Coaxial electrospun Ag-NPs-loaded endograft membrane with long-term antibacterial function treating mycotic aortic aneurysm.

doi:10.21203/rs.3.rs-2948529/v1

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Coaxial electrospun Ag-NPs-loaded endograft membrane with long-term antibacterial function treating mycotic aortic aneurysm.

https://doi.org/10.21203/rs.3.rs-2948529/v1

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published 01 Apr, 2024

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Endovascular stent-graft has been widely used in the treatment of aortic pathologies. The stent-graft is consisted of two main parts: one is artificial vascular membrane which was used to exclude the lesions, and the other is the nitinol stent skeleton. But the current endograft membrane which was PET or ePTFE has a poor ability to inhibit bacterial colonization, it is not fit for endovascular treatment of mycotic aneurysms, as the infection is prone to progress after endograft implantation. And even after endovascular repair for non-mycotic aortic pathologies, endografts could become infected in short term or in long term, especially for patients with diabetes mellitus or in immune insufficiency conditions. In order to solve the problem that aneurysms cannot resist infection for a long time in clinic, in this study, a kind of silver nanoparticles (Ag-NPs) -loaded endograft membrane was prepared for coating the outer layer of the current stent-graft. The Ag-NPs-loaded membrane adopts coaxial electrospinning technique, with Polycaprolactone (PCL) as the shell, Chitosan (CS), Polyethylene oxide (PEO) and Ag-NPs with broad-spectrum antibacterial effect as the core, forming fibers with core-shell structure. A series of physical and chemical properties and biological properties of the Ag-NPs-loaded membrane were characterized: The formation of the core-shell structure was confirmed by TEM; The drug release experiment revealed that the membrane could effectively control the release rate of Ag-NPs; The bacteriostasis experiment proved that the membrane coating had an obvious inhibitory effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Animal experiments confirmed that the Ag-NPs-loaded membrane is basically non-toxic, has good biocompatibility, and can play an effective antibacterial role in the mycotic aortic aneurysm model in porcine. In conclusion, the Ag-NPs-loaded membrane prepared by coaxial electrospinning had optimal anti-infection function and it realized slow-releasing of Ag-NPs to achieve long-term bacteriostasis. Thus, the Ag-NPs-loaded membrane might have potential in preventing infection progression and treating mycotic aortic aneurysm in endovascular way.

Endograft

Ag-NPs

Bacteriostasis

slow-release

coaxial electrospinning

aortic aneurysm

Aortic aneurysm is a serious pathology in aorta that, once ruptured, can cause severe pain, shock, and even sudden death. Endovascular treatment has already been an important option for aortic aneurysms. It excludes the aneurysm through the use of a covered stent, which consists of stent and artificial vascular membrane. The main material used for the vascular membrane was polyethylene terephthalate (PET) or expanded polytetrafluoroethylene (ePTFE)^{1, 2}. For a long time, bacterial infection onto the endograft has been a major threat after the endovascular procedure^3–5 .It could occur in the short term after the procedure, and it was also reported in years after⁶. So, the question of how to make the stent graft carry antibacterial capacity has therefore been investigated in several ways.

The preparation of a drug-loaded membrane combined with existing stent can solve the problem of the stent-graft carrying antibacterial ability. In recent years, many strategies to solve this problem have been reported, such as drug-loaded membrane prepared by pulsed laser deposition (PLD) technique⁷, modifying the surface of membrane through layer-by-layer (LBL) assembly or blend electrospinning⁸. These methods caused the drugs to adhere to the surface of membrane, which may lead to a burst release of drug and the inability to extend the drug action time⁹. The application of electrospun nanofibers in drug delivery system has characteristics of targeted drug delivery, sustained release, rapid onset and good pharmacokinetics¹⁰. This technology has been widely applied in the biomedical field.

Biomolecular materials, such as PCL, Polyactic acid (PLA), Polytetrafluoroethylene (PTFE), are usually selected in the preparation of vascular stent membrane by electrospinning, but their characteristics are different. PCL is a biodegradable polymer widely used in the biomedical field, which has excellent mechanical properties and biocompatibility and is often used in electrospinning^{11, 12}. However, as a relatively hydrophobic material, PCL has poor cell affinity, which is not conducive to cell adhesion¹³. Chitosan is a natural polysaccharide extracted from the chitin N-diacetyl group. It has good hydrophilicity, biodegradability, non-toxicity, and antibacterial properties and can be used to improve the hydrophilicity of PCL^14–16. In addition, chitosan has low solubility and high viscosity, so pure chitosan is difficult for electrospinning, so it is necessary to use blending and grafting methods to improve its spinnability. Duan¹⁷et al. found that adding an appropriate amount of polyoxyethylene (PEO) to the acetic acid solution of chitosan could greatly improve the spinnability of chitosan, which also improved its performance in coaxial electrospinning.

Chitosan does not exhibit good antimicrobial properties, but chitosan can be used to load antibacterial drugs^18–20, such as Amoxicillin, Penicillin, Vancomycin and Silver nanoparticles (Ag-NPs). Ag-NPs is a classic antibacterial agent, that has been studied for many years for its low toxicity and excellent antibacterial activity^21–23. The combination of chitosan and Ag-NPs has long been studied for medical applications because it is more effective against bacteria than pure chitosan. Another advantage of this combination is that chitosan is used as a stabilizer to prevent aggregation of Ag-NPs²⁴.

Therefore, this study aims to prepare the Ag-NPs-loaded membrane for binding to a current aortic stent-graft by coaxial electrospinning. The prepared Ag-NPs-loaded membrane has core-shell nanofibers, and Ag-NPs were wrapped in the core layer of nanofibers, which can effectively control the Ag-NPs release rate. In addition, the physicochemical properties, antibacterial properties and drug release properties of the Ag-NPs-loaded membrane were evaluated. And the biocompatibility and feasibility of Ag-NPs-loaded membrane were demonstrated by in vitro and in vivo experiments.

Preparation of the Ag-NPs-loaded Membrane

PCL (MW = 80,000 g/mol) was supplied by Solvay S.A.(Belgium), and Silver nanoparticles solution (10000 ppm) was purchased from Mingcheng Plastic Additives Chemical Co.Ltd (Dongguan, China). CS (molecular weight: approximately 300 kDa; degree of deacetylation:80–95%), PEO (molecular weight: approximately 600 kDa), Methylene chloride and Dimethyl for artificial and Acetic acid were both purchased from Sinopharm Chemical Reagent Co.Ltd (Shanghai, China). All materials are of analytical grade and are used as received.

Ag-NPs-loaded membranes were prepared by using coaxial electrospinning. By using a coaxial needle, the two components can be fed separately through the inner and outer coaxial capillary channels and integrated into the core/shell structure of the composite fiber^{25, 26}.Firstly, prepared the shell solution PCL was dissolved in DCM/DMF(7:3, v/v) and stirred for 4 h at room temperature. The solution with a PCL concentration of 15 wt% was prepared and used as the shell layer. Secondly, CS and PEO were dissolved in HAc to prepare CS and PEO solutions with a concentration of 3 wt%. After stirring separately at room temperature for 6 h, mix the two solutions, add silver nanoparticles, and stir for another 4 h. The mixed solution containing Ag-NPs was used as the core layer, and the concentration of Ag-NPs was set as 1 wt%, 2 wt% and 3 wt% for comparison. The preparation process is shown in Fig. 1.

The diameter of the coaxial needle was 1.0 mm for shell polymer and 0.4 mm for core polymer. The core and shell solutions were loaded into 10mL syringes and controlled by two syringe pumps, and the shell needle was connected to the syringe through a Teflon capillary catheter. The liquid supply velocity rations of the core and shell were set as 1:3, 1:2 and 1:1 for comparison. Applied a voltage of 15 kV to the coaxial needle and set the speed of the drum receiver to 200 r/min. The distance between the coaxial needle and the drum receiver was 15 cm, the ambient temperature was 30 ℃, and the humidity was 40%. After 8 h of electrospinning, remove the membrane from the drum receiver and put it into an electric heating blast drying oven (GZX-9070MBE, China). It was dried at 40 ℃ for 24 h to remove the surface solvent. A macroscopic view of CS-PEO-Ag-NPs/PCL core-shell electrospun membranes could be seen in Fig. 4(a).

Characterization methods

1. Scanning Electron Microscope (SEM) analysis

The morphology of the Ag-NPs-loaded membrane was observed via SEM (HITACHI SU-1500, Japan). The membranes were cut into 10 mm×5 mm pieces and pasted onto the copper grid, which was sprayed with gold before observation. Used Nano Measurer software to measure the fibers' diameter and then analyzed the frequency distribution of fibers. Energy spectrum analysis was also performed to detect whether Ag-NPs were present in the fibers.

2. Transmission Electron Microscopy (TEM) Analysis

Hold the 230-mesh carbon-coated copper grid with small tweezers and shake it slightly 15 cm away from the coaxial needle for 1 minute while electrospinning, and then coaxial electrospun fibers were deposited on the copper grid to verify the core-shell structure. Applying an accelerating voltage of 100 kV to the copper grid with TEM (Hitachi HT7800, Japan) and then taking TEM images to observe whether the core-shell structure was formed.

3. Fourier Transform infrared spectroscopy (FTIR)

The chemical composition of Ag-NPs-loaded membranes was analyzed by an infrared spectrometer (FT-IR 370, China). PCL, CS and PEO are ground into a fine enough powder, which is then diluted with Potassium Cyanide and a small sample was placed into the machine for testing. A small amount of Ag-NPs liquid was placed in two CaF2 cover slides and put into machine for testing. The membrane was pressed by a press until it was thin enough and then tested, the Each spectrum has a resolution of 2 cm^− 1 and a wave number range of 4000 to 500 cm^− 1.

4. Mechanical Properties

The Ag-NPs-loaded membranes were cut into rectangles of 50 mm×10 mm, clamp the membranes with the two ends of the material testing machine (WDW-1, Z2.5/TSIS 200Pro, Germany) and apply a 0.1kN pre-tightening force, stretch it at 10mm/min until the membranes breaks, take three samples from each Ag-NPs-loaded membrane and take the average value as the final result to obtain mechanical properties.

5. Contact Angle (CA) Measurements

The contact angle value of the Ag-NPs-loaded membranes was measured by a water contact angle measuring instrument (OCA 15EC, China). Take five samples from each electrospun membrane and the water contact angle was measured at 30 s, and take the average value as the final result.

Antibacterial experiment

The antibacterial activities of Ag-NPs-loaded membranes were examined in vitro; the inhibitory effect of the electrospun membrane on Escherichia coli (E. coli) and staphylococcus aureus (S. aureus). Cut the electrospun membranes containing different concentrations of Ag-NPs into a square of 1×1 cm and place each in solid petri dishes evenly coated with 30 µL E. coli and S. aureus. They were incubated at 37 ℃ for 24 h、72 h and 120 h. Then observed and measured the bacteriostatic circle.

Silver ions release

The absorbance of Ag-NPs was measured by a spectrometer, and the standard curve was drawn. Place the electrospun membranes to be tested into centrifuge tubes filled with 50 mL phosphate buffer. After soaking for 4 h, 8 h, 12 h, 24 h, 48 h and 72 h until 192 h, the appropriate amount of liquid to be measured was taken and the same amount of fresh phosphate buffer was added. Used a spectrophotometer to measure the absorbance of the obtained liquid, the standard curve plotted the release rate of silver nanoparticles.

In vitro cell experiment

1. Cytotoxicity test

Cytotoxicity of Ag-NPs-loaded membranes was detected using the Cell Counting Kit-8(CCK-8, Service) as a previously reported approach²⁷.The cell of choice is Human Umbilical Vein Endothelial Cells(HUVECs),the cell suspension was prepared using a treated medium at a cell density of 1×10⁵ cells/mL, and then 100 µL of the suspension was seeded in 96-well plates. After 1, 3 and 5 days of incubation,10 µL of CCK-8 kit was added into each well and placed in a humidified incubator for 4 h. Fresh medium and PCL-prepared membrane were used as a control group. The OD values were measured from a microplate reader (TecanGroupLtd., Switzerland) at wavelengths of 450 nm.

2. Cell adhesion

In order to evaluate the cytocompatibility and potential cytotoxicity of the Ag-NPs-loaded membranes, they were seeded in HUVECs. In brief, the prepared membranes were immersed into 75% ethanol for 1h, then membranes were removed and rinsed tree times with PBS. Finally, membranes were immersed into fresh medium under UV light for 4 h before inoculation. The prepared cell suspension(1×10⁶ cells/mL) was poured into petri dishes containing membrane until the membrane is completely covered, which were then placed into humidified incubator for 4h.After cell adhesion, fresh medium was added into dishes until the surface of membranes were covered, then moved them into the incubator for cultivation at 37 ℃ in 5% CO₂, with the culture medium refreshed everyday thereafter. After 1, 3 and 5 days of incubation, cell proliferation and growth were evaluated by live/dead staining. Live and dead cells were stained by calcein (AM) and PI, membranes were mounted on microscope slides for observation using an inverse fluorescence microscope (Eclipse Ti-U, Nikon Instruments Inc., Japan) at 1, 3 and 5 days, and then captured representative fluorescent images.

In vivo experiment in animal models of mycotic aortic aneurysms

Ag-NPs-loaded membrane were sewn on the outer side of metal vascular stents; The aneurysm grafts were selected from glutaraldehyde-treated bovine pericardium, aseptically processed and dispensed in refrigerated tubes containing PBS. All the experimental materials and instruments including membranes, stents and delivery devices are processed by irradiation sterilization. The integrity of the Ag-NPs-loaded membrane remained stable after irradiation sterilization and repeatly resheath in a 20F delivery system.

9 white pigs, divided into 3 groups, each weighing approximately 60 kg. General anesthesia with tracheal intubation was adopted. The flow chart of the operation on the white pigs was shown in the Fig. 2.

The glutaraldehyde-treated bovine pericardium was cut and sewn outside the body in the shape of a pocket, with an opening length of 4.0 cm. A median abdominal incision was made routinely into the abdomen to reveal the abdominal field. The posterior peritoneum was incised, surrounding tissues were bluntly separated, and several pairs of lumbar arteries were severed by ligation. The abdominal aortic sheath was cut open, the infrarenal abdominal aorta was sharply freed, and blocking bands were applied. After general heparinization, the infrarenal abdominal aorta was clamped. The right anterior lateral wall of the middle segment of the infrarenal abdominal aorta was incised longitudinally for approximately 4.0 cm. The aneurysm graft is sutured to the right anterior lateral wall incision of the abdominal aorta with continuous sutures so that the abdominal aorta forms an aneurysm. The blocking clamps were released, and the diameter of the abdominal aorta, the aneurysm, aneurysm neck and aneurysm length, and the diameter of both iliac arteries were measured. In this way, the model of infrarenal abdominal aortic aneurysm in pigs was constructed (Fig. 3).

A calibrated catheter was placed into the femoral artery, and abdominal aortography was performed, so that the aneurysm diameter, aneurysm neck and aneurysm length were measured, and the corresponding stent-graft was selected. The Ag-NPs-loaded membrane was sutured to the outer wall of the stent-graft (Fig. 3a2). The stent is delivered along the superstiff guidewire to the abdominal aorta and released in the predetermined position. Postoperative arteriography is conducted to check the exclusion completeness of the aneurysm.

Bacterial solution injection was then injected into the aneurysm sac. Pigs group 1 implanted aortic aneurysm, bare metal vascular stent and E. coli and S. aureus of 1.0 ml each; Pigs group 2 implanted aortic aneurysm, Ag-NPs-loaded membrane sutured stent and 1.0 ml of E. coli; Pigs group 3 implanted aortic aneurysm, Ag-NPs-loaded membrane sutured stent and 1.0 ml of S. aureus.

After the procedure, the pigs were administrated with ceftriaxone sodium and penicillin for 1 week. The pigs were tested for the temperature, serum leukocyte, blood biochemistry, etc. They were fed for 1 month, and then euthanized and sacrificed. Samples of the whole infra-renal aorta and iliac arteries were harvested. Gross observation, HE staining and Gram staining.

Characterization of membrane microstructure

As shown in Fig. 4(b-d), a, b and c are nanofibers of Ag-NPs-loaded membranes prepared with core-shell liquid supply ratios of 1:3,1:2 and 1:1, and the normal statistical graph of diameter distribution of three kinds of nanofibers also is shown in Fig. 4(b1-d1). The diameter of three kinds of nanofibers was 530 ± 88 nm,510 ± 89nm,520 ± 299nm. A variety of fiber diameters was also observed by Song et al²⁸.It can be seen from the figure that only a small amount of beads appear when the liquid supply ratio is 1:3 and 1:2. However, when the liquid supply ratio is 1:1, a large number of beads appear, and the nanofibers are disorderly and uneven, this may be due to the fact that the shell solution could not completely cover the core solution, resulting in the disorder of Taylor cone, and the disorderly and uneven electrospinning. It is concluded that the electrospinning effect is better when the liquid supply ratio is 1:3 and 1:2, and 1:2 is the best according to the normal analysis of filament diameter. Figure 4(h)is the energy spectrum analysis diagram of the Ag-NPs-loaded membrane, from which it can be seen that Ag-NPs are present and are distributed in the nanofibers, which can preliminarily prove that Ag-NPs are successfully loaded into the membrane.

TEM is the best method to observe the core-shell structure, Fig. 4(e-g) shows the TEM images of coaxial electrospun fibers. Furthermore, the core material is denser than the shell material and shows in black, whereas the shell material shows in gray. As shown in the figure, the core CS-PEO-Ag-NPs was completely encapsulated by PCL, the core-shell structure of nanofibers were well formed. Additionally, Under the three core-shell flow rates, the diameter difference between the core and shell is 29.36 nm, 84.22 nm, 11.74 nm, the rate of 1:2 was chosen in order to achieve the coverage of the shell, so that the Ag-NPs would be better coated, and membrane can achieve the effect of slow release.

Infrared spectral analysis

Figure 5(a) shows the infrared spectra of the sample membrane without Ag-NPs, PCL, CS and PEO. The stretching vibration peak of PCL was at 1727cm^− 1. 1655cm^− 1 and 1650cm^− 1 was the amide vibration peak of CS and PEO. These three peaks are the unique peaks of the above three materials^{29, 30}. In the sample membrane, there was a characteristic peak corresponding to PCL at 1729cm^− 1, which proved the existence of PCL. The characteristic peaks corresponding to CS and PEO were found at 1598cm^− 1. This conclusion proved the existence of CS and PEO. Figure 5(b) shows the infrared spectra of sample membranes with and without Ag-NPs. The infrared spectra of the two sample membranes were roughly the same, only in the range of 1359 − 1181 cm^− 1. The characteristic peak intensity of the membrane with Ag-NPs was slightly strengthened. The attenuation of the vibration absorption peak of -OH may be caused by the addition of Ag-NPs. In addition, there was a small characteristic peak of Ag-NPs at 2350cm^− 1; these results suggested that Ag-NPs may be physically trapped in the core layer of nanofibers³¹.

Mechanical Properties

Figure 5(c) shows the tensile test diagrams of PCL, CS-PEO/PCL and CS-PEO-Ag-NPs/PCL. It can be seen from the experimental results that the tensile strength and elastic modulus of PCL and CS-PEO/PCL were found as 3.057 ± 0.193 MPa,6.281 ± 0.862 MPa and 1.967 ± 0.071MPa,5.90 ± 0.727 MPa. Compared with pure PCL electrospun membrane, after the addition of CS and PEO with poor mechanical properties, the coaxial electrospun membrane had lower tensile strength but the same elastic modulus, so the rigidity had remained, and it was less prone to deformation. After the addition of Ag-NPs, the tensile strength of CS-PEO-Ag-NPs/PCL was 2.108 ± 0.158 MPa, which was slightly improved, it is speculated that the ionic interaction between positively charged silver atoms and partially negatively charged carbonyl groups of PCL leads to higher intermolecular bonding between polymer chains. Therefore, with the addition of Ag-NPs, the membrane obtains better strength and lower elongation at break³².

As shown in Fig. 5(d), the water contact angle of CS-PEO/PCL membrane is 71.98 ± 2.545°, which is much better than that of pure PCL membrane with water contact angle of 117.91 ± 3.074°. Furthermore, the addition of Ag-NPs did not affect the hydrophilicity of the membrane, the water contact angle is only slightly different after the addition. The water contact angle of the Ag-NPs-loaded membranes was about 70.87 ± 3.928°, showing neither too hydrophobic nor too hydrophilic, which is conducive to cell adhesion and growth.

Antibacterial performance

The antibacterial activity of the Ag-NPs-loaded membrane was determined against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, and these two are representatives of positive and negative bacteria³³. As shown in Fig. 6(a), when the concentration of Ag-NPs was 1wt%, the antibacterial effect on E. coli was not obvious, while the concentration increased to 2wt%, 3wt%, the antibacterial is obvious. Figure 6(b) shows that when the concentration of Ag-NPs was 1wt%,2wt%, the antibacterial effect on S. aureus was just a little. However, when the concentration was increased to 3wt%, the inhibition circle was more obvious than that of 1wt% and 2wt%. There was no significant change in these inhibition circles until 120 h. Due to the bacteriostatic mechanism of Ag-NPs, Ag-NPs do not disappear after killing the bacteria, it can be inferred that the Ag-NPs-loaded membrane containing Ag-NPs has a long-term antibacterial effect.

Ag-NPs release performance

The release rate of Ag-NPs was studied by measuring the absorbance of Ag-NPs in liquid at a time point by spectrophotometer, converting it into concentration by the standard curve of Ag-NPs, and then drawing the release curve as shown in Fig. 7(a). As can be seen from the figure, all three membranes showed sudden release in the first 24 h, in the case of Ag-NPs-loaded membranes, it may be that some fibers are not fully coated with Ag-NPs, causing Ag-NPs to be present on the surface and resulting in sudden release. However, after 24 h, the release rate of coaxial electrospun membranes obviously slowed down, and the release rate of the uniaxial electrospun membrane remained fast, and there was a significant difference in the release rate between them. The results show that the drug release rate could be controlled by coaxial electrospinning. With this function, membrane can prevent Ag-NPs sudden release, the sudden release of Ag-NPs dose is large, produce toxic side effects, failed to play a good therapeutic effect³⁴.The membranes prepared in this experiment have the function of drug-sustained release, and can be released for a long time, so as to achieve a long-term antibacterial effect.

Cytotoxicity and cell adhesion of Ag-NPs-loaded membranes

In order to observe the cell morphology and the distribution of live and dead cells on the membranes after 1, 3 and 5 days of culture, HUVECs was tested by live-dead staining, and results are shown in Fig. 7(b-g, b1-g1). In the 1 d, the number of cells on the two groups of membrane was balanced. However, up to 3 d and 5 d, cells on the Ag-NPs-loaded membrane proliferated more compared to the pure PCL membrane, because the Ag-NPs-loaded membrane has better hydrophilicity and is more conducive to cell adhesion. But there were a little more dead cells, which was within the normal range, cells death may be due to the cells gathered too densely, leading to the death of individual cells due to hypoxia. The results prove that the Ag-NPs-loaded membrane was not cytotoxic and was beneficial to cell adhesion.

Figure 7(h) shows the absorbance of each group, which is used to analyze cell survival rate. After 1, 3 and 5 days of culture, the OD value of each group gradually increased, and cell proliferation trend was basically the same. According to the significance difference analysis, there was a statistically significant difference between three groups only on the first day. By 3 days of culture, the Ag-NPs-loaded membrane and PCL membrane both had higher cell survival rate than control group, there was no statistically significant difference between them. Until 5 days of culture, the cell survival rate of Ag-NPs-loaded membrane and PCL membrane still higher than control group, and there was no statistically significant difference between them. This shows that Ag-NPs-loaded membrane was nontoxic, and the addition of appropriate amounts of Ag-NPs did not inhibit cell survival and proliferation.

In vivo experiments pathology and results

All experimental pigs survived well at 1 month after surgery. Ultrasonography showed patency of the abdominal aorta, renal artery and iliac artery. The stents were also well positioned. After euthanasia, the specimens and implanted stents those Ag-NPs loaded membrane remaining intact were collected properly.

General pathology showed that there is dirty fluid in the lumen of abdominal aortic aneurysm and other portions have formed mechanized thrombosis. In other words, bacterial infections appeared. Meanwhile cross-sectional morphology of all stents remained a basically regular circle, with plenty of thrombosis between stent and arterial wall.

As shown in Fig. 8(a1-c1, a2-c2), Microscopic pathological HE staining showed that there was no obvious inflammatory cell infiltration and no obvious inflammatory reaction in Pigs group 1. No significant inflammatory response was observed, reflecting good survival of E. coli and S. aureus. The inflammatory response of Pigs group 2,3 was persistent and intense, this proved that Ag-NPs released by Ag-NPs-loaded membrane have an inhibitory effect on bacteria. The inflammatory response of Pigs group 2 was stronger than that of Pigs group3 which reflected that the Ag-NPs covered vascular stent was less effective in antibacterial inhibition against S. aureus than against E. coli. In addition, the stent surface was covered by neoplastic endothelium composed predominantly of fibroblasts and the arterial wall was infiltrated by inflammatory cells. Meanwhile there was no significant dissection of smooth muscle in the middle layer of the artery.

The type and number of bacteria in the samples can be observed by Gram staining. As shown in Fig. 8(a3-c3, a4-c4), In the aneurysm wall and surrounding tissues of Pigs group 1, massive aggregations of E. coli were expressed in saffron color with staining assistance, the bare metal stent had no effect on preventing bacterial proliferation. E. coli was almost invisible in the aneurysm wall and surrounding tissues of Pigs group 2, which further proves that Ag-NPs released by Ag-NPs-loaded membrane have an inhibitory effect on bacteria. As the bacterial solution injected into the aneurysm of Pigs group 3 was S. aureus, it was difficult to observe S. aureus under the microscope due to the characteristics of Gram staining.

Stent-graft infection following endovascular repair of aortic diseases poses a challenging clinical problem, and the prognosis could be poor and lethal. There are two scenarios of stent-graft infection. First, the emergency endovascular repair of a rupture or pending-rupture mycotic aortic aneurysm. Stent-grafts are infected after implantation into the infection lesions. Staged drainage and long-term use of systemic antibiotics have been reported to be effective to control the stent-graft infection³⁵. If the infection cannot be controlled, open surgery conversion is recommended to remove the stent graft and the whole infected lesion even though the procedure is complicated³⁶. Second, there were studies reported that non-infected aortic diseases were treated by endovascular procedure, but the stent-graft got infected during the perioperative period or months even years after procedure³⁷. The prognosis in this case is even worse, as the infection is often caused by multi-resistance bacteria.

So, if we can develop an antimicrobial endograft, it may be applied in two scenarios in clinic. First, in mycotic aortic aneurysms, if local antimicrobial shield can be established by preventing the infection of endograft with Ag-NPs-loaded membranes, there would be a chance to conduct staged transcutaneous drainage and systemic antibiotics. And in this way, the mycotic aortic aneurysms might be cured by endovascular repair. Second, for non-infected aortic diseases, some patients are at high risk of developing endograft infection, such as the patients with diabetes, Immunodeficiency diseases, long-term use of glucocorticoids, poor fitness, or innutrition, et al. They would suffer from endograft infection at any time after the procedure or in long-term follow-up³⁸. The long-lasting of Ag-NPs is required to prevent the stent-graft infection. If Ag-NPs could exist in the affected area for a long time, it might prevent endograft infection in selected cases.

In this study, a drug-loaded vascular membrane with antibacterial function and sustained drug release function, called Ag-NPs-loaded membrane, was prepared by coaxial electrospinning technology. Drug-loaded nanofibers were constructed through electrospinning. Due to the drug-loaded membrane prepared by blend electrospinning can only attach the drugs to the surface of fibers, the drug will be quickly lost after blood erosion after implantation into the body, and the long-term antibacterial effect cannot be achieved. Coaxial electrospinning is a kind of electrospinning technique of preparing fibers with a core-shell structure³⁹. Compared with traditional electrospinning, coaxial electrospinning can encapsulate drug in the core layer of fibers to avoid the rapid loss of drug due to blood erosion and has better performance in drug loading and slow release⁴⁰. This technology has been widely used in the biomedical field⁴¹.

In the coaxial electrospinning process, the liquid supply ratio of core-shell solution directly affects the formation quality of nanofibers and the forming of core-shell structure. We set three groups of parameters, when the liquid supply ratio is 1:2, the distribution diameter of nanofibers in the membrane is the most uniform, and the core-shell structure is well formed, and the drug is well wrapped in the inner layer. Therefore, we chose the liquid supply ratio of 1:2 as the parameter for membrane preparation. In addition, the membrane covered on stent in human blood vessels would be subjected to pressure from blood flow; therefore, the mechanical properties of membranes are essential for tissue engineering applications⁴². With the addition of CS and PEO, the tensile strength of the membrane prepared by coaxial electrospinning reached 2.108 ± 0.158 MPa. Although the tensile strength of Ag-NPs-loaded membrane is lower than that of PCL membrane prepared by uniaxial electrospinning, Ag-NPs-loaded membrane can still meet the mechanical performance requirements of human arterial blood vessels.

Hydrophilicity is an important index to evaluate the potential biological properties of vascular stent membranes. The membrane with better surface hydrophilicity is conducive to cell adhesion and proliferation, but the hydrophilicity will also affect cell migration. Therefore, the surface hydrophilicity of the membrane should not be too good. Due to the good hydrophilicity of CS and PEO, their addition can improve the hydrophilicity of the membrane. The water contact angle of the Ag-NPs-loaded membranes was about 70.87 ± 3.928°, showing good hydrophilicity, but not too hydrophilic. The effectiveness of cell inoculation is one of the important factors to evaluate the biocompatibility of membrane. Cell adhesion test showed that Ag-NPs-loaded membrane was more conducive to cell adhesion and proliferation than PCL membrane. Meanwhile, nanoparticles that show antimicrobial activity, including Ag-NPs, especially in the case of high concentration will be toxic to human cell, and even harmful to human health⁴³. The cytotoxicity of the Ag-NPs-loaded membrane was detected by CCK-8. The results showed that the addition of Ag-NPs had little toxicity to HUVECs, the Ag-NPs-loaded membrane showed good biocompatibility and safety.

How to maintain the long-term drug release function and long-term bacteriostatic effect of the membrane is an urgent problem to be solved in tissue engineering. At present, amoxicillin, vancomycin and cephalosporin are commonly used as antibacterial drugs. In this study, we chose Ag-NPs as the drug loaded on the membrane. Studies had found that Ag-NPs produce antibacterial effects by binding to and penetrating the bacterial cell wall, after entering the cell wall, Ag-NPs will cause structural changes, disrupt the biochemical processes of DNA, cell enzymes and respiratory systems, and lead to bacterial death^{44, 45}. After killing bacteria, Ag-NPs will be free from bacteria to continue to produce antibacterial effects. When testing its antibacterial properties, we selected E. coli and S. aureus as the representatives of negative/positive bacteria. The results showed that when the addition of Ag-NPs reached 3 wt%, the antibacterial effect of the membrane was obvious, and the inhibition circle remained unchanged within 120 h. Due to the lack of longer observation, it could only be preliminarily inferred that the Ag-NPs-loaded membrane has long-term antibacterial function, which needs further verification in the future. In the release test of Ag-NPs, the release rate of Ag-NPs-loaded membrane prepared by coaxial electrospinning was much slower than that of the uniaxial electrospun membrane. The sustained drug release function of Ag-NPs-loaded membrane is attributed to the coaxial electrospinning, which encapsulates Ag-NPs in the inner layer of nanofibers. The sudden release phenomenon in the first 24 h may be due to the small amount of Ag-NPs were attached to the surface of nanofibers, which were quickly released into the PBS after washing^{46, 47}, and the remaining Ag-NPs wrapped by nanofibers were slowly released⁴⁸. However, the detection of the release of Ag-NPs-loaded membrane was only carried out in the PBS solution in vitro, and the detection cycle was only 196 h, which is not enough to prove that the Ag-NPs-loaded membrane has a long-term sustained release function after implantation in vivo, so further research is needed.

Because the porcine aorta has similarities with the human abdominal aorta in terms of morphology, physiology, and branch vessel distribution⁴⁹, it is appropriate model for studying abdominal aortic aneurysms and evaluating the efficacy and safety of metal vascular stents with Ag-NPs-loaded membranes⁵⁰. Results of HE staining of porcine aneurysm grafts showed that the pig implanted with stent covered with Ag-NPs-loaded membrane had a significant inflammatory reaction. It was proved that Ag-NPs-loaded membrane successfully released Ag-NPs into the aneurysm cavity and inhibited the bacteria. However, there was no obvious inflammatory cell infiltration in the pig implanted without covered stent, proving that the bacteria had a good living environment and were not affected. In addition, Gram staining results showed that bacteria were almost invisible on the aneurysm graft of pig implanted with stent covered with Ag-NPs-loaded membrane. On the contrary, a large number of aggregated bacteria were observed in the porcine aneurysm implanted without covered stent. Pathological findings are presented well and easy to learn. Finally, the results indicated that antibacterial effects and good biocompatibility were explored at the stents with Ag-NPs-loaded membranes.

Limitation:

The mechanical properties and thickness of the membrane should be further optimized, so that it not only can cover the existing covered stent, but also can realize the exclusion function of the artificial vascular membrane of the current stent-graft. Then it can be directly combined with nitinol stent skeleton. In this way, the profile of delivery system of this antibacterial stent-graft could be significantly reduced, so as to realize the delivery through peripheral arteries such as femoral artery. Moreover, the release behavior of Ag-NPs on the membrane can be optimized to make it controllable, such as PH, temperature or electromagnetic response, which would fulfill different antibacterial time-histories requirements in clinic.

Vascular membrane of aortic stent-graft loaded with Ag-NPs was constructed by coaxial electrospinning technology, which had the slow-release function of Ag-NPs as to exert long-term antibacterial function. The membrane nanofibers were composed of PCL as the shell, CS and PEO as the core, and Ag-NPs is added to the core. The inhibition function of the Ag-NPs-loaded membrane on E. coli and S. aureus was proved by bacteriostatic experiments. In vivo experiments showed that Ag-NPs-loaded membrane has optimal biocompatibility and antibacterial effects. It might provide a promising method for endovascular treatment of mycotic aortic pathologies.

PTFE: Polytetrafluoroethylene

PET: Polyethylene terephthalate

Ag-NPs: Silver nanoparticles

PCL: Polycaprolactone

CS: Chitosan

PEO: Polyethylene oxide

E. coli: Escherichia coli

S. aureus: Staphylococcus aureus

PLD: Pulsed laser deposition

LBL: Layer-by-layer

PLA: Polyactic acid

SEM: Scanning electron microscope

TEM: Transmission electron microscopy

Ethics approval and consent to participate:

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Shanghai Changhai Hospital Ethics Committee. Written informed consent was obtained from individual or guardian participants.

Consent for publication:

Not applicable.

Availability of data and materials:

All data and materials generated or analyzed during this study are included in this published article.

Competing interests：

The authors declare that they have no competing interests.

Funding:

This study was supported in part grants from National Natural Science Foundation of China (Grant Nos: 52175474, 52275498, 81970208, and 82270505).

Authors’ contributions：

Hg.Z , Pc.M , Jx.F and R.F conceived the idea; Qx.H and Zw.H designed the study and contributed to vitro experimental study; Pc.M, Jx.F R.F and Zw.H contributed to vivo experimental study; all authors contributed to the writing and revisions.

Acknowledgements:

The authors acknowledge funding support from the National Natural Science Foundation of China (Grant Nos: 52175474, 52275498, 81970208, and 82270505).

Authors’ information:

Qingxi Hu ^1,2,3, Zhenwei Huang ¹, Haiguang Zhang^1,2,3*, Pengcheng Ma ^5*, Jiaxuan Feng ^5*, Rui Feng^4*

Institute：

1 Rapid Manufacturing Engineering Center, School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China

2 Shanghai Key Laboratory of Intelligent Manufacturing and Robotics, Shanghai University, Shanghai 200072, China

3 National Demonstration Center for Experimental Engineering Training Education, Shanghai University, Shanghai 200444, China

4 Shanghai General Hospital, Shanghai Jiaotong University, Shanghai, People’s Republic of China

5 Department of Vascular Surgery, Changhai Hospital, Naval Medical University, Shanghai 200433, China

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Coaxial electrospun Ag-NPs-loaded endograft membrane with long-term antibacterial function treating mycotic aortic aneurysm.

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Preparation of the Ag-NPs-loaded Membrane

1. Scanning Electron Microscope (SEM) analysis

2. Transmission Electron Microscopy (TEM) Analysis

3. Fourier Transform infrared spectroscopy (FTIR)

4. Mechanical Properties

5. Contact Angle (CA) Measurements

Antibacterial experiment

Silver ions release

1. Cytotoxicity test

2. Cell adhesion

In vivo experiment in animal models of mycotic aortic aneurysms

Results

Characterization of membrane microstructure

Infrared spectral analysis

Mechanical Properties

Antibacterial performance

Ag-NPs release performance

Cytotoxicity and cell adhesion of Ag-NPs-loaded membranes

In vivo experiments pathology and results

Discussions

Limitation:

Conclusion

Abbreviations

Declarations

References

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