Stimuli-Responsive and Hemocompatible Pseudozwitterionic

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Stimuli-Responsive and Hemocompatible Pseudozwitterionic Interfaces Antoine Venault, Yong-Sheng Zheng, Arunachalam Chinnathambi, Sulaiman Ali Alharbi, Hsin-Tsung Ho, Yu Chang, and Yung Chang Langmuir, Just Accepted Manuscript • DOI: 10.1021/la505000m • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Stimuli-Responsive and Hemocompatible Pseudozwitterionic Interfaces Antoine Venaulta, Yong-Sheng Zhenga, Arunachalam Chinnathambi b, Sulaiman Ali Alharbi b, Hsin-Tsung Hoc, Yu Chang*,d,e , Yung Chang*,a a

R&D Center for Membrane Technology and Department of Chemical Engineering, Chung

Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan. b

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh -

11451, Kingdom of Saudi Arabia. c

Laboratory Medicine, Mackay Memorial Hospital, Taipei 104, Taiwan

d

Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Graduate

Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. e

Department of Obstetrics and Gynecology, E-Da Hospital, I-Shou University, No. 1, Yida

Road, Jiaosu Village, Yanchao District, Kaohsiung City, Taiwan.

ACS Paragon Plus Environment

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ABSTRACT We report a novel biomacromolecular formula for the design of hemocompatible gel interfaces of N-isopropylacrylamide (NIPAAm) and mixed-charge pairs of [2-(Methacryloyloxy)ethyl] trimethylammonium (TMA) and 3-sulfopropyl methacrylate (SA) with overall electrical neutrality. The study stresses on how well-defined compositions of nonionic NIPAAm and pseudozwitterionic TMA/SA in the poly(NIPAAm-co-TMA/SA) hydrogels along with environmental conditions (temperature, ionic strength, solution pH) affect swelling and adhesion of biofoulants on their interfaces. When challenged with plasma proteins, bacteria, recalcified platelets or whole blood, stimuli-responsive hydrogels better resisted their adhesion as the content of mixed-charges in the copolymer increased, to reach nonbiofouling for the gels made of 100% TMA/SA. The low hemolytic activity (0.5 %) associated to a long plasma clotting time (10 min) suggest excellent hemocompatibility of these hydrogels. Finally, hydrogels containing both NIPAAm and TMA/SA tend to exhibit preferential adhesion of leukocytes.

KEYWORDS Poly(NIPAAm-co-TMA/SA) hydrogels, tunable swelling, hemocompatibility, low-bifouling

INTODUCTION The development of antifouling polymer surfaces has received growing attention over the past 15 years, particularly in the design of membranes for water treatment or blood filtration, or


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antimicrobial coatings.1-5 A literature survey highlights that derivatives of poly(ethylene glycol) and zwitterionic polymers/copolymers are the two main kinds of surface modifiers for preparing antifouling surfaces,6-11 as they meet the important requirements defined earlier by Whitesides.12, 13

However, zwitterionic polymers are regarded as more efficient than PEG derivatives, which

may auto-oxidize and therefore eventually lose their antifouling properties.14 Zwitterionic polymers used in the design of antifouling surfaces or membranes are usually derivatives of sulfobetaine, carboxybetaine or phosphobetaine.15-20 These three pendant groups have proven their efficiency to repel proteins, bacteria or cells. However, as reminded in a recent study, zwitterionic-based hydrogels and films may partially lose their antifouling property when the surface chemistry is modified in order to add a supplementary bioactive function through the implementation of additional conjugate.21 Therefore, research is now also orientated toward the synthesis of mixed charged copolymers that can mimic the antifouling properties of zwitterionic polymers.21-23 They are composed of positively and negatively charged pendant groups which assembly can lead to the generation of a protective hydration layer, provided that a homogeneous arrangement along the polymer backbone has been achieved. For instance, a mixed-charge copolymer composed of positively charged [2-(methacryloyloxy)ethyl] trimethylammonium (TMA) and negatively charged 3-sulfopropyl methacrylate (SA) highlighted their potential excellent antifouling properties toward plasma proteins.23 Stimuli-responsive polymers, also termed intelligent polymers, are of major interest in the field of biomaterials.24-27 Poly(N-isopropylacrylamide) (polyNIPAAm) is one important temperatureresponsive polymers, and has been used alone or associated to other polymers in a number of biomedical applications including gene delivery,28 the development of gene vectors,29or as a drug carrier for chemotherapy.30 This is a nonionic polymer which is known to undergo a hydrophilic-


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hydrophobic transition in water at 32°C, referred as its lower critical solution temperature (LCST). In other words, expansion of the polymer chains resulting in swelling of the polymeric gel or on the contrary, collapsing of the chains leading to shrinkage of the gel, can be tuned by controlling the temperature. Therefore, diffusion of proteins and biomolecules within polyNIPAAm-based gels as well as interactions with these gels are strongly temperaturedependent, and so are the overall antifouling properties of these gels. Additionally, the change of conformation of polymer chains with temperature can permit to control the adhesion behavior of cells, which can even be further regulated through the incorporation of antifouling moieties.31 This tunable-bioadhesive behavior makes poly(NIPAAm) gels and their derivatives appropriate for biomedical applications. Based on these observations related to the potential excellent antibiofouling properties of pseudo-zwitterionic

copolymers

coupled

to

the

temperature-responsive

behavior

of

polyNIPAAm gels, we have conjugated mixed charge segments and polyNIPAAm, to obtain poly(NIPAAm-co-TMA/SA) hydrogels. The major goal of this study is to develop a novel multifunctional hydrogel having antifouling and environmental-responsive functionalities, as well as to study the effect of molar composition of hydrogels on these properties. Furthermore, our investigation will particularly stress on the adhesion behavior of blood cells onto the as-prepared hydrogels, as there is an important demand for the development of materials that could not only resist biofouling by proteins and bacteria, but also be hemocompatible.


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MATERIALS AND METHODS Materials. N-Isopropylacrylamide (NIPAAm) was purchased from Sigma-Aldrich and recrystallized with hexane before use in synthesis. Ammonium persulfate (APS), N,N,N′,N′tetraethylmethylenediamine (TEMED), and N,N′-methylenebisacrylamide (MBAA) were also purchased

from

Sigma-Aldrich.

3-sulfopropyl

methacrylate

potassium

salt

and

[2-

(methacryloyloxy) ethyl]trimethylammonium chloride and were bought from Sigma Chemical Co. Fibrinogen (fraction I from human plasma) was obtained from Sigma Chemical Co. Deionized water (DI water) was obtained by purifying water using a Millipore water purification system, with a minimum resistivity of 18.0 MΩ·cm. Preparation

of

Poly(NIPAAm),

Poly(NIPAAm-co-TMA/SA)

and

Poly(TMA/SA)

Hydrogels. A total solid content of 20 wt % with different molar ratios of NIPAAm (for poly(NIPAAm) gels), TMA/SA (for poly(TMA/SA) gels), or TMA/SA and NIPAAm (for poly(NIPAAm-co-TMA/SA) gels) as presented in Table 1 was dissolved in 10.2 mL of DI water containing 1.6 wt% MBAA. Then, nitrogen was bubbled through the monomers mixture to remove residual oxygen. 8.0 mg of APS and 8.0 mg (0.011 mL) of TEMED were used to initiate the copolymerization reaction. This corresponded to a [APS]/[TEMED] molar ratio of 1:2. The reaction was then carried out at 25 °C for 5 h between a pair of glass substrates, separated with a silicone rubber spacer with a thickness of 0.2 mm. Once polymerization completed, the gel was immersed in a large amount of water for 48 h, and the water was changed every 6 h to remove chemical residues. These disks were stored in DI water at 4°C until use. The chemical structure and the composition of hydrogels are presented in Scheme 1 and in Table 1, respectively.


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Scheme 1.

Molecular

structure of

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poly(NIPAAm),

poly(NIPAAm-co-TMA/SA) and

poly(TMA/SA) hydrogels. Table 1. Characteristics of poly(NIPAAm-co-TMA/SA) hydrogels Reaction ratios of comonomers (mol%)a

Compositions of hydrogelsb

Sample ID NIPAAm

TMA

SA

xNIPAAm

xTMA

xSA

N/S ratio

N100-TS0

100

0

0

1.00

0.00

0.00

-

N70-TS30

70

15

15

0.67

0.17

0.16

1.06

N50-TS50

50

25

25

0.52

0.25

0.24

1.04

N30-TS70

30

35

35

0.35

0.33

0.32

1.03

N0-TS100

0

50

50

0.00

0.51

0.49

1.02

a

Reaction mass ratios of TMA, SA and NIPAAm monomers used with fixed total monomer mass percentage of 20 wt% in the prepared reaction solution. bThe mole fraction of polyTMA (xTMA) and polySA (xSA) in the cross-linked poly(NIPAAm-co-TMA/SA) hydrogels was determined by XPS in dry state from the spectral area ratio of the atomic percentages based on N1s of polyTMA isopropyl groups and S2p of polySA side groups at the BE of approximately 401 eV and 168 eV, respectively. For N1s spectra, the characteristic peak of TMA, a quaternized ammonium, did not overlap with that of NIPAAM and MBAA, secondary amines, found at a maximum BE of about 398 eV.32


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Chemical Characterization of Hydrogels. Fourier-transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to analyze the chemical structure of dried gels surface, following procedures previously reported.31 The influence of temperature, ionic strength and pH, on swelling behaviors was assessed as follows. Concerning the effect of temperature, gels were first dried at 25°C under vacuum until they reached a constant weight. Dried gels were then weighed (WD) and subsequently immersed in DI water for 2 days at a controlled temperature: either 4°C, 25°C or 37°C. Afterwards, the excess of water onto the gels surface was gently wiped off, and the weight of wet gels recorded (WW). The swelling ratio corresponded to the ratio of the weight of water entrapped into the swollen polymer chains to the weight of the dried gel. To study the effect of ionic strength on swelling, a similar experimental procedure was followed, and the ionic strength of the immersion solution maintained at 37°C was adjusted by dissolving sodium chloride at a concentration in the range 0.1-2.5 M. Finally, in order to study the effect of pH, solutions having different pH in the range 1-11 were prepared by dissolving controlled amount of sodium hydroxide or hydrogen chloride. Hydrogels were then immersed and swelling evaluated similarly. Data presented correspond to the average of six independent measurements. Surface diiodomethane contact angles of gels were assessed at both 25°C and 37°C under water using an automatic contact angle meter (CAVP model, Kyowa Interface Science Co., Ltd, Japan). Diiodomethane was dropped at three different positions on each disk, and measurement was performed onto five disks for each testing condition. Eventually, the average of the fifteen values obtained for each condition was taken as the diiodomethane contact angle. Biofouling Tests – Protein Adsorption and Bacterial Adhesion/Detachment. Evaluation of resistance of gels to fibrinogen adsorption was assessed according to the ELISA test, which


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protocol has been reported many times. Therefore, one may want to refer to previous works for further details.17, 31 Bacterial attachment tests were also performed using three different bacteria: Escherichia coli, Staphylococcus epidermidis and Streptococcus mutans. The protocol of bacterial culture of these three species, incubation with surfaces and observation was described in former studies.31, 33 As incubation with species lasted 24 h, bacterial solution was replaced every 6 h by a fresh bacterial suspension, to ensure that bacteria contacting the gels were continuously in healthy state. Platelet Adhesion Tests. Adhesion of recalcified thrombocytes onto hydrogels was studied using the following experimental protocol. Each gel disk was first placed into individual well of a 24-well plate and equilibrated with 1 mL of PBS at a temperature of 25°C. A solution of platelet-rich plasma (PRP) was obtained by centrifuging for 10 min at 1200 rpm fresh blood. This solution, containing about 105 platelets/mL, was recalcified with 5 µL of calcium chloride solution 1M. Gels were then incubated for 2 hours at 37°C with 200 µL of recalcified PRP solution to allow adhesion of cells onto the surface of the gels. Thereafter, gels were washed twice with PBS and then immersed for 48 hours into a glutaraldehyde solution (2.5% v/v in PBS) which aimed at fixing adhering cells onto the surface of the gels. Then, gels were rinsed with PBS, and gradient dried in ethanol/PBS baths having increasing pure ethanol content (by volume) as follows: 0/100, 10/90, 50/50, 75/25, 90/10, 100/0. Immersion time in each ethanol/PBS solution was fixed to 20 minutes. Finally, gels were dried in air, and observed by scanning electron microscope (Hitachi S-3000 instrument, acceleration voltage: 7 keV). Prior to observation, gels were sputter-coated with gold. Whole Blood Adhesion Tests. Hydrogels (surface area: 0.4 cm2) disposed in a 24-well plates were equilibrated with 1 mL of PBS at 37°C for 24 hours. Then, 1 mL of fresh blood obtained


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from human volunteer and previously mixed with citrate phosphate dextrose adenine-1(7/1, v/v), was poured on each hydrogel. Incubation was performed for 2 hours at 37°C. Thereafter, gels were washed twice with PBS to remove loosely attached cells. Then, blood cells were fixed at 4°C using 300 µL of a solution of formaldehyde in PBS (4% v/v). Fixation step lasted 15 min, and a triple washing was then performed with PBS. Staining of the cells was done at 4 °C and performed for 15 min using a mixture of 3 µL CD3-FITC, 3 µL CD14-FTIC, and 3 µL CD45FITC in 270 µL of PBS with 2.5% (v/v) glutaraldehyde. Another triple washing with PBS was done before observing the surface of the gels by scanning confocal microscopy (Nikon A1R, Nikon, Japan). Images were taken at λex = 488 nm/λem = 520 nm. To monitor blood cells detachment, cells were first incubated with gels following the procedure detailed in previous paragraph. The relative percentages of cell attachment were then determined at different temperatures (4°C and 25 °C) and at different times (0, 30 min and 60 min) from the analysis of confocal microscope images obtained with the same instrument used in whole blood adhesion tests. Each test was repeated six times and percentages reported correspond to the average value obtained. Hemolytic Activity of Hydrogels. Determination of hemolysis ratio was done using isotonic PBS (10 mM, pH 7.4). Fresh blood was centrifuged to separate erythrocytes from other blood components. A saline solution at a concentration of 0.15 M was used to wash the obtained red blood cells solution. Then, gel disks were placed in a 24-well plate, and incubated at 37°C and for 1 hour with 500 µL of a PBS solution containing about 108 erythrocytes. Thereafter, blood cell solution was pipetted out of the wells, and centrifuged at 2000 rpm for 5 minutes in order to separate intact erythrocytes from disrupted cells. Hemolysis ratio, related to the amount of hemoglobin released after cell lysis, was evaluated by measuring the absorbance of the


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supernatant at 541 nm, using a PowerWave microplate spectrophotometer. Here, negative control (0% hemolysis ratio) corresponds to incubation of the cells with PBS, while positive control (100% hemolysis ratio) corresponds to the incubation of the cells with DI water. Each reported value is the average of six repeated measurements. Anticoagulant Activity of Hydrogels. Fresh human blood obtained from three healthy donors was centrifuged (1st run: 1200 rpm, 10 min, 25°C; 2nd run: 3000 rpm, 10 min, 25°C) to separate blood plasma from other blood components. Then, the supernatant phase (blood plasma) was recalcified at 37°C to a 20 mM CaCl2 concentration, using calcium from a 1M stock solution. Gels placed in individual well of a 24-well plate were incubated with 0.5 mL of recalcified plasma. To determine the plasma clotting time corresponding to a change in absorbance of the solution, a PowerWave microplate spectrophotometer equipped with a programmed temperature control was used to continuously record the absorbance at 660 nm of the plasma solution maintain at 37°C. Experiments were repeated six times for each gel, and reported value corresponds to the average obtained.

RESULTS AND DISCUSSION Characterization of Hydrogels – Influence of Environmental Stimuli on Their Swelling Behavior. Results presenting the composition of hydrogels, determined from XPS analysis performed on dry gels, are presented in Table 1, while Figure 1 displays spectra obtained from FT-IR analysis. The N/S ratio maintained to 1 highlight the efficient control of gel formation process. Furthermore, FT-IR spectra reveal that increasing the mass ratio of mixed charges gives rise to novel absorption bands at 949 cm-1, 1033 cm-1 and 1727 cm-1, attributed to TMA


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functional group,34 SO3- stretch and the carbonyl group composing both TMA and SA monomers.22, 34 The gradual increase of intensity of these three absorption bands with TMA and SA content is another indication of effective and well-controlled gel formation process.

N0-TS100

N30-TS70

Transmittance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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N50-TS50

N70-TS30

N100-TS0 -1

949 cm

800

-1

1727 cm

-1

1033 cm

1000

1200

1400

1600

1800

2000

-1

Wavenumber (cm )

Figure 1. FT-IR spectra of poly(NIPAAm), poly(NIPAAm-co-TMA/SA) and poly(TMA/SA) Hydrogels. Swelling behavior of hydrogels was studied in different conditions of temperature, ionic strength and pH. Figure 2a displays the influence of temperature. Swelling decreases with TMA/SA content, suggesting that mixed-charge groups prevent polymer chains expansion whatever the temperature tested. Secondly, there is an appreciable influence of temperature on swelling behavior for gels with high content of NIPAAm only: when temperature increases, swelling decreases. For high content of TMA and SA (from 50%), water uptake at 4°C, 25°C or 37°C was almost the same. By increasing the temperature, polymer domains of N100-TS0 and N70-TS30 collapse as hydrogen bonds between water molecules and polyNIPAAm moieties are


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weakened above the LCST of polyNIPAAm (i.e. 32°C).31 However, an important content of TMA and SA moieties (i) decreases the water uptake and (ii) cancels the influence of temperature on swelling behavior. The first observation is imparted to the cross-linking action of TMA and SA side chains. Combination of electropositive TMA and electronegative SA segments gives rise to a pseudo-zwitterionic structure, which despite its ability to trap water molecules, tends to prevent the expansion of polymer chains. An increase of temperature has no effect, because polyTMA and polySA are not temperature-sensitive. The ionic strength is another key parameter to control the water intake of the hydrogels. Results presented on Figure 2b reveal two main behaviors: gels with no pseudo-zwitterionic side chains (N100-TS0), or a low amount of pseudo-zwitterionic groups (N70-TS30) tended to shrink with the ionic strength, while those containing an appreciable content of ionic side chains (N50TS50, N30-TS70 and N0-TS100) swelled. Shrinkage of N100-TS0 and N70-TS30 is due to solvation effect of sodium and chloride ions by water molecules. Ions importantly interact with water molecules at the molecular level in all directions from a 3-dimensional space. Therefore, numerous water molecules break the species of the sodium chloride crystals apart. Therefore, the dissolved ions in the solution containing N100-TS0 and N70-TS30 tend to be solvated by the free water molecules along with water molecules originally entrapped within the polymeric structure, eventually arising in shrinkage. Obviously, the extent of shrinkage increases with the ion concentration in the immersion solution. However, the opposite phenomenon – that is swelling – was observed when increasing TMA/SA content in the polymeric structure. When no salt is added, electrostatic interactions between TMA and SA leads to a cross-linked structure (as also seen in Figure 2 on temperature effect). The presence of sodium chloride tends to weaken these interactions. As a result, TMA and SA become surrounded by water molecules (solvation


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effect), while sodium and chloride ions are entrapped in a solvated form between the electropositive and the electronegative groups, finally leading to the expansion of the polymer chains.

This

anti-polyelectrolyte

effect

was

previously

demonstrated

for

chitosan/carboxymethylcellulose hydrogels,35 and is now established for this pseudo-zwitterionic gel. Finally, a complex interplay of the pH and the gel composition on swelling was highlighted for every gel (Figure 2c) but polyNIPAAm one. In the range pH 1 to pH 9, swelling importantly decreased with TMA/SA content. The lowest swelling ratio were obtained in the pH range 3 - 9, in which pH is not significantly affecting the swelling behavior of hydrogels. At pH 11, results unveil a slight decrease of swelling ratio for all poly(NIPAAm-co-TMA/SA) hydrogels, to reach a plateau at about 4, while polyNIPAAm and poly(TMA/SA) hydrogels exhibit a similar swelling ratio (about 4.5). The pH-sensitivity behavior is ascribed to the presence of TMA and SA groups. Even though the pKa of polySA has not been determined yet, to the best of our knowledge, it is reasonable to assume that it is in the range of that of sulfurous acid, that is, 1.8. Below this value, SA would be protonated, and therefore neutral. In this respect, the gel would contain positive charges only, and electrostatic repulsions would give rise to swelling, as seen on the plot obtained at pH 1. A similar explanation based on electrostatic repulsions can be given for the evolution of swelling ratio at pH 11. At pH 11, we assumed that an excess of negative charges caused major electrostatic repulsions between side groups, responsible for swelling. In the range of pH 3-9, gels contain both positive and negative charges, no major repulsion occurs, and so, similar swelling ratios were measured.


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(a)

5.0 4.5

4°C

25°C

37°C

0M

4.5

0.1 M

1M

2.5 M

Swelling ratio (%)

4.0

3.5 3.0 2.5 2.0 1.5

3.5 3.0 2.5 2.0 1.5

1.0

1.0

0.5

0.5 N70-TS30 N50-TS50 N30-TS70 Mole ratio of one monomer to another

Swelling ratio (%)

0.0 N100-TS0

(b)

5.0

4.0 Swelling ratio (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.0 N100-TS0

N0-TS100

N70-TS30 N50-TS50 N30-TS70 Mole ratio of one monomer to another

N0-TS100

(c)

5.5 5.0 4.5 4.0 3.5

pH 1 pH 7

3.0 2.5 2.0 1.5 1.0 0.5 0.0 N100-TS0

pH 3 pH 9

pH 5 pH 11

N70-TS30 N50-TS50 N30-TS70 Mole ratio of one monomer to another

N0-TS100

Figure 2. Effect of environmental conditions on the swelling behavior of hydrogels. (a) Temperature and composition dependence. (b) Effect of ionic strength and hydrogels composition (c) Effect of pH and hydrogels composition.

Resistance to Nano-Biofouling. The adhesion of human fibrinogen, a sticky protein able to mediate bacterial attachment, or biological responses such as blood clotting was investigated. Results of ELISA test are presented in Figure 3. 100% adhesion corresponds to adhesion onto a polystyrene surface. If poly(NIPAAm) gel still exhibited 86% relative fibrinogen adsorption, a dramatic decrease of protein adsorption was observed when increasing the content of pseudozwitterionic side chains in the structure of polymeric gel, to reach 4.8% for N0-TS100 hydrogel. Figure 3 also exhibits a rise of diiodomethane contact angle, associated to TMA and SA content,


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as well as to the temperature. Furthermore, for gels mostly constituted of NIPAAm, oil repellency is more important at lower temperature, while the effect of temperature tends to decrease with TMA/SA content. Obviously, there are important relationships between protein adsorption and hydration properties of hydrogels surface, while the effect of temperature has to be associated with the temperature-responsive behavior of hydrogels presented in previous section. Even though all hydrogels present a hydrophilic nature, their composition importantly influences nano-biofouling. Poly(NIPAAm) is already known to be a low-biofouling material and it exhibits rather low protein adsorption levels, compared with hydrophobic ideal surfaces,32 but it is not an antibiofouling materials. However, by adding pseudo-zwitterionic side chains in the copolymer structure, hydrogels tend to exhibit extremely low levels of protein adsorption. The addition of TMA and SA in equimolar content results in an original structure exhibiting hydrophilic and antifouling properties similar to those of zwitterionic molecules. The diiodomethane contact angle under water increased with TMA/SA content, that is surface hydrophilicity was enhanced. Water is trapped between the electropositive and the electronegative groups, eventually arising in the repellency of organic matters approaching the polymeric structure. Also, for gels with high pseudo-zwitterionic side chains content (N30-TS70 and N0-TS100), temperature has minor impact on surface hydrophilicity, because temperatureresponsive behavior of polymer is minor for these gels (Figure 2). However, temperature does influence surface hydrophilicity for lower TMA/SA contents (N100-TS0, N70-TS30 and N50TS50), as it was demonstrated that cross-linking was less important, therefore promoting swelling. One can then predict that fibrinogen adsorption should be lower at 25°C than at 37°C, even though tests were not performed.


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86 %

140 Fibrinogen adsorption

O

25 C

O

37 C

130 80 120 60

110 100

40

90 18 %

20

10 %

0

N100-TS0

N70-TS30

N50-TS50

80 5.6 %

4.8 %

N30-TS70

N0-TS100

o

Relative protein adsorption (%)

100

Diiodomethane contact angle ( )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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70

Figure 3. Influence of hydrogel composition on their surface hydrophilicity at different temperature and their ability to resist protein adsorption. Fibrinogen adsorption test was

performed at 37°C. Resistance to Macro-Biofouling: Resistance to Bacterial Attachment. The use of microorganisms with different shapes and cell wall structures ensures a complete evaluation of the resistance to bacteria. Confocal images are shown in Figure 4 while Figure S1 presents the quantitative data associated. Attachment decreases with TMS/SA content. From N50-TS50 gel, very low level of adhesion is observed, and N0-TS100 does not exhibit any bacteria onto its surface. Decreasing of bacterial attachment directly results from resistance to nano-biofouling above discussed. Provided low physical entrapment on smooth surfaces, like those of hydrogels, bacteria chemically interact with surfaces through the establishment of hydrophobic interactions with the proteins constituting their cell walls. Then, bacteria adhering can mediate the attachment of other cells, therefore facilitating the development of biofilm. So, minimizing protein adsorption ability is critical. A number of remarkable results on resistance to bacterial attachment


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have been presented in recent studies using zwitterionic surface-modifiers.19, 36 Herein, we show that pseudo-zwitterionic polymers are also excellent materials to prevent bacterial adhesion on a surface.

Figure 4. Confocal images of bacteria adhering onto polyNIPAAm, poly(NIPAAm-co-SA/TMA) and poly(SA/TMA) hydrogels. (a) Escherichia coli, (b) Staphylococcus epidermidis, (c)

Streptococcus mutans. Hemocompatibility and Bio-Adhesion of Leukocytes. Hemocompatibility of gels is a prerequisite to their use in biomedical applications. A similar trend to that obtained for biofouling was seen on Figure 5: fewer platelets interact with surfaces by increasing TMA/SA content. From N100-TS0 gel to N50-TS50 gel, SEM image reveal important platelet adhesion and activation. Few platelets adhered to N30-TS70 gel while no platelets were found onto N0-TS100


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gel. Platelet adhesion and activation is mediated by fibrinogen adsorption,37 so that a correlation was logically found (Figure 3 and Figure 5). However, if fibrinogen adsorption on N50-TS50 gel was only 10% that measured on N100-TS0, it still presented an important level of platelet activation as seen on the SEM images, indicating that fibrinogen alone cannot explain the whole mechanisms of platelet adhesion and activation. Other proteins are involved as well, as reported in recent work.38 N30-TS70 and N0-TS100 provided better fibrinogen resistance since they contained more pseudo-zwitterionic moieties. One can reasonably assume that these two gels resist better the adsorption of every blood plasma protein, therefore explaining the quasi-absence of platelets onto their surfaces. Additionally, results of whole blood adhesion tests presented in Figure 6 show that N100-TS0 gel is totally covered by blood cells. Gels with pseudo-zwitterionic moieties exhibit improved resistance to all types of blood cell, and absolutely no cell adhered onto the surface of N0-TS100, containing 100% pseudo-zwitterionic segments, highlighting its very good hemocompatibility. Also, confocal images tend to show that pseudo-zwitterionic gels still present large cells (whose diameter is in the range 10-20 µm) onto their surface. These cells correspond to leukocytes. That would mean that despite their significantly lower concentration in whole blood, surfaces at play promote their adhesion.


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Figure 5. Effect of hydrogel composition on platelet adhesion and activation.

Figure 6. Interaction of whole blood cells with hydrogels: effect of hydrogel composition on resistance to blood cells.


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From our knowledge, pseudo-zwitterionic structures prevent the adsorption of cells according to a mechanism identical to that involved in bacterial attachment: hydration layer formed by trapping water molecules between electric charges repel hydrophobic proteins and cells. However, we also noticed that resistance to white blood cells was hard to achieve while nonfouling property by erythrocytes or thrombocytes seemed to be more readily reached. Despite concentrations in whole blood that can be several orders of magnitude larger than that of leukocytes, our qualitative data unveil that fewer “small” (erythrocytes and thrombocytes) cells could be observed. We assumed that factors including the large size of white blood cells (large cell-wall present more potential adsorption sites) and their various natures explained why these cells were still importantly adhering to the pseudo-zwitterionic gels. One could take advantage of this property to catch these cells from blood stream, leading to blood purification, since infected leukocytes can carry viruses and transmit it to uninfected cells.39 A possible supplementary explanation is that the remaining platelets still adsorbing onto the surfaces have mediated the adhesion and activation of white blood cells. Indeed, it has been reported in a number of previous papers the important interplay between platelet activation and leukocytes adhesion.40-42 Based on these works, our hypothesis is that these particular compositions (NIPAAm and TMA/SA) of hydrogels coupled to residual platelets adhering to the surface have mediated trapping of leukocytes. Further tests need to be performed to support this result though, but if confirmed, it would provide a novel direction to purify blood and remove unwanted cells from the blood stream. Hemocompatibility of pseudo-zwitterionic gels was further supported by results of Figure 7 evidencing a very low hemolysis ratio (0.5 ± 0.1%) and a long plasma clotting time (10.2 ± 1.0 min). On the contrary, poly(NIPAAm) gel (N100-TS0) showed higher hemolysis ratio, 5.0 ± 1.5


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%, close to the acceptable limit for a material to be considered as biocompatible (5%), and lower plasma clotting time (4.6 ± 1.4 min), revealing a quick biological response after the onset of blood contact with the gel. Thanks to the addition of TMA and SA in the polymeric structure, all poly(NIPAAm-co-TMA/SA) gels presented an hemolysis ratio much lower than 5%, which revealed the improvement of biocompatibility, while plasma clotting time gradually increased, the pseudo-zwitterionic heads allowing to delay the onset of blood activation. A gradual decreasing of the hemolysis ratio with the TMA/SA content was found, as mixedcharge improved blood cells repellency. Also, as the anti-thrombotic activity was improved when increasing the relative content of mixed charge brushes, the plasma clotting time was importantly delayed for the pseudo-zwitterionic gel (N0-TS100). It suggests that the pseudo-zwitterionic polymer could be used as an alternate biomaterial for systems and devices contacted with blood plasma, without important necrocytosis. Also, N30-TS70 and N0-TS100 exhibit a better anticoagulant activity than gels with lower content of TMA/SA, further supporting the role of mixed-charge polymers on hemocompatibility. Values obtained – 9.1 ± 1.0 min for N30-TS70 and 10.2 ± 1.0 min for N0-TS100 -compared with recent data reported for zwitterionic polymers.43 Finally, blood cells detachment (Figure S2) was enhanced at lower temperature, as well as with a higher content of TMA/SA brushes in the polymer backbone. The highest percentage of detachment (100%) was obtained at 4°C after 30 min and using the gels containing equal proportions of NIPAAm blocks and mixed-charge blocks. The detachment of cells from the gels surface is more efficient at 4°C than at 25°C, which is ascribed to the temperature-responsive behavior of poly(NIPAAm). At lower temperature, the hydration layer around the polymer backbones becomes thicker, weakening the hydrophobic interactions responsible for cells


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adhesion to the surface of the gels, eventually leading to their detachment. Detachment of cells is also promoted as the content of TMA/SA brushes increases, due to the nonfouling nature of these mixed-charge copolymers.

Hemolysis ratio

Plasma clotting time

Hemolysis ratio (%)

100

12 10

80 8 60 6 4 10 2

5 0

Plasma clotting time (min)

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PS

0 0 0 0 S50 -TS 0-TS3 S10 TS7 T 0 T 0 0 0 N0 N1 N3 N7 N5

0

Hydrogels

Figure 7. Evaluation of hemolytic activity of surfaces after contact with red blood cells, and effect of the nature of the substrate on plasma clotting time.

CONCLUSIONS This work presented stimuli-responsive hydrogels made of NIPAAm and mixed-charge pairs of TMA and SA. The tunable composition arose in essential properties. Hydrogels are temperature-responsive for high NIPAAm content and lose this property as TMA/SA content increases. High ionic force favors shrinkage of poly(NIPAAm) gels while anti-polyelectrolyte effect is responsible for swelling of poly(TMA/SA) gels. For pH below 3 and above 11, excess


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of positive and negative charges, respectively, gives rise to electrostatic repulsions between ionic groups, eventually responsible for swelling. Resistance to fouling due to plasma proteins and blood cells increased with molar ratio of mixed-charges in the polymer backbone. If lowbiofouling property was promoted with TMA/SA content in the molecular structure of the gels, important adhesion of leukocyte was generally observed for gels containing both NIPAAm and TMA/SA moieties. By changing the molar composition of poly(NIPAAm-co-TMA/SA) hydrogels, it is believed that gels exhibiting low-biofouling, selective biofouling or nonbiofouling properties can be prepared. Combined to low hemolytic activity and long plasma coagulation time, they can be used as potential hemocompatible material or materials promoting adhesion of leukocytes, potential carrier of viruses. In a prospective work, we intend to find the best combination of NIPAAm and TMA/SA moieties to catch white blood cells selectively.

AUTHOR INFORMATION

Corresponding Author *Yung Chang, E-mail: [email protected]; Phone: 886-3-265-4122, Fax: 886-3-265-4199. **Yu Chang, E-mail: [email protected]; Phone: 886-7-334-8732, Fax: 886-3-265-4199.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ASSOCIATED CONTENT


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Supporting Information Quantitative analysis of bacterial attachment. Blood cells detachment. This material is available free of charge via the Internet at http://pubs.acs.org/.

ACKNOWLEDGMENT Authors acknowledge the project of Outstanding Professor Research Program in Chung Yuan Christian University, Taiwan (CYCU-00RD-RA002-11757), and the Ministry of Science and Technology (MOST 103-2221-E-033 -078 -MY3) for financial support. Deanship of Scientific Research, College of Science Research Center, King Saud University, Kingdom of Saudi Arabia is also acknowledged. Yung Chang thanks King Saud University, Riyadh, Kingdom of Saudi Arabia, for the Visiting Professorship.

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TOC FIGURE


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Stimuli-Responsive and Hemocompatible Pseudozwitterionic

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