静电自组装壳聚糖/肝素多层膜修饰的聚对苯二甲酸丁二醇酯表面对红细胞及血小板的影响
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摘要
目的评价静电自组装壳聚糖和肝素固定于聚苯二甲酸丁二醇酯(PBT)膜表面对红细胞及血小板的影响。方法首先将PBT浸泡于聚乙烯亚胺溶液中氨解使其表面带氨基呈正电性,然后多次交替吸附带负电的肝素和带正电的壳聚糖,形成以肝素为最外层的多层膜结构;对该聚电解质多层膜的理化性能做表征,再做溶血率、红细胞变形性、红细胞渗透脆性、血小板黏附、血小板功能等评价实验。以未接触材料的红细胞或血小板为对照组,实验组分别为未改性PBT以及不同层数的静电自组装改性PBT膜,血小板黏附每组的标本数为5,其余指标每组标本数为9。结果水接触角、zeta电位、原子力显微镜等测量观察:PBT膜表面壳聚糖/肝素多层膜逐渐形成;不同层数的多层膜溶血率<5%,随组装层数增加,红细胞变形指数逐渐增加,10层静电自组装PBT膜与对照组相近,分别为0.545 7±0.010 7 vs 0.546 1±0.013 1(P>0.05);10层静电自组装PBT膜的红细胞渗透脆性与对照组相比明显降低,分别为4.270 1±0.089 5 vs 4.338 8±0.076 2(P<0.05),说明红细胞对低渗溶液的抵抗力增大。扫描电镜显示:未改性PBT膜表面黏附的血小板数明显多于改性膜表面,且观察到明显的伪足,随组装层数的增加,血小板在多层膜表面的黏附数减少。未改性PBT膜与2/5/10层静电自组装PBT膜表面黏附的血小板数分别为722 7±222 2 vs 468 2±158 5 vs 327 3±185 0 vs 227 3±103 1(P<0.05),未改性PBT膜与对照组的血小板最大聚集率(%)为82.36±2.11 vs87.22±2.10(P<0.05),低渗休克相对变化率(HSR)(%)为77.12±1.09 vs 79.33±1.27(P<0.05),CD62p表达率(%)为17.45±1.25 vs 9.78±0.58(P<0.05);随组装层数的增加,血小板最大聚集率(%)增加,HSR增大,CD62p表达率降低。其中,10层静电自组装PBT膜与未改性PBT膜相比,血小板最大聚集率(%)增加,分别为82.35±1.91 vs94.79±1.84(P<0.05),HSR相似,分别为77.32±0.95 vs 76.76±2.35(P>0.05),CD62p表达率降低,分别为17.67±1.25 vs 14.67±1.15(P<0.05)。结论壳聚糖/肝素静电自组装修饰PBT后,PTB膜亲水性改善,减少了血小板的黏附且对血小板功能和红细胞无明显影响。
Objective To evaluate the effect of modified polybutylene terephthalate( PBT) with immobilized bio-functional chitosan and heparin on red blood cells( RBCs) and platelets. Methods PBT was first soaked in a polyethyleneimine solution for aminolysis to form positive charge on the material surface. Negatively charged heparin and positively charged chitosan were then applied to the surface for alternating coating with the heparin layer being the final coating layer of the structure. The physicochemical properties of the polyelectrolyte multilayer were characterized,followed by tests on the hemolytic rate,erythrocyte deformability,erythrocyte osmotic fragility,platelet adhesion and platelet function to evaluate its effect on RBCs and platelets. The PBT contact-free RBCs and platelets were used as the control while the experiment group contained both unmodified PBT and modified ones with different coating layer counts. Each test was performed on a set of 9 samples except the platelet adhesion test( 5 samples). Results The results from water contact angle tests,zeta potential measurement and atomic force microscopy( AFM) showed that the chitosan/heparin multilayer formed gradually on the PBT surface. The hemolysis rate of multilayer films with different layer counts was less than 5%. The RBC deformability index increased as the coating layer increased. Nevertheless,a layer count of 10 presented no statistical different against the control( 0. 545 7 ±0. 010 7 vs 0. 546 1±0. 013 1,P>0. 05). The erythrocyte osmotic fragility dropped significantly for the ten-layer coating film compared to the control at 4. 270 1± 0. 089 5 vs 4. 338 8± 0. 0762( P< 0. 05),indicating increased erythrocyte resistance against low osmotic solution. Scanning electron microscopy( SEM) tests revealed that the intensity of platelet adhesion on the unmodified PBT was significantly higher than the modified ones. The adhesion activity dropped as the coating layer number increases: 722 7±222 2( unmodified) vs 468 2±158 5( 2 layers) vs 327 3± 185 0( 5 layers) vs 227 3±103 1( 10 layers)( P<0. 05). Referencing the control group,the unmodified PBT film presented negative impact on platelet function as the platelet aggregation rate [( 82. 36±2. 11) % vs( 87. 22±2. 10) %,P< 0. 05],the hypotonic shock relative( HSR) change rate [( 79. 33±1. 27) % vs( 77. 12±1. 09) %,P<0. 05],the expression rate of CD62 p [( 9. 78±0. 58) % vs( 17. 45±1. 25) %,P<0. 05]were in an inferior status. Comparing the unmodified and modified PBT,increased coating layers improved the maximum aggregation rate and HSR status while the expression of CD62 p were suppressed. The ten-layer coating PBT,in particular,presented an increased maximum aggregation rate( 82. 35 ± 1. 91 vs94. 79±1. 84,P<0. 05),similar HSR( 77. 32 ± 0. 95 vs 76. 76 ± 2. 35,P > 0. 05) and decreased CD62 p expression( 17. 67 ±1. 25 vs 14. 67±1. 15,P<0. 05). Conclusions With the chitosan/heparin multilayer coating,the hydrophilicity of the PBT surface has been improved with reduced platelet adhesion and the platelet/RBC function is not significantly affected.
Objective To evaluate the effect of modified polybutylene terephthalate( PBT) with immobilized bio-functional chitosan and heparin on red blood cells( RBCs) and platelets. Methods PBT was first soaked in a polyethyleneimine solution for aminolysis to form positive charge on the material surface. Negatively charged heparin and positively charged chitosan were then applied to the surface for alternating coating with the heparin layer being the final coating layer of the structure. The physicochemical properties of the polyelectrolyte multilayer were characterized,followed by tests on the hemolytic rate,erythrocyte deformability,erythrocyte osmotic fragility,platelet adhesion and platelet function to evaluate its effect on RBCs and platelets. The PBT contact-free RBCs and platelets were used as the control while the experiment group contained both unmodified PBT and modified ones with different coating layer counts. Each test was performed on a set of 9 samples except the platelet adhesion test( 5 samples). Results The results from water contact angle tests,zeta potential measurement and atomic force microscopy( AFM) showed that the chitosan/heparin multilayer formed gradually on the PBT surface. The hemolysis rate of multilayer films with different layer counts was less than 5%. The RBC deformability index increased as the coating layer increased. Nevertheless,a layer count of 10 presented no statistical different against the control( 0. 545 7 ±0. 010 7 vs 0. 546 1±0. 013 1,P>0. 05). The erythrocyte osmotic fragility dropped significantly for the ten-layer coating film compared to the control at 4. 270 1± 0. 089 5 vs 4. 338 8± 0. 0762( P< 0. 05),indicating increased erythrocyte resistance against low osmotic solution. Scanning electron microscopy( SEM) tests revealed that the intensity of platelet adhesion on the unmodified PBT was significantly higher than the modified ones. The adhesion activity dropped as the coating layer number increases: 722 7±222 2( unmodified) vs 468 2±158 5( 2 layers) vs 327 3± 185 0( 5 layers) vs 227 3±103 1( 10 layers)( P<0. 05). Referencing the control group,the unmodified PBT film presented negative impact on platelet function as the platelet aggregation rate [( 82. 36±2. 11) % vs( 87. 22±2. 10) %,P< 0. 05],the hypotonic shock relative( HSR) change rate [( 79. 33±1. 27) % vs( 77. 12±1. 09) %,P<0. 05],the expression rate of CD62 p [( 9. 78±0. 58) % vs( 17. 45±1. 25) %,P<0. 05]were in an inferior status. Comparing the unmodified and modified PBT,increased coating layers improved the maximum aggregation rate and HSR status while the expression of CD62 p were suppressed. The ten-layer coating PBT,in particular,presented an increased maximum aggregation rate( 82. 35 ± 1. 91 vs94. 79±1. 84,P<0. 05),similar HSR( 77. 32 ± 0. 95 vs 76. 76 ± 2. 35,P > 0. 05) and decreased CD62 p expression( 17. 67 ±1. 25 vs 14. 67±1. 15,P<0. 05). Conclusions With the chitosan/heparin multilayer coating,the hydrophilicity of the PBT surface has been improved with reduced platelet adhesion and the platelet/RBC function is not significantly affected.
引文
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[16]邢卫红,仲兆祥,景文珩,范益群.基于膜表面与界面作用的膜污染控制方法.化工学报,2013,64(1):173-181.
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[21]钱自奋.红细胞变形性的生理及临床意义.中国中西医结合杂志,1995,15(4):244-245.
[22]宋立川,喀蔚波.红细胞衰老过程中渗透脆性的变化.北京生物医学工程,1997,16(3):178-180.
[23]Lee JH,Lee HB.Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins.J Biomed Mater Res,1998,41(2):304-311.
[24]Yuan J,Zhu J,Zhu CH et al.Platelet adhesion on a polyurethane surface grafted with a zwitterionic monomer of sulfobetaine via a Jeffamine spacer.Polymer Intern,2004,53(11):1722-1728.
[25]刘更夫,黄飞,梅方超,等.血小板CD62p表达率与血小板输注效果的关联分析.中国输血杂志,2016,29(3):295-297.
[26]Rinder HM,Murphy M,Mitchell JG,et al.Progressive platelet activation with storage:evidence for shortenedsurvival of activated platelets after transfusion.Transfusion,1991,31(5):409-414.
[27]Slichter SJ,Bolgiano D,Jones MK,et al.Viability and function of8-day-stored apheresis platelets.Transfusion,2006,46(10):1763-1769.
[28]张丽,李静旗.体外保存血小板质量评价方法现状及研究进展.临床输血与检验,2016,18(5):509-512.
[2]Mari G,Veronesi S,Mathieu B.Filter for the removal of substances from blood products[P].US 7,524,425.2009-04-28.
[3]Mari G,Verri P,Ori A.Filter for the depletion of leukocytes from blood products[P].WO,018078.2004-03-04.
[4]Kim EJ,Yeo GD,Pai CM et al.Preparation of surface-modified poly(butylene terephthalate)nonwovens and their application as leukocyte removal filters.J Biomedi Materi Rese P B:App Biomater,2009,90(2):849-856.
[5]Cai K,Rechtenbach A,Hao J,et al.Polysaccharide-protein surface modification of titanium via a layer-by-layer technique:characterization and cell behaviour aspects.Biomaterials,2005,26(30):5960-5971.
[6]Richert L,Lavalle P,Payan E,et al.Layer by layer buildup of polysaccharide films:physical chemistry and cellular adhesion aspects.Langmuir,2004,20(2):448-458.
[7]Kang IK,Kwon OH,Kim MK et al.In vitro blood compatibility of functional group-grafted and heparin-immobilized polyurethanes prepared by plasma glow discharge.Biomaterials,1997,18(16):1099-1107.
[8]Kim YJ,Kang IK,Huh MW et al.Surface characterization and in vitro blood compatibility of poly(ethyleneterephthalate)immobilized with insulinand/or heparin using plasma glow discharge.Biomaterials,2000,21(2):121-130.
[9]Khor E,Lim LY.Implantable applications of chitin and chitosan.Biomaterials,2003,24(13):2339-2349.
[10]Berger J,Reist M,Mayer JM,et al.Structure and interactions inchitosan hydrogels formed by complexation or aggregation for biomedical applications.Eur J Pharm Biopharm,2004,57(1):35-52.
[11]国家质量技术监督局.中华人民共和国国家标准.医疗器械生物学评价GB/T 16886.12-2000.第12部分:样品制备与参照样品..
[12]国家食品药品监督管理局.中华人民共和国医药行业标准.一次性使用去白细胞滤器YY0329-2009.
[13]尚红,王毓三,申子瑜.溶血性贫血的检验//尚红,王毓三.全国临床检验操作规程.4版.北京:人民卫生出版社,2014:66-67.
[14]王红,刘嘉馨,雷宇,等.2种国产血小板滤器滤除白细胞对体外血小板功能的影响.中国输血杂志,2011,24(4):323-326.
[15]舒瑶,李全利,欧国敏,等.壳聚糖-肝素静电自组装多层膜在钛表面进行的生物化修饰.中国组织工程研究与临床康复,2010,14(21):3832-3838.
[16]邢卫红,仲兆祥,景文珩,范益群.基于膜表面与界面作用的膜污染控制方法.化工学报,2013,64(1):173-181.
[17]赵红,邹迪婧,李颖,等.聚氨酯/(羧甲基壳聚糖/壳聚糖)n复合涂层血液相容性.大连理工大学学报,2010,50(2):194-197.
[18]国家食品药品监督管理局.中华人民共和国国家标准.医用输液、输血、注射器具检验方法.GB/T 14233-2005.第2部分:生物学试验方法.
[19]Yao C,Huang Y,Li X,et al.Effects of p H on structure and function of single living erythrocyte.Chin Sci Bull,2003,48(13):1342-1346.
[20]Murthy N,Chang I,Stayton P,et al.p H-sensitive hemolysis by random copolymers of alkyl acrylates and acrylic acid.Macromol Symp 2001,172(1):49-56.
[21]钱自奋.红细胞变形性的生理及临床意义.中国中西医结合杂志,1995,15(4):244-245.
[22]宋立川,喀蔚波.红细胞衰老过程中渗透脆性的变化.北京生物医学工程,1997,16(3):178-180.
[23]Lee JH,Lee HB.Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins.J Biomed Mater Res,1998,41(2):304-311.
[24]Yuan J,Zhu J,Zhu CH et al.Platelet adhesion on a polyurethane surface grafted with a zwitterionic monomer of sulfobetaine via a Jeffamine spacer.Polymer Intern,2004,53(11):1722-1728.
[25]刘更夫,黄飞,梅方超,等.血小板CD62p表达率与血小板输注效果的关联分析.中国输血杂志,2016,29(3):295-297.
[26]Rinder HM,Murphy M,Mitchell JG,et al.Progressive platelet activation with storage:evidence for shortenedsurvival of activated platelets after transfusion.Transfusion,1991,31(5):409-414.
[27]Slichter SJ,Bolgiano D,Jones MK,et al.Viability and function of8-day-stored apheresis platelets.Transfusion,2006,46(10):1763-1769.
[28]张丽,李静旗.体外保存血小板质量评价方法现状及研究进展.临床输血与检验,2016,18(5):509-512.