Electrokinetic Analysis to Reveal Composition and Structure of

Article pubs.acs.org/ac

Electrokinetic Analysis to Reveal Composition and Structure of Biohybrid Hydrogels Ralf Zimmermann,*,† Susanne Bartsch,† Uwe Freudenberg,†,‡ and Carsten Werner†,‡ †

Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Hohe Strasse 6, 01069 Dresden, Germany ‡ Technische Universität Dresden, Center for Regenerative Therapies Dresden, Tatzberg 47, 01307 Dresden, Germany ABSTRACT: Biohybrid hydrogels combining electrically neutral synthetic polymers and highly anionic glycosaminoglycans (GAGs) offer exciting options for regenerative therapies as they allow for the electrostatic conjugation of various growth factors. Unraveling details of ionization and structure within such networks defines an important analytical challenge that requires the extension of current methodologies. Here, we present a mean-field approach to quantify the density of ionizable groups, GAG concentration, and cross-linking degree of such hydrogels based on experimental data from microslit electrokinetics and ellipsometry. An exemplary poly(ethylene glycol)− heparin system was analyzed to demonstrate how electrostatic f ingerprints of hydrogels obtained by the introduced strategy can sensitively display composition and structure of the polymer networks. elevated ion concentration within the hydrogel film (due to the counterions) which in turn causes an electrical potential difference between the gel and the electrolyte. For thick hydrogel films (i.e., the thickness is much larger than the Debye screening length), interfacial effects are negligible and the potential difference can be described analogue to the potential difference across a semipermeable membrane by the Donnan potential.10 To quantify the charge within hydrogel films, electrokinetic experiments can provide a valuable parameter related to the accumulation of mobile counterions: the excess conductivity at and within the hydrogel material (designated as surface conductivity, Kσ). For hydrogel films with low to intermediate charge densities and a homogeneous distribution of the polymer segments, the surface conductivity is related to the Donnan equilibrium between the hydrogel and an electrolyte composed of N monovalent ions according to the following equation:10,12

H

ydrogel matrixes of physically or chemically cross-linked polymers are instrumental to progress in tissue engineering.1 They can be tailored in mechanical properties to match the requirements of various types of tissues2 and incorporate multiple biomolecular components of naturally occurring extracellular matrixes (ECM) to stimulate embedded cells3 by specifically orchestrated signals. Biohybrid gel materials, consisting of combinations of synthetic and biologically derived polymeric constituents, are increasingly developed and employed for this purpose4−7 with systems containing polysaccharidic ECM components, glycosaminoglycans (GAGs) being particularly promising8 due to the effective binding, protection, and sustained release of numerous growth factors. The latter effect can be largely attributed to the sulfation patterns of the GAGs and is therefore largely determined by electrostatic interactions. Progress in advanced design concepts and novel synthetic strategies for biohybrid polymer matrixes critically depends on the comprehensive analysis of the obtained structures, including a thorough characterization of their physicochemical properties and interactions with biologically active molecules. Since GAGs give rise to strong electrostatic interactions, this has to comprise the evaluation of gel charging in aqueous environments as well as charge-induced structural features and electrostatic effects in the interactions of the gels with various signaling molecules. In addition, the degree of cross-linking of the polymer networks is of importance as it determines the accessibility of the gels for target molecules. To unravel these characteristics, electrokinetic measurements across a slit microchannel formed by two planar hydrogelcoated sample carriers in combination with in situ ellipsometry provide valuable options.9−13 The hydrogel film itself can be treated as a 3D-meshwork where ionizable groups are immobilized. The ionization of these groups is related to an © 2012 American Chemical Society

N

K σ = Fd ∑ c iu ie−z iyD i=1

(1)

where F is the Faraday constant, d is the hydrogel thickness, zi is the valence, ci is the concentration, ui is the mobility of ion species i (i = 1...N), and yD is the dimensionless Donnan potential (yD = FΨD/RT with ΨD being the Donnan potential, R the gas constant, and T the temperature). With the assumptions specified above, the dimensionless Donnan potential can be obtained from the electroneutrality condition: Received: September 3, 2012 Accepted: October 2, 2012 Published: October 2, 2012 9592

dx.doi.org/10.1021/ac302538j | Anal. Chem. 2012, 84, 9592−9595

Analytical Chemistry N

M

∑ z ic ie−y

D

i=1

+

∑ j=1

Article

cg,j 1 + 10−εj(pKj−pH)e εjyD

hydrogel films. The thickness and refractive index of the hydrogel films were determined on the basis of an optical model that involves 5 different layers (Si/SiO2/Teflon AF/ hydrogel/aqueous solution). The optical constants of the various layers of the interfacial system were taken from literature.19,20 Electrolyte Solutions. All electrolyte solutions used in this study were prepared from vacuum-degassed deionized water (Milli-Q gradient A10, Millipore Co., USA) by addition of 0.1 M KCl, KOH, and HCl stock solutions (VWR International GmbH, Darmstadt, Germany).

=0 (2)

In eq 2, the index j denotes the concentration cg and the pK of the j-th type of ionizable group in the hydrogel (j = 1...M, where M is the number of different types of ionizable groups). The parameter εj = −1 for anionic groups and εj = 1 for cationic groups.13 For molecular gel constituents of defined size containing known numbers of ionizable groups per molecule, the concentrations cg,j can be directly converted into the concentrations of these building blocks in the gel. Furthermore, if ionizable groups are chemically converted during the gel formation, the concentration of the remaining (nonreacted) groups can provide information on the cross-linking degree of the gel. Thus, the presented method not only allows for evaluating the hydrogel charge but also enables one to conclude on chemical conversions to describe the actual hydrogel network structure. Applying this concept, we have evaluated experimental results for a hydrogel film consisting of covalently linked star-shaped poly(ethylene glycol) (starPEG) and heparin.14,15 The analysis of the experimental data was underpinned by simulations of the Donnan equilibrium for varying hydrogel compositions and cross-linking degrees to explore the resulting electrical potentials and surface conductivities.

■

RESULTS AND DISCUSSION To unravel details of ionization and structure of the starPEG− heparin hydrogels, we determined the surface conductivity and layer thickness in 0.1 mM KCl solution at varied pH values (Figure 1). Under this condition, the surface conductivity

■

MATERIALS AND METHODS Hydrogel Films. Hydrogel films were prepared from an amine end-functionalized 4-arm star-shaped poly(ethylene glycol) (starPEG, MW 10000, Polymer Source Inc., Dorval, Canada) and heparin (MW 14 000, Calbiochem, Darmstadt Germany) onto planar glass and silicon carriers (sample area 20 mm × 10 mm). First, a thin Teflon AF film was spin-coated onto sample carriers according to a protocol described in detail elsewhere.16 The surface of the Teflon AF films was hydrophilized by means of argon plasma treatment,16 which then allowed for spin coating and covalent attachment of the hydrogel. To prepare the hydrogel film, heparin (254.6 mg/ mL) and a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 83.3 mg/mL) and N-hydroxysulfosuccinimide (sNHS, 47.4 mg/mL) were dissolved in phosphate buffered solution on ice. The heparin/EDC/sNHS solution was kept on ice for 15 min to activate the carboxyl groups of the heparin. In parallel, a heparin-free EDC/sNHS solution was prepared and used for the activation of the carboxyl groups at the Teflon AF surface. After 15 min, the starPEG (545.4 mg/ mL) was added to the heparin/EDC/sNHS solution. After complete mixing, the solution was spin-coated onto the activated Teflon AF surfaces. The resulting hydrogel films were stored for 24 h at 4 °C to allow for internal cross-linking and attachment at the Teflon AF surface. Afterward, the hydrogel films were rinsed at least 4 times with deionized water. The film thickness was found to be about 125 nm. Surface Conductivity Measurements. The surface conductivity of the hydrogel films was determined by streaming potential and streaming current measurements across a rectangular microchannel formed by two parallel sample carriers using the Microslit Electrokinetic Setup. Details of the measurements and of the setup are specified elsewhere.17,18 Ellipsometry. A M-44 ellipsometer (Woolam Co., Inc., USA) was used to investigate the pH-dependent swelling of the

Figure 1. Surface conductivity (red circles) and thickness (green squares/line) of a starPEG−heparin hydrogel film in 0.1 mM KCl solution of varied pH. The film was prepared at a molecular ratio of starPEG to heparin of 3 (in case of a quantitative reaction of the starPEG, a conversion of 12 of the 24 carboxyl groups of the heparin would occur). The experimental surface conductivity data were reproduced by the theory (red solid line). The red dashed line shows the surface conductivity caused by counterions compensating the charge of the sulfate groups in the gel. The blue and black dashed lines illustrate the contribution of the K+ ions (blue) and H3O+ ions (black) to the overall surface conductivity.

significantly contributes to the overall conductivity of the microslit channel in the electrokinetic experiment; i.e., the data allow the most accurate quantification of ionizable groups in the hydrogel. According to the nature of heparin, sulfate and carboxyl groups were considered in the modeling and evaluation of the experimental surface conductivity data. The surface conductivity of the hydrogel films was found to be strongly dependent on the solution pH (Figure 1). Below pH 5, the magnitude of Kσ is determined by the ions that compensate the charge of the strongly acidic sulfate groups. Without the presence of the less acidic carboxyl groups, a plateau would arise in the neutral and weak alkaline pH range (see red dashed line in Figure 1 and curve for the molar ratio of starPEG to heparin of γ = 6 in Figure 2, corresponding to a quantitative conversion of the carboxyl moieties of heparin). Therefore, the increase of the surface conductivity above pH 6 can be clearly attributed to the ionization of nonconverted carboxyl groups in the film. The possible range of variations of Kσ with varying amounts of COOH groups (related to varying 9593

dx.doi.org/10.1021/ac302538j | Anal. Chem. 2012, 84, 9592−9595

Analytical Chemistry

Article

mmol/L and pK = 4.0 for the carboxyl groups. The pK values very well agree with pK values reported in literature.22 The fact that the ionization of carboxyl groups in the hydrogel films occurs at significantly higher pH values than expected according to the pK value can be attributed to the electrical potential arising from the ionized sulfate groups (see inset of Figure 2). Because of the low number of carboxyl groups per heparin molecule (as compared to the number of sulfate groups), their ionization causes only a weak increase of the electrical potential (Figure 2). As an important prerequisite for the further analysis of the gel composition and its cross-linking degree, heparin of known molecular weight and number of sulfate and carboxyl groups per molecule was used for the formation of the hydrogel films (see experimental section and ref 8). As the sulfate groups are not involved in the gel formation, their number (as derived from the evaluation of the surface conductivity data) can be directly converted into the heparin concentration within the hydrogel films. With an average number of 65 sulfate groups per heparin molecule and a molecular weight of 14 000 g/mol, we obtain a heparin concentration of 11.4 μg/μL for the hydrogel film, which is comparable to values previously determined for similar (bulk) starPEG−heparin gels.15 The determined heparin concentration can be further used to quantify the cross-linking degree of the gels by simply dividing the concentration of carboxyl groups by the heparin concentration. This calculation is straightforward and gives an average number of 12.3 carboxyl groups per heparin molecule, which is in excellent agreement with the theoretical value expected from the gel composition (12 carboxyl groups per heparin molecule) and confirms the almost complete crosslinking of both gel components. Since other analytical approaches to determine the actual cross-linking degree via labeling of unreacted groups (e.g., trinitrobenzene sulfonic acid (TNBS) or Atto-labeling of free, nonconverted amine groups of starPEG)23,24 are prone to nonquantitative turnover or nonspecific side effects (e.g., electrostatic interactions with gel components), the sensitive and quantitative method described herein is clearly advantageous. The introduced method furthermore allows one to quantify a variation of the intrinsic sulfation pattern of the GAGs, as it can be obtained via selective desulfation of heparin25 pursued to tune its interaction with soluble signal molecules. The effect of desulfation on the surface conductivity is shown in the simulation results given in Figure 3. Compared to the reference curve for unmodified heparin (black curve in Figure 3), the chemical conversion of the 2-O-sulfate, 6-O-sulfate, and/or Nsulfate groups25 would cause significantly lower surface conductivity values. As desulfation results in a significant drop of the electrical potential within the hydrogel film (see inset of Figure 3), the ionization of the carboxyl groups occurs at lower pH values with increasing degree of desulfation. The sensitivity of the introduced approach for the determination of the GAG concentration within gels becomes obvious if we consider the sensitivity of the surface conductivity measurements under the experimental conditions. At the ionic strength of 0.1 mmol/L and the channel height of 30 μm, we can resolve details in the electrosurface characteristics corresponding to a surface conductivity of about 1 nS. In consequence, for the considered starPEG−heparin hydrogel with a heparin concentration of 11.4 μg/μL and the corresponding plateau value of the surface conductivity of

Figure 2. Simulation of the surface conductivity for varying molar ratios γ between starPEG and heparin applied during the gel formation (corresponding to varying cross-linking degrees of the gels). The blue curve corresponds to a highly cross-linked gel with a molar ratio between starPEG and heparin of 6; i.e., all COOH groups of the heparin are cross-linked with one arm of the starPEG. The black dashed line represents data for a hypothetical molar ratio of γ = 0 (pure heparin gel). This curve corresponds to the upper boundary for the variation of the surface conductivity. Weakly and intermediately cross-linked gels show an increase of the surface conductivity between these two extreme cases. The inset shows the pH dependence of the dimensionless Donnan potential for the same molar ratios. Simulation parameters used to obtain these curves: salt concentration (KCl), 0.1 mM; heparin concentration, 11.4 μg/μL; film thickness, 600 nm; pK of sulfate groups, 0.8; pK of carboxyl groups, 4.0.

cross-linking degrees) is illustrated by simulation results in Figure 2. In case of highly cross-linked hydrogels, no carboxyl groups remain at the heparin and, as already stated above, no increase of Kσ occurs in the neutral pH range. In contrast, the dashed line in Figure 2 represents the upper boundary for the increase of the surface conductivity. Soft starPEG−heparin hydrogels films of low cross-linking degree (the gelation starts at about γ = 0.7)15 would show an increase of Kσ somewhat below this hypothetical curve. The strong increase of Kσ observed below pH 5 in the experiment and simulations is caused by the exchange of the counterions in the hydrogel with decreasing pH (but not by an increase in the number of ionized moieties): At neutral and alkaline pH, the solution concentration of K+ ions is much higher than the concentration of the H3O+ ions. Consequently, the K+ ions compensate the negative charge of the heparin. With decreasing pH, the concentration of H3O+ ions in solution increases. According to eq 1, this increase is accompanied by the replacement of the counterions in the hydrogel which in turn causes an increase of Kσ due to the significantly higher ion mobility (u(H3O+) = 36.6 × 10−8 m2 V−1 s−1 vs u(K+) = 7.69 × 10−8 m2 V−1 s−1).21 The thickness of the hydrogel films was determined by ellipsometry to be about 600 nm in the weakly acidic pH range (pH 4−5). This corresponds to a swelling degree of 4.8 (thickness in swollen state/dry thickness). In line with the ionization of the carboxyl groups between pH 5.5 and 7.5, the swelling of the film slightly increased in the same pH range due to the increasing osmotic pressure (Figure 1). To draw conclusions about the gel composition and its crosslinking degree, we analyzed the experimental data (Kσ, d) quantitatively by solving the governing equations for the surface conductivity (eq 1) and Donnan potential (eq 2) numerically and made a comparison of the experimental data with the theory (Figure 1). The best fit of the data was obtained with cg = 53 mmol/L and pK = 0.8 for the sulfate groups and cg = 10 9594

dx.doi.org/10.1021/ac302538j | Anal. Chem. 2012, 84, 9592−9595

Analytical Chemistry

■

REFERENCES

(1) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345−1360. (2) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47−55. (3) Welzel, P.; Nitschke, M.; Freudenberg, U.; Zieris, A.; Götze, T.; Valtink, M.; Engelmann, K.; Werner, C. In Hydrogel Sensors and Actuators: Engineering and Technology; Gerlach, G., Arndt, K.-F., Eds.; Springer: Berlin, 2009; pp 249−266. (4) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47−55. (5) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352−1355. (6) Fischbach, C.; Mooney, D. J. In Polymers for regenerative medicine; Werner, C., Ed.; Springer: Berlin, Heidelberg, 2006; pp 191−221. (7) Yamaguchi, N.; Kiick, K. L. Biomacromolecules 2005, 6, 1921− 1930. (8) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 391− 412. (9) Duval, J. F. L.; van Leeuwen, H. P. Langmuir 2004, 20, 10324− 10336. (10) Yezek, L. P.; Duval, J. F. L.; van Leeuwen, H. P. Langmuir 2005, 21, 6220−6227. (11) Duval, J. F. L.; Zimmermann, R.; Cordeiro, A. L.; Rein, N.; Werner, C. Langmuir 2009, 25, 10691−10703. (12) Zimmermann, R.; Kuckling, D.; Werner, C.; Duval, J. F. L. Langmuir 2010, 26, 18169−18181. (13) Duval, J. F. L.; Küttner, D.; Nitschke, M.; Werner, C.; Zimmermann, R. J. Colloid Interface Sci. 2011, 362, 439−449. (14) Freudenberg, U.; Hermann, A.; Welzel, P. B.; Stirl, K.; Schwarz, S. C.; Grimmer, M.; Zieris, A.; Panyanuwat, W.; Zschoche, S.; Meinhold, D.; Storch, A.; Werner, C. Biomaterials 2009, 30, 5049− 5060. (15) Freudenberg, U.; Sommer, J.-U.; Levental, K. R.; Welzel, P. B.; Zieris, A.; Chwalek, K.; Scheider, K.; Prokoph, S.; Prewitz, M.; Dockhorn, R.; Werner, C. Adv. Funct. Mater. 2012, 22, 1391−1398. (16) Cordeiro, A. L.; Zimmermann, R.; Gramm, S.; Nitschke, M.; Janke, A.; Schäfer, N.; Grundke, K.; Werner, C. Soft Matter 2009, 5, 1367−1377. (17) Werner, C.; Körber, H.; Zimmermann, R.; Dukhin, S. S.; Jacobasch, H.-J. J. Colloid Interface Sci. 1998, 208, 329−346. (18) Zimmermann, R.; Osaki, T.; Schweiss, R.; Werner, C. Microfluid. Nanofluid. 2006, 2, 367−379. (19) Werner, C.; Eichhorn, K.-J.; Grundke, K.; Simon, F.; Grählert, W.; Jacobasch, H.-J. Colloids Surf., A 1999, 156, 3−17. (20) Zimmermann, R.; Dukhin, S.; Werner, C. J. Phys. Chem. B 2001, 105, 8544−8549. (21) Lide, D. R.; Frederikse, H. P. R. CRC Handbook of Chemistry and Physics, 78th ed; CRC Press: Boca Raton, 1995. (22) Mikhailov, D.; Mayo, K. H.; Pervin, A.; Linhardt, R. J. Biochem. J. 1996, 315, 447−454. (23) Cayot, P.; Tainturier, G. Anal. Biochem. 1997, 249, 184−200. (24) Teske, C. A.; Schroeder, M.; Simon, R.; Hubbuch, J. J. Phys. Chem. B 2005, 109, 13811−13817. (25) Takano, R. Trends Glycosci. Glycotechnol. 2002, 14, 343−354.

Figure 3. Simulation of the surface conductivity for varying amounts of sulfate groups per heparin. The black curve shows the surface conductivity of a hydrogel with a molar ratio between the starPEG and heparin of 3 (i.e., conversion of 12 of the 24 carboxyl groups per heparin molecule during the gel formation) and complete sulfation of the heparin (65 sulfates per molecule). The green, blue, and red curves correspond to gels with 1/3, 2/3, and complete desulfation of the heparin. All other simulation parameters are identical to those given for the curves in Figure 2. The inset shows the pH dependence of the dimensionless Donnan potential for the same conditions.

237 nS, the approach allows us to detect differences in the heparin concentration being as low as 0.05 μg/μL.

■

CONCLUSIONS We report a mean-field approach for the analysis of charge and structural features within biohybrid hydrogels. The method is based on microslit electrokinetics and in situ ellipsometry data obtained in aqueous media of varied electrolyte and pH. Current concepts of the electrohydrodynamics of soft interfaces were adapted to quantitatively determine concentration, degree of conversion, and ionization of GAG components within binary gels. The availability of these parameters allows us to interpret the characteristics of GAG-based hydrogels with unprecedented precision. Accordingly, ongoing work is dedicated to explore a series of PEG-based hydrogels and different persulfated GAGs, including heparan sulfate, chondroitin sulfate, and dermatan sulfate, and to correlate the results with uptake and release data of selected GAG-binding growth factors. Together, these studies will provide a valuable extension of the rational design of multibiofunctional polymer matrixes.

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 00 49 351 4658 258. Fax: 00 49 351 4658 533. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Nelly Rein, Woranan Panyanuwat, and Karina Schreiber (all Leibniz Institute of Polymer Research Dresden (IPF), Germany) for the support with the sample preparation and performing the electrokinetic measurements and ellipsometry as well as Andrea Zieris (also IPF) for the comments to the manuscript. U.F. and C.W. were supported by the Deutsche Forschungsgemeinschaft through Grants WE 2539-7/1 and FOR/EXC999 and by the Leibniz Association. 9595

dx.doi.org/10.1021/ac302538j | Anal. Chem. 2012, 84, 9592−9595