Ion and Temperature Sensitive Polypeptide Block Copolymer

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Ion and Temperature Sensitive Polypeptide Block Copolymer Jae Hee Joo, Du Young Ko, Hyo Jung Moon, Usha Pramod Shinde, Min Hee Park, and Byeongmoon Jeong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm500942p • Publication Date (Web): 02 Sep 2014 Downloaded from http://pubs.acs.org on September 3, 2014

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Ion and Temperature Sensitive Polypeptide Block Copolymer

Jae Hee Joo, Du Young Ko, Hyo Jung Moon, Usha Pramod Shinde, Min Hee Park, and Byeongmoon Jeong*

Department of Chemistry and Nano Science, Global Top 5 Program, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea

* E-mail: [email protected]

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ABSTRACT: Poly(ethylene glycol)/poly(L-alanine) multiblock copolymer incorporating ethylene diamine tetraacetic acid ([PA-PEG-PA-EDTA]m) was synthesized as an ion/temperature dual stimuli-sensitive polymer, where the effect of different metal ions (Cu2+, Zn2+, and Ca2+) on the thermogelation of the polymer aqueous solution was investigated.

The dissociation constants between the metal ions

and the multiblock copolymer were calculated to be 1.2x10-7 M, 6.6x10-6 M, 1.2x10-4 M, and for Cu2+, Zn2+, and Ca2+, respectively, implying that the binding affinity of the multiblock copolymer for Cu2+ is much greater than Zn2+ or Ca2+. Atomic force microscopy and dynamic light scattering of the multiblock copolymer containing metal ions suggested the micelle formation at low temperature, which aggregated as the temperature increased.

Circular dichroism spectra suggested that changes in the α-

helical secondary structure of the multiblock copolymer was more pronounced by adding Cu2+ than other metal ions.

The thermogelation of the multiblock copolymer

aqueous solution containing Cu2+ was observed at a lower temperature, and the modulus of the gel was significantly higher than the system containing Ca2+ or Zn2+, in spite of the same concentration of the metal ions and their same ionic valence of +2.

Above

results suggested that strong ionic complexes between Cu2+ and the multiblock copolymer not only affected the secondary structure of the polymer but also facilitated

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the thermogelation of the polymer aqueous solution through effective salt-bridge formation even in a millimolar range of the metal ion concentration.

Therefore,

binding affinity of metal ions for polymers should be considered first in designing an effective ion/temperature dual stimuli-sensitive polymer.

INTRODUCTION Addition of counter ions to ionic polymers induces electrostatic interactions between ions and polymers, therefore the precise control of ionic interactions can be a promising method in designing a novel functional biopolymer.1-2

Depending on the population

and strength of the ionic interactions, phase transition of a polymer aqueous solution may occur.

For example, the aqueous solution of bipyridine-conjugated dendritic

poly(ethylene glycol) (PEG) turned into a hydrogel upon addition of silver ions through the complex formation between bipyridines and silver ions.3

The aqueous solution of

diglutamate (E2) end-capped polypeptide, E2(SL)6E2GRGD, turned into a hydrogel by adding Mg2+, where β-sheet-based fibrous nanostructures were developed after addition of Mg2+ to the polypeptide aqueous solution. 4

A triazol cobalt complex underwent a

change in the coordination number when the temperature increased; sol-to-gel transition of the aqueous solution of the compound was induced through the transition of

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octahedral complex at low temperatures (27 oC).5

A poly(acrylic acid)-g-ferrocene aqueous solution turned into

a gel by adding poly(acrylic acid)-g-β-cyclodextrin only when the iron was in a reduced (Fe2+) state; the gel reversibly turned into a solution by the oxidation of Fe2+ into Fe3+.6 The ferrocene with Fe2+ could fit into the cavity of β-cyclodextrin, whereas the ferrocene with Fe3+ could not fit into the cavity of β-cyclodextrin.

Some biopolymers

such as pectin and alginate undergo sol-to-gel transition by Ca2+.7

The interchain

association, so called egg box dimerization, followed by lateral association of the dimers was suggested for the gelation mechanism.

The sol-gel transitions of all the

above systems are based on the metal ion mediated complexation or decomplexation mechanism. Thermogelation, where an polymer aqueous solution undergoes sol-to-gel transition triggered by thermal energy, has been extensively investigated due to its potentials for injectable drug delivery and tissue engineering applications.8-11

The sol-

to-gel transition temperature and the gel modulus are key parameters in designing a thermogelling biomaterial because they determine injectability, formulation temperature, applicability, and even the fates of incorporated cells or stem cells.9,11-14 In this study, we designed an ion/temperature dual stimuli-sensitive polymer.

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The polymer aqueous solution undergoes sol-to-gel transition which is supposed to be modulated by the specific cations as well as temperature.

In particular, we also assume

that the subtle difference in binding affinity of various metal ions for the polymer can affect the sol-to-gel transition behavior. To the best of our knowledge, there is no comparative study so far on thermogelation depending on the difference in binding affinity between metal cations and polymers. To prove this concept, we synthesized a poly(ethylene glycol)/poly(L-alanine) multiblock copolymer connected with ethylene diamine tetraacetic acid (EDTA) ([PA-PEG-PA-EDTA]m), and the thermogelation behavior was investigated for the selected metal ions of Ca2+, Zn2+, and Cu2+ which have different affinity for EDTA.15

EXPERIMENTAL SECTION Materials. α,ω-diamino-PEG (Mn = 2,000 Daltons) (ID Bio, Korea) and Ncarboxy anhydrides of L-alanine (KPX Life Science, Korea) were used as received. Anhydrous N,N-dimethyl formamide and ethylene diamine tetraacetic anhydrides, copper (II) chloride, zinc (II) chloride, and calcium chloride were used as received from Sigma-Aldrich, USA.

Chloroform (Sigma-Aldrich, USA) was treated with anhydrous

magnesium sulphate before use.

Toluene (Aldrich) was dried over sodium before use.

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Synthesis of Multiblock Copolymer. First, poly(L-alanine)-PEG-poly(L-alanine) (PA-PEG-PA) triblock copolymers were synthesized by the previously published method.12,16

Briefly, the PEG (6.10 g, 3.05 mmole) was dissolved in dried toluene (70

mL), and the residual water of PEG was removed by azeotropic distillation of the polymer solution to a final volume of 10 mL.

After the system was cooled to 40 oC

under a dry nitrogen atmosphere, N-carboxy anhydrides of L-alanine (3.01 g, 26.17 mmole) were added with the anhydrous chloroform/N,N-dimethyl formamide (29/1 v/v) cosolvent (50 mL).

The reaction mixtures were stirred at 40 oC for 12 hours. The

resulting PA-PEG-PA triblock copolymer was purified by fractional precipitation into diethyl ether/n-hexane, followed by evaporation of the residual solvent under vacuum. The composition and molecular weight of the PA-PEG-PA triblock copolymer were calculated by 1H-NMR spectra and gel permeation chromatogram. To prepare the multiblock copolymer of [PA-PEG-PA-EDTA]m, PA-PEG-PA (5.35 g, 2.07 mmole) was dissolved in anhydrous chloroform (60 mL), and the amino endgroups of the triblock copolymer reacted with an equivalent amount of ethylene diamine tetraacetic anhydrides dissolved in N,N-dimethyl formamide (10 mL).

The reaction

mixtures were stirred at 40 oC for 12 hours under dried nitrogen atmosphere. Triethylamine (1.30 mL, 9.32 mmole) was used as a catalyst.

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The resulting multiblock

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polymer was purified by fractional precipitation into diethyl ether/n-hexane, followed by evaporation of the residual solvent under vacuum. Then, the multiblock copolymer was dialyzed in water using a membrane with a molecular weight cut-off of 2,000 Daltons, and then freeze-dried.

The composition and molecular weight of the

multiblock copolymer were calculated by 1H-NMR spectra and gel permeation chromatogram. 1H-NMR

Spectroscopy. 1H-NMR spectra (in CF3COOD) of PEG, PA-PEG-PA and

[PA-PEG-PA-EDTA]m were investigated by the 500 MHz NMR spectrometer (Varian) to determine the composition and molecular weight of each block of the polymers. Gel Permeation Chromatography. A gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distributions of the polymers. formamide was used as an eluting solvent.

N,N-dimethyl

The PEGs and poly(ethylene oxide)

(PEOs) (Polysciences, Inc.) with a molecular weight range of 200-90,000 Daltons were used as the molecular weight standards.

A KW 802.5 column (Waters) was used.

Determination of Dissociation Constant. To the murexide aqueous solution (50 µM), Cu2+, Zn2+, and Ca2+ were added to a final concentration ranges of 0-400 µM, 04,000 µM, and 0-60,000 µM, respectively to get the UV-vis spectra of the murexide in

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free and bound states. To determine the dissociation constant between metal ion and [PA-PEG-PA-EDTA]m, the polymer aqueous solution (equivalent to 100 µM EDTA) was added to the murexide aqueous solution containing Cu2+, Zn2+, or Ca2+ (100 µM), and the murexide concentrations in free and bound states were calculated by UV-vis spectra.17-19 Atomic Force Microscopy (AFM). The multiblock copolymers aqueous solution (0.01 wt.%) containing Cu2+, Zn2+, or Ca2+ in an equivalent amount to the EDTA of the polymers were spin-coated on the silicone wafer at 5000 rpm for 60 seconds, and then dried in air. The images of the polymers on the silicone wafer were obtained in a tapping mode by AFM (Veeco Dimension 3100, Digital instruments Ltd, USA).

The

tapping mode AFM probe with a force constant of 20 N/m was scanned over the sample. Circular Dichroism (CD) Spectroscopy. Ellipticities of the multiblock copolymer ([PA-PEG-PA-EDTA]m) aqueous solution (5.0x10-3 wt% corresponding to 17.5 µM of EDTA) were investigated as a function of cupper (II) ion concentration at 0.0, 8.75, 11.7, and 17.5 µM at 5 oC. To eliminate any contribution of CD spectra by free metal ions, the ellipticity of the aqueous solution containing free metal ions at 17.5 µM was also checked as a control experiment.

In addition, ellipticity of the multiblock

copolymer aqueous solution (5.0x10-3 wt% ) containing equivalent amount (17.5 µM) of

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Cu2+, Zn2+, or Ca2+ were investigated by a CD instrument (J-810; JASCO, USA) at 5 oC. Dynamic Light Scattering. Apparent sizes of the multiblock copolymer ([PAPEG-PA-EDTA]m) aqueous solution (0.01 wt.%) containing equivalent amount of metal ions were studied by a dynamic light scattering instrument (ALV 5000-60x0) as a function of temperature. each temperature.

The aqueous solutions were equilibrated for 20 minutes at

A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm

was used as a light source. The results of dynamic light scattering were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient.

From the diffusion coefficient, the apparent

hydrodynamic size of a polymer aggregate could be obtained by the Stokes-Einstein equation. Dynamic Mechanical Analysis. Changes in modulus of the multiblock copolymer ([PA-PEG-PA-EDTA]m) aqueous solution (5.0 wt.%, corresponding to 17.5 mM of EDTA) at pH=7.4 containing various salts (17.5 mM) of copper (II) chloride, zinc (II) chloride, or calcium chloride were investigated as a function of temperature by the dynamic mechanical analyzer (Thermo Haake, Rheometer RS 1).

In addition, changes

in modulus of the multiblock copolymer aqueous solution containing various copper (II) chloride concentrations at 0.0 mM, 11.7 mM, or 17.5 mM was also studied as a function

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of temperature.

The above polymer/metal ion aqueous solutions in a sol state (4 oC)

were placed between parallel plates of 25 mm diameter with a gap of 0.5 mm. The data were collected under conditions of controlled stress (4.0 dyne/cm2) and a frequency of 1.0 rad./s.

The heating rate was 0.5 oC/min.

To minimize water evaporation

during the experiment, the plates were enclosed in a water saturated chamber.

RESULTS AND DISCUSSION The multiblock copolymer ([PA-PEG-PA-EDTA]m) was synthesized in two-step reactions (Scheme 1).

First, ring-opening polymerization of N-carboxy anhydrides of

L-alanine on α,ω-diamino-PEG (Mn = 2,000 Daltons) produced PA-PEG-PA triblock copolymers with α,ω-diamino end-groups.12,16 Second, the amino end-groups of the PA-PEG-PA reacted with ethylene diamine tetraacetic anhydrides to produce the multiblock copolymers incorporating EDTA.

Scheme 1

The composition and molecular weight of the multiblock copolymer were calculated from 1H-NMR spectra (in CF3COOD) (Figure 1a). First, the molecular

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weight of the PA-PEG-PA triblock copolymer was determined by the integration area of the peaks at 1.3-1.9 ppm (A1.3-1.9, methyl peak of PA) and 3.7-4.2 ppm (A3.7-4.2, ethylene glycol peak of PEG).12,16 The peaks appeared at 1.5-1.7 ppm and 1.8-1.9 ppm are methyl protons of internal alanine and terminal alanine of PA-PEG-PA, respectively.20 The x, number of repeating units of ethylene glycol, can be calculated by the molecular weight of PEG divided by the molecular weight of the repeating unit of 44.

Therefore,

x in scheme 1 is given by 44.1.

From the equation of A3.7-4.2/A1.3-1.9 = 4x/6y = 176.4/6y,

the y was calculated to be 4.1.

Therefore, the molecular weights of each block of PA-

PEG-PA were calculated to be 290-2000-290 Daltons, corresponding to the structure of (L-Ala)4.1-(EG) 44.1-(L-Ala)4.1.

The methyl peak of the terminal unit of the PA-PEG-PA

triblock copolymer appearing at 1.8-1.9 ppm moved to1.5-1.7 ppm after the coupling reaction between the PA-PEG-PA triblock copolymers and ethylene diamine tetraacetic anhydrides, indicating that the terminal alanine of PA-PEG-PA behaves as an internal alanine unit after the coupling reaction.

The connecting methylene groups of PA-PEG-

PA (d in the figure 1a) and methylene groups of EDTA (e in the figure 1a) appeared at 4.2-4.4 ppm and 4.5-4.6 ppm respectively.

The ethylene peak of EDTA (c in the figure

1a) was incorporated into the PEG peak at 3.8-4.2 ppm.

Considering the structure of

the multiblock copolymer ([PA-PEG-PA-EDTA]m) in scheme 1, the ratio of number of

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protons of PA at 1.5-1.7 ppm (methyl group of alanine, a in the figure 1a) to the number of protons of EDTA at 4.2-4.4 ppm (methylene group of EDAT, d in the figure 1a) is theoretically 6y/4= 1.5y = 6.15.

The integration ratio of methyl peak of PA at 1.5-1.7

ppm to methylene peak of EDTA at 4.2-4.4 ppm was 6.1, confirming that the multiblock copolymer ([PA-PEG-PA-EDTA]m) practically has one-to-one composition between EDTA and PEG-PA-PEG. The progress of reaction was also followed-up from the gel permeation chromatograms, where the retention time decreased from 19.8 -> 19.5 -> 11.5 minutes as the reaction proceeded PEG -> PA-PEG-PA -> [PA-PEG-PA-EDTA]m (Figure 1b). The gel permeation chromatogram of both PA-PEG-PA and [PA-PEG-PA-EDTA]m exhibited a little skewed shape, which might come from the interactions of carboxylic acids or amines of the polymer with the surface of stationary phase of the GPC column. Retention time of the multiblock copolymer corresponds to that of PEG with a molecular weight of 35,000 Daltons.

Considering the molecular weight (2840

Daltons) of a repeating unit of the [PA-PEG-PA-EDTA]m, the number of repeating units (m) of the multiblock copolymer described in the scheme 1 might be approximated to 12. The unimodal distribution of the molecular weights of both PA-PEG-PA triblock copolymers and [PA-PEG-PA-EDTA]m multiblock copolymers indicated that the

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polymers were well-prepared.

Figure 1

The dissociation constants between [PA-PEG-PA-EDTA]m and metal cations were calculated using UV-vis spectroscopy of a dye (murexide).

Free murexide exhibited an

absorption band at 524 nm, and a blue shift to 463 nm was observed after binding to Cu2+. The absorption spectra of the dye after (thick brown curve) and before (thin brown curve) adding [PA-PEG-PA-EDTA]m (100 µM, S100) to an aqueous solution containing dye (50 µM) and Cu2+ (100 µM) were compared (Figure 2a).

After adding

[PA-PEG-PA-EDTA]m to the aqueous solution containing both the dye and Cu2+, free dyes were liberated due to the stronger binding affinity of the Cu2+ to EDTA of the multiblock copolymer than murexide. For Zn2+ and Ca2+, the dissociation constants of the metal ions for [PA-PEG-PA-EDTA]m were similarly obtained (Supporting Information: Figure S1a and S1b). The absorption band at 524 nm of the free dye shifted to 456 nm and 484 nm by adding Zn2+ and Ca2+, respectively.

The detailed

procedure to calculate the dissociation constants of between metal ions and [PA-PEGPA-EDTA]m was described in the supporting information (Supporting Information-

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Calculation of KD).

Based on the plot of absorbance against metal ion concentration,

the dissociation constants between metal ions and [PA-PEG-PA-EDTA]m were also calculated to be 1.2x10-7 M, 6.6x10-6 M, 1.2x10-4 M, and for Cu2+, Zn2+, and Ca2+, respectively (Figure 2b).

EDTA is a hexadentate ligand and the one-to-one binding

between metal ion and EDTA is preferred because the entropy loss (ΔS Zn2+ > Ca2+, indicating the smaller dissociation constant, or higher binding affinity of EDTA for Cu2+ than Zn2+ or Ca2+.15

The differences in binding affinity of EDTA for

the divalent metal ions come from the differences in size, polarizability, hydration, and the involvement of d-orbitals of the metals. The binding affinity of [PA-PEG-PAEDTA]m for above metal cations was much lower than free EDTA due to the fact that two neutral amide bonds of the multiblock copolymer were involved, instead of the two carboxylate anions of the free EDTA.

In addition, it might be energetically demanding

for metal ions to complex with EDTA incorporated along the high molecular weight

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polymers due to the steric hindrance.

However, the trend of higher binding affinity of

EDTA for Cu2+ than Zn2+ or Ca2+ was evident for the multiblock copolymer of [PAPEG-PA-EDTA]m.

Figure 2

The binding of polymer to metal ions has been extensively studied for water purification purpose as an ultrafiltration membrane. The dissociation constants of poly(ethylene imine) for Cu2+ and Zn2+ were reported to be 2.0x10-7 M and 1.6x10-6 M, respectively.21

A metal complexing sorbent prepared by the polymerization of N,N’-

dimethyl-N,N’-bis (4-vinylphenyl)-3-oxapentanediamide through the imprinting method of Ca2+ exhibited the dissociation constant of 2.95x10-3 M for Ca2+.22

The

stronger binding of Cu2+ compared with Zn2+, Pb2+, Co2+, and Ca2+ was also reported for poly(acrylic acid) and poly(N-vinyl imidazole).23,24 The metal binding might affect the nanostructure of the multiblock copolymer of ([PA-PEG-PA-EDTA]m).

We investigated the self-assembly and the secondary

structure of the polymer by AFM, dynamic light scattering, and CD spectroscopy.

The

multiblock copolymers consisting of hydrophilic PEG and hydrophobic PA formed

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micelles in water. The AFM images of the multiblock copolymers containing different metal ions were developed from their aqueous solutions by the same protocol for the comparative purpose.

The AFM images of the multiblock copolymers clearly

exhibited the spherical micellar nanostructures with an apparent size of 20-80 nm (Figure 3a). Dynamic light scattering study also suggested that [PA-PEG-PA-EDTA]m aqueous solution (0.01 wt.%) containing equivalent amount of metal ions exhibited micelle formation of 10-50 nm in size with a narrow distribution at 10 oC. However, the polymers aggregated to 20-950 nm in size with a broad distribution at 40 oC. At 20 o

C, the apparent size and size distribution were intermediate of the values at 10 and 40

o

C (Figure 3b).

Figure 3

CD spectra of the multiblock copolymer ([PA-PEG-PA-EDTA]m) (the legend of PCu-0.0 in Figure 4a) showed two minima at 205 nm and 221 nm, indicating that the major secondary structure of the PA in the multiblock copolymer is α-helix.

The ratio

of the two minima at 221 nm and 205 nm (221/205) was suggested as a measure of the coiled coil superstructures of polypeptides.25,26 The ratio (221/205) decreased as the

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concentration of Cu2+ increased from 0.0, 8.75, 11.7, and 17.5 µM at a fixed multiblock copolymer concentration equivalent to 17.5 µM of EDTA (inserted plot of Figure 4a). As a control, the CD spectra of Cu2+ aqueous solution (17.5 µM) in deionized water (Cu in the figure 4a) were also shown. They did not show any significant band in this concentration range. Therefore, the CD spectra suggested that the Cu2+ affected the secondary structure of the multiblock copolymer.

Once the metal ions bind to the

EDTA moieties which is located in the middle of the poly(L-alanine) block, it might pull the polypeptide chain, otherwise it exhibits more extended conformation due to ionic repulsion between anionic carboxylate groups. And thus, the strong binding of Cu2+ to EDTA moieties of the [PA-PEG-PA-EDTA]m decreases the ionic repulsion and shrinks the polypeptide conformation along the chain, which might facilitate formation of a coiled α-helical structure. GEAK-[LAEIEAK]2-LAEIYAm

PEG-diblock copolymers with a structure of PEGwas

also

reported

to

develop

coiled

coil

superstructures in a concentration range of 9-100 µM.26 The ratio (221/205) of the multiblock copolymers containing metal ions at the same concentration decreased in the decreasing order of Ca2+, Zn2+, and Cu2+ (inserted plot in Figure 4b).

This trend also suggested that the Cu2+ was the most effective in

changing the secondary structure of the multiblock copolymer, which might come from

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the strong complex formation between EDTA units of [PA-PEG-PA-EDTA]m and Cu2+.

Figure 4

Changes in modulus of the multiblock copolymer aqueous solutions (5.0 wt.%) containing the metal ions of Cu2+, Zn2+, or Ca2+ were investigated as a function of temperature. As the concentration of Cu2+ increased from 0.0 mM to 11.7 mM and 17.5 mM at a fixed concentration of EDTA in the multiblock copolymer ([PA-PEG-PAEDTA]m) at 17.5 mM, the modulus increased (Figure S2). As the concentration of the Cu2+ increased to 21 mM, amounting to 1.2 equivalent of EDTA, precipitation of salts, Cu(OH)2, was observed.

Therefore, we investigated the changes in modulus of the

multiblock copolymer aqueous solutions at the molar ratio of cation to EDTA of the multiblock copolymer ([PA-PEG-PA-EDTA]m) being one-to-one.

As the temperature

increased, the modulus of the multiblock copolymer aqueous solution containing various metal cations increased, indicating that thermogelation occurred (Figure 5). The sol-gel transition was reversible.

The thermosensitivity comes from the delicate

balance between hydrophobicity and hydrophilicity which are provided by hydrophobic PA block and hydrophilic PEG block, respectively.27

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The storage modulus (G”) and the loss modulus (G”) are measures of an elastic component and a viscous component of a complex modulus, respectively. Therefore, crossing of G’ over G” can considered to be an evidence of sol-to-gel transition.

G’

was greater than G” for all the multiblock copolymer aqueous systems as the temperature increased over 25 oC, suggesting that all systems were in a gel state above 25 oC.

The modulus of the gel incorporating Cu2+, Zn2+, and Ca2+ was 110 Pa, 69 Pa,

and 28 Pa, respectively, at 37 oC. The modulus of polymer gel prepared in deionized water (PDW) exhibited 50 Pa at 37 oC. Addition of calcium ion increased the sol-togel transition temperature and lowered the gel modulus, suggesting that thermogelation of the [PA-PEG-PA-EDTA]m aqueous solution was interfered by Ca2+.

This fact might

be related to the change in the secondary structure of the multiblock copolymer by adding the cation.

Even though the incorporated amount of metal ion was the same,

sol-to-gel transition temperature and modulus of the system were dependent on the type of the metal cations. The highest gel modulus was observed in the system containing Cu2+, and the gel maintained its physical integrity at 37 oC for more than two weeks under in vitro condition, similar to PEG-PAF diblock copolymer thermal gel.28

Figure 5

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Based on above study, ionic complexes between metal ions and [PA-PEG-PAEDTA]m can be discussed as follows.

The externally added metal cations complex

with EDTA moieties of the multiblock copolymer to form ionic complexes.

Thick

brown helical curves, thin blue curves, and dot lines indicate PA blocks, PEG blocks, and ionic complexes among the multiblock copolymers of [PA-PEG-PA-EDTA]m, respectively (Figure 6).

The binding of metal ions to EDTA units of the multiblock

copolymers leads to the formation of ionic complexes or intra/inter molecular saltbridges, which in turn affect the secondary structure of the multiblock copolymer. There can be three types of ionic complexes between the metal ion and EDTA of the multiblock copolymer.

Type I is a simple intramolecular ionic complex between the

divalent cation and two carboxylic acids of an EDTA unit along the polymer. is an intramolecular complex with coordination number of 6.

Type II

Type III is the

intermolecular ionic complex acting as a salt-bridge between the polymer chains. Type II and type III can contribute the formation of a gel through intra- and/or intermolecular salt bridges which stiffen the polymer chains.

The sol-to-gel transition

temperature decreases as the molecular stiffness increases as in the case of PEG-poly(Llactic acid) and PEG-poly(DL-lactic acid) multiblock copolymers, PEG-poly(L-alanine)

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and PEG-poly(DL-alanine).27,29

The change in gel modulus might be correlated with

the binding affinity of the metal cation to [PA-PEG-PA-EDTA]m as well as the change in the secondary structure of the multiblock copolymer.

The high affinity of Cu2+ for

the multiblock copolymers leads to the formation of strong intra/inter molecular saltbridges, which in turn affect the secondary structure of the multiblock copolymer.

The

complexes also act as intra/inter micellar bridges and lead to the increase in the gel modulus as well as the decrease in the sol-to-gel transition temperature.

Figure 6

CONCLUSIONS Multiblock copolymers of [PA-PEG-PA-EDTA]m were synthesized by the coupling reactions between α,ω-diamino end groups of PA-PEG-PA and ethylene diamine tetraacetic anhydrides.

The binding affinity of [PA-PEG-PA-EDTA]m for Cu2+, Zn2+,

and Ca2+ exhibited parallel trends with that of free EDTA for the metal ions.

AFM

images exhibited the multiblock copolymers containing Cu2+ formed intermicellar aggregates more seriously than other metal ions.

CD spectroscopy showed that Cu2+

affected the secondary structures of the multiblock copolymer more than Zn2+ and Ca2+.

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The multiblock copolymer aqueous solution containing Cu2+, Zn2+, or Ca2+ underwent sol-to-gel transition as the temperature increased.

The sol-to-gel transition temperature

of the polymer aqueous solutions (5.0 wt.%) decreased and the gel modulus increased after addition of Zn2+, and Cu2+, whereas Ca2+ showed opposite effects on the gelation. The binding affinity of [PA-PEG-PA-EDTA]m for metal ions exhibited parallel trends with the gel modulus.

Here, we are suggesting that the strong intra- and/or inter-

molecular ionic complexes between cupper ions (Cu2+) and EDTA units of the multiblock copolymers lower the sol-to-gel transition temperature and increase the gel modulus even at a low metal ion concentration in a mM range.

Current study not only

emphasized the importance of the type of metal ions in designing an ion/temperature dual stimuli-sensitive polymer but also demonstrated that a subtle difference in ion/ligand interactions determined the macroscopic thermogelling properties.

ASSOCIATED CONTENT Supporting Information UV-vis spectra of murexide as a function of Zn2+ and Ca2+ concentrations, and change in modulus of aqueous polymer solution containing Cu2+ as a function of temperature and Cu2+ concentration.

This material is available free of charge via the Internet at

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http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes These authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2012M3A9C6049835).

REFERENCES (1) Yoshida, T.; Lai, T. C.; Kwon, G. S.; Sako, K. Expert Opin. Drug Del. 2013, 10, 1497-1513. (2) Hennink, W. E.; van Nostrum C. F. Adv. Drug Del. Rev. 2012, 64, 223-236. (3) Kim, H. J.; Lee, J. H.; Lee, M. Angew. Chem. Int. Ed. 2005, 44, 5810-5814. (4) Bakota, E. L.; Wang ,Y.; Danesh, F. R.; Hartgerink, J. D. Biomacromolecules 2011,

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12, 1651-1657 (5) Kuroiwa, K.; Shibata, T.; Takada, A.; Nemoto, N.; Kimizuka, N. J. Am. Chem. Soc. 2004, 126, 2016-2021. (6) Peng, F.; Li, G.; Liu, X.; Wu, S.; Tong, Z. J. Am. Chem. Soc. 2008, 130, 1616616167. (7) Fang, Y.; Al-Assaf, S.; Phillips, G. O.; Nishinari, K.; Funami, T.; Williams, P. A. Carbohydate Polym. 2008, 72, 2319-2327. (8) Li, J.; Loh, X. Expert Opin. Ther. Patents 2007, 17, 965-977. (9) Park, M. H.; Joo, M. K.; Choi, B. G.; Jeong, B. Acc. Chem. Res. 2012, 45, 423-433. (10) Yu, L.; Ding, J. Chem. Soc. Rev. 2008, 37, 1473-1481. (11) Moon, H. J.; Ko, D. Y.; Park, M. H.; Joo, M. K.; Jeong, B. Chem. Soc. Rev. 2012, 41, 4860-4883. (12) Choi, Y. Y.; Jang, J. H.; Park, M. H.; Choi, B. G.; Chi, B.; Jeong, B. J. Mater. Chem. 2010, 20, 3416-3421. (13) Lee, H. S.; Choi, B. G.; Moon, H. J.; Park, K.; Jeong, B.; Han, D. K. Macromol. Res. 2012, 20, 106-111. (14) Pek, Y. S.; Wan, A. C. A.; Yang, J. Y. Biomaterials 2009, 31, 385-391. (15) Skoog, D. A.; West, D. M. In Fundamentals of Analytical Chemistry; 4th ed.;

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Saunders College Publishing, PA: USA, 1982; pp 284-286. (16) Kim, J. Y.; Park, M. H.; Joo, M. K.; Lee, S. Y.; Jeong, B. Macromolecules 2009, 42, 3147-3151. (17) Mannel-Crois, C.; Meister, C.; Zelder, F. Inorg. Chem. 2010, 49, 10220-10222 (18) Grudpan, K.; Jakmunee, J.; Vaneesorn, Y.; Watanesk, S.; Maung, U. A.; Sooksamiti, P. Talanta 1998, 46, 1245-1257. (19) Izadmanesh, Y.; Ghasemi, J. B.Talanta 2014, 128, 511-517. (20) Yun, E. J.; Yon, B.; Joo, M. K.; Jeong, B. Biomacromolecules 2012, 13, 1106-1111. (21) Juang, R. S.; Chen, M. N. Ind. Eng. Chem. Res. 1996, 35, 1935-1943. (22) Rosatzin, T.; Andersson, L. I.; Simon, W.; Mosbach, K. J. Chem. Soc. Perkin Trans. 1991, 2, 1261-1265. (23) Tomida, T; Hamaguchi, K; Tunashima, S; Katoh, M; Masuda, S. Ind. Eng. Chem. Res. 2001, 40, 3557-3562. (24) Pekel, N.; Guven, O. Colloid Polym. Sci. 1999, 277, 570-573. (25) Su, J. Y.; Hodges, R. S.; Kay, C. M. Biochemistry 1994, 33, 15501-15510. (26) Vandermeulen, G. W. M.; Tziatzios, Klok, A. A. Macromolecules 2003, 36, 41074114. (27) Choi, Y. Y.; Joo, M. K.; Sohn, Y. S.; Jeong, B. Soft Matter 2008, 4, 2383-2387.

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(28) Jeong, Y.; Joo, M. K.; Bahk, K. H.; Choi, Y. Y.; Kim, H. T.; Kim, W. K.; Lee, H. J.; Sohn, Y. S.; Jeong, B. J. Controlled Rel. 2009, 137, 25-30. (29) Joo, M. K.; Sohn, Y. S.; Jeong, B. Macromolecules 2007, 40, 5111-5115.

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Page 27 of 37

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Scheme 1. Synthetic scheme of the multiblock copolymer of [PA-PEG-PA-EDTA]m. x=44.1 (vendor), and y is calculated to be 4.1 by 1H-NMR spectra.

m is approximated

to be 12.3 by gel permeation chromatography.

O

H2N

NH2

x

O O HN O O H2N

O N H

O x

y

NH2

N H

y

O

O O

N

N

O O

O H N O

O

O N H

O y

N H

x

H N

N

N

y

m

O

O

O

-

O-

27


O

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Page 28 of 37

Figure Captions

Figure 1. (a) 1H-NMR spectra ((CF3COOD) of the PEG, PA-PEG-PA, and [PA-PEGPA-EDTA]m. Each peak was assigned to the molecular structure.

(b) Gel permeation

chromatograms of the PEG, PA-PEG-PA, and multiblock copolymer ([PA-PEG-PAEDTA]m). PEG with 35,000 Daltons (PEG-35000) was also compared.

N,N-

dimethyl formamide was used as a solvent. Figure 2. (a) UV spectra of murexide (50 µM) as a function of Cu2+ concentrations. The legends are the concentration of metal ions in µM. The absorption spectra of the dye after (thick brown curve) and before (thin brown curve) adding the multiblock copolymer ([PA-PEG-PA-EDTA]m) (corresponding to 100 µM EDTA, S100) to an aqueous solution containing dye and metal ions (100 µM) is also compared. Absorbance of metal ions alone in 100 µM in the absence of dye is negligible.

(b) Plot

of absorbance of metal bound murexide at peak maxima (at 463 nm, 456 nm, and 484 nm for Cu2+, Zn2+, and Ca2+, respectively), as a function of metal ion concentration. Figure 3. (a) AFM images of the self-assembled multiblock copolymers ([PA-PEG-PAEDTA]m) developed from the aqueous solution (0.01 wt.%) containing metal ions. Size distribution, determined by dynamic light scattering, of the self-assembled

28


(b)

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multiblock copolymers ([PA-PEG-PA-EDTA]m) aqueous solution (0.01 wt.%) containing equivalent amount of metal ions.

Gray, black dotted, and black curves

indicate size distribution curves at 10 oC, 20 oC, and 40 oC, respectively.

Ca, Zn, and

Cu indicate the polymer aqueous solutions containing Ca2+, Zn2+, and Cu2+, respectively. Figure 4. (a) Circular dichroism spectra of the multiblock copolymers ([PA-PEG-PAEDTA]m) aqueous solutions as a function of Cu2+ concentration at a fixed EDTA concentration (17.5 µM) of the multiblock copolymer. The Cu2+ concentration varied over 0.0 µM (PCu-0.0), 8.75 µM (PCU-8.75), 11.7 µM (PCu-11.7), and 17.5 µM (PCu17.5).

The ratio of ellipticity at 221 nm to 205 nm is inserted as a plot. Cu (control)

in the legend indicates the Cu2+ aqueous solution (17.5 µM) in the absence of the multiblock copolymer.

(b) Circular dichroism spectra of the multiblock copolymer

([PA-PEG-PA-EDTA]m) aqueous solutions containing specific cations. The concentrations of both EDTA in the multiblock copolymer and metal ions were fixed at 17.5 µM. PCu, PZn, and PCa indicate the polymer aqueous solutions containing Cu2+, Zn2+, and Ca2+, respectively.

Ca, Zn, and Cu indicate the aqueous solutions containing

Cu2+, Zn2+, and Ca2+, respectively, in the absence of the polymer.

The ratio of

ellipticity at 221 nm to 205 nm is inserted as a plot. Figure 5. Changes in modulus of the multiblock copolymer ([PA-PEG-PA-EDTA]m)

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Page 30 of 37

aqueous solutions (5.0 wt.%) containing cations as a function of temperature. Both concentrations of the EDTA in the polymer and metal ions were fixed at 17.5 mM. PCu, PZn, PCa, and indicate the polymer aqueous solutions containing Cu2+, Zn2+, and Ca2+, respectively. PDW indicates a [PA-PEG-PA-EDTA]m aqueous solution (5.0 wt.%) in deionized water. G’ and G” are the storage component and the loss component of a complex modulus, respectively. Figure 6. Scheme of the self-assembly of the multiblock copolymer ([PA-PEG-PAEDTA]m) in the presence of the metal ions.

Thick brown helical curves, thin blue

curves, and dot lines indicate PA blocks, PEG blocks, and ionic complexes among the multiblock copolymers of [PA-PEG-PA-EDTA]m, respectively.

Metal ions with high

binding affinity for EDTA form intra/inter molecular salt-bridges, which affect the secondary structure of polymers and lead to the formation of intermicellar bridges. The potential models of intrachain (type I and Type II) and/or interchain (Type III) ionic complexes are also shown.

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Figure 1.

d

H N O

O

f

O N H

O y

b

a

b x

N H

b,c

N

y

a

c c

d

H N

f

f e d

a)

N

e

e

O

m

O

O OH

HO

a

[PA-PEG-PA-EDTA]m

PA-PEG-PA PEG

6.0 Detector response (a.u.)

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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5.0

4.0 3.0 ppm

2.0

1.0

b) PEG-35000 PEG/PA Multiblock

PA-PEG-PA PEG 2000

0

5 10 15 20 Retention time (min.)

31


25

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Figure 2.

log([A-Amin]/[Amax-A])

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

Page 32 of 37

3.0 Cu

Zn

b)

Ca

2.0 1.0 0.0 -1.0 -2.0 -6

-5

-4 -3 log [M 2+]

32


-2

-1

Page 33 of 37

Figure 3.

a)

Ca

50 nm

Zn

200 nm

200 nm

50 nm

Cu

50 nm

200 nm

80

b) Intensity (a.u.)

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

Biomacromolecules

60

Cu

40 Zn 20 Ca 0 1

10

100 1000 Size (nm)

33


10000

Biomacromolecules

40 Cu 30 a) PCu-0.0 PCu-8.75 20 PCu-11.7 PCu-17.5 10 0 -10 0.95 0.90 -20 0.85 -30 0.80 -40 Incr. [Cu2+] 0 8.75 11.7 15.5 Conc. (uM) -50 190 200 210 220 230 240 250 260 270 Wavelength (nm) 2 2 1 / 2 0 5

40 Cu 30 b) Zn Ca 20 PCu PZn 10 PCa 0 -10 0.84 -20 0.82 -30 0.80 -40 PCa PZn PCu -50 190 200 210 220 230 240 250 260 270 Wavelength (nm) 2 2 1 / 2 0 5

Ellipticity (mdeg)

Figure 4.

Ellipticity (mdeg)

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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Figure 5.

140 G' (Pa), G'' (Pas)

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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PCu-G' PZn-G' PCa-G' PDW-G'

120 100

PCu-G" PZn-G" PCa-G" PDW-G"

80 60 40 20 0 0

5

10 15 20 25 30 Temperature (℃)

35


35 40

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Figure 6.

M2+

O H N

N

H N

N O

-

O

O

O

-

N H

M2+

Type I

M

2+

O O

36


OO-

N

Type II

N

O O

N

O

O O

H N

O

O O N H

N O-

O

M2+

OO

N

N

Type III

Page 37 of 37

Table of Content

Ion and Temperature Sensitive Polypeptide Block Copolymer

Jae Hee Joo, Hyo Jung Moon, Du Young Ko, Usha Pramod Shinde, Min Hee Park, and Byeongmoon Jeong*

140

G' (Pa), G'' (Pas)

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

Biomacromolecules

H N

120

O

100

O

O Ny H

O x

N H

H N O

Binding Affinity for EDTA => Cu2+ >Zn2+ >Ca2+

80

N

N

y

O

O-

m

-

O

O

Cu2+

60

Zn2+ water

40

Ca2+

20 0 0

5

10 15 20 25 30 35 Temperature (℃)

37


40

Ion and Temperature Sensitive Polypeptide Block Copolymer

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