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Dual-Thermoresponsive Phase Behavior of Blood CompatibleZwitterionic Copolymers Containing Nonionic Poly(

-isopropylacrylamide)

Yung Chang,*

,†

Wen-Yih Chen,

‡

Wetra Yandi,

†

Yu-Ju Shih,

†

Wan-Ling Chu,

†

Ying-Ling Liu,

†

Chih-Wei Chu,

Ruoh-Chyu Ruaan,

‡

and Akon Higuchi

‡

R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan, Department of Chemical and Materials Engineering, National Central University, Jhong-Li, Taoyuan 320, Taiwan, and Research Center for Applied Sciences, Academia Sinica 128 Sec. 2, Academia Road, Nankang, Taipei 11529, Taiwan Received February 17, 2009; Revised Manuscript Received May 29, 2009

Thermoresponsive statistical copolymers of zwitterionic sulfobetaine methacrylate (SBMA) and nonionic

-isopropylacrylamide (NIPAAm) were prepared with an average molecular weight of about 6.0 kDa viahomogeneous free radical copolymerization. The aqueous solution properties of poly(SBMA-

-NIPAAm) weremeasured using a UV

visible spectrophotometer. The copolymers exhibited controllable lower and upper criticalsolution temperatures in aqueous solution and showed stimuli-responsive phase transition in the presence of salts.Regulated zwitterionic and nonionic molar mass ratios led to poly(SBMA-

-NIPAAm) copolymers having double-critical solution temperatures, where the water-insoluble polymer microdomains are generated by the zwitterioniccopolymer region of polySBMA or nonionic copolymer region of polyNIPAAm depending on temperature. Ahigh content of the nonionic polyNIPAAm in poly(SBMA-

-NIPAAm) exhibits nonionic aggregation at hightemperatures due to the desolvation of polyNIPAAm, whereas relatively low content of polyNIPAAm inpoly(SBMA-

-NIPAAm) exhibits zwitterionic aggregation at low temperatures due to the desolvation of polySBMA. Plasma protein adsorption on the surface coated with poly(SBMA-

-NIPAAm) was measured witha surface plasmon resonance (SPR) sensor. The copolymers containing polySBMA above 29 mol % showedextremely low protein adsorption and high anticoagulant activity in human blood plasma. The tunable and switchablethermoresponsive phase behavior of poly(SBMA-

-NIPAAm), as well as its high plasma protein adsorptionresistance and anticoagulant activity, suggests a potential for blood-contacting applications.

Introduction

Blood compatibility is highly recommended for blood-contacting materials in important biomedical applications, suchas antithrombogenic implants, hemodialysis membranes, andbiosensors.

However, only a small number of synthesizedbiomaterials are regarded as good blood-compatible candidates.Zwitterionic polymers containing the pendant groups of phos-phobetaine, sulfobetaine, and carboxybetaine have receivedgrowing attention for use in the new generation of blood-contacting materials because of their good plasma proteinresistance.

4,7

In the last several years, poly(sulfobetainemethacrylate) (polySBMA) with a methacrylate main chain andan analogue of the taurine betaine pendant group (CH

-(CH

)

) has become the most widely studiedzwitterionic polymer due to its ease of synthetic prepara-tion.

4,8,10,12

It was reported that the surfaces grafted withpolySBMA reduced ﬁbrinogen adsorption to a level comparablewith the adsorption on poly(ethylene glycol)-grafted ﬁlms inour previous studies.

8,15

We also grafted a dense polymer brushof polySBMA on a gold surface via surface-initiated atomtransfer radical polymerization and suggested that zwitterionicpolySBMA is an effective and stable nonbiofouling material toprovide a surface for use in human blood and implants.

In general, intelligent biocompatible polymers can representdiverse forms, which might be dissolved as unimers or micellesin an aqueous medium, adsorbed or grafted on aqueous

solidinterfaces, or cross-linked in the form of physical or chemicalhydrogels.

These polymers can undergo large physicalchain conformation changes to small environmental stimuli of physical, chemical, or biochemical nature. Poly(

-isopropyl-acrylamide) (polyNIPAAm) is the most widely studied ther-moresponsive polymer.

This nonionic polymer undergoesa sharp hydrophilic

hydrophobic transition in water at 32

C;this temperature is called the lower critical solution temperature(LCST).

The solution properties of zwitterionic polymers differconsiderably from those of nonionic polymers. In aqueoussolution, polySBMA, like other zwitterionic polymers, exhibitsan upper critical solution temperature (UCST) that increaseswith the molar content.

This is attributed to the charge

chargeor dipole

dipole interactions between the betaine groups. Thecombination of the UCST of the zwitterionic polymers withthe LCST of the polyNIPAAm displays intriguing temperature-induced self-assembly behavior of different types of polymericaggregates in aqueous solution.

24,29

To further develop the zwitterionic-based materials forbiomedical applications, we were inspired to study smartpolymer systems carrying both controllable biocompatibility andstimuli-responsive functions. Recently, some research worksreported physical micellization of synthesized diblock copoly-

* To whom correspondence should be addressed. E-mail: ychang@cycu.edu.tw.

†

Chung Yuan Christian University.

‡

National Central University.

Research Center for Applied Sciences.

Biomacromolecules

XXXX,

xxx,

000

10.1021/bm900208u CCC: $40.75



XXXX American Chemical Society

D o w n l o a d e d b y T A I W A N C O N S O R T I A o n J u l y 2 6 , 2 0 0 9 P u b l i s h e d o n J u l y 2 , 2 0 0 9 o n h t t p : / / p u b s . a c s . o r g | d o i : 1 0 . 1 0 2 1 / b m 9 0 0 2 0 8 u

mers with thermoresponsive and zwitterionic properties.

29,30,32

These block copolymers were found to exhibit double ther-mosensitive phase transition of LCST and UCST behaviors inwater. However, these studies did not extend to the use orevaluation of these copolymers as biological or biomedicalmaterials. An early work reported the biocompatible nature of SBMA-based statistical copolymer coatings as potential anti-bioadherent surface coatings.

In this work, an interestingcombination of the zwitterionic polySBMA and nonionicthermoresponsive polyNIPAAm was studied as an example of intelligent biocompatible polymers, especially for their human-blood-contacting properties. The effect of copolymer concentra-tions, solvent polarities, and ionic strengths on the LCST andUCST of poly(SBMA-

-NIPAAm) in aqueous solution withdifferent monomer ratios of SBMA and NIPAAm are discussedin detail. This study also demonstrates the adsorption of plasmaproteins on the surface coated with poly(SBMA-

-NIPAAm)from human blood plasma via surface plasmon resonance (SPR),and the anticoagulant activity of the copolymers in a platelet-poor plasma solution by recalciﬁed plasma clotting tests. Thiswork is aimed at addressing two important issues of poly(SBMA-

-NIPAAm), that is, (i) systematic measurement of the LCSTor UCST characteristics from various copolymer compositionsat different copolymer concentrations, solvent polarities, andionic strengths and (ii) in vitro evaluation of blood compatibilityof the fully coated copolymer surface and copolymer suspensionusing human plasma solution.

Materials and Methods

Materials.

[2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)-am-monium hydroxide (sulfobetaine methacrylate, SBMA) macromonomerwas purchased from Monomer-Polymer and Dajac Laboratories, Inc.,U.S.A.

-Isopropylacrylamide (NIPAAm) from Sigma-Aldrich wasrecrystallized with hexane. Ammonium persulfate (APS),

′

-tetraethylmethylenediamine (TEMED), copper(I) bromide (99.999%),2-bromoisobutyryl bromide (BIBB, 98%), pyridine (98%), 2-hydroxy-ethyl acrylate (97%), 2,2-bipyridine (BPY, 99%), triethylamine (99%),tetrahydrofuran (THF HPLC grade), and ethanol (absolute 200 proof)were purchased from Sigma-Aldrich. 1-Undecanethiol (99

%), (1-mercapto-11-undecyl)tetra(ethylene glycol) (99

%), and 11-mercapto-1-undecanol (99

%) were purchased from Asemblon INC in Redmond,Washington, U.S.A. Fibrinogen (fraction I from human plasma),

-globulin (fractions II, III, 99%), and human serum albumin (HSA,96

99%) were purchased from Sigma Chemical Co. Acetone andmethanol were of analytical grade, purchased from Sigma ChemicalCo. Deionized water (DI water) used in experiments was puriﬁed usinga Millipore water puriﬁcation system with a minimum resistivity of 18.0 M

Ω

m. THF for reactions and washings was dried by sodiumbefore use.

-mercaptoundecyl bromoisobutyrate was synthesizedthrough a reaction of BIBB using a method published previously byour group.

4,10

H NMR (300 MHz, CDCl

): 4.15 (t,

)

6.9, 2H, OCH

),2.51 (q,

)

7.5, 2H, SCH

), 1.92 (s, 6H, CH

), 1.57

1.72 (m, 4H,CH

), and 1.24

1.40 (m, 16H, CH

Preparation of Poly(SBMA-

-NIPAAm) in Aqueous Solu-tion.

A total solid content of 8 wt % for different mass ratios of SBMAand NIPAAm (Table 1) was dissolved in 10.2 mL of DI water, andnitrogen was bubbled through to remove residual oxygen. Thecopolymerization of poly(SBMA-

-NIPAAm) was initiated using 8.0mg of APS and 8.0 mg (0.011 mL) of TEMED. The relative molarratio of [APS]/[TEMED] was 1:2. The reaction was stirred underpositive nitrogen pressure for 6 h at 23

C. After polymerization, theresulting reaction solution was cooled to 4

C for 3 h and then addedslowly into acetone and redissolved into DI water repeatedly toprecipitate the polymer out of the reaction solution and to removeresidual chemicals. Finally, the copolymer was dried in a vacuum ovenat room temperature (23

C) to yield a white powder.

Preparation of Self-Assembled Monolayers on Gold Sur-faces.

Two self-assembled monolayers (SAMs) were formed on thesubstrates: (1) methyl-terminated (CH

) and (2) initiator

-mercap-toundecyl bromoisobutyrate (Br) SAMs. Glass chips were ﬁrst coatedwith an adhesion-promoting chromium layer (thickness 2 nm) and asurface plasmon active gold layer (48 nm) by electron beam evaporationunder vacuum. Before SAM preparation, the gold-coated glass substratewas cleaned by washing with pure ethanol and DI water in sequence,dried with N

, then left in a UV light cleaner for 20 min at a sourcepower of 110 W, followed by rinsing with DI water and ethanol, andﬁnally dried again by N

. For preparation of CH

SAMs, the cleanedchip was soaked in a 2 mM ethanol solution of 1-undecanethiol or(1-mercapto-11-undecyl) and tetra(ethylene glycol) thiols for 24 h toform SAMs on the gold surface, and the chip was rinsed in sequencewith ethanol and water and then dried in a stream of N

. For thepreparation of an initiator SAM on a gold surface, the cleaned chipwas soaked in a 2 mM ethanol solution of

-mercaptoundecylbromoisobutyrate for 24 h to form Br-SAMs on the gold surface andthen rinsed with pure ethanol followed by THF and dried in a streamof N

4,10

Preparation of SBMA and NIPAAm Polymer Brushes onGold Surfaces.

Dense polymer brushes of polySBMA and polyNIPAAmon an SPR sensor chip were achieved via the surface-initiated ATRPmethod, which were prepared by the following method, as reportedpreviously.

4,10,36

Polymer brushes were polymerized on gold substrateswith immobilized initiators of Br-SAMs based on our previousreports.

4,10

The reaction solutions of CuBr and BPY were ﬁrst placedinto a sealed glass reactor in a drybox under nitrogen atmosphere. 200mM of degassed solution (pure water and methanol at a 1:3 volumeratio) with SBMA or NIPAAm monomers was transferred to the reactor,and the gold surface with immobilized initiators was then placed intothe reactor under nitrogen. After polymerization, the substrate wasremoved and rinsed with ethanol and water, and the samples were keptin water overnight. The prepared substrates were usually rinsed withPBS buffer to remove unbound polymers before any experiments. Thethickness of the substrates was measured by ellipsometry. The thickness

Table 1.

Characteristic Data of Poly(SBMA-

-NIPAAm) Statistical Copolymersreaction ratios ofcomonomers

(wt %)compositions ofcopolymers

(mol %)characterization ofcopolymers

(g/mol)critical solutiontemperature

(

C)sample ID SBMA NIPAAm polySBMA polyNIPAAm

UCST LCSTS100-N0 100 0 100.0 0.0 6336 3.6 27S70-N30 70 30 45.3 54.7 5818 3.4 18S50-N50 50 50 29.0 71.0 6262 3.5 15 41S30-N70 30 70 15.0 85.0 6649 3.5 37S0-N100 0 100 0.0 100.0 6951 3.5 32

Reaction mass ratios of SBMA and NIPAAm monomers used with ﬁxed total monomer mass amount of 0.8 g in the prepared reaction solution.

Thecomposition of the poly(SBMA-

-NIPAAm) copolymers was estimated by

H NMR in D

O from the relative peak area of (CH

)

proton resonance ofthe polySBMA side groups at

)

3.2 ppm and that of the methyl proton resonance of the polyNIPAAm isopropyl groups at

)

1.14 ppm.

Weight-averagemolecular weights (

) and molecular weight distributions (

) were estimated by GPC and calibrated with PEO.

UCST and LCST were determinedby reading the absorbance at 230 nm on a UV

visible spectrophotometer.

Biomacromolecules, Vol. xxx, No. xx, XXXX

Chang et al.

of polySBMA brushes and polyNIPAAm brushes was able to becontrolled from 10 to 15 nm, as measured by ellipsometry. The chemicalcharacterization of polymer brushes is described in detail via X-rayphotoelectron spectroscopy and surface contact angle measurementsin our previous publications.

4,10

Characterization of the Copolymers.

The structure of poly(SBMA-

-NIPAAm) statistical copolymers was characterized by

H NMRspectra using a 500 MHz spectrometer and D

O as the solvent. Thecomposition of the poly(SBMA-

-NIPAAm) copolymers was esti-mated by

H NMR in D

O from the relative peak area of (CH

)

proton resonance of the polySBMA side groups at

)

3.2 ppm andthat of the methyl proton resonance of the polyNIPAAm isopropylgroups at

)

1.14 ppm. Molecular weights of prepared statisticalcopolymers were determined by aqueous gel permeation chromatog-raphy (GPC) using one column of Viscogel TM GMPW

K0105 (themolecular weight range was from 500 Da to 800 kDa) connected to aWaters 2414 refractive index detector at a 1.0 mL/min ﬂow rate and acolumn temperature of 23

C. The eluent was an aqueous solutioncomposed of 0.1 M NaCl at pH 7.4. Poly(ethylene oxide) (PEO)standards from Polymer Standard Service, Inc. (Warwick, U.S.A.) wereused for calibration.

Determination of UCST and LCST for the Copolymers.

Thephase transition temperatures (UCST or LCST) of the aqueous solubleand insoluble copolymers at various copolymer concentrations, solventpolarities, and ionic strengths of the solution conditions were determinedby reading the absorbance at 230 nm on a UV

visible spectropho-tometer using SpectraMax M5 from Molecular Devices. A givenconcentration of prepared copolymer was dissolved in an aqueoussolution at 23

C. The temperature of polymer solution in a well wascontrolled using a heating circulator and a cooler. The temperature wasﬁrst cooled from 23 to 1

C and then gradually raised from 1 to 70

C,and the absorbance value was read for every 1

C increment of eachsample after a 10 min thermal equilibration. The UCST or LCST in aparticular solution condition was defined as the temperature where themaximum slope for the absorbance versus temperature curve occurs.The confidence in the accuracy of the measured values of UCST orLCST at 230 nm was justified by the phase transition temperatures of polyNIPAAm determined at 600 nm. It was shown that the LCST valuesof polyNIPAAm determined by reading the absorbance at 230 and 600nm were identical over a range of concentrations of sodium chloridefrom 0.01 to 2.0 M, consistent with the observation by Hoffman et al.that an increase in the sodium chloride concentration led to a decreasein LCST values of polyNIPAAm.

Plasma Protein Adsorption.

A custom-built SPR biosensor with afour-channel Teﬂon ﬂow cell, designed by the Institute of Photonicsand Electronics Academy Sciences (Prague, Czech Republic) basedon wavelength interrogation, was used to monitor protein adsorptionon the coated substrate.

An SPR chip was attached to the base of theprism, and optical contact was established using a refractive indexmatching fluid (Cargille). A protein solution of 1.0 mg/mL humanfibrinogen in a phosphate buffer saline (PBS, 0.15 M, pH 7.4) wasdelivered to the surfaces at a flow rate of 0.05 mL/min at 37

C. Inthis study, platelet poor plasma (PPP) solution containing plasmaproteins was also tested on the coated substrate. A surface-sensitiveSPR detector was used to monitor protein

surface interactions in realtime. The wavelength shift was used to measure the change in thesurface adsorption amount (mass per unit area). The calibration of thewavelength shift from SPR data associated with the amount of adsorbedprotein was calculated based on equations established by Campbell andco-workers.

The calibration follows the standard calculation for thesame custom-built SPR system, with a 1 nm wavelength shift resultingin an SPR response equivalent to about 15 ng/cm

of adsorbedproteins.

7,38

Plasma Clotting Time.

The anticoagulant activity of preparedcopolymers in this work was evaluated by testing plasma clotting timein human plasma solution. PPP solution was prepared by centrifugationof the human blood at 3000 rpm for 10 min at 23

C. Next, 160

L of the human PPP solution was mixed with the polymer solution (10 mg/ mL, 46

L) in a 96-well plate. The solution was then recalciﬁed byaddition of calcium (1 M CaCl

, 4

L) and agitation for 30 s. Twoincubation temperatures were tested, 23 and 37

C. The clotting timeof the plasma was determined at the time when the onset of theabsorbance transition occurs by reading the absorbance at 660 nm usinga PowerWave microplate spectrophotometer with programmed tem-perature control. Each clotting time is an average value of ﬁve samplesfrom repeated measurements.

Results and Discussion

Three zwitterionic-based copolymers with differing SBMAmolar contents of copolymers (S70-N30, S50-N50, and S30-N70) were prepared from various monomer compositions in thereaction solution. Molecular weight distributions of the synthe-sized poly(SBMA-

-NIPAAm) copolymers from GPC werecalculated using Empower Pro from Waters, as shown in Table1. All copolymer samples were controlled with a similar averagemolecular weight of about 6.0 kDa and the same broadmolecular weight distribution (i.e.,

)

3.4

3.6). Theincreasing amount of NIPAAm monomers in the reactionsolution increased the molar mass ratio of polyNIPAAm in theprepared copolymer. The composition of the poly(SBMA-

-NIPAAm) copolymers was estimated by

H NMR in D

O. Atypical spectrum for S50-N50 is shown in Figure 1. Resultsshowed that a pure poly(SBMA-

-NIPAAm) copolymer wasobtained. It is noted that the molar ratio of polySBMA in theprepared copolymers is only 42 mol % even though the amountof SBMA monomers used in the reaction solution is as high as70 wt %, indicating higher polymerized reactivity of NIPAAmmonomers than of SBMA monomers in water. For comparison,two homopolymers of polySBMA (S100-N0) and polyNIPAAm(S0-N100) were also synthesized as references. To determinethe soluble-insoluble phase transition of zwitterionic-basedcopolymers, the optical absorbance of dilute copolymer solutionwas measured using a UV

visible spectrophotometer withprecise temperature control from 1 to 70

C. All preparedsamples exhibited clear phase transitions associated with thecompositions of copolymers, even though their molecular weightdistributions were poorly controlled by conventional free radicalpolymerization. It is generally acceptable that self-assembledmonolayers (SAMs) presenting hydrophobic methyl groupsusually induce large amounts of protein adsorption. Therefore,a self-assembly method was used to create one dense surfacewith CH

SAMs as references for the SPR study. To characterizethe blood compatibility of zwitterionic-based copolymers, preparedcopolymers were ﬁrst physically adsorbed onto the SPR sensorsurfaces covered by CH

-terminated SAMs, followed by the insitu evaluation of plasma protein adsorption on the surfaces withself-assembled poly(SBMA-

-NIPAAm) copolymers. For com-parison, the surface-initiated atom transfer radical polymerization(ATRP) method was also used to create two well-packed graftedsurfaces with polySBMA and polyNIPAAm polymer brushes asreferences.

Phase Transition Temperatures of Poly(SBMA-

-NIPAAm) Copolymers in Aqueous Solution.

The phasetransition temperature of the copolymer solution was measuredusing UV

visible spectrophotometer coupled to a temperaturecontroller at 1

C/10 min. No thermal hysteresis was observedat equilibrium temperature from heating or cooling the copoly-mer solution. The maximum slope of a phase transition curvefrom the change of absorbance (

) signal as a function of temperature (d

)

0) was determined by referring to theUCST or LCST that was recorded for each sample. As shown

Blood Compatible Zwitterionic Copolymers

Biomacromolecules, Vol. xxx, No. xx, XXXX