STUDY OF STRUCTURE-FUNCTION RELATIONSHIPS OF ZWITTERIONIC
POLYMERS
A Dissertation
Presented to
The Graduate Faculty of the University of Akron
In Partial Fulfillment
of the Requirement for the Degree
Doctor of Philosophy
Chen-Jung Lee
May 2018
STUDY OF STRUCTURE-FUNCTION RELATIONSHIPS OF ZWITTERIONIC
POLYMERS
Chen-Jung Lee
Dissertation
Approved: Accepted:
Advisor Department Chair Dr. Hongbo Cong Dr. Michael Cheung
Committee Member Dean of the College Dr. Gang Cheng Dr. Donald P. Visco Jr.
Committee Member Dean of the Graduate School Dr. Lingyun Liu Dr. Chand Midha
Committee Member Date Dr. Jie Zheng
Committee Member Dr. Xiong Gong
Committee Member Dr. Yang Liu
ii
ABSTRACT
This dissertation is to study the structure-property relationships of zwitterionic materials from the aspect of molecular level with integrating its antifouling ability into other functions including buffering ability, ionic conductivity and electronic conductivity.
In the first project (chapter II), a tertiary amine-based polycarboxybetaine (PCB) were synthesized to compare with the conventional quaternary ammonium-based PCB in order to study how tertiary amine group affect the properties of the resulting polymer.
Replacing quaternary ammonium with tertiary amine not only endowed this tertiary amine- based PCB with buffering capability at neutral condition, but also led to a weaker carboxylate that leads a less acidic buffering range. It was found that tertiary amine is favorable to obtain good lactone ring stability in switchable PCB materials. More importantly, the tertiary amine cation does not compromise antifouling properties of zwitterionic materials.
In the second project (chapter III), three typical but structurally different zwitterionic materials, polycarboxybetaine, polysulfobetaine, and polyphosphorylcholine, were studied on their ionic conductivity along with other materials. Recently, zwitterions have shown its potential on enhancement of the ionic dissociation. To gain a better understanding on how immobilized zwitterionic functional group interact with the mobile ions in the solution, a series of polyelectrolyte hydrogels, including zwitterionic, cationic,
iii anionic hydrogels, were examined and compared on their ionic conductivity and volume
change in three types of salt solutions with various concentration. Zwitterionic hydrogels
showed much higher ionic conductivity than that of the nonionic poly(ethylene glycol)
methyl ether methacrylate hydrogel in all tested solutions. For both cationic and anionic
hydrogels, the presence of mobile counterions led to high ionic conductivity in low salt solutions; however, the ionic conductivity of zwitterionic hydrogels surpassed that of cationic and ionic hydrogels in high salt solutions.
In the third and fourth projects (chapter IV and V), zwitterionic sulfobetaine
functional group was incorporated with poly(ethylenedioxythiophene) (PEDOT) to create
a series of zwitterionic conjugated polymers, and the effect of sulfobetaine group on the
conjugated polymer chain was studied. To address the challenge of the long-term stability in implantable bioelectronics, zwitterionic material with superior antifouling and conducting ability is highly desired for the interface between biological system and electronic system. Therefore, Poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT) was designed to combine the advantages of zwitterionic material and conjugated material.
The PSBEDOT-coated surface exhibited low interfacial impedance, good cyclic stability and switchable antifouling/antimicrobial properties. In addition, two PSBEDOT derivatives with different spacer length, PSBEDOT-4 and PSBEDOT-5, were synthesized to compare with PSBEDOT to study how the zwitterionic sulfobetaine side group affects the reactivity of monomer and the properties of the resulting polymer. It was found that
PSBEDOT-4 and PSBEDOT-5 showed significant improvement on interfacial impedance and cyclic stability without compromising antifouling properties.
iv
DEDICATION
To my family and coffee, you are God’s gift to me.
v
ACKNOWLEDGMENTS
First and foremost, I would like to express my deepest gratitude to my supervisor
Dr. Gang Cheng for all the support and guidance. Without him, this dissertation would simply have not happened.
I would like to thank all my committee members, Dr. Lingyun Liu, Dr. Jie Zheng,
Dr. Xiong Gong, Dr. Yang Liu, and Dr. Hongbo Cong for their valuable advices.
I would like to give my special thanks to my friends. It is my honor to have you guys accompany me along this long journey. All the happiness and sadness will be the best memory in my life.
Last but not least, I must thank Haiyan Wu. All the memories of my PhD life have you inside.
vi
TABLE OF CONTENTS
Page
LIST OF TABLES ...... x
LIST OF FIGURES ...... xi
LIST OF SCHEMES ...... xiv
CHAPTER ...... 1
I. INTRODUCTION ...... 1
II. STRUCTURE-FUNCTION RELATIONSHIPS OF A TERTIARY AMINE-BASED POLYCARBOXYBETAINE ...... ….13
2.1 Introduction ...... 13
2.2 Experimental section ...... 15
2.2.1 Chemicals...... 15
2.2.2 Synthesis of Monomers...... 16
2.2.3 Synthesis of Polymer Brushes via Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP) ...... 22
2.2.4 Synthesis of PCBMAA-1T, PCBMAA-2T, and PCBAA-1 ...... 23
2.2.5 Molecular Weight Measurement of PCBMAA-1T, PCBMAA-2T, and PCBAA-1...... 23
2.2.6 Protein Adsorption Study ...... 24
2.2.7 Monomer and Polymer Titrations of CBMAA-1T, CBMAA-2T, and CBAA-1 ...... 24
2.3 Results and discussion ...... 25
2.4 Conclusion ...... 33
vii III. IONIC CONDUCTIVITY OF POLYELECTROLYTE HYDROGELS ...... 35
3.1 Introduction ...... 35
3.2 Experimental section ...... 38
3.2.1 Chemicals ...... 38
3.2.2 Synthesis of the hydrogel ...... 39
3.2.3 Equilibrium water content assay ...... 39
3.2.4 The volume ratio of the hydrogels assay ...... 40
3.2.5 Ionic conductivity assay ...... 40
3.3 Results and discussion ...... 41
3.4 Conclusions ...... 54
IV. ELECTROACTIVE POLY(SULFOBETAINE-3,4- ETHYLENEDIOXYTHIOPHENE) (PSBEDOT) WITH CONTROLLABLE ANTIFOULING AND ANTIMICROBIAL PROPERTIES ...... 56
4.1 Introduction ...... 56
4.2 Experimental section ...... 58
4.2.1 Chemicals and general instrumentation ...... 58
4.2.2 Synthetic procedures ...... 59
4.2.3 Electropolymerization of SBEDOT ...... 62
4.2.4 X-Ray photoelectron spectroscopy (XPS) study ...... 63
4.2.5 Electrochemical characterization of PSBEDOT ...... 64
4.2.6 BAEC and NIH-3T3 cell adhesion study ...... 65
4.2.7 Protein adsorption study – Surface Plasmon Resonance ...... 66
4.2.8 Bacterial adhesion, antimicrobial and releasing study ...... 67
4.3 Results and discussion ...... 70
viii 4.4 Conclusions ...... 81
V. STRUCTURE-FUNCTION STUDY OF POLY(SULFOBETAINE 3,4-ETHYLENE- DIOXYTHIOPEHEN) (PSBEDOT) AND ITS DERIVATIVES ...... 83
5.1 Introduction ...... 83
5.2 Experimental Section ...... 86
5..2.1 Chemicals ...... 86
5.2.2 Synthesis of SBEDOT-4 and SBEDOT-5 ...... 87
5.2.3 Electro-polymerization and electrochemical analysis of PSBEDOT, PSBEDOT-4 and PSBEDOT-5 ...... 92
5.2.4 Fibrinogen adsorption of PEDOT, PSBEDOT, PSBEDOT-4 and PSBEDOT-5 ...... 92
5.2.5 Surface morphology characterization and thickness measurement ...... 93
5.3 Result and discussion ...... 94
5.4 Conclusion ...... 106
VI. CONCLUSION AND FUTURE WORK ...... 107
REFERENCES ...... 110
ix
LIST OF TABLES
Table Page
1. pKa of Carboxylate and Amine of CBAA-1, CBMAA-1T and CBMAA-2T Monomers and Polymers ...... 32
2. The water content of hydrogels in deionized water ...... 43
3. Percentage of the attached cells on PSBEDOT surfaces relative to PEDOT coated surfaces (n=3)...... 66
4. Film thickness of PSBEDOT, PSBEDOT-4, and PSBEDOT-5 on ITO-coated and gold-coated substrates. All samples were electro-polymerized by the galvanostatic method: 0.5 mA/cm2 for 120s...... 96
5. Frequency change and the percentage of protein adsorption of PEDOT, PSBEDOT, PSBEDOT-4 and PSBEDOT-5...... 104
x
LIST OF FIGURES
Figure Page
1. 1H NMR spectrum of N-(2-((2-hydroxyethyl)amino)ethyl)methacrylamide ...... 17
2. 1H NMR spectrum of CBMAA-1T ...... 18
3. 13C NMR spectrum of CBMAA-1T ...... 18
4. 1H NMR spectrum of CBMAA-2T ...... 20
5. 13C NMR spectrum of CBMAA-2T ...... 20
6. 1H NMR spectrum of CBAA-1 ...... 21
7. 13C NMR spectrum of CBAA-1 ...... 21
8. 1H NMR overlay plot of ring formation of CBMAA-1T in D-acetonitrile (A). Conversion kinetics of ring formation of CBMAA-1T in D-acetonitrile (B) and D-acetic acid (C). Conversion kinetics of ring opening of CBMAA-1T in D2O (D)...... 27
9. 2D NMR analysis of the compound of CBMAA-1T ...... 28
10. Protein adsorption test on PCBTAA-1T polymer brush surface with 1 mg/mL fibrinogen solution, undiluted human plasma and serum...... 30
11. Switchable charged state of CBMAA-1T in different pH condition...... 31
12. pH titrations of CBAA-1, CBMAA-1T, and CBMAA-2T monomers (A) and polymers (B) from pH 1 to 13...... 32
13. The chemical structure of monomers and crosslinkers: (A) 2-((3- acrylamidopropyl) dimethylammonio)acetate (CBAA), (B) 2-methacryloyloxyethyl phosphorylcholine (MPC), (C) [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide (SBMA), (D) poly(ethylene glycol) methyl ether methacrylate (PEGMA), (E) [2- (acryloyloxy)ethyl]trimethylammonium chloride (TMA), (F) 2-acrylamido-2-methyl-1-propanesulfonic acid sodium (AMPS), (G) 2-aminoethyl methacrylate hydrochloride (AEMA), (H) 2-(dimethylamino)ethyl methacrylate (DMAEMA), (I) methacrylic acid (MAA), (J) N,N’-methylenebisacrylamide (MBAA) and (K) tetraethylene glycol dimethacrylate (TEGDMA)...... 42
xi 14. The volume ratio of neutral and ionic hydrogels in various solutions: Neutral hydrogels in (A) deionized water, NaCl solution and PBS, (C) MgSO4 solution and (E) MgCl2 solution; ionic hydrogels in (B) deionized water, NaCl solution and PBS, (D) MgSO4 solution and (F) MgCl2 solution. (Standard deviation is less than 5%) ...... 45 15. The ionic conductivity of neutral hydrogels in (A) deionized water and 2 mM electrolyte solutions, (B) 10 mM electrolyte solutions and (C) 100 mM electrolyte solutions. *P < 0.05...... 47
16. The ionic conductivity of ionic hydrogel in (A) deionized water and 2 mM electrolyte solutions, (B) 10 mM electrolyte solutions and (C) 100 mM electrolyte solutions...... 49
17. The ionic conductivity of neutral and ionic hydrogels in (A) 300 mM MgCl2 solution, (B) 1 M MgCl2 solution and (C) 3 M MgCl2 solution. *,**,***P < 0.05...... 53 18. 1H spectrum of EDOT-DMA ...... 60
19. 13C spectrum of EDOT-DMA ...... 60
20. 1H spectrum of SBEDOT ...... 61
21. 13C spectrum of SBEDOT ...... 61
22. Optical images of PSBEDOT coating on ITO (left) and gold (right) substrates...... 62
23. The XPS profiles of PSBEDOT coating. Survey spectrum (left), high- resolution spectrum of S 2p (right) ...... 63
24. Cyclic voltammograms of PSBEDOT film at different scan rate...... 65
25. BAECs adhesion test with PSBEDOT coated ITO-PET. (A) PSBEDOT coated region, (B) Region across coating boundary and (C) uncoated region...... 66
26. Protein (FITC-Fg) adsorption test on surface visualized under fluorescence microscope at the same excitation light intensity and exposure time. (A) PSBEDOT coated surface, (B) PEDOT coated surface, (C) bare gold sensor chip surface...... 67
27. Electrochemical characterization of PSBEDOT coated substrates. (A) Comparison of the cyclic voltammograms of PSBEDOT on gold from the first cycle (black) and after 500 cycles (red) of the applied potential, (B) electrochemical impedance spectra (Bode plots) of the bare gold substrate (black squares) and PSBEDOT coated gold substrate (blue triangles) ...... 73
28. Representative SPR sensorgrams of PSBEDOT coated sensor chips, showing the low protein adsorption from 100% human blood plasma (red) and 30% human blood serum (black)...... 75
xii 29. Cell adhesion tests of PSBEDOT-coated gold substrates incubated with (A) BAECs and (B) NIH3T3 fibroblast cells, and the PEDOT-coated gold substrates incubated with (C) BAECs and (D) NIH3T3 fibroblast cells for 24 hours...... 76
30. Representative fluorescence microscopy images of the bacterial adhesion, antimicrobial and release studies on PSBEDOT and control surfaces. Attached E. coli K12 from a suspension with 109 cells per mL on gold (A) and oxidized PSBEDOT (D); the viability of the attached E. coli K12 on gold (B) and oxidized PSBEDOT (E) after subjection to 0.6 V for 1 hour; and the remaining E. coli K12 on gold (C) and oxidized PSBEDOT (F) after subjection to 0 V for 1 hour. In the viability study, bacterial cells were stained using a LIVE/DEAD BacLight Bacterial Viability assay kit. Cells with a damaged cytoplasm membrane are in yellow and red, and cells with an intact cytoplasm membrane are in green...... 78
31. Quantitative bacterial adhesion, antimicrobial and release studies on PSBEDOT and control surfaces. (A) Attachment of E. coli K12 from a suspension with 109 cells per mL on oxidized (Ox) PSBEDOT, reduced (Red) PSBEDOT and control surfaces; (B) bactericidal activity results of PSBEDOT and the control surface against E. coli K12 after subjection to 0.6 V for 1 hour; and (C) detachment of E. coli K12 from oxidized PSBEDOT and gold after subjection to 0 V for 1 hour...... 79
32. 1HNMR of SBEDOT-4 ...... 89
33. 13CNMR of SBEDOT-4 ...... 90
34. 1HNMR of SBEDOT-5 ...... 91
35. 13CNMR of SBEDOT-5 ...... 91
36. Surface morphology of (A) PSBEDOT, (B) PSBEDOT-4, and (C) PSBEDOT- 5 synthesized on gold substrates at 0.5 mA for 120s, 240s, and 360s (Y-axis) imaging at the magnification of 200x and 5000x (X-axis) ...... 98
37. Surface morphology of (A) PSBEDOT, (B) PSBEDOT-4, and (C) PSBEDOT- 5 synthesized on ITO-coated glass substrates at 0.5, 1.0, 1.5, 2.0 mA for the same total deposition charge (Y-axis) imaging at the magnification of 200x and 5000x (X-axis) ... 99
38. Electrochemical impedance spectra (EIS) of (A) PSBEDOT and (C) PSBEDOT-4 on ITO substrates in different polymerization condition. Comparison of EIS of PSBEDOT, PSBEDOT-4, and PSBEDOT-5 on (B) gold substrates and (D) ITO substrates. All EIS experiment performed in 100 mM LiClO4 solution...... 100 39. Cyclic voltammograms of (A) PSBEDOT, (B) PSBEDOT-4 and (C) PSBEDOT-5at 20th, 220th, 520th, and 1000th cycle. (D) Percentage of total charge of PSBEDOT, PSBEDOT-4 and PSBEDOT-5 at 20th, 520th, and 1000th cycle...... 102
40. Fibrinogen adsorption of PEDOT, PSBEDOT, PSBEDOT-4 and PSBEDOT-5 tested by electrochemical quartz crystal microbalance (eQCM)...... 104
xiii
LIST OF SCHEMES
Scheme Page
1. Synthetic routes of CBMAA-1T and CBMAA-2T. Reaction condition: (i) ethanol, water, NaOH; (ii) methanol, ethyl bromoacetate, N,Ndiisopropylethylamine (DIPEA); (iii) methanol, ethyl acrylate; (iv) NaOH...... 25
2. Synthetic route of PSBEDOT. Reaction conditions: (i) 3-chloropropane-1,2- diol, p- toluenesulfonic acid, toluene; (ii) dimethylamine, water, acetonitrile; (iii) 1,3- propanesultone, tetrahydrofuran; (iv) electropolymerization in aqueous solution...... 70
3. Synthetic route of PSBEDOT-4 and PSBEDOT-5. Reaction condition: (i) 1,4- dibromobutane for m=4 and 1,5-dibromopentane for m=5, tetrabutylammonium bromide, dichloromethane/50 wt% of sodium hydroxide solution (1:1); (ii) dimethylamine solution (40 wt% in water), acetonitrile; (iii) 1,3-propanesultone, anhydrous tetrahydrofuran; (iv) electro-polymerization in aqueous solution of 40 mM monomer and 100 mM LiClO4 by galvanostatic method...... 95
xiv
CHAPTER I
INTRODUCTION
Fouling is the process in which those undesired substances attach and settle onto
the surface. As the accumulation of the undesired substance, the process results in adverse
effects on the subject. For example, as ships sail in the ocean that contains microorganisms, plants, and animals, the unwanted settlement on the hull can create a rough surface with high frictional resistance leading to a reduction of ship velocity and loss of maneuverability.
Therefore, higher fuel consumption is unavoidable that cause more air pollution and more voyage cost.[1] Additionally, in the biomedical field, it is widely believed that protein adsorption is the first step to the inflammatory response which can cause potential platelet adhesion and thrombosis. Thus, antifouling materials which can prevent the nonspecific protein adsorption have attracted enormous attention as the next generation of biomaterials.
Among antifouling materials, zwitterionic materials exhibit the unique chemical
properties and the excellent resistance to nonspecific protein adsorption, bacteria adhesion
and biofilm formation, and have been used in various applications such as batteries,[2]
drug delivery,[3, 4] oil-water separation,[5] optical imaging,[6] optoelectronic devices,[7]
wound dressing.[8] The antifouling ability of a zwitterionic material is attributed to the
well-structured water layer. This water layer formed by electrostatically ionic interactions
between charged groups of zwitterionic materials and water molecules is more effective to
1 resist protein adsorption than poly(ethylene glycol) based materials (PEGs) which only
bear hydroxyl groups.[9] On the other hand, zwitterionic polymer which is considered as
an alternative biomaterial to currently widely used PEGs is a neutral polymer bearing a
positive charge and a negative charge on the same repeating unit. Unlike PEGs containing
the same hydroxyl group, zwitterionic polymers ideally can have infinite number of
combinations of functional groups. This unique structural property imparts zwitterionic
polymer functional diversity. Over recent decades, lots of effort has been put in the
structure-function study. For example, the carboxylate anion groups of polycarboxybetaine
(pCB) can immobilize amine-containing biomolecules and the sulfonate group of polysulfobetaine (pSB) can incorporate antimicrobial silver cations, which makes them not just an antifouling polymer, but also a good carrier for drug delivery.[8, 10] Additionally, by adding a hydroxyl group, the pCB derivatives can switch reversibly between antifouling zwitterionic form and antimicrobial cationic lactone form in response to the pH value of the environment.[11-13] In future, to address various challenges of a complex biological system, synergism and multifunctionality are important for the designing of novel biomaterials. However, this requires a solid and systematic fundamental knowledge such as how zwitterionic functional group interacting with other functional groups and how the structural variation affecting the properties of zwitterionic polymers. To pursue the above- mentioned goal, this dissertation focuses on studying the structure-function relationships of zwitterionic polymers, especially pCB and pSB, and integrates other functions, such as buffering ability, ionic conductivity and electrical conductivity, with antifouling property.
1.1 Structure-function relationships of a tertiary amine-based polycarboxybetaine
2 Polycarboxybetaine has been studied for a variety of biomedical applications due
to its several advantages such as structural versatility,[14, 15] ease of synthesis,[16]
possibility of functionalization,[17-19] ultra-low biofouling property[20, 21] and
biocompatibility.[22-25] Since pCBs contain the carboxylate group that exhibits buffering
capability under acidic environment, this renders pCBs possessing potential use for wound healing applications. The effects of pH on wound healing have been studied over recent decades, and it has been proved that the acidic environment can help the process of wound healing by inhibiting the growth of pathogenic bacteria, recruitment of macrophages to the wound site,[26] regulating protease activity, promoting the release of oxygen, and facilitating epithelization and angiogenesis.[27, 28] Moreover, in a series of complex wound healing process, it involves a large number of enzymes which each activity can be affected by pH value and require optimum pH for optimal performance. For example, the optimal activity of elastase, matrix metalloproteinase-2 and plasmin can be achieved at pH
8 and neutrophil elastase require pH 8.3,[29, 30] while stratum corneum thiol protease have the best performance at an acidic pH milieu.[31] Additionally, several investigations have shown that the skin-graft treatment for chronic wound demonstrated higher take-rate at alkaline environment.[32-34] Thus, a biomaterial with buffering capability can be useful for wound healing applications.
In first project, I propose to create an integrated tertiary amine-based
polycarboxybetaine (pCB) with antifouling, antimicrobial and buffering capability. There
are several hypotheses in this work. First, a tertiary amine-based pCB can exhibit buffering capability not only under acidic environment but also basic condition. Second, since the acidity of the carboxylate group can be affected by the adjacent amine group, replacing the
3 quaternary ammonium group with tertiary amine group can lead to a weaker acidity of the
carboxylate group, which is more suitable for biomedical applications. Third, previous
studies have shown that the length of methylene spacer between the cationic amine group
and the anionic carboxylic group of zwitterionic polymers strongly influence the properties including pKa. Two tertiary amine-based pCBs with different length of methylene spacer were synthesized for the comparison of their properties.
1.2 Ionic conductivity of polyelectrolyte hydrogels
Polyelectrolyte hydrogel which combines the advantage of electrolyte and hydrogel, i.e. a solid gel with soft mechanical property and ability to conduct ions, has drawn interest in a variety of applications, including electronic devices, tissue engineering scaffolding, coatings,[35] batteries, fuel cells, water purification, and drug delivery. As one type of solid
electrolyte, polyelectrolyte hydrogel provides many advantage over liquid electrolyte such
as free of electrolyte leaking, improvement of chemical stability and ease of miniaturization.
Among all the properties of polyelectrolytes, ionic conductivity is an important parameter for a range of applications, because ions function as the charge carriers and the ionic mobility in polyelectrolyte directly affects the performance and sensitivity of the devices.
Zwitterionic polymer which is classified in polyampholyte is well known for the ability of high water retention due to the strong interaction between the zwitterionic functional groups and water molecules. Another unique property of zwitterionic materials is the anti- polyelectrolyte effect. In contrast to conventional polymer, this effect endows zwitterionic polyelectrolyte with good solubility in a solution with high salt concentration. Both properties make zwitterionic polyelectrolyte a great potential candidate for fast ion conductor. Moreover, zwitterion have attracted special attention in last decade as a
4 dissociation enhancer in polyelectrolyte[36, 37] and ionic liquid[38], and the conductivity
of the tested systems have been significantly improved with the addition of a small amount of zwitterions. In another work, zwitterions were used to modify the spin-coated films of
PEDOT: PSS which showed improved conductivity. The conductivity enhancement was attributed the charge-screening effect induced by zwitterion, which reduce the influence of
PSSH on the charge transport across the conjugated PEDOT chains[39]. However, although these studies clearly stated the benefit of zwitterions, the effect of the immobilized zwitterionic functional group on the ionic and electrical conduction is still unclear.
Thus, in the second project, I propose to conduct a comprehensive study on ionic
conductivity of various polyelectrolytes, including cationic, anionic, zwitterionic and non-
ionic polyelectrolytes, in various salt solutions with different concentrations. A deeper
understanding on how the electrolyte groups of polyampholytes affect the conductivity of
polyampholytes can be helpful to design new ionic conducting materials for biomedical,
environmental, and energy applications.
As mentioned before, since the effect of the immobilized zwitterionic functional
group on the electrical conduction remains unclear, this topic was investigated in the next
two projects.
1.3 Zwitterionic conductive polymer Poly(sulfobetaine 3,4-ethylenedioxythiophene)
(PSBEDOT) with controllable antifouling and antimicrobial properties
Conjugated polymers (CPs) are organic polymers containing a backbone chain of
alternating single and double bonds, which can conduct electricity due to the system of delocalized π electron. In addition to the electrical conduction, these metal-like conducting
5 polymers possess the advantage of organic materials including processability, design
flexibility and ease of manufacture, and have been attracted enormous attention for a
variety of applications. As one of the extensively studied field for CPs, bioelectronics is a
new and rapidly developing subject in the convergence of biology and electronics. In the
past, implantable device, which is one of main topic of research in bioelectronics, was
fabricated with hard metal or inorganic materials. However, due to the mismatched
mechanical properties between biological tissue and implanted device, adverse reactions
can arise.[40] The use of organic conducting polymer on the hard electronic materials can not only improve the stability due to the similar mechanical properties, but also lower the
impedance resulting in high quality and performance of implants.[41] Moreover, due to the high requirement of signal-to-noise ratio, classical metal electrodes are unsuitable for recordings of brain activity. Although a more advanced electrolyte/oxide/silicon field- effect transistor has been developed, the oxide layer blocks ion transport between the electrolyte and the channel, which is still not as efficient as organic electrochemical transistor. Organic electrochemical transistor in which the channel directly contact with an electrolyte can receive ions from biological environment that promotes the signal amplification. Nevertheless, before CPs can be fully exploited, there are still several major challenges to address such as electrochemical stability, biocompatibility and mechanical durability, and, moreover, the lack of functional group to conjugate bioactive moieties restrains their potential in biomedical applications.
As one of the important conducting polymers, poly (3,4-ethylenedioxythiophene)
(PEDOT), a derivative of polythiophene, has the external heterocyclic rings which endows the polymer with low oxidation potential, improved solubility and high electrochemical
6 stability. This external heterocyclic ring significantly decreases the amount of α-β coupling in the resulting polymer which is the main reason that cause the poor long-term stability in polypyrene (PPy). Compared to PEDOT, another limitation for PPy is the unfavorable reduction reaction by using relatively weak but biologically relevant reducing agent due to the high oxidational potential. PEDOT had been considered as a promising CP in biomedical applications for a long period of time. However, due to the poor water solubility, the electropolymerization of EDOT is usually performed in organic solvent. Since water is the most economical solvent and friendly to environment and human body, this limited solubility can impede the further applications of EDOT. For example, the local in vivo electropolymerization of conducting polymer in brain have been tested in mice to rebuild charge transport pathways across the glial scars, which the electropolymerization is performed in PBS solution.[42] Additionally, although PEDOT has been considered as a high biocompatible material comparing to other CPs, PEDOT is still likely to trigger foreign body reaction when implanted. In a previous study, the increase in the impedance of the implanted PEDOT-coated silicon microelectrode over time was attributed to the protein adsorption, which can eventually lead to the fibrous encapsulation and the failure of the device.[41]
In the third project, I propose to synthesize a sulfobetaine-functionalized PEDOT.
As mentioned before, zwitterionic materials have been well-known for the excellent resistant to protein adsorption and high hydrophilicity. I hypothesize that by introducing zwitterionic sulfobetaine group to PEDOT, the abovementioned two drawbacks of PEDOT can be addressed.
7 1.4 Structure-function study of Poly(sulfobetaine 3,4-ethylenedioxythiophene)(PSBEDOT) and its derivatives
Indeed, conjugated polymers are of interest for a range of bioelectronic devices due to the ionic and electronic conductivity and soft mechanical properties. However, to optimize the performance and fulfill the requirements for different applications, the functionalization of conjugated polymers is highly desired for tuning the properties such as solubility, electrochromic properties, hydrophilicity and morphology. To date, CPs have been incorporated with diverse functional groups such as hydroxy group,[43] carboxylic acid,[44, 45] cyanobiphenyl group[46] and sulfonate[47] to meet the various ends.
Sulfobetaine is one of the zwitterionic functional groups that exhibit excellent antifouling properties which have been applied in many applications. In recent years, sulfobetaine have been incorporated with conjugated polymers such as fluorene[48] and thiophene[49] to create materials for interlayers. Interlayers, in organic electronics, are thin layers placed between organic active layers and metal electrodes to enhance the performance of the electronic devices such as organic light-emitting diodes (OLEDs),[48, 50] organic field- effect transistors (OFETs) and organic photovoltaics (OPVs).[51] It has been reported that using sulfobetaine-functionalized conjugated polymers as interlayers significantly improved the performance of OPV and OLED devices due to the reduced work function of the electrode.
However, closely attaching functional groups to conjugated backbone can strongly
affect the kinetics of polymerization of CPs and the properties of the resulting polymer.
Zwitterionic groups with large polarity may introduce strong electronic effect on the
conjugated polymer backbone. It is reported, for example, that a zwitterionic
8 phosphorylcholine-functionalized EDOT was unable to be polymerized in aqueous
solution due to the electronic effect, and then was polymerized in acetonitrile with the help of a surfactant.[52] In the previous project, sulfobetaine was used to functionalize EDOT to address the water solubility and the biocompatibility. Owing to the antifouling ability of sulfobetaine group, PSBEDOT showed great resistant to proteins, cells, and bacteria.
Although SBEDOT can be polymerized in aqueous solution, the required monomer concentration for electropolymerization is higher than EDOT. In addition, there were soluble oligomers generated during the electropolymerization, which was observed by the discoloration of the monomer solution.
In the fourth project, I propose to further study how the structure of zwitterionic
side chains affect the reactivity of the monomer and the properties of zwitterionic PEDOT
polymers. By introducing the methoxyalkyl spacer between PEDOT backbone and
zwitterionic sulfobetaine side chain, both electronic and steric effect can be reduced.
Therefore, two SBEDOT derivatives with different length of spacer will be synthesized to
examine the effect of spacer length on the electrochemical properties, antifouling and
surface morphology.
1.5 Overview of the dissertation
In this dissertation, I am proposing to study the structure-function relationship of
zwitterionic materials from various aspects. In Chapter II, a tertiary amine-based
polycarboxybetaine (PCB) were synthesized to study how the tertiary amine group affect
the properties of the resulting polymer including the kinetics of reversible ring formation,
antifouling property, and buffering ability. In contrast to the conventional zwitterionic
9 materials possessing the permanent charged quaternary ammonium group, a tertiary amine-
based PCB is able to change the charged state in response to the pH of the environment. In addition, the weaker basicity of tertiary amine group increased the pKa of carboxylate group toward a more suitable buffering range for wound healing application. More importantly, the tertiary amine-based PCB still showed great resistant to fibrinogen solution and undiluted human plasma and serum in the zwitterionic state. In Chapter III, a comprehensive study on how the function group of polyelectrolyte hydrogels affect the ionic conductivity in three salt solutions with various concentration was conducted. Total nine materials were synthesized into hydrogel including three zwitterionic hydrogels, three cationic hydrogels, two anionic hydrogels, and one nonionic hydrogel. The volume ratio and ionic conductivity of all hydrogels were examined in NaCl, MgSO4 and MgCl2
solutions with concentration varying from low (2mM) to high (3M). And zwitterionic
hydrogels showed much higher ionic conductivity than that of the nonionic poly(ethylene
glycol) methyl ether methacrylate hydrogel in all tested solutions. In Chapter IV, a
zwitterionic conducting polymer, poly(sulfobetaine-3,4-ethylenedioxythiophene)
(PSBEDOT), was developed to address the drawbacks of the conventional conducting
polymer. The antifouling properties of PSBEDOT were studied thoroughly from human
blood to mammalian cells to bacteria. Additionally, the switchability between cationic
antimicrobial state and zwitterionic antifouling state was demonstrated, which is useful in
bacterium catching, killing and releasing. In Chapter V, two SBEDOT derivatives,
SBEDOT-4 and SBEDOT-5 where the number indicates the spacer length, were
synthesized to study the effect of zwitterionic sulfobetaine group on the properties of the
resulting polymer. Along with PSBEDOT, the systematic characterizations on these
10 materials were conducted including surface morphology, film thickness, interfacial
impedance, cyclic stability and protein adsorption. Two SBEDOT derivatives have showed improved electrochemical properties with antifouling property comparable to PSBEDOT.
Through these studies, I would like to provide insights and knowledge about zwitterionic materials for the development of next-generation biomaterials.
11
12
CHAPTER II
STRUCTURE-FUNCTION RELATIONSHIPS OF A TERTIARY AMINE-BASED
POLYCARBOXYBETAINE
2.1 Introduction
Due to the excellent properties to resist biofouling, zwitterionic polycarboxybetaine
(PCB) materials have attracted noticeable interest for a variety of biomedical applications
such as wound healing/tissue engineering,[53-55] medical implants[13, 56] and
biosensors.[57-59] The first generation zwitterionic PCBs, such as poly(carboxybetaine methacrylate) (PCBMA)[16] and poly(carboxybetaine acrylamide) (PCBAA),[14] exhibit excellent antifouling properties to resist protein adsorption, cell attachment, and biofilm formation. However, these conventional zwitterionic polymers lack antimicrobial properties.[60] To address this shortcoming, several PCB derivatives, which contain hydroxyl group(s) and can form lactone rings, were developed.[11, 12, 61] These polymers can switch between zwitterionic antifouling state at their ring-open form and cationic antimicrobial states at their ring form in response to the pH changes in the environment.
For wound healing applications, polymers with buffering capabilities under acidic and neutral conditions are particularly useful. It has been proved that the acidic environment inhibits the growth of the pathogenic bacteria,[62-64] regulates protease activity,[30] promotes the release of oxygen,[65, 66] and facilitates epithelization and angiogenesis.[67]
13 Integrated material with antifouling, antimicrobial and buffering capacity are highly
desired for wound healing applications.
Since the quaternary ammonium cation is permanently charged, quaternary
ammonium-based PCBs only exhibit buffering capability attributed to the carboxylate
group under acidic conditions. Permanently charged quaternary ammonium groups also
increase the acidity of the adjacent carboxylate group.[68] Unlike most quaternary
ammonium-based zwitterionic materials, zwitterionic materials, which contain primary,
secondary or tertiary amine(s), can respond to a much broader pH range. Additionally,
studies have shown the length of the methylene spacer between the cationic amine group
and the anionic carboxylic group of zwitterionic polymers made a significant influence on
their properties and characteristics.[69] Here we hypothesized that the buffering capacity at neutral and basic conditions could be introduced to PCB by replacing the quaternary ammonium with the tertiary amine. We also expected that the tertiary amine group would lead to a weaker acidity of carboxylate compared to quaternary ammonium cations so that carboxylate could have the buffering capability at more biologically relevant conditions.
A better understanding of the structure−function−stability relationships of the
zwitterionic polymers is also necessary to design a single material integrating all desired properties, such as antifouling, antimicrobial and buffering properties. The objectives of this work are to design and synthesize tertiary amine-based zwitterionic materials with uncompromised antifouling, switchable properties and buffering capacity, and to investigate the effect of the molecular structure on these properties. To test our hypothesis, we designed and synthesized two tertiary amine-based carboxybetaine derivatives, 2-((2- hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate ( C B M A A – 1 T ) and 3 - ( ( 2
14 – h y d r o x y e t h y l ) ( 2 -methacrylamidoethyl)ammonio)propanoate (CBMAA-2T).
The antifouling properties of PCBMAA-1T and PCBMAA-2T polymer brushes were studied using a surface plasmon resonance (SPR) sensor. The ring formation kinetics and titration of CBMAA-1T and CBMAA-2T were investigated.
2.2 Experimental section
2.2.1 Chemicals.
Ethanol (200 proof) was purchased from DeconLabs (King of Prussia, PA).
Hydrochloric acid solution (1 M) was purchased from Ward’s Science (West Henrietta,
NY). Sodium hydroxide solution (1 M) and anhydrous ethyl ether were purchased from
EMD Millipore (Billerica, MA). Acetonitrile-d was purchased from Cambridge Isotope
Laboratories (Andover, MA). Methanol, dichloromethane, diethyl ether, acetonitrile,
tetrahydrofuran (THF), acetic acid-d, (HAc-d),2,2′-bipyridine, copper(I) bromide, 2- hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, N,N′-methylenebis(acrylamide), sodium hydroxide, silica gel 60, phosphate-buffered saline (PBS), Amberlite IRA-400 OH form resin, and methacrylic anhydride were purchased from Sigma-Aldrich (St. Louis,
MO). Copper(II) bromide was purchased from Acros Organics (Hampton, NH). N-(2-
Hydroxylethyl) ethylene diamine, ethyl bromoacetate, and N,N-diisopropylethylamineand ethyl acrylate were purchased from Alfa Aesar (Ward Hill, MA). Human fibrinogen (Fg) was purchased from Calbiochem (San Diego, CA). Pooled human blood plasma and serum were purchased from BioChemed Services (Winchester, VA). GPC analytical standard polyethylene glycol/poly(ethylene oxide) was purchased from SigmaAldrich. Deionized
15 (DI) water used in all experiments was purified using a Milli-Q Direct 8 Ultrapure Water
system (Millipore, Billerica, MA) with a minimum resistivity of 18.2 MΩ·cm.
2.2.2 Synthesis of Monomers.
Synthesis of N-(2-((2-Hydroxyethyl)amino)ethyl)-methacrylamide.
An amount of 4.23 g (0.106 mol) of NaOH was dissolved in a mixture of 30 mL of
water and 70 mL of ethanol in a 250 mL three-neck round-bottom flask, followed by the
addition of 10 g (0.096 mol) of N-(2-hydroxylethyl)ethylene diamine. The mixture was cooled down in an ice bath. Next, 15.73 mL (0.106 mol) of methacrylic anhydride was added dropwise under nitrogen protection. After stirring at 0 °C for 2 h, the reaction continued at room temperature for 3 h. The crude product was purified by silica gel column chromatography (dichloromethane/methanol, 9/1 (v/v)). The pure product was obtained as a colorless liquid and analyzed by nuclear magnetic resonance (NMR). 1H NMR (300 MHz,
D2O) (Figure 1) δ 5.75 (s, 1H), 5.49 (s, 1H), 3.72 (t, J = 11.4 Hz, 2H), 3.44 (t, J = 12.9 Hz,
2H), 2.85-2.77 (m, 4H), 1.97 (s, 3H).
16
Figure 1. 1H NMR spectrum of N-(2-((2-hydroxyethyl)amino)ethyl)methacrylamide
Synthesis of 2-((2-Hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate (CBMAA-
1T).
The amounts of 5.76 g (0.0334 mol) of N-(2-((2-
hydroxyethyl)amino)ethyl)methacrylamide, 6.14 g (0.037 mol) of ethyl bromoacetate, and
21.60 g (0.167 mol) of N,N-diisopropylethylamine (DIPEA) were dissolved in 60 mL of
methanol. The reaction was carried at 40 °C under nitrogen protection for 24 h. The crude
product was precipitated by diethyl ether, and then was purified by silica gel column
chromatography (dichloromethane/methanol, 92/8 (v/v)). The hydrolysis of the ester group on the carboxylate was carried out by dissolving the product in a solution containing equal amount of sodium hydroxide in mole. Water and released ethanol were removed via a rotary evaporator, and remaining water was further removed by using a freeze-dryer. The final product was obtained as a transparent liquid, and it was analyzed by NMR. The overall yield was 33%. The singlet appearing at 3.28 ppm is a trace of methanol. 1H NMR (300
MHz, D2O) (Figure 2) δ 5.67 (s, 1H), 5.40 (s, 1H), 3.57 (t, J = 12 Hz, 2H), 3.30 (t, J = 12.3
17 Hz, 2H), 3.16 (s, 2H), 2.73-2.67 (m, 4H), 1.88 (s, 3H). 13C NMR (75 MHz, D2O) (Figure
3) δ 172.42, 169.94, 138.35, 122.03, 56.59, 56.04, 55.34, 54.63, 34.76, 17.56.
Figure 2. 1H NMR spectrum of CBMAA-1T
Figure 3. 13C NMR spectrum of CBMAA-1T
The 1H[13C] gHMBC NMR spectrum of CBMAA-1T was measured by using a
Varian VNMRS 500 MHz spectrometer. Each NMR sample was prepared by dissolving
30 mg of product in about 600 μL of chloroform-d solvent contained in NMR sample tubes.
Quantitative 1H NMR spectra were collected with 3 s acquisition time, 5-10 s relaxation
18 delay, 32 transients, and 90° pulse width of 22.9 μs. The data was zero-filled 131 k points,
exponentially weighted with a line broadening of 0.5 Hz and Fourier transformed.
Qualitative 13C NMR spectra were collected with 1H decoupling, 1.3 s acquisition time, 1
s relaxation delay, 512-1024, transients and 90° pulse width of 10.2 μs. The data was zero-
filled 131 k points, exponentially weighted with a line broadening of 1.5 Hz and Fourier
transformed. The 1H[13C] gHMBC spectra were obtained with 90° pulse widths for 1H and
13C of 9.0 and 8.1 μs, respectively, a 1.0 s relaxation delay, Δ = 1.8 ms (based on 1JCH =
140 Hz and nJCH = 8 Hz) and a 0.128 s acquisition time. Eight transients were averaged
for each of 512 increments during t1. The spectral width in the f1 and f2 dimensions are
23.8k and 4k Hz, respectively. Data were zero-filled to a 2k × 4k matrix, and weighted with a Gaussian function before Fourier transformation.
Synthesis of 3-((2-Hydroxyethyl)(2-methacrylamidoethyl)ammonio)-propanoate
(CBMAA-2T).
The amounts of 6.26 g (0.036 mol) of N-(2-((2-
hydroxyethyl)amino)ethyl)methacrylamide and 4.36 g (0.044 mol) of ethyl acrylate were
dissolved in 50 mL of methanol. The reaction was carried at 40 °C under nitrogen
protection for 48 h. Then the solvent was removed by the rotary evaporator. Without any
further purification, the hydrolysis of the ester group on the carboxylate was carried out by dissolving the product in a solution containing an equal amount of sodium hydroxide in moles. Water and released ethanol were removed via rotary evaporator, and remaining water was further removed by using a freeze-dryer. The overall yield was 47.5%. The pure
1 product was analyzed by NMR. H NMR (300 MHz, D2O) (Figure 4) δ 5.76 (s, 1H), 5.51
(s, 1H), 3.75 (t, J = 12.3 Hz, 2H), 3.46 (t, J = 13.5 Hz, 2H), 2.93 (t, J = 15.3 Hz, 2H),
19 13 2.82−2.77 (m, 4H), 2.43(t, J = 15.3 Hz, 2H), 1.98 (s, 3H). C NMR (75 MHz, D2O) (Figure
5) δ 181.17, 171.65, 138.96, 121.16, 58.60, 54.64, 51.79, 50.61, 36.79, 34.10, 17.68
Figure 4. 1H NMR spectrum of CBMAA-2T
Figure 5. 13C NMR spectrum of CBMAA-2T
Synthesis of 2-((3-Acrylamidopropyl)dimethylammonio)acetate (CBAA-1).
The amounts of 15 g (0.096 mol) of N,N-Dimethylaminopropylacrylamide and
19.24 g (0.115 mol) of ethyl bromoacetate were reacted in 150 mL of acetonitrile at 60 °C with nitrogen gas for 24 h. Most of the solvent was removed by rotary evaporator. Then the raw product was precipitated against ethyl ether. After removing ethyl ether, the processed product was dissolved in water, and went through an Amberlite IRA-400 anion
20 exchange column. The final product was obtained as a white powder after removing water
1 by freeze-drying, and was analyzed by NMR. H NMR (300 MHz, D2O) (Figure 6) δ
6.38−6.22 (m, 2H), 5.86−5.82 (m, 1H), 3.92 (s, 2H), 3.68−3.62 (m, 2H), 3.46−3.42 (m,
13 2H), 3.28 (s, 6H), 2.14−2.04 (m, 2H). C NMR (75 MHz, D2O) (Figure 7) δ 168.86, 168.62,
129.65, 127.44, 63.54, 61.94, 51.23, 35.98, 22.27.
Figure 6. 1H NMR spectrum of CBAA-1
Figure 7. 13C NMR spectrum of CBAA-1
21 2.2.3 Synthesis of Polymer Brushes via Surface-Initiated Atom Transfer Radical
Polymerization (SI-ATRP)
Polymer brushes were synthesized on gold-coated SPR substrates via SI-ATRP
method.[70] The ATRP initiator, ω-mercaptoundecyl bromoisobutyrate, was synthesized by following the procedure reported previously.[71] Gold-coated SPR sensor chips were generously provided by Dr. Shaoyi Jiang at the University of Washington Seattle. Each substrate before use was cleaned by using an ultrasonic cleaner to remove undesired substance in each of the following solvents: acetone, isopropanol, and water. In each solvent, substrates were sonicated twice for 5 min. After being treated with UV ozone for
20 min, substrates were rinsed with DI water and ethanol and then dried with air. The self- assembled monolayer (SAM) of initiator was prepared by submerging cleaned gold substrates in 1 mM ATRP initiator in ethanol 24 h at room temperature. The initiator-
immobilized substrates were then rinsed with ethanol and dried with air.
An amount of 0.6 g of CBTAA-1 (2.6 mmol) was dissolved in 6 mL of the mixture of DI water and methanol (2:8, V/V) in a glass test tube. The above solution was transferred to another test tube containing 44.77 mg of 2,2′-bipyridine (0.287 mmol), 18.69 mg of copper(I) bromide (0.13 mmol), and 2.91 mg of copper(II) bromide (0.013 mmol). The solution of polymerization was finally transferred to a third test tube containing the gold substrate with immobilized SAM of the initiator. All three tubes sealed with rubber septum stopper were purged with nitrogen gas before transferring. After reaction of 48 h, Gold- coated SPR sensor chips was taken out and rinsed with ethanol and water. The gold-coated
SPR sensor chips then were submerged in PBS solution for at least four hours before use.
22 2.2.4 Synthesis of PCBMAA-1T, PCBMAA-2T, and PCBAA-1
All polymers were synthesized by free radical polymerization. In a typical
procedure, an aqueous solution of CBAA-1 (0.5 g, 2.33 mmol) in DI water was prepared and purged with nitrogen gas for 10 min. Then VA- 044 (7.5 mg, 0.02 mmol) was added
to the aqueous solution and was purged with nitrogen gas for another 10 min. The
polymerization was reacted at temperature 50 °C for 48 h. After 48 h, the polymer solution was transferred to a dialysis membrane tube with 10 000 Da molecular weight cutoff. DI water was changed twice each day. The solvent of the polymer solution was removed by a freeze-dryer after the dialysis. PCBMAA-1T and PCBMAA-2T were synthesized using the same procedure above.
2.2.5 Molecular Weight Measurement of PCBMAA-1T, PCBMAA-2T, and PCBAA-1
The relative molecular weight of PCBMAA-1T, PCBMAA-2T, and PCBAA-1
were measured by the aqueous gel permeation chromatography system containing Water
1515 isocratic HPLC pump (Waters, Milford, MA), Water 2414 refractive index detector
and PL aquagel-OH MIXED-M 8 μM 300 × 7.5 mm column (Agilent Techologies, Santa
Clara, CA). The calibration curve was established by running PEO/PEG standard (Sigma,
St. Louis, MO) in the aqueous solution of 0.01 M NaH2PO4 and 0.3 M NaNO3 at a flow
rate of 1 mL/min at 30 °C. The samples were measured in the same manner as the analytic
standard PEO/PEG. The molecular weights (MW) of PCBMAA-1T, PCBMAA-2T, and
PCBAA-1 are 41881, 43398, and 94691 Da, respectively.
23 2.2.6 Protein Adsorption Study
The protein adsorption of PCBMAA-1T polymer brush was evaluated with a
custom-built four-channel SPR sensor, which measures the change in the resonant
wavelength at a fixed angle[72]. The SPR chip of PCBMAA-1T polymer brush was rinsed with ethanol and water and dried with air before the protein adsorption measurement. At the beginning of the measurement, a baseline was obtained by running PBS solution at a flow rate of 0.05 mL/min for 20 min. Each following protein solution flowed in an independent channel through the SPR sensor for 10 min: 1 mg/mL fibrinogen solution, undiluted human blood plasma, and undiluted human serum. PBS solution then was used to wash off any unbound proteins. Each SPR spectrum was recorded and used to calculate the absorbed amount of each kind of protein.
2.2.7 Monomer and Polymer Titrations of CBMAA-1T, CBMAA-2T, and CBAA-1
The buffering range of monomers and polymers were determined by titration
method. The initial concentration for all samples was 0.1 M. The initial concentration of
polymers was calculated according to the amount of the repeating unit. At first, the pH
value of each sample solution was adjusted to 1 by adding 1 M hydrochloric acid solution.
Then 50 μL of 0.25 M sodium hydroxide solution was added each time. The change in pH
of the sample solution was measured by using an OAKTON ion 510 series pH meter. In
the whole titration process, the sample solution was stirred to maintain a homogeneous
solution. The equivalence point and pKa were calculated by CurTiPot software, which is
developed by Prof. Ivano Gebhardt Rolf Gutz.
24 2.3 Results and discussion
2-((2-Hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate (CBMAA-1T) and
3-((2-hydroxyethyl)(2-methacrylamidoethyl)ammonio)propanoate (CBMAA-2T) were designed to reach the goal of integrated antifouling materials with both the buffering capability and the switchability between the cationic form and the zwitterionic form. −1 and −2 refer as to one and two methylene spacers between amine and carboxylate groups, respectively, and T refers as to the tertiary amine group as the cation. The synthesis routes of CBMAA-1Tand CBMAA-2T are shown in Scheme 1. The chemical structure and purity of both monomers were confirmed by NMR.
Scheme 1. Synthetic routes of CBMAA-1T and CBMAA-2T. Reaction condition: (i)
ethanol, water, NaOH; (ii) methanol, ethyl bromoacetate, N,Ndiisopropylethylamine
(DIPEA); (iii) methanol, ethyl acrylate; (iv) NaOH.
25 It was expected that CBMAA-1T would form six membered lactone-ring under suitable conditions, since the lactone ring formation was observed for the quaternary ammonium-based zwitterionic monomers with similar structures using acids as catalysts.[13, 56, 61] Compared to the quaternary ammonium-based monomers, CBMAA-
1T can form the lactone ring under various conditions. In the second step of CBMAA-1T
synthesis, the lactone ring was formed via a transesterification reaction where the ethyl group on the carboxylate was replaced by the hydroxyethyl group that attached to the tertiary amine group. Surprisingly, CBMAA-1T at its zwitterionic form can form the lactone ring in acetonitrile without using acid or base as the catalyst under room temperature; however, the reaction rate is slow. The 1H NMR overlap plot shown in Figure
8A was collected at different time points to provide an insight of the ring formation process of CBMAA-1T. The signature peaks of red circle-marked protons and vinylic protons were selected to give a clear view of the conversion. The conversion of the lactone form was calculated based on the ratio of the integral value of vinylic protons in each form. It was found that 85% of CBMAA-1T was converted to its ring form in acetonitrile after 3 months
(Figure 8B). In acetic acid (Figure 8C), the ring formation of CBMAA-1T is slower compared to its quaternary ammonium-based analogue, 2-((2-hydroxyethyl) (2- methacrylamidoethyl) (methyl)ammonio)acetate (CBMAA-1), which was reported in our previous study.[13, 56] About 30% of CBMAA-1T was converted to ring form within 10 h. As shown in Figure 8D, the kinetics of ring opening of CBMAA-1T was recorded.
CBMAA-1T was fully converted from its ring form to zwitterionic form after 192 h in
D2O. Surprisingly, the rate of the ring opening of CBMAA-1T is much slower than
CBMAA-1, which achieved 20% in 6 h.[56] The slower ring-open rate might be caused by
26 the lower ring tension of CBMAA-1T, since CBMAA-1T has three substitution groups on the amine, which leads to less steric hindrance and less ring tension. The result indicates that the lactone ring of tertiary amine is more stable compared to its quaternary ammonium- based analogues. The more stable lactone ring is preferred for antimicrobial applications, since it can maintain its antimicrobial state for a longer time to kill microorganisms more efficiently.
Figure 8. 1H NMR overlay plot of ring formation of CBMAA-1T in D-acetonitrile (A).
Conversion kinetics of ring formation of CBMAA-1T in D-acetonitrile (B) and D-acetic acid (C). Conversion kinetics of ring opening of CBMAA-1T in D2O (D).
27
Figure 9. 2D NMR analysis of the compound of CBMAA-1T
To further confirm the ring structure, CBMAA-1T was studied by heteronuclear multiple-bond correlation (gHMBC) 2D-NMR, which provides correlations of two- and three-bond between 1H and 13C. In Figure 9, the methylene group marked with a red circle
has the most different chemical environment when the chemical structure changes between ring form and ring-opening form. The two red dots marked with black circles at 4.4 ppm of F2 chemical shift provide the evidence that two hydrogens of red-circle-marked carbon have correlations to two carbons marked with blue circles. 1D-NMR analysis with integral value of peaks is also given to accompany with 2D NMR to provide a better view. Vinylic protons have two significant peaks located at the range of 5−6 ppm.
28 One of the important properties of zwitterionic materials is their capability to resist
the adsorption of biomacromolecules. To evaluate intrinsic antifouling properties of the
material, a well-packed surface is needed. In this work, PCBMAA-1T polymer brushes
were synthesized on gold-coated SPR chips using the SI-ATRP method. The thickness of
the gold chip coated with CBMAA-1T polymer brush was measured by an ellipsometer,
and the thickness of the polymer brush film is 13 ± 2.3 nm. The antifouling property of
PCBMAA-1T polymer brush was studied using 1 mg/mL fibrinogen, 100% plasma, and
100% serum by SPR sensor. Figure 10 shows PCBMAA-1T polymer brush is highly resistant to the protein adsorption. The amount of the adsorbed proteins on PCBMAA-1T is less than 0.8 ng/cm2 for fibrinogen and 0.3 ng/cm2 (the detection limit of SPR sensor)
for undiluted blood plasma and serum, which are two most challenging protein solutions
in nature. It should be noted that only a limited number of materials could resist protein
adsorption from blood serum and plasma. A previous study unveiled that blood-contacting
materials, which have less than 5 ng/cm2 fibrinogen adsorbed from the blood, are less likely to trigger the blood coagulation.[73] PCBMAA-1T has three different charged states, depending on the pH of the environment. At low pH (below the pKa of carboxylate), both carboxylate and tertiary amine are protonated and it leads to the positively charged
PCBMAA-1T. At high pH (above the pKa of tertiary amine), tertiary amine and
carboxylate are deprotonated and the side chains of PCBMAA-1T are negatively charged.
At the intermediate pH range, carboxylate is deprotonated and amine is protonated.
PCBMAA-1T carries balanced charges, which is one of the critical properties for materials to prevent biofouling in the complex biological systems.[74] In this study, the protein adsorption study was conducted under the neutral conditions in PBS at pH 7.4. The data
29 indicates that tertiary amine cation does not compromise the antifouling property as long
as it is at the charged state. A previous study has demonstrated the idea of that zwitterionic
PCB containing the tertiary amine can switch between antifouling state and fouling state under different pH conditions.[69] It is expected that the PCBMAA-1T surface (Figure 11) will switch to its fouling state under either low pH or high pH conditions. Although of quaternary ammonium-based PCBs show the similar antifouling properties as tertiary amine-based PCBs at low and neutral pH conditions, only tertiary amine-based PCBs can respond under high pH conditions.
Figure 10. Protein adsorption test on PCBTAA-1T polymer brush surface with 1 mg/mL fibrinogen solution, undiluted human plasma and serum.
30
Figure 11. Switchable charged state of CBMAA-1T in different pH condition.
Since the pKa of carboxylate and amine groups determines the range of the
antifouling state, the antimicrobial state and the buffering capability, the titration study was conducted to measure their pKa values. Since the pKa of the carboxylate group is determined by its distance to the cation and the type of the cation, CBMAA-2T and CBAA-
1 monomers and their polymers were used as the controls. CBMAA-2T contains the same tertiary amine structure as CBMAA-1T, but it has a two-methylene spacer between carboxylate and amine groups. CBAA-1 has the same spacer between carboxylate and amine as CBMAA-1T, but its cation is the permanently charged quaternary ammonium.
Figure 6 shows the titration curves of CBMAA-1T and PCBMAA-1T. The pKa values of all monomers and polymers are shown in Table 1. CBMAA-2T has the highest pKa value
(3.01) of carboxylate and CBAA-1 has the lowest pKa value (less than 2.00). As we expected, the tertiary amine cation leads to a weaker carboxylate compared to quaternary ammonium cations. The pKa values of amine and carboxylate of the polymers are following the same trend as the corresponding monomer. The data indicates that the length
31 of spacer has a greater impact on the pKa of carboxylate than the type of the cation. The
pKa values of CBMAA-1T and CBMAA-2T are 7.78 and 8.12, respectively. It is surprising that the length of the spacer between carboxylate and amine does not have much influence on the basicity of the amine groups. We also found that both the acidity of carboxylate and the basicity of amine increase in PCBMAA-1T and PCBMAA-2T compared to the corresponding monomer. This phenomenon might be caused by the relatively higher density of counterions in the polymer form, which induces the charge separation. In general, tertiary amine-based carboxybetaine monomers and polymers have much broader buffering windows than quaternary ammonium based analogues.
Table 1. pKa of Carboxylate and Amine of CBAA-1, CBMAA-1T and CBMAA-2T
Monomers and Polymers.
Figure 12. pH titrations of CBAA-1, CBMAA-1T, and CBMAA-2T monomers (A) and polymers (B) from pH 1 to 13.
32 2.4 Conclusion
An integrated zwitterionic polymeric material, PCBMAA-1T, was synthesized to
carry desired properties (antifouling, switchability, and buffering capability) for
biomedical applications. In PCBMAA-1T, the quaternary ammonium cation was replaced by the tertiary amine, which provides the material with the buffering capability at neutral conditions. Titration study showed that PCBMAA-1T could resist pH changes at both acidic (pH 1−3) and neutral/basic (pH 7−9) conditions. Antifouling study data demonstrate that PCBMAA-1T is highly resistant to protein adsorption from undiluted human plasma and serum and PCBMA-1T is qualified as the ultralow fouling material. Through this study, a better understanding of the structure−property relationship of zwitterionic materials was obtained. It is found that the tertiary amine leads to better lactone ring stability in switchable PCB materials and a weaker carboxylate, which has the buffering capability under less acidic conditions. To the best of our knowledge, such an all-in-one material has not been reported. We believe this material will be very useful for tissue engineering, chronic wound healing, and medical device coatings that prefer an environment with stable pH.
33
34
CHAPTER III
IONIC CONDUCTIVITY OF POLYELECTROLYTE HYDROGELS
3.1 Introduction
Polyelectrolytes, composed of repeating electrolyte units, combine the properties
of both electrolytes and polymers. Depending on the type of the electrolyte group,
polyelectrolytes can be classified into three groups: polycations, polyanions and
polyampholytes (also called zwitterionic polyelectrolytes) that carry both cationic and
anionic repeating groups. Due to the unique property of interacting with salts, solvents and macromolecules, polyelectrolyte hydrogels have drawn enormous attention and have been used as critical components for a range of applications, including: electronic devices,[75,
76] tissue engineering scaffolding,[77] coatings,[78] batteries,[79] fuel cells,[80] water purification[81] and drug delivery.[82-84] For example, Tarek R. F. and Paula T. H. reported a fuel cell using a poly(ethylene oxide)/poly(acrylic acid) (PEO/PAA) composite membrane, which showed a maximum power density close to some commercial products.
In a fuel cell, polyelectrolyte (PE) membrane sandwiched between bipolar plates plays an important role which requires high ionic conductivity and the ability to prevent the crossover of fuel-cell gases for good performance. In their works, many composite membrane systems fabricated by layer-by-layer deposition (LBL) of different PE couples on the supporting substrate had been reported such as LPEI/PSS, PDME/PAMPS, and
35 PEO/PAA (LPEI: linear polyethyleneimine; PSS: poly(styrene sulfonic acid, sodium salt);
PDME: poly(dimethylamine-co-epichlorohydrin); PAMPS: poly(2-acrylamido-2-methyl-
1-propane sulfonic acid)). Although the hydrophilicity of composite membrane is essential to the conductivity, the stability of the LBL film and the size of the polymer molecule can also affect the performance of the fuel cell. Since different polyelectrolyte couples show different physical and chemical properties, finding the right PE couple is vital to optimize the performance.[85]
Recently, zwitterionic polyelectrolytes, polycarboxybetaine,[13, 56, 59, 61]
polysulfobetaine[16, 61] and polyphosphobetaine[86, 87] have become very attractive for
a broad spectrum of biological applications due to their unique properties. Zwitterionic
polyelectrolytes have the strongest inter- and intra-molecular attractions at low ionic strength conditions due to the shorter distance between cations and anions. The strong hydration property has drawn special interest for polyampholytes for biomedical applications. Recent studies also discovered that zwitterionic polyelectrolytes can strongly bind water which leads to strong hydration,[88] biocompatibility,[53] antifouling properties[13, 56, 61, 89] and compatibility with biomacromolecules.[90] Among all properties of polyelectrolytes, ionic conductivity is an important parameter for bioelectronics, neural tissue engineering, electrochemical sensing, and so on because ions function as the charge carriers and the ionic mobility in polyelectrolyte directly affects the performance and sensitivity of the devices. A previous study discovered that small molecular zwitterions can significantly increase the conductivity of ionic liquids[2] and it leads to the question whether the immobilized zwitterionic functional groups will affect the ionic conductivity of an electrolyte solution. Additionally, in 2015, Peng, Xu, et al.
36 developed a graphene-based solid-state supercapacitor with zwitterionic sulfobetaine
hydrogel electrolyte, which showed a significant improvement in the electrochemical
performance. The abilities of gel electrolytes such as transporting ions, separating and
binding the electrodes are important for solid-state supercapacitors. This zwitterionic gel
electrolyte enhanced the capacitance, rate capacity, and durability of the supercapacitor
compared to the polyvinyl alcohol (PVA) gel electrolyte which is widely used in solid-
state supercapacitors. The improvement was attributed to higher ionic conductivity which
is due to the synergetic effect of high water retention ability and the ion migration
channel.[91] Although some cationic or anionic polyelectrolytes have been studied for their ion transport properties, the effect of zwitterionic side chains of the polyelectrolytes on ionic transport is still unclear. A deeper understanding of how the electrolyte groups of polyampholytes affect the conductivity of polyampholytes is critical to design new ionic conducting materials for biomedical, environmental and energy applications.
In addition to the polyelectrolytes, non-ionic poly(ethylene glycol) (PEG) was also
widely used as a matrix for nearly all applications that were previously mentioned. Because
PEG is considered to be non-inflammatory and non-immunogenic, it has been used in a variety of neural and bio-electrochemical applications, including neural regeneration scaffolds,[92] electrochemical biosensors[93] or coating for neural probes,[94] to function as the anti-biofouling moiety. Although PEG materials are assumed to not interfere with the ion transport that is critical in these applications, no systematic study has been done to support such an assumption.
The objective of this study is to understand how the functional electrolyte group of polyelectrolyte sidechains affect the ionic conductivity of polyelectrolytes. Cationic,
37 anionic and zwitterionic polyelectrolytes were studied in the form of the hydrogel to
observe the change in ionic conductivity in various salt solutions with different
concentrations. Three types of zwitterionic polyelectrolytes poly(carboxybetaine
acrylamide) (PCBAA), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(sulfobetaine methacrylate) (PSBMA) were studied since they represent a group of the most promising polyelectrolyte biomaterials. Poly(ethylene glycol) methyl ether methacrylate (PEGMA) was used as a reference material. Through this comprehensive and systematic study, we can gain a better understanding of the effects of electrolyte groups of polymers on the ion transport of various salt solutions.
3.2 Experimental section
3.2.1 Chemicals
2-methacryloyloxyethyl phosphorylcholine (MPC), sulfobetaine methacrylate
(SBMA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), 2-
(acryloyloxy)ethyl]trimethylammonium chloride (TMA), 2-acrylamido-2-methyl-1-
propanesulfonic acid sodium (AMPS), 2-aminoethyl methacrylate hydrochloride (AEMA),
2-(dimethylamino)ethyl methacrylate (DMAEMA); methacrylic acid (MAA), N,N'-
Methylenebisacrylamide (MBAA) 2-Hydroxy-4′-(2-hydroxyethoxy)-2- methylpropiophenone, tetraethylene glycol dimethacrylate, methyl bromoacetate and acetonitrile, sodium chloride (NaCl), magnesium chloride (MgCl2), magnesium sulfate
(MgSO4) and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). N-(3-dimethylaminopropyl)acrylamide was purchased from TCI
America (Portland, OR, USA). Carboxybetaine acrylamide or 2-((3-
38 acrylamidopropyl)dimethylammonio)acetate (CBAA) was synthesized using a previously
published method[95].
3.2.2 Synthesis of the hydrogel
All hydrogels except Zw-PSBMA-EG were synthesized with the same molar
concentration of monomer, the crosslinking agent (N, N′-methylenebis(acrylamide), and the photoinitiator (2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone).[96] For
Zw-PSBMA-EG, tetraethylene glycol dimethacrylate was used as the crosslinking agent.
The following is a brief description of the synthesis of hydrogels using CBAA as an example. 3 mmole of CBAA, 0.15 mmole of N, N′-methylenebis(acrylamide), and 10 mg of 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone were dissolved in 2 mL of ethanol/water mixture (v/v, 3/7). Two quartz slides were clipped on both sides of a PTFE mold. The PTFE mold has an interior volume of 5.9 cm × 1.2 cm × 0.33 cm and a small opening on the top for solution injection. The well-mixed pre-polymerized solution was injected with a syringe into the mold, and the opening on the top was sealed with parafilm.
The polymerization was carried out under a 365 nm UV lamp (UVL-28 EL series UV lamp,
UVP Inc.) for two hours. The obtained hydrogel was immersed in DI water that was changed every 12 h for three days to remove unreacted monomers. Then the hydrogel was punched into a cylindrical shape by a biopsy punch with an 8mm inner diameter.
3.2.3 Equilibrium water content assay
The hydrogel sample was first equilibrated in DI water for 72 h. Then, the wet weight of the hydrogel sample was measured after the removal of excess water. The dry weight of each hydrogel was recorded after the sample was freeze-dried for 48 h. The
39 equilibrium water contents of the hydrogels were calculated by (Wet weight – Dry weight)
/ Wet weight × 100%.
3.2.4 The volume ratio of the hydrogels assay
The thickness and diameter of the hydrogel samples were measured using an AOS
digimatic caliper (Mitutoyo, Taiwan). The volume ratio of the hydrogels was calculated by the following equation.
(%) = × 100 0 𝑉𝑉 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑉𝑉 where V is the volume of the tested hydrogel in various saline solutions and V0 is the volume of the tested hydrogel in DI water.
3.2.5 Ionic conductivity assay
Four different electrolytes (NaCl, MgCl2, MgSO4 and PBS) were selected for
ionic conductivity measurements. All hydrogel samples were equilibrated for 24 hours in
three different concentrations (2 mM, 10 mM, and 100 mM) of NaCl, MgCl2 and MgSO4
solutions. After measuring the thickness and diameter, the hydrogel samples were
sandwiched between two 316 stainless steel electrodes for electrochemical impedance
spectroscopy (EIS) measurements using a Gamry Reference 600 Plus potentiostat (Gamry,
USA). The EIS measurements with a frequency range of 0.1 Hz to 100 kHz were conducted under open-circuit conditions with an excitation voltage of 10 mV. The software ZView
(Scribner, USA) was used to fit the impedance data with the Randles equivalent circuit which contains an electrolyte resistance (Rs) in series with the parallel combination of a
40 constant phase element (CPE) and a charge transfer resistance (Rp). The obtained Rs values
were used to calculated the ionic conductivities of the hydrogel samples using the following equation,
= × 𝒍𝒍 𝝈𝝈 𝒔𝒔 𝑹𝑹 𝑨𝑨 where σ is ionic conductivity, A is the cross-sectional area of the sample, and l is
the thickness of the sample. The conductivities of the solutions were measured using a
VWR Digital Conductivity Meter (Radnor, PA, USA). All experiments were repeated three times.
3.3 Results and discussion
As we can see in figure 13, four types of chemically crosslinked hydrogels,
zwitterionic hydrogels (Zw-PCBAA, Zw-PMPC, Zw-PSBMA and Zw-PSBMA-EG), cationic hydrogels (Cat-PTMA and Cat-PAEMA and Cat-PDMAEMA), anionic hydrogels
(An-PAMPS and An-PMAA), and non-ionic hydrogel (PEGMA) were synthesized by the copolymerization of the corresponding monomer and the crosslinker. MBAA was used as the crosslinker for all hydrogels except Zw-PSBMA-EG hydrogel for which a different crosslink agent, tetraethylene glycol dimethacrylate (TEGDMA) was used, and the molar ratio of the crosslinker to the monomer was kept constant at 5%. All of the hydrogels are transparent except for Zw-PSBMA hydrogel. Zw-PSBMA hydrogel was an opaque white color due to the formation of aggregates and clusters which can inhibit the movement of dipoles.[97]
41
Figure 13. The chemical structure of monomers and crosslinkers: (A) 2-((3-
acrylamidopropyl) dimethylammonio)acetate (CBAA), (B) 2-methacryloyloxyethyl phosphorylcholine (MPC), (C) [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide (SBMA), (D) poly(ethylene glycol) methyl ether methacrylate (PEGMA), (E) [2-(acryloyloxy)ethyl]trimethylammonium chloride (TMA),
(F) 2-acrylamido-2-methyl-1-propanesulfonic acid sodium (AMPS), (G) 2-aminoethyl methacrylate hydrochloride (AEMA), (H) 2-(dimethylamino)ethyl methacrylate
42 (DMAEMA), (I) methacrylic acid (MAA), (J) N,N’-methylenebisacrylamide (MBAA) and
(K) tetraethylene glycol dimethacrylate (TEGDMA).
The high-water content is an important parameter for hydrogels, which enables similar chemical/physical properties with tissues and biological systems. Higher water content has also been found to increase the ionic conductivity of hydrogel and leads to high ion transfer rate[98]. In this study, the water contents of different hydrogels were measured and are shown in Table 2. Cationic and anionic hydrogels showed much higher water content (>93%) than zwitterionic hydrogels in DI water. For cationic or anionic crosslinked hydrogels, the stronger electrostatic repulsion between cationic or anionic side chains leads to a stretched state and larger pore size[99]. On the contrary, the mixed anionic and cationic groups of zwitterionic polymers have a stronger electrostatic attraction in DI water, leading to collapse polymer chains and smaller pores. Among zwitterionic hydrogels with the
MBAA crosslinker, the water content of Zw-PCBAA hydrogel was highest (91.5%), followed by the moderate Zw-PMPC hydrogel (88.7%) and the lowest Zw-PSBMA hydrogel (74.3%). Due to the same synthetic strategy and crosslinker, all three hydrogels possess similar crosslinking density. According to the reported literature[100], the order of hydrophilicity is PCBAA > PMPC > PSBMA. This indicates that the inherent hydrophilicity of constituent polymer mostly determines the water content of the hydrogel.
Because the water content is an important factor for hydrogel conductivity,[98] to increase the water content of PSBMA hydrogel, we synthesized Zw-PSBMA-EG hydrogel that used more hydrophilic TEGDMA as the crosslinker. The water content of Zw-PSBMA-EG increased to 84.1% from 74.3% by switching the crosslinker from MBAA to TEGDMA.
Table 2. The water content of hydrogels in deionized water.
43
As shown in Figure 14, the volume changes of hydrogels in different saline solutions were further evaluated and the volume of the hydrogel in each conduction was normalized to the volume of the same hydrogel in DI water. For zwitterionic hydrogels,
their volumes did not change significantly in low-concentration salt solutions (2 mM and
10 mM), except that the volume of the PSBMA-EG hydrogel increased with increasing saline concentration. The significant difference of volume change between Zw-PSBMA and Zw-PSBMA-EG hydrogels in in PBS and high ionic strength solutions (> 100 mM of
NaCl and MgCl2) might be caused by the higher hydrophilicity and the longer chain length of TEGDMA crosslinker compared to MBAA. Interestingly, Zw-PSBMA-EG hydrogels shrink in MgSO4 solutions; however, the mechanism of this phenomenon is unclear.
Zwitterionic hydrogels are neutral in DI water and keep the balanced charges with the
addition of salts. Salts shield the charges, reduce the electrostatic attraction, and lead to the
swelling of zwitterionic hydrogels. On the contrary, except for PDMAEMA hydrogels,
cationic hydrogels and anionic hydrogels shrank significantly with increasing salt
concentration (Figure 14B, 14D and 14F). This can be attributed to the reduction of the
electrostatic repulsion between the charged groups of the ionic polymer with the addition
of salts. The swelling of PDMAEDMA in PBS might be caused by high binding affinity
of multivalent phosphate ions to the onto the polyelectrolyte chains. High MgCl2, solutions
may also lead to an adsorption of chloride ions onto the polyelectrolyte chains and cause
the reswelling.
44
Figure 14. The volume ratio of neutral and ionic hydrogels in various solutions: Neutral
hydrogels in (A) deionized water, NaCl solution and PBS, (C) MgSO4 solution and (E)
MgCl2 solution; ionic hydrogels in (B) deionized water, NaCl solution and PBS, (D)
MgSO4 solution and (F) MgCl2 solution. (Standard deviation is less than 5%)
The ionic conductivity of all hydrogel samples was measured by impedance spectroscopy using a potentiostat (Figure 15 and 16). In deionized water, all ionic
hydrogels showed much higher conductivity than DI water, and non-ionic and zwitterionic hydrogels have lower conductivity than the cationic and anionic hydrogels. Cationic and anionic hydrogels have a high concentration of counterions and the mobile counterions function as charge carriers and lead to high conductivity. Among cationic hydrogels, Cat-
PDMAEMA has the lowest conductivity since it has no added counterion. However, Cat-
PDMAEMA becomes partially ionized in DI water and generates hydroxide counterions that serve as the charge carrier. Among zwitterionic hydrogels, the ionic conductivity of
45 Zw-PCBAA remains the highest one, Zw-PMPC exhibits a lower ionic conductivity than
Zw-PSBMA, which is different from the behavior in other tested electrolyte solutions. This phenomenon may be caused by the different mobility of the polymer chains, which is still under investigation. Zwitterionic hydrogels also have much higher conductivity than the non-ionic PEGMA hydrogel. An increase in water content of hydrogels usually increases molecular mobility, and the water content of hydrogel is affected by many factors such as crosslinking density and hydrophilicity[101]. However, although the water content of Zw-
PSBMA hydrogel was lower than that of PEGMA, the ionic conductivity of Zw-PSBMA
was statistically significant higher than that of PEGMA. Our previous study also observed
that poly(carboxybetaine thiophene) hydrogel has much higher ionic conductivity than
reported PEG/poly(thiophene) hydrogels.[59] This phenomenon is likely caused by the
highly polar side groups in zwitterionic polymers that promote fast ion dissociation and
transportation.[102]
46
Figure 15. The ionic conductivity of neutral hydrogels in (A) deionized water and 2 mM electrolyte solutions, (B) 10 mM electrolyte solutions and (C) 100 mM electrolyte solutions.
*P < 0.05.
47 For zwitterionic hydrogels (Figure 15), Zw-PCBAA and Zw-PMPC showed
higher ionic conductivity than Zw-PSBMA in various electrolyte solutions (except in
deionized water and 2 mM NaCl solution). To increase the water content of Zw-PSBMA hydrogel, more water-soluble TEGDMA was used to replace MBAA crosslinker. Due to the different cross-linker, Zw-PSBMA-EG hydrogel was much more elastic and has a higher water content (84%) than Zw-PSBMA. As shown in Figure 15A, Zw-PSBMA-EG hydrogel shows similar ionic conductivity to Zw-PCBAA at 2 mM salt solutions. As the ionic strength increased, the ionic conductivity of Zw-PSBMA-EG in NaCl and MgCl2
solutions became higher than other tested zwitterionic hydrogels. In the 100 mM MgCl2
solution, the ionic conductivity of Zw-PSBMA-EG was 33% higher than that of Zw-
PCBAA. The increase in electrolyte concentration also significantly increased the size of
TEGDMA-crosslinked Zw-PSBMA-EG hydrogel but not the size of MBAA-crosslinked
Zw-PSBMA hydrogels in NaCl and MgCl2 solutions. As a result, the increased water
content and mobility of polymer chain led to enhanced ionic conductivity. Additionally,
the appearance of Zw-PSBMA-EG hydrogel changed gradually from semi-transparent to
transparent with increasing ionic strength, which can be attributed to the anti-
polyelectrolyte effect of the zwitterionic polymer. However, the size of Zw-PSBMA-EG
hydrogel remained the same in all tested MgSO4 solutions (Figure 14C), therefore, the ionic conductivity of Zw-PSBMA-EG is similar to that of Zw-PCBAA and Zw-PMPC. It is still unclear why the swelling behavior of Zw-PSBMA-EG hydrogel is more responsive to
NaCl and MgCl2 solutions than the MgSO4 solution. According to the Hofmeister series,
- - - the order of the salting-in effect for different anions is as follows: SCN > I > ClO4 >
- - - - - 2- NO3 > Br > Cl > F > IO3 > ½ SO4 , and the anions have a much stronger influence on
48 the solubility of macromolecules than cations.[103, 104] In this case, we can consider the polyelectrolytes as the macromolecules.
Figure 16. The ionic conductivity of ionic hydrogel in (A) deionized water and 2 mM electrolyte solutions, (B) 10 mM electrolyte solutions and (C) 100 mM electrolyte solutions.
49 As shown in Figure 15 and 16, cationic hydrogels (Cat-PTMA and Cat-PAEMA)
showed the highest ionic conductivity among all hydrogels in the tested solutions, followed by anionic An-PAMPS hydrogel. In addition to the cations and anions of electrolytes, mobile counterions of the ionic polymers also function as the charge carrier, and electrolytes and polymer counterions together contribute to higher ionic conductivity in all cationic and anionic hydrogels. Although Cat-PTMA showed higher ionic conductivity than An-PAMPS in all electrolyte solutions, the ionic conductivity of Cat-PTMA and An-
PAMPS in DI water is at the comparable level. In DI water, the lower ionic conductivity of Cat-PTMA than Cat-PAEMA can be caused by the stronger interaction of Cl- with the
quaternary amine of Cat-PTMA than with the primary amine of Cat-PAEMA. Compared
to An-PAMPS, the higher conductivity of Cat-PTMA and Cat-PAEMA hydrogels in NaCl solution can be caused by the higher ion mobility of chloride ion (7.91 x 10−8 m2 V−1 s−1)
[105]than that of sodium ion (5.19 × 10−8 m2 V−1 s−1)[106]. Moreover, An-PMAA⋅ showed⋅
a relatively lower ionic conductivity than the⋅ other⋅ ionic hydrogels, due to the lower
ionization capability of the carboxylic group than sulfonate group. However, the increased size of An-PMAA hydrogel in PBS enhanced the ionic conductivity to the point that it is
almost equal to the ionic conductivity of An-PAMPS. It had been reported that the
dimension of An-PMAA hydrogel significantly increased when the solution changed from
acidic to neutral conditions that cause the deprotonation of carboxylate and increase the
solubility of the PMAA.
In low electrolyte solutions, cationic and anionic hydrogels show higher conductivity because of the high counterion concentration in the gel. The conductivity of the hydrogel is controlled by two parameters: the mobility and concentration of ions. In
50 low salt solutions, the concentration of total ions plays a dominant role in conductivity. In
the high salt solutions, the fraction of counterions to the total ions is significantly reduced, so the mobility of the ions become the dominant parameter. To verify this hypothesis, the high salt solutions were further used to evaluate their ionic conductivity. Figure 17 shows the ionic conductivity of all hydrogels in high MgCl2 solutions. In 300 mM MgCl2 solution,
Cat-PAEMA and Cat-PTMA hydrogels still possessed higher ionic conductivity than other
hydrogels. For example, the ionic conductivity of Cat-PAEMA hydrogel was about 50% higher than that of Zw-PCBAA hydrogel, and about 15% higher than that of Zw-PSBMA-
EG. In 1 M MgCl2 solution, Zw-PSBMA-EG hydrogel surpassed that of cationic/anionic hydrogels that contain counterions and had the highest ionic conductivity, which is about
10% higher than Cat-PAEMA. Furthermore, the ionic conductivity difference between
Cat-PAEMA and other hydrogels became smaller; and the ionic conductivity of Cat-
PAEMA hydrogel was only 12% higher than that of Zw-PCBAA hydrogel. In 3 M MgCl2,
the ionic conductivity of Zw-SBMA-EG is about 13% higher than that of Cat-PDMAEMA.
Zw-PCBAA and Cat-PDMAEMA showed comparable ionic conductivity. In the high
MgCl2 solution (i.e., 3 M), the fraction of counterions in cationic and anionic hydrogels
among total mobile ions of the system become lower and the stronger interactions between mobile ions and cationic/anionic polymers than zwitterionic polymers decreases the ionic conductivity. Additionally, the increase in ionic conductivity of An-PMAA was small in high electrolyte solutions, which can be attributed to the two reasons. Firstly, the unionized carboxylic groups form hydrogen bonds, but this does not have a strong influence on low electrolyte solutions. It was reported that the number of hydrogen-bond linkages increases with increasing ionic strength due to the decreased ionization level of the carboxylic
51 group[107]. Second, ionized carboxylate groups electrostatically repel each other in low
electrolyte solutions. However, as the electrolyte concentration increases, cations can
shield this electrostatic repulsion[108]. These two mechanisms can cause a denser hydrogel network in An-PMAA and decrease the growth of ionic conductivity.
52
Figure 17. The ionic conductivity of neutral and ionic hydrogels in (A) 300 mM MgCl2 solution, (B) 1 M MgCl2 solution and (C) 3 M MgCl2 solution. *,**,***P < 0.05.
53 3.4 Conclusions
In this work, a series of polyelectrolyte hydrogels, including zwitterionic, cationic, and anionic hydrogels, were synthesized. Owing to the electrostatic repulsion of the ionic functional groups on the lateral chain, the ionic hydrogels show higher water content than zwitterionic hydrogels in DI water. Interestingly, the volume of the ionic hydrogels shrank and that of zwitterionic hydrogels swelled in saline solutions. Although PEG has been used in many energy and bioelectronic applications, this study observed that PEG impairs the ion conductivity in aqueous systems. Under all tested conditions, zwitterionic hydrogels show much higher ionic conductivity than that of the PEGMA hydrogel. The results indicate that zwitterionic materials are more suitable for energy and bioelectronic applications. The presence of counterions in cationic/anionic hydrogels leads to higher ionic conductivity in low concentration electrolyte solutions than that in zwitterionic hydrogels. However, stronger interaction between mobile ions and cationic/anionic hydrogels also compromises ionic conductivity. The ion conductivity of Zw-PSBMA-EG hydrogel surpasses cationic/anionic hydrogels in high concentration electrolyte solutions
(≥1 M MgCl2). The present work contributes to a better understanding of the influence of electrolyte groups of polymers on the ionic conductivity of polyelectrolyte hydrogels. Our results indicate the zwitterionic polyelectrolytes are more favorable for ion transportation.
54 License of re-use for Chapter IV
Author reusing their own work published by the Royal Society of Chemistry.
You do not need to request permission to reuse your own figures, diagrams, etc, that were originally published in a Royal Society of Chemistry publication. However, permission should be requested for use of the whole article or chapter except if reusing it in a thesis. If you are including an article or book chapter published by us in your thesis please ensure that your co-authors are aware of this. Reuse of material that was published originally by the Royal Society of Chemistry must be accompanied by the appropriate acknowledgement of the publication. The form of the acknowledgement is dependent on the journal in which it was published originally, as detailed in
'Acknowledgements'.
55
CHAPTER IV
ELECTROACTIVE POLY(SULFOBETAINE-3,4-ETHYLENEDIOXYTHIOPHENE)
(PSBEDOT) WITH CONTROLLABLE ANTIFOULING AND ANTIMICROBIAL
PROPERTIES
4.1 Introduction
Conjugated polymers (CPs) hold great promise for next-generation bioelectronics,[109, 110] because of their good compatibility with biological systems, design flexibility, ease of fabrication and relatively low costs. Previous studies found that
CPs could improve the communication between electrochemical devices and biological systems initially;[111-113] however, CPs such as polyacetylene,[114] polyaniline
(PANi),[115] polypyrrole (PPy),[116] polythiophene (PTh) and poly(3,4-
thylenedioxythiophene) (PEDOT)[117] were not originally designed for complex
biological applications. When these polymers are used in biological systems, one major
challenge is to maintain a “clean” and biocompatible biotic–abiotic interface to minimize
foreign body reactions, reduce infections and prolong the service life of the device, while
maintaining the material’s conductivity, stability and functionalities. Conventional CPs
consist of hydrophobic or charged side chains. Biomolecules, mammalian cells and
bacteria tend to attach to hydrophobic or charged surfaces. The adsorption of biomolecules and attachment of unwanted cells will reduce the sensitivity or lead to the failure of
56 embedded devices.[118, 119] To increase the biocompatibility, PTh,[120] PANi[121] and
PPy[122] hydrogels have been developed to combine the electrical properties of CPs with
the properties of hydrogels.[123] To gain biocompatibility, CPs are blended or physically
crosslinked with biocompatible and non-conducting polymers. For example, polyethylene
glycol (PEG) was used to crosslink PANi for glucose sensing.[124] However, non-
conducting components compromise the electrochemical properties of the CPs.[125]
Furthermore, the non-conducting components of current conducting hydrogels are not
effective enough to prevent long-term biofouling and foreign body responses. Previous
studies discovered that zwitterionic polymers could effectively resist non-specific protein
adsorption and cell attachment.[13, 56, 61] In our previous study, an integrated
poly(carboxybetaine thiophene) (PCBTh) with both conducting and antifouling properties
was developed.[59] Due to the loosely packed polymer networks in the hydrogel, the
electron conductivity needs to be further improved to meet the requirements of applications
that demand high electron/current transport properties.
Herein, we have designed and synthesized a novel sulfobetaine-functionalized CP platform PSBEDOT, using poly(3,4-ethylenedioxythiophene) (PEDOT) as the conducting backbone due to its exceptional conductivity,[126] low oxidation potential,[127] relatively high chemical and thermal stability,[128] and optical properties.[129] SBEDOT monomers were polymerized on electrodes to form a densely packed film through electropolymerization in 100% aqueous solution. The PSBEDOT surfaces were designed to have electro-switchable antimicrobial/antifouling properties and excellent electrical conducting properties, to minimize infection, increase biocompatibility and improve the performance of bioelectronics. The conductivity, stability and antifouling properties
57 against both proteins and cells, and the antimicrobial properties of the PSBEDOT surface,
were systematically investigated during this work.
4.2 Experimental section
4.2.1 Chemicals and general instrumentation
3,4-Dimethoxythiophene was purchased from Matrix Scientific (Columbia, SC,
USA). (±)-3-chloro-1,2-propanediol, toluene, p-Toluenesulfonic acid monohydrate,
dimethylamine solution (40 wt. % in H2O), acetonitrile, 1,3-propanesultone, anhydrous
magnesium sulfate, anhydrous tetrahydrofuran(THF), chloroform, methanol,
dichloromethane, ethyl acetate, phosphate-buffered saline(PBS) and fluorescein diacetate
were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were used as
received without further purification. Bovine aorta endothelial cell (BAEC) and Mouse
NIH 3T3 fibroblast cell were purchased from American Type Culture Collection
(Manassas, MD, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased
from Life Technologies (Carlsbad, CA, USA). Water used in all experiments was purified
using a Millipore Milli-Q Direct 8 Ultrapure Water system (Billerica, MA, USA). Electro- polymerization and other electrochemical characterizations were performed on a Solartron
Modulab XM ECS test system or a Gamry Reference 600 potentiostat. XPS spectra were obtained from a PHI VersaProbe II Scanning XPS Microprobe. All NMR experiments were performed at 303.2 K unless stated otherwise and on Varian Mercury 300 MHz spectrometers.
58 4.2.2 Synthetic procedures
Chloromethyl-EDOT was first synthesized following a method published
previously.[130] EDOT-dimethylamine was synthesized as a versatile intermediate, which was used to synthesize zwitterionic EDOT derivatives bearing carboxybetaine or sulfobetaine side chains.
Synthesis of EDOT-dimethylamine (EDOT-DMA)
Chloromethyl-EDOT (3.8 g, 20 mmol) was added to a solution of dimethylamine
(40 wt. % in H2O) (22.5 mL, 200 mmol) and acetonitrile (22.5 mL). The mixture was sealed in a schlenk flask and heat at 80 oC for 2 days. Another 22.5 mL of dimethylamine (40 wt. %
o in H2O) was added after it cooled down. Then the solution was heated at 80 C for another
36 hours. After the solution cooled to room temperature, it was concentrated with a rotary evaporator, extracted with ether, dried with MgSO4. Product was purified with silica gel
column chromatography (MeOH/CH2Cl2/ethyl acetate, 1/10/10 (v/v/v)). Pure product was
1 obtained as a light yellowish liquid (Yield: 65 %). H NMR (300 MHz, CDCl3) (Figure 18)
δ 6.30-6.36 (m, 2H), 4.20-4.30 (m, 2H), 3.90- 3.97 (m, 1H), 2.41-2.64 (m, 2H), 2.31 (s,
13 6H). C NMR (75 MHz, CDCl3) (Figure 19) δ 141.84, 99.90, 99.55, 71.95, 67.52, 59.65
(one carbon not seen due to overlapping signal)
59
Figure 18. 1H spectrum of EDOT-DMA
Figure 19. 13C spectrum of EDOT-DMA
Synthesis of SBEDOT
1,3-Propanesultone (1.46 g, 12 mmol) was slowly added into a solution of EDOT- dimethylamine (2.0 g, 10 mmol) in 50 mL of anhydrous THF. The mixture was heated at
55 oC for 36 hours under a positive nitrogen flow. After filtration, washed with THF and vacuum dried, pure product was obtained as a white powder (Yield: 82%). 1H NMR (300
60 MHz, D2O) (Figure 20) δ 6.58-6.65 (m, 2H), 5.00 (m, 1H), 4.14-4.31 (m, 2H), 3.62-3.85
(m, 4H), 3.27 (s, 3H), 3.25 (s, 3H), 2.98 (t, 2H, J = 7.2 Hz), 2.22 (m, 2H). 13C NMR (Figure
21) (75 MHz, D2O) δ 139.94, 138.45, 101.66, 101.00, 67.80, 65.90, 63.69, 62.55, 51.97,
51.69, 47.16
Figure 20. 1H spectrum of SBEDOT
Figure 21. 13C spectrum of SBEDOT
61 4.2.3 Electropolymerization of SBEDOT
Electropolymerization was performed on a Solartron Modulab XM ECS test system or a Gamry Reference 600 potentiostat equipped with a three-electrode electrochemical set-up, using a Pt electrode as the counter electrode and an Hg/HgCl2 electrode (sat. KCl)
as the reference electrode. One of the great advantages of this SBEDOT monomer is it
could be directly polymerized in aqueous solution, which significantly facilitates its future applications in vivo. SBEDOT was polymerized on either ITO coated PET films or Gold coated SPR sensor chips (Figure 22), with cyclic voltammetry from -0.6 V to 1.3 V, or galvanostatic method at 0.1 mA/s, from an aqueous solution containing 60 mM monomer and 100 mM LiClO4 as electrolyte.
Figure 22. Optical images of PSBEDOT coating on ITO (left) and gold (right) substrates.
62 4.2.4 X-Ray photoelectron spectroscopy (XPS) study
XPS was also used to examine the composition profile of electropolymerized
PSBEDOT film, using a PHI VersaProbe II Scanning XPS Microprobe. All data processing was performed using the software provided with the instrument. The PSBEDOT samples were run for both the survey and the high-resolution spectra (Figure 23). The survey spectra of PEDOT sample was used for comparison. All data processing was performed using the software provided with the instrument. Peak areas, line shapes, and intensities of C 1s, O
1s, N 1s and S 2p high-resolution spectra were monitored. As shown in Figure S6, the atomic ratios were in agreement with molecular compositions. From the S 2p high resolution spectra of PSBEDOT, two types of S were observed with equivalent peak intensity, indicating its elemental and chemical composition was the same as expected.
Figure 23. The XPS profiles of PSBEDOT coating. Survey spectrum (left), high-resolution
spectrum of S 2p (right).
63 4.2.5 Electrochemical characterization of PSBEDOT
Electrochemical impedance spectroscopy (EIS) and Cyclic Voltammetry (CV)
were performed in PBS using a Gamry Reference 600 potentiostat in PBS buffer. Stability measurement of PSBEDOT film was carried out with CV (-0.3 V to 0.6 V). Although
PSBEDOT was hydrophilic, it showed excellent stability in aqueous solution, even after applying a potential sweep for over 500 cycles (Figure 24). The PSBEDOT films for measurements were coated on gold coated SPR sensor chips. For EIS, the frequencies were spaced from 10 kHz to 1 Hz with a low amplitude voltage (~10 mV). Before EIS experiment, samples were equilibrated in PBS buffer for 10 minutes. Potentiodynamic study of PSBEDOT film was recorded with CV, from -0.8 V to 1.0 V, at different scan rates of 10, 20, 50, 100, 200 mv/s.
64 Figure 24. Cyclic voltammograms of PSBEDOT film at different scan rate.
4.2.6 BAEC and NIH-3T3 cell adhesion study
Bovine aorta endothelial cell (BAEC) and NIH-3T3 were purchased from
American Type Culture Collection (Manassas, MD, USA). Cell attachment study was carried out following a similar procedure to that used in a previous work. PSBEDOT and
PEDOT was electro-deposited on ITO coated PET substrates, then equilibrated in DI-water for 24 hours and transferred to sterilized PBS. All samples were exposed under UV for half an hour before the cell adhesion experiment.
BAECs and NIH-3T3 were separately seeded on different substrates at 105 cells/mL with DMEM medium consisting of DMEM, 10% fetal bovine serum (FBS), and 1%
o penicillin–streptomycin and kept in an incubator with 5% CO2 at 37 C for 24 hours. After the incubation, medium was removed from the wells. After very gently rinsed with sterilized PBS, it was changed to the staining solution that prepared in sterilized PBS as follows. Fluorescein diacetate was dissolved at a concentration of 10 mg mL-1 in acetone, then 50 μL of the solution was diluted in 10 mL sterilized PBS and used for staining the cells. After incubated for 5 min with the staining solution, surface cell coverage and cell morphology was visualized and imaged with an Olympus IX81 fluorescence microscope
(Olympus, Japan) equipped with a FITC filter at 4× or 10× magnification (Figure 25).
65
Figure 25. BAECs adhesion test with PSBEDOT coated ITO-PET. (A) PSBEDOT coated
region, (B) Region across coating boundary and (C) uncoated region.
Table 3. Percentage of the attached cells on PSBEDOT surfaces relative to PEDOT coated
surfaces (n=3)
4.2.7 Protein adsorption study – Surface Plasmon Resonance
A custom-built four-channel SPR sensor was used to measure protein adsorption
on PSBEDOT surface. Firstly, PBS solution at a 50 μL min-1 flow rate was used to obtain
a baseline signal. 100% human blood plasma and 30% diluted human blood serum were
then injected into different channels for 10 minutes followed by a PBS wash to remove any loosely bound proteins. The amount of adsorbed proteins was calculated as the change in wavelength before and after protein injection.
FITC-labeled fibrinogen adsorption study - fluorescence microscopy
After equilibrated in PBS, the substrates were gently rinsed with DI-water and then transferred into a sterile 12-well plate. 4 mL of FITC-labelled fibrinogen (FITC-Fg)
66 solution (0.1 mg/mL) was added into each well. All samples were immersed in the solution for 30 minutes to allow protein adsorption on substrate surfaces. To remove loosely adsorbed proteins on sample surfaces, all samples were gently rinsed with PBS. Protein adsorption on each substrate surface was visualized with an Olympus IX81 fluorescent microscopy (Olympus, Japan) with 4x objective lens through FITC filter at a fixed exposure time for all samples, so the different protein adsorption will lead to different fluorescent intensity on images. ImageJ software was used to quantify the fluorescent intensity of each sample. The results are shown in Figure 26.
Figure 26. Protein (FITC-Fg) adsorption test on surface visualized under fluorescence
microscope at the same excitation light intensity and exposure time. (A) PSBEDOT coated surface, (B) PEDOT coated surface, (C) bare gold sensor chip surface.
4.2.8 Bacterial adhesion, antimicrobial and releasing study
The method for evaluating the antibacterial efficiency of polymer surfaces was
modified from a previously published method.[131] E. coli K12 was first cultured in
separate pure cultures overnight at 37 oC on Luria-Bertani (LB) agar plates. One colony
was used to inoculate 5 mL of LB medium (20 g/L). These initial cultures were incubated
67 at 37 oC with shaking at 200 rpm for 12 hours. This culture was then used to inoculate a
second culture in 25 mL of LB medium. When the second suspended culture reached an
optical density of 0.8 at 600 nm, bacteria were collected by centrifugation at 8,000 x g for
10 min at 4 oC. Cell pellets were washed three times with sterile PBS (pH 7.4) and
subsequently suspended in PBS to get a final concentration of 109 cells/mL.
Bacterial attachment study
Before the bacterial attachment study, PSBEDOT coated Au substrates were
equilibrated under 0.6 V and 0 V in PBS for 20 minutes to obtain surface at the oxidized
state and reduced state respectively. A 0.1 mL suspension of E. coli at a concentration of
109 cells/mL was pipetted onto each PSBEDOT coated Au substrate and then covered with a glass cover slip. The sample was incubated at room temperature for 1 hour. The cover slide was removed and the sample was rinsed in 50 mL of PBS. Then, the sample in PBS was stained with 1 mL of water containing 20 µM of red fluorescent nucleic acid stain propidium iodide (Life Technologies, Carlsbad, CA). The number of cells was determined with a CCD-CoolSNAP camera (Roper scientific, Inc., USA) mounted on Olympus IX81 fluorescent microscopy (Olympus, Japan) with 40x objective lens through FITC filter.
Three separate samples were analyzed for each coating.
Antimicrobial study
After the cell attachment study, same PSBEDOT substrates with attached cells were transferred to PBS solution and 0.6 V potential was applied for 1 h. The sample was rinsed in 50 mL of PBS. Then, the sample in PBS was stained with 1 mL of water containing
20µM of red fluorescent nucleic acid stain propidium iodide and 3.34 µM green fluorescent
68 nucleic acid stain SYTO9 (Life Technologies, Carlsbad, CA). The number of live and dead cells was determined with a CCD-CoolSNAP camera (Roper scientific, Inc., USA) mounted on Olympus IX81 fluorescent microscopy (Olympus, Japan) with 40x objective lens through FITC filter and Texas Red filter at a fixed exposure time for all samples. Three separate samples were analyzed for each coating.
Bacterial release study
After the antimicrobial study, same PSBEDOT substrates with attached cells were
transferred to PBS solution and 0 V potential was applied for 1 h. The sample was rinsed
in 50 mL of PBS. The number of live and dead cells was determined with a CCD-
CoolSNAP camera (Roper scientific, Inc., USA) mounted on Olympus IX81 fluorescent
microscopy (Olympus, Japan) with 40x objective lens through FITC filter and Texas Red
filter at a fixed exposure time for all samples. Three separate samples were analyzed for
each coating.
69 4.3 Results and discussion
Scheme 2. Synthetic route of PSBEDOT. Reaction conditions: (i) 3-chloropropane-1,2-
diol, p-toluenesulfonic acid, toluene; (ii) dimethylamine, water, acetonitrile; (iii) 1,3-
propanesultone, tetrahydrofuran; (iv) electropolymerization in aqueous solution.
As shown in Scheme 2, SBEDOT was synthesized using a three-step method.
EDOT-Cl was firstly synthesized using reported procedures.[130] A straightforward
amination of EDOT-Cl, followed by quaternization with 1,3-propanesultone, produced the target zwitterionic compound, SBEDOT, with a good overall yield. The pure product was characterized using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. We used electropolymerization methods to prepare the CP surface, since they provide precise control of the polymer film growth on the electrode surfaces by simply adjusting the potential/current and reaction time.[132] Both cyclic voltammetry (CV) and galvanostatic
(GS) methods were used to polymerize SBEDOT on various substrates in an aqueous
70 solution containing 60 mM monomer and 100 mM LiClO4 as the electrolyte. Zwitterionic
PSBEDOT was successfully coated on both an indium tin oxide coated polyethylene
terephthalate (ITO-PET) substrate and gold coated SPR sensor chips. The surfaces
prepared using the GS method showed much better homogeneity than those generated from the CV method. It was noticed that, during the GS electropolymerization process, the working potential decreases smoothly with the increase in reaction time, indicating a decrease of the overall impedance and reflecting the excellent electrical conductivity of the deposited PSBEDOT films. EDOT monomers were also polymerized using a similar method on the same substrates and the PEDOT surface was used as a control throughout this study. It should be pointed out that much effort has been devoted to developing flexible, stable and biocompatible CP-based bioelectronic devices, but the conventional monomers are either poorly water soluble or require the addition of surfactants to improve the aqueous processability.[52] One major advantage of the SBEDOT monomer is that it is highly water-soluble and can be directly polymerized in aqueous solution without using organic solvent or surfactants, which significantly facilitates its future applications in vivo. The successful film deposition of PSBEDOT might be a result of the high polymerization rate, high molecular weight and anti-electrolyte effect of PSBEDOT polymers. A similar phenomenon was also observed by Dr. Yu and co-workers.[52]
To confirm the successful surface deposition of the material, the detailed chemical composition of the PSBEDOT surface was analyzed with X-ray photoelectron spectroscopy (XPS). The peak areas, line shapes and intensities of the C 1s, O 1s, N 1s and
S 2p high resolution spectra were monitored. In the survey spectrum of PSBEDOT (Figure
23), the presence of N and a doublet of S, which were not present in PEDOT, indicated that
71 PSBEDOT was successfully deposited onto the substrate. The atomic ratios were also in
agreement with the molecular composition. The detailed high-resolution spectrum of S 2p
shows that two types of S coexisted with nearly equivalent peak intensities, which
confirmed the presence of immobilized PSBEDOT homopolymer with equal amounts of
sulfur atoms in both the thiophene rings and ionic sulfonate side chains.
To deliver/detect low electrical signals, both high electrochemical stability and low interfacial impedance are required for bioelectronics. CV and electrochemical impedance spectroscopy (EIS) methods were used to analyze the electrochemical properties of the coated films. The PSBEDOT films showed good stability, with a slight decrease of electro- activity after CV sweeping for 500 cycles from 0.3 to 0.6 V vs. Hg/HgCl2 electrode (Figure
27A). To determine the interfacial impedance of the PSBEDOT film, EIS was performed on both coated and uncoated substrates. The impedance of the PSBEDOT-coated substrate was about 10 times lower than the uncoated gold at low frequencies (Figure 27B), which is comparable to that of PEDOT and suggests that a densely packed polymer layer was formed. Our result indicates that the PSBEDOT can significantly decrease the interfacial impedance of the gold electrode, which is highly desired and may significantly improve the signal collection and charge delivery of bioelectronics.[133] It should be noted that dopants may affect the conductivity of the conjugated polymer films. To increase the conductivity of PEDOT, the polymer is usually doped with strongly acidic polystyrene sulfonate (PSS), which may potentially cause degradation of the adjacent non-noble metals/polymers or trigger inflammation. Since PSBEDOT was designed for biological applications, no leachable or acidic dopants were added. The electropolymerization of
SBEDOT was conducted using lithium perchlorate as an electrolyte. The perchlorate anion
72 was incorporated into the film as a dopant. To remove perchlorate, all surfaces are
equilibrated in PBS solution, so perchlorate can be exchanged with other anions in the
solution.
Figure 27. Electrochemical characterization of PSBEDOT coated substrates. (A)
Comparison of the cyclic voltammograms of PSBEDOT on gold from the first cycle (black) and after 500 cycles (red) of the applied potential, (B) electrochemical impedance spectra
(Bode plots) of the bare gold substrate (black squares) and PSBEDOT coated gold substrate
(blue triangles).
The adsorption of protein onto the surface of submerged objects in biological
systems is one of the root causes of many biofouling phenomena, which eventually lead to
the failure of bioelectronics. A four-channel surface plasmon resonance (SPR) sensor was used to evaluate the antifouling properties of the PSBEDOT coated gold chips using 100% human blood plasma and 30% human blood serum, which are two of the most complex protein solutions. As shown in Figure 28, the PSBEDOT-coated gold surface can effectively resist protein adsorption from both 100% human blood plasma and 30% human
73 blood serum. The protein adsorption on the surface was calculated from the SPR wavelength shift before the protein injection and after the buffer wash. The adsorption amounts are about 28 ng cm-2 for plasma and 33 ng cm-2 for serum, which are slightly higher than those of non-conducting zwitterionic polymer brush surfaces, such as poly(carboxybetaine methacrylate)[89] and its derivatives.[13, 56] For coatings, the packing density and surface roughness are two important factors for their antifouling properties. Compared to polymer brushes generated from atom transfer radical polymerization (ATRP), polymer films obtained from electrochemical polymerization may not be densely packed and well oriented, so this may increase the specific surface area of the substrate. Therefore, it is possible that the polymer architecture and high surface area resulting from electrochemical polymerization slightly compromise the antifouling performance; however, from the aspect of application, electropolymerized surfaces are easier to prepare and allow for more flexible control of the film thickness than polymer brush-based surfaces.
74
Figure 28. Representative SPR sensorgrams of PSBEDOT coated sensor chips, showing
the low protein adsorption from 100% human blood plasma (red) and 30% human blood
serum (black).
To further evaluate the antifouling properties of the PSBEDOT surfaces, cell
attachment studies were performed using both bovine aorta endothelial cells (BAECs) and
mouse NIH 3T3 fibroblast cells. PSBEDOT and PEDOT surfaces were prepared from GS
electropolymerization of SBEDOT and EDOT monomers on both ITO-PET surfaces and
gold-coated SPR sensor chips. All cells were incubated with the substrates at 37 oC for 24
hours before imaging. For the PSBEDOT coated ITO-PET, a significant difference was
observed between the coated and uncoated regions across the coating edge. A large amount of cells was found on the uncoated area of ITO-PET, while very few cells were found on the PSBEDOT-coated site (Figure 25). Both PSBEDOT and PEDOT were also coated on gold SPR substrates. Nearly a full coverage of BAECs and NIH 3T3 fibroblast cells was
75 seen on the PEDOT surfaces, while there was almost no cell attachment on the PSBEDOT surfaces (Fig. 3). The densities of the adhered BAECs and NIH 3T3 fibroblast cells on the
PSBEDOT surfaces were 0.7% and 0.9% of that on the PEDOT surfaces (Table S1†).
These results demonstrate that the PSBEDOT surface highly resists nonspecific cell attachment.
Figure 29. Cell adhesion tests of PSBEDOT-coated gold substrates incubated with (A)
BAECs and (B) NIH3T3 fibroblast cells, and the PEDOT-coated gold substrates incubated with (C) BAECs and (D) NIH3T3 fibroblast cells for 24 hours.
One major challenge of implantable devices/materials is the surgical infection. To prevent infections, antifouling and antimicrobial strategies are commonly used. Due to the
unique structure of the zwitterionic conjugated PSBEDOT, we expect that PSBEDOT can
switch between antifouling and antimicrobial states under different potentials. In the
oxidized state, the PSBEDOT backbone is positively charged and the overall polymer
76 becomes cationic. In the reduced state, the PSBEDOT backbone is neutral, so the polymer
remains in its zwitterionic state. To evaluate PSBEDOT's potential to minimize infections,
bacterial adhesion, antimicrobial and releasing studies on the PSBEDOT surfaces were
conducted using E. coli K12 as a model species. Before the attachment study, the
PSBEDOT substrates were equilibrated at 0.6 or 0 V in PBS for 30 minutes to generate
oxidized and reduced PSBEDOT surfaces respectively. The bacterial attachment study (Fig.
30 and 31A) showed that the reduced PSBEDOT surfaces were highly resistant to the attachment of E. coli K12 at a very high concentration (109 cells per mL). After 1 hour, the cell density on the reduced PSBEDOT surface was less than 1.9% of that on the gold surface. The attachment of E. coli K12 on the oxidized PSBEDOT surface increased to
33.4% relative to the gold surface. The densities of the attached E. coli K12 cells on both the oxidized and reduced PEDOT surfaces were high (46.6% and 38.4% relative to the gold surface). The excellent antifouling properties of being able to resist bacterial attachment on the reduced PSBEDOT surface are due to its strong hydration properties and a similar phenomenon was also observed for poly(sulfobetaine methacrylate) (PSBMA) polymer brush surfaces.[89] Previous studies demonstrated that there was a direct correlation between bacterial attachment and biofilm development.[89, 134] The lower bacterial attachment on both the oxidized and reduced PSBEDOT surfaces can potentially minimize infections.
77
Figure 30. Representative fluorescence microscopy images of the bacterial adhesion, antimicrobial and release studies on PSBEDOT and control surfaces. Attached E. coli K12 from a suspension with 109 cells per mL on gold (A) and oxidized PSBEDOT (D); the viability of the attached E. coli K12 on gold (B) and oxidized PSBEDOT (E) after subjection to 0.6 V for 1 hour; and the remaining E. coli K12 on gold (C) and oxidized
PSBEDOT (F) after subjection to 0 V for 1 hour. In the viability study, bacterial cells were stained using a LIVE/DEAD BacLight Bacterial Viability assay kit. Cells with a damaged cytoplasm membrane are in yellow and red, and cells with an intact cytoplasm membrane are in green.
78
Figure 31. Quantitative bacterial adhesion, antimicrobial and release studies on PSBEDOT and control surfaces. (A) Attachment of E. coli K12 from a suspension with 109 cells per
mL on oxidized (Ox) PSBEDOT, reduced (Red) PSBEDOT and control surfaces; (B)
bactericidal activity results of PSBEDOT and the control surface against E. coli K12 after
79 subjection to 0.6 V for 1 hour; and (C) detachment of E. coli K12 from oxidized PSBEDOT and gold after subjection to 0 V for 1 hour.
To confirm that oxidized PSBEDOT can kill attached bacterial cells, PSBEDOT
substrates with attached cells were submerged in PBS and a 0.6 V potential was applied
for 1 hour. Before and after applying the potential, the viability of the attached bacterial
cells was analyzed with LIVE/DEAD® Cell Viability Assays using a fluorescence
microscope. The results in Fig. 31B show that the PSBEDOT surfaces caused membrane
damage to 89% of the E. coli in one hour and the gold substrate killed >97.9% of the
attached cells. In solution, over 95.8% of E. coli K12 cells were still viable after one hour.
One advantage of CP surfaces is that the surface potential can be actively controlled. By
applying a lower potential (0 V), the oxidized cationic PSBDEOT surface can switch to a
reduced zwitterionic surface. Due to the repulsive force generated by strong hydration of
the zwitterionic side chains and the disappearance of the attractive force between the
negatively charged bacteria and positively charged PSBDEOT surfaces, the killed bacterial cells can be released. To confirm the hypothesis, a bacterial cell releasing experiment was conducted using PSBEDOT and gold surfaces that carried killed bacterial cells from the antimicrobial study. As shown in Figure 31C, 96.7% of E. coli K12 cells on the PSBDEOT surface were released within 1 hour under the static conditions after the potential was decreased to 0 V from 0.6 V, while only 30% of the cells on the gold surface were released.
The final cell density on PSBEDOT was less than 3% of that on the gold substrate. It should be noted that the release of killed bacterial cells is critical for implanted materials, since the attached dead cells may cause chronic inflammation and lead to the failure of implanted materials/devices. Previously several switchable antifouling/antimicrobial materials have
80 been reported.[11, 13, 56] These zwitterionic polymers can undergo ring formation to become cationic under low pH conditions (pH < 5) and can switch back to their zwitterionic state under neutral or basic conditions. In this work, the electrochemical approach allows for more rapid and active control of the state of the zwitterionic materials. Through this study, we have demonstrated that PSBEDOT surfaces could effectively resist cell attachment in their reduced state, kill the small amount of attached cells in their oxidized state and release the dead cells after switching back to the reduced state.
Numerous applications, ranging from the field of solid state technology[135, 136]
to biomedical engineering,[135, 137, 138] need to use high performance CPs as the key components that determine the function and properties of the devices, so the development of novel multifunctional CPs is of great importance. One of the most attractive features of
CPs over traditional biomaterials is that they could allow electrical stimulation of the attached tissues and cells.[139] It is expected that the novel PSBEDOT could be used to manipulate cell attachment through electrochemical control and also could serve as a protective coating to reduce protein adsorption and cell attachment thus prolonging the lifetime of implanted devices. Although there is much work to be done to fully understand
and realize the potential of zwitterionic conjugated polymers, we believe this work will
fundamentally advance the development of bioelectronics.
4.4 Conclusions
In this work, we designed and synthesized a novel antifouling and electroactive
PSBEDOT material. Zwitterionic PSBEDOT can be facilely polymerized in aqueous
solution through an electrochemical method. The PSBEDOT polymer films exhibit
81 excellent electrochemical properties, low interfacial impedance, stability and switchable
antifouling/antimicrobial properties. The interfacial impedance of PSBEDOT was less than
10% of that of bare gold at low frequency. It also showed superior antifouling properties against whole blood, mammalian cells and bacteria. The PSBEDOT surface can also be switched between cationic antimicrobial and zwitterionic antifouling states by applying different potentials. It can kill over 89% of attached cells in one hour at 0.6 V and release over 96.7% of the dead cells in one hour at 0 V under static conditions. It shows great promise for applications in bioelectronics. This new material may significantly increase the performance and service life, minimize the foreign body reaction, improve the biocompatibility and reduce the infection of bio-electronic devices.
82
CHAPTER V
STRUCTURE-FUNCTION STUDY OF POLY (SULFOBETAINE 3,4-ETHYLENE-
DIOXYTHIOPEHEN) (PSBEDOT) AND ITS DERIVATIVES
5.1 Introduction
After Dr. Alan Heegar et al. discovered that polymers with alternating single and double bonds can function as conductors,[140] various conjugated polymers (CP)s, such as such as polyacetylene (PA),[141] polyaniline (PANi),[142] polypyrrole (PPy),[143]
polythiophene (PTh)[144] and poly(3,4-ethylenedioxythiophene) (PEDOT),[145] have
been studied for a range of applications including such as solar cells,[146, 147] lightweight batteries,[148] electrochromic devices,[149, 150] chemical sensors,[151, 152] and biosensors and molecular electronic devices.[153-155] CPs have attracted significant interests in biomedical and biotech applications due to their great design flexibility,[156] and they have been explored as biomaterials to replace metals or modify metal surfaces in biosensing,[133, 157, 158] tissue engineering,[159, 160] wound healing,[161] and biofuel cell.[162] In these applications, electrodes are a core component for the delivery of charge and/or recording of electrical signal. Metal-based electrodes are usually fabricated from platinum, platinum alloys and gold with a good chemical stability, but they suffer from poor biocompatibility, low surface area and low flexibility. Surface modification of metallic electrodes has been extensively explored to increase the integration with biological
83 tissue and minimize foreign body encapsulation at the electrode/tissue interface.[133, 157,
158] Surface modification of metallic electrodes with biocompatible but non-conducting materials compromises electron and ion transport between electrode and biological system, because non-conducting materials can block electronic and ionic conduction. The improved communication between synthetic and biological systems is critical for medical devices to perform more efficiently by permitting the use of smaller charges or detect very low electrical signal [163].
For biosensing applications, CPs possess several advantages, such as enhanced
sensitivity, versatility, being a suitable matrix for enzymes entrapment, and direct
deposition of polymer on the electrode while encapsulating the protein molecules
simultaneously.[164, 165] It was found that CPs could improve impedance characteristics
and provide a softer mechanical interface when compared to metallic electrodes,[157] but
several studies indicated that the benefits provided by CP coatings following implantation
are not maintained in the long-term.[166, 167] In vivo studies have shown that CPs provide low impedance with minimal bilayer capacitance, but as fibrosis proceeds, the growth of non-conductive scar tissue dominates.[168, 169] Current CPs also lack functional groups to conjugate bioactive moieties, tunable mechanical properties, antimicrobial properties to prevent surgical infection and sensitive response to environmental stimuli (pH, ionic strength and redox potential). These drawbacks must be addressed before the potential of
CPs can be fully realized.[170] Among CPs, PEDOT is considered as the best biocompatible CP for a long period of time, since EDOT has the external heterocyclic rings designed to yield electroactive and electrochromic polymers that switch at low potentials[171] and the heterocyclic ring also increase the solubility of both monomer and
84 polymer.[172, 173] Although a previous study have shown that PEDOT-coated probe can
enhance the signal-to-noise ratio in vivo at the initial stage, as the fibrous encapsulation
grows, the benefit of PEDOT coating decreased.[41] To increase the biocompatibility of
CPs, CPs were also blended or physically crosslinked with non-conducting anti-fouling
materials. For example, Polyethylene glycol (PEG) was used to crosslink PANi for glucose
sensing.[174] However, non-conducting components compromise electrochemical
properties of conducting hydrogels.[175, 176] In addition, non-conducting components of
current conducting hydrogels is not effective to reduce the foreign body response. To
address the critical challenge of CPs, two water soluble CPs, poly(phosphorylcholine 3,4-
ethylenedioxythiophene) (PEDOT-PC)[177] and poly(sulfobetaine 3,4-
ethylenedioxythiophene) (PSBEDOT),[76] were recently reported and both CPs contain
conducting backbone and zwitterionic anti-fouling side chains. PSBEDOT exhibited
excellent resistant to proteins, cells, and bacteria while maintaining the low interfacial
impedance.[76] PSBEDOT holds great potential for the next generation of conducting
polymer that not only possess excellent biocompatibility but also switchable antimicrobial
and antifouling properties by applying different electric potentials.
It is known that closely attached functional or substitution groups can have
electronic effect, steric effect and solubility effect on the electro-polymerization kinetics and the property of the resulting polymer.[178, 179] Zwitterionic side chain that bears both positive and negative ions may introduce a strong electronic effect on the thiophene ring and significantly influence the solubility of the polymer. For example, zwitterionic
PEDOT-PC film was unable to be polymerized in aqueous solution because of the closely functionalized phosphorylcholine group and the electro-polymerization reaction could only
85 be conducted in acetonitrile.[177] The property of not being able to be polymerized in
aqueous solution may impede the utility of the CPs. In contrast to EDOT-PC, SBEDOT
can be readily polymerized in aqueous solution; however, the polymerization required the
relatively higher monomer concentration (40 mM) compared to EDOT. We hypothesize
that the sulfobetaine side chain of SBEDOT affect electronic density, reactivity of the
conjugated backbone, the oxidation potential of SBEDOT and conductivity of the polymer.
To have further understanding on how the structure of zwitterionic side chains affect the reactivity of the monomer and properties of zwitterionic PEDOT polymers, herein, we introduced the methoxyalkyl spacer to reduce both electronic and steric effect between
PEDOT backbone and zwitterionic sulfobetaine side chain. Two SBEDOT derivatives,
SBEDOT-4 and SBEDOT-5 where the number indicates the spacer length, have been synthesized to examine the effect of spacer length on the electrochemical properties and surface morphology.
5.2 Experimental Section
5.2.1 Chemicals
3,4-Dimethoxythiophene was purchased from Matrix Scientific (Columbia, SC, USA). 1,4- dibromobutane, 1,5-dibromopentane, potassium hydroxide, tetra-n-butylammonium bromide, dimethylamine solution (40 wt. % in water), 1,3-propanesultone were purchased from Alfa Aesar (Ward Hill, MA, USA). (±)-3-chloro-1,2-propanediol, toluene, p- toluenesulfonic acid monohydrate, dichloromethane, dimethyl sulfoxide (DMSO), sodium acetate (anhydrous), hexane, methanol, tetrahydrofuran (anhydrous), acetonitrile, lithium perchlorate, sodium hydroxide, diethyl ether, magnesium sulfate (anhydrous), phosphate-
86 buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fibrinogen was purchased from Calbiochem (San Diego,CA, USA). All chemicals were
used as received without further purification.
5.2.2 Synthesis of SBEDOT-4 and SBEDOT-5
2-(Chloromethyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine (chloromethyl-EDOT)
and (2,3-Dihydrothieno[3,4-b][1,4]dioxin-2-yl) methanol (hydroxymethyl-EDOT) was
first synthesized following the method published previously[76, 180].
Synthesis of 2-((4-bromobutoxy)methyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine. (4- bromobutoxy methyl-EDOT)
The substitution reaction of the hydroxymethyl-EDOT was carried out by following a published literature.[181] 5 g of hydroxymethyl-EDOT, 89.71 g of 1,4-dibromobutane and 2.74 g of tetrabutylammonium bromide were added and stirred in 100 ml dichloromethane (DCM) for 5 minutes. Then, 100 ml solution of sodium hydroxide (50 wt. %) was added, and the reaction was carried out at room temperature for 2 hours with the protection of nitrogen gas. The crude product was extracted in DCM against water, and then the solvent was removed by rotary evaporator. After being purified by flash column chromatography (DCM : Hexane = 3 : 2), the product was verified via NMR.1H NMR
(300MHz, CDCl3) δ 6.35-6.31 (m, 2H), 4.30-4.20 (m, 2H), 4.08-4.01 (m, 1H), 3.71-3.57
(m, 2H), 3.53 (t, 2H), 3.43 (t, 2H), 1.95 (quin, 2H), 1.73 (quin, 2H). 13C NMR (75MHz,
CDCl3) δ 141.75, 141.69, 99.83, 99.76, 72.80, 71.08, 69.40, 66.31, 33.65, 29.70, 28.33
87 Synthesis of 2-(((5-bromopentyl)oxy)methyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine. (5- bromopentoxy methyl-EDOT)
The synthesis procedure was the same with the above one. The only difference is
1 1,4-dibromobutane was changed to 1,5-dibromopentane. H NMR (300MHz, CDCl3) δ
6.33-6.31 (m, 2H), 4.33-4.21 (m, 2H), 4.08-4.02 (m, 1H), 3.71-3.56 (m, 2H), 3.50 (t, 2H),
3.41 (t, 2H), 1.88 (quin, 2H), 1.66-1.57 (m, 2H), 1.55-1.45 (m, 2H)
Synthesis of 4-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxy)-N,N-dimethylbutan-
1-amine. (dimethylamine-EDOT-4)
4 g of 4-bromobutoxy methyl-EDOT was added to a solution of 16 ml dimethylamine (40 wt. % in water) and 16 ml acetonitrile in a Schlenk flask, and then, the
reaction was carried out at 80oC for 24 hours. Another 16 ml of dimethylamine was added, and the reaction continued at 80oC for another 24 hours with the protection of nitrogen gas.
The solvent was removed by rotary evaporator. The crude product was dissolved in acetonitrile, and then was precipitated in diethyl ether. After being dried with magnesium sulfate, the product was purified with flash column chromatography (DCM:MeOH = 4:1).
Synthesis of 5-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxy)-N,N-
dimethylpentan-1-amine. (dimethylamine-EDOT-5)
The synthesis procedure was the same with the above one. The only difference is
4-bromobutoxy methyl-EDOT was changed to 5-bromopentoxy methyl-EDOT.
Synthesis of 3-((4-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-
yl)methoxy)butyl)dimethylammonio)propane-1-sulfonate. (SBEDOT-4)
88 1.9 g 1,3-propanesultone was slowly added to a solution of 3.53 g dimethylamine-
EDOT-4 in 20 ml of dry THF. The reaction was carried out at 55oC for 24 hours with the protection of nitrogen gas. The crude product was concentrated by removing excess solvent and then was precipitated in diethyl ether. The pure product was obtained as the white
1 powder. H NMR (Figure 32) (300MHz, D2O) δ 6.65-6.61 (m, 2H), 4.53-4.51 (m, 1H),
4.43-4.39 (m, 1H), 4.23-4.16 (m, 1H), 3.85 (d, 2H), 3.73 (t, 2H), 3.59-3.54 (m, 2H), 3.50-
3.43 (m, 2H), 3.21 (s, 6H), 3.09 (t, 2H), 2.39-2.31 (m, 2H), 1.95 (quin, 2H), 1.78 (quin,
13 2H). C NMR (Figure 33) (75MHz D2O) δ 140.94, 140.86, 100.43, 100.42, 72.99, 70.63,
68.80, 65.75, 63.93, 62.28, 50.65, 47.39, 25.48, 19.02, 18.29 (One carbon is not seen due
to the overlapping signal.)
Figure 32. 1HNMR of SBEDOT-4
89
Figure 33. 13CNMR of SBEDOT-4
Synthesis of 3-((5-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-
yl)methoxy)pentyl)dimethylammonio)propane-1-sulfonate (SBEDOT-5)
The synthesis procedure was the same with the above one. The only difference is
dimethylamine-EDOT-4 was changed to dimethylamine-EDOT-5.1H NMR (Figure 34)
(300MHz, D2O) δ 6.48-6.45(m, 2H), 4.38-4.33 (m, 1H), 4.27-4.22 (m, 1H), 4.06-4.00 (m,
1H), 3.68 (d, 2H), 3.58-3.52 (m, 2H), 3.45-3.39 (m, 2H), 3.29-3.24 (m, 2H), 3.06 (s, 6H),
2.95 (t, 2H), 2.24-2.13 (m, 2H), 1.80-1.69 (m, 2H), 1.66-1.57 (m, 2H), 1.41-1.31 (m,
13 2H). C NMR (Figure 35) (75MHz D2O) δ 140.76, 140.65, 100.29, 100.21, 72.77, 71.06,
68.51, 65.58, 63.96, 61.98, 50.51, 47.20, 27.95, 22.11, 21.59, 18.12 (One carbon is not seen due to the overlapping signal.)
90
Figure 34. 1HNMR of SBEDOT-5
Figure 35. 13CNMR of SBEDOT-5
91 5.2.3 Electro-polymerization and electrochemical analysis of PSBEDOT, PSBEDOT-4
and PSBEDOT-5
The galvanostatic electro-polymerization was performed by Gamry Reference 600
potentiostat with the three-electrode method in which Pt wire is used as the counter
electrode and Ag/AgCl2 electrode (3M NaCl) is used as the reference electrode. If not noted, the monomer solutions contained 40 mM monomer and 100 mM lithium perchlorate
(LiClO4) as the electrolyte. Two types of substrates were used in the experiments, indium tin oxide (ITO)-coated glass substrate and gold-coated glass substrate. On gold substrates, the polymers were synthesized with different total deposition charges by maintaining the constant current (0.5 mA) for different length of time: 120 s, 240 s, and 360 s. On ITO substrates, total deposition charge remained the same while different currents (0.5 mA, 1.0 mA, 1.5 mA, and 2.0 mA) were applied.
Electrochemical impedance spectroscopy (EIS) was performed using a sinusoidal
excitation signal with an excitation amplitude of 10 mV at frequencies logarithmically
spaced from 100 kHz to 0.1 Hz. Cyclic stability measurement was carried out via cyclic
voltammetry for 1000 cycles between -0.2 V to 0.8 V at the scan range of 100 mV/s.
5.2.4 Fibrinogen adsorption of PEDOT, PSBEDOT, PSBEDOT-4 and PSBEDOT-5
The antifouling properties of PEDOT, PSBEDOT, PSBEDOT-4, and PSBEDOT-
5 were characterized by using quartz crystal microbalance (Gamry eQCM 10M) along with
Gamry software: resonator. The polymers were electro-polymerized on gold-coated 10M eQCM quartz chips in the static eQCM cell via the galvanostatic method of 1 mA/cm2. The
monomer concentration is 40 mM for of SBEDOT derivatives and 10 mM for EDOT. Since
92 the reactivity of each monomer is different, so the mass of the polymer that deposited on
the QCM chip is controlled by stopping the electro-polymerization at 20000 Hz. Prior to
the protein adsorption test, the polymer-coated QCM chip was transferred into the flow cell and was equilibrated in a flow of PBS (pH=7.4) to establish a stable baseline at the flow rate of 0.1 ml/min. Then, PBS was replaced by 1 mg/ml fibrinogen in PBS. After 20 minutes, PBS was flowed again to wash off the loosely adsorbed proteins for 20 minutes.
The adsorption of fibrinogen on the surface of the polymer was recorded as the frequency change. The mean frequency for each sample before protein injection and after PBS rinse is calculated, and Δfs is the frequency change before and after the protein adsorption.
5.2.5 Surface morphology characterization and thickness measurement
The surface morphology of PSBEDOT, PSBEDOT-4, and PSBEDOT-5 were
characterized by scanning electron microscope (TESCAN LYRA3). Every sample was
dried by air before imaging.
The film thickness of the polymers was measured by the surface profiler (Bruker
DektakXT). Three parallel slits were made on each sample by using a point tweezer gently scratching off the polymer. The probe of surface profiler scan in the direction that is perpendicular to the slits to create a two-dimensional profile. The height of the bottom of the slits was normalized to zero. The software of surface profiler (Vision64) was used to calculate the mean height of the polymer surface between two adjacent slits. Three different regions of each sample were scanned, and five mean heights were chosen for each sample to calculate the mean of the film thickness.
93 5.3 Result and discussion
Scheme 3 shows the synthesis route of PSBEDOT-4 and PSBEDOT-5.
Hydroxymethyl EDOT was first synthesized as the starting material and then reacted with
1,4-dibromobutane or 1,5-dibromopentane to introduce the butyl group or pentyl spacer
into SBEDOT. The rest of the synthesis reactions were carried out by following the
published synthesis route of SBEDOT.[76] The final product of the monomers was
characterized by 1H and 13C nuclear magnetic resonance spectroscopy. In contract to the
chemical polymerization approach, electro-polymerization was a more attractive approach
for surface modification because of no need for further conjugation reaction, small amount of a monomer needed, and well controlled polymerization condition. All monomers
(SBEDOT, SBEDOT-4, and SBEDOT-5) were successfully electro-deposited on both
ITO-coated and gold-coated glass substrates via a galvanostatic method. Initially 40 mM monomer was used since it is the minimal concentration for the electro-deposition of
SBEDOT. However, it was discovered that both SBEDOT-4 and SBEDOT-5 can be electro-polymerized at the concentration as low as 10 mM. It should be noted that electro- polymerized polymer/oligomer may not deposit on the electrode. Based on the mechanism of the anodic electro-polymerization, the key parameters determining the success of electro-deposition are the intrinsic stability of the radical cation, the solubility of the polymer/oligomer and the adhesive force between the polymer and the electrode. The highly stable radicals can diffuse away from the electrode, while the highly unstable radicals can rapidly react with solvent or anions, which the soluble products will be produced in both cases.[182] Therefore, there is a stability range for radicals to perform the electropolymerization. Compared to EDOT, SBEDOT has slightly higher oxidation
94 potential. Because of the stronger dipole caused by a shorter distance of the zwitterionic
side chain to the backbone, the quaternary amine group in SBEDOT may affect the radical cation stability. In addition, it is observed that soluble products formed close to the working electrode surface during the galvanostatic deposition and led to the discoloration of the reaction solution. This phenomenon may be caused by the higher solubility of PSBEDOT in water compared to PEDOT.
Scheme 3. Synthetic route of PSBEDOT-4 and PSBEDOT-5. Reaction condition: (i) 1,4-
dibromobutane for m=4 and 1,5-dibromopentane for m=5, tetrabutylammonium bromide,
dichloromethane/50 wt% of sodium hydroxide solution (1:1); (ii) dimethylamine solution
(40 wt% in water), acetonitrile; (iii) 1,3-propanesultone, anhydrous tetrahydrofuran; (iv)
electro-polymerization in aqueous solution of 40 mM monomer and 100 mM LiClO4 by
galvanostatic method.
95 Table 4. Film thickness of PSBEDOT, PSBEDOT-4, and PSBEDOT-5 on ITO-coated and gold-coated substrates. All samples were electro-polymerized by the galvanostatic method:
0.5 mA/cm2 for 120s.
PSBEDOT PSBEDOT-4 PSBEDOT-5
Film thickness on ITO (nm) 262 ± 45 1456 ± 77 2169 ± 172
Film thickness on gold (nm) 210 ± 42 661 ± 27 734 ± 83
The film thickness of three polymers on both ITO and Gold substrates, which
synthesized under the same condition (0.5 mA/cm2 for 120 s), was measured and shown in table 4. The type of substrate has a strong influence on the film thickness and the thickness of all polymers on the gold substrate is much lower than on ITO substrate, especially for
PSBEDOT-4 and PSBEDOT-5. Since the total deposition charges for all samples are the same, the thinner film thickness on gold substrates may be caused by the higher packing density of the polymer due to the low resistance of the gold substrate. In addition, the thinner PSBEDOT film than PSBEDOT-4 and PSBEDOT-5 may be caused by several reasons. First, due to its higher solubility, some PSBEDOT did not deposit on the electrode surface. Second, because of the electronic effect, the degree of polymerization for
PSBEDOT is relatively low, which low-MW polymer have a more ordered and packed film.[183] Third, due to the large side chain of PSBEDOT-4 and PSBEDOT-5, the asymmetry of the molecule with a bulky side chain can cause the molecules to twist resulting in the imperfect packing.[183]
96 The effect of the size of the pendent side chain on the surface morphology was
observed by scanning electron microscope (SEM). In the first study (shown in Fig 36),
each polymer was electropolymerized on gold substrates at the same current density (0.5
mA/cm2) for different periods (120 s, 240 s, and 360 s) to form three polymer films with
different thickness. At the low magnification, the surface of PSBEDOT appeared relatively flat, and both PSBEDOT-4 and PSBEDOT-5 surfaces showed wrinkled feature. At the higher magnification, it can be observed that as increasing the time of electro- polymerization, the diameter of bumps on the surface of PSBEDOT largely grew from 1.1
μm to 4.7 μm, and the wrinkling density of PSBEDOT-4 and PSBEDOT-5 increased. For the further examination, each polymer was synthesized on ITO-coated substrate with various deposition current density (0.5, 1.0, 1.5, and 2.0 mA/cm2). As shown in Fig 37, we can observe that the deposition current also affects the morphology. At low deposition current density (0.5, 1.0, and 1.5 mA/cm2), PSBEDOT has a uniform film, but the film
formed at 2.0 mA appeared defect as fibers with the width of ca. 0.6 μm. For PSBEDOT-
4, high deposition current density (2.0 mA/cm2) led to a less wrinkled surface comparing
to the other three currents. Similar to PSBEDOT-4, PSBEDOT-5 had a less wrinkled surface using high deposition current density as well. The surface of PSBEDOT-5 was
covered with grains which diameter is around 1.5 μm. Moreover, we can observe the effect of substrate on the surface morphology by comparing the first row of Fig 36 and Fig 37.
The morphology of PSBEDOT showed a significant difference between two types of substrates. The polymer film on ITO substrates presented more uniform than on gold substrates. For PSBEDOT-4 and PSBEDOT-5, the wrinkling feature appeared on both gold and ITO substrates, but the density on ITO was higher than on gold. As we can see from
97 the result, the surface properties of substrates can affect the growth rate and deposition during the electropolymerization, which led to the final morphology appeared differently to some extent.[184, 185]
Figure 36. Surface morphology of (A) PSBEDOT, (B) PSBEDOT-4, and (C) PSBEDOT-
5 synthesized on gold substrates at 0.5 mA for 120s, 240s, and 360s (Y-axis) imaging at the magnification of 200x and 5000x (X-axis)
98
Figure 37. Surface morphology of (A) PSBEDOT, (B) PSBEDOT-4, and (C) PSBEDOT-
5 synthesized on ITO-coated glass substrates at 0.5, 1.0, 1.5, 2.0 mA for the same total deposition charge (Y-axis) imaging at the magnification of 200x and 5000x (X-axis)
99
Figure 38. Electrochemical impedance spectra (EIS) of (A) PSBEDOT and (C) PSBEDOT-
4 on ITO substrates in different polymerization condition. Comparison of EIS of
PSBEDOT, PSBEDOT-4, and PSBEDOT-5 on (B) gold substrates and (D) ITO substrates.
All EIS experiment performed in 100 mM LiClO4 solution.
Low interfacial impedance between electrolyte and electrode is important to design an electrode for many biomedical applications such as impedance-based biosensing, neuroprotheses, etc. All polymer films were examined by EIS. In Figure 38 (A) and (C),
EIS of PSBEDOT and PSBEDOT-4 films on ITO substrates with various current density were studied. The total deposition charge was kept at 120 mC to maintain the same thickness. The selected current density for electro-polymerization did not show a
100 considerable influence on the impedance of all polymers. Therefore, in the further
impedance comparison of PSBEDOT, PSBEDOT-4 and PSBEDOT-5, the current density
used for electro-polymerization was chosen to be 0.5 mA/cm2. For the impedance
comparison, each polymer was synthesized at 0.5 mA/cm2 for 120 seconds on both gold
and ITO substrates, and the results are shown in Fig 38 (B) and (D), respectively. On both
substrates, PSBEDOT-4 and PSBEDOT-5 showed around 20 times lower impedance than
PSBEDOT at low frequency (below 1 Hz). For example, at 1 Hz, the impedance of
PSBEDOT-4 on ITO and gold are 82 Ω and 55 Ω, respectively, while the impedance of
PSBEDOT is around 1630 Hz on both types of substrate. The impedance curve of
PSBEDOT has similar trend to that of the control group, which its impedance starts
increasing around 500 Hz. For PSBEDOT-4 and PSBEDOT-5, the frequency change did
not strongly affect their impedance until 1 Hz, which means that these two polymers can
be more useful for application at low frequencies. Moreover, the effect of substrate on the
impedance was subtle. For bare substrate, the impedance of ITO is twice than that of gold
at 100 Hz and three times at 0.1 Hz. Although, in general, the impedance of the polymers
on gold is lower than on ITO, the impedance difference of the polymers between these two substrates was not significant. This study demonstrates PSBEDOT-4 and PSBEDOT-5 can further reduce the interfacial impedance and significantly improve the signal transduction at the interface.
101
Figure 39. Cyclic voltammograms of (A) PSBEDOT, (B) PSBEDOT-4 and (C)
PSBEDOT-5at 20th, 220th, 520th, and 1000th cycle. (D) Percentage of total charge of
PSBEDOT, PSBEDOT-4 and PSBEDOT-5 at 20th, 520th, and 1000th cycle.
For electrodes to deliver or collect stable signal for an extended period, another important factor is its electrochemical stability. In this work, we tested each polymer film by running CV for 1000 cycles in 100 mM LiClO4. Although each polymer film was electrodeposited on gold-coated substrates with the same galvanostatic condition: 0.5 mA/cm2 for 120 s, as shown in Figure 39A, 39B, and 39C, which PSBEDOT have relatively small current response (Y axis) that means its interfacial impedance is higher than the other two. In the comparison of the anodic peak of three polymers, PSBEDOT
102 have a broader peak with higher oxidation potential that may be caused by two reasons.
First, according to the published literature, increasing oxidation potential indicates the
shortening of the conjugation length.[186, 187] Second, the width of peak can be
interpreted as the distribution of conjugated length, which means PSBEDOT-4 and
PSBEDOT-5 with a sharper peak have a more uniform distribution of polymer. Moreover, as the sweeping of voltage goes, the oxidation potential of PSBEDOT-4 and PSBEDOT-5 increased, but PSBEDOT did not show this behavior. In Fig 39D, we compared the total charge of each material by integrating the curve area. We chose three curves, 20th, 520th
and 1000th, to observe the degradation behavior, and normalized 20th cycle to 100%. At
520th cycle, three materials remained over 80% of total charge. However, at 1000th cycle,
total charge of PSBEDOT decreased to 60% while that of PSBEDOT-5 and PSBEDOT-4
is around 70% and 80%, respectively. So, among three polymers, PSBEDOT-4 shows the highest stability.
103
Figure 40. Fibrinogen adsorption of PEDOT, PSBEDOT, PSBEDOT-4 and PSBEDOT-5 tested by electrochemical quartz crystal microbalance (eQCM).
Table 5. Frequency change and the percentage of protein adsorption of PEDOT, PSBEDOT,
PSBEDOT-4 and PSBEDOT-5.
PEDOT PSBEDOT PSBEDOT-4 PSBEDOT-5
Δfs (Hz) -777.3 -31.6 -40.9 -21.8
Percentage of protein 100 4.07 5.26 2.80 adsorption (%)
104 One of major challenges for implantable device is the inflammatory reaction and
foreign body reaction. Right after implantation, the nonspecific protein adsorbed on the
implanted device and lead to the adhesion of platelet or macrophages,[188, 189] and
eventually leads to the failure of the device. Therefore, the ability to resist nonspecific
protein adsorption is critical to implantable medical devices. Our previous study
demonstrated that PSBEDOT highly resist the adsorption of protein and the adhesion of
mammalian cells and bacteria. Herein, we chose same model protein, fibrinogen, to
evaluate PSBEDOT-4 and PSBEDOT-5, since fibrinogen is a highly abundant plasma
glycoprotein that involves in many physiological processes such as coagulation, platelet
activation and aggregation, inflammatory response and leucocyte binding.[190] The
change in mass of each polymer surface was monitored by eQCM and the results are shown in Fig 40. Since the change in mass is proportional to the frequency change, the frequency change (Δfs) before and after the protein adsorption were reported in table 5. The frequency change of PSBEDOT-4 and PSBEDOT-5 are -40.9 Hz and -21.8 Hz, respectively, which are comparable to PSBEDOT (-31.6 Hz). This shows that PSBEDOT-4 and PSBEDOT-5 remains a good resistance to protein adsorption after introducing carbon spacer while non- zwitterionic PEDOT is not able to prevent the adsorption of fibrinogen that results in -
777.3 Hz of frequency change. However, it should be noted that in addition to the nature of zwitterionic group, the packing density of polymer is also a vital factor to the antifouling properties of the resulting polymer. We believe the antifouling property of PSBEDOT-4 and PSBEDOT-5 can be further improved by optimizing the film thickness and packing.
105 5.4 Conclusion
In this study, we synthesized two zwitterionic conjugated polymers, PSBEDOT-4
and PSBEDOT-5 by introducing the carbon spacers between the zwitterionic sulfobetaine
group and the conjugated polymer backbone of PSBEDOT. The oxidation potential and
surface morphology of PSBEDOT-4 and PSBEDOT-5 are significantly different from
PSBEDOT. The diminution in hindrance effect facilitate the stable electro-polymerization resulting in the increase of mean conjugated length, cyclic stability, and packing.
Additionally, PSBEDOT-4 and PSBEDOT-5 show better antifouling properties compared to PSBEDOT, which indicate the spacer does not compromise their antifouling properties.
PSBEDOT-4 and PSBEDOT-5 with improved electrochemical stability, lower interfacial impedance and good antifouling ability may provide a better solution for the challenges of implanted bioelectronics.
106
CHAPTER VI
CONCLUSION AND FUTURE WORK
In this dissertation, I had showed that how the structural variation affects the
properties of zwitterionic materials from the view of molecular level by integrating its
antifouling ability with other functions including buffering ability, ionic conductivity and
electronic conductivity.
In Chapter II, a tertiary amine-based polycarboxybetaine (PCB) were synthesized.
By replacing the quaternary ammonium to tertiary amine, this tertiary amine-based PCB
(PCB-T) integrated multiple desire properties including antifouling, switchability, and buffering capability. Owing to the lactone formation of hydroxy group and carboxylate group, PCB-T can reversely switch between cationic ring form and zwitterionic form in response to pH of the environment. Compared to the traditional switchable PCB, tertiary amine increases the stability of the lactone ring. This switchability is particularly useful to
catch and kill the bacteria in cationic form and then release and prevent further attachment in zwitterionic form. On the other hand, the tertiary amine group of PCB-T not only showed buffering capability at basic conditions but also led to a weaker carboxylate with a less acidic buffering range. More importantly, this material exhibited high resistant to protein adsorption in complex human blood. I believe this study provides general strategy and knowledge for designing multifunctional biomaterials.
107 In Chapter III, A series of polyelectrolyte hydrogels, including zwitterionic,
cationic, anionic hydrogels, were examined and compared on their ionic conductivity and volume change in three types of salt solutions with various concentration. This systematic investigation provides a deeper understanding on how immobilized zwitterionic functional group interact with the mobile ions in the solution. The volume change of zwitterionic hydrogels in tested solutions is relatively stable compared to ionic hydrogels in which the electrostatic repulsion strongly affects their properties such as water content and volume change. Due to the neutral charge, zwitterionic hydrogels have weak interaction with the mobile ions in the solution, which does not impede the ionic transport in the salt solution with high concentration. Additionally, zwitterionic hydrogels showed much higher ionic conductivity than that of PEGMA hydrogel which is a widely used material in a variety of applications. This work has shown that zwitterionic materials can be a good candidate for applications that need an antifouling platform with a favorable ionic transport environment.
In Chapter IV and V, Poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT) and two derivatives were designed and developed to integrate antifouling, ionic conductivity and electronic conductivity. Owing to the hydrophilicity and antifouling of sulfobetaine group, PSBEDOTs were able to be readily polymerized in aqueous solution, and exhibited high resistant to proteins, cells and bacteria. I believe the antifouling conducting polymers can be invaluably helpful for designing organic bioelectronics with long-term stability.
Zwitterionic materials have been studied on their antifouling properties for a long
period of time. In recent year, it has been found that the unique properties of zwitterionic
materials such as free of counter ions and strong dipole moment provides great help in
108 electronic devices. In addition, although the mechanism is not fully revealed, the zwitterions have been demonstrated that it can facilitate the dissociation of lithium ions which is extremely useful in the development of batteries. Thus, I believe that, in future, zwitterionic materials will play an important role in both biology and electronics field.
109
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