Dual-functional anticoagulant and antibacterial blend coatings based on gemini quaternary ammonium salt waterborne polyurethane and heparin

Abstract

Complications such as thromboembolism and bacterial infection, arising from the widespread use of implanted medical devices, are serious problems for current clinical treatment. In this work, dual-functional anticoagulant and antibacterial blend coatings were designed and fabricated from gemini quaternary ammonium salt waterborne polyurethane (GWPU) emulsion and heparin aqueous solution. The results demonstrate that the L-lysine-derivative gemini quaternary ammonium salt (GQAS) in GWPU endows the coating with excellent non-releasing antibacterial ability and simultaneously the release of heparin provides superior anticoagulant activity. Moreover, the controlled release of heparin is achieved by the ionic interaction between the positive charge in GQAS and the negative charge in heparin. On the basis of the dual-functional anticoagulant and antibacterial properties of GWPU/heparin blend coatings, this work proposes a facile strategy to achieve syncretic performances in biomaterials, and these coatings would have the potential to improve the application of implanted devices.

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1. Introduction

The high incidence of serious complications caused by the increasing clinical applications of implanted medical devices (such as vascular catheters, vascular scaffolds, heart valves, artificial hearts, etc.) arouses considerable attention.^1–3 Blood coagulation and bacterial infection are the most common ones for blood-contacting materials. Implanted devices coated with a layer of anticoagulant and antibacterial coatings may be the most promising and valuable method to moderate the complications. When the implanted device comes in contact with blood, adsorption of blood proteins takes place within a few seconds at the solid–liquid interface, followed by the adhesion and aggregation of platelets and the activation of the intrinsic coagulation and complement system, finally resulting in the formation of a thrombus.⁴ Regarding to thromboembolism, heparin is an important and the most popular anticoagulant for clinical applications. It is believed that heparin can strongly interact with antithrombin III to prevent the formation of fibrin clot.⁵ Chemical immobilization of heparin and the construction of heparin-releasing system are two general methods exploited to improve hemocompatibility of materials.^6–8 For instance, Lv et al. proposed a new solvent system for blending polyurethane and heparin.⁷ Young et al. introduced heparin onto poly(ethylene terephthalate)–acrylic acids (PET-AA) by using PEO as a spacer.⁹ However, simply blending matrix with heparin cannot keep long lasting effect. In contrast, when heparin is fixed on the chain, the efficient anticoagulant activity will decrease. Therefore, the contradiction of two competitive aspects remains to be solved.

Another associated significant consideration is anti-bacterial infection. Initial adhesion of bacteria on device surfaces is the first and critical step in the development of an infection, then bacteria proliferate to form a colony or biofilm and further hematogenous spread, and lastly systemic toxicity may unfortunately happen.^10,11 In respect of bacterial infection, preventive strategies are mainly in view of three aspects: “anti-adhesion”, “release-killing” and “contact-killing”.^12,13 Anti-adhesion ability is generally obtained from the modification of polymeric materials with antifouling agents (e.g., poly(ethylene glycol), zwitterions), as an auxiliary means, which can repel the adhesion of bacteria.^10,14 Release-killing capability is introduced into materials by impregnation with bacteria-killing chemicals, such as antibiotics, biocides (e.g., phenols, halogens), and heavy metals (e.g., silver ions).^15,16 Apparently, this approach suffers from several inherent disadvantages owing to the release of antibacterial agents, for example, limited service time as the amounts of active agents decrease gradually and adverse effects resulting from the released antibacterial chemicals in the surrounding environment.^17,18 Recently, polymeric coatings with contact-killing ability attract broad attention because of the advantages such as good environmental and chemical stability, low systemic toxicity, reduced likelihood of generating resistance to the active agents, long-term durability as well as increased antibacterial efficiency and selectivity thanks to higher local concentration. Two main methods have been developed for the synthesis of non-leaching biocidal polymers with contact-killing ability. One is the bonding of biocidal functional moieties to the preformed polymers, and the other is the synthesis of biocidal functional monomers and their subsequent polymerization.^19,20 Among them, quaternary ammonium salts (QASs, cationic surfactants) as antibacterial agents, by disrupting the integrity of bacterial membranes and inducing the leakage of intracellular components from bacterial cells, are most widely explored.^21–23 They exhibit high antibacterial abilities without bacterial resistance forming and good stability of their antibacterial activities even after they were immobilized into polymer chains.

Polyurethanes (PUs) have been extensively applied in the biomedical field, such as coating material, on account of the excellent mechanical properties and good biocompatibility compared to other polymers.^24,25 However, the coagulation and bacterial infection are still the issues remained to be solved, and many researchers have developed numerous approaches to improve hemocompatibility or/and antibacterial performance of polyurethane coatings over the past decades.^26–30 For example, Bernacca et al. studied the antithrombogenicity of surface-modified PUs included covalent attachment of anticoagulant agents (e.g., heparin, taurine).³¹ Grapski et al. reported the contact biocidal PUs by incorporating pyridine in the chain extender followed by quaternization.¹¹ However, to the best of our knowledge, few approaches could simultaneously improve the anticoagulant and antibacterial performances of polyurethane coatings. The double release coating system of anticoagulant and biocidal agents may suffer the defect of long-term efficacy, and the anticoagulant and antibacterial multilayer films via layer-by-layer assembly need tedious preparation procedures.^26,27

Herein, to simultaneously avoid the thrombogenesis and prevent the bacterial infection involved in the application of indwelling medical devices, we propose a simple design of blend coatings based on the nontoxic biodegradable waterborne polyurethanes (WPUs) drawn partly from previous researches.^32,33 The novel anticoagulant and antibacterial coatings are made up of heparin and WPU containing gemini quaternary ammonium salts (GQASs). The GQAS components incorporated into the polymer backbone not only endow the coatings with outstanding and long-term bactericidal ability, but also be used to anchor heparin for long-lasting antithrombogenic property by ionic interaction with negatively-charged heparin.^34,35 The preparation of desired products is compose of three processes: firstly, the synthesis of L-lysine-derivative gemini quaternary ammonium salt chain extender (EG12) with bactericidal function; secondly, the synthesis of nontoxic biodegradable waterborne polyurethane containing hydrophilic poly(ethylene glycol) (PEG) and the synthetic cationic diamine monomer via a conventional process without any organic solvent involved; lastly, the preparation of the blends based on gemini quaternary ammonium salt waterborne polyurethane (GWPU) emulsion and heparin aqueous solution. The synthetic polyurethanes were characterized by proton nuclear magnetic resonance (¹H NMR) spectroscopy. The surface properties of these blend coatings were measured by zeta potentials (ZPs) and water contact angles (WCAs). The release test of heparin from GWPU/heparin coatings was determined by toluidine blue method. Their hemocompatibility was evaluated via protein adsorption, platelet adhesion, and anticoagulant activity. The antibacterial performance was identified through plate counting method. In short, the feasibility of these blends as anticoagulant and antibacterial coatings was preliminary investigated.

2. Experimental section

2.1. Materials

Heparin sodium salt (molecular weight 6000–20 000, ≥180 USP units per mg), toluidine blue (BS) and sodium dodecyl sulfate (SDS, 98.5%) were obtained from Aladdin Reagent (China) and used without additional purification. Fibrinogen (FG, from bovine plasma, Type I–S, 65–85% protein, ≥75% of protein is clottable) was purchased from Sigma (U.S.A) and used without purification. PEG (molecular weight 1450, Dow Chemical, U.S.A, 99%) and polycaprolactone (PCL, molecular weight 2000, Dow Chemical, U.S.A) were dehydrated at 90–100 °C under vacuum for 2 h before use. Isophorone diisocyanate (IPDI, BASF, Germany, 99.5%) was redistilled under vacuum before use. L-Lysine was used as received. Micro BCATM Protein Assay Reagent kits were obtained from PIERCE (U.S.A). Unless otherwise noted, other chemical reagents and solvents of reagent grade obtained from commercial suppliers were used without additional purification.

2.2. Synthesis and preparation of materials

2.2.1. Synthesis of L-lysine-derivative GQAS chain extender (EG12). The L-lysine-derivative GQAS chain extender with two hydrophilic head groups and two long hydrophobic alkyl tails was synthesized according to a previously reported procedure.³³ In brief, N,N,N′,N′-tetramethyl-L-lysine ethyl ester was firstly quaternized with alkyl halide (1-bromododecane) in isopropanol to gain the alkylammonium endowed with biocidal activity. Then, the ethyl ester in resultant was replaced by 1,3-propane diamine as a spacer arm. Subsequently, Boc-lysine was introduced through the classic activated ester condensation reaction. Finally, the cationic diamine chain extender was obtained after the Boc-protected amine groups were deprotected and converted to primary amine groups.

2.2.2. Synthesis of nontoxic biodegradable GWPU emulsion with the synthetic alkylammonium chain extender. A series of nontoxic biodegradable waterborne polyurethanes were synthesized employing a two-step polymerization “prepolymerization/emulsification methods” based on IPDI, PEG, PCL and chain extenders (L-lysine and EG12). The feed molar ratios are listed in Table 1. In the prepolymerization stage, IPDI and 0.1% organic bismuth were added into stirred anhydrous PEG and PCL at 70 °C under a dry nitrogen atmosphere. After reaction for 1 h, the mixture was cooled to 30 °C, and then EG12 was added into the reaction mixture with continuous stirring for 10 min. In the second emulsifying process, the prepolymer was poured into L-lysine aqueous solution for emulsification with high-speed stirring (1000 rpm) under ambient temperature for 3 h, and meanwhile dilute sodium hydroxide solution to neutralize the carboxyl groups of L-lysine was added into aqueous emulsion dropwise. WPUs without GQAS as a contrast were prepared with a similar process as described above.

Table 1 Contents of different coatings

Samples^a	Feed molar ratio (mmol)					Heparin in coatings (μg)
	IPDI	PCL	PEG	EG12	L-Lysine
a The GWPUs are denoted as WPUX and GWPUX/h, where X is the molar content of EG12 in total chain extenders.
WPU	3.00	0.75	0.25	0.00	1.70	0
WPU/h	3.00	0.75	0.25	0.00	1.70	70
GWPU30	3.00	0.75	0.25	0.50	1.20	0
GWPU30/h	3.00	0.75	0.25	0.50	1.20	70
GWPU50	3.00	0.75	0.25	0.85	0.85	0
GWPU50/h	3.00	0.75	0.25	0.85	0.85	70

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2.2.3. Preparation of heparin blended gemini quaternary ammonium salt waterborne polyurethane (GWPU/heparin) coatings. The ultimate blend coatings were prepared via mixing the various WPU emulsions with isochoric heparin aqueous solution under ultrasonic vibration and stirring at room temperature. The concentration of heparin was 1 mg mL⁻¹. As a comparison, the samples without heparin were made by using double distilled water instead. The corresponding coatings were fabricated by casting the mixture onto clean hydrophilic glass plates. To obtain smooth coatings without other defects, the water was firstly evaporated in air at room temperature, and then the coatings were dried in a drying oven at 60 °C for 24 h, and lastly under vacuum at 37 °C for 24 h. The thickness of the coatings is about 200 μm tested by vernier caliper.

2.3. Methods

2.3.1. Characterization and instruments. ¹H NMR (400 MHz) spectra were obtained with a Varian unity Inova-400 spectrometer, using tetramethylsilane (TMS) as an internal standard in DMSO-d₆. Zeta potentials of GWPU emulsions diluted 10 times were measured with phase analysis light scattering (ZETA-SIZER, MALVERN Nano-ZS90, United Kingdom), using a DTS 1060C clean disposable zeta cell as the container and the Smoluchowski module in water at 25 °C at an angle of 90°. Each group of samples tested at least three times for the similar results. Water contact angles of the coating surfaces were performed with a Drop Shape Analysis System DSA 100 (Kruss, Hamburg, Germany).³³ The measurement was carried out by putting a drop of deionized water (3 μL) on the air-facing side of the coating at room temperature, and the static water contact angle as a function of the contact time was recorded in the stable temperature and humidity environment via TraceMan functional module. This procedure for each sample was performed three times and a typical curve was presented for each sample.^36,37

2.3.2. Release test of heparin from GWPU/heparin coatings. The GWPU/heparin samples (1 × 1 cm²) were immersed in phosphate buffered saline (PBS) solution (10 mL, pH 7.4) at 37 °C. Periodically, taking out the supernatant (2 mL) of heparin release solution and adding a fresh one so as to maintain a sink condition. Then, the quantitative release solution (2 mL) was added to toluidine blue solution (3 mL) with adequately vibrating for 2 h. Subsequently, hexane (3 mL) was added and the mixture was shaken well to make blue–heparin complex be extracted into the organic layer. At last, the toluidine blue in the aqueous phase was determined with UV-1800PC spectrophotometer (Shanghai Mapada Instrument Co. Ltd., China) by measuring the absorbance at 631 nm, and the released amount of heparin in the aqueous solution was calculated in accordance with the standard curve. Three parallel specimens were carried out for each sample and the results were the statistical averages with the standard deviation of different specimens.⁷

2.3.3. Biocompatibility assays.
2.3.3.1. Protein adsorption. Human fibrinogen (Fg) as the model protein was utilized to study the protein adsorption on the blend coating surfaces, traditional biomedical polyether urethane (PEU) as a control. The samples (1 × 1 cm²) were immersed in PBS solution and equilibrated at 37 °C for 1 h. Subsequently, they were incubated in the protein solution (1 mg mL⁻¹ in PBS) for 2 h at 37 °C. After adsorption, the coatings were slightly rinsed with PBS and deionized water successively, and then transferred to 2 wt% SDS (0.05 M NaOH) solution at 37 °C under mechanical oscillation for 30 min to remove the adsorbed protein. The desorption solution followed was colored with a Micro BCA™ protein assay reagent kit in water bath at 60 °C for 1 h, and the absorbance of light intensity at 562 nm was measured by UV spectrophotometer mentioned above. Finally, the amount of protein adsorption was calculated with reference to the standard curve of Fg. The assays were performed in triplicate for each sample and the results were the mean values of three replicates with the standard deviation.³⁸
2.3.3.2. Platelet adhesion. Fresh blood samples were collected from a healthy experimental rabbit provided by the Huaxi Animal Center and collected into vacuum tubes containing anticoagulant sodium citrate (anticoagulant to blood ratio, 1 : 9, v/v). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was obtained from blood centrifuged at 1000 rpm and 4000 rpm for 15 minutes, respectively. Platelet adhesion experiments were carried out according to the process as described briefly below. At first, the samples (1 × 1 cm²) were immersed in phosphate buffered saline and equilibrated at 37 °C for 1 h. After removing the PBS, 1 mL of PRP was introduced and the samples were incubated with PRP at 37 °C for 2 h. Then, the PRP was removed and the samples were rinsed five times with PBS. The adhering platelets on the samples were fixed with 2.5 wt% glutaraldehyde in PBS at 25 °C for 2 h afterwards. Finally, after being washed with PBS solution, the samples were dehydrated with a series of gradient ethanol aqueous solutions (30, 50, 70, 90, and 100 vol% ethanol; 30 min in each mixture), and then dried in vacuum for 24 h. Platelet adhesion and activation were recorded under a scanning electron microscope (SEM, Inspect F, FEI Company, U.S.A). Each sample was observed 3 times by using three individual specimens.³⁹ The animal studies were carried out with the approval from the Ethics Committee of Sichuan University and in compliance with the Principles of Laboratory Animal Care of the National Institutes of Health, China.
2.3.3.3. Anticoagulant activity. The antithrombogenicity of GWPU/heparin coatings was evaluated by measuring the vitro coagulative time tests including activated partial thromboplastin time (APTT) and prothrombin time (PT) with a semi-automatic blood coagulation analyzer (CA-50, Sysmex, Japan). The tests were carried out as follows: the samples (0.5 × 0.5 cm²) were firstly incubated with 0.2 mL of PPP for 30 min at 37 °C in 96-well plates. Then, 50 μL of the incubated PPP for APTT was added into a test cup and warmed in the analyzer for 60 seconds, followed by the addition of 50 μL APTT agent (Dade Actin Activated Cephaloplastin Reagent, Siemens; incubated 10 min before use). Next, they were mixed well and incubated at 37 °C for 3 min. Afterwards, 50 μL of 0.025 M CaCl₂ solution was added. Finally, the APTT was measured by the analyzer. For the PT test, 50 μL of the incubated PPP solution was mixed well with 100 μL of PT agent (Thromborel® S, Siemens; incubated 10 min before use) at 37 °C for 2 minutes, and then the PT was obtained. Five replicate specimens were measured for each sample.⁴⁰

2.3.4. Antibacterial identification. The representative organism Staphylococcus aureus (S. aureus, gram-positive, ATCC 6538) were used to assess the antibacterial activity of different coatings. The nutrient media were prepared from the Mueller–Hinton broth (Hangzhou Microbial Reagent), dissolved in distilled water and sterilized in an autoclave (LDZX-30FBS, Beijing Shenan Instrumentarija, China) at 121 °C, 103 kPa for 20 min. Firstly, a single colony was inoculated in the nutrient media overnight at 37 °C in an incubator (DHP-9082, Shanghai Qixin Scientific Instrument Co., Ltd, China). Then, the obtained bacterial cells were diluted to 10⁶ CFU mL⁻¹. Next, 2 mL of diluted bacteria was added into the well of 96-well plate that contains corresponding sample (0.5 × 0.5 cm²). Then, the 96-well plate was kept in the incubator for 24 h at 37 °C. After the coatings were rinsed 3 times with the nutrient media, some samples were immersed in 2 mL of media and treated with ultrasonic oscillation. A 100 μL portion of each shaken bacterial solution was diluted appropriately and respectively spread evenly on Luria–Bertani medium agar plates. The incubation period was conducted in the incubator at 37 °C for 24 h, and then the visible cells of each plate were counted by quantifying the CFUs. The assessment was performed in triplicate. To observe the morphology of the bacteria on the surface, the rest coatings were treated with 2.5 wt% glutaraldehyde in PBS at 4 °C for 1 night to fix the bacteria. Then, the coatings were washed with PBS, and subjected to a drying process by immersing in a series of gradient alcohol aqueous solutions (30, 50, 70, 90, and 100 vol% ethanol) for 15 min each time, and freeze dried at last. Bacteria adhesion on the coating surfaces was observed by SEM.⁴¹

3. Results and discussion

3.1. Characterization of GQAS waterborne polyurethanes (GWPUs)

A series of nontoxic biodegradable GWPUs were synthesized without any organic solvent using IPDI, PEG, PCL, L-lysine and EG12, as shown in Fig. 1. The content of GQAS was controlled by changing the feed molar ratios of EG12 in chain extenders, as listed in Table 1. ¹H NMR results demonstrate that the GWPUs have been successfully synthesized, as presented in Fig. S1.†

Fig. 1 The structure components of GWPU. Therein, PEG and PCL are used as the soft segments; EG12 and L-lysine are used as the chain extenders.

Firstly, zeta potentials (ZPs) of these synthetic GWPU emulsions are measured and the data are summarized in Table 2. The zeta potential value of WPU is −26.4 ± 1.0 mV, while the values of GWPU30 and GWPU50 are 19.9 ± 0.5 mV and 22.9 ± 1.2 mV, respectively. It is quite clear that GQAS can change the electronegativity of the emulsions for its intrinsic cationic. Furthermore, these zeta potentials are increased along with the increment in EG12 content within the scope of the research. Nevertheless and interestingly, the zeta potential values do not show any apparent changes after the emulsions mix with heparin aqueous solution, for instance, the zeta potential of GWPU30/h is 18.8 ± 0.7 mV when that of GWPU30 is 19.9 ± 0.5 mV. It is considered that the low content of heparin could not effectively affect the zeta potential.

Table 2 Zeta potentials and blood clotting times of various samples

Samples	Blood	WPU	WPU/h	GWPU30	GWPU30/h	GWPU50	GWPU50/h
a ZP is used as the abbreviation of zeta potential; -: untested; *: no coagulation.
ZP^a (mV)	-	−26.4 ± 1.0	−27.2 ± 0.3	19.9 ± 0.5	18.8 ± 0.7	22.9 ± 1.2	23.0 ± 0.3
APTT (s)	20.5 ± 0.3	20.9 ± 1.1	*	527.8 ± 2.6	*	148.5 ± 5.2	*
PT (s)	8.0 ± 0.2	9.0 ± 0.4	19.2 ± 0.6	43.2 ± 1.4	55.8 ± 1.7	*	*

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To further understand the surface properties of the prepared coatings, the water contact angles (WCAs) are tested and plotted in Fig. 2. In summary, the WCAs all decrease dynamically with the increase of contact time. At the beginning of water contacting, the initial WCAs of the samples increase with the increasing content of GQAS. For example, the initial WCA of WPU/h is 42°, while the initial WCAs of GWPU30/h and GWPU50/h are 54° and 65°, respectively. After 35 seconds, the WCAs of all samples become much lower than the initial ones. It is interesting that the decreasing amplitude of WCAs for GWPUs is much larger than that of samples without GQAS. For instance, the decreasing amplitude of WCA for GWPU50/h is more than 50° (from 65° to 11°), while the WCA for WPU/h decreases about 17° (from 42° to 25°). On the other hand, after the GWPUs mix with hydrophilic heparin, the blend samples exhibit better hydrophilicity. For example, the initial WCAs of GWPU30 and GWPU30/h are 69° and 54°, and the balance WCAs (at 35 s) of GWPU30 and GWPU30/h are 20° and 9°, respectively. Above all, both GQAS and heparin are in favor of enhancing their hydrophilicity. The reason might be that the hydrophilic PEG segments, GQAS and heparin migrate and aggregate on the surfaces. The migration and aggregation of amphipathic GQAS in surface of GWPU to decrease the WCA has been observed and demonstrated by our previous work, which is due to the GQAS chains tend to gradually move to the water–air interface during drying process.⁴² The rapid decrease of WCAs recorded at the short exposure times might be thanks to the rearrangements of the structural units (such as the hydrophilicity groups of quaternary ammonium salts) on the surfaces, which are beneficial to the application of blood-contacting materials.³⁶

Fig. 2 Static water contact angles dependent on time of various coatings.

3.2. Hemocompatibility of GWPU/heparin coatings

As the coatings of blood-contacting implanted devices, the hemocompatibility is the primary consideration. Protein adsorption, platelet adhesion and anticoagulant activity are widely used to evaluate the blood compatibility of materials from different perspectives. Fibrinogen (Fg) is one of the most abundant proteins present in blood and can mediate the platelet adhesion and aggregation process due to its binding to the platelet GP IIb/IIIa receptor. In addition, platelet adhesion and activation on the surfaces will trigger blood coagulation, leading to thrombus formation.^43,44 Thus, protein adsorption and platelet adhesion are used to investigate the hemocompatibility of GWPU/heparin coatings. The results of Fg adsorption are plotted in Fig. 3. Compared with Fg adsorption (2.0 μg cm⁻²) of PEU, WPU and WPU/h nearly show no protein adsorption. However, the amounts of Fg adsorption dramatically increase to about 8.5 μg cm⁻² for GWPU30 and GWPU50, which are considerably higher than the control sample. Fortunately, the absorption evidently decreases when heparin is incorporated into the coatings. It is well known that protein adsorption is closely related to the hydrophilicity and charge of material surfaces.⁴⁵ Generally, the Fg adsorption will decrease when the hydrophilicity increases and the electropositive decreases. Although the WCAs of GWPUs are smaller than that of WPU, the GWPUs bring out higher amounts of Fg adsorption. It's highly possible that EG12 cationic surfactants on the coating surfaces lead to the electropositivity of the surfaces, and then the negatively charge Fg is strongly attracted. When the coatings blend with heparin, the corresponding Fg adsorption of blend coatings decreases compared with GWPUs, which is consistent with its better hydrophilicity and also the surface electropositivity decreases by neutralizing with the negatively charged heparin. It should noticed that the effect of heparin on surface electropositivity is different from the zeta potential of bulk emulsion. The immigration and aggregation of heparin on the surface of blend coatings will strongly affect the surface electropositivity while the little amount of heparin in whole bulk emulsion will slightly affect the zeta potential.

Fig. 3 FG absorption of different coatings with or without GQAS.

Interestingly, the samples containing EG12 show excellent antiplatelet adhesion property as other samples, even though they exhibit high Fg adsorption which is believed that could intensely promote platelet adhesion.⁴³ The SEM images of platelet adhesion and activation on the surface of the blend coatings are shown in Fig. 4. For controlled PEU, a substantial number of platelets adhere and aggregate on the PEU surfaces (shown at a magnification of 1000×). In addition, platelets on the surface are mostly discrete, wherein some even show irregular shapes and grow up to the pseudopodia at a magnification of 10 000×. These phenomena are indicative of platelet aggregation and activation. However, in the experimental groups, only few platelets are observed on the surface of the coatings without heparin and less platelets are found on the heparin-containing surfaces in the SEM images. That is to say, all the coatings possess outstanding antiplatelet adhesion property, which might result from the good hydrophilic surface, the anti-adhesion property of PEG and the volume rejection of hydrophobic long chain in GQAS.

Fig. 4 SEM images of PEU, WPU, GWPU, WPU/h and GWPU/h coatings to evaluate platelet adhesion.

To understand anticoagulant property of the blend coatings, the release of heparin is investigated firstly. The percentages of released heparin from various blend coatings over time are showed in Fig. 5. The relatively apparent initial burst release of heparin is only observed for WPU/h from the cumulative release curves. After 4 weeks of releasing, 18 percent and 14 percent of heparin only release from GWPU30/h and GWPU50/h, respectively. It's even less than the release percentage of WPU/h within two days (almost 20 percent), which indicates the wonderful controlled-release property of GWPU30/h and GWPU50/h attributed to the effect of GQAS. It is conjectured that electronegative heparin combines with GQAS by ionic interaction, thus slowing down the heparin release of GQAS-containing coatings.^34,46 Meanwhile, it is obvious that the release performance of heparin is further enhanced with the increase content of GQAS, because the cumulative release percentage of GWPU30/h is always higher than that of GWPU50/h. Compared with the results of previous literatures, which exhibit apparent initial burst phenomena and uncontrolled release rate, these blend coatings share the advantages: long-term, sustainable and slow release of heparin, which makes them suitable for long-term applications.^7,47

Fig. 5 Heparin release of WPU/heparin and GWPU/heparin coatings.

On account of different release performances, the APTTs and PTs of the blend coatings are estimated and the data are given in Table 2. The results show that APTT and PT of rabbit blood are approximately 20.5 ± 0.3 s and 8.0 ± 0.2 s, respectively. For WPU, the clotting times are extremely close to those of rabbit blood, indicating no inconspicuous changes on the antithrombogenicity. In contrast, the APTTs for GWPU30 and GWPU50 are significantly increase to 527.8 ± 2.6 s and 148.5 ± 5.2 s, respectively, suggesting that the incorporation of GQAS could improve the property of anticoagulant activity. Furthermore, all the heparin blended coatings exhibit outstanding anticoagulant ability in view of their significantly prolonged APTTs that have exceed the instrument limit set of 600 s. Also, the PTs for the GWPU/heparin coatings extend in a large degree with increasing the content of GQAS by comparison to the blank control groups. Thus, the obtained data do suggest ideal anticoagulation of GWPU/heparin coatings.

In view of the results of protein adsorption, platelet adhesion and anticoagulant performance, good hemocompatibility of GWPU/heparin coatings has realized. Even though Fg adsorption of the blend coatings containing GQAS is higher than the controlled sample, the succedent anti-platelet adhesion and ultima anti-thrombogenicity exhibit excellent properties unexpectedly. Further investigations are needed to expound the series of consequences.

3.3. Antibacterial evaluation

The antibacterial evaluation results of GWPU/heparin coatings are shown in Fig. 6 and 7. Obviously, in Fig. 6, bacteria of WPU are abundantly present in the nutrient media, and bacterial numbers significantly increase in the heparin-containing but without GQAS sample of WPU/h, which indicates that heparin could promote the apparent growth of S. aureus and improve the incidence of bloodstream infection.⁴⁸ In contrast, almost no bacteria could be found in the samples with GQAS, which confirms that the GQAS has strong inhibitory effect on the proliferation of S. aureus, and the efficient antibacterial activity is maintained after GQAS is incorporated into waterborne polyurethane backbone.³³ The adherence of bacteria onto the coating surfaces is typically observed based on WPU and GWPU30/h, as shown in Fig. 7. Vividly, plenty of S. aureus adhere to the surface of WPU, whereas few are found on the surface of GWPU30/h, indicating that the anti-adhesion properties for S. aureus of the coatings are improved by importing GQAS. Thus, the good anti-adhesion would further enhance the antibacterial activity of GWPU/heparin coatings, reducing the incidence of bacteria infection. Herein, it should be noticed that S. aureus is one of gram positive bacteria, which is the predominant cause of infection within the bloodstream upon implantation in vivo.²⁶ In addition, the antibacterial EG12 in GWPU have also been proved to show excellent antibacterial properties to gram negative bacteria, such as Escherichia coli, as reported in our previous work.³³

Fig. 6 Live bacteria attached on the coating surfaces.

Fig. 7 SEM of bacteria adhesion on the surfaces of WPU and GWPU30/h.

4. Conclusions

In summary, we have successfully synthesized the nontoxic biodegradable waterborne polyurethanes containing gemini quaternary ammonium salts by prepolymerization/emulsification methods, and subsequently prepared the objective functional coatings through the sample blending of GWPU emulsions and heparin aqueous solution. Depending on the corresponding measures, we find that the blend coatings possess superb surface hydrophilicity and antiplatelet adhesion, and the heparin release from blend coatings could be controlled due to the ionic interaction between GQAS and heparin, which endows them the potential for long-term applications. The GWPU/heparin coatings achieve the goals as the initial design, since they exhibit preferable anticoagulant performance and pronounced antibacterial activity of S. aureus. Thus, the excellent dual-functional anticoagulant and antibacterial of GWPU/heparin coatings and facile design strategy would hold high potential to apply in implanted medical devices.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (51173118, 51273126 and 51273124), the National Science Fund for Distinguished Young Scholars of China (51425305), the Youth Science and Innovation Team of Sichuan Province (2015TD0001), and the Sichuan Province Fund for Provincial Academic Leaders (2013DTPY0004).

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR results of GWPU. See DOI: 10.1039/c5ra27081b

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