Preparation of Vascular Endothelial Cadherin Loaded-Amphoteric

Oct 30, 2017 - National and Local Joint Engineering Research Center of Biomedical Functional Materials, School of Chemistry and Materials .... Company...
2 downloads 10 Views 5MB Size
Download PDF
Article Cite This: Langmuir XXXX, XXX, XXX-XXX

pubs.acs.org/Langmuir

Preparation of Vascular Endothelial Cadherin Loaded-Amphoteric Copolymer Decorated Coronary Stents for Anticoagulation and Endothelialization Huan Chen,†,§ Xiaobo Wang,†,§ Qian Zhou,† Ping Xu,† Yang Liu,† Mimi Wan,† Min Zhou,*,‡ and Chun Mao*,† †

National and Local Joint Engineering Research Center of Biomedical Functional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, P.R. China ‡ Department of Vascular Surgery, Nanjing Drum Tower Hospital, The Affliated Hospital of Nanjing University Medical School, Nanjing, 210008, P.R. China ABSTRACT: A new strategy for preparation of blood-contact materials, with their short-term anticoagulation depending on zwitterionic structure and long-term hemocompatibility based on endothelialization, was proposed, performed, and proved. The copolymer made of sulfonamide zwitterionic and acrylic acid was designed and synthesized, and grafted to the surface of the bare metal coronary stent. Then, the vascular endothelial cadherin (VECad), one of the specific antibodies of endothelial progenitor cells (EPCs), was fixed onto the copolymer chain. Finally, it is proved by in vitro blood tests that the coronary stent decorated with VECad loaded-amphoteric copolymer displayed good platelet antiadhesion characteristic. This anti-adhesion characteristic was attributed to the zwitterionic structure and the biofunctionality of specifically capturing EPCs confirmed by the results that the antibody-decorated coronary stent was trapped with EPCs. Finally, the in vivo implantation experiments of the antibody-decorated coronary stent in rabbit for 4 weeks were carried out. Results indicated that the endothelium and smooth surface of the antibody-loaded stent was found to be due to the covered effect of EPCs, without obvious intimal hyperplasia. The strategy we proposed has great potential in the design and preparation of blood-contact biomedical materials and devices.

■

INTRODUCTION Thrombosis and intimal hyperplasia will occur on the surface of cardiovascular implants including vascular stents, artificial blood vessels, and artificial heart valves due to the incompatible interactions between the contact interfaces of blood (or tissue) and materials, eventually leading to treatment failure.1−4 Systemic use of the antiplatelet and anticoagulant drugs can cause significant side effects and have little effect when used in small-caliber vascular grafts. These problems seriously influence and restrict the further application and efficacy of artificial cardiovascular implants in clinic.5−8 Many studies indicated that endothelialization of the biomaterial surface before implantation can prevent acute and subacute thrombosis and inhibit intimal hyperplasia.9,10 Considering the convenience of placing an implant and the sterilization problem, the endothelialization of implant surface in vivo is more in line with the need for biomedical treatment than that of endothelialization in vitro, if it is possible to solve the problem of rapid endothelialization in vivo. Adverse endothelialization can cause local subacute or delayed thrombosis, leading to coronary artery occlusion.9,11 At the same time, it will make the stimulating factor of serum continuous proliferate, thus causing delayed restenosis. Vascular © XXXX American Chemical Society

endothelial cells play an important role in the coagulation and fibrinolysis system.12 However, we know it takes some time to complete the endothelialization of the implant surface in vivo. Thus, how to obtain the anticoagulant properties of an implant becomes a key issue before its surface endothelialization is achieved. It is well-known that the zwitterionic structure can bring good anticoagulant properties to the biomaterial surface.13−20 Shen and his co-workers devoted a lot of effort to the study of the influence of the different types of zwitterionic ions and the different surface-construction modes on the anticoagulant properties of the blood-contact material surface based on a hypothesis of maintaining the natural state of protein.16,17 Our research group also paid attention to the construction of zwitterionic surfaces for different biomaterials.19,20 However, the surface-grafted layer with zwitterionic structure can delay the healing process of the endothelial layer due to the antibiofouling property of zwitterionic structure.21 In summary, Received: August 29, 2017 Revised: October 30, 2017 Published: October 30, 2017 A

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 1. Preparation routes of (A) BMS-PDA, (B) poly(DMAPS-co-AA), and (C) BMS-poly(DMAPS-co-AA).

■

the design idea of blood-contact materials, if we can overcome the problems of coagulation and thrombosis during the unfinished process of endothelialization, and solve the rejection of endothelial cell adhesion to the endothelium in the presence of zwitterionic structure, can really meet the biomedical needs. How to obtain the blood-contact material surface with the comprehensive effect including short-term anticoagulation and long-term endothelialization has become the subject of this case. Here, a new type of vascular endothelial cadherin (VE-Cad) loaded-amphoteric copolymer decorated coronary stent was designed, prepared for improving short-term anticoagulation depending on zwitterionic structure and long-term endothelialization based on the selective and specific capture of advanced endothelial progenitor cells (EPCs), and evaluated by in vivo and in vitro experiments. EPCs, the precursor cells of endothelial cells, can be involved in the repair of damaged vascular from the bone marrow mobilization to peripheral blood under the stimulation of physiological or pathological factors.22−24 It can be attributed to the fact that EPCs belong to stem cells, which possess the potential to proliferate and differentiate into endothelial cells.25,26 Recent studies showed that EPCs play an important role in the biomedical treatment of cardiovascular and cerebrovascular disease, peripheral vascular disease, tumor vessel formation, and wound healing.27,28 At the site of the vascular injury, the recruitment of EPCs can enhance the healing of the blood vessels and inhibit intimal hyperplasia and restenosis.27 The anti-CD34 antibody decorated stent is the first generation of commercial EPC captured-stents (OrbusNeich’s Genous Bioengineered R stent).29 However, it still has some limitations due to CD34 not being a specific marker of EPCs. Its positive cells can be differentiated into many kinds of cells, including inflammatory cells and vascular smooth muscle cells, which can increase the risk of restenosis.30,31 In order to overcome this limitation, the new type of antibody and related suitable surface modification method are required. Advanced EPCs can contribute to angiogenesis based on their high proliferation and differentiation capacity.27 It is found that VE-Cad can express in late EPCs, especially in the junction of endothelial cells. It is the major protein of the adherent junctions of endothelial cells.32−34 Therefore, in this paper, a new type of VE-Cad loaded-amphoteric copolymer decorated coronary stent was investigated in vitro and in vivo.

EXPERIMENTAL SECTION

Materials. Dopamine hydrochloride (DA), copper(I) bromide (CuBr), bipyridine (bpy), triethylamine (TEA), sodium acrylate, N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDTETA), ammonium persulfate, 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate (DMAPS), tris(hydroxymethyl)aminomethane (Tris, 97%), nitrophenyl disodium (pNPP), glutaraldehyde, 2-bromoisobutyryl bromide (BIBB), copper bromide (CuBr2), and hydrogen peroxide (H2O2) were obtained from Sigma-Aldrich. Ethanol, methanol, acetone, sodium chloride, disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate, and concentrated sulfuric acid (H2SO4) were purchased from Shanghai Sinopharm Reagent Company (China). The C3a enzyme immunoassay kit was received from American BD Company, the EGM-2 medium set was gained from Lonza Inc. USA, and anti-VE-cad antibodies were acquired from Beijing Biosynthesis Biotechnology Company. Human umbilical cord blood stem cells and medium were obtained from Saiye Biotechnology Company, China. The stainless steel bare metal stent (BMS,1.5 mm diameter, 18 mm length) was purchased from Yinyi Company (Dalian, China). A C3a enzyme immunoassay kit and BD FACSCalibur were obtained from BD Biosciences Company (USA). Preparation and Characterization of BMS-Poly(DMAPS-coAA). The synthetic routes for the sample were described in Figure 1. Preparation of BMS-PDA. As we know, there is not enough active functional group on the BMS surface. Therefore, the polydopamine (PDA) layer with rich amino groups was introduced onto the BMS surface by self-polymerization of dopamine. The BMS was washed three times with acetone, ethanol, and distilled water in an ultrasonic cleaner, respectively, and then was activated by piranha solution for 30 min. After activation, the BMS was put into 2 mg/mL of dopamine Tris−HCl solution (pH 9, 0.05 M) for 24 h under dark conditions (Figure 1A). Finally, the BMS-PDA product was washed with ethanol and distilled water, respectively, and then dried under a vacuum. Preparation of Poly(DMAPS-co-AA). To remove the inhibitor, acrylic acid was redistilled under reduced pressure at 4 °C in the dark. A 5.24 g portion of DMAPS (0.0188 mol) and a 1.35 g portion of acrylic acid (0.0188 mol) monomer were dissolved in 40 mL of ultrapure water in the three-necked flask. A 0.057 g portion of ammonium persulfate was dissolved in 10 mL of ultrapure water, and the solution was transferred to a 50 mL constant pressure drop funnel that was placed in the above three-necked flask under argon. The initiator solution was dropped into the reaction system under stirring at 60 °C (Figure 1B). After 5 h of reaction, the mixed solution was poured into a large amount of THF solution, and repeated two times. The obtained solution was dialyzed by deionized water with a dialysis membrane (Mw = 10000 Da) for 1 week, and then, the final product of poly(DMAPS-co-AA) was obtained by freeze-drying. B

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir Preparation of BMS-Poly(DMAPS-co-AA). After the BMS-PDA was immersed in EDC/NHS solution containing the dissolved poly(DMAPS-co-AA) at room temperature for 2 h, the BMSpoly(DMAPS-co-AA) was obtained (Figure 1C). Then, the product was washed thoroughly with ultrapure water and then dried under a vacuum at room temperature. In this case, the AA component was necessary for the design of the copolymer. Its carboxyl group not only can react with the amino group of the BMS-PDA (Figure 1C) by help of EDC/NHS solution but also can react with the amino group of antiVE-cadherin antibody, which occurs in the next step (culture of endothelial progenitor cells). It was like a bridge that connected BMS and zwitterionic structure, as well as zwitterionic structure and antiVE-cadherin antibody. The stability of the poly(DMAPS-co-AA) coating was very good based on the chemical bonding between the carboxyl group of AA and the amino group of the BMS-PDA. Characterization. The structure of poly(DMAPS-co-AA) was confirmed by nuclear magnetic resonance (1H NMR) (400 M Hz, Avance III, Bruker, Billerica, MA). The sample for 1H NMR characterization was prepared by adding poly(DMAPS-co-AA) into D2O with tetramethylsilane as an internal standard. The molecular weight of poly(DMAPS-co-AA) we synthesized was characterized by gel permeation chromatography (GPC). The surface chemical composition of BMS-poly(DMAPS-co-AA) was studied by X-ray photoelectron spectra (XPS). The XPS measurements were performed using a Thermo ESCALAB 250 instrument with Al Kα (1486.6 eV). The surface morphologies of stents were revealed with scanning electron microscopy (JSM Model 6300 SEM, JEOL). In a typical experiment, different samples were introduced onto a copper plank and gold-sprayed on the carrier stage. Each sample had three parallel samples. In Vitro Testing of BMS and BMS-Poly(DMAPS-co-AA). Whole Blood Cell and Platelet Adhesion Test. The BMS and BMSpoly(DMAPS-co-AA) were placed in a 24-well plate, and each well was soaked with 1 mL of phosphate buffer solution (PBS, pH 7.4) for 12 h. After the PBS was removed, each hole was added with 1 mL of fresh rabbit whole blood at 37 °C with an incubation time of 90 min. After the above samples were washed three times with 1 mL of PBS, they were immersed in 600 μL of 2.5 vol % glutaraldehyde PBS solution and fixed for 30 min. Then, after rinsing immediately in 1 mL of PBS three times, the BMS and BMS-poly(DMAPS-co-AA) were put in 50, 60, 70, 80, 90, 95, 100% (V/V) ethanol−water solutions progressively for 30 min and dried in air. For observation, the morphology of cohered blood cells on the substrates was determined by SEM. In the platelet adhesion test, platelet rich plasma (PRP), collected with centrifugal blood for 10 min at 1200 rpm, was incubated with the stents for 90 min using the procedures described above. Complement Activation and Platelet Activation Test. To evaluate the complement activation, a C3a enzyme immunoassay kit was employed to measure the formation of its activation peptides (the cleavage of complement component C3 and C3a des-Arg). The BMS and BMS-poly(DMAPS-co-AA) were incubated with the poor platelet plasma for 1 h at 37 °C, respectively. Each sample was provided with three parallel samples. To estimate the platelet activation, the BMS-poly(DMAPS-co-AA) was hatched at 37 °C with platelet rich plasma. And 30 min later, the incubation mixture was removed to examine the activation condition of the platelets by flow cytometry. The ratio of platelet activation was detected by measuring the expression of anti-CD62P (as the fluorescently labeled platelet activation marker) and anti-CD42a (as the platelet pan-marker) by a BD FACSCalibur. Each sample was provided with three parallel samples. Growth of Endothelial Progenitor Cells on the Stent Surface. Culture of Endothelial Progenitor Cells. The BMSpoly(DMAPS-co-AA) was incubated with excess anti-VE-cadherin antibodies for 2 h in an analeptic processing room, and then, the product was named BMS-poly(DMAPS-co-AA)-antibody. The BMS, BMS-poly(DMAPS-co-AA), and BMS-poly(DMAPS-co-AA)-antibody were seeded with EPCs (obtained from generation and differentiation of human umbilical cord blood stem cells) for 1 week, respectively. The growth status of cells on the stent surfaces was observed by SEM.

Endothelial Progenitor Cell Viability Test-pNPP Method. For quantitative determination of the adhesion of endothelial cells on the surface of BMS, BMS-poly(DMAPS-co-AA), and BMS-poly(DMAPSco-AA)-antibody, the survival rates of EPCs in the three different stents were measured by the pNPP method. This method was able to determine the content of alkaline phosphatase, which can reflect the amount of cell adhesion on the stent surface.35 First, the stent samples were put into the 24-well plate, and then, after the cell density was adjusted to 5 × 104 cells/well, the EPCs were seeded in the 24-well plate under starvation treatment for one night and cultured for 96 h. The samples were removed to another 24-well plate, and washed with PBS twice. A 1 mg/mL pNPP solution was added into the 24-well plate. After culturing for 3 h, sodium hydroxide solution was added to stop the reaction. The light absorption at 405 nm was detected by a microplate reader based on the absorbance value being proportional to the number of cells. Rabbit Model for in Vivo Implantation Test. BMS-poly(DMAPSco-AA)-antibody samples were sent to Nanjing Drum Tower Hospital for in vivo experiment, including the implantation of rabbit carotid artery 4 weeks later, the coverage of endothelial progenitor cells and the degree of platelet adhesion, B ultrasound, angiography, and pathological section experiment. The animal testing was approved by the ethics committees of Nanjing Drum Tower Hospital, Nanjing, China. Statistical Analysis. Statistical data analysis was performed using SPSS (v 11.5, SPSS Inc.). The results were shown as the mean ± standard deviation (SD), and at least three repeated experiments were conducted. Statistical analysis of the differences between two groups was performed using a Student’s two-tailed t test. Statistical significance was defined as p < 0.001.

■

RESULTS AND DISCUSSION Characterization. NMR is useful for studying molecular motions, interactions, and conformations. As shown in Figure 2

Figure 2. 1H NMR spectrum of poly(DMAPS-co-AA).

of the synthesized poly(DMAPS-co-AA), the peak (δ = 4.70 ppm) belonged to the solvent of D2O, and these peaks (δ = 1.1, 2.3, 2.9 3.2, 3.5, 3.8, and 4.5 ppm) corresponded to these protons (a, h, i, e, f, g, e, d) of DMAPS, respectively.36,37 Moreover, the peak (δ = 2.66 ppm) was attributed to the proton marked of the block of AA. The molecular weights (Mn = 34811, Mw = 38988) of poly(DMAPS-co-AA) were determined by gel permeation chromatography. The polymer dispersity index (PDI) was about 1.12, which indicated that the molecular weight distribution of poly(DMAPS-co-AA) was uniform. The polymerization degree of DMAPS was 56, and the polymerization degree of AA was 44. In the synthesis of poly(DMAPSC

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir co-AA), the starting amount of DMAPS (5.24 g, 0.0188 mol) was the same as that of acrylic acid monomer (1.35 g, 0.0188 mol). This particular zitterionic polymer/AA ratio was chosen on the basis of three factors: (1) the anticoagulant effect of the modified stent results from the zwitterionic structure of DMAPS; (2) the stability of poly(DMAPS-co-AA) decorated the BMS surface, with acrylic acid acting as a bridge; (3) the capture effect of EPCs based on the surface of anti-VE-cadherin antibodies grafted poly(DMAPS-co-AA). X-ray photoelectron spectroscopy (XPS) is used to characterize the chemical composition, the element contents of the material surface, and the changes of the chemical bond after each step reaction for surface modification. The XPS data for poly(DMAPS-co-AA) grafted onto the BMS-PDA surface (BMS-poly(DMAPS-co-AA) were shown in Figure 3. It can be

Figure 5. Rabbit whole blood adhesion tests of BMS and BMSpoly(DMAPS-co-AA) (A and B) and the platelet adhesion tests of BMS and BMS-poly(DMAPS-co-AA) (C and D).

be attributed to the biofouling function of the zwitterionic structure of BMS-poly(DMAPS-co-AA). As shown in Figure 5C and D, when the BMS samples were contacted with the platelet-rich plasma for 90 min, many platelets activation and aggregation with pseudopodias on the BMS surface adhesion were observed. On the contrary, the surface of BMS-poly(DMAPS-co-AA) showed no adhered platelets. As we know, the activated platelets play an important role in the formation of thrombosis and atherosclerosis. Thus, the results of the platelet adhesion test indicated that the BMSpoly(DMAPS-co-AA) had the biofunctions including antiplatelet adhesion, and inhibition of platelet activation, which are a great advantage in the prevention of thrombosis. Complement Activation Test. The complement system is an enzyme cascade that plays an important role in the defense against pathogens in the defense system of the human body. Activation of the complement system can produce allergic toxins, which lead to immune system activation. After the BMS and BMS-poly(DMAPS-co-AA) samples were incubated with the anticoagulated plasma for 1 h at 37 °C, compared to the generated C3a content (0.945 ± 0.05 ng/mL) of the negative control, the datum of BMS and BMS-poly(DMAPS-co-AA) samples were 1.02 ± 0.04 and 0.955 ± 0.05 ng/mL, respectively (Figure 6). It indicated that the BMS-poly(DMAPS-co-AA) samples did not initiate complement activation.

Figure 3. XPS full scan spectrum of the surface of BMS-poly(DMAPSco-AA).

clearly observed in S 2p, S 2s, and N 1s signal peaks. The emergence of S element was an obvious proof of poly(DMAPSco-AA) grafted onto the surface of BMS-PDA because S element was a characteric element of poly(DMAPS-co-AA), not existing in the original substrate and PDA layer. Observation of the Stent Surfaces by SEM. From the SEM images, the BMS surface appeared smooth (Figure 4A),

Figure 4. SEM micrographs of the surfaces of (A) BMS*, (B) BMSPDA, and (C)BMS-poly(DMAPS-co-AA). (*There are some original holes on the BMS surface.)

and there were some small bumps (as shown in the white circle) on the BMS-PDA surface that were attributed to the polymerization of dopamine (Figure 4B). Further, the obvious surface-grafting effect (as shown in the white circle) of BMSpoly(DMAPS-co-AA) was observed (Figure 4C). In Vitro Testing of BMS and BMS-Poly(DMAPS-co-AA). Platelet and Whole Blood Adhesion Test. The preliminary hemocompatibilities of stent samples were investigated by the platelet and whole blood adhesion tests. The quantity and morphology of platelets and blood cells adhesion were analyzed by SEM images. As shown in Figure 5A and B, when the surface of BMS samples was contacted with the fresh rabbit blood for 30 min, some blood cells adhesion and aggregation was observed from the BMS surface; however, the surface of BMSpoly(DMAPS-co-AA) showed no blood cells attachment. It can

Figure 6. Complement activation assay of BMS and BMS-poly(DMAPS-co-AA). PBS was used as a negative control. D

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 7. Platelet activation assay of BMS and BMS-poly(DMAPS-co-AA). PBS was used as a negative control.

Platelet Activation Test. The interaction of BMS-poly(DMAPS-co-AA) with platelet was determined by the expression of the activation markers of the platelet surface. Here, different samples were analyzed by flow cytometry after incubation with whole blood in 30 min; the percentage of platelet activation was calculated by the percentage of antiCD62P-FITC marked platelet.38 As shown in Figure 7, the platelet activation rate of negative control was 2.57%, while that datum of the BMS-poly(DMAPSco-AA) was only 0.9%. The results indicated that copolymer with zwitterionic structure played an important role in inhibiting platelet activation. The Growth of Endothelial Progenitor Cells on the Stent Surface. As mentioned earlier, the BMS-poly(DMAPSco-AA) was incubated with excess anti-VE-cadherin antibodies for 2 h in an analeptic processing room. It meant that the BMSpoly(DMAPS-co-AA) was adequately reacted with anti-VEcadherin antibodies. However, in this case, the copolymer of poly(DMAPS-co-AA) was fixed to the BMS surface, and the subsequently loaded antibodies were not arranged in parallel on the BMS surface due to the flexible long chain structure of poly(DMAPS-co-AA). It indicated that the relation of the surface coverage of antibodies and the trapping effect of EPCs cannot be obtained just by calculating the coverage of antibodies on the BMS surface. Therefore, the subsequent EPC capture experiments were used to validate the load efficacy of the anti-VE-cadherin antibodies. The cultivation test of EPCs on the material surface, which can provide a reference for the endothelium degree, is an essential evaluation for the stent implantation of the human body. The BMS, BMS-poly(DMAPS-co-AA), and BMS-poly(DMAPS-co-AA)-antibody were exposed to the EPCs for a week (Figure 8). The results showed that there was only a very

Figure 8. Growth of endothelial progenitor cells on the different surfaces of (A) BMS, (B) BMS-poly(DMAPS-co-AA), and (C) BMSpoly(DMAPS-co-AA)-antibody.

small amount of EPCs that adhered the BMS surface. Similarly, as shown by the SEM image in Figure 8B, after the BMSpoly(DMAPS-co-AA) seeded with EPCs for a week, the cells were also difficult to adhere and grow on the surface of the BMS-poly(DMAPS-co-AA) due to the antibiofouling effect of the zwitterionic structure.19 This effect of antibiofouling caused by the zwitterionic groups may have a prominent inhibiting effect on platelet or cell adhesion. However, only by the rapid endothelialization of the stent surface, the risk of thrombus formation and neointimal hyperplasia can be reduced significantly. In this case, we fully took into account the important influence factors of the rapid endothelium for the stent surface. Then, the blocks of AA were introduced into the decorated layer of the BMS surface, which the carboxylic acid of AA can be bonded with the anti-VE-cadherin antibody after activation, and then the EPCs can be specific captured rapidly. Results showed that the BMS-poly(DMAPS-co-AA)-antibody surface was packaged completely by EPCs, which illustrates that both the load efficacy of the anti-VE-cadherin antibodies and the combination between antibodies and EPCs were very effective (Figure 8C). E

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir Endothelial Progenitor Cells Viability test-pNPP Method. The situations of survival and proliferation of EPCs on the stent surfaces were also evaluated by the pNPP method. After the incubation, the stent samples were removed from the 24-well plate and washed with sterile PBS, and then, pNPP reagent was added and NaOH was used for the termination of the experiments. Finally, the UV absorbance tests were carried out. The results of this study can directly reflect the adhesion and growth of EPCs on the stent surface. As shown in Figure 9, the UV absorbance values of BMS, BMS-poly(DMAPS-co-AA), and BMS-poly(DMAPS-co-AA)-

Figure 10. (A) Carotid artery stenting of BMS-poly(DMAPS-co-AA)antibody. (B) The suture process of blood vessels after implantation. (C) B ultrasound image of carotid artery. (D) Vasography image after 4 weeks of implantation of BMS-poly(DMAPS-co-AA)-antibody of experimental rabbits and after 4 weeks of implantation of BMSpoly(DMAPS-co-AA)-antibody of experimental rabbits. (E) SEM micrograph of the BMS-poly(DMAPS-co-AA)-antibody surface. (F) Vascular slice image.

Figure 9. Viabilities of endothelial progenitor cells and cells grown on the surfaces of BMS, BMS-poly(DMAPS-co-AA), and BMS-poly(DMAPS-co-AA)-antibody.

antibody samples were 0.281, 0.134, and 0.482, respectively. Due to the UV absorbance data being proportional to the numbers of EPCs that adhered and grew on the stent surface, the absorbance value of BMS-poly(DMAPS-co-AA)-antibody sample was the largest of the data obtained from the three stent samples, indicating that this kind of stent surface had specific capture ability for EPCs. Further, the absorbance datum of the BMS sample was higher than that of BMS-poly(DMAPS-co-AA) due to the effect of antibiofouling that was caused by the zwitterionic groups of poly(DMAPS-co-AA). These experimental results were consistent with the SEM results of EPCs culture experiments. From the point of statistics t test, the p value between BMS and BMSpoly(DMAPS-co-AA) was below 0.001, which showed a significant difference between BMS and the BMS-poly DMAPS-co-AA. Meantime, the p value for BMS absorbance and BMS-poly DMAPS-co-AA-antibody absorbance was also p < 0.001 with significant difference, illustrating the loading of antibody was very effective. Rabbit Model for in Vivo Implantation Test. The in vivo implantation experiments of BMS-poly(DMAPS-co-AA)-antibody were carried out by the vascular surgeons of Nanjing Drum Tower Hospital that long-time-cooperated with our group. After the rabbits were anesthetized, the sterilized stents were delivered to the carotid artery of rabbits through a stent deliver system, and then, the blood vessels and external tissues were sutured (Figure 10A and B). Then, the implanted rabbits were fed for 1 month, B ultrasound, angiography, endothelial coverage, the degree of platelet adhesion and the pathological section of the blood vessel were adopted to analyze the implanted situation. From B ultrasound images of the carotid artery of experimental rabbits, it can be seen that the arterial lumen

was filled with blood, and there was no obvious mural thrombus formation (Figure 10C). At the same time, the contrast picture showed that the stent carotid artery was filled with the contrast agents, and had no stent stenosis (Figure 10D). After the experimental rabbits were sacrificed, the fresh carotid artery was taken out, and the degree of endothelial coverage and platelet adhesion were analyzed by SEM pictures. As shown in Figure 10E, the surface of BMS-poly(DMAPSco-AA)-antibody showed smooth coverage of endothelial cells and no significant adhesion of platelets and was fibrous. Meanwhile, from the slice picture of BMS-poly(DMAPS-coAA)-antibody, the stent implantation after 4 weeks without obvious intimal hyperplasia was observed (Figure 10F). Results of the comprehensive animal experiments in 4 weeks after the implantation of stents showed no platelet adhesion, intimal hyperplasia, and vascular patency, indicating that the placement of BMS-poly(DMAPS-co-AA)-antibody can play a positive role for the normal flow of blood.

■

CONCLUSION Zwitterionic structures are capable of resisting nonspecific protein adsorption, bacterial adhesion, and blood coagulation. EPCs can differentiate into mature endothelial cells and accelerate re-endothelialization after vascular injury, preventing both stent thrombosis and restenosis. In this paper, the copolymer, poly(DMAPS-co-AA) with zwitterionic and acrylic acid, was designed and synthesized, and then, the EPC-cadherin antibodies were fixed on the surface of BMS-poly(DMAPS-coAA) by chemical grafting for preparing complex functions coronary stents. Its good antibiofouling and EPC-capture properties were confirmed by the in vitro experiments. By F

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Outcomes of Decellularized Vascular Grafts via Incorporation of a Novel Extracellular Matrix. Biomaterials 2017, 141, 63−73. (8) Sugiura, T.; Agarwal, R.; Tara, S. H.; Yi, T.; Lee, Y. U.; Breuer, C. K.; Weiss, A. S.; Shinoka, T. Tropoelastin Inhibits Intimal Hyperplasia of Mouse Bioresorbable Arterial Vascular Grafts. Acta Biomater. 2017, 52, 74−80. (9) Werner, N.; Junk, S.; Laufs, U.; Link, A.; Walenta, K.; Böhm, M.; Nickenig, G. Intravenous Transfusion of Endothelial Progenitor Cells Reduces Neointima Formation After Vascular Injury. Circ. Res. 2003, 93, e17−e24. (10) Kong, D.; Melo, L. G.; Mangi, A. A.; Zhang, L. N.; LopezIlasaca, M.; Perrella, M. A.; Liew, C. C.; Pratt, R. E.; Dzau, V. J. Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells. Circulation 2004, 109, 1769−1775. (11) Stone, G. W.; Ellis, S. G.; Cox, D. A.; Hermiller, J.; Shaughnessy, C. O.; Mann, J. T.; Turco, M.; Caputo, R.; Bergin, P.; Greenberg, J.; Popma, J. J.; Russell, M. E. A Polymer-Based, Paclitaxel-Eluting Stent in Patients with Coronary Artery Disease. N. Engl. J. Med. 2004, 350, 221−231. (12) Rao, S. V.; Shaw, R. E.; Brindis, R. G.; Klein, L. W.; Weintraub, W. S.; Peterson, E. D. On-Versus Off-Label Use of Drug-Eluting Coronary Stents in Clinical Practice (Report From The American College of Cardiology National Cardiovascular Data Registry [NCDR]). Am. J. Cardiol. 2006, 97, 1478−1481. (13) Chen, Y. J.; Han, H. J.; Tong, H. G.; Chen, T. T.; Wang, H. B.; Ji, J.; Jin, Q. Zwitterionic Phosphorylcholine−TPE Conjugate for pHResponsive Drug Delivery and AIE Active Imaging. ACS Appl. Mater. Interfaces 2016, 8, 21185−21192. (14) Liu, X. S.; Chen, Y. J.; Li, H.; Huang, N.; Jin, Q.; Ren, K. F.; Ji, J. Enhanced Retention and Cellular Uptake of Nanoparticles in Tumors by Controlling Their Aggregation Behavior. ACS Nano 2013, 7, 6244− 6257. (15) Liu, X. S.; Li, H.; Jin, Q.; Ji, J. Surface Tailoring of Nanoparticles via Mixed-Charge Monolayers and Their Biomedical Applications. Small 2014, 10, 4230−4242. (16) Liu, P. S.; Chen, Q.; Li, L.; Lin, S. C.; Shen, J. Anti-Biofouling Ability and Cytocompatibility of The Zwitterionic Brushes-Modified Cellulose Membrane. J. Mater. Chem. B 2014, 2, 7222−7231. (17) Wang, X. B.; Miao, J. J.; Shao, X. B.; Mao, C.; Shen, J. Zwitterionic Hyperbranched Polyester Functionalized Cardiovascular Stent and Its Biocompatibility. J. Colloid Interface Sci. 2014, 420, 88− 96. (18) Mizuhara, T.; Saha, K.; Moyano, D. F.; Kim, C. S.; Yan, B.; Kim, Y. K.; Rotello, V. M. Acylsulfonamide-Functionalized Zwitterionic Gold Nanoparticles for Enhanced Cellular Uptake at Tumor pH. Angew. Chem., Int. Ed. 2015, 54, 6567−6570. (19) Wang, X. B.; Miao, J. J.; Zhao, H. L.; Mao, C.; Chen, X. Q.; Shen, J. Fabrication of Nonbiofouling Metal Stent and in vitro Studies on Its Hemocompatibility. J. Biomater. Appl. 2014, 29, 14−25. (20) Hu, H.; Wang, X. B.; Xu, S. L.; Yang, W. T.; Xu, F. J.; Shen, J.; Mao, C. Preparation and Evaluation of Well-Defined Hemocompatible Layered DoubleHydroxide-Poly (Sulfobetaine) Nanohybrids. J. Mater. Chem. 2012, 22, 15362−15369. (21) Ren, X. K.; Feng, Y. K.; Guo, J. T.; Wang, H. X.; Li, Q.; Yang, J.; Hao, X. F.; Lv, J.; Ma, N.; Li, W. Z. Surface Modification and Endothelialization of Biomaterials as Potential Scaffolds for Vascular Tissue Engineering Applications. Chem. Soc. Rev. 2015, 44, 5680− 5742. (22) Bonfim-Silva, R.; Souza, L. E. B.; Melo, F. U. F.; Oliveira, V. C.; Magalhaes, D. A. R.; Oliveira, H. F.; Covas, D. T.; Fontes, A. M. Bone Marrow-Derived Cells are Recruited by The Melanoma Tumorwith Endothelial Cells Contributing to Tumor Vasculature. Clin. Transl. Oncol. 2017, 19, 125−133. (23) Shi, X.; Zhang, W. H.; Yin, L. Y.; Chilian, W. M.; Krieger, J.; Zhang, P. Vascular Precursor Cells in Tissue Injury Repair. Transl. Res. 2017, 184, 77−100. (24) Li, Q.; Cui, H. H.; Yang, Y. J.; Li, X. D.; Chen, G. H.; Tian, X. Q.; Jin, C.; Dong, Q. T.; Huang, P. S.; Xu, J. Quantitative Proteomics

implanting in rabbits for 4 weeks, the pathological section of the stent loaded cadherin antibodies showed totally endothelialization with no significant intimal hyperplasia, which can maintain vascular patency. This surface modification strategy for short-term anticoagulation and long-term endothelialization of coronary stents that we proposed has great potential in biomedical applications.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Chun Mao: 0000-0003-4085-3414 Author Contributions §

H.C., X.W.: These authors contributed equally to this work.

Author Contributions

C.M., M.Z., and M.W. designed the experiments; H.C., X.W., Q.Z., P.X., and Y.L. synthesized materials and collected the experimental data; all authors participated in the interpretation of results and wrote the paper. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The work was supported by Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National Natural Science Foundation of China (51641104, 21603105, 21571104), Chinese Postdoctoral Science Foundation (2015M580446), Medical Science and Technology Development Foundation, Nanjing Department of Health (JQX14002), Social Development Project of Jiangsu (BE2015603), Jiangsu Key Technology RD Program (BE2016010), the Priority Academic Program Development of Jiangsu Higher Education Institution, and Major projects of Natural Sciences of University in Jiangsu (14KJA150006).

■

REFERENCES

(1) Mao, L.; Chen, J. H.; Zhang, X. B.; Kwak, M.; Wu, Y.; Fan, R.; Zhang, L.; Pei, J.; Yuan, G. Y.; Song, C. L.; Ge, J. B.; Dng, W. J. A Promising Biodegradable Magnesium Alloy Suitable for Clinical Vascular Stent Application. Sci. Rep. 2017, 7, 46343−46354. (2) Stefopoulos, G.; Giampietro, C.; Falk, V.; Poulikakos, D.; Ferrari, A. Facile Endothelium Protection from TNF-α Inflammatory Insult with Surface Topography. Biomaterials 2017, 138, 131−141. (3) Zang, D.; Yi, H.; Gu, Z. D.; Chen, L.; Han, D.; Guo, X. L.; Wang, S. T.; Liu, M. J.; Jiang, L. Interfacial Engineering of Hierarchically Porous NiTi/Hydrogels Nanocomposites with Exceptional Antibiofouling Surfaces. Adv. Mater. 2017, 29, 1602869−1602875. (4) Cheng, S. Y.; Jin, Y.; Wang, N. X.; Cao, F.; Zhang, W.; Bai, W.; Zheng, W. F.; Jiang, X. Y. Self-Adjusting, Polymeric Multilayered Rollthatcan Keep the Shapes of the Blood Vessel Scaffolds during Biodegradation. Adv. Mater. 2017, 29, 1700171−1700178. (5) Schneider, K. H.; Aigner, P.; Holnthoner, W. G.; Monforte, X.; Nürnberger, S.; Rünzler, D.; Redl, H.; Teuschl, A. H. Decellularized Human Placenta Chorion Matrix as A Favorabl Esource of SmallDiameter Vascular Grafts. Acta Biomater. 2016, 29, 125−134. (6) Gong, W. H.; Lei, D.; Li, S.; Huang, P.; Qi, Q.; Sun, Y. J.; Zhang, Y. J.; Wang, Z.; You, Z. W.; Ye, X. F.; Zhao, Q. Hybrid Small-Diameter Vascular Grafts: Anti-Expansion Effect of Electrospun Poly εCaprolactone on Heparin-Coated Decellularized Matrices. Biomaterials 2016, 76, 359−370. (7) Kristofik, N. J.; Qin, L. F.; Calabro, N. E.; Dimitrievska, S.; Li, G. X.; Tellides, G.; Niklason, L. E.; Kyriakides, T. R. Improving in vivo G

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and The Protective Role of Tongxinluo. Cell. Physiol. Biochem. 2017, 41, 1503−1518. (25) Kukumberg, M.; Yao, J. Y.; Neo, D. J. H.; Yim, E. K. F. Microlens Topography Combined with Vascular Endothelial Growth Factor Induces Endothelial Differentiation of Human Mesenchymal Stem Cells Into Vasculogenic Progenitors. Biomaterials 2017, 131, 68− 85. (26) Asahara, T.; Takahashi, T.; Masuda, H.; Kalka, C.; Chen, D. H.; Iwaguro, H.; Inai, Y.; Silver, M.; Isner, J. M. VEGF Contributes to Postnatal Neovascularization by Mobilizing Bone Marrow-Derived Endothelial Progenitor Cells. EMBO J. 1999, 18, 3964−3972. (27) Mannarino, E.; Pirro, M. Endothelial Injury and Repair: A Novel Theory For Atherosclerosis. Angiology 2008, 59, 69S−72S. (28) van Beusekom, H. M. M.; Gokhan, E.; Sorop, O.; Patrick, W. S.; Willem, J. G. The Genous Endothelial Progenitor Cell Capture Stent Accelerates Stent Re-Endothelialization But Does Not Affect Intimal Hyperplasia in Porcine Coronary Arteries. Catheter. Cardio. Inte. 2012, 79, 231−242. (29) Nakazawa, G.; Granada, J. F.; Alviar, C. L.; Tellez, A.; Kaluza, G. L.; Guilhermier, M. Y.; Parker, S.; Rowland, S. M.; Kolodgie, F. D.; Leon, M. B.; Virmani, R. Anti-CD34 Antibodies Immobilized on the Surface of Sirolimus-Eluting Stents Enhance Stent Endothelialization. JACC-Cardiovasc. Inte. 2010, 3, 68−75. (30) Hans, P. W.; Meltem, A. A.; Gerhard, Z. Endothelial Progenitor Cell Capture Stents–Hype or Hope? Int. J. Cardiol. 2009, 06, 18−20. (31) Garg, S.; Duckers, H. J.; Serruys, P. W. Endothelial Progenitor Cell Capture Stents: Will This Technology Find Its Niche in Contemporary Practice? Eur. Heart J. 2010, 31, 1032−1035. (32) Bernard, S.; Loffroy, R.; Sérusclat, A.; Boussel, L.; Bonnefoy, E.; Thévenon, C.; Rabilloud, M.; Revel, D.; Moulin, P.; Douek, P. Increased Levels of Endothelial Microparticles CD144 (VE-Cadherin) Positives in Type 2 Diabetic Patients with Coronary Noncalcified Plaques Evaluated by Multidetector Computed Tomography (MDCT). Atherosclerosis 2009, 203, 429−435. (33) Lee, J. M.; Choe, W. S.; Kim, B. K.; Seo, W. W.; Lim, W. H.; Kang, C. K.; Kyeong, S.; Eom, K. D.; Cho, H. J.; Kim, Y. C.; Hur, J.; Yang, H. M.; Cho, H. J.; Lee, Y. S.; Kim, H. S. Comparison of Endothelialization and Neointimal Formation with Stents Coated with Antibodies Against CD34 and Vascular Endothelial-Cadherin. Biomaterials 2012, 33, 8917−8927. (34) Lim, W. H.; Seo, W. W.; Choe, W.; Kang, C. K.; Park, J.; Cho, H. J.; Kyeong, S.; Hur, J.; Yang, H. M.; Cho, H. J.; Lee, Y. S.; Kim, H. S. Stent Coated with Antibody Against Vascular Endothelial-Cadherin Captures Endothelial Progenitor Cells, Accelerates Re-Endothelialization, and Reduces Neointimal Formation. Arterioscler., Thromb., Vasc. Biol. 2011, 31, 2798−2805. (35) Chen, W. Z.; Shen, X. K.; Hu, Y.; Xu, K.; Ran, Q. C.; Yu, Y. L.; Dai, L. L.; Yuan, Z.; Huang, L.; Shen, T. T.; Cai, K. Surface Functionalization of Titanium Implants with Chitosan-CatecholConjugate for Suppression of ROS-Induced Cells Damage andImprovement of Osteogenesis. Biomaterials 2017, 114, 82−96. (36) Tian, M.; Wang, J. M.; Zhang, E.; Li, J. J.; Duan, C. M.; Yao, F. L. Synthesis of Agarose-Graft-Poly[3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate] Zwitterionic Graft Copolymers via ATRP and Their Thermally-Induced Aggregation Behavior in Aqueous Media. Langmuir 2013, 29, 8076−8085. (37) Gui, Z. L.; Qian, J. W.; Zhao, Q.; Ji, Y. L.; Liu, Y. X.; Liu, T.; An, Q. F. Controllable Disintegration of Temperature-Responsive SelfAssembled Multilayer Film Based on Polybetaine. Colloids Surf., A 2011, 380, 270−279. (38) Li, B.; Juenet, M. Y.; Aid-Launais, R.; Maire, M.; Ollivier, V.; Letourneur, D.; Chauvierre, C. Development of Polymer Microcapsules Functionalized with Fucoidan to Target P-Selectin Overexpressed in Cardiovascular Diseases. Adv. Healthcare Mater. 2017, 6, 1601200−1601210.

H

DOI: 10.1021/acs.langmuir.7b03064 Langmuir XXXX, XXX, XXX−XXX