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The exploration on blood coagulation of Nalkyl chitosan nanofiber membrane in vitro Xiaoyan Wang, Guan Jing, Xupin Zhuang, Zhihong Li, Shujie Huang, Jian Yang, Changjun Liu, Fan Li, Feng Tian, Jimin Wu, and Zhan Shu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01492 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018
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Biomacromolecules
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The exploration on blood coagulation of N-alkyl chitosan
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nanofiber membrane in vitro
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Xiaoyan Wang1,2, Jing Guan1*, XuPin Zhuang2*, Zhihong Li1, Shujie Huang1, Jian Yang1, ChangJun
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Liu1, Fan Li1, Feng Tian1, Jimin Wu1, and Zhan Shu1.
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1 Key laboratory of Medical Equipment, Academy of Military Medical Sciences, No. 106 Wandong
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Road, Hedong District, Tianjin, 300161, China
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2 Department of Textile, Tianjin Polytechnic University, No. 399 Binshui West Road, Xiqing District,
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Tianjin, 300387, China.
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Abstract:
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N-alkylated chitosan (NACS) may improve blood clotting efficiency of chitosan (CS). To study its
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blood coagulation capability, a series of NACS with various carbon chain lengths and degrees of
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substitution (DS) of alkyl groups were synthesized and characterized by FTIR, NMR, elemental
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analysis, and X-Ray diffraction (XRD). The corresponding NACS nanofiber membranes (NACS-NM)
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were subsequently fabricated by electronic spinning technique. SEM, XRD, DSC, surface area,
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porosity, contact angle, blood absorption and mechanical property were used to characterize the basic
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property of CS-NM/NACS-NM. Moreover, cytotoxicity assay, coagulation, activated partial
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thromboplastin time, plasma prothrombin time, thrombin time, and platelet aggregation tests were
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performed to evaluate the biocompatibility and blood coagulation property of NACS-NM. The result
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showed that NACS-NM was no cytotoxic. NACS-NM with DS of 19.25% for N-hexane CS (CS6b),
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17.87% for N-dodecane CS (CS12b), and 8.97% for N-octadecane CS (CS18a) exhibited good blood
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clotting performance. Moreover, NACS-NMs favored the activation of coagulation factors and platelets.
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In addition, intracellular Ca2+ was not related to platelet activation. Above results suggested that NACS-
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NM would be an effective hemostatic agent.
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Keywords: N-alkyl chitosan, Nanofiber membrane, Coagulation, Cytotoxicity, Platelet.
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1.INTRODUCTION
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Uncontrolled trauma hemorrhage, one of the three major causes of unpreventable death, causes high
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mortality rate and severe complications, Statistics data shows that more than 85% of the deaths is from
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potentially survivable wounds in battlefields and more than half of trauma-related deaths in civilian
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mortalities1-2. Hence, developing effective hemostatic agents is considerably important. Topical
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hemostatic agents that are recently available include oxidized cellulose fabrics, absorbable gelatin
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sponge, fibrin glue, topical thrombin and microfibrillar collagen powder. However, each product has
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exhibited potential drawbacks so that no single product has dominantly emerged as preference3.
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Therefore, safe, fast, and effective hemostatic materials still need investigating.
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Chitosan (CS), β-(1-4) linked 2-amino-2-deoxy-β-D-glucopyranose, is a N-deacetylated derivative of
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chitin obtained by transforming the acetamide groups into primary amino groups4. CS shows good
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biocompatibility, tissue regeneration, antibacterial, anti-inflammatory and hemostatic properties5,
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which involves the agglutination of blood proteins and platelet activation for encouraging fibrin clot
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formation6. Previous work proved that CS derivatives prepared by grafting hydrophobic groups could
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improve hemostatic property greatly7-8. N-alkylated chitosan (NACS) becomes one of the most
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desirable materials for fabrication of ideal hemostatic dressings because of its outstanding hemostatic
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activity and excellent plugging ability to hemorrhaging wound surface9-11. NACS could transform
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whole liquid blood into a gel quickly, and therefore it can stop bleeding rapidly from both minor and
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severe injuries in small and large animals12. Further studies showed that NACS with suitable carbon
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length and degree of substitution (DS) of alkyl groups could accelerate blood clotting significantly7.
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Until now only four kinds of NACS-based hemostatic materials, namely, powder, sponge, foam, and
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gauze, are currently available13-15. Nanofiber membrane (NM) is well known as possessing larger
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surface area than above forms of materials due to its nanoscale size effect16-18, thus, NACS-NM could
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have the potential suggested to accelerate platelet aggregation.
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It’s commonly known that CS and its derivatives can be fabricated by electrospinning19-21, while
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NACS-NM has not been fabricated publicly up to date. In the present study, we modified CS on -NH2
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group and prepared CS-NM and NACS-NM as novel materials by electrospinning. The basic properties
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of NACS were characterized by FTIR, NMR, elemental analysis and X-Ray diffraction (XRD), and
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those of NACS-NMs were further assessed by SEM, XRD, DSC, surface area, porosity, contact angle,
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blood absorption and mechanical properties. The cytotoxicity of NACS-NM was evaluated by MTT
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and fluorescence labeling method. The clotting performance was subsequently measured by in vitro
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coagulation time and thromboelastograph (TEG). Finally, the effect of NACS-NM on blood
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components was assayed by blood plasma coagulation, platelet aggregation and intracellular Ca2+
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concentration tests.
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2.EXPERIMENTAL SECTION
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2.1 Materials
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CS (Huantai county golden shell products Co, Ltd, Zibo, Shandong, China), with high molecular
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weight of 1.97×106 and deacetylation degree (DD) of 90.0%, was used without further purification.
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Sodium hydroxide, ice acetic acid, ethanol, and other reagents were all of analytical grade. The
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activated partial thromboplastin time (APTT), plasma prothrombin time (PT), and thrombin time (TT)
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reagents and MTT kits were bought from Shanghai Sun biotechnology Co. Ltd. Hexane aldehyde,
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dodecyl aldehyde, and octadecane aldehyde were purchased from Sigma (St. Louis, MO, USA).
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Fluorescent reagents FITC, anti-P selectin, Fluo-4 AM, SYTO 9, and propidium iodide (PI) were
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obtained from Invitrogen (Carlsbad, CA, USA).
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2.2 Fabrication of CS-NM/NACS-NM
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NACS was dissolved in acetic acid (90%, v/v) to prepare a solution of 1.5% (w/v), then the solution
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was moved into a 20mL syringe, with which a circular needle (inner diameter: 0.5 mm; length: 20 mm)
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was connected. In addition, a syringe pump was applied to supply the solution to the needle at a rate of
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1.5 mL/h and a collector was fixed at distance of 15 cm from the needle of syringe. During
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electrospinning, the needle was submitted to a voltage of 25 kV. All the experiments were carried out at
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room temperature under a humidity of less 30%. After electrospinning, the CS-NM/NACS-NM were
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removed from the collector, dried in an oven at 60 ℃, and neutralized by an aqueous solution of K2CO3
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for 2 h at 25 ℃. Subsequently, CS-NM/NACS-NM was washed with distilled water until pH 7 was
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obtained and then vacuumed in an oven at 50 ℃ for 24 h.
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2.3 The properties of CS-NM/NACS-NM
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2.3.1 Morphology
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The surface morphology of the CS-NM/NACS-NM were observed by SEM (S-4800, HITACH, Japan).
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The diameters of fibers and their distributions in the CS-NM/NACS-NM were assessed by using the
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image pro-plus software.
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2.3.2 Crystallinity
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The XRD patterns of CS-NM/NACS-NM were also determined according to the same method as the
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CS/NACS. The thermal behavior of CS-NM and NACS-NM were characterized by differential
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scanning calorimetry (DSC; DSC-200 F3, NETZSCH Company, Germany) in the temperature range of
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30–600 °C at a heating rate of 10 °C/min.
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2.3.3 Surface area and porosity
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The surface area of CS-NM/NACS-NM was tested by automatic gas adsorption analyzer (Autosorb iQ,
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America). Preprocessing parameters: decompression drying at 200 °C, 2 h, nitrogen gas for gas
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absorption, absorption temperature of -196 °C. The porosity of CS-NM/NACS-NM was calculated by
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referring the literature22-23.
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2.3.4 Water contact angle and blood absorption
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Contact angle was measured by a contact angle goniometer (JYSP-180, Jinshengxin Testing Machine
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Co. Ltd., China). To evaluate the hydrophilic-hydrophobic property of CS-NM/NACS-NM, all of the
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membranes were completely dried prior to measurement to avoid water interaction issue24. Then a 5.0
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mL pure water droplet was placed onto the surface of CS-NM/NACS-NM using a microsyringe, after
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which contact angle data were recorded by routine procedure. At least three readings from different
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surface locations were obtained for each measurement.
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A (2×2 cm2 CS-NM/NACS-NM) weight (Md) of nanofiber was immersed into the 1 mL anticoagulant
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whole blood and soaked in the blood for 2 min. After that, the weight of the soaked nanofiber (Mw)
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was measured using a balance. Each sample was tested three times. The blood absorption (A) of
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nanofiber was calculated by the following equation:
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A=
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2.3.5 Mechanical property
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The mechanical property of NM with a width of 1.5 mm and length of 14 mm was tested by an
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electronic single-fiber strength tester. The test condition was: advance tension (0.4 cN), gauge length
ெೢ ିெ ெ
× 100%
(1)
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(10 mm), stretching speed (10 mm/min). Each sample was tested three times.
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2.4 Cytotoxicity
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2.4.1 MTT
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The in vitro cytotoxicity of NM was estimated by MTT assay according to ISO 10993-5. Samples
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immersed in DMEM（6 cm2/mL）were kept in an incubator (5% CO2, 37 ℃) for 24 h, then the
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samples were removed and the excess medium was named as liquid extract of experimental group.
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Pure DMEM maintained under the same condition was named as liquid extract of control group. At the
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same time, the human fibroblast cells (MRC5) were first seeded into 96-well plates at a seeding density
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2×104 cells/mL and then incubated in DMEM supplemented with penicillin (100 U/mL), streptomycin
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(100 g/L), and 10% fetal bovine serum overnight. Subsequently, the DMEM medium was replaced with
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the liquid extract of the experiment/control group (100 µL). The cells were then cultured continuously
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for 72 h. Afterwards, MTT solution (5 g/L) was added, and the cells were incubated for another 4 h.
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Subsequently, the incubation medium was replaced with 150 µL of DMSO to dissolve the formazan
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crystals. The amount of MTT-formazan complex was determined by measuring the absorbance of
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dissolved crystals at 570 nm, with 655 nm as a reference7. The relative growth rate (RGR) was
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calculated as follow:
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RGR =
A ×100% A0
(2)
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Where A is the difference of sample groups’ absorbance at 570 nm and 630 nm, and A0 is the difference
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of control groups’ absorbance at 570 nm and 630 nm.
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2.4.2 Live/dead assay
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The live/dead double-staining method was introduced to determine the cytotoxicity of NACS-NM. The
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MRC5 cells were incubated in liquid extract of experimental and control groups, which was similar to
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the MTT assay. Then 10 µL of PI and SYTO 9 mixtures were added to stain the cells, after which the
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cells were incubated at 37 °C for 15 min. Subsequently, the cells were observed and captured under a
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fluorescence microscope (F-4600, Hitachi, Tokyo, Japan).
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2.5 Blood coagulation properties of CS-NM/NACS-NM
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All blood used in the following experiments was sampled from rabbits, which were bred by the Tianjin
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medicine research institute. The anticoagulant whole blood was prepared according to the ratio of blood
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to sodium citrate equaling 9:1(v/v).
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2.5.1 Blood coagulation time (BCT)
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First, 2×2 cm2 CS-NM/NACS-NM was placed in the test tube in a 37 ℃ water-bathing constant
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temperature vibrator for 3 min. Anticoagulant whole blood (1 mL) was subsequently added into the
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tube. After 1 min, 775 µL of CaCl2 (0.025mol/L) was added, and the time for blood clotting was then
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recorded. The control group (blood only) was also evaluated by the same procedure. Each sample was
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tested three times.
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2.5.2 TEG
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To monitor the whole dynamic process of thrombus forming, TEG was used to analyze the platelet
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aggregation, blood coagulation, and fibrinolysis. First, 20 µL of CaCl2 (0.2 mol/L) solution was added
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to the polyethylene cup. Afterward, 340 µL of mixed blood (experimental group, CS/NACS:
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anticoagulant blood=10 mg: 1 mL) or pure blood (control group) was also added into the cup. The cup
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was loaded then into defined position to collect data.
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2.5.3 APTT, PT and TT
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The test equipment is a semiautomatic blood coagulation analyzer (Clotec-II, Sysmex, Kobe, Japan).
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First, the anticoagulant whole blood was centrifuged at 3000 rpm for 15 min. The supernatant was
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drawn as platelet-poor plasma (PPP). In experimental groups, each CS-NM/NACS-NM (1×1 cm2) was
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mixed with 200 µL PPP and warmed at 37 ℃ water bath for 15 min. Subsequently, 100 µL of mixture
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(sample and PPP) was centrifuged, and the supernatant was mixed with 200 µL of prewarmed PT
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reagents. The elapsed time from the addition of PT until blood clotting was recorded as PT value. When
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TT was tested, a procedure similar to that of PT measurement was performed, except that the amount of
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TT reagent was 100 µL. APTT was measured according to the TT test method, except for the 100 µL of
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CaCl2 (0.025mol/L) required to initiate clotting. PPP (control group) was also evaluated by using these
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methods. Each test was conducted at least three times.
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2.6 Platelet aggregation
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Anticoagulant whole blood was centrifuged at 1500 rpm for 15 minutes, and the platelet-rich plasma
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(PRP) was aspirated using a straw. Approximately 200 µL PRP was added to the CS-NM/NACS-NM (1
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x 1 cm2) and incubated at 37 ℃ for 1 h. To eliminate the surface adhesion of blood platelet on NACS-
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NM/CS-NM, the samples were gently washed three times by a gradient series of ethanol (50%, 75%,
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85% and 100%). Finally, the samples were immersed in 2.5% glutaraldehyde fixation fluid overnight,
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dried and observed under SEM.
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2.7 Fluorescence intensity (FI) of Ca2+
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Anticoagulant whole blood was obtained and centrifuged at 1200 x g for 20 min. The upper plasma was
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aspirated and centrifuged at 1200 x g for 15 min again. The bottom platelets were transferred into an
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EP tube containing 10 µL Fluo-4 AM. After incubation at 37 ℃ for 15 min, the CS-NM/NACS-NM (1 x
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1 cm2) was added, and the mixtures were incubated for another 15 min. Subsequently, 0.1% Trion –
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100 was added into each EP tube with severe concussion for 5 min. Each EP tube was centrifuged with
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1200 x g at 4 ℃ for 5 min and 100 µL of supernatant was added into the black 96-well plates. The
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Ex/Em = 485 nm/505 nm wavelength was evaluated by a microplate reader.
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2.8 Statistical analysis
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All data were expressed as the mean ± standard deviation. The significance of differences was
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determined by one-way ANOVA. Results were considered significant at P
