A Method for Preparation of an Internal Layer of Artificial Vascular

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Biological and Medical Applications of Materials and Interfaces

A method for preparation of an internal layer of artificial vascular graft co-modified with Salvianolic acid B and heparin Haizhu Kuang, Yao Wang, Junfeng Hu, Chunsheng Wang, Shuyang Lu, and Xiumei Mo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02602 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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ACS Applied Materials & Interfaces

A Method for Preparation of an Internal Layer of Artificial Vascular Graft Co-modified with Salvianolic Acid B and Heparin

Haizhu Kuanga,1, Yao Wangb,1, Junfeng Hu a, Chunsheng Wang b, Shuyang Lu b, *, Xiumei Mo a, *

a

State Key Lab for Modification of Chemical Fibers and Polymer Materials, College

of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China.

b

Department of Cardiovascular Surgery，Zhongshan Hospital, Fudan University,

Shanghai 200032, China.

* Corresponding author at: a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua

University, 2999 North Renmin Road, Shanghai 201620, P. R. China. E-mail address: [email protected] (Xiumei Mo);

b

Department of Cardiovascular Surgery，Zhongshan Hospital, Fudan University,

Shanghai 200032, China. E-mail address: [email protected] (Shuyang Lu)

Haizhu Kuang and Yao Wang contributed equally to this work

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Studies have shown that salvianolic acid B (SAB), which is derived from Chinese salvia (Salvia miltiorrhiza), a plant used in traditional Chinese medicine, can promote the proliferation and migration of endothelial cells. The inner layer of an artificial vascular graft was fabricated using the coaxial electrospinning method and was loaded with the anticoagulant heparin and SAB. The release of heparin and SAB was sustained for almost 30 days, and without an initial burst release of SAB. Furthermore, the combined effect of SAB and heparin contributed to promoting human umbilical vein endothelial cell (HUVEC) growth and improved the blood compatibility of the graft. In addition, upregulation of GRP78 by SAB protected human endothelial cells from oxidative stress-induced cellular damage. In vivo evaluation through Masson's trichrome and H&E staining was performed after the graft was subcutaneously embedded in SD rats for two weeks, and indicated that the graft possessed satisfactory biocompatibility and did not cause a significant immune response. Hence, the functional inner layer is promising for preventing acute thrombosis and promote rapid endothelialization of artificial vascular grafts. Key words: salvianolic acid B, heparin, coaxial electrospinning, artificial vascular graft, blood compatibility, biocompatibility, rapid endothelialization. 1. Introduction Coronary heart disease and peripheral vascular disease have become a serious burden on human health. According to statistics, there are approximately 10 million cases of vascular surgery that have been performed worldwide.1 Autologous and allogeneic vascular transplantation cannot provide sufficient blood vessels for implantation in the human body, and thus, there is a very important clinical significance in developing artificial blood vessels.2 The implantation of small-caliber blood vessels often fails and embolization often occurs after a vascular graft is transplanted into the body, as it cannot rapidly endothelialize.3-4 Re-stenosis and embolization are two hurdles result from small caliber artificial blood vessels in clinical practice, and the problems cannot be sufficiently resolved. In recent years, there has been much research focusing on two aspects regarding small-caliber artificial blood vessels: (i) they improve the blood compatibility and biocompatibility ACS Paragon Plus Environment

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and increase the patency rate of blood vessels by the artificial vascular graft surface modification; and (ii) artificial blood vessels provide a natural barrier to anti-thrombosis by promoting rapid endothelialization of the artificial vascular graft surface.5 Our research covers both of these aspects in our preparation of an internal layer

for

artificial

vascular

graft.

Mixing

the

synthetic

material

poly

(L-lactide-co-caprolactone) (PLCL) with natural materials composed of collagen (COL) enhances the biocompatibility of synthetic materials.6-8 Also, the manner through which SAB and heparin are loaded into the fiber, by the coaxial electrospinning technique, promotes rapid endothelialization and prevent acute thrombosis of artificial vascular surfaces. Heparin is a powerful anticoagulant, and many studies have reported that its effect is positive.9-13 Salvia (Salvia miltiorrhiza) is a traditional Chinese herb that has been widely used in China for thousands of years. A gradual accumulation of evidence has revealed that it offers some remedial relief for angina pectoris, hyperlipidemia, and acute ischemic stroke. Salvia has also been used in Japan, the United States, and other European countries, for the treatment of cardiovascular and cerebrovascular diseases.14-15 Salvia promotes endothelial cell adhesion,16 and it also protects endothelial cells via anti-oxidative apoptosis, and can promote the migration of endothelial cells.17-19 Additionally, salvia can affect the adhesion of platelets and can retard platelet activity by inhibiting platelet aggregation and thrombus formation.20-21 It also affects some disease of the nerves and bones.22-25 Salvianolic acid B (SAB) is btained by extraction and purification from salvia, and it has anti-oxidative, anti-inflammatory, anti-hypoxic, anti-arteriosclerotic and anti-apoptotic properties. This substance can also ameliorate brain injury or neurodegenerative diseases.26 Furthermore, upregulation GRP78 by SAB protects human endothelial cells from oxidative stress-induced cellular damage.27 Tan IIA, which is extracted from Salvia , exerts a similar effect as that of SAB, and can protect endothelial cells against oxidative injury induced by H2O2 through PXR-dependent mechanisms. Those studies suggested that SAB may constitute a promising intervention against cardiovascular disorders. However, because SAB easily decomposes in aqueous solution, it is ACS Paragon Plus Environment


necessary to find a suitable drug-loading medium for loading SAB. Mesoporous silica nanoparticles (MSN) have satisfactory stability and dispensability, and excellent biocompatibility, showing great potential for drug delivery applications.28-30 Coaxial electrospinning technology can be used to easily prepare a core-shell structure of fiber with a large pore interval, and porosity large specific surface area , that can be widely applied in tissue engineering, controlled drug release, chemical, and other fields.31-34 Our laboratory has conducted a large amount of research in the fabrication of drug carrying fibers by coaxial electrospinning and improving the biocompatibility of grafts by blending polymer materials and natural materials35-36. Vascular regeneration is a continuous process. Drugs are commonly administered because they can play a long-term role in the promotion of endothelial cell growth, and the drugs should be released through a sustained and stable manner over a long period of time. In the current study, we prepared a composite scaffold composed of heparin encapsulated nanofibers and SAB loaded MSN. SAB is adsorbed on the MSN, and blended into the coaxial fiber shell. Through this modified fiber structure, we can achieve greater controlled release than that of nanofibers with a core-shell to control the delivery of SAB37. 2. Materials and methods 2.1 Materials Poly (L-lactide-co-caprolactone) (PLCL, Mw = 80 000 g mol-1) with an L-lactic acid/ε-caprolactone ratio of 50: 50 was supplied by Jinan Daigang Biotechnology Co., Ltd. (Jinan, China). Collagen type I was purchased from Shandong International Biotechnology Park. (molecular weight 3×105 Da). The MSN were synthesized by Donghua University Institute of Biology. SAB was purchased from Shanghai Lugu Pharmaceuticals Co. (Shanghai, China), and 1,1,1,3,3,3- hexafluoro -2-propanol (HFIP) was acquired from Shanghai Darui Fine Chemical Co., Ltd. (Shanghai, China). Concentrated ethyl alcohol (Changshu Hongsheng Fine Chemical Co., Ltd) was used. Human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, China). Counting Kit-8 (CCK-8), RPMI Medium Modified (RPMI-1640), fetal bovine serum (FBS), ACS Paragon Plus Environment

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glutaraldehyde, and phosphate buffer saline (PBS, pH ¼ 7.4) were purchased from Shanghai Limin Industrial Co., Ltd. (Shanghai, China). Male SD (Sprague Dawley) rats were purchased from Shanghai Slaccas Experimental Animal Ltd. (Shanghai, China). All chemicals were used as received. 2.2 Preparation of SAB-MSN First, SAB was dissolved in alcohol to obtain mixed solutions with different SAB concentrations (1.0, 2.0, 3.0, and 4.0 mg/mL) at room temperature. Then, the MSN were dispersed in alcohol at different concentrations (1.0, 2.0, 3.0, and 4.0 mg/mL) and subjected to ultrasound to obtain MSN dispersed solutions. Next, the SAB and MSN mixture was vigorously stirred for 24 h at room temperature to form MSN loaded SAB (SAB-MSN). The formed SAB-MSN were separated by centrifugation (13000 rpm, 30 min), and an aliquot was removed from the supernatant to measure the SAB concentration using a 752N UV-Vis spectrophotometer (Jingke Industrial Co., Ltd, Shanghai, China) at 286 nm using a standard SAB concentration absorbance calibration curve was obtained. Finally, SAB-MSN was desiccated in a vacuum dryer for 3 hours at room temperature. The efficiency of SAB loading by the MSN was calculated using the following equation: S(%)= (M1-M2)/M1×100, where M1 denotes the initial amount of SAB, M2 denotes the amount of SAB in the supernatant after centrifugation, and S% indicates the loading efficiency. 2.3 Preparation of the nanofibers 2.3.1 For the preparation of the PLCL/COL (PC) fiber, PLCL and COL blending (w/w, 8:2) was performed by dissolving in HFIP at a concentration of 10%. The applied voltage and solution flow were 10 kV and 1.0 mL/h, respectively. 2.3.2 For the preparation of the core (heparin)-shell (PLCL/COL) fiber (Hep@PC), heparin was dissolved in reverse osmosis (RO) water as a core solution at a concentration of 15%. The shell solution was the same as that used in the preparation of the PC fiber. The coaxial electrospinning was performed using a high voltage of 14 kV and flow rates of 1.0 mL/h for the shell and 0.1 mL/h for the core. 2.3.3 For the preparation of the PLCL/COL blend with SAB-MSN (PC/SAB-MSN)



fiber, SAB-MSN were separately dispersed in HFIP (3 mg/ml) and then processed by ultrasound vibration for 30 minutes to ensure uniform dispersion. For the preparation of the spinning solution, PLCL and collagen were dissolved in the solution of HFIP with SAB-MSN, 3 mg/mL. The applied voltage and a solution flow were 10 kV and 1.0 mL/h respectively. 2.3.4 For the preparation of the core (heparin)-shell (PLCL/COL blend with SAB-MSN) fiber (Hep@PC/SAB-MSN), heparin was dissolved in RO water as a core solution. PLCL and collagen were dissolved in a solution of HFIP with SAB-MSN, at 3mg/mL as a shell. The applied voltage and solution flow of the sheath were 14 kV and 1.0 mL/h, respectively, the core solution flow rate was 0.1 ml/h. The progress of the coaxial electrospinning was showed as Figure.1. The temperature was 25°C and the humidity was maintained at 50% in all electrospinning processes. Fiber membranes underwent to cross-linking in a sealed desiccator with glutaraldehyde (GA) vapor (evaporating from 10 mL of 25% GA aqueous solution) at room temperature for 20 minutes. Then all samples were placed in vacuum dryer for 7 days at room temperature. Finally, the samples were stored in dark place with low temperature for ultimate use.


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Figure 1. The process of fabricating the core (heparin)-shell (PC/SAB-MSN) fiber

2.4 Scaffolds characterization The morphology of the nanofibers was observed by scanning electron microscopy (SEM) (JSM-5600, Japan). The image analysis software (ImageJ, National Institutes of Health, USA) was utilized to analyze the average fiber diameter (n-100) under lower magnification (1000×). The structure of the core-shell fiber was investigated by transmission electron microscopy (TEM) (JEOL, JEM-2100, Japan). Numerous fiber samples were collected in carbon-coated copper grids before TEM observation, and then TEM imaging was achieved by passing a beam of electrons through copper grids containing the fibers without staining. The contact angle of each sample was measured three times using a contact angle instrument (OCA40, Dataphysics, Germany). 2.5 In vitro drug release The concentration of heparin and SAB was separately determined by the toluidine blue method12 and a 752N UV-Vis spectrophotometer (Jingke Industrial Co., Ltd, Shanghai, China) at 286 nm. The theoretical amount of heparin in the fiber membrane



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was calculated using the following equation of A=V×(v1/v2)×H%, where V denotes the shell solution volume,v1 and v2 denote the solution flow of the core (0.1 mL/h) and shell (1.0 mL/h) respectively, and H% indicates the concentration of core solution (15%). The theoretical amount of SAB was calculated using the following equation of B=V×R×S%, where V indicates the shell solution volume (10 mL), R indicates the amount of SAB-MSN blended per milliliter shell solution (3 mg/mL), and S% indicates the loading percentage of SAB loaded MSN. For the heparin and SAB release experiment, 0.2 g fiber membrane of Hep@PC/SAB-MSN was immersed in a 10 mL centrifuge tube containing 2 mL PBS solution. The tube was incubated in a horizontal continuous shaker at 37°C and 120 cycles/min. For analysis, 2 mL medium was removed, and 2 mL fresh PBS was added for subsequent incubation at the pre-scheduled time. The samples in the experiment were divided into two groups, one group was used to test the amount of heparin, and the other group for test the amount of SAB. Known concentrations of SAB and heparin were used to obtain the standard curves, and each time point was tested three times. The amount of heparin was measured by standard methods using toluidine blue (TB, AMERCO, Solon, OH) solution (0.005% TB in 0.2% sodium chloride solution). The percentage of accumulated release can be calculated with the following formula. R% =

∑ × 100% ×

∑ denotes the mass of accumulated release, m denotes the mass of the fiber mat,

and R denotes the percentage of the drug within the fiber. 2.6 Cell culture in vitro The

cytocompatibility

of

fiber

PC,

Hep@PC,

PC/SAB-MSN,

and

Hep@PC/SAB-MSN were evaluated using the CCK-8 cell viability assay. HUVECs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum and 1% antibiotic- antimycotic in a 37°C atmosphere containing 5% CO2, and the medium was replenished every two days. All samples were individually placed in 24-well plates and secured by stainless steel rings. Before seeding cells, all scaffolds and cover slips were sterilized with alcohol vapor for 48 hours, and then ACS Paragon Plus Environment

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washed three times with phosphate-buffered saline solution (PBS). The cell adhesion test was performed when the density of HUVECs reached 2.0 ×104 cells/well. After culturing for 4 h, 8 h, and 12 h, HUVECs were washed with PBS and then evaluated by CCK-8 assay. The cell proliferation test was performed when the density of HUVECs reached 1.0 ×104 cells/well, and was then evaluated on the 1st, 4th and 7th day. The cells viability in the cell adhesion test and cell proliferation test on different scaffolds was measured by CCK-8 assay. The cells and matrices were incubated 400 ml Cell Counting Kit-8 (CCK-8) solution, containing 10% CCK-8 and 90% RPMI-1640, for 2 h. Then 100 mL solution of each sample was transferred into separate wells of a 96-well plate to test the OD value at 450 nm using a microplate reader (MK3, Thermo, USA). The mean and standard deviation from triplicate wells for each sample are provided. Additionally, the viability and morphology of the cells were observed by fluorescence microscopy after using calcein reagent staining. 2.7 Blood compatibility 2.7.1 Measurement of plasma recalcification profiles Whole blood was collected from adult New Zealand experimental rabbits because it had been previously reported that they preserve platelet activity. Plasma recalcification times were determined by the method described by Motlagh. Samples of PC, Hep@PC, PC/SAB-MSN, and Hep@PC/SAB-MSN were placed in a 24-well plate covering the entire bottom surface of the dish, and 500µl of PPP (the platelet poor plasma; blood was spun at 2000 × g for 15 minutes to obtain platelet poor plasma) was added to each well. Controls consisting of tissue culture-treated plastic (TCP), were exposed to PPP with CaCl2 (positive control) and without CaCl2 (negative control). After incubating for 1 hour at 37°C, 100 µL of PPP was transferred from each well plate to a 96-well plate, each of which was prepared in 5 parallel, followed by the rapid addition of 100 µL of 0.025 M CaCl2 in each well except for the negative control. The 96-well plate was placed in a fully automated microplate reader, and absorbance was measured every 30 seconds for 45 minutes at 405 nm38. 2.7.2. Platelet adhesion ACS Paragon Plus Environment


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For PRP (platelet rich plasma), the whole blood was centrifuged at 250 g for 15 min to obtain the PRP supernatant. The PRP preparation and PBS used for this experiment t have been previously described. Samples of PC, Hep@PC, PC/SAB-MSN, and Hep@PC/SAB-MSN were incubated with 500 µl of PRP at 37°C for 2 h. These samples were gently rinsed with warm PBS after incubation to remove the non-adherent cells from the surface. The adherent cells and the biomaterials were fixed for at least 2 hours with glutaraldehyde and paraformaldehyde. Afterward, the samples were dehydrated using an ethanol gradient at 30%, 50%, 70%, 80%, 90%, and 100%. Then, the samples were sputter-coated with a 7-nm layer of gold, and the morphology of adhered platelets was assessed via SEM (Scanning Electron Microscope

3400N,

Electron

Probe

Instrumentation

Center,

Northwestern

University)39. 2.8 Quantitative RT-PCR and Western blot analyses HUVECs were cultured for 24 hours on PC/SAB-MSN fiber membranes and on PC fiber membranes, and then cultured for 4 hours in a standard medium containing 500 µM H2O2. The mRNA expression levels of CHOP, GRP78, cytochrome c (CytC), and caspase-12 in HUVECs were analyzed in triplicate for each sample using quantitative RT-PCR. Protein expression levels of CHOP, GRP78, CytC, and caspase-12 were analyzed by Western blotting (WB) in triplicate for each sample.40-41 2.9 Biocompatibility in vivo Twelve healthy male SD rats were selected, and the body weight was 50-60 g, SPF level. Grafts of PLCL and Hep@PC/SAB-MSN were cut with a 5-mm length and then sterilized with ethylene oxide. Then, the grafts were subcutaneously embedded into SD rats. Finally, the grafts were removed after 3, 7, and 14 days and subsequently stained with hematoxylin and eosin (H&E) and Masson's trichrome for evaluation. The number of inflammatory cells was counted using Image-Pro Plus 6.0 software.

3.0 Statistical analysis The experimental data were evaluated using the one-way ANOVA statistical method. Image analyses were carried out using ImageJ software. A value of 0.05 was ACS Paragon Plus Environment

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selected as the significance level, and the data are indicated with (*) for p < 0.05. 3. Results and discussion 3.1 Loading percentage The surface of MSN is capable of loading drugs by physical adsorption. The loading rate of MSN was tested by adjusting the concentration of MSN and SAB, and the other experimental conditions remained the same. As shown in Figure 2, at the beginning of the test, the drug-loading rate of MSN quickly increased with the increase in drug concentration, and then the rate decreased. This stability can be attributed to the limited number of pores in the MSN surface. Therefore, the adsorption capacity of MSN did not increase with higher drug concentration. The maximum amount of SAB that the MSN could load was approximately 11%.

Figure 2. Percentage of SAB loaded on the MSN; the concentration of the MSN in the SAB solutions was 1, 2, 3, and 4 mg/ml.

3.2 Morphology and structure The surface morphology, diameter distribution, and contact angle of the samples of PC, Hep@PC, PC/SAB-MSN, and Hep@PC/SAB-MSN fiber were obtained (Fig. 3). The average diameter of the fibers without MSN was 564 ± 89 (Fig. 3a), and the fiber with MSN was 633 ± 94 (Fig. 3b); therefore, an increase of 67 nm was observed in the fiber diameter. For coaxial electrospinning, the diameter of the fiber with heparin was 714 ± 87, whereas the average diameter of fiber with heparin and MSN was observed as 821 ± 162 (Fig. 3c-d). Therefore, an increase of approximately 100 nm was observed in this case, and the uniformity of the coaxial electrospinning fiber



(with SAB-MSN) diameter was reduced. The contact angle of PLCL and COL is 118° (Fig. 3a). As shown in Figure 3b, the fiber membrane contact angle decreased to 81°, which may have been caused by the impact on the hydrophilicity of SAB-MSN mixed in the shell material. Because heparin had been loaded in the fiber (Fig. 3c), the hydrophilicity of the fiber membrane changed and the contact angle decreased to 65°. Figure 3d shows that after MSN was loaded with SAB and then spun into fibers, the hydrophilicity of the fiber membrane further increased, and the contact angle decreased to 31°. The hydrophilicity increased due to the exposed SAB-MSN in the fiber surface and the heparin that had been loaded in the fiber.

Figure 3. The contact angle and diameter distribution of samples: (a) PC, (b) PC/SAB-MSN, (c) Hep@PC，and (d) Hep@PC/SAB-MSN.

Single fiber and MSN (Fig. 4) were characterized via TEM, and the dendritic structure of the MSN can be observed. There are many holes in the surface of the MSN (Fig. 4a). The surface and internal structure of the fiber were characterized via TEM (Fig. 4b-4c). Observation of the core structure of the fiber revealed that heparin was uniformly loaded into the fiber (Fig. 4b). Most SAB-MSN were uniformly encapsulated in the shell, and a small amount of SAB-MSN was exposed in the surface of the fiber (Fig.4c). Additionally, it was confirmed through energy dispersive spectrometry (SEM-EDS) that the exposed SAB-MSN was uniformly distributed on the surface of the fiber membrane (Fig. 4d), thus confirming the structure of the fibers.


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Figure 4. TEM images of MSN and core (heparin)–sheath (PLCL and COL with SAB-MSN) fiber: (A) structure of MSN, (B) core-shell structure of the fiber, (C) SAB-MSN in the fiber shell, and (D) SEM-EDS image of the fiber membrane.

3.3 In vitro drug release SAB was continuously released for 30 days (Fig. 5). To construct the vehicle, 0.2 g fiber membrane of Hep@PC/SAB-MSN was immersed in 2 mL PBS solution. SAB was gently released, and no burst release was observed. The cumulative release reached up to 38% in the first 10 days, and then the release process slowed. The total release attained within 30 days reached 56%. SAB was adsorbed on the MSN and blended into the coaxial fiber shell. Through the modified fiber structure, SAB was stable, and sustained released was achieved. An initial burst release of heparin (36%) was observed on the first day, and then a steady increase in release was observed up to 30 days. At the end of the test, the cumulative amount of heparin released was 68%.



Figure 5. Curve of SAB and heparin released from the Hep@PC/SAB-MSN membrane.

3.4 Cell culture in vitro The viability of adhesive cells demonstrated that SAB had a positive impact on improving the rate of cell adhesion (Fig.6A). After culturing HUVECs for 8 h, the HUVEC adhesion on PC/SAB-MSN and Hep@PC/SAB-MSN was significantly greater (p

A Method for Preparation of an Internal Layer of Artificial Vascular

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