Determination of the Composition for Binary Mixtures of Polyanions

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Determination of the Composition for Binary Mixtures of Polyanions: The Case of Mixed Solutions of Alginate and Hyaluronan Ilaria Geremia,† Massimiliano Borgogna,† Andrea Travan,† Eleonora Marsich,‡ Sergio Paoletti,† and Ivan Donati*,† †

Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, I-34127 Trieste, Italy Department of Medical, Surgical, and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, I-34129 Trieste, Italy

‡

S Supporting Information *

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INTRODUCTION Three dimensional systems based on polymer mixtures are gaining attention in the field of biomaterials as potential extracellular matrix (ECM) mimics. In fact, it is considered that the structural complexity of the latter can only be matched by multiple structural and interdigitated functional components.1 For this reason, tissue engineering approaches strongly rely on the development of synthetic ECM which, by mimicking the biochemistry and the biophysical stimuli of the natural one, creates the proper niche for cell recognition and mechanotransduction driving healthy tissue development. Among the vast class of polymers well-suited for biological applications, polysaccharides rank as ideal candidates as synthetic ECM mimics for their generally accepted biocompatibility and their commercial availability complying with good laboratory practice (GLP) and good manufacturing practice (GMP) requirements. Indeed, polysaccharides have been used for biomedical applications such as articular viscosupplementation2 and cell transplantation3,4 above all. Along this line, it was shown that binary systems of alginate and a lactose-modified chitosan (short-named chitlac) enhance chondrocyte proliferation and production of glycosaminoglycans.5 This result was traced back to both the synergistic interactions among the two and the bioactive properties of chitlac;6 improvement in cell proliferation was also observed when hyaluronan was used as third component of the mixture.7 Mixed systems composed of (sodium) alginate and (sodium) hyaluronate (hyaluronan, HA) are of particular interest because they combine the gel-forming ability of the former (biologically inert) polysaccharide with the biological activity of the latter one. Mixed beads of alginate and hyaluronan have been proposed for in vitro cartilage engineering.8 Similarly, enzymatically cross-linked hydrogels based on modified alginate and hyaluronan have been proposed as cell delivery vehicles.9 In addition, scaffolds based on the combination of the two polysaccharides have been suggested for cartilage engineering.10 Tissue engineering applications based on the combined use of alginate and hyaluronan rely on the controlled release of the latter component to the cell environment, where it exerts its biological activity. Moreover, when scaffolds are proposed for medical use, the time-scale matching between their degradation and ingrowth of newly formed functional tissue is of fundamental importance for restoring mechanical integrity and biological activity of the tissue. To monitor in detail these aspects, a quantitative method to determine the concentration © 2014 American Chemical Society

in solution of mixtures of polysaccharides is needed. Indeed, Lindenhayn and co-workers8 attempted to evaluate the release of hyaluronan from mixed alginate/HA scaffolds by using biosynthesized tritiated HA. However, this process requires a rather complex preparation of the sample and it might represent an issue for the determination of both polysaccharides in the mixture at the same time. The present manuscript deals with the setup and evaluation of two analytical methods, namely Circular Dichroism (CD) and 23Na longitudinal relaxation, to determine the composition of binary mixtures of the sodium salts of alginate and hyaluronan in solution. The quantitative results obtained were confirmed by 1H NMR, and the developed methods were applied to the determination of the composition and polymer release of mixed hydrogels. Although focused on the specific case of binary mixtures of alginate and hyaluronan, the approach drawn in the present manuscript can find applications in different binary systems composed of natural or synthetic noninteracting polymers.

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MATERIALS AND METHODS

Alginate Pronova UP LVG (molecular weight, MW ∼ 120 000; fraction of guluronic G residues, FG = 0.69; fraction of guluronic diads, FGG = 0.59; number average of G residues in G-blocks, NG>1 = 16.3) and sodium hyaluronate (hyaluronan) Pharma grade HA80 (MW ∼ 800000) were kindly provided by Novamatrix/FMC Biopolymer (Sandvika, Norway). Alginate isolated from Macrocystis pyrifera (M. Pyr., MW ∼ 160 000; FG = 0.42; FGG = 0.21; NG>1 = 5) was kindly provided by Prof. Gudmund Skjåk-Bræk, Institute of Biotechnology, University of Trondheim (NTNU) (Norway). Hyaluronan HA24 (MW ∼ 240 000) was provided by SIGEA S.r.L. (Trieste, Italy). Sodium chloride, calcium carbonate (CaCO3), δ-D-Gluconolactone (GDL) and ethylenediaminetetraacetic acid tetra sodium salt (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Simulated Body Fluid (SBF) was prepared according to the procedure reported by Kokubo.11 2.1. Circular Dichroism (CD). Circular Dichroism spectra of the sodium form of alginate (LVG or M. Pyr.), hyaluronan, and their binary mixtures were recorded in deionized water (polymer concentration, cpolym, 0.05−0.25 g/L) with a Jasco J-700 spectropolarimeter. A quartz cell of 1 cm optical path length was used, and the following setup was maintained: bandwidth, 1 nm; time constant, 2s; scan rate, 20 nm/min. Raw data were normalized by the concentration Received: December 13, 2013 Revised: January 21, 2014 Published: January 23, 2014 1069

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Figure 1. Comparison between the experimental () and the theoretical (− −, eq 1) CD curve for a binary mixture of hyaluronan (HA24) and (a) LVG or (b) M.Pyr. alginate. (c) Comparison between experimental (■) and theoretical (□, eq 1) composition of binary HA/LVG Alg mixtures as determined by CD measurements. of the polymeric solution, and they were expressed as reduced ellipticity, θred. 2.2. 1H NMR. 1H NMR spectra of LVG alginate, hyaluronan, and their binary mixtures were recorded at 60 °C with a JEOL 270 NMR spectrometer (6.34 T) operating at 270 MHz for proton. Samples were dissolved in D2O and 40 μL of NaOD 20% in D2O (final NaOD concentration 0.3 M) were added prior to the analysis. The chemical shifts are expressed in ppm downfield from the signal for 3(trimethylsilyl)propanesulfonate. 2.3. 23Na-T1. Longitudinal relaxation time (T1) of sodium ions in the presence of alginate (LVG or M. Pyr.), hyaluronan, and their binary mixtures were performed at 25 °C on a JEOL 270 NMR spectrometer (6.34 T) operating at 71.38 MHz for sodium in the presence of D2O (10% V/V) and at different concentrations of added sodium chloride. The relaxation rate of 23Na in polymer solutions (R1,polym = 1/T1,polym) was measured by means of the inversion recovery sequence using 19 different τ intervals. The relaxation rate of 23 Na in the absence of the polymer was indicated as R1,solv. ΔR1was defined as R1,polym − R1,solv. When SBF was used, EDTA (tetrasodium salt) to a final concentration of 20 mM and D2O (10% V/V) were added. 2.4. Binary HA/LVG Alginate Hydrogel Preparation and Polysaccharide Release. A binary solution of hyaluronan HA24 (final concentration, cpolym = 15 g/L) and LVG alginate (final concentration, cpolym = 15 g/L) in deionized water was blended with CaCO3 (final concentration 50 mM) and degassed. GDL was then added to a final concentration of 100 mM. The slurry was poured on a Petri dish and allowed to gel for 18 h at room temperature. For the release studies, a disk-shaped hydrogel was poured in 10 mL of deionized water. The system was maintained at room temperature for 24 h under gentle stirring. The supernatant was collected and analyzed. 2.6. Hydrogel and Supernatant Treatment Prior to Analysis. The HA/LVG alginate hydrogel and the supernatant, respectively, were transferred in a dialyzing tube (Mw cutoff 10,000). Deionized water was added for the former sample. The system was dialyzed

against aqueous HCl 0.1 M (3 shifts) and against deionized water until the conductivity was below 4 μS. The pH was adjusted to 7.2, the solution was filtered through 0.45 μm Millipore filters and freeze-dried. The freeze-dried material was weighted, and a known amount was used for the determination of the polysaccharide composition.

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RESULTS AND DISCUSSION The determination of the composition for binary solutions containing two different polyanions poses a challenge since methods based on size exclusion have limited chance of success due to the polydispersity of the samples, unless they are little polydisperse and their molecular weight (or, more exactly, their hydrodynamic volume) differs by orders of magnitude. A quantitative approach needs to be based on a reduced (i.e., normalized by the total polymer mass) experimental parameter, ψ, which is sensitive to the composition of the binary mixture and can be expressed as a linear combination of the reduced contribution of each component considered alone, δ0x (eq 1). ψ = δ10ϕ1 + δ20ϕ2 = δ20 + (δ10 − δ20)ϕ1 = δ20 + (Δδ 0)ϕ1 (1)

where ϕ1and ϕ2 are the weight fractions of component 1 and component 2 in the binary mixture, respectively, and Δδ0 is the difference in the reduced properties of the two pure components. This approach assumes that no change (or at least but a negligible change) of the physical-chemical properties of the pure components takes place upon moving to the binary solution. More specifically, this approach then assumes that the physical-chemical (e.g., temperature, solvent, and cosolute composition) conditions in which the mixture properties are measured do not affect the individual properties of the two components and there is no interaction between the 1070

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Figure 2. (a) Dependence of the reduced relaxation rate (ΔR1/cpolym) on the fraction of alginate (ϕAlg) in HA/Alg binary mixtures with a total polymer concentration (cpolym) of 0.75 g/L (□ LVG), 1.5 g/L (■ LVG, ▲ M. Pyr.) and 3 g/L (● LVG, Δ M. Pyr.) in the presence of NaCl 0.05 M. Lines represent the linear fitting of the experimental data (R2 > 0.99). (b) Dependence of the relaxation rate of the 23Na ion on alginate concentration in binary mixtures of HA24/LVG alginate in the presence of NaCl 0.05 M. Dotted line represents the linear best-fit of the experimental data (R2 > 0.98). (c) Dependence of Δδ0 on NaCl concentration in binary HA/LVG alginate solutions at total polymer concentration of 3g/L. Dotted line represents the best fit of the experimental data according to y = axb (R2 > 0.99). (d) Dependence of the reduced relaxation rate (ΔR1/cpolym) on the fraction of alginate (ϕAlg) in HA/LVG alginate binary mixtures with a total polymer concentration (cpolym) of 3 g/L in SBF.

In the specific case of a binary mixture of alginate and hyaluronan in the presence of added Na+ ions, eq 1 holds since a rheological analysis (not reported) evidenced the lack of interactions between the two polyanions.19 When considering CD spectra, the experimental parameter ψ in eq 1 is the reduced ellipticity of the mixture, as this markedly differs for the two polysaccharides (Figure 1S in the Supporting Information). Equation 1 was expressed as a system of equations over the whole range of wavelengths explored that was solved in ϕAlg by minimizing the sum of the squared errors between experimental and theoretical data points. By applying this procedure to a binary mixture of HA/Alginate, a good fitting of the experimental curve is achieved for both LVG and M.Pyr. samples (Figure 1a,b). When CD at the wavelength of the minimum (210 nm) was used to determine the mixture composition of HA and alginate systems, |Δδ0| from the fitting equals 202 ± 5 deg cm2 g−1 for LVG, whereas |Δδ0| equals 235 ± 12 deg cm2 g−1 when the binary mixture contains alginate from M. Pyr. This difference clearly stems from the inherent difference of the respective CD spectra of the two alginates: the quoted references12,13 not only showed that the CD spectra of alginates depend on the monomer and diad composition, but even exploited such difference to successfully arrive at their

two components or, if any interaction develops, it does not affect such individual properties (linear additivity). Therefore, the presence of ions known to induce conformational modifications or chain aggregations in the polymer components of the mixture has to be avoided unless a specific cheleating agent is added to the system. Given this premise, in the present work, binary mixtures composed by alginate and hyaluronan were considered and two techniques were selected for the determination of their composition, namely circular dichroism (CD) and the longitudinal relaxation time determination of 23Na (23Na-T1) studied by NMR. CD is a versatile technique that has already been used to determine the dyadic composition of alginate samples. 12,13 The second technique (23Na longitudinal relaxation) was selected based on the differences in the linear charge density, ξ,14,15 between the two polysaccharides. Specifically, ξ equals 1.53 and 1.48 for LVG and M. Pyr. alginate, respectively,16 thus in both cases it is higher than the critical value for the condensation of the monovalent ions, i.e., ξcr = 1.17,18 At variance, hyaluronan is characterized by a ξ of 0.71. Hence, 23Na longitudinal relaxation rate is expected to strongly depend on the composition of the binary mixtures. 1071

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Figure 3. (a) Composition percentage of a hydrogel composed of a binary mixture HA/LVG alginate by means of 1H NMR (black bars), CD (white bars) and 23Na-T1 (gray bars). Dotted line represents the theoretical composition percentage. (b) Release (%) of LVG alginate (white bars) and hyaluronan (gray bars), with respect to their initial amount, from an HA24/LVG alginate hydrogel incubated in water as determined by means of CD, 1H NMR, and 23Na-T1, respectively.

2c, with the higher effect of alginate on ΔR1 recorded for the lower concentration of the added Na+ ions. In order to assess the possibility of using 23Na-T1 for the determination of the composition of HA/alginate binary mixtures in the presence of complex mixtures of ions, simulated body fluid (SBF) was used. In this case, the presence of calcium ions in the medium, although to a limited extent, impairs the determination of alginate in solution, as the divalent ion will be coordinated by the polysaccharide. However, when a chelating agent, such as EDTA, is added in solution, a linear dependence is found for the reduced relaxation rate of 23Na (ΔR1/cpolym) on alginate weight fraction (Figure 2d). The reliability of the two quantitative methods for the determination of the composition of binary mixtures was confirmed by means of 1H NMR. In this latter case, the signal arising from the anomeric protons of the G residues in alginate (G1-GAlg) and the signal of the methyl protons of the N-acetylglucosamine units of hyaluronan (GlcNAcHA) were used. A linear correlation (R2 > 0.99) was found between the ratio of these two signals and the ratio between the weight fractions of the two polysaccharides (Figure 3S in Supporting Information). The quantitative determination of the composition of the alginate/HA binary mixture by means of 1H NMR was carried out in alkaline conditions. The advantage of this approach is that the analysis can be carried out without prior degradation of the sample. However, its limitation is that only anomeric proton of G residues of alginate can be detected as the H1 arising from hyaluronan and from the M residues in alginate, as well as the H5G of the GG diads in the latter, overlap with water. Figure 3a reported the composition of a binary hydrogel containing alginate and hyaluronan as determined by CD, 23NaT1 and 1H NMR. Prior to the analysis, the sample was dissolved by removing the calcium ions (see Materials and Methods section). A very good agreement was achieved in the determination of the composition by means of the three methods used. In view of these results, CD and 23Na-T1 were used to determine the composition of the polysaccharide mixture released from a HA/LVG alginate hydrogel and the results were compared with those obtained by means of 1H NMR (Figure 3b). It can be seen that, also in this case, a good agreement was found. Due to the high amount of the HA in the supernatant and its low effect on the sodium ion relaxation,

determination. This point should be always kept in mind when dealing with a compositionally complex biopolymer like alginate. However, in both cases, no detectable dependence on the total polymer concentration, i.e., cpolym, in the range 0.05−0.25 g/L was noticed. Figure 1c, reports the good agreement between the experimental compositions of the binary mixtures of LVG alginate and hyaluronan and those calculated on the basis of eq 1. However, it should be noted that, when the fraction of hyaluronan overcomes by and large the one of alginate, a small error (approximately 7%) is associated with the theoretical determination. The approach sketched in eq 1 can also be applied upon considering the reduced relaxation rate of 23Na (ΔR1/cpolym) as the experimental parameter ψ. In this case, due to the (albeit limited) contribution of the viscosity to the ion relaxation rate, it could expected that Δδ0 = Δδ0(c). However, in the case of the HA/LVG Alg binary mixtures, there is a minor effect of total polymer concentration (cpolym) in the range 0.75−3 g/L (Figure 2a). On average, the value of Δδ0 in this concentration range is 13.9 ± 0.7 L s−1 g−1. When M. Pyr. is used in the binary mixtures with HA (Figure 2a), the reduced relaxation rate is lower. This is due to the lower charge density of the alginate from M. Pyr., compared with that of LVG alginate, as a consequence of its lower fraction of G residues. In this latter case, Δδ0 in the concentration range from 1.5 g/L to 3 g/L is 8.9 ± 0.7 L s−1 g−1, also showing a slightly higher dependence on the total polymer concentration than the one detected for LVG alginate. It should be stressed that quantitative determination of binary mixtures by means of 23Na-T1 was not affected by the molecular weight of HA. In fact, Figure 2S in the Supporting Information showed that two calibration curves with HA24 and HA150 did not display significant differences. At variance with the effect of the composition of the alginate samples used, due to its comparatively low ξ, HA has little impact on the relaxation rate of sodium ions. Indeed, the experimental data points recorded for HA/LVG alginate binary mixtures display a linear dependence on alginate concentration in solution (Figure 2b). Since ξ of alginate induced monovalent ion condensation, it is expected that the overall concentration of the supporting salt, i.e., NaCl, influences the 23Na longitudinal relaxation. Indeed, the parameter Δδ0 showed a power-law dependence from the aqueous NaCl concentration in solution, as reported in Figure 1072

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23

(11) Kokubo, T.; Hanakawa, M.; Kawashita, M.; Minoda, M.; Beppu, T.; Miyamoto, T.; Nakamura, T. J. Mater. Sci.: Mater. Med. 2004, 15, 1007−1012. (12) Donati, I.; Gamini, A.; Skjåk-Braek, G.; Vetere, A.; Campa, C.; Coslovi, A.; Paoletti, S. Carbohydr. Res. 2003, 338, 1139−1142. (13) Morris, E. R.; Rees, D. A.; Thom, D. Carbohydr. Res. 1980, 81, 305−314. (14) Manning, G. S. Biophys. Chem. 1977, 7, 95−102. (15) Manning, G. S. Biophys. Chem. 1978, 9, 65−70. (16) Donati, I.; Asaro, F.; Paoletti, S. J. Phys. Chem. B 2009, 113, 12877−12886. (17) Donati, I.; Benegas, J. C.; Cesár o, A.; Paoletti, S. Biomacromolecules 2006, 7, 1587−1596. (18) Paoletti, S.; Cesàro, A.; Delben, F.; Crescenzi, V. R. R. Polyelectrolyte aspects of conformational transitions and interchain interactions in ionic polysaccharide solutions: Comparison of theory and microcalorimetric data. In Microdomains in Polymer Solutions; Dubin, P. L., Ed.; Plenum Press: New York, 1985; pp 159−189. (19) Travan, A.; Fiorentino, S.; Grassi, M.; Borgogna, M.; Marsich, E.; Paoletti, S.; Donati, I. manuscript in preparation.

Na-T1 tends to slightly overestimate its concentration in the mixture: in this case, the error percentage is below 6%.

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CONCLUSIONS The need of quantitative methods for the determination of the composition of mixed polymer solutions is particularly stringent whenever such mixtures are used in tissue engineering applications where one of the components exerts a biological effect on cells and tissues. The present manuscript has proven that CD and 23Na-T1 represent a fast and reliable alternative to 1 H NMR for the determination of the composition of binary mixtures of alginate and hyaluronan. However, this approach could find wide applicability for the determination of binary mixtures composed of different and noninteracting polymers, in particular in those cases where 1H NMR signals of the polymers are not well resolved or overlapping.

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ASSOCIATED CONTENT

S Supporting Information *

CD spectra of LVG alginate and hyaluronan, relaxation rate for 23 Na in binary mixtures as a function of HA molecular weight, and 1H NMR of HA/LVG alginate binary mixtures are included. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; tel: +39 040 558 8733. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Miss Giulia Ganzini is thanked for her skillful assistance in the experimental part. This study was supported by the EU-FP7 Project “Development of a resorbable sealing patch for the prevention of anastomotic leakage after colorectal cancer surgical treatment - AnastomoSEAL” (Contract number 280929).

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REFERENCES

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