TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier

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TEMPO-oxidized nanofibrillated cellulose as a high density carrier for bioactive molecules Ramon Weishaupt, Gilberto Siqueira, Mark Schubert, Philippe Tingaut, Katharina ManiuraWeber, Tanja Zimmermann, Linda Thöny-Meyer, Greta Faccio, and Julian Ihssen Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 28 Sep 2015 Downloaded from http://pubs.acs.org on October 4, 2015

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TEMPO-oxidized nanofibrillated cellulose as a high density carrier for bioactive molecules Ramon Weishaupt†, Gilberto Siqueira§, Mark Schubert§, Philippe Tingaut§, Katharina ManiuraWeber†, Tanja Zimmermann§, Linda Thöny-Meyer†, Greta Faccio†,* and Julian Ihssen† †Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland §

Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for

Applied Wood Materials, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland KEYWORDS TEMPO-oxidized nanofibrillated cellulose functionalization, peptide and protein immobilization, enzyme immobilization

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ABSTRACT

Controlled and efficient immobilization of specific biomolecules is a key technology to introduce new, favorable functions to materials suitable for biomedical applications. Here, we describe an innovative and efficient, two-step methodology for the stable immobilization of various biomolecules, including small peptides and enzymes onto TEMPO oxidized nanofibrillated cellulose (TO-NFC). The introduction of carboxylate groups to NFC by TEMPO oxidation provided a high surface density of negative charges able to drive the adsorption of biomolecules and

take

part

in

covalent

crosslinking

reactions

with

1-ethyl-3-[3-

dimethylaminopropyl]carbodiimide (EDAC) and glutaraldehyde (Ga) chemistry. Up to 0.27 μmol of different biomolecules per mg of TO-NFC could be reversibly immobilized by electrostatic interaction. An additional chemical crosslinking step prevented desorption of more than 80% of these molecules. Using the cysteine-protease papain as model, a highly active papain-TO-NFC conjugate was achieved. Once papain was immobilized, 40% of the initial enzymatic activity was retained, with an increase in kcat from 213 s-1 to >700 s-1 for the covalently immobilized enzymes. The methodology presented in this work expands the range of application for TO-NFC in the biomedical field by enabling well-defined hybrid biomaterials with a high density of functionalization.

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Introduction Biomimetic materials engineered to elicit specific physiological responses have sparked increasing attention in both research and development1. In this respect, the use of sustainable2, 3, biodegradable4-6, and non-toxic7 biopolymers is crucial. Plant-derived cellulose8 is the most abundant biopolymer on earth and a valuable alternative to synthetic polymers due to its renewable source, physical strength9, 10, inherent low toxicity7, 11-13. Disintegration of the higher order structures of the plant cell wall to get access to the basic cellulose fibril units (3-5 nm in diameter)14 is achieved via mechanical, enzymatic15 or chemical treatment16. Different terminologies exist for the resulting disintegrated material like micro-, nanofibrillated cellulose or cellulose nanofibrils. Here, the term nanofibrillated cellulose (NFC) is used. NFC offers superior properties compared to conventional cellulosic materials. It provides a superior surface to volume ratio17, physical resistance18 and a high density of surface-exposed primary hydroxyl groups17 that are available for subsequent modification9,

10

. The surface

properties of NFC can be changed gradually, from hydrophilic to hydrophobic or from anionic to cationic by chemical modification19-21. Its beneficial chemical properties and high specific surface area make NFC a promising carrier material for the immobilization of biomolecules such as proteins, peptides and enzymes. These biomolecules are used in numerous biomedical applications ranging from diagnostics22,

23

to

therapeutic treatments24 and to functional surfaces25. Immobilization not only enables specific bioactivity to a substrate but offers several additional advantages over free soluble biomolecules, i.e., enhanced structural stability26, re-use27, prevention of release and limitation of product or analyte contamination28. The interaction between the protein or peptide and the material can be: (i) non-covalent binding by adsorption and thus possibly subject to leaching of the biomolecule

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from the surface, (ii) covalent bonding, or (iii) via specific affinity based interaction of ligand/tag pairs29. In this study, we investigate both non-covalent and covalent immobilization strategies. The TEMPO-mediated oxidation of the primary hydroxyl groups on the nanofibers surface introduces carboxylate groups at the C6 position17. It has been previously shown that the activation of nanofibrillated cellulose via oxidation allows the use of popular coupling chemistry such

as

s-NHS/EDAC

(N-hydroxysulfosuccinimide

/1-ethyl-3-[3-dimethylaminopropyl]

carbodiimide) for immobilization of proteins30, 31. Further, multistep coupling chemistry for the immobilization of short peptides onto NFC based conjugate materials has been reported32. Covalent immobilization by chemical coupling is often laborious and may lack of efficiency. A combination with the much simpler immobilization by physical adsorption could provide solutions with advantages of both approaches26. Filpponen, et al.33 recently described the combination of adsorption (“physical click”) and chemical coupling (“chemical click”) for the grafting of BSA (bovine serum albumin) onto cellulosic substrate with azine/alkyne functionality19. Adsorption and affinity ligand/tag based immobilization was combined by Orelma, et al.31 in order to facilitate human immunoglobulin G (hIgG) detection based on cellulose interfaces. However, modification/engineering of the biomolecule was required before immobilization in both studies, potentially limiting the general applicability of the introduced immobilization strategy. The goal of this study is to investigate and achieve the tunable and controllable functionalization of NFC employing up-scalable chemistry without depending on laborious engineering of biomolecules or the substrate. We thus studied the electrostatic interaction of TEMPO-oxidized NFC; here referred to as TO-NFC, with model biomolecules differing in size (1.9–23.4 kDa) and isoelectric point (IEP), selected due to their beneficial optical properties. The attached heme cofactor facilitated the detection at high sensitivity using

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spectrophotometric analysis. The spontaneous assembly of the biomolecules with the TO-NFC via electrostatic interaction was investigated and conditions for the highest loading capacity were determined. These conditions were then used for the development of the second step, which is the covalent immobilization. In contrast to previous work in the field, the impact of the immobilization chemistry on the specific catalytic properties was investigated with the model enzyme papain, a cysteine-protease that has potential application not only in biotechnology34-36 but also in biomedical applications, such as biosensors37 and drug carriers24.

Materials and Methods Never-dried Elemental Chlorine Free (ECF) cellulose fibers from bleached softwood pulp (Picea abies and Pinus spp.) were obtained from Stendal GmbH (Berlin, Germany) and used for the NFC production. 2,2,6,6-Tetramethyl-1-piperidinyloxyl (TEMPO) and sodium hypochlorite (NaClO) solution (12-14% chlorine) were purchased from VWR international. Sodium bromide (NaBr ≥ 99%) and sodium hydroxide (NaOH ≥ 99%) were supplied by Carl Roth GmbH & Co. L-cysteine, 4-Morpholineethanesulfonic acid (MES), DMSO, glutaraldehyde (Ga 25% grade I), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride

(EDAC),

pGlu-Phe-Leu-p-

nitroanilide (pGFLNa) Bovine serum albumin (BSA) standard, cytochrome c (Equus caballus, geneID: 100053958), myoglobin (Equus caballus, geneID: 100054434), microperoxidase mp11 (derived from E. caballus cytochrome c) papain (Carica papaya, P4762), and all other chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland) in analytical grade, and used without further purification.

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TEMPO mediated oxidation of cellulose and TO-NFC production. The TEMPO mediated oxidation procedure of the never dried cellulose fibers was performed after the method described by Saito, et al.17. The cellulose fibers were suspended in water in order to form a suspension with a concentration of 2% (w/w). TEMPO and sodium bromide (NaBr) were dissolved in water to concentrations of 0.1 and 1.0 mmol per gram of cellulose pulp, respectively, and mixed with the fiber suspension. The pH of the suspension was adjusted to 10 with NaOH solution (1 mol L-1). A concentration of 10 mmol NaClO was chosen per gram of cellulose pulp. The TEMPOoxidized cellulose fibers were thoroughly washed until the conductivity was similar to that of distilled water. The oxidized and purified cellulose fibers were dispersed in water to a concentration of 2% (w/w) and ground using a Supermass Colloider (MKZA10-20J CE Masuko Sangyo, Japan) to obtain cellulose nanofiber suspension. The degree of carboxylation of the obtained TO-NFC was determined via electric conductivity titration as described by Saito and Isogai38 and Katz, et al.39. The degree of oxidation (DO) referring to the average number of carboxyl groups per anhydroglucose unit was calculated using the following equation40:

∗ ∗

∗

(1)

∗

in which V1 is the volume of NaOH added for the neutralization of the strong acid, V2 the volume for the neutralization of the weak acid, c is the concentration of NaOH (molL-1) and w is the dry weight of the cellulose nanofibers (mg). Specific surface area. The NFC suspension (in water) was vacuum filtered to achieve never dried aqueous NFC cakes. The NFC cakes obtained after filtration were solvent exchanged to ethanol, and super critically dried in a critical point dryer chamber. Liquid carbon dioxide was injected into the chamber at pressure of 50 bar. After solvent exchange, from ethanol to liquid CO2, the material was dried at the CO2 critical point (ca. 37 oC and 100 bar). The specific surface

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area was determined for NFC- and TO-NFC aerogels using a multipoint Brunauer-EmmettTeller (BET)-method. Nitrogen physisorption on the surface of the specimens was measured with a surface- and pore size analyzer (Coulter SA3100). The average diameter d was derived from the BET specific surface area (SSA) values of the NFC, using the following equation (assuming a cylindrical shape): ∗

2

A density ρ of 1’500 kg/m3,41 was used for the nanofibrillated cellulose. Atomic Force Microscopy (AFM). Suspensions of bare TO-NFC and TO-NFC saturated with protein cytochrome c (0.207 μmol/mg TO-NFC) were re-suspended in an ultra-sonication water bath for 30 min with 20% intensity. AFM performed on a Veeco Multimode Scanning Probe Microscope with a Nanoscope V, using an etched silicon tip with a nominal spring constant of 5 N/m and a nominal frequency of 70 kHz. Height phase and amplitudes images were obtained in tapping mode at the fundamental resonance frequency of the cantilever with a scan rate of 0.5 line/s. Prior to AFM analysis, a drop of a diluted suspension of TO-NFC was deposited on a mica surface and allowed to dry at room temperature. The fibril diameter (n>60) was analyzed using the ImageJ Software (version 2.0.0 64-bit, National Institute of Health, USA). Adsorption of proteins and peptides to TO-NFC. A series of increasing protein concentrations (cytochrome c; and myoglobin) in the range of 0-0.23 μM (0-2.5 mg/mL) or peptide concentrations (microperoxidase mp11) in the range of 0-0.19 μM (0-0.36 mg/mL) were added to 0.24 mg/mL of TO-NFC or unmodified NFC a fixed concentration in 1.5 mL polypropylene tubes to determine the adsorption capacity via the change of absorbance in the supernatant. The unmodified NFC was treated in an ultrasonic water bath for 30 min at 20% intensity immediately before the experiments to regenerate a homogenous suspension. The

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citrate/phosphate buffers with distinct pH values were prepared according to McIlvaine42 and modified to the indicated ionic strength by adding KCl as described by Elving, et al.43 or diluted in Milli-Q H2O. All buffers were filtered using 0.2 µm PES (Polyether sulfone) membranes Sartolab 180C5 (Sartorius, Göttingen Germany) after adjustment of the pH (±0.05). The samples were incubated for 1h at 25 °C on a thermomixer comfort (Eppendorf, Hamburg Germany) with horizontal agitation (900 rpm). After centrifugation at 14’000 x g for 3 min the protein concentration in the supernatant was determined with a spectrophotometer (Synergy Mx, Biotek, VT USA) in 96 well plates (Greiner, UV-Star 655801) using the Gen5 software (version 2.01, Biotek, VT USA) for analysis. Protein and peptide concentrations were calculated as follows: . . ∗ ∗

. %

(3)

with Absorbanceλmax (a.u.) = spectrophotometric readout at λmax (see Figure S1) in arbitrary units (a.u.), l = length of light path of 100 μL sample = 0.32 cm, k = dilution factor of the supernatant and Aλmax(0.1%) the experimentally determined absorbance coefficient for a 1% protein solution (1 mg/mL) at the given conditions (Table S2). The resulting concentration in (Csupernatant) was subtracted from a reference containing an identical concentration of protein (Creference), treated by the same procedure but without containing TO-NFC resulting in Cadsorbed. Potential unspecific binding of the biomolecules to the plastic walls or precipitation was verified. Experiments were carried out at least in triplicates. The stated pH level was measured directly within the final sample preparation. The Langmuir-type adsorption isotherm according to the Tóth model44,45 was fitted to the data using the Matlab® software (version 8.4.0, MathWorks, R2014b, USA) in order to determine the Tóth equilibrium constant KT and the Tóth maximum adsorption capacity Nmax (μmol/mg TO-NFC).

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∗

(4)

/

P is the concentration of the adsorbent in the solution (μmol/mg TO-NFC), N is the amount adsorbed at the equilibrium (μmol/mg TO-NFC), and t is the Tóth model exponent. The determined values for these parameters conditions are summarized in Table S3. Dependency of the adsorption capacity on ionic strength and pH of the buffer. To study the influence of the ionic strength on the adsorption capacity the model biomolecules were added to TO-NFC in non-saturating concentrations, eg. 0.154 μmol/mg TO-NFC (1.9 mg/mg) for cytochrome c, 0.113 μmol/mg TO-NFC (2.00 mg/mg) for myoglobin and 0.118 μmol/mg TONFC (0.22 mg/mg) for mp11, in McIllvaine buffer at a fixed pH 6.0 but varying ionic strength. The influence of the pH on the adsorption capacity was studied using cytochrome c at an ionic strength of 20 mM at pH 3.4, 4.5, 6.0 and 7.4. Incubation and quantification were carried out as described above. Conjugation of proteins and peptides to TO-NFC. Cytochrome c, microperoxidase mp11 and papain were immobilized to TO-NFC in suspension with a two-step procedure adapted for the use on NFC in suspension as illustrated in Scheme 1. A reaction mixture containing TO-NFC (0.24 mg/mL) in 10 mM MES at pH 4.5 (cytochrome c/mp11) or pH 5.2 (papain), protein solution of cytochrome c: 0.086 μmol/mg TO-NFC (1.06 mg/mg), mp11: 0.140 μmol/mg TONFC (0.26 mg/mg), and papain: 0.073 μmol/mg TO-NFC (1.7 mg/mg) was incubated at 25 °C for 1 hour with horizontal agitation (900 rpm) facilitating homogenous adsorption. After washing in an equal volume of Milli-Q H2O and centrifugation, the reaction mixtures were re-dispersed by sonication in a water bath for 10 min. Subsequently the crosslinking reagents were added to final concentrations of 1% glutaraldehyde (II) and 5 mM EDAC (cytochrome c/mp11) or 20 mM for papain (III). The reaction mixtures were incubated at 4 °C under agitation (900 rpm) for 16

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hours. Desorption was performed twice in an equal volume of desorption buffer (McIlvaine buffer at pH 5.2 and ionic strength of 1000 mM for 1 h) to remove remaining reagents and adsorbents. The immobilized amount of cytochrome c and microperoxidase mp11 was determined as described above. In the case of papain this value was determined in similar experiments, using the Bradford assay46 (n=6) and a BSA dilution series for quantification. The initially adsorbed amount of protein/peptide was set as 100% for evaluation of a potential loss of protein/activity during the subsequent immobilization procedure. Amidolytic activity of free- and immobilized papain. The enzymatic activity of free- and TONFC-immobilized papain was measured using the small protease substrate pGFLNa as described by Filippova, et al.47. Enzyme reaction buffer (10 mM MES, 1.43 mM EDTA pH 6 34% DMSO), was freshly supplemented with stock solutions of the papain activating agent24 Lcysteine (1.11 M in Milli-Q, final concentration: 3.5 mM) and pGFLNa (40 mM in DMSO, final concentration 1 mM). The enzymatic reaction was started by adding 20 μL of papain-TO-NFC suspension to 180 µL reaction buffer that had been equilibrated to 25 °C. Papain reaction rates were determined by measuring the release of p-Nitroanilide as the increase in absorbance at λ=410 nm over 10 min every 30 s with a spectrophotometer (Synergy Mx, Biotek, VT USA). Activity was calculated by the least squares linear regression using at least 5 time points . The specific activity of papain (specific papain activity units PAU/mg protein) was calculated as follows:

∆ ∗

∗∆ ∗

∗

∗

∗∗

(5)

where Vtot is the total volume of the reaction (200 μL), Vn the volume of the enzyme containing sample (20 μL), ε is the molecular extinction coefficient at 410 nm for p-Nitroanilide (0.0088 μM-1cm-1 ), k is the dilution factor and l is the light path length of the well with 200 μL (0.64 10


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cm). The activity yield per mg of TO-NFC was calculated by PAU/mg TO-NFC. The molar enzyme content was calculated based on the molecular weight Mw for papain (Table S1). For the determination of the kinetic parameters, a series of substrate concentrations from 5-1000 mM pGFLNa (n=3) was prepared and the activity assay performed as described above. Initial reaction rates were fitted to the Michaelis-Menten equation using the Matlab® software (version 8.4.0, MathWorks, R2014b). The long-term stability of enzyme-TO-NFC conjugates was investigated by incubation at pH 9.0 and a 65 °C in Tris-HCl buffer with an ionic strength of 20 mM. Activity was measured periodically over 105 hours and compared to a reference of free soluble enzyme.

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Results Characterization of the cellulose materials. The functionalization of the NFC materials was characterized by specific surface area measurements (SSA) and conductimetric titration of the carboxylate group content38, 39. The degree of substitution (DO) of primary hydroxyl groups by carboxylate groups and the mean diameter of the nanofibrillated cellulose fibers was calculated based on the obtained results (see Table 1) Table 1: Characteristics of nanofibrillated cellulose species. NFC species

SSA (m2/g)a

pristine NFC TO-NFC

166.31 235.45

mean diameter carboxylate (nm)b content (mmol/g) 16.03 0.04c 11.33 1.91

surface coverage (μmol COO-/m2)

DOd

0.24c 8.11

0.05, Tukey HSD: Tukey honestly significant different) compared to the initially adsorbed fraction (adsorbed) nor the initially 22


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added (free soluble) one. The route employing Ga (III) resulted in the highest yield: 0.073 ± 0.012 μmol/mg TO-NFC (1.72±0.29 mg/mg) followed by adsorbed (no desorption was applied) with 0.065 ± 0.010 μmol/mg TO-NFC (1.52±0.23 mg/mg). The route employing EDAC (III) had the lowest distinctive yield in terms of protein mass with 0.049 ± 0.012 μmol/mg TO-NFC (1.15±0.29 mg/mg). The initial specific activity (1576±76 PAU/mg of papain) was reduced to 40% upon adsorption. Chemical coupling did not further affect the immobilized specific activity (white bars in Figure 4). The immobilization procedure resulted in a TO-NFC-papain conjugate material with very high average immobilization density of 945±118 PAU per mg TO-NFC. In order to investigate the effect of immobilization on the activity of papain in more detail, the initial reaction rates at increasing substrate concentrations were recorded and fitted to the model of Michaelis-Menten for single substrate catalysis (Figure S5). The resulting kinetic parameters are summarized in Table 3.

Table 3: Michaelis-Menten constants of free soluble- and TO-NFC bound papain. Fraction

KM (µM)a

Vmax (µM/min) b

kcat (s-1)c

kcat/KM (s-1M-1*106)d R2e

free soluble

57.03±6.14

227.60±8.40

213.18±7.86

3.74±0.29

0.91

adsorbed

58.34±15.95

89.60±13.03

95.26±13.86

1.63±0.27

0.78

EDAC

372.10±42.08

705.50±66.54

991.20±93.49

2.66±0.36

0.96

Ga

466.40±55.45

776.90±55.40

745.42±53.16

1.60±0.08

0.96

a

KM is the Michaelis constant at the substrate concentration where the reaction rate is and b an inverse measure for the enzyme’s substrate affinity, Vmax is the maximal substrate turnover rate under saturated conditions. The linear correlation between enzyme concentration and substrate turnover rate under the given conditions was verified in advance in at least triplicates (see Figure S4), ckcat is the turnover number describing the maximal substrate turnovers per second per enzyme molecules. dthe specificity constant as the ratio of kcat over KM expresses the 23


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efficiency of an enzyme to bind and convert a substrate molecule. ethe goodness of the fit after the least square method (R2).

Immobilization led to a more than 3-fold increase in the substrate turnover rate of papain (kcat) with both covalent coupling agents compared to free soluble papain. By contrast, kcat of papain was decreased by 55% after adsorption. Interestingly, the affinity of papain for its substrate (1/ KM) was influenced in an inverse manner by covalent crosslinking following the adsorption. Treatment with crosslinking agents reduced the affinity several folds compared to the free soluble reference of 12.2% (Ga) and 15.3% (EDAC). However, the untreated papain-TO-NFC suspension (adsorbed) exhibited no significantly altered substrate affinity compared to free soluble papain (p 80 %. The temporal and spatial separation of the electrostatic binding from the chemical coupling process allowed circumventing traditional drawbacks like poor yield and low reproducibility. A high density of specific amidolytic activity, up to 945±118 PAU (papain activity units) per mg of TO-NFC could be immobilized, in a form resistant to desorption and stable under harsh conditions. The substrate turnover rate of immobilized papain was increased several fold while its substrate specificity slightly decreased compared to its free soluble form. With this work, a significant improvement could be achieved in the functionalization of TO-NFC with proteins and peptides compared to previous work in terms of yield and density. We propose the strategy presented in this study for further development of more efficient TO-NFC-protein conjugates especially applicable in biomedical applications such as enzyme- or antibody based diagnostic biosensors and smart wound dressings, facilitating controlled release of protein drugs or other versatile bioactive coatings.

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ASSOCIATED CONTENT Supporting information contains: Relevant properties and absorbance spectra of the model biomolecules (Table S1 & Figure S1), their absorbance and extinction coefficients (Table S2) and adsorption isotherm parameters (Table S3). The analysis of the time- (Figure S2) and pH (Figure S3) dependent adsorption process. The further characterization of the model enzyme papain (Figure S4, S5 & S6). AFM measurement of fibril network morphology of bare TO-NFC and TO-NFC saturated with adsorbed protein (Figure S7 & 8). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel.: +41 58 765 72 62. Fax: +41 58 765 74 99. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest

ACKNOWLEDGMENT We thank Esther Strub, Stefanie Altenried, Karl Kehl, Thomas Ramsauer & Luzia Wiesli for Technical Assistance. Solenne Desseaux is thanked for her help with AFM. Houssine Sehaqui is

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gratefully acknowledged for the support with SSA analysis. Michèle Senn is thanked for the precious contribution during her internship.

ABBREVIATIONS TO-NFC; TEMPO-oxidized nanofibrillated cellulose, pristine NFC; untreated nanofibrillated cellulose; TEMPO: 2,2,6,6-Tetramethyl-1-piperidinyloxy, PAU; Papain activity units, pGFLNa; pGlu-Phe-Leu-p-nitroanilide, small chromogenic peptide substrate to papain.

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For Table of Contents Use Only

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TEMPO-Oxidized Nanofibrillated Cellulose as a High Density Carrier

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