Mesoporous Silica Nanoparticles-Encapsulated Agarose and Heparin

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Mesoporous Silica Nanoparticles-Encapsulated Agarose and Heparin as Anticoagulant and Resisting Bacterial Adhesion Coating for Biomedical Silicone Fan Wu, tingting xu, Guangyao Zhao, Shuangshuang Meng, Mimi Wan, Bo Chi, Chun Mao, and Jian Shen Langmuir, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

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Mesoporous

Silica

Nanoparticles-Encapsulated

Agarose and Heparin as Anticoagulant and Resisting Bacterial Adhesion Coating for Biomedical Silicone Fan Wu,† Tingting Xu,†,‡ Guangyao Zhao,† Shuangshuang Meng,† Mimi Wan,*,† Bo Chi,‡ Chun Mao,*,† and Jian Shen† †

National and Local Joint Engineering Research Center of Biomedical Functional Materials,

Jiangsu Key Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China ‡

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science

and Light Industry, Nanjing Tech University, Nanjing, 211816, China KEYWORDS: silicone, mesoporous silica nanoparticles, heparin, agarose, anticoagulant effect, resisting bacterial adhesion

ABSTRACT: Silicone catheter has been widely used in peritoneal dialysis. The research missions of improving blood compatibility and the ability of resisting bacterial adhesion of silicone catheter have been implemented for the biomedical requirements. However, most of modification methods of surface modification were only able to develop the blood-contacting biomaterials with good hemocompatibility. It is difficult for the biomaterials to resist bacterial

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adhesion. Here, agarose was selected to resist bacterial adhesion and heparin was chosen to improve hemocompatibility of materials. Both of them were loaded into mesoporous silica nanoparticles (MSNs), which were successfully modified on the silicone film surface via electrostatic interaction. Structures of the mesoporous coatings were characterized in detail by dynamic light scattering, transmission electron microscopy, Brunauer-Emmett-Teller surface area, thermogravimetric analysis, fourier transform infrared spectroscopy, scanning electron microscope, and water contact angle. Platelet adhesion and aggregation, whole blood contact test, hemolysis and related morphology test of red blood cells, in vitro clotting time tests, and bacterial adhesion assay were performed to evaluate the anticoagulant effect and the ability of resisting bacterial adhesion of the modified silicone films. Results indicated that silicone films modified by MSNs had good anticoagulant effect and could resist bacterial adhesion. The modified silicone films have potential as blood-contacting biomaterials that attributed to their biomedical properties.

Introduction

Many natural and synthetic polymeric materials with good blood compatibility have been used as artificial grafts in vivo. The blood-contacting artificial grafts should possess ideal surface bioproperties including high hemocompatibility and the ability to enhance antibacterial property.1-5 Surface modification, one of the most effective tactics, was utilized to improve the hemocompatibility of artificial blood-contacting grafts.6,7 Until now, these methods included surface modification of the blood-contacting artificial grafts by heparin, PEG, zwitterionic groups and corresponding polymer based on different chemical or physical methods.8-10 These


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methods are only able to enhance the hemocompatibility of the artificial blood-contacting grafts, but cannot provide the ability of inhibiting bacterial adhesion. However, the biological failures of medical operation are often associated with microbial plaque accumulation and bacterial infections. So it is also necessary to provide the ability of inhibiting bacterial adhesion for the biomaterials by some additional ways, such as the integration of biomaterial and antibacterial drugs, the decoration of antibacterial silver nanoparticles on the surface of biomaterials, and so on.11-13 In order to meet the requirement of more medical treatment, novel surface modification methods are still the focus of the biomedical research. Conventionally used coating such as calcium phosphate, polymer and nanotubes can realize the release of drugs from material surface. However, high drug-loaded amount and long-term release (more than 10 days) of the anti-microbial drugs cannot be guaranteed by these above coating methods owing to lack of pores and high surface area.14-16 Mesoporous silica coating can potentially realize sustained release of drugs and high drug-loaded amount, as mesoporous silica had been proven to be ideal drug releaser that can be attributed to its tunable pore size, high surface area, and long-range ordered pore structure.17-21 Therefore, mesoporous silica coating with heparin and agarose was introduced on the surface of silicone film, realizing high drug-loading amount and slow-release of specific drugs in the presence of mesoporous structure. In this case, agarose was chosen to realize the purpose of inhibiting bacterial adhesion and heparin was selected as anticoagulant drug.22,23 Both of them were encapsulated successively by MSNs, and then immobilized on silicone films by electrostatic interaction. The bioactivity of the released drug was evaluated by testing the biocompatibility of the samples. Platelet, whole blood cell attachment, hemolysis and related morphology test of red blood cells (RBCs), in vitro


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clotting time tests, and bacterial adhesion test were carried out to study the effect of MSNs coating on the silicone films, one kind of blood-contacting biomaterial. Experimental Section Materials. Cetyltrimethylammonium bromide (CTAB) was obtained Shanghai Lingfeng Chemical Reagent Co., Ltd. Sodium hydroxide, agarose, tetraethoxysilane (TEOS), sodium chloride, potassium phosphate monobasic, disodium hydrogen phosphate, potassium chloride, ethanol, ammonium nitrate, sulfuric acid, nitric acid, glutaraldehyde were received by Sinopharm Chemical Reagent Co., Ltd. (3-Aminopropyl) triethoxysilane (APTES) and heparin were bought from Aladdin Chemistry Co., Ltd. The blood used in this work was from rabbit supplied by Nanjing Jiangning District Qinglong animal breeding farm. Synthesis of Agarose Loaded Mesoporous Silica Nanoparticles (AMSNs). The synthesis of AMSNs was similar to the procedure from previous literature.24 Briefly, 480 mL distilled (DI) water was mixed with 3.5 mL NaOH (2 M) solution. Afterwards, 1.02 g CTAB and 0.5 g agarose dissolved in NaOH solution by magnetic stirring and increasing the temperature to 80 ℃. After CTAB and agarose were completely dissolved, 4.67 g TEOS was added dropwised into above solution, then stirred at 80 ℃ for 2 h. The precipitations were obtained by centrifugation and washed by ethanol and DI water for several times, respectively. The precipitations were dried at 60 ℃ overnight. Ammonium nitrate (1.6 g) was added to 610 mL ethanol, and then the precipitations were dispersed in it, heated for 40 min, which ensured the removal of CTAB. After washing with ethanol and DI water several times, the final products can be obtained. MSNs without agarose were prepared by a similar procedure for comparison.


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Synthesis of Agarose and Heparin Loaded Mesoporous Silica Nanoparticles (AHMSNs). The heparin loading was performed by adding 0.5 g AMSNs into heparin solution (50 ml, 10 mg/mL) under magnetic stirring at 37 ℃ for 12 h. In order to remove unloaded heparin, the mixtures were washed DI water several times and dried overnight at 37 ℃ in vacuum.

Preparation of AHMSNs Coating Immobilized on Silicone Films (AHMSNs-Si). The preparation process of AHMSNs modified silicone films was described in Figure 1. Silicone films (Si, 1*1 cm2) were immersed in sulfuric acid solutions and nitric acid solutions with the volume ratio of 3:1 to produce hydroxyl groups on films surfaces.25 APTES solution was prepared by 5 mL APTES mixed with 95 mL ethanol. The as-treated silicone films were soaked in the APTES solution of ethanol at room temperature for 24 h under argon gas.26 The obtained samples were treated by ultrasonic washing several times with DI water. Amination of silicone films were put into a culture dish and then added 10 mL AHMSNs suspension (5 mg/mL)， which was kept in an oven at 37 ℃ for 5 h. AHMSNs can self-assemble into the surfaces of amination of silicone films via electrostatic interaction. The prearation of MSNs or AMSNs coating immobilized on silicone films were similar to this procedure for comparison. The obtained samples were named as MSNs-Si and AMSNs-Si.


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Figure 1. A schematic illustration for the coating AHMSNs on Si surface and the anti-fouling properties of modified surface. Characterization. Nanoparticle morphologies were observed by using a transmission electron microscopy (TEM, HITACHI H-7650, Japan). Nano-ZS 90 Nanosizer (Malvern Instruments, UK) was used to analyze the size distribution of nanoparticles. The Brunauer-Emmett-Teller (BET) surface area and pore size were tested by applying ASAP2050 system. The fourier transform infrared spectroscopy (FTIR) of the samples were detected by using a Varian Cary 5000 fourier transform infrared spectrophotometer, and the samples were mixed with KBr at a ratio of 3:97 (w/w) and then pressed as a thin disk for testing. Thermogravimetric analysis was performed using the Perkin Elmer Instruments (TGA, Diamond TG/DTA, USA) with a 10 K/min heating rate in N2 atmosphere. To gain the information of surface morphology, the silicone films were observed on a scanning electronic microscope (SEM, JEOL 6300, Japan). Static water contact angles were got by measuring water dropler (3 µL) on the silicone films (Rame-Hart, Inc.,USA).


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Drug Release Performance. The release profile of the heparin was studied by carring out toluidine blue method.27 Firstly, the calibration curve was obtained by the following procedure: 2 mL heparin aqueous solution with sure concentration was mixed with the 3 mL toluidine blue solution and then shaken vigorously for 3 min. Hexane (3 mL) was added to the mixed solution and then shaken violently ensured that the toluidine blue–heparin complex was collected by organic phase. The absorbance of the rest of toluidine blue in the aqueous solution was measured at 631 nm with a visible spectrophotometer (Agilent 8453, Agilent Technologies) and the concentration of heparin solution could be known. To evaluate the release of heparin, AMSNs-Si was placed in an individual tube with 5 mL of phosphate buffer solution (PBS, pH=7.4) at 37 ℃. At specific time, 3 mL PBS was collected and then fresh PBS was supplemented to keep a sink condition. PBS was collected until there was no longer any heparin release. Platelet Adhesion Assay. Platelet-rich plasma (PRP) was obtained from fresh rabbit blood which was centrifuged at 1000 rpm for 15 min. Then, the samples were immersed in PRP at 37 ℃ for 1 h. After washing with PBS for three times, the samples were immersed in 2.5% glutaraldehyde solution (in PBS) at 4 ℃ for 2 h. After washing with PBS, the samples were dehydrated through ethanol soltuions with DI water (50, 60, 70, 80, 90, 95, and 100%) for 0.5 h at each concentration and dried in drying condition at room temperature. The morphology of platelets attached to the samples was analyzed by using SEM.28,29 Whole Blood Adhesion Tests. Si, MSNs-Si, AMSNs-Si, AHMSNs-Si incubated with 1.0 mL of fresh rabbit whole blood for 1 h at 37 ℃ in the 24-well tissue culture plate. After washing with PBS several times, the samples were immersed in 2.5% glutaraldehyde solution (in PBS) at 4 ℃ for 2 h. PBS was used to remove the residual glutaraldehyde, and then the samples were


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dehydrated through ethanol soltuions with DI water (50, 60, 70, 80, 90, 95, and 100%) for 30 min at each concentration and dried in a desiccator at 25 ℃. Finally, the samples were goldcoated using a sputter-coater and analysed by SEM.9,30 Hemolysis and Related Morphology Test of RBCs. In order to separate RBCs, fresh rabbit blood was centrifuged at 2500 rpm for 10 min, and the supernatant was discarded. RBCs were washed with NaCl solution (0.9%) several times and resuspended in NaCl solution (0.9%). 2.5 mL of RBC suspensions (2%) were mixed with the same volume of saline water, and the samples were added to the mixed solution. The mixtures were incubated at 37 ℃ for 3 h. Negative control and positive control were produced by adding 2.5 mL of diluted 2% RBC suspensions to the same volume of NaCl solution (0.9%) and DI water, respectively. After incubation, the samples were separated by centrifugation. Microplate reader (Multiskan FC, Thermo, USA) was used to measure the optical density of the supernatant at 545nm. The calculation formula of percent of hemolysis was listed as follows: % hemolysis=[(ODtest – ODneg)/(ODpos – ODneg)]×100%. ODtest, ODpos and ODneg are the absorption values of the test sample, positive control (water), and negative control (NaCl solution, 0.9%), respectively.30,31 Morphological changes of RBCs were observed by using light microscopy. Si, MSNs-Si, AMSNs-Si, AHMSNs-Si were put in centrifuge tubes. 4 mL RBC suspensions (2%) were added into each centrifuge tube. The samples were incubated with RBC suspensions (2%) for 1.5 h, and then were centrifuged to obtain RBCs pellet. RBCs pellet was diluted in NaCl solution (0.9%), and dropped on glass slides, and observed under Olympus BX41 microscope and photographed with a Olympus E-620 camera (Olympus Ltd., Japan).32,33


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In Vitro Clotting Time Tests. The influence of sample surface on the coagulation system was evaluated by measuring activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). For the vitro coagulation time tests, the platelet-poor plasma was obtained from fresh rabbit blood by centrifugation at 3000 rpm for 15 min. The samples were soaked in platelet-poor plasma and incubated for 60 min at 37 ℃. The coagulation time was measured in an semi automated coagulometer (RT-2204C, Rayto, USA).34,35 Bacterial Adhesion Assay. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used to test the ability of resisting bacterial adhesion of the samples. Si, MSNs-Si, AMSNsSi, AHMSNs-Si were put in a 24-well plate. Each well was added bacterial suspension (2 mL). The samples cultured with bacteria at 37 ℃ for 24 h. Afterwards, the samples were washed by PBS and immobilized using 2.5% glutaraldehyde solution at 4 ℃ for 2 h. After washing with PBS, the samples were dehydrated through ethanol soltuions with DI water (25, 50, 75, and 100%) for 30 min at each concentration, respectively. The dried samples were sputter-coated with gold and the morphology was observed by SEM.36,37 Results and Discussion Characterization of AHMSNs. The structures of MSNs and drug-loaded MSNs were displayed clearly by TEM images. Figure 2a, b and c showed the representative TEM image of MSNs, AMSNs and AHMSNs. All of them had good mesoporous structures. Long-ranged ordered mesoporous channels can be observed on MSNs (Figure S1). Meantime, the addition of agarose and heparin didn’t change the mesoporous structure of AMSNs and AHMSNs. Dynamic light scattering (DLS) examination (Figure 2d) revealed that MSNs, AMSNs, AHMSNs all had a


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narrow size distribution. Average sizes of samples were bigger than that of TEM results, because DLS gave hydrodynamic size of nanoparticles.38 The nitrogen adsorption–desorption isotherms and pore size distributions of the samples were shown in Figure S2. Furthermore, the physiochemical properties of MSNs, AMSNs and AHMSNs were summarized in Table S1. BET surface area of MSNs was 686 m2g-1, which decreased to 532 m2 g-1 with the addition of agrose. When heparin was loaded, the final BET surface area nearly had no change with AMSNs. These results indicated that added drugs having a few effects on mesoporous structure.

Figure 2. TEM images of (a) MSNs, (b) AMSNs, and (c) AHMSNs. (d) Size distribution of different samples. In order to illustrate that the agarose and heparin were encapsulated into MSNs successfully, the samples were measured with FTIR spectra and thermogravimetric analysis. As can be seen in Figure 3, MSNs, AMSNs, AHMSNs all showed the characteristic peaks at around 1620 and


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3500 cm-1 which can be ascribed to O-H group.39 The peaks at 465, 800, 1050 cm-1 should come from the stretching vibrations of Si-O-Si bonds.39,40 The absorption peak around 933, 1075 cm−1 were observed on AMSNs and AHMSNs samples owing to the 3, 6-anhydrogalactose and C–O stretching vibration from agarose.41 Absorption peak of the group C=O in heparin at around 1730 cm-1 was observed on AHMSNs.23 These results indicated that both drugs of agarose and heparin were introduced in AHMSNs and the thermogravimetric analysis curves (Figure 3c) also confirmed it. The MSNs nanoparticles yielded weight loss of 11.1% attributed to the existence of water in the mesopores. Comparing the MSNs curve with the AMSNs curve showed that the content of agarose accounts for about 21.3% of the total. An additional weight loss of 5.7% could be observed for AHMSNs which indicated that heparin was loaded successfully.

Figure 3. (a) FTIR spectra of MSNs, AMSNs, and AHMSNs, (b) an amplified portion of FTIR spectra. (c) Thermogravimetric analysis curves of MSNs, AMSNs, and AHMSNs, respectively. Characterization of AHMSNs-Si. It was found by SEM (Figure 4a, b) that the surface of pure silicone film was smooth, and the MSNs with nanoscale can be observed clearly on silicone surface. The same happened to the AMSNs-Si and AHMSNs-Si films (Figure 4c, d). The thickness of the coating was detected by using SEM technique. Figure S3 showed that the thickness of the AHMSNs-Si coating was about 30 µm. Furthermore, the AHMSNs-Si was


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immersed in PBS solution for 7 days at 37 ℃ to test the stability of the AHMSNs coating. The surface morphology of the AHMSNs-Si didn’t change after being immersed in PBS solution for one week (Figure S4). The water contact angle of MSNs-Si (95.6±3.9°) decreased in comparison with blank Si (121±2.9°) (Figure 4e, f). The MSNs-grafted silicone film was more hydrophilic. After drug loaded, the water contact angle of AMSNs-Si and AHMSNs-Si decreased greatly (Figure 4g, h), which attributed to the hydrophilicity of agarose and heparin.22


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Figure 4. SEM images of (a) Si, (b) MSNs-Si, (c) AMSNs-Si, and (d) AHMSNs-Si, respectively. The static water contact angle of (e) Si, (f) MSNs-Si, (g) AMSNs-Si, and (h) AHMSNs-Si. Drug Release Performance. The release behaviour of heparin from the AHMSNs-Si was investigated in PBS solution. Figure 5 displayed that the release of heparin was rather fast at the first day and around 40% of heparin can be released. The burst release phenomenon can be attributed to heparin located on the outer surface of the coating can easily diffuse to the medium. After one day, a lower releasing rate was observed and heparin release in this system can last as long as 30 days. According to the literatures, the dissolving temperature of agarose was usually above 60 ℃.41,42 Therefore, agarose remained in the materials under 37 ℃. The purpose of immobilizing of agarose in our manuscript was to resist bacterial adhesion. Hence, the reservation of agarose on the surface of silicone film instead of releasing in the blood can protect the surface of silicone film from bacterial adhesion. The purpose of immobilizing heparin in the materials was to prevent coagulation of the whole blood, so the release of heparin to the blood was necessary.

Figure 5. The release profiles of heparin in PBS at 37℃.


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Platelet Adhesion. Platelets played a significant role in the coagulation process. The combination of mutually fused platelets and the insoluble fibrin will cause thrombus.43,44 Biomaterials which showed low platelet adhesion property were in great demand as an anticoagulation idea.45 To examine hemocompatibility of AHMSNs-Si surface, platelet or blood cell adhesion property were evaluated.29,46,47 Figure 6a-c showed SEM pictures of the original Si, MSNs-Si and AMSNs-Si after 60 min contact with the PRP. All of them adsorbed many platelets, and the adhered platelets were distorted with pseudopodia. Yet, under the similar test conditions, it was also clearly observed that there was almost no platelets on the AHMSNs-Si surface (Figure 6d), indicating that the AHMSNs-Si samples do not affect platelets activation.

Figure 6. SEM images of (a) Si, (b) MSNs-Si, (c) AMSNs-Si, and (d) AHMSNs-Si exposed to PRP of rabbit for 1 hour, respectively. Whole Blood Adhesion. Same tendency was observed when the silicone were contacting with fresh rabbit whole blood. Large amount of adherent blood cells on the Si, MSNs-Si and AMSNsSi film were observed (Figure 7a-c). None of blood cell adhesion was found on the AHMSNs-Si


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film (Figure 7d). It can be deduced that thrombus was not easy to form on the surface of AHMSNs-Si, which was attributed to the anticoagulant effect of heparin.

Figure 7. SEM images of (a) Si, (b) MSNs-Si, (c) AMSNs-Si, and (d) AHMSNs-Si after being contacting with rabbit whole blood for 1 hour. Hemolysis and Related Morphology Test of RBCs. Hemolysis rate of the blood cells is a significant index to estimate the biocompatibility of biomaterials. Hemolysis has been known as the breakage of the RBC’s membrane. It will cause the hemoglobin release into the surrounding blood.27 And the released adenosine diphosphate can accelerate the formation of clotting and thrombus due to it is in favor of the assembly of blood platelets.48 Actually, silicone film and silica are regarded as “Generally Recognized As Safe” by the FDA due to their good biocompatibilities. As shown in Figure 8a, Si, MSNs-Si and AMSNs-Si had low hemolysis rates (1.36% ,1.24% and 1.33%). After encapsulated heparin, the hemolysises of the AHMSNs-Si were much lower than the medical materials standard (

Mesoporous Silica Nanoparticles-Encapsulated Agarose and Heparin

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