胆红素水溶性分子吸附剂的制备及性能研究

发布时间：2018-06-11 21:49

本文选题：吸附透析 + 胆红素　；参考：《大连理工大学》2012年博士论文

【摘要】：分子吸附再循环系统(Molecular Adsorbents Recycling System, MARS)是目前临床治疗重症肝病效果优良的非生物型人工肝支持系统。在以人血清白蛋白(Human serum albumin, HSA)为分子吸附剂的吸附透析过程中,添加到透析液中的HSA与血液中的蛋白质竞争性结合疏水性毒素并被透析液带走,从而有效、广谱去除患者血液中的蛋白质结合毒素及水溶性毒素,实现改善患者体内环境、促进肝脏功能恢复和为肝移植争取时间的目的。MARS的治疗效果已经得到医生和患者的广泛认可,在国外被推荐作为各类肝衰竭的标准治疗方案。由于HSA是MARS实现解毒功能的关键分子吸附剂,用量较大,而作为注射制剂的HSA来源有限,价格昂贵。HSA来源受限及其再生设备复杂等问题导致MARS临床治疗费用高昂,其应用人群范围受限。鉴于此,本论文设计合成了一种水溶性胆红素分子吸附剂替代HSA用于对肝病病人血液的吸附透析,该吸附剂在实现对胆红素等毒素高效去除的同时,可以简化治疗设备,显著降低治疗成本。在本论文中,首先针对胆红素分子的结构特点,选择壳聚糖、右旋糖酐、聚乙烯亚胺(Polyethyleneimine, PEI)等水溶性高分子为载体,制备了带有不同吸附功能基的系列胆红素分子吸附剂。对其胆红素吸附能力评价的结果表明：以PEI为载体、以β-环糊精(β-cyclodextrin,β-CD)为功能基的水溶性吸附剂P-CD-PEI在相同条件下对胆红素的吸附透析能力最强。进一步以P-CD-PEI为研究对象,对其合成过程进行了优化。最终获得的β-CD-PEI的平均分子量约为157kD,平均每个β-CD-PEI分子上具有366个分枝点,偶联β-CD的数量约为51个。选择Nipro Sureflux-130G透析器,采用200mL含1%(w/v) p-CD-PEI的透析液对150mL含300mg／L胆红素的人血浆进行吸附透析,其对血浆中胆红素的1h吸附量达到3.55mg／g。采用分子模拟的方法研究了分子吸附剂功能基p—CD与胆红素的结合方式以及结合能,并与α-CD、γ-CD及HSA与胆红素的相互作用进行了比较。研究结果表明：在环糊精家族中,α-CD由于疏水内腔太小,不能与胆红素形成包合物；β-CD及Y-CD与胆红素形成的2：1包合物比1：1包合物更稳定,其中p—CD胆红素2：1包合物稳定性高于HSA与胆红素的复合物,意味着β-CD与胆红素的结合能力强于HSA,有能力与HSA竞争结合胆红素。偶联反应对β-CD分子结构的改变不会减弱其与胆红素的结合能力。进一步通过将α、β、γ-CD分别偶联到PEI载体上合成了三种分子吸附剂,吸附透析实验结果表明β-CD-PEI对胆红素的结合能力最强；使用Benesi-Hildebrand法分析β-CD与胆红素包合后胆红素紫外可见光谱变化,进一步确认了β-CD与胆红素以2：1的比例形成包合物,从而证实了分子模拟研究的结果。在吸附透析治疗中,对血浆胆红素的去除效果取决于透析膜的特性、血浆流速、透析液流速、血浆中的胆红素浓度、透析液中水溶性吸附剂的浓度以及吸附透析时间等因素。在吸附透析系统的参数优化中发现：当血浆及透析液流速分别为300和50mL/min时,β-CD-PEI吸附透析对血浆胆红素的去除效果最佳；超滤形成的跨膜液流产生的浓差极化现象增加了胆红素的传质阻力,降低了β-CD-PEI在透析膜内的分子扩散,不利于β-CD-PEI吸附透析对胆红素的去除；以聚醚砜为膜材质的Lengthen LST140透析器对于胆红素的传质效果强于以三醋酸纤维素为膜材质的Nipro Sureflux-130G透析器；透析膜表面及孔道中吸附的白蛋白能够对胆红素进行协助传递,进而提高透析膜对胆红素的传质效果。在吸附透析过程中,β-CD-PEI的吸附量随着血浆中胆红素浓度的升高而增加,采用1L浓度为1%的β-CD-PEI透析液对200mL胆红素初始浓度分别为80.3、140.4、214.8、267.3、305.2mg／L的肝病病人血浆进行吸附透析,6h的总胆红素去除率均能达到30-40%,适用于不同患病程度的肝病病人。增加分子吸附剂β-CD-PEI的用量可以提高吸附透析对胆红素的去除效果。使用1L4%的β-CD-PEI透析液可以去除血浆中44.8%的胆红素(初始浓度140.4mg／L),比相同情况下4L1%分子吸附剂的胆红素去除率提高9个百分点。β-CD-PEI对血浆胆红素的清除率比相同质量分数的牛血清白蛋白高9.5个百分点,同时β-CD-PEI对血浆中的总胆汁酸、芳香族氨基酸等疏水毒素也具有明显的去除能力,说明β-CD-PEI具有替代白蛋白进行吸附透析的潜力。通过基于物料衡算的数学模型描述了吸附透析过程中血浆胆红素浓度变化规律。通过实验数据回归,计算得到了Nipro Sureflux-130G血液透析膜对胆红素的总传质系数Dt为1.7L/min, p-CD-PEI与胆红素的吸附平衡常数Ka1为22.7L/μmoL。进一步使用该模型预测了同一体系中血浆胆红素初始浓度和吸附剂用量改变时胆红素的吸附透析效果,平均误差5%。上述研究结果证明,胆红素水溶性分子吸附剂β-CD-PEI对肝病患者血浆中的胆红素及其它疏水性毒素具有较高的清除能力,同时具有价格低廉、治疗设备简单的优势。具有替代白蛋白应用于临床治疗的潜力。
[Abstract]:Molecular Adsorbents Recycling System (MARS) is an excellent abiotic artificial liver support system for the treatment of severe liver disease. In the process of adsorption and dialysis with human serum albumin (Human serum albumin, HSA) as a molecular adsorbent, HSA and protein in the blood are added to the dialysate. Competitive combination of hydrophobic toxin and dialysate is taken away, which is effective and broad-spectrum removal of protein binding toxins and water-soluble toxins in the patient's blood to improve the environment of the patient, promote the recovery of liver function and the time for liver transplantation for the purpose of.MARS treatment has been widely recognized by doctors and patients abroad. It is recommended as the standard treatment for all kinds of liver failure. Because HSA is a key molecular adsorbent for MARS to realize detoxification, the amount of HSA is limited, the limited source of.HSA and the complexity of the regenerating equipment, such as the limited source of the price and the complexity of the regeneration equipment, are very expensive for the clinical treatment of MARS, and the scope of its application is limited. In view of this, In this paper, a water soluble bilirubin molecular adsorbent is designed and synthesized to replace HSA for the absorption and dialysis of the blood of patients with liver disease. The adsorbent can be used to effectively remove the bilirubin and other toxins. It can simplify the treatment equipment and reduce the cost of treatment significantly.
In this paper, a series of bilirubin molecular adsorbents with different adsorption functional groups were prepared by selecting the water soluble polymers such as chitosan, dextran, Polyethyleneimine, PEI, and other water-soluble polymers, such as chitosan, dextran, and polyethyleneimine (PEI). The results of the evaluation of the adsorption capacity of the bilirubin showed that PEI was used as the carrier. The adsorption and dialysis ability of beta cyclodextrin (beta -cyclodextrin, beta -CD) as a functional group, P-CD-PEI, was the strongest for bilirubin under the same condition. The synthesis process was optimized with P-CD-PEI as the research object. The average molecular weight of beta -CD-PEI was about 157kD, with an average of 366 on each beta -CD-PEI molecule. At the branch point, the number of the coupling beta -CD was about 51. The Nipro Sureflux-130G dialyzer was selected and the 200mL containing 1% (w/v) p-CD-PEI dialysate was used to adsorb the human plasma containing 300mg / L bilirubin in 150mL, and the 1H adsorption capacity of the bilirubin in plasma reached 3.55mg / g..
The binding energy and binding energy of the functional group P CD with bilirubin are studied by molecular simulation, and the interaction between the alpha -CD, gamma -CD and HSA with bilirubin is compared. The results show that in the cyclodextrin family, the alpha -CD can not form a inclusion complex with bilirubin because the hydrophobic inner cavity is too small; beta -CD and Y-CD are found in the cyclodextrin family. The 2:1 inclusion complex formed by bilirubin is more stable than the 1:1 inclusion complex, and the stability of the p - CD bilirubin 2:1 inclusion complex is higher than that of the HSA and bilirubin complex, which means that the binding ability of beta -CD to bilirubin is stronger than that of HSA, and it has the ability to compete with the bilirubin in competition with HSA. The change of the coupling reaction to the molecular structure of beta -CD does not weaken the binding of bilirubin to the bilirubin. Three kinds of molecular adsorbents were synthesized by coupling the alpha, beta, and gamma -CD to the PEI carrier. The adsorption dialysis experiment results showed that the binding ability of beta -CD-PEI to bilirubin was the strongest. Benesi-Hildebrand method was used to analyze the visible spectrum changes of bilirubin after beta -CD and bilirubin inclusion, and further confirmed the beta -CD and bilirubin. The inclusion complex was formed in the ratio of 2:1, which confirmed the result of molecular simulation.
In the treatment of adsorption dialysis, the removal of plasma bilirubin depends on the characteristics of the dialysis membrane, the flow velocity of the plasma, the flow velocity of dialysate, the concentration of bilirubin in the plasma, the concentration of water soluble adsorbents in the dialysate and the time of adsorption dialysis. For 300 and 50mL/min, the removal of plasma bilirubin by beta -CD-PEI adsorption was the best. The concentration polarization caused by the ultrafiltration of the transmembrane fluid increased the mass transfer resistance of bilirubin, reduced the molecular diffusion of beta -CD-PEI in the dialysis membrane, and was not conducive to the removal of bilirubin by beta -CD-PEI adsorption dialysis; polyethersulfone was used as the membrane material. The mass transfer effect of Lengthen LST140 dialyzer on bilirubin is better than that of the Nipro Sureflux-130G dialyzer with three cellulose acetate membrane. The albumin adsorbed on the surface of the dialysis membrane and the pore channel can assist the transmission of bilirubin, and then improve the quality of the dialysate to bilirubin.
In the process of adsorption dialysis, the adsorption amount of beta -CD-PEI increases with the increase of the concentration of bilirubin in plasma. The plasma of the patients with the initial concentration of 200mL bilirubin is 80.3140.4214.8267.3305.2mg / L with the concentration of 1L 1%, and the removal rate of total bilirubin in 6h can reach 30-40%, and the application of the total bilirubin removal rate of 6h can reach 30-40%. Increasing the dosage of molecular adsorbent beta -CD-PEI can improve the removal of bilirubin by adsorption dialysis. The use of 1L4%'s beta -CD-PEI dialysate can remove 44.8% of the bilirubin (initial concentration 140.4mg / L) in plasma, and increase the bilirubin removal rate by 9 percent than that of 4L1% sub sorbents under the same condition. The scavenging rate of beta -CD-PEI to plasma bilirubin is 9.5 percentage points higher than that of the same mass fraction of bovine serum albumin, while beta -CD-PEI also has the ability to remove hydrophobic toxins such as total bile acid and aromatic amino acids in plasma, indicating that beta -CD-PEI has the potential to replace albumin for adsorption dialysis.
The change of plasma bilirubin concentration in the process of adsorption dialysis was described by a mathematical model based on material balance. The total mass transfer coefficient of Nipro Sureflux-130G hemodialysis membrane to bilirubin was calculated to be 1.7L/min, and the adsorption equilibrium constant Ka1 of p-CD-PEI and bilirubin was further used for 22.7L/ u moL. by regression of experimental data. The model predicted the adsorption dialysis effect of bilirubin in the same system when the initial concentration of plasma bilirubin and the dosage of adsorbent changed. The average error was 5%.
The above results show that bilirubin water soluble molecular adsorbent beta -CD-PEI has high scavenging ability for bilirubin and other hydrophobic toxins in plasma of patients with liver disease, and has the advantages of low price and simple treatment equipment. It has the potential to replace albumin in clinical treatment.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.0

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