Structure and Dynamics of Alginate Gels Cross-Linked by Polyvalent

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The Structure and Dynamics of Alginate Gels Crosslinked by Polyvalent Ions Probed via Solid State NMR Spectroscopy Ji#í Brus, Martina Urbanová, Jiri Czernek, Miroslava Pavelkova, Katerina Kubova, Jakub Vyslouzil, Sabina Abbrent, Rafal Konefal, Jiri Horsky, David Vetchy, Jan Vysloužil, and Pavel Kulich Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00627 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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The Structure and Dynamics of Alginate Gels Crosslinked by Polyvalent Ions Probed via Solid State NMR Spectroscopy Jiri Brus1*, Martina Urbanova 1, Jiri Czernek1, Miroslava Pavelkova2, Katerina Kubova2, Jakub Vyslouzil2, Sabina Abbrent1,Rafal Konefal1, Jiri Horský,1 David Vetchy,2 Jan Vysloužil3, Pavel Kulich4

1)

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic 2)

University of Veterinary and Pharmaceutical Sciences, Faculty of Pharmacy, Department of

Pharmaceutics, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic 3)

Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 267/2, 611

37, Brno, Czech Republic 4)

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 296/70,

621 00, Brno, Czech Republic.

ACS Paragon Plus Environment

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TOC

KEYWORDS: alginate gels, microbead formulations, external gelation, segmental dynamics, molecular-level structure, solid-state NMR spectroscopy

ABSTRACT: Alginate gels are an outstanding biomaterial widely applicable in tissue engineering, medicine, and pharmacy for cell transplantation, wound healing and efficient bioactive agent delivery, respectively. This contribution provides new and comprehensive insight into the atomic-resolution structure and dynamics of polyvalent ion-crosslinked alginate gels in microbead formulations. By applying various advanced solid-state NMR (ssNMR) spectroscopy techniques, we verified the homogenous distribution of the crosslinking ions in the alginate gels and the high degree of ion exchange. We also established that the two-component character of the alginate gels arises from the concentration fluctuations of residual water molecules that are preferentially localized along polymer chains containing abundant mannuronic acid (M) residues. These hydrated M-rich blocks tend to self-aggregate into subnanometer domains. The resulting co-existence of two types of alginate chains differing in segmental dynamics was revealed by 1H-13C dipolar profile analysis, which indicated that the average fluctuation angles of the stiff and mobile alginate segments were ca. 5-9° or 30°, respectively. Next, the

13

C CP/MAS NMR spectra indicated that the alginate


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polymer microstructure was strongly dependent on the type of crosslinking ion. The polymer chain regularity was determined to systematically decrease as the crosslinking ion radius decreased. Consistent with the 1H-1H correlation spectra, regular structures were found for the gels crosslinked by relatively large alkaline earth cations (Ba2+, Sr2+ or Ca2+), whereas the alginate chains crosslinked by bivalent transition metal ions (Zn2+) and trivalent metal cations (Al3+) exhibited significant irregularities. Notably, however, the observed disordering of the alginate chains was exclusively attributed to the M residues, whereas the structurally welldefined gels all contained guluronic acid (G) residues. Therefore, a key role of the units in Mrich blocks as mediators promoting the self-assembly of alginate chains was experimentally confirmed. Finally, combining 2D

27

Al 3Q/MAS NMR spectroscopy with density functional

theory (DFT) calculations provided previously unreported insight into the structure of the Al3+ crosslinking centers. Notably, even with a low residual amount of water, these crosslinking units adopt exclusively six-fold octahedral coordination and exhibit significant motion, which considerably reduces quadrupolar coupling constants. Thus, the experimental strategy presented in this study provides a new perspective on crosslinked alginate structure and dynamics for which high-quality diffraction data at the atomic resolution level are inherently unavailable.

INTRODUCTION Alginates, naturally occurring biopolymers obtained from brown sea algae, exhibit a suitable combination of physicochemical properties, such as biocompatibility, biodegradability, nontoxicity, non-immunogenicity, and excellent mucoadhesion, that render them a nearly ideal and long-sought-after material for use in tissue engineering, medicine and pharmacy for cell transplantation, wound healing and efficient bioactive agent delivery, respectively.1-4 This


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outstanding position and the popularity of alginates particularly result from the ability of alginate polymers to easily undergo temperature-independent physical crosslinking and solgel transition, which is induced either by co-adding polyvalent ions or by changing pH.5,6 The pH-responsive behavior of the resulting alginate gel microbeads is thus due to their low pHinduced shrinkage, which ensures that any encapsulated drug is retained and protected against enzymes in the acidic pH of the stomach.7 Consequently, alginate gels have been extensively investigated for a long time, and this effort currently resulted in attempts to formulate novel injectable and 3D bioprinted hydrogels.8,9,10 However, the application of alginates is much wider and goes beyond the field of biomedicine. For instance an application of alginate gels as materials suitable for capturing toxic metal ions such as Pb2+, Cd2+ from waste water seems to be very promising.11 On a molecular level, alginate polymers consist of α-L-guluronic acid (G) and β-Dmannuronic acid (M) residues. These residues are linked by 1-4 glycosidic bonds, forming homopolymeric blocks of G units (GG blocks) or M units (MM blocks) and heteropolymeric sequences of randomly coupled G and M units (GM blocks).12.13 The ratio of M to G and their arrangements considerably vary with the algae source.14-17 When the polymer chains are crosslinked by polyvalent ions, typically by Ca2+ ions, increasing the number of G residues was found to markedly increase selective binding. By contrast, poly-M blocks and alternating MG blocks exhibit lower selectivity,18,19 and the efficient complexation of MM and MG blocks requires a rather high polyvalent ion concentration. Consistent with these findings, comprehensive X-ray diffraction studies suggested that the tight electrostatic (ionic) binding of G-rich blocks in two parallel alginate chains; thus, these studies resulted in the egg-box model being formulated.20,21,22 Currently, the egg-box model is the most accepted and easiest approach for visualizing the local geometry along alginate chains. As the chemical composition of alginate polymers significantly varies, other possible structures involving the


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presence of MM blocks and enabling interchain hydrogen bonding have also been suggested.21 A series of high-level molecular dynamics (MD) and quantum chemical (e.g., density functional theory, DFT) computational studies providing in-depth insight into the mechanism of alginate chain folding23, the local and global structures of alginate and the nature of the interactions between uronic units and crosslinking ions have been published.24 Bivalent alkaline earth cations (Mg2+, Ca2+, and Sr2+) were revealed to form ionic bonds, whereas bivalent transition metal ions (Mn2+, Co2+, Cu2+, and Zn2+) and trivalent metal cations (Fe3+, Cr3+, Al3+, Ga3+, Sc3+, and La3+) were found to complex uronates via strong coordinatecovalent bonds. Furthermore, the binding strength of the trivalent cations was significantly stronger than that of the divalent cations, and the bivalent alkaline earth cations had the lowest binding energy. In this regard, a good correlation was found between alginate’s affinity25,26 for bivalent transition metal ions and the DFT-calculated interaction energies. Similarly, alginate had a greater affinity for trivalent metal cations than for divalent cations, which was consistent with the DFT-calculated binding energies. By contrast, the trend of the calculated interaction energies of the alkaline earth cations in the ionic complexes was opposite to the alginate affinity order. This finding shows that binding strength is not a limiting factor in alginate gelation and that the compositional and conformational flexibility/diversity of alginate polymer chains are also important factors that affect the tendency of alginates to form aggregates and macroscopic gels.24,27 According to recent MD studies, greater fractions of M monomers increase heteropolymer chain flexibility and promote chain association.24 Although many advanced computational studies of alginate polymers have been published,20,24,28 surprisingly few experimental investigations verifying and characterizing the


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structures of these systems at the atomic resolution level have been performed to date.25-27 Therefore, to bridge this gap, we present our investigation of the local structure and segmental dynamics of a series of alginate gels crosslinked by various polyvalent ions (Ca2+, Zn2+, Al3+, Mn2+, Sr 2+, Cu2+, and Ba2+). Particular attention is devoted to experimentally verifying the different roles of M and G residues in forming alginate gels and describing the local geometries around trivalent Al3+ ions, the structure of which has never been experimentally investigated. In addition, we probed the degree of Na+ ion exchange during alginate gel crosslinking, the location of water molecules along the polymer chains and the homogeneity of the resulting alginate beads. For these studies, we used several state-of-the-art solid-state NMR (ssNMR) spectroscopy techniques, which have recently evolved into powerful tools for studying the structure of various organic, inorganic and hybrid materials, including complex multicomponent drug-delivery systems.32-34

Materials and Methods

Materials. Sodium alginate (ALG_Na, Sigma Aldrich, St. Louis, USA) of medium viscosity grade (5.0 – 40.0 cps, for 1 wt.% dispersion in purified water) was used as the starting material for preparing the crosslinked alginate gels. Aqueous solutions of CaCl2, ZnCl2, MnCl2, SrCl2, BaCl2, and AlCl3 (1 mol/dm3) were used as crosslinking agents. The primary structure of the ALG_Na polymer, i.e., fractions of individual G and M monomer units and fractions of the corresponding dyads and triads, as determined by highresolution 1H NMR spectroscopy35 (Figure 1), is as follows: FG = 0.43; FM = 0.57; FGG = GG/(M + G) = 0.21; FMM = MM/(M + G) = 0.35; FGM = FMG = MG/(M + G) = 0.22; FGGG = GGG/(M + G) = 0.10; FMGM = MGM/(M + G) = 0.11; FGGM = FMGG = GGM/(M + G) = 0.11


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The degree of randomness is B = 0.9 (B = 1 reflects a random distribution of monomer units in the copolymer chain, whereas B = 0 corresponds to a strictly homopolymeric chain).36 The molecular weight of the ALG_Na polymer was estimated from the intrinsic viscosity [η] measured on an Ubbelohde capillary viscometer and modified for gradual dilution. The value of [η] was obtained by extrapolation according to the Huggins equation. Following the approach described by Martinsen et al.,37 we derived two sets of Mark-Houwink parameters for the determined [η] = 255 cm3g-1 in 0.1 M NaCl at 25 °C: K = 0.00069 cm3g-1 and a = 1.13 for alginate with a molar fraction of M (fM) = 0.28, and K = 0.0073 cm3g-1 and a= 0.92 for alginate with fM = 0.58. Both these parameters give similar molecular weights: Mw = 94 000 and 99 000, respectively.

O

4

6 OH

O

3

OH

5

(G)

O

2 OH 1

(M)

HO O

OH O

O

MM-1

O OH

MG-1 G1

MGM-5 GG-5 GGM-5

5.1

5.0

4.9

4.8

4.7

4.6

4.5

ppm

IB IA

IC

5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 ppm

Figure 1. Expanded regions of the 1H NMR spectrum of the ALG_Na polymer in D2O and a schematic representation of GM diads.

Alginate bead preparation: Crosslinked alginate microbeads were prepared via external ionic gelation. A 6 wt.% sodium alginate dispersion was prepared by homogenizing ALG_Na in purified water. The polymer was swelled in purified water (5 min) at room temperature and subsequently homogenized at 13,000 rpm for 5 min using an Ultra-Turrax (T25 basic, IKA-


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Werke, Staufen, Germany). The dispersion volume was ultimately adjusted to 100 mL with purified water. The prepared homogenous dispersions were then extruded through a 0.7 mm diameter needle at a dropping rate of 2.0 mL/min into a 1.0 M hardening aqueous solution. The distance between the edge of the needle and the surface of the solution was 5.0 cm. Microbeads formed instantaneously and then were left in the crosslinking solution for 1 hr. The resulting beads were subsequently washed three times with purified water and dried at 25 °C for 24 hr in a cabinet drier (HORO – 048B, Dr. Hofmann GmbH, Ostfildern, Germany).

Table 1. Composition of reaction mixtures (concentration of sodium alginate wALG_Na and molar concentration of crosslinking agents in water cion); number of crosslinking ions, as determined by atomic absorption spectroscopy, in alginate gels; and basic physical characteristics of crosslinking ions (molar weight M and ionic radius r).

Sample

wALG_Na, (%)

Crosslinking agent

cion, (mol/dm3)

wion, (g/kg)

r, (pm)

M, (g/mol)

ALG_Ba

6

BaCl2

1.0

379 ± 4

135

137.27

ALG_Sr

6

SrCl2

1.0

250 ± 7

113

87.62

ALG_Ca

6

CaCl2

1.0

209 ± 5

99

40.08

ALG_Zn

6

ZnCl2

1.0

207 ± 6

74

65.38

ALG_Cu

6

CuCl2

1.0

203 ± 8

73

63.55

ALG_Mn

6

MnCl2

1.0

178± 9

67

54.94

ALG_Al

6

AlCl3

1.0

59 ± 1

50

26.98

NMR spectroscopy: High-resolution NMR spectra were measured on a Bruker Avance III 600 spectrometer operating at 600.2 MHz. 1H NMR spectra were acquired at 355 K in D2O. The width of 90° pulse was 10 µs, relaxation delay was 10 s, acquisition time was 2.18 s, and number of scans acquired was 100. The integrated intensities were determined with


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spectrometer integration software with an accuracy of ±1 %. Chemical shifts were calibrated based on the 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) signal at δ = 0.00 ppm. ssNMR spectra were measured at 11.7 T on a Bruker Avance III HD 500 US/WB NMR spectrometer (Karlsruhe, Germany, 2013), and the following techniques were applied: i) 1H, 13

C, 23Na and 27Al magic angle spinning (MAS) and cross-polarization (CP) MAS NMR

experiments; ii) 1H-1H single-quantum/single-quantum (SQ/SQ) spin-diffusion experiments with DUMBO homodecoupling38 in both detection periods; iii) 1H-1H single-quantum/doublequantum (SQ/DQ) correlation experiments with an SPC5 DQ recoupling period and DUMBO homodecoupling in both detection periods; iv) 1H-13C wide-line separation (WISE)39 experiments, v) 23Na and 27Al triple-quantum (TQ) MAS NMR experiments, and vi) 1H-13C phase-inverted Lee-Goldburg recoupling under MAS (PILGRIM) experiments.40 The frictional heating of the samples spinning at frequencies of 10-12 kHz was compensated by active cooling. Temperature calibration41 was performed with Pb(NO3)2. Detailed experimental parameters are provided in Supporting Information S1.

Scanning Electron Microscopy (SEM): Particle size, morphology and surface topography were analyzed using SEM. To avoid charging artifacts, the samples were coated with a 10 nm thick layer of platinum/palladium (Pt/Pd) under argon using the ion sputtering coating method. Pt/Pd was sputtered at a current of 20 mA for 20 s, 15× (Cressington sputter coater 208HR, England). The samples were anchored onto an SEM sample holder using carbon conductive double-sided adhesive discs (EMS, USA). Images were on a Hitachi SU8010 (Hitachi High-Technologies, Japan) scanning electron microscope at an accelerating voltage of 14.0 kV for 40 s.

Atomic absorption spectrometry (AAS) and thermogravimetric analysis (TGA): The Zn, Mn, Al, Ca, Ba, Cu and Sr ion content in the prepared beads was determined on a contrAA 700 atomic absorption spectrometer (Analytik Jena, Germany). The residual water was determined


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by TGA on a PerkinElmer Pyris 1 thermogravimetric analyzer in the temperature range of 30– 300 °C at a rate of 10 °C/min. In all the experiments, the sample size was 4–7 mg, and the flow rate of the nitrogen purge gas was fixed at 25 cm3/min. The water content was evaluated as the mass loss in the temperature interval from 30 to 150 °C (see Supporting Information S2, Figure S5).

Quantum chemical calculations: The DFT-based B3LYP/6-311G** method was applied to model both the structural and NMR spectroscopic features of the complexes formed between a single Al3+ cation and mannuronate-type hydrated disaccharide chains. Thus, numerous initial orientations corresponding to either mono- or bidentate binding modes were prepared by employing the one-chain DFT structure kindly provided by Prof. T. Mineva,28 and the local minima of the potential energy surfaces (PES) were determined. Subsequently, the 27Al NMR parameters were computed (the gauge-independent atomic orbital (GIAO)42,43 technique was used to overcome the gauge problem) for the representative structures, which, within the respective models, differed in protonation states. The Gaussian 0944 suite of quantum chemical programs was used with default settings. The quadrupolar coupling parameters were extracted from the Gaussian output files using EFGShield program (version 4.0) for parsing and summarizing the results of electric field gradient and nuclear magnetic shielding tensor calculations. The DFT approach specified above was previously applied to define the NMR parameters of a quadrupolar nucleus45,46 and of oligopeptide models.47


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RESULTS AND DISCUSSION Selection of metal ions used in our study was primarily made on the basis of literature, 8,11,20,24,27,28,48-51

where alginate gels crosslinked by bivalent alkaline earth cations (Mg2+,

Ba2+, Ca2+, and Sr2+), bivalent transition metal ions (Mn2+, Co2+, Cd2+, Cu2+, and Zn2+), bivalent and trivalent metal cations (Pb2+, Fe3+, Cr3+, Al3+, Ga3+, Sc3+, and La3+) were described. Consequently, as the aim of our work was detailed atomic-resolution study of local structures of alginate chains induced by different crosslinking ions with increasing ionic radii, we prepared and intended to investigate alginate systems cross-linked by the above-mentioned metal ions regardless their potential toxicity. Unfortunately, some of these ions are paramagnetic (Mn2+, Co2+, Cu2+, Fe3+, Cr3+), which makes high-resolution measurements of NMR spectra basically impossible. In this regard, we performed preliminary tests with alginate gels crosslinked by Mn2+ and Cu2+ ions. However, as expected, the obtained NMR spectra were featureless (see Supporting Information S3, Figures S6 and S7). The paramagnetic character of many of the investigated ions thus considerably reduces the number of alginate gels suitable for detailed solid-state NMR studies. Further, in selecting suitable ions the size of ionic radii were considered, because our aim was to cover sufficiently broad range of ionic radii in order to monitor systematic structural changes of polymer chains induced by the crosslinking process. Therefore we selected Ba2+, Sr2+, Ca2+, Zn2+ and Al3+ ions as representative crosslinking units covering the sufficiently broad range of ionic radii (135-50 pm, Table 1). Zn2+ ion was used instead of Cu2+ as it has similar ionic radius (73 and 74 pm, respectively). Other potentially considered ions such as La3+, Sc3+ and Mg2+ with ionic radii 103, 75 and 72 pm, respectively, were thus not involved in our study. Overall, seven types of alginate beads crosslinked by different polyvalent ions (Ca2+, Zn2+, Ba2+, Sr2+, Mn2+, Cu2+, Al3+) were prepared by external ionic gelation.1-3 The prepared beads were grayish brown, macroscopically homogeneous, and


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relatively hard. The molecular-level structure and dynamics of the prepared gels were investigated by ssNMR, and the surface morphology was probed by electron microscopy.

Composition of alginate gels NMR structural characterization was performed on all the prepared alginate gels except ALG_Mn and ALG_Cu as the strongly paramagnetic Mn2+ and Cu2+ ions render highresolution NMR measurements impossible (see Supporting Information S3, Figures S6 and S7). To probe the degree of sodium ion exchange during the crosslinking process, we used 23

Na MAS NMR spectroscopy. Because the amount of alginate gels packed in the ZrO2 rotors

was kept constant for all samples (20 mg), the residual Na+ ion content was directly determined by comparing the absolute intensities of the detected 23Na MAS NMR signals (Figure 2; for a direct comparison of the signal intensities, see Supporting Information S4, Figures S8 and S9). 1

23

H NMR

Na MAS NMR

5.5 kHz

ALG_Na

ALG_Na +2.8 ppm

4.5 kHz

ALG_Ba

Offset Y values

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NaCl

3.5 kHz

ALG_Sr

ALG_Sr

+2.3 ppm NaCl

3.1 kHz

ALG_Ca

ALG_Ba

+3.4 ppm

ALG_Ca -1.2 ppm

4.9 kHz

ALG_Zn

ALG_Zn

2.5 kHz

+1.7 ppm

ALG_Al 40

20

0

-20

-40

chemical shift, ppm

40

20

ALG_Al

0

-20

-40

chemical shift, ppm

Figure 2. 1H NMR and 23Na MAS NMR spectra of the reference ALG_Na polymer and the prepared alginate gels. The spectra are arranged according to ionic radii, from the largest (Ba2+) to the smallest (Al3+) (bottom). The upper spectra correspond to the reference ALG_Na system. The 23Na MAS NMR spectra of crosslinked gels are multiplied by a factor of 128.


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The obtained results showing ca. 3-7 % residual Na+ ions indicates that almost complete ion exchange occurs during the crosslinking process. This finding, together with the quantitative data obtained from AAS analysis (Table 1), indicates high occupation of uronic acid residues by polyvalent ions, reaching ca. 60 % for bivalent ions and 40 % for trivalent ions. More interestingly, however, the recorded 23Na MAS NMR spectra of the prepared alginate gels show significant changes in isotropic NMR chemical shifts compared with the reference ALG_Na system. These changes, which reach up to 3.4 ppm for the ALG_Ca system, reflect significant differences in the local structures near Na+ ions in different alginate gels. As residual Na+ ions can be considered as an independent probe for the prevailing structures of crosslinked alginate gels, we also recorded 2D 23Na 3Q/MAS NMR spectra, which generally offer enhanced spectral resolution because of the efficient suppression of quadrupolar broadening in the indirect dimension (Figure 3). In the obtained spectra displayed in Figure 3, we observed considerable narrowing and systematic walking along the isotropic chemical shift (CS) axis of the 2D signals, indicating significant differences in the arrangement of alginate polymer in differently crosslinked gels. The appearance of narrow and multiple resonances in the 23Na 3Q/MAS NMR spectra suggests the co-existence of several structurally distinct local geometries near the residual sodium ions in the crosslinked alginates, in contrast to the fairly continuous distribution of sodium ions in the reference ALG_Na system. This finding also indicates the absence of large-scale translational diffusion motion of the Na+ ions. Moreover, the observed substantial changes in the 23Na NMR resonances confirms the homogenous crosslinking and fairly uniform spread of residual Na+ ions throughout the alginate gels. However, when segregated domains of unaltered alginate polymers are formed during heterogeneous crosslinking, the corresponding Na+ ions should provide essentially identical 23Na NMR resonances.


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Na 3Q/MAS NMR

F2

ALG_Na ALG_Ba ALG_Sr ALG_Ca ALG_Al ALG_Zn

ppm

F1 ALG_Zn

CS

ALG_Al

0

ALG_Ca ALG_Sr ALG_Ba

ALG_Na

5

5

0

-5

-10

-15

ppm

Figure 3. 2D 23Na 3Q/MAS NMR spectra of the reference ALG_Na system and the synthesized crosslinked alginate gels.

Subsequently, by combining TGA and 1H NMR spectroscopy, we probed the content, distribution and dynamics of the residual water in the alginate gels. TGA revealed that the amount of residual water was not negligible and covered a range from 5 to 15 wt.%, which approximately corresponds to 1-3 water molecules per uronic acid residue (see Supporting Information S2, Figure S5). The recorded 1H NMR spectra (Figure 2) clearly show the twocomponent character of the alginate systems: i) the narrow signals with ca. 3-5 kHz linewidth reflect the alginate gel mobile fractions consisting mainly of water molecules, and ii) the broad signal with ca. 25 kHz linewidth corresponds to rigid segments of alginate gels. Thus, the translational and rotational diffusion motion exhibited by residual water molecules entrapped in alginate gels is hindered by stiff alginate macromolecules.

Structure of alginate polymer chains 13

C CP/MAS NMR spectroscopy provides an ideal tool for probing the atomic-resolution

structure of alginate gels via analysis of the line shapes and resonance frequencies of the recorded signals. This is particularly because the 13C CP/MAS NMR spectra of alginate


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polymers are well resolved showing resonances of individual carbon atoms of the M and G units. Moreover, the highly precise and reliable signal assignment obtained by the combination of solid-state and liquid-state NMR spectroscopy of alginate systems differing in ratio of M and G units is presented in recent literature30,31,52. Consequently, applying the reported signal assignment the structural changes in the prepared alginate gels can be directly monitored. The clear differences in the local structures of alginate polymer chains shown in the 13C CP/MAS NMR spectra of the prepared gels (Figure 4) are induced by ion exchange and subsequent crosslinking by different polyvalent ions. The most prominent feature of these spectra is the specific broadening of certain signals. This phenomenon, which is clearly apparent for the signals of carbonyl units ca. 170-180 ppm and of M4 and M5 of the M unit pyranose rings ca. 75 ppm, indicates distinct differences in the local structures of the synthesized alginate gels. Detailed inspection of these spectra and deconvolution analysis revealed nearly exclusive broadening of the M unit signals, whereas the G segment signals remain unchanged (Table 2). Specifically, the signal linewidths of the M units in ALG_Zn and ALG_Al are nearly twice as large as those of the M units in the reference ALG_Na system. Overall, the observed broadening of M signals systematically increases as decreasing crosslinking ion size decreases.


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R

O O4

3

6 O

6 OH

HO

OH OH

O

5

OH

(M)

HO

(G)

O OH

2

3

O

1

5

4 HO

O OH

O

2

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(G)

OH

O

HO O

OH O

1

OH

O

(M)

(G)

O

(M)

O HO

OH

OH O

O

OH

O

O

R

O OH

13

C CP/MAS NMR M4,M5

G G6, M6

M3,M2 G3,G5 G2 G

G1, M1

G

G4

G

ALG_Na

ALG_Ba ALG_Sr ALG_Ca ALG_Zn ALG_Al 190

180

170

110 100

90

80

70

60

chemical shift, ppm

Figure 4. Chemical structure of M (mannuronate) and G (guluronate) units, atom numbering used for signal assignment, and 13C CP/MAS NMR spectra of the reference ALG_Na polymer and the prepared ALG gels. The spectra are arranged according to ionic radii, from the largest (Ba2+) ion to the smallest (Al3+) ion (bottom). The upper spectrum corresponds to the reference ALG_Na system. The signal assignment was adopted from literature.30,31,52

Generally, when analyzing signal broadening in 13C CP/MAS NMR spectra, the most likely mechanism is an increase in the dispersion of chemical shifts, which is induced by variations in local structures, including conformation non-uniformity and variable hydrogen-bonding strength. Consequently, the observed changes in the carbonyl signal line shapes probably reflect differences in the strength, geometry and nature of ion binding in the alginate gels. Along with the broadening of the carbonyl signals, we observe similar broadening of the signals of M units. In particular, for the ALG_Zn and ALG_Al gels crosslinked by the


16

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Biomacromolecules

smallest ions, this broadening nearly leads to a complete disappearance of the M4 and M5 signals. Thus, structural changes in the crosslinked alginate chains are strongly dependent on the chemical properties of the crosslinking ions and impact the M segments nearly exclusively, whereas the local structures of G units are essentially unaffected. This phenomenon likely relates to the different conformational diversity of the MG, MM and GG blocks and their capability to form stable complexes.23-25,53

Table 2: Linewidth of the signals of individual structural units determined by deconvolution of 13C CP/MAS NMR spectra of ALG_Na and the prepared alginate beads. The signal assignment was adopted from literature.30,31,52

Linewidth of units (Hz)

Sample G6 177 ppm

M6 176 ppm

G1 102 ppm

M1 99 ppm

G4 81 ppm

M4/M5 76 ppm

M3/M2 71 ppm

G3/G5 68 ppm

G2 65 ppm

ALG_Na

311

233

430

419

598

450

380

251

228

ALG_Ba

329

290

460

477

599

497

321

361

298

ALG_Sr

347

340

361

607

567

494

382

288

232

ALG_Ca

374

322

402

583

552

501

384

407

274

ALG_Zn

381

486

459

474

693

880

402

326

280

ALG_Al

280

507

397

545

499

950

607

283

249

In general, alginates are stiff macromolecules because of the restricted rotation around the glycosidic linkage. However, chain stiffness was found to depend on alginate composition and increase as the number of GG blocks in the polymer chain increases. By contrast, greater fractions of M monomer units increase alginate chain flexibility.23,25 In addition, because of the orientation of the carboxylate and hydroxyl groups, GG-rich blocks exhibit a considerably higher tendency to form stable complexes with polyvalent ions than poly-M and alternating


17

Biomacromolecules

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MG blocks, which have much flatter structures with more shallow nests of the MM and MG blocks.25 Consequently, according to the proposed egg-box model, GG sequences, particularly when occupied by cationic species, are geometrically well defined, energetically stabilized and uniformly arranged along the alginate chains. The resulting structural uniformity of Grich blocks is reflected by the narrow 13C CP/MAS NMR signals, which are observed in the corresponding spectra (Figure 4) regardless the type of cationic species involved in gel crosslinking. By contrast, because of their more open geometry, the GM and MM segments are likely less occupied by cation species and thus more readily allow interchain aggregation.24 Consequently, after ion exchange, the corresponding local structures around these M units exhibit structural diversity, which is indicated by the broadening of the corresponding 13C CP/MAS NMR signals. As observed in the recorded 13C CP/MAS NMR spectra, this geometrical variability of M-rich blocks further depends on the size, valence and amount of interacting cationic species. Large cations such as Ba2+, Sr2+ or Ca2+ fill the majority of the free volume of the polymer gel, thus preventing significant conformational variations in the polymer segments. Consequently, the corresponding M units adopt a uniform arrangement. By contrast, small cations such as Al3+ or Zn2+ occupying a much smaller fraction of the free volume allow the polymer segments to adopt a much higher number of local arrangements. Overall, the higher conformation flexibility of M-rich blocks, combined with their reduced affinity for binding with crosslinking ions, considerably augments the irregular arrangement of uronic units in alginate chains. This conformational disorder is intensified for M-rich alginates, for alginate gels crosslinked by small-radius species, and particularly for trivalent cations, for which a smaller quantity is required for efficient crosslinking.


18

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Biomacromolecules

Segmental dynamics of polymer chains in alginate gels In addition to the irregularities in polymer chain arrangement, the broadening of 13C CP/MAS NMR signals can also originate from segmental dynamics. Although alginate polymer chains are rigid, some degree of internal motion is assumed, particularly in strongly hydrated domains. Therefore, considering the two-component character of the prepared alginate gels revealed by 1H NMR spectroscopy (Figure 2), we probed the local dynamics of alginate gels in detail using 2D 1H-13C separated-local-filed PILGRIM experiments, which enable site-specific measurements of 1H-13C dipolar couplings.40,54 As any segmental motion with a correlation time shorter than ca. 40 µs causes the averaging of one-bond 1H-13C dipolar interactions, the ratio of the motionally averaged dipolar coupling constant (DCH) to the rigidlimit value (DCH,rig) defines the order parameter (S), which can be converted to the segmental motion amplitude.55 In the simplest case, assuming small-amplitude (θ) axially symmetric motion, the order parameter reflects the average fluctuation angle of the CH segment (root mean square amplitude) 〈 〉 according to the relationship: = 1 − 3⁄2〈 〉.


19

Biomacromolecules

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1

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13

H- C PILGRIM NMR

a) ALG_Al

M2

G1 M1

G2

ppm

G1

M1

M2 G2

-10 0 10

140

b)

M1

120

100

80

G1

12.2 kHz

ppm

12.2 kHz

ALG_Na 7,2 kHz

ALG_Ba

ALG_Sr

ALG_Ca

ALG_Zn 12.0 kHz

8.8 kHz

ALG_Al

20

0

ppm

20

0

ppm

Figure 5. 2D 1H-13C PILGRIM NMR spectrum of the ALG_Al gel (a) and the 1H-13C dipolar profiles, together with the corresponding splitting, extracted for the M1 and G1 units of all the synthesized alginate gels.

The order parameters shown in Figure 5 were estimated from the splitting of dipolar profiles extracted for each spectroscopically resolved CH group of the alginate segment. As indicated by the high values of the determined order parameters S = 0.96-0.98, alginate segments are basically rigid, executing low-amplitude motion with an average fluctuation angle of ca. 5-9°. No significant dependence on the type of crosslinking cation was found. Thorough inspection of the recoded dipolar profiles, however, revealed inner low-intensity dipolar doublets (shoulders), which indicate alginate polymer fractions exhibiting enhanced


20

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Biomacromolecules

dynamics. As reflected by the corresponding order parameters ranging from ca. 0.6 to 0.7, the amplitudes of these released motions can reach up to ca. 30°. Notably, however, the presence of inner doublets is observed predominantly in the dipolar profiles of M units, whereas nearly exclusively outer dipolar doublets are found in G segments. Thus, this finding suggests that M-rich blocks in part undergo phase separation, which is likely accompanied by preferential hydration, enabling the release of segmental motion.

Distribution of water molecules To support our assumptions about the inhomogeneous distribution of water, and because residual water generally composes a considerable part of alginate gels, we performed a series of 2D 1H-13C WISE and heteronuclear correlation (HETCOR) experiments. In principle, a 2D WISE experiment56-59 is used to probe local molecular mobility. Molecular motion is monitored by the 1H wide-line signals, which are separated in the second dimension according to the 13C chemical shifts for each resolved structure unit. Whereas rigid segments are reflected by broad 1H lines, mobile units exhibit narrow signals. However, when 1H-1H spin diffusion is active, this experiment can also be used to monitor the propagation of 1H line narrowing from external spins such as water.60 Therefore, obtaining information about the preferential localization of water molecules is possible. Moreover, if the 1H frequency is set off-resonance with respect to the 1H NMR signal of water, the presence of water molecules in the proximity of any structural unit resolved in the 13C CP/MAS NMR spectrum is reflected by a narrow doublet in the corresponding 1H dipolar profile. In our particular case, a series of 2D 1H-13C WISE experiments with variable spin-diffusion mixing times was performed for a representative alginate gel crosslinked by Al3+ ions (ALG_Al). For a short spin-diffusion time (CP period of 500 µs), the extracted 1H dipolar spectra of all the resolved G and M units are dominated by a broad component reflecting rigid


21

Biomacromolecules

alginate chins. By contrast, narrow doublets appear preferentially in the wide-line spectra of carbonyl groups and M units. No splitting was observed for the G species signals (Figure 6, doublets marked by arrows). As the spin-diffusion mixing times are increased (CP > 750 µs), however, this doublet is rapidly propagated into the G unit wide-line signal. This finding signifies that along the polymer chain, water molecules exhibit slight concentration fluctuations, which render M units more hydrated than G-rich blocks and supports the previous results of 1H-13C PILGRIM spectroscopy, suggesting some degree of phase separation of the M-rich blocks accompanied by preferential hydration.

1

H-13C WISE NMR (500 µs)

1

H-13C WISE NMR (1000 µs)

0

0

50

G2

-50

M2/M3

ppm

-50

M4/M5

ppm

G5

M1/G1

COO

G2

M2/M3

M4/M5

G5

M1/G1

COO

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 22 of 36

50 180

160

1

140

120

100

80

ppm

180

160

13

1

H- C HETCOR (100 µs)

ppm

140

120

100

80

ppm

13

H- C HETCOR (1750 µs)

ppm

Cn-Hn

2

Cn-Hn

2

COO-Hn 4

4

C1-H1

6

6

8

8

10

10

Cn-H2O

COO-H2O 180

160

140

120

100

80

ppm

180

160

140

120

100

80

ppm

Figure 6. 2D 1H-13C WISE (a) and FSLG HETCOR NMR (b) spectra of the ALG_Al gel measured at different CP mixing times. The 1H wide-line projections for each resolved 13C NMR signal are in upper part of the figure.


22

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Subsequently, we performed a series of complementary 1D and 2D 1H DUMBO NMR experiments.61,62 Because of the amorphous character of the prepared alginate gels, the 1H NMR resonances of individual protons essentially overlap, and only three types of 1H signals can be easily resolved (Figure 7). According to the data obtained for crystalline maltose anomers,63,64 the directly bonded CH protons on glucopyranosyl rings resonate in the 2.0-4.5 ppm frequency region, whereas the signals of water molecules and hydroxyl groups are located ca. 5.0-6.5 ppm. Consistent with the 1H-13C WISE and FSLG HETCOR spectra (Figure 6), the 1H signal at 7.0-8.0 ppm observed in the ALG_Al spectrum is attributable to the relatively highly acidic protons of the COOH and H2O species involved in chemical exchange.

1

H DUMBO NMR

δ(1H) < 3+

Al 50 ppm

12

10

Structure and Dynamics of Alginate Gels Cross-Linked by Polyvalent

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