonization and stent-related infections.

2.1Materials and instruments

The amino acids used for peptide synthesis were obtained from GL Biochemical Company (China). Phosphate buffered saline (PBS) and crystal violet staining solution were purchased from Sigma-Aldrich. The live/dead bacterial viability kit was purchased from Thermo Fisher Scientific (MA, USA). E. coli (ATCC 8739) was purchased from ATCC company. Trypsin soy peptone liquid medium (TSB), trypsin soy peptone agar medium (TSA), broth (LB), and LB agar were purchased from Hangzhou Microbial Reagent Co. Ltd. (Hangzhou, China). All chemicals were of analytical grade and not subjected to other treatments. All the experiments were conducted using ultrapure water.

Particle size and potential were determined using a potentiometer (Zetasizer Nano ZS90). A UV spectrophotometer (UV-3600, Shimadzu, Japan) was used to confirm UV absorption. The bacterial concentration was confirmed using Synergy NEO, BioTek, and Vermont. Bacteria were cultured in a constant temperature shaker (THZ-300C, Yiheng Technology Instrument Co., Ltd., Shanghai, China), super clean workbench (SW-CJ-2FD, Sujing Antai Co., Ltd., Suzhou, China), and biochemical incubator (SPX-150BSH-II, CIMO Medical Device Manufacturing Co., Ltd., Shanghai, China). Measure pH using a sewing pH meter (PB-10, sewing, G ö ttingen, Germany). Fluorescence images were captured using a Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Japan). The morphology of the hydrogel was observed using transmission electron microscopy (TEM; JEM 2100, JEOL Ltd., Japan). The peptides were purified using HPLC (Agilent 1260, Agilent Technologies (China) Co., Ltd.) and preparative liquid phase (LC-8A, Shimadzu Co., Ltd.) with ultrapure water from a water purification machine (H20pro UV-T, Millipore). Ureteral stents were purchased from Bard Medical Technology Co., Ltd. (Shanghai, China). A pull-up coating machine (SYDC-100, Shanghai Sanyan Technology Co., Ltd., Shanghai, China) was used for coating. X-ray photoelectron spectroscopy (PHI 5000 VersaProbe III) was used to detect the surface elements of the coatings. The contact angle tester (5C2000D1, Shanghai Zhongchen Digital Technology Equipment Co., Ltd., Shanghai, China) was used to detect the hydrophilicity of the coating surface.Female rabbit purchased from Huabukang Biological Products Center in Xuanwu District, Nanjing City.

2.2 Synthesis and purification of peptides

J-1 (PFKLSLHL) was synthesized using a solid-state method. Immerse 100 mg of MBHA resin in DMF and stirred for 3 h to remove the protective groups from the resin. In DMF, HBTU and HOBT were used to activate 5-fold equivalent amino acids, with each coupling reaction lasting 45 min. The protective groups were removed using piperidine/DMF (20%, v/v) for 30 min. The above steps were repeated to add monomeric amino acids until the desired peptide sequence was synthesized. J-1 was cleaved from the MBHA resin using a mixture of TFA/EDT/water/TIS (94:2.5:2.5:1, v/v/v/v) cutting fluid at room temperature. The peptide was precipitated in ice ether, purified by HPLC (Shimadzu LC-20A, JEOL, Japan), and determined by LC-MS. Using a C18 chromatographic column (Shimadzu), gradient elution was performed with a mobile phase of water and acetonitrile (containing 0.1% TFA).

2.3 Synthesis of J-1-ADP@Cu hydrogel

J-1 was dissolved in dimethyl sulfoxide (DMSO) to form a 100 mM stock solution. J-1 stock solution was added to 10 mM ADP sodium salt solution (pH 7.4) and quickly polymerized to obtain the J-1-ADP hydrogel without adding any other chemical crosslinking agent. The hydrogel was immersed in an excess of 0.1 mol/L CuSO₄ solution for three days, centrifuged to remove the excess CuSO₄ solution after three days, and then immersed in deionized water for five days. During this period, the copper sulfate solution was replaced with deionized water every day to Obtain Cu²⁺-loaded metal nanoparticles J-1-ADP@Cu Hydrogel.

2.4 Preparation of ureteral stent coating

The ureteral stent was placed in anhydrous ethanol for ultrasonic vibration for 5 min, and then in physiological saline for 30 min. An immersion coating machine (SYDC-100) was used to coat the ureteral stent. The coated stent was dried at room temperature for 2 h and then cut into 1 cm segments for storage.

2.5 Determination of Hydrated Particle Size and Zeta Potential of Hydrogel

Two milliliters of J-1-ADP and J-1-ADP were ultrasonically diluted with deionized water (J-1-ADP@Cu), and the hydrogel was loaded into a 2 mL centrifuge tube. After even mixing, a Marvin particle size analyzer was used to measure the hydration particle size and potential of the sample. One milliliter of each sample。 was taken to measure the hydration particle size, and the remaining 1 mL of each sample was used to measure the zeta potential. Three parallel samples were set for each group, and the average value was calculated.

2.6 Characterization of Hydrogels

To verify J-1-ADP hydrogel successfully encapsulates with Cu²⁺,and observe J-1-ADP and J-1-ADP@Cu Morphological characteristics of hydrogels. Suck 10 µL samples were dropwise added to the carbon-supported copper mesh, repeated 2–3 times, and left at room temperature overnight. After the copper mesh was completely dried, the morphology of the samples was observed using a transmission electron microscope. Then,using X-ray photoelectron spectroscopy to detect the surface elements of the J-1-ADP and J-1-ADP@Cu coated. Absorb 100 µL sample, spin-coated on a 0.5×1 cm glass slide etched with Indium tin oxide (ITO), placed at room temperature, dried, and then test with an X-ray photoelectron spectroscopy.

2.7 Hydrophilicity of J-1-ADP/ J-1-ADP@Cu coating

To detect the J-1-ADP/ J-1-ADP@Cu Hydrophilicity of the coating, 200 µL samples were spin coated on a 2×2 cm glass slide, dried, and a contact angle detector was used to detect J-1-ADP/J-1-ADP@Cu and the contact angle between the coating and H₂O/CH₂I₂.

2.8 Biocompatibility of J-1-ADP@Cu coated stent

The coated bracket was placed in a 24-hole plate, including the bare bracket group and J-1-ADP@Cu Coating bracket group, with each group having three holes. Add 1 mL of DMEM containing L929/HUVEC cells per well (5×10⁴/mL) and culture in a cell incubator with 5% carbon dioxide at 37°C. After incubation for 24 h, the sample was gently cleaned thrice to remove cells that did not adhere to the stent. The cleaned bracket was then placed into the new hole. Pancreatic enzyme digestion solution (300 µL) was added to each well µL) incubated for 3 min, and 600 µL of DMEM was added to terminate the digestion culture. The cells were flushed down on the surface of the stent and 100 cells were removed each time the suspension was added six times to 96 well plates. Then, 10 µL of the CCK-8 solution was incubated in a cell incubator for 4 h, and the OD value was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA).

2.9 In vitro antibacterial activity of J-1-ADP@Cu coated stent

E. coli was cultivated in LB liquid medium at 37°C for 12 h. Then, the bacterial solution (1 mL) was placed in a centrifuge tube (1.5 mL) and centrifuged at 4000 rpm for 2 min. After removing the supernatant, the E. coli was suspended in phosphate-buffered saline (PBS, pH 7.4). The E. coli suspension was diluted until the OD value detected by the UV spectrophotometer at 625 nm was 0.1, and the concentration of E. coli was approximately 1.5×10⁸ CFU/mL. The Luwei Microbiology Company (Shanghai, China) provided a comparison table for the approximate concentration and absorbance of bacterial solutions at 625 nm. Select a bracket was length of 10 mm. The stent was divided into bare stent and J-1-ADP@Cu Coating bracket groups. The brackets were placed separately into sterile holes of a 24-well plate (one hole per piece). Add 1mL/hole (1.5×Drip 10⁵ CFU/mL of E. coli was added to a 24-well plate. The orifice plate was placed in a bacterial incubator at 37°C for 24 h. After the incubation, the stent was removed and added to a new well. It was washed three times with 2 mL of sterile PBS and placed in a 1.5 mL sterile centrifuge tube. Place 1 mL of sterile PBS in each centrifuge tube and placed in a 40 kHz ultrasound bath for 5 min. Then diluted 10 times and 100 µL was cultured in LB solid medium at 37°C for 18 h, with three parallel samples set in each group. The number of E. coli colonies on the LB agar plates was calculated, and the results were recorded.

2.10 Stability of J-1-ADP@Cu coated stent

A 50 mL shaking tube (containing 40 mL artificial urine) was selected as the in vitro bladder model, and take 1 ml 1.0×10⁵ CFU/mL E. coli bacterial liquid was added to the in vitro bladder model. The stent was divided into two groups (bare stent group and J-1-ADP@Cu coating bracket group), with three brackets per group. The stent was placed in the bladder model mentioned above, and the extracorporeal bladder model was placed in a 37°C constant-temperature shaking table (60 rpm) for one month. During this period, 100 µL of artificial urine was collected weekly for plate coating to detect the infection.

2.11 Ability to inhibit biofilms

To verify J-1-ADP@Cu coated stent can inhibit biofilm formation, take 1.5×10⁸ CFU/mL of 1 mL of E. coli solution was adding to 24-hole plates. The experiment was divided into two groups (bare stent group J-1-ADP@Cu coated stent group), with three stent in each group. The stent were placed in the wells containing the bacterial solution for 3 h and incubated in a biochemical incubator for 48 h. After the incubation, the stent was removed and added to a new well. It was washed three times with 2 mL of sterile PBS and placed in a 1.5 mL sterile centrifuge tube. One milliliter of sterile PBS was placed in each centrifuge tube, a 40 kHz ultrasound bath was taken for 5 min, diluted 10 times, centrifuged for 10 min (5000 rpm, 4°C), the sediment was collected, and the supernatant was discarded. Finally, 20 µL of the prepared live/read staining solution was added, vortexed until completely uniform, and allowed to stand at room temperature in the dark for 20 min. The staining solution (10 µL) was dropped onto a slide, covered with a cover glass, observed using an inverted fluorescence microscope, and photographed for recording.

2.12 In vivo experiments

Uncoated stent was used as the control group. Therefore, the bare bracket and J-1-ADP@Cu coated stent were used in female rabbit bladder models (n = 3). The animal experiment involved in this study was approved by the Ethics Committee of Changzhou City, Jiangsu Province. Female rabbits were anesthetized by intravenous injection of 10% chloral hydrate with a live weight of 0.3 mL/100 g. After anesthesia, the stent was placed into the bladder of female rabbits using surgical methods, and the entire surgical process was sterile. Female rabbits were fed continuously for one month. After continuous feeding for one month, the female rabbits were sacrificed. The bladder was then completely removed. After removing the stent from the bladder, the bladder sample was completely stored in 10% formalin solution (1.1% methanol and 3.7% formaldehyde) for 6 h and then removed and dehydrated with ethanol. The ethanol was removed with xylene, and the specimen was soaked in paraffin to facilitate tissue slicing. After fixing the thin samples on a glass slide, xylene and xylene substitutes were selected for dewaxing. Hematoxylin-eosin and hematoxylin were selected for hematoxylin-eosin staining (H&E) for further histopathological evaluation. The bladder stent was removed and placed in a 5 mL sterile centrifuge tube, washed three times with 2 mL sterile PBS, and placed in a 1.5 mL sterile centrifuge tube. One milliliter of sterile PBS was placed in each centrifuge tube, placed in a 40 kHz ultrasound bath for 5 min, incubated in a shaker for 8 h, then dilute 20000 times, and 100 µL was cultured in LB solid medium at 37°C for 18 h. The number of E. coli colonies on LB agar plates was calculated, and the results were recorded and plotted by using the Origin software.

3.1 Characterization of antimicrobial peptides and coatings

As shown in Fig. 1A, after embedding Cu, the particle size slightly increased, indicating that Cu was successfully embedded in the hydrogel. The PDI was ~ 0.058, indicating Gel and Gel@Cu stabilities. After embedding Cu, the potential increased, indicating that the positively charged Cu²⁺ was successfully embedded in the hydrogel (Fig. 1B). The Gel and Gel@Cu appearance characteristics were also observed (Fig. 1C, D). Mass spectrometry analysis (Figure S1) showed that the peptide sequence obtained was the desired target product.

3.2 Contact angle of coating

From Fig. 2, it can be seen that the addition of copper to J-1-ADP has no significant effect on the contact angle between the coating and H₂O/CH₂I₂, J-1-ADP@Cu. The contact angle with H₂O is less than 90° (Fig. 2A and B), our coating is hydrophilic, can gradually dissolve and release in H₂O. Meanwhile, J-1-ADP@Cu. The contact angle with CH₂I₂ is much less than 90° (Fig. 2C), indicating that our coating is more lipophilic and can bind to the lipid components on the surface of E. coli, making it easier to exert antibacterial effects.

3.3 Biocompatibility

Antibacterial peptides may be cytotoxic to normal cells; therefore, we tested the biocompatibility of the coated stent. As shown in Fig. 3, the survival rates of HUEVCs and L929 cells in the uncoated stent group were 100%, and the cell survival rates in the coated stent group were greater than 80%. Therefore, it can be considered that the coated stent was not biologically toxic.

3.4 Antibacterial effect

Considering that E. coli is the most common pathogen causing urinary tract infections^[29], we selected E. coli to evaluate the antibacterial activity of the coated stent. Figure 4 shows the J-1-ADP Hydroge@Cu²⁺. The number of E. coli colonies in the coated stent was significantly lower than that in the control stent. This indicates that the coating has efficient and strong antibacterial activity.

3.5 Coating stability

As shown in Fig. 5, until the fourth week, the J-1-ADP Hydrogel@Cu^{2 +} coated stent exhibited good antibacterial activity, with an increase in bacterial colonies starting from the fifth week, indicating a decrease in antibacterial materials and a decrease in antibacterial effectiveness. Until the sixth week, the bacterial count of the coated stent was comparable to that of the uncoated stent, indicating that the coated stent had no antibacterial activity. The coating material was found to be stable in urine and maintained its antibacterial activity for at least one month. Because most ureteral stents remain in the body for 2–4 weeks^[30], the stability of all coatings can meet basic clinical needs.

3.6 Inhibition of biofilm formation

By observing the biofilm on the ureteral stent through immunofluorescence staining, live E. coli were stained green, while dead bacteria were stained red or red-green. As shown in (Fig. 6), the uncoated group showed a large amount of green fluorescence, indicating that E. coli formed a large amount of biofilm on the surface of the stent. However, only a small amount of green fluorescence and a trace of red fluorescence were observed on the surface of the coated stent. The surface of the coated stent generated only a small amount of biofilm and killed some E. coli. The resulting proof (A) J-1-ADP@Cu coated stent can effectively inhibit biofilm formation by E. coli.

3.7 In vivo experiments

After being placed in the bladder of female rabbits for one month, comparing the surface infection of the two groups of stents, the number of E. coli colonies on the surface of coated stents decreased by 83% compared to uncoated stents (Fig. 7A, B). It has been proven that coated stent can exert excellent antibacterial effects in female rabbits. At the same time, it can be seen from the pathological sections of the female rabbit bladder (Fig. 7C, D) that both groups of stents showed no significant abnormalities in the female rabbit bladder, whereas the control group had significantly more bacterial colonies on the surface of the stents than the coated stent. This may be because E. coli has already colonized the surface of the stent but has not yet affected the female rabbit bladder tissue.

In summary, after we added ADP to the antibacterial peptide J-1, J-1 self-assembled to form a hydrogel, and used it as a carrier to wrap Cu²⁺ nanoparticles to make the antibacterial material we needed, and obtained ureteral stent coating by dip coating. Our antibacterial stent coating has been validated through in vivo and in vitro experiments to have low biological toxicity, good antibacterial effect, and can be maintained for at least one month while effectively inhibiting biofilm formation. Significantly reduced risk of stent-related tract infections. Provide new strategies to reduce ureteral stent-related infections.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.

Author Contributions

ZhiXiong Hu:Design and implementation of the research, data curation, riting-original draft,.Xun Wang: implementation of the experiment, data curation, and writing the original draft. HongLei Shi:conceived the original, Resources,Supervision, Roject Administration, Funding Acquisition, Writing-Review and Editing.

Acknowledgements

Acknowledge the funding support by 2023 Changzhou Basic Research Program(No.CJ20230006).The authors also acknowledge Professor YaFei Wang from the School of Materials at Changzhou University for providing assistance in the detection experiment of this study.

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No competing interests reported.

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S1. J-1-ADP@Cu Mass spectrometry analysis
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S2. (A)XPS image of J-1-ADP@Cu, (B)XPS image of J-1-ADP

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Inhibition of Bacterial Biofilm Formation on Ureteral Stent Surface by Jelleine-1 Hydrogel Coating Doped with Copper Nanoparticles

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Abstract

Figures

1. Introduction

2. Materials and Experiments

2.1Materials and instruments

2.2 Synthesis and purification of peptides

2.3 Synthesis of J-1-ADP@Cu hydrogel

2.4 Preparation of ureteral stent coating

2.5 Determination of Hydrated Particle Size and Zeta Potential of Hydrogel

2.6 Characterization of Hydrogels

2.7 Hydrophilicity of J-1-ADP/ J-1-ADP@Cu coating

2.8 Biocompatibility of J-1-ADP@Cu coated stent

2.9 In vitro antibacterial activity of J-1-ADP@Cu coated stent

2.10 Stability of J-1-ADP@Cu coated stent

2.11 Ability to inhibit biofilms

2.12 In vivo experiments

3. Results and Discussion

3.1 Characterization of antimicrobial peptides and coatings

3.2 Contact angle of coating

3.3 Biocompatibility

3.4 Antibacterial effect

3.5 Coating stability

3.6 Inhibition of biofilm formation

3.7 In vivo experiments

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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