Biohybrid Networks of Selectively Desulfated Glycosaminoglycans for

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Biohybrid networks of selectively desulfated glycosaminoglycans for tunable growth factor delivery Andrea Zieris, Ron Dockhorn, Anika Röhrich, Ralf Zimmermann, Martin Mueller, Petra B. Welzel, Mikhail V. Tsurkan, Jens-Uwe Sommer, Uwe Freudenberg, and Carsten Werner Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5012294 • Publication Date (Web): 20 Oct 2014 Downloaded from http://pubs.acs.org on October 26, 2014

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Biohybrid networks of selectively desulfated glycosaminoglycans for tunable growth factor delivery Andrea Zieris#†, Ron Dockhorn#†,‡, Anika Röhrich§, Ralf Zimmermann†, Martin Müller†, Petra B. Welzel†, Mikhail V. Tsurkan†, Jens-Uwe Sommer†,‡, Uwe Freudenberg†,$, and Carsten Werner*,†,$. †

‡

Leibniz Institute of Polymer Research Dresden, Hohe Strasse 6, 01069 Dresden, Germany

Technische Universität Dresden, Institute for Theoretical Physics, Zellescher Weg 17, 01069 Dresden, Germany

§

B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstrasse 18, 01307 Dresden, Germany $

Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstrasse 105, 01307 Dresden, Germany

KEYWORDS. Hydrogels, growth factors, mechanical properties, mean field, drug delivery.

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ABSTRACT (Word Style “BD_Abstract”). Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore the related options, selectively desulfated heparin derivatives were prepared, thoroughly characterized and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cellinstructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.

INTRODUCTION The glycosaminoglycan (GAG) heparan sulfate (HS) of the mammalian extracellular matrix (ECM) regulates storage and presentation of various growth factors,1 as spatially matching electrostatic interactions between the negatively charged N-, 2-O- and 6-O-sulfates of the GAG and basic amino acid residues of growth factors result in effective association.1 Heparin, a highly sulfated type of HS, is widely used in GAG-protein binding studies2 and heparin-containing materials are increasingly utilized for the biomimetic presentation of growth factors.3-12 For the latter purpose, a recently introduced set of biohybrid hydrogels was prepared by reacting the


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carbodiimide/N-hydroxysulfosuccinimide (EDC/s-NHS)-activated carboxylic acid group of heparin with amine functionalized four-armed (star-shaped) poly(ethylene glycol) (starPEG)13,14 and applied for sustainable administration of various therapeutically relevant growth factors.14-17 To further extend this approach, biohybrid gel matrices widely tunable in both the delivery of functional proteins and biomechanical properties are of particular interest. As the specific heparin used (MW approx. 14 kDa) contains ~ 24 carboxylic acid groups per molecule1, it can act as multivalent crosslinker in the reaction with end-functionalized starPEG. The viscoelastic properties of the obtained gels can be effectively tuned across a broad range upon variation of the molar ratio of the building blocks.13,14 However, the analysis of underlying differences in the local network structure so far remained challenging.18-20 Selective desulfation of heparin21 offers a versatile means for the approximation to charge patterns of naturally occurring, less sulfated GAGs1 (chondroitin sulfate, heparan sulfate and hyaluronic acid) and is therefore considered appropriate for modulating the growth factor delivery from GAG-based polymer hydrogel matrices (Figure 1A). However, altered GAG sulfation patterns not only affect growth factor conjugation1,2 but similarly influence the network properties of the hydrogels: Swelling of biohybrid GAG-starPEG networks, which was previously demonstrated to depend on the interplay of elastic retraction forces and osmotic and excluded volume driven expansion forces,14,22 is strongly affected by alterations of the GAG charge density. Moreover, the protonation of the heparin carboxylic acid group23,24, the miscibility and the spatio-temporal distribution of network forming polymer precursors can be expected to be influenced by the GAG desulfation.25-32 In consequence, the impact of GAG sulfation patterns on network formation and resulting hydrogel properties cannot be easily predicted from solution pKa values nor from model reactions. Combining swelling measurements


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and rheometry with a mean field analysis of physical network properties14,22 and GAG ionization23,24,33,34 we herein explore biohybrid polymer networks of heparin derivatives differing in their sulfation patterns. The results allow for the targeted design of hydrogels affording an extensive modulation of physicochemical characteristics and growth factor binding, representing a new class of thoroughly defined bioactive matrices for various applications in bioengineering.

EXPERIMENTAL SECTION Preparation of desulfated heparin derivatives. Pyridinium heparinate28,35,36: 35 g of Amberlite IR-120 H+ ion exchange resin were filled in a glass column and washed with deionized water, regenerated for 30 minutes with 10 % aqueous HCl, and washed with water until neutral pH. A solution of 1 g heparin (14,000 g/mol; sodium salt, porcine intestinal mucosa, Calbiochem) in 45 ml deionized water was applied. The combined filtrate and washings were adjusted to pH 6.0 with pyridine. After solvent removal by evaporation the product was lyophilized. Subsequently, the pyridinium salt of heparin served as the starting compound for the N- and 6-O-desulfation procedure. N-desulfation (N-DS) procedure36: The N-DS, 6-O-DS, 2-O-DS and NAc procedure were combined to obtain completely desulfated heparin, cDSH, NAc). Therefore, 25 ml DMSO containing 5 % water were added to the pyridinium salt of heparin and the mixture was stirred for 1.5 hours at 50°C. After dilution with 25 ml water pH was adjusted to pH 9.5 with 1 N aqueous NaOH. The mixture was dialyzed (MWCO = 8 kDa) against deionized water for 3 days, concentrated and lyophilized. To remove the resulting free amino group, which could possibly lead to heparin-heparin crosslinks upon activation by EDC/s-NHS for gel formation, the product was subjected to N-acetylation procedure.


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6-O-desulfation (6-O-DS) procedure35: 420 mg of the pyridinium salt of heparin were dissolved in 50 ml N-methyl-2-pyrrolidone containing 10 % water. The reaction was carried out for 24 hours at 90°C. After cooling to room temperature 50 ml deionized water were added and pH adjusted to 9.0 by addition of 1 M aqueous sodium hydroxide. The reaction mixture was dialyzed (MWCO = 8 kDa) against deionized water for three days, concentrated and lyophilized. As this process additionally removes the N-sulfate, the product was subjected to the Nresulfation procedure to obtain exclusively 6-O-desulfated heparin. 2-O-desulfation (2-O-DS) procedure28: 500 mg heparin were dissolved in 10 ml 0.4 M aqueous sodium hydroxide. The solution was frozen and lyophilized over night. Dissolving and lyophilizing was done twice. The pH was adjusted to 9.0 with 20 % acetic acid. After dialysis (MWCO = 8 kDa) against deionized water for three days the reaction mixture was concentrated and lyophilized. N-acetylation (NAc) procedure28: 20 ml 10 % methanol containing 50 mM Na2CO3 were added to the reaction mixture. While stirring 200 µl acetic anhydride were added at 0°C. The pH was adjusted to 7-8 with saturated Na2CO3 solution. Every 30 min 200 µl acetic anhydride were added for three hours. The reaction mixture was dialyzed (MWCO = 8 kDa) against deionized water for three days, concentrated and lyophilized. Characterization of desulfated heparin derivatives. Polyelectrolyte titration37: The anionic charge of the different heparin samples (unmodified and selectively desulfated heparin; 1 mg/ml in MilliQ, pH adjusted to 10) was determined by the Particle Charge Detector (PCD, Mütek GmbH, Herrsching, Germany) via titration with PDADMAC (poly(diallyldimethylammonium chloride), respectively. Based on the exact charge compensation of the polyelectrolyte heparin by dropwise added PDADMAC solution (as indicated by streaming potential measurements), the


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titration of the heparin samples reveals a quantitative and reproducible estimation of its charges. To determine the heparin sulfate content, all the data were corrected for the charge contribution of the carboxylic acid group by subtracting one quarter of the overall charge of the nondesulfated heparin from all measured values (considering only the major sequence of heparin). Finally, the decrease of the anionic charge for the different desulfated heparins caused by the sulfate removal is expressed with respect to the non-desulfated heparin, which served as the control sample (sulfate content is set to 100 %). Infrared (IR) spectroscopy37: IR measurements on the different heparin samples (unmodified and selectively desulfated heparin; 1 mg/ml in MilliQ) were performed on an IFS 55 (EQUINOX, Bruker-Optics GmbH, Ettlingen, Germany) Fourier transform IR (FTIR) spectrometer in the attenuated total reflection (ATR) mode. The ATR-FTIR spectra were recorded on a special mirror setup using the “single-beam sample reference” concept. For that, the particular heparin solution was spread on the upper half (sample) of the silicon internal reflection element (50 x 20 x 2 mm3) and the lower uncoated half was used as the reference. Shuttling the two halves repeatedly in the IR beam, recording the respective intensity spectra (IReference(v), ISample(v)), and computing = − log

(1)

resulted in well-compensated ATR-FTIR absorbance spectra (A(v)). For determination of the heparin sulfate content, the intensity peaks of the sulfate and the carboxylic acid group were recorded and their ratio was calculated. The decrease of this ratio for the different desulfated heparins indicated the sulfate removal, so that the remaining sulfate content could be determined with respect to the non-desulfated heparin, which served as the control sample (set to a sulfate content of 100 %).


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Preparation of starPEG-heparin hydrogels. StarPEG-heparin hydrogels were formed by crosslinking amino end-functionalized four-arm starPEG with EDC/s-NHS activated carboxylic acid groups of heparin.13 A total polymer content of 11.76 % and a 2:1:1 ratio of EDC:sNHS:NH2-groups of starPEG [mol/mol] were used. The molar ratio of starPEG to heparin was set to 3. Heparin and starPEG (10,000 g/mol Polymer Source Inc., Dorval, Canada) were each dissolved in one third of the total volume of ice-cold MilliQ by ultrasonication and afterwards kept on ice (~ 2-4 °C). Similarly, s-NHS (Fluka) and EDC (Sigma-Aldrich) were separately dissolved in the sixth part of the total volume of ice-cold MilliQ. Subsequently, s-NHS and EDC solutions were added to heparin, mixed well and incubated for 15 min on ice to activate heparin carboxylic acid groups. Finally, the star-PEG solution was added to the activated heparin and quickly mixed by vortexing (Minishaker MS2, IKA, Staufen, Germany). For preparation of freestanding gel disks, 104.7 µl of the liquid gel mixture were placed onto a 1 cm² hydrophobic glass cover slip and covered with a second hydrophobic one. Surface-bound gels were prepared on aminosilanized glass cover slips (3.13 µl/cm²) as described elsewhere.13 After polymerization overnight at 22 °C in a humidified atmosphere, the hydrophobic cover slips were removed. Each gel sample was washed in phosphate buffered saline (PBS, SigmaAldrich) to remove s-NHS/EDC and any non-bound starPEG/heparin. PBS (pH 7.4) was exchanged five times (once per hour) and once again after storage for 24 h. Subsequently, the swollen gels were immediately used for further experiments. Characterization of starPEG-heparin hydrogels. Determination of volumetric swelling degree: For determination of the volumetric swelling degree, the initial diameter of the freestanding disks (n = 8) was measured with a digital vernier calliper (MMO, Börnicke, Germany), while the same procedure was repeated after swelling in PBS for 24 h at room temperature.


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Volumetric swelling degree Q (change in gel volume after swelling compared to the dry volume of the gels, V0) was calculated by: " = ⁄ ∙ ⁄$

(2)

where dt is the diameter of the disk after the washing process, dreact is the diameter of the unswollen gel disk (cured reaction mixture), Vreact the volume of the cured reaction mixture and: $ = %&'() ⋅ +&'() + %

-./

⋅+

-./

(3)

where vstarPEG and vheparin denote the molar volume and nstarPEG and nheparin the moles of the compounds, respectively. Conversion efficiency analysis by residual starPEG detection38: Quantification of starPEG in the washing solution (n = 4) was performed by acidic hydrolysis and subsequent high performance liquid chromatography (HPLC) analysis as described elsewhere.38 Briefly, PBSswollen hydrogel volume samples were subjected to vapor hydrolysis in vacuo using 6 M HCl at 110 °C for 24 h and subsequently neutralized. Extraction of starPEG/heparin-fragments from the samples was accomplished by repeated rinsing with a definite volume of 50 mM sodium acetate buffer at pH 6.8. The released starPEG/heparin-fragments were chromatographically separated after precolumn derivatization with ortho-phthalaldehyde on a Zorbax SBC18 column (4.6 x 150 mm, 3.5 mm, Agilent Technologies, Boeblingen, Germany) using an Agilent 1100 LC system (Agilent) with fluorescence detection. StarPEG was quantified using external standards. Determination of rheological characteristics: Storage and loss modulus of free-standing starPEG-heparin hydrogel disks (n = 4) were determined using oscillating measurements on a rotational rheometer (Ares LN2, TA Instruments, Eschborn, Germany) with plate-plate geometry (plate diameter 25 mm, gap width 1.2-1.5 mm). Dynamic frequency sweep tests under strain control were carried out at 25 °C in a shear frequency range of 100-102 rad/s. The strain


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amplitude was set to 3 % and storage and loss modulus were measured as a function of the shear frequency. Final storage moduli are expressed as the mean value over the shear frequency range. Characterization of FGF-2 delivery by radiolabeling studies and Enzyme-Linked Immunosorbent Assay (ELISA). Surface-bound gels (n = 3) were placed in custom-made incubation chambers that decreased the exposure of the protein to surfaces not originating from the hydrogels to a minimum. Native FGF-2 protein solution (Miltenyi Biotech, Bergisch Gladbach, Germany) was spiked with

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I-labeled FGF-2 (Chelatec SAS, Nantes, France) as a

percentage of total protein. This mixture containing 1 µg/ml FGF-2 in PBS was added to surfacebound hydrogels (200 µl per cm²) and the protein was adsorbed over night at 22 °C. After the incubation period, gels were rinsed two times with an excess volume of PBS. Radioactivity was measured twice per sample using gamma counting (LB 123, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Immobilized protein was quantified using

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I-FGF-2

standards. To perform FGF-2 release studies, surface-bound gels (n = 3) were placed in custom-made incubation chambers and 200 µl of protein (1 µg/ml) solution were added per cm². Immobilization was performed over night at 22 °C. The FGF-2 solution was removed followed by two washes with PBS. Each of these solutions was collected and assayed in duplicate using an ELISA Quantikine kit (R&D Systems, Minneapolis, USA). After immobilization, FGF-2 was allowed to release from these gels at 22 °C into 250 µl/cm² of serum-free (SF) endothelial cell growth medium (ECGM; Promocell GmbH, Heidelberg, Germany) supplemented with 0.02 % ProClin 300 (Sigma-Aldrich). Samples were always taken at the same intervals (after 3, 6, 24 and 96 h) and stored at – 80 °C until analyzed by ELISA. An equal volume of fresh medium was added back at each time point.


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Data analysis. Statistical analysis was performed by one-way analysis of variance (ANOVA) and post-hoc Tukey–Kramer multiple comparison test using InStat software (GraphPad). Any p value of less than 0.05 was considered statistically significant. All data are presented as mean ± standard deviation with the indicated numbers (n) of independent repeats.

RESULTS AND DISCUSSION Desulfated heparin (DSH) derivatives devoid of 2-O-, 6-O- and N-sulfate groups (Nreacetylated) were obtained as reported previously.21,35,36,39-41 Figure 1B summarizes the resulting derivatives and gives their expected and experimentally determined sulfate contents. Polyelectrolyte titration and ATR-FTIR spectroscopy were combined for quantification. Figure 1C gives ATR-FTIR spectra of thin casted films of heparin samples. Ratios of the intensities of the ν(O=S=O) at 1220 cm-1 and that of the ν(COO-) band at 1615 cm-1 were used to quantify the sulfation degree and diagnostic IR signals for the amide I and II band of the N-acetyl group were observed at around 1650 and 1550 cm-1 for the N-DSH, NAc, the 6-O-DSH, NAc and the completely desulfated heparin cDSH, NAc derivatives. This analysis confirmed desulfation degrees exceeding 80 %. Therefore, the average number of sulfate groups per disaccharide unit is close to the expected (theoretical) values (see Figure 1, panel B): 2.1 vs. 2 for the N-DSH, NAc, 1.3 vs. 1 for the 6-O-DSH, NAc and 0.3 vs. 0 for the cDSH, NAc. For simplification, we refer in the figures to the theoretical data.


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Figure 1. Selectively desulfated heparin derivatives used in the formation of biohybrid hydrogels for adjusting growth factor release profiles. A: Modulated growth factor delivery from hydrogels with desulfated heparin building blocks – schematic view. B: Functional groups at 2-O, 6-O and N-position of heparin and its desulfated derivatives, expected residual sulfate contents (assuming full desulfation) and analytically determined sulfate contents (mean ± standard deviation from n


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≥ 3 for both polyelectrolyte titration and ATR-FTIR). C: ATR-FTIR spectra on casted films of heparin and its desulfated derivatives. Arrows indicate diagnostic IR bands of the carboxylic acid (red) and sulfate (blue) groups, whose peak intensity ratio was used to quantify the overall sulfate content. The black arrow points to the intensity peak originating from NAc.

The prepared set of selectively desulfated heparin derivatives (Figure 1) was used in the formation of biohybrid polymer networks with amine-terminated starPEG using EDC/s-NHS activation of the GAG carboxylic acid groups.42-44 The influence of the charge density on the swelling behavior of the binary gels was analyzed utilizing a mean field type model (for details see14,22). For this, we consider that nHEP molecules heparin and nPEG molecules of four-functional star-shaped poly(ethylene glycol) form a hydrogel with a final molar ratio γ = nPEG/nHEP. Under ideal conditions, the final molar ratio is given by the molar ratio of the components before the reaction occurs. For the reference system containing unmodified heparin a relation between the degree of swelling Q and γ (supporting information, section 1.2) was set up to describe the equilibrium swelling in different electrolyte solutions by the molar volume fraction of both components and the charge density of heparin.14,22 Based on this, the degree of swelling Q in PBS as a function of γ was predicted for the various desulfated GAGs (see Figure 2 (I)) using the experimentally determined sulfate content (supporting information, section 1.2). Upon stepwise removal of charged moieties by desulfation the mean field approach predicts a slightly decreased Q (maximal reduction of the swelling degree from 40 to 32, see Figure 2 (I)) due to the reduced osmotic contributions of counterions to the swelling.


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Figure 2. Influence of GAG sulfation pattern on biohybrid network formation. Several input parameters were combined in a mean field approach: (I) Charge related network expansion; (II) Amount/distribution of network-forming partners; (III) Potential for heparin activation (protonation of COOH); (IV) Experimental swelling data. This model was used to describe the final network structure, n = 8. (V) The mean field approach covers the overall osmotic pressure in the hydrogel Π as a result of the (expansive) osmotic pressure of counterions Πch, the (expansive) osmotic pressure of excluded volume interaction Πev and the (retractive) osmotic pressure of network elasticity Πel. The overall osmotic pressure of the system is balanced (Π = 0) at equilibrium swelling conditions Qeq, i.e. the hydrogel does not swell or shrink. Statistics: mean ± standard deviation, Tukey-Kramer multiple comparisons test (InStat, GraphPad), *indicates p < 0.05, NS indicates non significant differences.

The charge density of the GAG additionally influences the protonation of reactable groups and therefore the formation of covalent bonds within the hydrogel networks. For the crosslinking reaction leading to hydrogel formation, the protonated heparin carboxylic acid group has to be activated by EDC/s-NHS at a pH between 5 and 7.5.43 Upon gradual desulfation, the availability of protonated residues for activation decreases (maximal pKa reduction from 5.7 to 4; Figure 2 (III) and supporting information, section 1). Accordingly, network formation is impaired for heparins lacking sulfate groups, resulting in higher degrees of swelling, opposite to the trend expected for the osmotic swelling due to reduced charging. Amount, distribution and interaction of the polymeric gel precursors ultimately determine network formation and have to be considered in detail. Therefore, the miscibility of starPEG with heparin and the spatiotemporal correlation of the reactive heparin carboxylic acid groups and the


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amino groups of starPEG g(r, t) were investigated (Figure 2 (II)). Since the correlation function g(r, t) cannot be accessed directly25-32 further experimental data are needed to describe the variations in the network structure resulting from desulfation of the heparin component. Equilibrium swelling data is used to describe possible defect structures as it is directly correlated to the elastic properties of the hydrogel network (see Figure 2 (V).45-47 For gels made from fully sulfated heparin a swelling of QSHexp = 50 was obtained, while for hydrogels formed with NDSH, NAc (QN-DSH,

NAc

= 38) exhibited a significantly lower swelling (see Figure 2 (IV)). In

contrast, a nearly unaltered swelling was determined for hydrogels made out of 6-O-DSH, NAc (Q6-O-DSH,

NAc

=48), while a significantly higher swelling for cDSH, NAc containing networks

(QcDSH, NAc = 76) (see Figure 2 (IV)) was found. Since these trends cannot be explained by the reduction of the counterion osmotic pressure (see Figure 2 (I)) only network defects can be concluded to occur in dependence on the type of heparin derivative. Such network defects result whenever not all of the four arms of a starPEG molecule are linked to different GAG molecules (schematic illustration in Figure 3 (I)).


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Figure 3. Network structure, mechanical properties and growth factor release in dependence on GAG sulfation pattern of desulfated heparin derivatives used for gel formation. Variations in the elastically active network strands (I, right) as well as the experimentally determined and theoretically calculated storage moduli revealed the presence of defect structures within the network (I, left, n=4). Based on the results of the mean field model, the molar ratio of the hydrogel building blocks was adjusted to obtain networks with similar mechanical properties but different sulfation patterns (SH vs. heparin derivates, see arrows, n=4, II). Altered sulfation


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patterns thus enable a far-going modulation of FGF-2 release from the produced hydrogel matrices (n=3, III). Statistics: mean ± standard deviation, Tukey-Kramer multiple comparisons test (InStat, GraphPad), *indicates p < 0.05, NS indicates non significant differences.

Multiple bonds to the same GAG molecule or dangling ends are defects that reduce the mechanical integrity (elasticity) of the hydrogel network and can be caused by the availability of several crosslinking points at one GAG molecule and the restricted mobility of the remaining arms of a starPEG molecule, respectively.48-52 As a measure for effective crosslinks, the swelling of the gels for the different heparin derivatives obtained by the mean field approach was compared with the experimentally observed swelling (see supporting information, section 1.2) to provide the molar ratio of elastically active network strands in the hydrogel (γeff, supporting information, Figure S.1.1 and Table S 1.1). These values expressed as percental change (γeff//γSH1) reflect the ‘effective crosslinking degree’ which is significantly influenced by the contained type of GAG component (Figure 3 (I)): gels made from N-DSH, NAc were found to exhibit elevated molar ratios of elastically active network strands (+58 %). In contrast, the use of 6-ODSH, NAc led to a slight decrease of -22 %, while gels prepared out of cDSH, NAc displayed a strong decrease of elastically active network strands (-55%). These results are in line with HPLC analyses of the PBS solutions applied for rinsing/swelling the gels (supporting information, section 2, Figure S2.1): For hydrogels prepared from fully sulfated heparin about 8 % and from networks prepared with cDSH, NAc 12 % of the starPEG was removed upon swelling. In contrast, N-DSH, NAc-containing hydrogels displayed the lowest amount of non-bound starPEG (3 %), indicating that the introduced acetyl group reduces multiple bond formation and/or dangling ends upon network formation (see Figure 3 (I)). These observations can be attributed to


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the modified hydrophilic balance, causing altered excluded volume interactions of the heparin derivatives and to variations in their miscibility with starPEG.25-32 Furthermore, the storage modulus of the swollen hydrogels was experimentally determined by oscillatory rheometry (for details see supporting information sec. 1.2) as this parameter provides direct access to the elastic network properties.45-47 The theoretically predicted storage modulus obtained by the mean field approach considering γeff for the different hydrogels (Figure 3 (I) and supporting information, section 1.2) was quantitatively confirmed by experimental data. Similar to the changes in the content of elastically active network strands, networks formed with N-DSH, NAc displayed a dramatic increase in the storage modulus (14.7 kPa, compared to 7.3 kPa for gels made from unmodified heparin). In contrast, when 6-O-DSH, NAc was used as a gel precursor, a comparable stiffness of 6.8 kPa was observed and hydrogels prepared out of cDSH, NAc showed a strong decrease of storage modulus to values as low as 1.3 kPa (Figure 3 (I)). Altogether, experimental data and theoretical analysis of elastically effective network strands indicate that differences in the network characteristics observed for the compared heparin derivatives result from both differences in the network structure and in the counterion osmotic pressure. Based on the coinciding results from experiment and theory, γeff can be adjusted for producing gels with similar network characteristics from heparin derivatives with different sulfation patterns (see Figure 3 (II)) using γeff to calculate the corresponding molar ratio of the precursors (γeff*, supporting information, section 1.2). As a proof of concept, gels of similar stiffness were formed from all compared heparin derivatives, matching the characteristics of gels made from unmodified heparin at γ = 3 (7.3 kPa) within the experimental error (± 2.5 kPa, Figure 2 (VI)) by adjusting the molar ratio to γeff* for the different derivates (see supporting information


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section/table S1.2). Thus, our introduced approach allows for the targeted design of biohybrid hydrogels with precisely adjusted growth factor affinity. As an example, the release of fibroblast growth factor 2 (FGF-2) from the hydrogels was modulated by using heparin derivatives of varying sulfation patterns (see Figure 3 (III)). Removal of only one sulfate residue (sulfation degree ~ 69 % of heparin, on average 2.1 sulfate groups per disaccharide unit) involved in the FGF-2 interaction with heparin already increased the cumulative protein release by a factor of 3 when compared to gels made of heparin with 100 % sulfate content. This effect was further enhanced by removing both the 6-O- and N-sulfate (sulfation degree ~ 44 % of heparin, on average 1.3 sulfate groups per disaccharide unit) resulting in a 5 fold increase in FGF-2 release levels. Additional elimination of the 2-O-sulfate for cDSH (remaining sulfation degree ~ 11 %, on average 0.3 sulfate groups per disaccharide unit) boosted the release efficiency up to an 11 fold increase compared to the standard heparin. While generally lower amounts of FGF-2 were immobilized with decreasing sulfate content (supporting information, section 2, Figure S2.2), the absolute quantities of released growth factors were found to be gradually elevated: For a given loaded amount of FGF-2 up to 10-fold higher release rates were observed when using gels containing desulfated heparin derivates instead of the fully sulfated heparin. Remarkably, hydrogels formed out of cDSH, NAc with only 0.3 sulfate groups per disaccharide unit remaining (11% compared to the amount found in SH) were observed to bind 34 % of the amount of FGF-2 compared to gels made of SH (supporting information, section 2, Figure S2.2). The high total heparin content of the investigated hydrogel system is thought to cause this significant affinity for FGF-2 even if the low sulfated cDSH, NAc-derivate is used as gel building block.


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In sum, heparin derivatives with customized desulfation not only allow for a far-going adjustment of release profiles of biohybrid hydrogels but afford a more efficient cytokine administration.

CONCLUSION In sum, a rational design strategy for biohybrid hydrogels with tailored biomolecular and physicochemical properties was developed and applied. A set of selectively desulfated heparin derivatives was utilized for customizing the growth factor delivery from gels with independently adjusted physical properties. The obtained materials offer an unprecedented level of control over cell-instructive matrix characteristics and can therefore be instrumental in applications relying on the microenvironmental regulation of cellular fate decisions.

ASSOCIATED CONTENT Supporting Information A theoretical part describing the ionization pattern of carboxyl groups at the heparin, the swelling behavior and the storage modulus of the hydrogels synthesized of the different derivates (section 1), HPLC analysis of the StarPEG content of the washing solutions after hydrogel swelling and FGF-2 binding to the hydrogels (radiolabeling) (section 2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION


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Corresponding Author *Phone: +49 351 4658531. Fax: +49 351 4658 533. E-mail: [email protected]. Notes. The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. #These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft through grants SFB-TR 67, WE 2539-7 and FOR/EXC999, by the Leibniz Association (SAW-2011-IPF-2 68) and by the European Union through the Integrated Project ANGIOSCAFF (7th Framework Program). A.Z. was supported by a stipend of the Dresden International Graduate School for Biomedicine and Bioengineering. We thank Monique Marx and David Scharnetzki (Leibniz Institute of Polymer Research Dresden) for CD measurements of desulfated heparins and Tina Lenk (Leibniz Institute of Polymer Research Dresden) for HPLC analysis and Michael Lang (Leibniz Institute of Polymer Research Dresden) for helpful discussions.


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Biohybrid networks of selectively desulfated glycosaminoglycans for tunable growth factor delivery TOC Figure


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Biohybrid Networks of Selectively Desulfated Glycosaminoglycans for

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