Quantification of Substitution of Gelatin Methacryloyl: Best Practice

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Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls Christiane Claaßen, Marc H. Claaßen, Vincent Truffault, Lisa Sewald, Günter E.M. Tovar, Kirsten Borchers, and Alexander Southan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01221 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls Christiane Claaßen1, Marc H. Claaßen2, Vincent Truffault2, Lisa Sewald1, Günter E. M. Tovar1, 3, Kirsten Borchers1, 3 and Alexander Southan1* 1

Institute of Interfacial Process Engineering and Plasma Technology IGVP, University of

Stuttgart; Nobelstr. 12, 70569 Stuttgart, Germany; 2

Max Planck Institute for Developmental Biology; Spemannstrasse 37, 72076 Tübingen,

Germany; 3

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB; Nobelstr. 12, 70569

Stuttgart, Germany. KEYWORDS NMR spectroscopy; modified gelatin; direct quantification; degree of methacryloylation; degree of acetylation.

ABSTRACT

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physico-

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chemical material properties. In this contribution, 1H-13C-HSQC-NMR-spectra are used to gain detailed structural information of GMs and of two-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the 1H-NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple 1H-NMR with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D-NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.


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INTRODUCTION Biomaterials mimicking features of the extracellular matrix (ECM) have been studied extensively for the past decades as artificial scaffolds for tissue engineering1-3 and for controlled drug delivery4-6. In the context of soft hydrogel materials, gelatin – a hydrolysis product of collagen – is particularly interesting due to its inherent biocompatibility and biodegradability.7-12 However, the physical hydrogels formed by gelatin in aqueous solution lack mechanical stability at human body temperature, making chemical cross-linking of gelatin hydrogels often indispensable.13 Chemically cross-linked gelatin-based hydrogels were for example obtained by addition of cross-linkers such as glutaraldehyde14 or carbodiimide (e. g. 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride)15, by direct enzymatic cross-linking (e. g. transglutaminase16, or laccase and tyrosinase17) or by chemical modification of amino acid residues, e. g. for enzymatic cross-linking with horseradish peroxidase18, or for radical crosslinking by introducing methacryl groups19. The latter approach results in gelatin methacryloyl (GM) which has gained more and more interest in recent years as documented by the rapidly increasing publication numbers shown by Yue et al.20 GM-based hydrogels represent a versatile platform for tissue engineering a broad variety of tissues, e. g. adipose tissue21, cartilage22 or bone23, 24, and were also used for bioprinting25 or cell culture coatings26, for recent reviews see references 27, 28. These diverse application perspectives are based on the possibility to fine-tune the physico-chemical properties of the GM-based hydrogels by adjusting the degree of methacryloylation (DM) of GMs, resulting in tailored gelling behavior, solution viscosity, equilibrium degree of swelling, and mechanical strength.25, 29 However, in order to be able to fully utilize the advantageous properties of GMs and to be able to reproduce results given in the numerous literature reports, it is essential to have


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precise information about the DM of the GMs used. This is not the case so far because current analytical techniques are inappropriate for correct characterization and quantification of the chemical nature of the occurring chemical groups as also indicated by the different designation of the GMs as gelatin methacrylamide25, 26, 29-32, gelatin methacrylate33-35 and methacryloyl gelatin36-39. Common techniques for quantifying the DM of GMs include monitoring the decrease of free lysine amino groups upon modification. This was achieved either by 1H-NMR spectroscopy, integrating the ε-CH2 signal of lysine25, 29, 34, or by a photometric assay using 2,4,6trinitrobenzene-sulfonic acid (TNBS)29, 30, 32, 33, 38, 40. However, such data only considers methacrylamide groups in GMs (methacryl groups bound to amino residues), while methacrylate groups (methacryl groups bound to hydroxyl residues) are fully neglected, in spite of the knowledge that not only amino but also hydroxyl groups react in the modification reactions of gelatin25, 32, 38. Therefore this method underestimates the total amount of methacrylic functions. Another method using 1H-NMR spectroscopy relies on the signal caused by aromatic protons which can be used to calculate the total methacryl group content33, 41, provided that the aromatic amino acid content of the respective gelatin raw material is known e. g. based on specific analysis of the amino acid composition. However, this method does not allow a differentiation between methacrylamides and methacrylates, as there is a lack of detailed signal assignments in 1

H-NMR spectra of GMs. Most recently, Yue et al. combined a quantification of amino group

conversion via a fluoroaldehyde assay and a quantification of methacrylate groups via a Fe(III)hydroxamic acid complex to determine the ratio of methacrylate to methacrylamide groups.20 Although this is certainly a promising approach, the procedure is time-consuming and demands quantitative conversion of amino groups and methacrylate groups in the applied wet chemical


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assay. The situation becomes even more complex if different modifications are applied to gelatin, such as modification with cross-linkable methacryl groups and non-cross-linkable acetyl groups, resulting in GMAs, which is useful for tailoring solution viscosities independent from the DM25. In this case, no method for quantification of the acetylation and methacryloylation has been proposed so far. From our point of view it would be desirable to achieve direct spectroscopic quantification of the total DM of GMs, distinction between gelatin-bound methacrylamide and methacrylate groups as well as unbound impurities such as methacrylic acid, and additionally quantification of acetyl groups in GMAs without the need to know the exact amino acid composition of the gelatin used. Therefore, we aimed to derive more detailed information about the signals present in the 1

H-NMR spectrum of GMs by conducting 2D-NMR experiments. We hypothesized that it is

possible to identify signals in the 1H-NMR spectrum which allow an easy distinguishing between methacrylamide and methacrylate groups. By additionally using an internal standard, a correct quantification of DM should be possible, laying the foundation for a better understanding of GM structure and also a better comparability of GMs used in different studies. MATERIALS AND METHODS Materials Acetic anhydride (AcAnh), methacrylic anhydride (MAAnh), sodium hydroxide (NaOH), 2,4,6-trinitrobenzene-sulfonic acid solution (TNBS), sodium dodecyl sulfate solution (SDS), hydrochloric acid solution (HCl) and glycine were purchased from Sigma Aldrich (Germany). Other reagents were purchased from the following sources (given in parentheses): Sodium 3trimethylsilyl-propionate-2,2,3,3-d4 (TMSP; Merck; Germany), deuterium oxide (Deutero;


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Germany), gelatin type A (MedellaPro®, bloom 233, pig skin North America; Gelita; Germany) and sodium hydrogen carbonate (Carl Roth; Germany). Dialysis was conducted using dialysis membranes (MWCO 12 kDa–14 kDa) from Medicell International Ltd (UK). Amino acid analysis of gelatin type A Amino acid analysis was conducted according to the European Commission Regulation (EC) No 152/2009 III F at the University of Hohenheim, Germany. Briefly, gelatin was hydrolyzed with hydrochloric acid and amino acids were separated via ion chromatography. The separated amino acids were converted with ninhydrin in a post-column derivatization and amino acid derivatives were detected with a photometric detector. Quantification was done using amino acid standards. Synthesis of biopolymer derivatives Synthesis of methacryl modified gelatin (GM2, GM5, GM10): Methacryl modified gelatin was prepared according to a previously described procedure.29, 30 Briefly, gelatin (23.06 g) was dissolved in deionized water (250 mL) at 37 °C; pH was adjusted to 7.3 with 4 M NaOH solution. A 2-fold, 5-fold or 10-fold molar excess of MAAnh (2.49 g / 6.22 g / 12.44 g) was added dropwise after gelatin was completely dissolved within 30 min. The excess was calculated with respect to free amino groups of gelatin based on a content of 0.35 mmol amino groups per gram as reported by Van den Bulcke et al.30. The reaction mixture was vigorously stirred for 5 h and the pH of the solution was kept between 7.0 and 7.4 constantly by adding 4 M NaOH solution with an automatic titration device. Afterwards the solution was filtrated, the pH was adjusted to 9.5 (in order to obtain neutral GM solutions upon redissolution after freeze-drying), and the solution was dialyzed for 4 days against deionized water at room temperature and then


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freeze-dried. The procedure typically yielded 19.14 g (83 %) for GM2, 18.68 g (81 %) for GM5 and 18.51 g (80 %) for GM10 as a white foam-like solid. Synthesis of methacryl and acetyl modified gelatin (GM2A8, GM5A5):Two-fold functionalized gelatin was prepared according to a previously described procedure.25 Gelatin solution was prepared as described above and a 2-fold or respectively 5-fold molar excess of MAAnh (2.49 g / 6.22 g) was added dropwise within 30 min. For the 2 h of methacryloylation reaction, the pH was adjusted between 7.0 and 7.4 with 4 M NaOH with an automatic titration device. After this time, AcAnh was added dropwise within 30 min. The amount of AcAnh was calculated to be 6.59 g for GM2A8 to yield an 8-fold molar excess or respectively 4.12 g for GM5A5 to yield a 5-fold molar excess. During 3 h acetylation reaction the pH was adjusted as previously described. Postprocessing was done as described above. The procedure typically yielded 17.30 g (75 %) for GM2A8 and 18.68 g (81 %) for GM5A5 as a white foam-like solid. NMR-spectroscopy of gelatin and gelatin derivatives 1

H-13C-HSQC (heteronuclear single quantum coherence)-NMR spectroscopy: For 2D-NMR

spectroscopy of gelatin and gelatin derivatives, samples of approx. 15 mg biopolymer were dissolved in 600 µL deuterium oxide with TMSP as internal standard (1 mg/mL). Highresolution 800 MHz NMR-spectra were measured at 37 °C on a Bruker Avance-III spectrometer. Quadrature selection was achieved using echo/anti-echo coherence selection; by this efficient water suppression was already achieved. The residual water signal was suppressed by the mean of a water pre-saturation applied during the relaxation delay. Prior to the interpretation, phase correction was applied to all spectra to obtain purely absorptive peaks, baselines were corrected


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before integrating the signals of interest, and the chemical shift scales were adjusted to the TMSP signal (δ(1H) = 0 ppm / δ(13C) = 0 ppm). 1

H-NMR spectroscopy: For 1H-NMR spectroscopy of gelatin and gelatin derivatives, samples

of approx. 15 mg biopolymer were dissolved in 600 µL deuterium oxide with TMSP as internal standard (1 mg/mL). The exact biopolymer and TMSP concentrations have to be known as they are used for the quantification of DM (see equation 1). Proton NMR-spectra were measured at ambient temperature on a Bruker Avance 500 spectrometer (500 MHz). No water suppression was applied, in order to prevent effects of water suppression onto the signal intensities42. Prior to the interpretation, phase correction was applied to all spectra to obtain purely absorptive peaks, baselines were corrected before integrating the signals of interest, and the chemical shift scale was adjusted to the residual solvent signal (D2O δ(1Η) = 4.79 ppm). Determination of methacryloylation degree The DM in this study was defined as the molar amount of methacryl groups per gram biopolymer derivative. DM was determined via 1H-NMR spectroscopy (DMTMSP, DMphe, DMlys) or colorimetric assay (DMTNBS). DM based on TMSP as internal standard: DMTMSP was calculated with the double bond protons of the methacryl groups (5.0 ppm-6.5 ppm / nominally integrating for 2 protons) and based on the TMSP integral (0 ppm / integrating for 9 protons), see equation 1. More specifically, the signals occurring in the NMR-spectra from the methacryl groups (5.0 ppm6.5 ppm; three in case of high methacryloylated gelatins; two in case of low methacryloylated gelatins) were integrated individually. The signal with the highest chemical shift (6.3 ppm5.9 ppm) gives the methacrylate amount present in the gelatin derivatives, while the signal in the


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middle (5.9 ppm-5.5 ppm) gives the total amount of methacryl groups present in the gelatin derivative. The signal with the smallest chemical shift (5.5 ppm-5.2 ppm) was not used for quantification (see explanation in the Results and Discussion section).

DM =

×

×

(1)

Remaining methacrylic acid was sometimes observed in the spectra and was identifiable by a separate signal with a low intensity at 5.35 ppm. The other methacrylic acid signals at 5.66 ppm and 1.88 ppm, which can be found in the 1H NMR spectrum of neutralized methacrylic acid in D2O (see supporting information), were superimposed with the methacrylamide and methacrylate signals (5.9 ppm-5.5 ppm) and the gelatin backbone signals, respectively. If there were remaining traces of methacrylic acid from the synthesis procedure, ∫methacryl in equation 1 was corrected for that amount. This was done by integrating over the non-superimposed methacrylic acid signal and substracting this from ∫methacryl, to yield the intensity caused by only the methacryl groups bound to the gelatin. For the methacrylate amount the integral of the signal with the highest chemical shift (6.3 ppm-5.9 ppm) was inserted as ∫methacryl. This signal is nominally integrating for one proton, while the TMSP signal is integrating for nine protons. For the total amount of methacryl groups (methacrylate and methacrylamide) the integral of the signal in the middle (5.9 ppm5.5 ppm) was inserted as ∫methacryl. It is caused by an overlay of signals belonging to one proton of the methacrylate and one proton of the methacrylamide groups and is therefore nominally integrating for one proton as well to obtain the total methacryl group content.


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DM based on phenylalanine content: DMphe was calculated with the double bond protons of the methacryl groups (5.0 ppm-6.5 ppm / nominally integrating for 2 protons) and based on the phenylalanine integral (6.9 ppm-7.5 ppm / nominally integrating for 5 protons) of the gelatin as described previously33, 41, see equation 2. Integration of the methacryl signals was done as described for DMTMSP. The phenylalanine content of the raw gelatin was determined as described above.

DM! =

!

"#

× # × phenylalanine content

(2)

For the DMphe the integral of the signal in the middle (5.9 ppm-5.5 ppm) was inserted as ∫methacryl (if necessary corrected for traces of methacrylic acid as described above). It is caused by an overlay of signals belonging to one proton of the methacrylate and one proton of the methacrylamide groups and is therefore nominally integrating for one proton to obtain the total methacryl group content. The phenylalanine signal is nominally integrating for five aromatic protons. DM calculated via decrease of lysine integral: DMlys was calculated as reported by our group before29. NMR-spectra were normalized to the phenylalanine integral (6.9 ppm-7.5 ppm / integrating for 5 protons) and lysine methylene signals of non-modified gelatin and gelatin derivatives were integrated (2.8 ppm-2.95 ppm) and integrals compared, see equation 3. The lysine content of the raw gelatin has been determined as described above.

DM/ = 1 −

/ 2 / 3 4

× lysine content

]

(3)

DM calculated via TNBS assay: Remaining amino groups after derivatization were determined using TNBS with the Habeeb method.43 Briefly, 25 µL of gelatin solutions (concentration for


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unmodified gelatin: 5 mg/mL; concentration for modified gelatin derivatives: 20 mg/mL) were pipetted into 96-well plates, 25 µL sodium hydrogen carbonate solution (4 % w/v, pH 8.5) and 25 µL TNBS (0.1 % v/v) were added. Micro well plates were incubated for 2.5 h at 37 °C in the dark with gentle shaking. Then 25 µL SDS solution (10 % w/v) and 12.5 µL HCl solution (1 mol/L) were added and absorption was measured at 330 nm using a fluorescence micro well plate reader (Tecan Reader Synergy 2) from BioTek (Germany). Calculation of amino group content was done using a glycine standard curve (0.02 mmol/L-2.5 mmol/L). DMTNBS was calculated as shown in equation 4.

DM67 = NH: content gelatin

− NH: content GM

(4)

Determination of acetylation and total modification degree The degree of acetylation (DA) in this study was defined as the molar amount of acetyl groups per gram biopolymer derivative. DA was determined by comparing the intensities of acetyl and methacryl methyl groups in the 1H-13C-HSQC-NMR-spectra and correlating this with the DMTMSP see equation 5.

DA =

>#? >#?

(5)

× DM

The total degree of modification (DMA) was calculated by summing up DM and DA. Statistical analysis Statistical analysis was done using a two-sided Student T-test. P values less than 0.05 were considered statistically significant. Significance levels are stated as follows: p

Quantification of Substitution of Gelatin Methacryloyl: Best Practice

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