Article pubs.acs.org/Langmuir
Hemocompatibility of Polyampholyte Copolymers with Well-Defined Charge Bias in Human Blood Yu-Ju Shih,†,‡ Yung Chang,*,†,§ Damien Quemener,‡ Hui-Shan Yang,† Jheng-Fong Jhong,† Feng-Ming Ho,†,∥ Akon Higuchi,§,⊥ and Yu Chang*,#,△ †
R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan ‡ IEM (Institut Europeen des Membranes), UMR 5635 (CNRS-ENSCM-UM2), Universite Montpellier 2, Place E. Bataillon, F34095 Montpellier, France § Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia ∥ Department of Health, Tao-Yuan General Hospital, Jhong-LiTaoyuan 320, Taiwan ⊥ Department of Chemical and Materials Engineering, National Central University, Jhong-Li, Taoyuan 320, Taiwan # Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C. △ Department of Obstetrics and Gynecology, E-Da Hospital, I-Shou University, No. 1, Yida Road, Jiaosu Village, Yanchao District, Kaohsiung City, Taiwan, R.O.C, ABSTRACT: In this work, the hemocompatibility of polyampholyte copolymers from the mixed-charge copolymerization of negatively charged 3-sulfopropyl methacrylate (SA) and positively charged [2-(methacryloyloxy)ethyl] trimethylammonium (TMA) was studied. Charge-bias variation of the prepared poly(SA-co-TMA) copolymers can be controlled using the regulated SA and TMA monomer ratio via homogeneous free radical copolymerization. A systematic study of how charge-bias variations in poly(SA-co-TMA) copolymers affect the hemocompatibility in human blood plasma was reported. The hydrodynamic size of prepared polymers and copolymers is determined to illustrate the correlations between intermolecular cationic/anionic associations and the blood compatibility of polySA, poly(SA-co-TMA), and polyTMA suspensions in human blood plasma. It was found that the protein resistance and hydration capability of prepared copolymers can be effectively controlled by regulating the charge balance of the SA/TMA compositions in poly(SA-co-TMA). The results suggest that polyampholyte copolymers of poly(SA-coTMA) with overall charge neutrality have a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities as well as zwitterionic sulfobetaine-based homopolymers when in contact with blood plasma at human body temperature.
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INTRODUCTION Hemocompatible polymers resisting the adsorption of plasma proteins is important in the development of blood-contacting biomaterials in such applications as blood-collection devices, antithrombogenic implants, hemodialysis membranes, drugdelivery carriers, and diagnostic biosensors.1−6 Specific plasma protein, such as fibrinogen in blood plasma, is particularly important for platelet activation and blood clotting since it can bind to the platelet GP IIb/IIIa receptor.7−9 Thus, good nonspecific plasma protein resistance is one of the most important requirements for blood-contacting polymers.10 The general properties of the functional groups in these polymer chains are hydrophilic, electrically neutral, and hydrogen-bond acceptors rather than hydrogen-bond donors.11 This set of properties has become a general consideration to guide the © 2014 American Chemical Society
design of new hemocompatible polymers that resist the adsorption of plasma proteins in human blood.12−14 Recently, biomimetic polymers containing a zwitterionic structure similar to that of PC, such as phosphobetaine, sulfobetaine, and carboxybetaine, have received growing attention for use in the new generation of blood-contacting materials because of their good plasma protein resistance.15−24 Previous studies showed that the control of surface charge neutrality is important for a zwitterionic surface with effective protein-resistance properties.3,6,19,20 Importantly, it was further reported that the mixed-charge formulation in self-assembled monolayers, copolymer hydrogels, and polymeric brushes Received: October 8, 2013 Published: May 15, 2014 6489
dx.doi.org/10.1021/la5015779 | Langmuir 2014, 30, 6489−6496
Langmuir
Article
Table 1. Characteristic Data of PolySA, Poly(SA-co-TMA), and PolyTMA (Co)Polymers reaction ratios of comonomers (mol %)a
characterization of copolymersb
compositions of copolymers (mol %)c
degree of polymerization
physical properties of copolymersd
sample ID
SA
TMA
Mw (kDa)
Mw/Mn
SA
TMA
SA (m)
TMA (n)
polymerization conversion (%)
Cbias
SA100-TMA0 SA80-TMA20 SA70-TMA30 SA50-TMA50 SA30-TMA70 SA20-TMA80 SA0-TMA100
100 80 70 50 30 20 0
0 20 30 50 70 80 100
19.3 21.1 22.4 18.3 20.1 19.9 23.2
1.33 1.46 1.43 1.22 1.64 1.81 1.90
100 75 55 48 23 15 0
0 25 45 52 77 85 100
79 67 54 39 21 14 0
0 22 44 42 71 79 111
78.3 88.4 92.5 80.6 91.6 92.3 98.7
−100.0 −50.6 −10.2 3.7 54.3 69.9 100.0
φ (mV) −13.2 −9.8 −2.2 1.3 3.6 7.9 9.6
± ± ± ± ± ± ±
1.2 0.9 0.4 0.3 0.2 0.8 1.2
DH (nm) ∼12.21 ∼15.33 ∼16.26 ∼13.47 ∼15.68 ∼13.56 ∼17.69
a
The molar ratio of initiator/comonomers was 1:100. bWeight-average molecular weights (Mw) and molecular weight distributions (Mw/Mn) were estimated by GPC and calibrated with PEO. cThe composition of the poly(SA-co-TMA) copolymers was estimated by 1H NMR in D2O from the relative peak area of the (CH3)2SO3− proton resonance of SA side groups at δ = 2.13 ppm and the (CH3)2N+ proton resonance of the TMA side groups at δ = 4.52 ppm. dThe φ (zeta potential) and DH (hydrodynamic diameter) of suspended poly(SA-co-TMA) copolymers in PBS at 37 °C were estimated by dynamic light scattering (DLS) using a Zetasizer Nano-ZS 90. After polymerization, the resulting reaction solution was cooled to 4 °C for 3 h and then added slowly to ethanol and redissolved in deionized water repeatedly to precipitate the polymer out of the reaction solution and to remove residue chemicals. Then, the poly(SAco-TMA) copolymer was dried in a freeze-dryer at −45 °C to yield a white powder. Characterization of Mixed-Charge Poly(SA-co-TMA) Copolymers. The structure of poly(SA-co-TMA) copolymers was characterized by 1H NMR spectra using a 500 MHz spectrometer and D2O as the solvent. The chemical composition of the poly(SA-co-TMA) copolymers was estimated by 1H NMR in D2O from the relative peak area of (CH3)2N+ proton resonance of the PTM side groups at δ = 4.52 ppm and that of the CH2CH2CH2SO3− proton resonance of the PSA side groups at δ = 2.13 ppm. The molecular weights of the prepared poly(SA-co-TMA) copolymers were determined by aqueous gel-permeation chromatography (GPC) using two Viscogel columns, a G4000 PWXL and a G6000 PWXL (the range of molecular weight was 2 to 8000 kDa), connected to a model Viscotec refractive-index detector at a flow rate of 1.0 mL/min and a column temperature of 25 °C. The eluent was an aqueous solution composed of 0.1 M NaCl at pH 7.4. Poly(ethylene oxide) (PEO) standards from Polymer Standard Service, Inc. (Warwick, RI) were used for calibration. Hydrodynamic Size of Mixed-Charge Poly(SA-co-TMA) Copolymers in Plasma Protein Solution. In general, aggregation of the colloid polymer association and/or protein adsorption onto suspended polymers will result in an increase in the measured poly(SA-co-TMA) random polymers’ hydrodynamic size. The dynamic measurement of hydrodynamic size using dynamic light scattering (DLS) was applied to monitor nonspecific plasma protein adsorption from a single protein solution and a plasma solution onto the prepared poly(SA-co-TMA) random polymer polymers. A single protein solution of 1.0 mg/mL human fibrinogen in phosphate-buffered saline (PBS, 0.15M, pH 7.4) was prepared at 37 °C. A platelet-poor plasma (PPP) solution was prepared by centrifugation of human blood at 3000 rpm for 10 min at 37 °C. One hundred microliters of the fibrinogen solution (1.0 mg/mL) or PPP solution (100%) was mixed with 100 μL of the polymer solution (10 mg/mL) at 37 °C. The hydrodynamic diameter of poly(SA-co-TMA) random polymers in plasma protein solution was determined by DLS using a Zetasizer Nano ZS90 from Malvern, and the measured value was read for every 1 min increment of each sample for a period of 40 min at a constant temperature of 37 °C. Plasma Clotting Time and Red Blood Cell Hemolysis. The plasma clotting time and red blood cell hemolysis of the prepared copolymers were evaluated according to the standard protocol previously described.14 Water Sorbed into Mixed-Charge Poly(SA-co-TMA) Copolymers at Equilibrium. Previous studies have pointed out that the
provide a new avenue to achieving nonfouling surfaces with the prevention of nonspecific protein adsorption if the charge balance can be well controlled.4,25−27 However, at present it is still unclear how changes in the positive or negative charge bias deviation from the overall charge neutral of polyampholyte copolymers would influence their hemocompatible nature in human blood plasma. One current challenge is to develop molecular insight into pseudozwitterionic structures in the new formulation with effective antithrombogenic capabilities when they are in contact with human blood. In this work, polyampholyte copolymers with a sulfobetaine-like structure of mixed charged groups were synthesized via homogeneous free radical copolymerization, including seven prepared copolymers containing various ratios of positively and negatively charged moieties of poly(3-sulfopropyl methacrylate)-co-poly([2-(methacryloyloxy)ethyl] trimethylammonium) (poly(SA-co-TMA)). There are two unique aspects of this work: (a) the demonstration of how charge variations in poly(SA-co-TMA) copolymers affect plasma−protein association, blood-plasma clotting, and blood-cell hemolysis in human blood plasma and (b) the fundamental understanding of how polymer-chain hydration changes in poly(SA-co-TMA) copolymers affect the hemocompatible character of human blood plasma.
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MATERIALS AND METHODS
Materials. 3-Sulfopropyl methacrylate (SA), [2-(methacryloyloxy)ethyl] trimethylammonium chloride (TMA, 75 wt % solution in H2O monomer), ammonium persulfate (APS), ethanol (absolute 200 proof), poly(propylene oxide) (PPO) with an average molecular weight of 1 kDa, and poly(ethylene glycol) (PEG) with an average molecular weight of 4 kDa were purchased from Sigma-Aldrich. Fibrinogen (fraction I from human plasma) was purchased from Sigma Chemical Co. Deionized water (DI water) used in experiments was purified using a Millipore water-purification system with a minimum resistivity of 18.0 MΩ·m. Phosphate buffered saline (PBS) was purchased from Sigma. Preparation of Mixed-Charge Poly(SA-co-TMA) Copolymers in Aqueous Solution. A total solid content of 40 wt % for seven different molar ratios of SA, TMA, and APS initiator ([SA]/[TMA]/ [APS] = 100:0:1, 80:20:1, 70:30:1, 50:50:1, 30:70:1, 20:80:1, and 0:100:1 respectively, as shown in Table 1) was dissolved in 15 mL of cosolvent solution (deionized water and methanol in a 1:3 volume ratio), and nitrogen was bubbled to remove residual oxygen. The reaction was stirred under positive nitrogen pressure for 6 h at 70 °C. 6490
dx.doi.org/10.1021/la5015779 | Langmuir 2014, 30, 6489−6496
Langmuir
Article
Figure 1. Simplified model of charge-bias control in a (co)polymer conformation for (a) polySA, (b) poly(SA-co-TMA), and (c) polyTMA.
molecular weight of about 21.3 ± 0.2 kDa with similar molecular weight distributions (i.e., Mw/Mn = 1.6 ± 0.3). The degree of polymerization (Dp: m or n) of each component (SA or TMA) was regulated by the initial molar ratio of monomer to monomer and monomer to initiator. To determine the quantitative analysis of charge distribution further in the prepared poly(SA-co-TMA) copolymers, the degree of charge bias level (Cbias) is defined as the percentage difference in the degree of polymerization between n and m divided by the total summation of n and m. The positive values of Cbias (54.3− 100%) were obtained as n > m, indicating the prepared copolymers (SA30-TMA70, SA20-TMA80, and SA0-TMA100) with different degrees of the positive charge bias level. In contrast, the negative values of Cbias (−10.2 to −100%) indicated the prepared copolymers (SA70-TMA30, SA80TMA20, and SA100-TMA0) with different degrees of the negative charge bias level. The increasing number of TMA monomers in the reaction solution increased the molar mass ratio of polyTMA in the prepared poly(SA-co-TMA) copolymer. The composition of poly(SA-co-TMA) copolymers was estimated by 1H NMR in D2O from the relative peak area of (CH3)2SO3− proton resonance of the SA side group at δ = 2.13 ppm and the (CH3)2N+ proton resonance of the TMA side groups at δ = 4.52 ppm. A typical spectrum for SA50-TMA50 is shown in Figure 2. Results showed that a pure poly(SA-co-TMA) copolymer was obtained. It is noted that the molar ratio of polyTMA in the prepared copolymers is about 52 mol % when the number of TMA monomers used in the reaction solution is controlled at 50 mol %, indicating the similar chemical reactivity of TMA monomers and SA monomers in aqueous solution. The zeta potential and hydrodynamic diameter of the copolymers in the soluble unimer state were estimated by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) at a polymer concentration of 10 mg/mL in PBS solution. The hydrodynamic diameter of all prepared copolymers in the soluble unimer state (
