Preparation of new amphiphilic macroporous nonwoven polymeric adsorbents aimed for selective removal of low-density lipoprotein from plasma Xiaodong Hou, Tao Zhang, Amin Cao Lab of Materials Science, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China Received 4 August 2013; revised 25 March 2014; accepted 5 April 2014 Published online 25 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33190 Abstract: In the present work, new amphiphilic macroporous polymeric adsorbent (AMPA) membranes for LDLapheresis were prepared by 60Co g-ray irradiation-induced grafting copolymerization of polypropylene (PP) nonwoven fabric with acrylic acid, followed by bonding cholesterol through linkers of different length. The new AMPA membranes were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscope (SEM), and contact angle microscopy. Static adsorption and hemo-perfusion tests show these new adsorbents could efficiently remove LDL from human plasma. Meanwhile, the AMPA displayed good adsorption capacity for triglyceride (TG) as well. The static
adsorption performance of the AMPA membranes depends on the length of linker. In addition, a balance between the amount of bonded cholesterol and remaining carboxyl group was found necessary to reach the optimal adsorption performance. The best result was achieved by the AMPA membrane PA15C623, by which 62.8 6 3.8 lg of LDLC, 16.5 6 0.71 lg of HDL-C, 132.4 6 3.0 lg of TG are C 2014 removed from human plasma per square centimeter. V Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 52–61, 2015.
Key Words: low density lipoprotein, irradiation, grafting copolymerization, nonwoven, adsorbent
How to cite this article: Hou X, Zhang T, Cao A. 2015. Preparation of new amphiphilic macroporous nonwoven polymeric adsorbents aimed for selective removal of low-density lipoprotein from plasma. J Biomed Mater Res Part B 2015:103B:52–61.
INTRODUCTION
Cardiovascular disease, the leading cause of death in the world, is predominantly triggered by a series of clinical events relevant to atherosclerosis.1 It is already widely accepted that atherosclerosis is initially induced by the abnormal high level of human plasma lipid bioconjugates as well as cholesterol and triglyceride (TG). Among the lipoprotein conjugates, the level of low-density lipoproteincholesterol conjugate (LDL-C) is one of the key factors responsible for the development of atherosclerosis. In contrast, high-density lipoprotein-cholesterol conjugate (HDL-C) plays a positive role against atherosclerosis.2 As for the patients suffering from severe hyperlipemia, their high LDLC level could not be effectively treated only by the dietary and drugs, and LDL-apheresis therapy is generally applied due to its less side-effects and relatively low costs.3 The LDL-apheresis therapy is a technique of extracorporeal elimination of LDL-C pathologically accumulated in human plasma. It has already been proven a powerful tool for clinically treating patients with either homozygous familial hypercholesterolemia (FH) or severe heterozygous hypercholesterolemia.4–8 The key of LDL-apheresis technique is
to look for high performance adsorbents bearing excellent adsorption capacities, high LDL-C adsorption selectivity, good hemocompatibility, low cell toxicity, necessary mechanical strength, and economic preparation cost from a practical viewpoint. Up to now, a number of LDL adsorbents have already been developed mainly including nonspecific adsorbents, selective adsorbents mediated by electro-static attraction between the adsorbents and plasma lipoproteins, and immuno-adsorbents which are designed to selectively adsorb lipoproteins via specific immuno interaction. Most of these adsorbents have been generally prepared by immobilizing the LDL adsorbents on a substrate carrier such as microscopic inorganic or polymeric beads.8–15 For example, the most popular LDL adsorbent at present, the Liposorber system (Kaneka, Japan) which has been used since 1987, consists of porous cellulose beads coupled with dextran sulfate.3 In addition to bead, another form of adsorbent carrier for LDL apheresis membrane has been attracted increasing attention in the past few decades.5,8,16–18 Very recently, Li et al. reported a heparin modified polysulfone membrane for selective removal of LDL.5
Correspondence to: X. Hou (e-mail: [email protected]) or A. Cao (e-mail: [email protected]) Contract grant sponsor: Shanghai Nanotechnology Promotion Center
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SCHEME 1. Pathway to prepare AMPA.
With regard to biomolecular structure of human LDL-C conjugate, cholesterol is rich in the cores of the LDL-C conjugate microparticles.19,20 In addition, cholesterol is wellknown as an essential component of human cell membrane. Chemically, cholesterol is a rigid, hydrophobic molecule with abundant natural origins. Because of its excellent biocompatibility, cholesterol has been frequently applied in the preparation of biomedical materials.21–24 The strong hydrophobic interaction between cholesterol molecules can be applied to design alternative LDL adsorbents as well. For example, Wang et al.14 recently prepared an amphiphilic LDL adsorbent by anchoring cholesterol molecules onto the surface of sulfonated dextran micro beads and found that the resultant adsorbents showed better selectivity than the surface cholesterol-free dextran micro beads in removing plasma LDL-C, total cholesterol (TC) and triglyceride. Nonwoven fabric has been known to bear macroporous internal and surface structures, and can be easily designed and tuned for different applications such as hygiene, family, and suitable biomedical products with cheap preparation cost,25–28 but the extremely low hydrophilicity limited its applications. To improve the hydrophilicity of a non-woven fabric, g-ray irradiation induced grafting copolymerization with hydrophilic acrylic acid has been recognized as a simple and effective way,29–33 and the resulting macroporous
polymeric nonwoven fabrics with improved hydrophilicity and tunable averaged pore sizes may provide a new route to design functional adsorbents for the LDL-apheresis therapy to realize selective removal of abnormally high level human plasma LDL-C. Our research interests were focused on development of new functional polymer materials for biomedical applications such as drug delivery,34–37 fluorescent imaging,38 bioscaffold,39 blood purification,40 and so on. The established approaches for selective removal of LDL are mainly based on electrostatic adhesion between the negatively charged adsorbent, such as dextran sulfate of Liposorber or polyacrylate, and the positively charged LDL. We recently prepared a heparin modified adsorbent membrane for selective removal of LDL from plasma by following the principle of electrostatic adhesion.40 To further improve the adsorption capacity and selectivity of the adsorbent membrane, we aim to prepare a new LDL adsorbent by taking advantage of two different principles of electrostatic adhesion and cholesterol–cholesterol hydrophobic interaction. In this work, we prepared a series of new amphiphilic macroporous polypropylene (PP) nonwoven fabric adsorbents (AMPA) for the LDL-apheresis with optimized pore sizes and covalently anchored cholesterol adsorption ligands as shown in Scheme 1. At first, biomedical grade PP non-woven fabrics
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were chemically modified through 60Co g-ray irradiation induced in situ graft-polymerization with acrylic acid. Subsequently cholesterol molecules as functional adsorption ligands were coupled to the amphiphilic macroporous surfaces via a flexible linker with tunable hydrocarbon chain length. Then the content of carboxyl group on the adsorbent surface was measured by titration, and surface composition of the prepared amphiphilic macroporous PP nonwoven adsorbents was further characterized by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectrometer, contact angle meter and scanning electron microscope (SEM). Finally, static adsorption and in vitro human hemoperfusion experiments were implemented to investigate the LDL adsorption behavior of the new prepared AMPA.
anhydrous sodium sulfate. The removal of solvent afforded a crude product which was further purified by flash column chromatography (silica gel, ethyl acetate/hexane 1:20) to afford a pure product 7.5 g (yield 75%) 1H NMR (CDCl3, d ppm): 0.67–2.40 (m, 47H, ACH3, ACH(CH3) A, ACHA, ACH2A), 3.12 (m, 1H, ACHOA), 3.42–3.46 (t, 2H, ACH2AOACHA), 3.62–3.66 (t, 2H, ACH2AOH), 5.33–5.35 (d, 1H, AC(CH2A)@CHA). Chol-O-(CH2)6-OH (6-Cholesteryloxyhexanol, L6) was prepared following the above procedure with corresponding 6-hexadiol (7.6 g, 64.5 mmol, SCRC, Shanghai). The yield was 6.8 g (yield 63%). 1H NMR (CDCl3, d ppm): 0.67–2.40 (m, 51H, ACH3, ACH(CH3) A, ACHA, ACH2A), 3.12 (m, 1H, ACHOA), 3.42–3.46 (t, 2H, ACH2AOACHA), 3.62–3.66 (t, 2H, ACH2AOH), 5.33–5.35 (d, 1H, AC(CH2A)@CHA).
MATERIALS AND METHOD
Preparation of PAx membrane Biomedical grade PP non-woven fabrics (WuHu, AnHui province, China) with an average pore size of 0.45 lm were cut into 5 3 5 cm2 of membranes, and washed sequentially with acetone, ethanol and distilled water with ultrasonication, and then dried under vacuum. Subsequently, the dried sample was soaked in 5, 10, or 15 wt % degassed aqueous acrylic acid (be distilled under vacuum before use, Sinopharm Chemical Reagent (SCRC), Shanghai) solution containing 1M sulfuric acid, and kept shaking gently for 24 h to been wetted and saturated completely. About 1.0 wt % Mohr’s salt was added into the aqueous solution to inhibit homo-polymerization prior to irradiation. Then the nonwoven fabric membranes were irradiated by 60Co g-ray at ambient condition, with total irradiation dose 20 kGy at a dose rate of 1.2 kGy h21. The grafted PP membranes (denoted as PAx, P stands for PP nonwoven, Ax is x wt % acrylic acid used in the grafting copolymerization) were thoroughly washed using distilled water with ultrasonication to remove homopolymer and unreacted monomer. Synthesis of cholesteryl tosylate(Chol-Tos) Cholesterol (25 g, 64.5 mmol, SCRC, ShangHai) and tosyl chloride (25 g, 131.5 mmol) were dissolved in 200 mL of pyridine and refluxed for 24 h. The mixture was then poured into 1.8 L of 5% aqueous potassium carbonate in an ice bath and kept stirring for 1 h. The precipitate was filtered and redissolved in 400 mL of dichloromethane, washed with water and dried with anhydrous sodium sulfate. The removal of solvent afforded a crude product which was further recrystallized in acetone to yield the titled compound 33.0 g (88.0%). Synthesis of cholesterol adsorption ligands with hydrocarbon linkers (Ln) of different length Chol-O-(CH2)4-OH (4-Cholesteryloxybutanol, L4) was prepared from Cholesteryl tosylate (7 g, 12.9 mmol) and 4butadiol (5.8 g, 64.5 mmol, SCRC, ShangHai) in 300 mL of dry dioxane for 48 h.35 The mixture was concentrated in vacuum and the crude product was dissolved in 200 mL of dichloromethane, washed three times by saturated sodium bicarbonate and distilled water alternatively and dried over
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Preparation of AMPA bearing functional cholesteryl surface ligands The as-synthesized Chol-O-(CH2)4-OH (or Chol-O-(CH2)6-OH) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC, SCRC, Shanghai) were dissolved in 100 mL of dichloromethane at a feeding ratio of [COOH]:[OH]:[EDC]515:1.25:1, followed by a catalytical amount of 4-dimethylaminopyridine (DMAP, SCRC, Shanghai). Then, an acrylic acid-grafted PP nonwoven membrane was immersed into the above solution, and kept stirring at ambient temperature for 24 h. The resultant membrane was Soxhlet extracted in dichloromethane for 24 h to remove all unreacted reactants and dried in vacuum. Consequently, the AMPA membrane were denoted as PAxCy-n where y is the carbon atom number of cholesteryl hydrocarbon linker, and n refers to a feeding ratio of [ACOOH] to [AOH] during the coupling reaction. Characterization procedures Titration of carboxyl density. The amounts of functional carboxyl groups on surface of the grafted membranes were measured by back titration as reported before.40 1
H NMR. 1H NMR spectra were measured in CDCl3 at ambient temperature on a Varian VXR 300 FT-NMR spectrometer, operating at 300.0 MHz for 1H nuclei with tetramethylsilane (TMS) as the internal chemical shift reference. Fourier-transform infrared spectroscopy. An attenuated total reflectance FTIR (FTS-185, Bio-Rad, America) was utilized to analyze the surface chemistry structure. All samples were shredded into strips (0.8 3 5 cm2). Water contact angle. Static contact angles were measured on a contact angle goniometer KH-0601 equipped with video capture (Institute of Chemistry CAS, Beijing). Nearly 5 lL of water was dropped on a dry membrane and the contact angle was recorded immediately. The results were reported as the mean of ten measurements. Scanning electron microscope. The topographies of membranes were observed on a JSM-6390 SEM (JEOL, Japan). All samples were cut into chips (1 6 0.2 mm) and sputter-
SELECTIVE REMOVAL OF LOW-DENSITY LIPOPROTEIN FROM PLASMA
ORIGINAL RESEARCH REPORT
FIGURE 1. 1H NMR spectrum of cholesterol coupled with linker.
coated with gold-palladium (Denton Vacuum Desc II) for 2 min prior to SEM observation. Assay of lipoprotein adsorption capacity Lipoprotein adsorption capacities of the as-made membranes were evaluated under two different states of plasma, in static adsorption and in vitro plasma perfusion. In static adsorption, a 5 3 5 cm22 of membrane (0.2– 0.5 g) was sheared into fragments and put into a vial, then 6 mL of physiological saline was added to wet and thoroughly saturate these fragments. The excessive saline was decanted, and the suction-dried sample was incubated with 1 mL of plasma and shaken at 37 C for 3 h. LDL-C, HDL-C, TC, and TG in the plasma were measured by using a UV spectrophotometer U-2800 (HITACHI, Japan) with the corresponding kits. Plasma of adult patients with hyperlipemia was supplied by BaiMao Hospital (Changshu, China) with informed signed consent from the patients and the authority of the hospital. Lipoprotein adsorption capacity of the membrane was calculated according to the following equation: AC(lg cm22) 5 Vp 3 (Cb 2 Ca)/A, where AC stands for adsorption capacity, and Ca and Cb are the concentration of lipoprotein after and before adsorption, Vp and A are the volume of plasma and the area of sample respectively. In vitro plasma perfusion was performed in a kit of perfusion equipment, which consisted of a peristaltic pump, a tailor-made adsorbent column, two bags for plasma before and after perfusion, and some soft rubber tubing. Three slips of the membranes (diameter 5 cm) were saturated previously with physiology saline, and then mounted into the tailor-made adsorbent column. The whole system was also rinsed with physiology saline before each perfusion. An
appropriate volume of plasma was circularly perfused and the changes of lipoproteins level were detected at regular intervals. RESULTS
Chemical characterization Synthesis of functional cholesterol ligands with different hydrocarbon linkers Ln. Figure 1 shows 1H NMR spectra of the synthesized Chol-O(CH2)4OH and Chol-O(CH2)6OH, and the two compounds show similar 1H NMR spectrum except the number of hydrogen atoms at 0.67–2.40 ppm (e). The appearance of signal at chemical shift 3.42–3.46 ppm (c) confirms the generation of ether linkage between the linker and cholesteryl moiety. The signals at 3.62–3.66 ppm (b) is assigned to the end group of ACH2OH for the linker L4 or L6, which excludes the possibility of one linker molecule bonding two cholesterol molecules. Preparation of PAx membrane. The grafting of acrylic acid is first confirmed by FTIR-ATR spectroscope as illustrated in Figure 2. Figure 2(a) displays the typical polypropylene FTIR-ATR spectrum with adsorption bands which are respectively due to asymmetrical and symmetrical stretching of the ACH2, ACH3, and ACH group at 2850–2950 cm21. A new strong adsorption band appears at 1700 cm21 in Figure 2(b) corresponding to the characteristic adsorption bands of carboxyl group from the grafting poly (acrylic acid). The successful preparation of PAx is indirectly proved as well by using the method of water contact angle. By comparing the samples P, PA5, PA10, and PA15 (see Table I), it can be seen that chemical structure and hydrophilicity on
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FIGURE 2. FTIR-ATR spectra of P0.45 nonwoven grafted with acrylic acid of various concentrations and coupled with cholesterol. (a) P (b) PA15 (c) PA5C0–1 (d) PA10C0–1 (e) PA15C0–1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
surface significantly change after grafting with acrylic acid. When the grafting yield of acrylic acid increases from 0 to 11.5% 6 0.2%, to 59.2% 6 2.5%, and to 88.6% 6 4.8%, the corresponding amounts of carboxyl are 0, 18.2 6 0.3, 59.4 6 5.7, and 109.8 6 10.2 lmol cm22, respectively, and while water contact angle decreases sharply from 123.5 6 3.7 , 106.2 6 1.5 , 95.4 6 2.6 , to 74.2 6 4.8 , respectively. Obviously, the hydrophilicity of grafted poly (acrylic acid) was responsible for the change of the surface property of PP nonwoven. Preparation of AMPA bearing functional cholesteryl surface ligands. As seen in Figure 2, compared with the sample (b) that is only grafting with acrylic acid, the absorption band of samples (c)–(e) at 1700 cm21 becomes dramatically weaker; meanwhile a new band appears at 1650 cm21, which should be attributed to the overlapped bands of the newly formed carboxylic ester and residual carboxylic group. In addition, no obvious absorption of
hydroxyl at 3400 cm21 is observed in Figure 2(c–e), which indicates cholesterol molecules were anchored to the membrane by covalent bonds. A sharp band appearing at 1150 cm21 is certainly assigned to the stretching of CAOAC, whose intensity enhanced accordingly with the increase of the amount of carboxyl group from Figure 2(c–e). All the aforementioned results confirmed the formation of new ester bond between carboxyl group from acrylic acid and hydroxyl from cholesterol ligand. The results of water contact angle listed in Table I further supported the above explanation. Owing to the tremendous water-repelling capability of cholesterol, the water contact angles of samples bonding cholesterol greatly increases comparing to the corresponding precursor (from 123.0 6 3.1 , l16.1 6 5.5 , and 110.1 6 3.4 to 106.2 6 1.5 , to 95.4 6 2.6 and 74.2 6 4.8 , respectively). When carbon atom number of the linker increases from 0 (PA15C023) to 4 (PA15C423), and to 6 (PA15C623), the corresponding water contact angle increases from 89.3 6 4.8 to 123.5 6 3.2 , and to 129.7 6 5.4 . An increase of 40 in water contact angle is observed from PA15C623 to PA15C023, indicating the length of hydrophobic linker can strongly affect the surface hydrophilicity of the nonwoven fabric adsorbents. Topographies of AMPA before and after plasma static adsorption. Figure 3 shows the topographies of AMPAs grafting with different amount of polyacrylic acid before and after plasma static adsorption. After plasma static adsorption, a membrane-like substance, which may be the viscous lipoprotein in the plasma, is adsorbed to the fibers of the AMPA membranes, and the fibers have more uneven diameters, much coarser surfaces, and larger interfiber space. Noticeably, with the increase of polyacrylic acid grafting yield from left to right in Figure 3(b), the interfiber space becomes more saturated by the adsorbed lipoprotein. The increase in the adsorbed capacity of lipoprotein can be attributed to the strengthened affinity between lipoprotein
TABLE I. Water Contact Angles of Unmodified PP Nonwoven Fabric and AMPA Sample
GY (%)a
COO2 (lmol cm22)b
Water Contact Angle ( )c
P PA5 PA10 PA15 PA5C0-1 PA10C0-1 PA15C0-1 PA15C0-3 PA15C4-3 PA15C6-3
0 11.5 6 0.2 59.2 6 2.5 88.6 6 4.8 NAd NA NA NA NA NA
0 18.2 6 0.3 59.4 6 5.7 109.8 6 10.2 –e – – – – –
123.5 6 3.7f 106.2 6 1.5f 95.4 6 2.6f 74.2 6 4.8f 123.0 6 3.1 116.1 6 5.5 110.1 6 3.4 89.3 6 4.8 123.5 6 3.2 129.7 6 5.4
Note a Grafting yield is calculated by equation GY (wt %) 5 (Wg 2 Wi)/Wi 3 100, where Wg and Wi refer to the weight of initiative and grafted weight respectively, reported as the mean of three times measurements with standard deviation. b Carboxyl density (lmol cm22) 5 Mc/A, where Mc is the carboxyl amount calculated by titration and A the surface area of the sample. c Reported as the mean of 10 times measurements with standard deviation. d Not applicable. e To avoid hydrolysis of ether bond by strong basic, carboxyl densities on these samples (–) are not measured by titration. f Data was cited from previous publication40 for convenient comparison.
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ORIGINAL RESEARCH REPORT
FIGURE 3. SEM images of AMPAs before (a) and after (b) plasma static adsorption. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and adsorbent membrane due to the improved hydrophilic property of the AMPA. The hydrophilic property of the AMPA membranes is demonstrated in Table I. Figure 4 reflects the topographies of AMPAs bonding cholesterol through linkers of different length before (a) and after (b) plasma static adsorption. Similar changes of the fiber topographies as in Figure 3 are observed in each group of samples after plasma static adsorption. As the length of the linker increased, the membrane-like lipopro-
tein layers become thicker. That is in good accordance with the results of lipoprotein adsorbing capacity test in Table II. DISCUSSION
The static adsorption performance of the AMPA In static plasma adsorption, the adsorption performance of adsorbent membrane is decided mainly by the following factors: ligand, plasma concentration, adsorption time, and linkers.
FIGURE 4. SEM images of AMPAs bonding cholesterol through linkers of different length before (a) and after (b) plasma static adsorption. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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TABLE II. Hydrocarbon Chain Length Dependence of Static Plasma Lipid Adsorption Capacities for AMPAs Sample
LDL (lg cm22)
HDL (mg cm22)
TC (mg cm22)
TG (mg cm22)
P PA15C0-1 PA15C4-1 PA15C6-1 PA15C0-3 PA15C4-3 PA15C6-3 PA15C0-5 PA15C4-5 PA15C6-5
5.6 6 0.5 40.8 6 2.4 44.3 6 1.4 55.8 6 0.9 37.0 6 1.6 38.7 6 2.0 62.8 6 3.8 17.3 6 2.1 29.2 6 1.1 47.9 6 3.5
4.3 6 0.4 15.4 6 1.1 14.5 6 2.3 13.4 6 2.0 20.0 6 2.7 22.2 6 0.6 16.5 6 0.7 12.1 6 1.2 17.0 6 4.4 10.2 6 2.2
10.4 6 0.8 62.4 6 3.5 76.5 6 4.0 50.5 6 3.8 57.7 6 2.7 60.4 6 1.5 80.3 6 5.7 32.6 6 3.2 53.0 6 1.4 58.1 6 2.3
53.7 6 2.4 134.5 6 7.3 157.4 6 4.9 136.8 6 5.3 128.8 6 4.8 98.9 6 5.1 132.4 6 3.0 104.7 6 4.4 100.7 6 2.9 96.3 6 3.4
Figure 5 reveals the change of lipoprotein level with static adsorption time using the AMPA membrane PA15C0-1. The two lipoprotein levels, LDL-C and HDL-C, quickly decrease in the first 30 min, and then slowly decrease to a plateau in 2 h. Interestingly, HDL-C level has a minimum before reaching the equilibrium which is different from the sustaining decline of LDL-C level. Haofeng Yu et al.41 ascribed this behavior to the larger diffusion speed of HDLC due to its smaller size (HDL 3.5–9 nm; LDL, 20–25 nm). With the stronger competitive adsorption capability, LDL would eventually recapture the adsorption sites which had been occupied previously by HDL particles. The effect of plasma concentration on the adsorption capability of the absorbent membranes is demonstrated by the adsorption isotherm of LDL-C using the AMPA membrane PA15C6-3 in Figure 6. Apparently, higher plasma concentration helps to the adsorption of LDL-C, which can be easily explained by the adsorption mechanism. As the equilibrium concentration increases, the diffusion of LDL-C particles enhances; therefore, more LDL-C particles are bound
FIGURE 5. Effects of adsorption time on lipoprotein levels. (PA15C0–1, COO2, 109.8 lmol/L) (a) LDL-C, (b) HDL-C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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by the active sites on the membrane. When all the active sites are devitalized, excessive LDL-C particles do not help improve the adsorption capability; that is, adsorption capability of the adsorbent reaches the peak value. In static adsorption test, therefore, higher adsorption capability and better selectivity performance can be achieved by using hypercholesterolemia plasma (TC >6.5 mmol L21) than by using normal plasma, which has been proved by the previous work.41 In the current work, the concentration of TC available is 2.2–2.8 mmol L21, actually far below the hypercholesterolemia plasma, the corresponding maximum adsorption capability of AMPA-membranes for LDL-C reaches 21.8 lg cm22 which is higher than that of the heparin modified adsorbent membrane we prepared before.40 The static adsorption capacity of the adsorbent membranes bonded with different surface ligands is shown in Figure 7. It is clear that the modified PP nonwoven membranes have better plasma static adsorption performance than the unmodified ones. Among the modified membranes, better adsorption performance is achieved by AMPA membranes than the ones only grafted with acrylic acid. It is true for both poly (acrylic acid) and cholesterol that the higher the amount of ligand bonded on the membranes, the
FIGURE 6. Adsorption isotherm of LDL-C by AMPA PA15C623 at 37 C.
SELECTIVE REMOVAL OF LOW-DENSITY LIPOPROTEIN FROM PLASMA
ORIGINAL RESEARCH REPORT
FIGURE 7. Plasma static adsorption capacity of the prepared adsorbent membranes bonded with different surface ligands. Results are reported as the mean of three measurements with standard deviation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
better lipoprotein capacity it has. This observation can be explained on the basis of the structure of lipoprotein and membrane. Poly (acrylic acid) increases adsorption of LDL-C by electrostatic interaction between the polyanion and the basic amino acid residues that are abundant in some regions of LDL particle surfaces. The cholesterol bonded on the absorbent membranes help anchor cholesterol molecules existing in the core of LDL-C particles through cholesterol–cholesterol hydrophobic interaction. These two interactions synergistically produce the good adsorption performance of AMPA membranes. The effect of length of linker on static adsorption capacity of AMPA-membranes is listed in Table II. Under a fixed feeding ratio of carboxyl group to cholesterol, as the
FIGURE 8. The lipoprotein levels at different in vitro hemoperfusion durations using AMPAs. The initial lipoprotein level (mmol/L): LDL (3.3 6 0.10), HDL (1.40 6 0.10), TC (5.0 6 0.10) and TG (4.5 6 0.2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
length of linker increases, static adsorption capability of AMPA membranes for LDL-C increases and selectivity for LDL-C/HDL-C also improves accordingly. The best adsorption performance was achieved by the PA15C6-3 membrane by which 62.8 6 3.8 lg of LDL-C, 16.5 6 0.71 lg of HDL-C, 80.35.7 lg TC and 132.4 6 3.0 lg of TG are removed from human plasma per square centimeter. It is well understood that aliphatic hydrocarbon chains, acting as spacer arms, improves the mobility of adsorption sites which is equivalent to increase the amount of adsorption sites, resulting in enhanced interactions between ligands and target molecules and consequently improved adsorption performance.42,43 On the other hand, for the same length of linker, the static adsorption performance does not increase with the feeding ratio of carboxyl group to cholesterol but has an optimum. This observation can be explained as following: although the larger amount of cholesterol bonded on the surface of AMPA membranes might anchor more LDL-C, excessive cholesterol can sharply increase the hydrophobic property of the membranes, which will weaken the electrostatic interaction between lipoptroteins and membranes thereby decrease the static adsorption performance. It implies that there is a balance between the amount of cholesterol and remaining carboxyl group on the surface of the AMPA membranes. In our case, the best result was obtained at the feeding ratio 3 of carboxyl group to cholesterol. Although it is hard to directly compare our results of static adsorption with the related reports5,41,44 due to the different forms of adsorbent, human plasma and adsorption conditions, the present adsorbent membrane provided satisfactory static adsorption property, especially taking into account the normal lipoprotein (instead of hyper-cholesterol) plasma level used in our test. For example, the adsorption capacity of membrane adsorbents reported by Li et al.5 is at a level of
adsorption performance of the AMPA membranes depends on the length of linker. In addition, a balance between the amount of bonded cholesterol and remaining carboxyl group was found necessary to reach the optimal adsorption performance. The best result was achieved by the AMPA membrane PA15C623, by which 62.8 6 3.8 lg of LDLC, 16.5 6 0.71 lg of HDL-C, 132.4 6 3.0 lg of TG are C 2014 removed from human plasma per square centimeter. V Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 52–61, 2015.
Key Words: low density lipoprotein, irradiation, grafting copolymerization, nonwoven, adsorbent
How to cite this article: Hou X, Zhang T, Cao A. 2015. Preparation of new amphiphilic macroporous nonwoven polymeric adsorbents aimed for selective removal of low-density lipoprotein from plasma. J Biomed Mater Res Part B 2015:103B:52–61.
INTRODUCTION
Cardiovascular disease, the leading cause of death in the world, is predominantly triggered by a series of clinical events relevant to atherosclerosis.1 It is already widely accepted that atherosclerosis is initially induced by the abnormal high level of human plasma lipid bioconjugates as well as cholesterol and triglyceride (TG). Among the lipoprotein conjugates, the level of low-density lipoproteincholesterol conjugate (LDL-C) is one of the key factors responsible for the development of atherosclerosis. In contrast, high-density lipoprotein-cholesterol conjugate (HDL-C) plays a positive role against atherosclerosis.2 As for the patients suffering from severe hyperlipemia, their high LDLC level could not be effectively treated only by the dietary and drugs, and LDL-apheresis therapy is generally applied due to its less side-effects and relatively low costs.3 The LDL-apheresis therapy is a technique of extracorporeal elimination of LDL-C pathologically accumulated in human plasma. It has already been proven a powerful tool for clinically treating patients with either homozygous familial hypercholesterolemia (FH) or severe heterozygous hypercholesterolemia.4–8 The key of LDL-apheresis technique is
to look for high performance adsorbents bearing excellent adsorption capacities, high LDL-C adsorption selectivity, good hemocompatibility, low cell toxicity, necessary mechanical strength, and economic preparation cost from a practical viewpoint. Up to now, a number of LDL adsorbents have already been developed mainly including nonspecific adsorbents, selective adsorbents mediated by electro-static attraction between the adsorbents and plasma lipoproteins, and immuno-adsorbents which are designed to selectively adsorb lipoproteins via specific immuno interaction. Most of these adsorbents have been generally prepared by immobilizing the LDL adsorbents on a substrate carrier such as microscopic inorganic or polymeric beads.8–15 For example, the most popular LDL adsorbent at present, the Liposorber system (Kaneka, Japan) which has been used since 1987, consists of porous cellulose beads coupled with dextran sulfate.3 In addition to bead, another form of adsorbent carrier for LDL apheresis membrane has been attracted increasing attention in the past few decades.5,8,16–18 Very recently, Li et al. reported a heparin modified polysulfone membrane for selective removal of LDL.5
Correspondence to: X. Hou (e-mail: [email protected]) or A. Cao (e-mail: [email protected]) Contract grant sponsor: Shanghai Nanotechnology Promotion Center
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ORIGINAL RESEARCH REPORT
SCHEME 1. Pathway to prepare AMPA.
With regard to biomolecular structure of human LDL-C conjugate, cholesterol is rich in the cores of the LDL-C conjugate microparticles.19,20 In addition, cholesterol is wellknown as an essential component of human cell membrane. Chemically, cholesterol is a rigid, hydrophobic molecule with abundant natural origins. Because of its excellent biocompatibility, cholesterol has been frequently applied in the preparation of biomedical materials.21–24 The strong hydrophobic interaction between cholesterol molecules can be applied to design alternative LDL adsorbents as well. For example, Wang et al.14 recently prepared an amphiphilic LDL adsorbent by anchoring cholesterol molecules onto the surface of sulfonated dextran micro beads and found that the resultant adsorbents showed better selectivity than the surface cholesterol-free dextran micro beads in removing plasma LDL-C, total cholesterol (TC) and triglyceride. Nonwoven fabric has been known to bear macroporous internal and surface structures, and can be easily designed and tuned for different applications such as hygiene, family, and suitable biomedical products with cheap preparation cost,25–28 but the extremely low hydrophilicity limited its applications. To improve the hydrophilicity of a non-woven fabric, g-ray irradiation induced grafting copolymerization with hydrophilic acrylic acid has been recognized as a simple and effective way,29–33 and the resulting macroporous
polymeric nonwoven fabrics with improved hydrophilicity and tunable averaged pore sizes may provide a new route to design functional adsorbents for the LDL-apheresis therapy to realize selective removal of abnormally high level human plasma LDL-C. Our research interests were focused on development of new functional polymer materials for biomedical applications such as drug delivery,34–37 fluorescent imaging,38 bioscaffold,39 blood purification,40 and so on. The established approaches for selective removal of LDL are mainly based on electrostatic adhesion between the negatively charged adsorbent, such as dextran sulfate of Liposorber or polyacrylate, and the positively charged LDL. We recently prepared a heparin modified adsorbent membrane for selective removal of LDL from plasma by following the principle of electrostatic adhesion.40 To further improve the adsorption capacity and selectivity of the adsorbent membrane, we aim to prepare a new LDL adsorbent by taking advantage of two different principles of electrostatic adhesion and cholesterol–cholesterol hydrophobic interaction. In this work, we prepared a series of new amphiphilic macroporous polypropylene (PP) nonwoven fabric adsorbents (AMPA) for the LDL-apheresis with optimized pore sizes and covalently anchored cholesterol adsorption ligands as shown in Scheme 1. At first, biomedical grade PP non-woven fabrics
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were chemically modified through 60Co g-ray irradiation induced in situ graft-polymerization with acrylic acid. Subsequently cholesterol molecules as functional adsorption ligands were coupled to the amphiphilic macroporous surfaces via a flexible linker with tunable hydrocarbon chain length. Then the content of carboxyl group on the adsorbent surface was measured by titration, and surface composition of the prepared amphiphilic macroporous PP nonwoven adsorbents was further characterized by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectrometer, contact angle meter and scanning electron microscope (SEM). Finally, static adsorption and in vitro human hemoperfusion experiments were implemented to investigate the LDL adsorption behavior of the new prepared AMPA.
anhydrous sodium sulfate. The removal of solvent afforded a crude product which was further purified by flash column chromatography (silica gel, ethyl acetate/hexane 1:20) to afford a pure product 7.5 g (yield 75%) 1H NMR (CDCl3, d ppm): 0.67–2.40 (m, 47H, ACH3, ACH(CH3) A, ACHA, ACH2A), 3.12 (m, 1H, ACHOA), 3.42–3.46 (t, 2H, ACH2AOACHA), 3.62–3.66 (t, 2H, ACH2AOH), 5.33–5.35 (d, 1H, AC(CH2A)@CHA). Chol-O-(CH2)6-OH (6-Cholesteryloxyhexanol, L6) was prepared following the above procedure with corresponding 6-hexadiol (7.6 g, 64.5 mmol, SCRC, Shanghai). The yield was 6.8 g (yield 63%). 1H NMR (CDCl3, d ppm): 0.67–2.40 (m, 51H, ACH3, ACH(CH3) A, ACHA, ACH2A), 3.12 (m, 1H, ACHOA), 3.42–3.46 (t, 2H, ACH2AOACHA), 3.62–3.66 (t, 2H, ACH2AOH), 5.33–5.35 (d, 1H, AC(CH2A)@CHA).
MATERIALS AND METHOD
Preparation of PAx membrane Biomedical grade PP non-woven fabrics (WuHu, AnHui province, China) with an average pore size of 0.45 lm were cut into 5 3 5 cm2 of membranes, and washed sequentially with acetone, ethanol and distilled water with ultrasonication, and then dried under vacuum. Subsequently, the dried sample was soaked in 5, 10, or 15 wt % degassed aqueous acrylic acid (be distilled under vacuum before use, Sinopharm Chemical Reagent (SCRC), Shanghai) solution containing 1M sulfuric acid, and kept shaking gently for 24 h to been wetted and saturated completely. About 1.0 wt % Mohr’s salt was added into the aqueous solution to inhibit homo-polymerization prior to irradiation. Then the nonwoven fabric membranes were irradiated by 60Co g-ray at ambient condition, with total irradiation dose 20 kGy at a dose rate of 1.2 kGy h21. The grafted PP membranes (denoted as PAx, P stands for PP nonwoven, Ax is x wt % acrylic acid used in the grafting copolymerization) were thoroughly washed using distilled water with ultrasonication to remove homopolymer and unreacted monomer. Synthesis of cholesteryl tosylate(Chol-Tos) Cholesterol (25 g, 64.5 mmol, SCRC, ShangHai) and tosyl chloride (25 g, 131.5 mmol) were dissolved in 200 mL of pyridine and refluxed for 24 h. The mixture was then poured into 1.8 L of 5% aqueous potassium carbonate in an ice bath and kept stirring for 1 h. The precipitate was filtered and redissolved in 400 mL of dichloromethane, washed with water and dried with anhydrous sodium sulfate. The removal of solvent afforded a crude product which was further recrystallized in acetone to yield the titled compound 33.0 g (88.0%). Synthesis of cholesterol adsorption ligands with hydrocarbon linkers (Ln) of different length Chol-O-(CH2)4-OH (4-Cholesteryloxybutanol, L4) was prepared from Cholesteryl tosylate (7 g, 12.9 mmol) and 4butadiol (5.8 g, 64.5 mmol, SCRC, ShangHai) in 300 mL of dry dioxane for 48 h.35 The mixture was concentrated in vacuum and the crude product was dissolved in 200 mL of dichloromethane, washed three times by saturated sodium bicarbonate and distilled water alternatively and dried over
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Preparation of AMPA bearing functional cholesteryl surface ligands The as-synthesized Chol-O-(CH2)4-OH (or Chol-O-(CH2)6-OH) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC, SCRC, Shanghai) were dissolved in 100 mL of dichloromethane at a feeding ratio of [COOH]:[OH]:[EDC]515:1.25:1, followed by a catalytical amount of 4-dimethylaminopyridine (DMAP, SCRC, Shanghai). Then, an acrylic acid-grafted PP nonwoven membrane was immersed into the above solution, and kept stirring at ambient temperature for 24 h. The resultant membrane was Soxhlet extracted in dichloromethane for 24 h to remove all unreacted reactants and dried in vacuum. Consequently, the AMPA membrane were denoted as PAxCy-n where y is the carbon atom number of cholesteryl hydrocarbon linker, and n refers to a feeding ratio of [ACOOH] to [AOH] during the coupling reaction. Characterization procedures Titration of carboxyl density. The amounts of functional carboxyl groups on surface of the grafted membranes were measured by back titration as reported before.40 1
H NMR. 1H NMR spectra were measured in CDCl3 at ambient temperature on a Varian VXR 300 FT-NMR spectrometer, operating at 300.0 MHz for 1H nuclei with tetramethylsilane (TMS) as the internal chemical shift reference. Fourier-transform infrared spectroscopy. An attenuated total reflectance FTIR (FTS-185, Bio-Rad, America) was utilized to analyze the surface chemistry structure. All samples were shredded into strips (0.8 3 5 cm2). Water contact angle. Static contact angles were measured on a contact angle goniometer KH-0601 equipped with video capture (Institute of Chemistry CAS, Beijing). Nearly 5 lL of water was dropped on a dry membrane and the contact angle was recorded immediately. The results were reported as the mean of ten measurements. Scanning electron microscope. The topographies of membranes were observed on a JSM-6390 SEM (JEOL, Japan). All samples were cut into chips (1 6 0.2 mm) and sputter-
SELECTIVE REMOVAL OF LOW-DENSITY LIPOPROTEIN FROM PLASMA
ORIGINAL RESEARCH REPORT
FIGURE 1. 1H NMR spectrum of cholesterol coupled with linker.
coated with gold-palladium (Denton Vacuum Desc II) for 2 min prior to SEM observation. Assay of lipoprotein adsorption capacity Lipoprotein adsorption capacities of the as-made membranes were evaluated under two different states of plasma, in static adsorption and in vitro plasma perfusion. In static adsorption, a 5 3 5 cm22 of membrane (0.2– 0.5 g) was sheared into fragments and put into a vial, then 6 mL of physiological saline was added to wet and thoroughly saturate these fragments. The excessive saline was decanted, and the suction-dried sample was incubated with 1 mL of plasma and shaken at 37 C for 3 h. LDL-C, HDL-C, TC, and TG in the plasma were measured by using a UV spectrophotometer U-2800 (HITACHI, Japan) with the corresponding kits. Plasma of adult patients with hyperlipemia was supplied by BaiMao Hospital (Changshu, China) with informed signed consent from the patients and the authority of the hospital. Lipoprotein adsorption capacity of the membrane was calculated according to the following equation: AC(lg cm22) 5 Vp 3 (Cb 2 Ca)/A, where AC stands for adsorption capacity, and Ca and Cb are the concentration of lipoprotein after and before adsorption, Vp and A are the volume of plasma and the area of sample respectively. In vitro plasma perfusion was performed in a kit of perfusion equipment, which consisted of a peristaltic pump, a tailor-made adsorbent column, two bags for plasma before and after perfusion, and some soft rubber tubing. Three slips of the membranes (diameter 5 cm) were saturated previously with physiology saline, and then mounted into the tailor-made adsorbent column. The whole system was also rinsed with physiology saline before each perfusion. An
appropriate volume of plasma was circularly perfused and the changes of lipoproteins level were detected at regular intervals. RESULTS
Chemical characterization Synthesis of functional cholesterol ligands with different hydrocarbon linkers Ln. Figure 1 shows 1H NMR spectra of the synthesized Chol-O(CH2)4OH and Chol-O(CH2)6OH, and the two compounds show similar 1H NMR spectrum except the number of hydrogen atoms at 0.67–2.40 ppm (e). The appearance of signal at chemical shift 3.42–3.46 ppm (c) confirms the generation of ether linkage between the linker and cholesteryl moiety. The signals at 3.62–3.66 ppm (b) is assigned to the end group of ACH2OH for the linker L4 or L6, which excludes the possibility of one linker molecule bonding two cholesterol molecules. Preparation of PAx membrane. The grafting of acrylic acid is first confirmed by FTIR-ATR spectroscope as illustrated in Figure 2. Figure 2(a) displays the typical polypropylene FTIR-ATR spectrum with adsorption bands which are respectively due to asymmetrical and symmetrical stretching of the ACH2, ACH3, and ACH group at 2850–2950 cm21. A new strong adsorption band appears at 1700 cm21 in Figure 2(b) corresponding to the characteristic adsorption bands of carboxyl group from the grafting poly (acrylic acid). The successful preparation of PAx is indirectly proved as well by using the method of water contact angle. By comparing the samples P, PA5, PA10, and PA15 (see Table I), it can be seen that chemical structure and hydrophilicity on
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FIGURE 2. FTIR-ATR spectra of P0.45 nonwoven grafted with acrylic acid of various concentrations and coupled with cholesterol. (a) P (b) PA15 (c) PA5C0–1 (d) PA10C0–1 (e) PA15C0–1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
surface significantly change after grafting with acrylic acid. When the grafting yield of acrylic acid increases from 0 to 11.5% 6 0.2%, to 59.2% 6 2.5%, and to 88.6% 6 4.8%, the corresponding amounts of carboxyl are 0, 18.2 6 0.3, 59.4 6 5.7, and 109.8 6 10.2 lmol cm22, respectively, and while water contact angle decreases sharply from 123.5 6 3.7 , 106.2 6 1.5 , 95.4 6 2.6 , to 74.2 6 4.8 , respectively. Obviously, the hydrophilicity of grafted poly (acrylic acid) was responsible for the change of the surface property of PP nonwoven. Preparation of AMPA bearing functional cholesteryl surface ligands. As seen in Figure 2, compared with the sample (b) that is only grafting with acrylic acid, the absorption band of samples (c)–(e) at 1700 cm21 becomes dramatically weaker; meanwhile a new band appears at 1650 cm21, which should be attributed to the overlapped bands of the newly formed carboxylic ester and residual carboxylic group. In addition, no obvious absorption of
hydroxyl at 3400 cm21 is observed in Figure 2(c–e), which indicates cholesterol molecules were anchored to the membrane by covalent bonds. A sharp band appearing at 1150 cm21 is certainly assigned to the stretching of CAOAC, whose intensity enhanced accordingly with the increase of the amount of carboxyl group from Figure 2(c–e). All the aforementioned results confirmed the formation of new ester bond between carboxyl group from acrylic acid and hydroxyl from cholesterol ligand. The results of water contact angle listed in Table I further supported the above explanation. Owing to the tremendous water-repelling capability of cholesterol, the water contact angles of samples bonding cholesterol greatly increases comparing to the corresponding precursor (from 123.0 6 3.1 , l16.1 6 5.5 , and 110.1 6 3.4 to 106.2 6 1.5 , to 95.4 6 2.6 and 74.2 6 4.8 , respectively). When carbon atom number of the linker increases from 0 (PA15C023) to 4 (PA15C423), and to 6 (PA15C623), the corresponding water contact angle increases from 89.3 6 4.8 to 123.5 6 3.2 , and to 129.7 6 5.4 . An increase of 40 in water contact angle is observed from PA15C623 to PA15C023, indicating the length of hydrophobic linker can strongly affect the surface hydrophilicity of the nonwoven fabric adsorbents. Topographies of AMPA before and after plasma static adsorption. Figure 3 shows the topographies of AMPAs grafting with different amount of polyacrylic acid before and after plasma static adsorption. After plasma static adsorption, a membrane-like substance, which may be the viscous lipoprotein in the plasma, is adsorbed to the fibers of the AMPA membranes, and the fibers have more uneven diameters, much coarser surfaces, and larger interfiber space. Noticeably, with the increase of polyacrylic acid grafting yield from left to right in Figure 3(b), the interfiber space becomes more saturated by the adsorbed lipoprotein. The increase in the adsorbed capacity of lipoprotein can be attributed to the strengthened affinity between lipoprotein
TABLE I. Water Contact Angles of Unmodified PP Nonwoven Fabric and AMPA Sample
GY (%)a
COO2 (lmol cm22)b
Water Contact Angle ( )c
P PA5 PA10 PA15 PA5C0-1 PA10C0-1 PA15C0-1 PA15C0-3 PA15C4-3 PA15C6-3
0 11.5 6 0.2 59.2 6 2.5 88.6 6 4.8 NAd NA NA NA NA NA
0 18.2 6 0.3 59.4 6 5.7 109.8 6 10.2 –e – – – – –
123.5 6 3.7f 106.2 6 1.5f 95.4 6 2.6f 74.2 6 4.8f 123.0 6 3.1 116.1 6 5.5 110.1 6 3.4 89.3 6 4.8 123.5 6 3.2 129.7 6 5.4
Note a Grafting yield is calculated by equation GY (wt %) 5 (Wg 2 Wi)/Wi 3 100, where Wg and Wi refer to the weight of initiative and grafted weight respectively, reported as the mean of three times measurements with standard deviation. b Carboxyl density (lmol cm22) 5 Mc/A, where Mc is the carboxyl amount calculated by titration and A the surface area of the sample. c Reported as the mean of 10 times measurements with standard deviation. d Not applicable. e To avoid hydrolysis of ether bond by strong basic, carboxyl densities on these samples (–) are not measured by titration. f Data was cited from previous publication40 for convenient comparison.
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SELECTIVE REMOVAL OF LOW-DENSITY LIPOPROTEIN FROM PLASMA
ORIGINAL RESEARCH REPORT
FIGURE 3. SEM images of AMPAs before (a) and after (b) plasma static adsorption. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and adsorbent membrane due to the improved hydrophilic property of the AMPA. The hydrophilic property of the AMPA membranes is demonstrated in Table I. Figure 4 reflects the topographies of AMPAs bonding cholesterol through linkers of different length before (a) and after (b) plasma static adsorption. Similar changes of the fiber topographies as in Figure 3 are observed in each group of samples after plasma static adsorption. As the length of the linker increased, the membrane-like lipopro-
tein layers become thicker. That is in good accordance with the results of lipoprotein adsorbing capacity test in Table II. DISCUSSION
The static adsorption performance of the AMPA In static plasma adsorption, the adsorption performance of adsorbent membrane is decided mainly by the following factors: ligand, plasma concentration, adsorption time, and linkers.
FIGURE 4. SEM images of AMPAs bonding cholesterol through linkers of different length before (a) and after (b) plasma static adsorption. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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TABLE II. Hydrocarbon Chain Length Dependence of Static Plasma Lipid Adsorption Capacities for AMPAs Sample
LDL (lg cm22)
HDL (mg cm22)
TC (mg cm22)
TG (mg cm22)
P PA15C0-1 PA15C4-1 PA15C6-1 PA15C0-3 PA15C4-3 PA15C6-3 PA15C0-5 PA15C4-5 PA15C6-5
5.6 6 0.5 40.8 6 2.4 44.3 6 1.4 55.8 6 0.9 37.0 6 1.6 38.7 6 2.0 62.8 6 3.8 17.3 6 2.1 29.2 6 1.1 47.9 6 3.5
4.3 6 0.4 15.4 6 1.1 14.5 6 2.3 13.4 6 2.0 20.0 6 2.7 22.2 6 0.6 16.5 6 0.7 12.1 6 1.2 17.0 6 4.4 10.2 6 2.2
10.4 6 0.8 62.4 6 3.5 76.5 6 4.0 50.5 6 3.8 57.7 6 2.7 60.4 6 1.5 80.3 6 5.7 32.6 6 3.2 53.0 6 1.4 58.1 6 2.3
53.7 6 2.4 134.5 6 7.3 157.4 6 4.9 136.8 6 5.3 128.8 6 4.8 98.9 6 5.1 132.4 6 3.0 104.7 6 4.4 100.7 6 2.9 96.3 6 3.4
Figure 5 reveals the change of lipoprotein level with static adsorption time using the AMPA membrane PA15C0-1. The two lipoprotein levels, LDL-C and HDL-C, quickly decrease in the first 30 min, and then slowly decrease to a plateau in 2 h. Interestingly, HDL-C level has a minimum before reaching the equilibrium which is different from the sustaining decline of LDL-C level. Haofeng Yu et al.41 ascribed this behavior to the larger diffusion speed of HDLC due to its smaller size (HDL 3.5–9 nm; LDL, 20–25 nm). With the stronger competitive adsorption capability, LDL would eventually recapture the adsorption sites which had been occupied previously by HDL particles. The effect of plasma concentration on the adsorption capability of the absorbent membranes is demonstrated by the adsorption isotherm of LDL-C using the AMPA membrane PA15C6-3 in Figure 6. Apparently, higher plasma concentration helps to the adsorption of LDL-C, which can be easily explained by the adsorption mechanism. As the equilibrium concentration increases, the diffusion of LDL-C particles enhances; therefore, more LDL-C particles are bound
FIGURE 5. Effects of adsorption time on lipoprotein levels. (PA15C0–1, COO2, 109.8 lmol/L) (a) LDL-C, (b) HDL-C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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by the active sites on the membrane. When all the active sites are devitalized, excessive LDL-C particles do not help improve the adsorption capability; that is, adsorption capability of the adsorbent reaches the peak value. In static adsorption test, therefore, higher adsorption capability and better selectivity performance can be achieved by using hypercholesterolemia plasma (TC >6.5 mmol L21) than by using normal plasma, which has been proved by the previous work.41 In the current work, the concentration of TC available is 2.2–2.8 mmol L21, actually far below the hypercholesterolemia plasma, the corresponding maximum adsorption capability of AMPA-membranes for LDL-C reaches 21.8 lg cm22 which is higher than that of the heparin modified adsorbent membrane we prepared before.40 The static adsorption capacity of the adsorbent membranes bonded with different surface ligands is shown in Figure 7. It is clear that the modified PP nonwoven membranes have better plasma static adsorption performance than the unmodified ones. Among the modified membranes, better adsorption performance is achieved by AMPA membranes than the ones only grafted with acrylic acid. It is true for both poly (acrylic acid) and cholesterol that the higher the amount of ligand bonded on the membranes, the
FIGURE 6. Adsorption isotherm of LDL-C by AMPA PA15C623 at 37 C.
SELECTIVE REMOVAL OF LOW-DENSITY LIPOPROTEIN FROM PLASMA
ORIGINAL RESEARCH REPORT
FIGURE 7. Plasma static adsorption capacity of the prepared adsorbent membranes bonded with different surface ligands. Results are reported as the mean of three measurements with standard deviation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
better lipoprotein capacity it has. This observation can be explained on the basis of the structure of lipoprotein and membrane. Poly (acrylic acid) increases adsorption of LDL-C by electrostatic interaction between the polyanion and the basic amino acid residues that are abundant in some regions of LDL particle surfaces. The cholesterol bonded on the absorbent membranes help anchor cholesterol molecules existing in the core of LDL-C particles through cholesterol–cholesterol hydrophobic interaction. These two interactions synergistically produce the good adsorption performance of AMPA membranes. The effect of length of linker on static adsorption capacity of AMPA-membranes is listed in Table II. Under a fixed feeding ratio of carboxyl group to cholesterol, as the
FIGURE 8. The lipoprotein levels at different in vitro hemoperfusion durations using AMPAs. The initial lipoprotein level (mmol/L): LDL (3.3 6 0.10), HDL (1.40 6 0.10), TC (5.0 6 0.10) and TG (4.5 6 0.2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
length of linker increases, static adsorption capability of AMPA membranes for LDL-C increases and selectivity for LDL-C/HDL-C also improves accordingly. The best adsorption performance was achieved by the PA15C6-3 membrane by which 62.8 6 3.8 lg of LDL-C, 16.5 6 0.71 lg of HDL-C, 80.35.7 lg TC and 132.4 6 3.0 lg of TG are removed from human plasma per square centimeter. It is well understood that aliphatic hydrocarbon chains, acting as spacer arms, improves the mobility of adsorption sites which is equivalent to increase the amount of adsorption sites, resulting in enhanced interactions between ligands and target molecules and consequently improved adsorption performance.42,43 On the other hand, for the same length of linker, the static adsorption performance does not increase with the feeding ratio of carboxyl group to cholesterol but has an optimum. This observation can be explained as following: although the larger amount of cholesterol bonded on the surface of AMPA membranes might anchor more LDL-C, excessive cholesterol can sharply increase the hydrophobic property of the membranes, which will weaken the electrostatic interaction between lipoptroteins and membranes thereby decrease the static adsorption performance. It implies that there is a balance between the amount of cholesterol and remaining carboxyl group on the surface of the AMPA membranes. In our case, the best result was obtained at the feeding ratio 3 of carboxyl group to cholesterol. Although it is hard to directly compare our results of static adsorption with the related reports5,41,44 due to the different forms of adsorbent, human plasma and adsorption conditions, the present adsorbent membrane provided satisfactory static adsorption property, especially taking into account the normal lipoprotein (instead of hyper-cholesterol) plasma level used in our test. For example, the adsorption capacity of membrane adsorbents reported by Li et al.5 is at a level of
