Preparation of the Silicone-Modified Antimicrobial Polyethylene Endotracheal Tube (PE ETT)

Ventilator-associated pneumonia (VAP) is a common complication for mechanical ventilation patients in ICU and trachea cannula, repeated cannula and mechanical ventilation way and mechanical ventilation time are one VAP vulnerable factor [1]. Endotracheal tube (ETT) is artificial biomaterial and it does not have antibacterial property. Once inserted into human body, it becomes the potential source of infection because pathogenic bacteria is absorbed on its surface for growing and breeding to form bacterial biofilm which can adhere dissociated bacteria and avoid bacteria from killing and phagocytosing under the effect of antibiotics and body’s immune system [2]. In order to prevent infection after biomaterial placement, the antibacterial coatings are painted on its surface, such as nanoZnO [3], TiO2 photocatalytic materials [4], Ag-loaded coating [5], etc to inhibit bacteria from adhering on the surface of cannula to form bacterial biofilm.


Introduction
Ventilator-associated pneumonia (VAP) is a common complication for mechanical ventilation patients in ICU and trachea cannula, repeated cannula and mechanical ventilation way and mechanical ventilation time are one VAP vulnerable factor [1]. Endotracheal tube (ETT) is artificial biomaterial and it does not have antibacterial property. Once inserted into human body, it becomes the potential source of infection because pathogenic bacteria is absorbed on its surface for growing and breeding to form bacterial biofilm which can adhere dissociated bacteria and avoid bacteria from killing and phagocytosing under the effect of antibiotics and body's immune system [2]. In order to prevent infection after biomaterial placement, the antibacterial coatings are painted on its surface, such as nanoZnO [3], TiO 2 photocatalytic materials [4], Ag-loaded coating [5], etc to inhibit bacteria from adhering on the surface of cannula to form bacterial biofilm.
Among that, there is more research for Ag-loaded coatings in antibacterial materials that Ag+ ion can destroy cell division and mutual respiratory function of bacteria and virus, having stronger bactericidal ability and inhibiting pathogen activity [6,7]. However, there is less research for Ag-loaded antibacterial coating in biomedical materials. The majority research is concentrated on modified or composited antibacterial materials and it is rearly verified for moldability and biocompatibility of antibacterial coatings. Furthermore, Ag + ion is sedimented on the surface of biomaterial or in the meso pore through ion exchange and physical absorption [8]. The poor binding force with base material leads to antibacterial coating shedding, influencing on its antibacterial property and safety. So people prepare antibacterial coatings by recombinating polymer and Ag + ion to enhance binding force between antibacterial coating and base material and improve compactibility, such as polyvinylpyrrolidone [9], polyurethane [10], chitosan [11], etc. However, there is less research for silicone materials. Silicone materials contain many kinds of active group and present excellent cohesiveness and film-processing property [12]. The hydrophobic groups of Si-CH 3 and -CH 3 could reduce coating surface energy and improve chemical inertness. Its polymer is compatible with tissue and blood of human body and has certain anticoagulation in effect so it is widely applied in biomaterials [13].
In this study, silicon compound of SiO 2 /KH570 and SiO 2 /KH570/ MTES prepared through the sol-gel process with TEOS as inorganic silicon source, KH570 and MTES as the organicsilicon source, were used to prepare series silicone-modified antimicrobial films on conventional PE ETT by mixing silver antibacterial agent. Effects of different alkoxysilanes of KH570 and MTES content on the properties of silicon compound solutions and their films were investigated. We also study the influence of different Ag-SiO 2 content on the stability of the silicone-modified antimicrobial solution and the sterilizing rate of their films. A transparent silicone-modified antimicrobial PE ETT and their properties were introduced in this paper. Its biosecurity is verified in pyrogen test, hemolysis test and oral mucous irritation test. Such silicone-modified antibacterial coatings are hoped to be used in surface coating field of medical materials or medical apparatus and instruments to improve infection situation in the hospital.

Preparation of antimicrobial coating
The silicone-modified antimicrobial solution were synthesized by sol-gel method and mechanical mixing as described in Figure 1

Characterization of the materials
The hardness and adhesion of all the films were measured by pencil hardness tester and Squaring-off, respectively. Infrared spectrums (IR) of two gels of TEOS/KH570/MTES and TEOS/KH570/MTES/ Ag-SiO 2 were performed on IR spectrometer. The micrograph and transmittance of the resulting films were measured by scanning electronic microscope (SEM) and a UV spectrometer. The antimicrobial property was evaluated by the method described as GBT 21866-2008 Test method and effect for antibacterial capability of paints film, and the precipitation of Ag + ion was measured by inductively coupled plasma mass spectrometry (ICP-MS). The biosecurity used in the field of medical materials was evaluated by Pyrogen test, hemolysis test and oral mucous irritation test.

The hardness and adhesion of the films and the stability of the solutions
Organosilicone with various active groups like Si-OH, R1-COO-R, Si-O-Si, etc, is especially preferred to enhance the adhesion of hybrid films on many substrates. KH570 with long chain is an excellent organic modified silicone, which can improve the flexibility of films, and MTES with two stiff groups of CH 3 gives the film an improved hardness and is considered to have influence on reducing the interface energy to prevent protein adhesion. So we investigated the ratio of TEOS, KH570 and MTES in order to make better performance films and solutions. The hardness and adhesion of the obtained films including TEOS/KH570, TEOS/KH570/MTES and TEOS/KH570/ MTES/Ag-SiO 2 , and the storage stability of their solutions are showed in Table 1. The result silicon solution of TEOS/KH570 was flocculated and their film has worse adhesion when the ratio of TEOS and KH570 is 18:3. With increasing the KH570 content, the TEOS/KH570 films exhibit strong adhesion, and their solutions have good storage stability at room temperature for 72 h, but the hardness reduce from 7H to 1H. The films with TEOS:KH570 ratio of 15:6 and 13:8 exhibited desirable hardness of 3H and 5H and excellent adhesion, were investigated further to prepare a higher-quality basic film for the antimicrobial film on PE ETT. MTES used to prepared TEOS/KH570/MTES films can exactly enhance the hardness of the films but sacrifice the adhesion property.

Constituent phase of the films
The IR spectrum of resulting films of TEOS/KH570/MTES (15:6:1) and TEOS/KH570/MTES/Ag-SiO 2 (15:6:1:0.8) are shown in Figure 3(a) and 3(b), respectively. Obviously, the IR spectrums are similar and the analyses are gave in Table 2. The broad peak at 3440 cm -1 was assigned to O-H bonds, the strength of that peak in (b) is stronger, because of much more Si-OH groups on the surface of Ag-SiO 2 . The presence of a strong peak between 1000 cm -1 and 1110 cm -1 was attributed to  the stretching vibration of Si-OH or Si-O-Si group, which indicated that TEOS, KH570 and MTES had been reacted sufficiently. The C-H stretching peak at 2850～3000 cm -1 in the spectrum can be an indication of Si-OC 2 H 5 and-CH 3 groups mainly from KH570 and MTES. The IR spectrums reflect that TEOS/KH570/MTES and Ag-SiO 2 have well compatibility, which is consistent with the SEM analysis.

Surface morphology of the films
The surface of coatings is uniform and flat in Figure 4(a) without granule and phase separation. Meanwhile, Ag antibacterial agent content is little in Figure 4(b) coatings imbedded by silicone network polymer, it is not found Ag antibacterial agent particles and the surface is flat. It shows SEM micrographs of the films of TEOS/KH570/MTES and TEOS/KH570/MTES/Ag-SiO 2 , which were prepared by dipcoating the solution on ETT and baked at 80°C for 1 h. The films is smooth and crack-free, the SEM results show that the films are uniform and amorphous, there is no significant inorganic domain size shown in the two series of films. It indicates that there is a crosslinking of silica sol with alkoxysilanes, and Ag-SiO 2 has been homogeneously dispersed in TEOS/KH570/MTES/Ag-SiO 2 film. Figure 5 shows the optical transmittance spectra of ETT (a), ETT coated with TEOS/KH570/MTES film (b) and ETT with TEOS/KH570/ MTES/Ag-SiO 2 film (c). The PE ETTs before or after coating with the solution maintained the high transmittance, which are 87.55%, 87.45% and 87.39%, respectively. There is no absorption can be observed in the figure, which indicates that nanoparticles have been homogeneously dispersed in the films, all the films on ETT's surfaces are homogeneous, and the condensation reaction between the hydroxyl groups of the hydrolysates of the alkoxysilanes and the hydroxyl groups on the surface of the colloidal silica from SiO 2 sol and Ag-SiO 2 have occurred. It means that antibacterial coating can increase transmittance which keeps the original appearance of ETT and improves its view definition in the application process, convenient for clinical observation.

Analysis for the stabilities of Ag + ion
In order to research the stability of silver, the antimicrobial film was dipped in hot water for 24 h, then the extraction of Ag + ion was measured by ICP-MS, and the results are showed in the Table 4. The Ag + ion concentration of the deionized water and the antimicrobial solution of TEOS/KH570/MTES/Ag-SiO 2 are 0.002 µg/L and 0.025×1000 µg/L, respectively. We can see that silver in the antimicrobial solution is about 10000 times more than deionized water. But, in the sample A and sample B, the Ag + ion concentration are 0.005 µg/L and 0.009 µg/L, respectively, only a little higher than deionized water. That affirms that the Ag-SiO 2 has been homogeneously dispersed in the antimicrobial film and firmly chemically bonded with the silicon compound solution. It means that the firm chemical bond is established between Ag antibacterial agent and composite silicone sol and the cohesiveness between antibacteraial coating and ETT base material surface is strong. It can keep antibacterial property for a long time to extend ETT's use time to certain extent and decrease drawing times thus to reduce inflammation occurance probability.

Biosecurity
Pyrogen test, hemolysis test and oral mucous irritation test are conducted for the blank ETT and the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 in order to investigate security of the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 . The results of pyrogen test are shown as data in Table 5. The total rose temperature is 0.7°C for blank ETT and 0.03°C for the siliconemodified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 , which are fit for pyrogen inspection range regulated in the standard of Chinese Pharmacopoeia (Version 2010). It is indicated no pyrogenic effect caused by materials for the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 and better cohesion between antibacterial coatings and ETT which is not easy for dipping and precipitation. The  The solution diluted 1000 times with deionized water C 0.025   hamster oral mucous irritation test of the blank ETT and the siliconemodified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 are shown in Figure 6. The extracts of the blank ETT and the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 showed no irritation reactions to golden hamsters oral mucous. Both the right and left sides of oral mucosals were epithelial integrity, they had no proliferation of epithelial cells, flattened, degeneration or necrosis, submucosal had no congestion, edema, inflammatory infiltration and necrosis and or other pathological changes. The two groups' oral mucosal tissue reactions of acute exposure were scored 0, showing no irritation.
Above all, the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 is fit for biosecurity use requirement for biological materials.

Discussion
In this paper, the silicon compound of SiO 2 /KH570 and SiO 2 /  dipping liquid injecting into rabbit blood is guaranteed not to cause temperature rise reaction and keeps rabbit's temperature.
The results of hemolysis test are shown as data in Table 6. Absorbance A in the table is the average absorbance of three same samples. The average absorbance of negative control liquid and positive control liquid is 0.0110 and 0.8097 respectively. The test complies with requirements and the results are valid. The hemolysis ratio of the blank ETT (H1) and the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 (H2) is 0.5885% and 0.7512% respectively. The hemolysis ratio should be less than 5% according to the standard of Biological Evaluation of Medical Devices: GB/T16886.4-200 Temolysis Test Inspection. It is found that hemolysis test of the silicone-modified antimicrobial PE ETT with ratio of 2.6% Ag-SiO 2 is qualified and it is testified that antibacterial ETT is safe to red cell of human body.
According to GB/T16886.10-2005 standard, the results of the golden KH570/MTES prepared through the sol-gel process with TEOS as inorganic silicon source, KH570 and MTES as the organicsilicon source, were used to prepare series silicone-modified antimicrobial films on conventional PE ETT. By researching and analysising the characterization of antimicrobial PE ETT, we determine the proportion of the TEOS/KH570/MTES/Ag-SiO 2 (15:6:1:0.6~1.0) films with ratio at least 2.6% to prepare silicone-modified antimicrobial PE ETT. The silicone-modified antimicrobial PE ETT coated by TEOS/ KH570/MTES/Ag-SiO 2 (15:6:1:0.6~1.0) films with ratio at least 2.6% has the high transmittance of above 87%, high hardness of 5H, and strong adhesion, exhibits excellent antimicrobial property with the sterilization rate up to 93.5%. What's more, it is proven in the pyrogen test, hemolysis test and oral mucous irritation test that the siliconemodified antimicrobial PE ETT has excellent biocompatibility and biosecurity of requirement for biological materials and equipped with excellent physical and biological properties. It is hoped to be used in the field of coated materials for medical materials or medical equipment to improve infection situation in the hospital.