nanomaterials
Article
Colloidal Lignin Particles as Adhesives
for Soft Materials
Maija-Liisa Mattinen 1, * , Guillaume Riviere 1 , Alexander Henn 1 ,
Robertus Wahyu N. Nugroho 1 , Timo Leskinen 1 , Outi Nivala 2 ,
Juan José Valle-Delgado 1 , Mauri A. Kostiainen 3 and Monika Österberg 1
1
2
3
*
Bioproduct Chemistry, Department of Bioproducts and Biosystems, School of Chemical Engineering,
Aalto University, P.O. Box 16300, FI-00076 Aalto, Espoo, Finland;
[email protected] (G.R.);
[email protected] (A.H.);
[email protected] (R.W.N.N.);
[email protected] (T.L.);
[email protected] (J.J.V.-D.);
[email protected] (M.Ö.)
VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, FI-02044 VTT Espoo, Finland;
[email protected]
Biohybrid Materials, Department of Bioproducts and Biosystems, School of Chemical Engineering,
Aalto University, P.O. Box 16100, FI-00076 Aalto, Espoo, Finland;
[email protected]
Correspondence:
[email protected] or
[email protected]; Tel.: +358-50-302-3511
Received: 4 November 2018; Accepted: 29 November 2018; Published: 3 December 2018
Abstract: Lignin has interesting functionalities to be exploited in adhesives for medicine, foods and
textiles. Nanoparticles (NPs) < 100 nm coated with poly (L -lysine), PL and poly(L -glutamic acid)
PGA were prepared from the laccase treated lignin to coat nanocellulose fibrils (CNF) with heat.
NPs ca. 300 nm were prepared, β-casein coated and cross-linked with transglutaminase (Tgase) to
agglutinate chamois. Size exclusion chromatography (SEC) and Fourier-transform infrared (FTIR)
spectroscopy were used to characterize polymerized lignin, while zeta potential and dynamic light
scattering (DLS) to ensure coating of colloidal lignin particles (CLPs). Protein adsorption on lignin
was studied by quartz crystal microbalance (QCM). Atomic force microscopy (AFM) was exploited
to examine interactions between different polymers and to image NPs with transmission electron
microscopy (TEM). Tensile testing showed, when using CLPs for the adhesion, the stress improved ca.
10 and strain ca. 6 times compared to unmodified Kraft. For the β-casein NPs, the values were 20 and
8, respectively, and for the β-casein coated CLPs between these two cases. When NPs were dispersed
in adhesive formulation, the increased Young’s moduli confirmed significant improvement in the
stiffness of the joints over the adhesive alone. Exploitation of lignin in nanoparticulate morphology is
a potential method to prepare bionanomaterials for advanced applications.
Keywords: lignin; nanoparticle; protein; nanocellulose; fibril; enzyme; heat; self-assembly; cross-link
1. Introduction
Technologies focusing on the preparation of adhesives utilizing natural polymers such as proteins
and cellulosics for medical, textile and food applications are emerging research fields [1–3] However,
biorefinery industry produces also lignin by-product, which is still underutilized, even though this
aromatic, antioxidative and antimicrobial biopolymer could be an interesting raw material for many
value-added applications [4].
Nanocellulose can be produced by several methods [5–8]. It is a lightweight, transparent and
biodegradable polymer. Hence, nanocellulose fibrils (CNF) as well as bacterial nanocellulose (BC)
are excellent raw materials for tissue regeneration and replacement [9–12]. Major challenges for the
exploitation of CNF include the ability to disperse colloidal material with different formulations.
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Surface functionalization of the fibrils could be an attractive method to improve stability, functionality
and compatibility of the nanomaterial with selected matrices. For example, poly (L -lysine, PL) coated
wax particles assembled on CNF surface yield hydrophobic fibrils [13]. Furthermore, capability to
obtain tight bonding between tissue edges to prevent bleeding with excellent gas barrier properties and
to achieve strong mechanical strength for the sealant, are crucial properties for the medical adhesives
to support wound healing and tissue deformation during the recovery [14–16].
Due to excellent solubility and biocompatibility, regenerated silk proteins have been used in
medical applications such as textiles, implants and materials for controlled drug release. Deposition
of silk fibrin on polymeric surfaces is a remarkable challenge [2]. Enzymatic cross-linking with
transglutaminase (Tgase, EC 2.3.1.13) catalyzing cross-links between glutamines and lysines has been
used to stabilize proteins against chemicals and proteases [17]. Furthermore, Tgase has been used
to graft silk proteins onto damaged wool fibers to improve strength of the surfaces and to degrease
felting shrinkage during washing [18].
Foods such as edible coatings are nearby medical applications. Food packages based on
petroleum-based raw materials are not biodegradable. They have poor oxygen barrier properties
possibly leaching harmful compounds into foods. Water-soluble edible coatings based on dairy proteins
could be excellent alternatives for these packages. Protein coatings have good gas barrier properties
and no bad flavor or taste [19–21]. For example, nanospheres prepared from caseins form opaque
films and could be used to coat foods as well as biological tissues [22]. However, poor water resistance
and mechanical strength of the casein coatings needs to be improved to meet full applicability of the
nanomaterial in above applications [19].
Silkworm adhesive is an excellent biomimetic model for the preparation coatings for
nanoparticulate bonding agents [23] since many technologies for tissue engineering and surgery rely
on nanoparticle (NP) based adhesion [24]. Strong and rapid adhesion between hydrogels is feasible at
room temperature by spraying hard NPs on the surfaces and bringing them into contact. Tight adhesion
between the soft materials is based on the NPs’ ability to adsorb tightly onto surfaces, where they act
as connectors between polymer chains dissipating energy under stress [24]. Thus, tailored colloidal
lignin particles (CLPs) prepared from technical lignin could be interesting nanomaterials to be used
as additives in adhesives and coatings. Different CLPs could be produced in the laboratory and
semi-industrial scale [25–36]. Enzymatic cross-linking could be an attractive method to increase
porosity of NPs in addition to stability improvement against organic solvents [37–39]. Including small
molecules in the hydrophobic core of CLPs antioxidant and antimicrobial property of the particles could
be enhanced [40,41]. Specificity of the particles could be tailored via surface modification [34,42–44].
In this contribution, tiny CLPs including bilayer polypeptide modifications were prepared from
Kraft using self-assembling to tailor CNF surfaces with heat treatment. Furthermore, larger protein
coated CLPs were prepared and enzymatically cross-linked for adhering skin tissue (chamois). Finally,
water-soluble adhesive formulation was used to demonstrate effect of various NPs for the adhesion of
soft chamois specimens. It was concluded that nanoparticle architecture could be an interesting general
platform for the preparation technical lignin-based nanobiomaterials for advanced applications.
2. Materials and Methods
2.1. Chemicals
Reagent grade chemicals and solvents for the CLP preparation and modifications were purchased
from Sigma-Aldrich (Steinheim, Germany). Water soluble Pritt adhesive (Henkel AG & Co, Düsseldorf,
Germany) was purchased from a department store in Finland. Throughout the study, Milli-Q water
was used in the aqueous solutions.
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2.2. Proteins
Mixture of serum proteins (pI 5.2–7.8), casein from bovine milk (mixture of α-, β-, λ- and
κ-subunits), gelatin (MW 47 kDa, pI 7.0–9.0), bovine serum albumin (BSA, 66 kDa, pI 4.8–5.6)
and purified β-casein (MW 24 kDa, pI 4.6–5.1) were purchased from Sigma-Aldrich (Germany).
For analyses, gelatin was dialyzed (cut-off 21 kDa) and freeze-dried. Due to low solubility, β-casein
was first dissolved in H2 O and vortexed at room temperature following dilution with H2 O (1 mg mL−1 )
and pH adjustment (pH 3.0). After 2 h, the solution was vortexed, ultrasonicated and filtrated. Collagen
IV (Col IV) from human placenta (Sigma Aldrich, USA) was treated according to Goffin et al. [45]
PL peptide (0.1 m-% in H2 O w/v, MW 150–300 kDa, pI 9.0) and sodium salts of poly(L-glutamic
acid, PGA) peptides (MW 50–100 kDa and 15–50 kDa) diluted with water (1 mg mL−1 ) were ordered
from Sigma-Aldrich (Germany). Chamois for the adhesion experiments was purchased from Biltema
(Espoo, Finland).
2.3. Enzymes
Low redox Melanocarpus albomyces laccase (MaL, pH-range 5.0–7.5) was overproduced in
Trichoderma reesei. High redox Trametes hirsuta laccase (ThL, pH-range 4.5–5.0) was produced in
its native host following chromatographic purification [46,47]. The reactivities of the enzyme
preparations were determined against 2.2-azinobis-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) at pH
4.5 in 25 mM Na-succinate buffer [46] using Perkin Elmer Lambda 45 spectrophotometer (USA) at
436 nm (ε = 29.300 M−1 cm−1 ). For ThL (3.5 mg mL−1 ), the activity was 5270 nkat mL−1 and, for MaL
(8.1 mg mL−1 ), it was 2050 nkat mL−1 . Tgase (pH-range 4–9) [17] was purchased from Activa MP
Ajinomoto (Japan). After further purification, the enzyme activity (8764 nkat mL−1 ) was determined
as previously described [48].
2.4. Nanocellulose
The preparation of CNF exploited in this study was described by Valle-Delgado et al. [49] CNF was
produced using mechanical fibrillation of never-dried, bleached Kraft hardwood birch pulps obtained
from Finnish pulp mills using a high-pressure fluidizer (Microfluidics M-110Y) from Microfluidics Int.
Co. (Westwood, MA, USA). No pre-treatments were used prior to fibrillation. The number of passes
through the microfluidizer was 12 and the final dry matter content was 1.35 wt-%. The operating
pressure was 2000 bar. The average width of the fibrils was 8–9 nm, length several micrometers and a
zeta potential ca. −3 mV. CNF thin films for the CLP coatings were prepared on the silica plates as
recently described [49].
2.5. Preparation of CLPs
Lignin nanoparticles were prepared from LignoboostTM purchased from Domtar plant (Plymouth,
NC, USA) with minor changes in the procedure [25]. First, lignin (2 g) was dissolved in the mixture
of THF and H2 O (3:1, v/v). Then, H2 O was added in the filtrated solution, filtrated again and finally
dialyzed (Spectra/Por® 1, RC dry dialysis tube, 6–8 kDa) for removal of THF. Concentration of the
CLP dispersion was ca. 1.5 mg mL−1 , average particle size ca. 300 nm and zeta potential ca. −33 mV.
For the preparation of tiny CLPs, lignin was enzymatically oxidized using low and high redox
potential laccases. Powdered lignin (1 g) was dissolved in 0.1 M NaOH (700 mL) under constant
magnetic stirring at pH 12.5. Then HCl (1 M) was slowly added to adjust the pH 6.0 and 8.0 for the
ThL and MaL treatments, respectively. Then, the solution was transferred into a 1 L measuring flask.
Due to low reactivity of ThL in alkaline reaction conditions, pH 8.0 was omitted for this enzyme. Then,
lignin solutions (330 mL) were oxidized with laccases (500 nkat g−1 ) and magnetically stirred (20 h).
The enzymatic reactions were terminated using acid precipitation (1 M HCl). The supernatant (pH
2) was removed using ultracentrifugation (OptimaTM L Series, rotor type 70 Ti, Beckman Coulter,
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Bromma, Sweden) at 6000 rpm (G-force 1000) for 20 min at 25 ◦ C. The precipitate was collected with
H2 O (pH 5.5) and dried (80 ◦ C) prior preparation of tiny CLPs.
After lignin oxidation, the method modified from Lievonen et al. [25] was used to prepare CLPs
below 100 nm. Enzymatically treated lignin (0.5–1 mg mL−1 ) and the references (2.1 mg mL−1 ) were
solubilized in THF:H2 O (3:7, v/v) and the mixture was stirred (3 h) following filtration with 0.7 µm
Whatman GF/F (Sigma-Aldrich, Germany). Then, H2 O was fast poured into the solution under
constant stirring following vigorous mixing (15 min). THF was removed the solution using dialysis
(cut-off: 6–8 kDa) under constant flux for 3 days. The aqueous CLP dispersion was filtrated and
characterized as previously described [25].
2.6. Adsorption of Proteins on Lignin
Adsorption of model proteins on lignin surface was studied by quartz crystal microbalance
with dissipation (QCM-D, Q-Sense E4, Sweden) at different pHs [42]. For the analysis, golden plates
were oxidized with UV-light (10 min), spin-coated with polystyrene (PS) and lignin (WS 650, Laurell
Technologies Corp., North Wales, PA, USA) [50]. PS was dissolved in toluene (0.5 mg mL−1 ) applied
twice (50 µL) and dried at 80 ◦ C (30 min). Lignin was coated from the dioxane-H2 O mixture (85:15
v/v, 0.5 mg mL−1 ) and applied four times on a plate and dried as above. Spin-coating sequence was
300 rpm (3 s), 1000 rpm (5 s) and finally 2000 rpm (30 s). Protein samples were dissolved in water (10 µg
mL−1 ) at 40 ◦ C and filtrated. For the pH optimization of the β-casein adsorption, it was dissolved in
the buffers (50 mM, 0.1 mg mL−1 ): pH 3.0 and 5.0 (citrate), 6.5 (phosphate), 7.4 (PBS) and 8.5 (Tris-HCl).
Then, lignin films were stabilized with the buffers (1 h) and exposed to β-casein adsorption (100 µL
min−1 , 25 ◦ C) until stable baseline was detected following rinsing with the buffer (30 min). Masses
of the adsorbed β-casein were calculated from the frequencies according to Johannsmann et al. [51].
After β-casein adsorption on lignin film at the optimized pH, the protein coating was cross-linked
with Tgase using enzyme dosages 5, 25 and 50 nkat in the measuring cell (40 µL).
2.7. Coating CLPs with Proteins
Surfaces of CLPs (1 mg mL−1 ) were coated with β-casein at pH 3.0 using β-casein to CLP mass
ratio of 0.00001 to 1. Extent of surface charge modifications and changes in the average particle size of
CLPs were analyzed after stabilization of the samples at room temperature overnight. Bilayer protein
coated CLPs were prepared by modifying only slightly negatively charged particles first with PL and
then with acidic of sodium salts of PGA. In the end of the experiment excess of PGA was added in
the solution to ensure maximal coverage of single PL coated CLPs and presence of large amounts of
carboxylic acid groups for the esterification reaction, crucial for CNF coating.
2.8. Stabilization of Protein Coated CLPs
Surfaces of β-casein coated CLPs were enzymatically stabilized using Tgase. To avoid
cross-linking and aggregation of the particles, the enzyme dosage was optimized. In the reactions,
Tgase activities varied 5–40 nkat g−1 . After overnight incubation at room temperature, the enzyme
activity was terminated using ultracentrifugation (5000 rpm, 30 min). Supernatant was removed and
the precipitate, cross-linked β-casein coated CLPs, were redispersed in H2 O at pH 3 and pH 7.5 for the
stability studies.
2.9. Physicochemical Characterization of CLPs
Average particle sizes and zeta potential values of CLP dispersions were analyzed using a
Zetasizer (Malvern, Nano-ZS90 instrument, Malvern, UK). The zeta potential values were calculated
from the electrophoretic mobility data using Smoluchowski model. Three scans were collected for
zeta potential and five scans for the average particle size measurement using dynamic light scattering
(DLS) to evaluate the reproducibility of the measurements.
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2.10. SEC
Polymerization of lignin by laccases was studied by aqueous high-performance gel permeation
size exclusion chromatography (HP-GPC/SEC). For the analyses, enzymatically polymerized and
cross-linked lignins including molecular weight standards (194 Da to 0.1 kDa) were dissolved in NaOH
(0.1 M) in two concentrations (0.1 and 0.5 mg mL−1 ). Weight-average molar mass (MW) of the samples
were analyzed by Agilent 1260 Infinity (Agilent Technologies, Espoo, Finland) equipped with a UV
detector operating at 280 nm as previously [39,52].
2.11. FTIR
Fourier-transform infrared (FTIR) spectra of the lignin samples were recorded using Thermo
Nicolet iS50 FTIR spectrometer with iS50 ATR-crystal (Thermo Fisher Scientific, Vantaa, Finland).
Analysis of the spectral area (3800–600 cm−1 ) was carried out as duplicate measurements with 32 scans
from each sample and averaged prior normalization, which was based on peak area using Excel
(Microsoft, Espoo, Finland).
2.12. AFM
Atomic force microscopy (AFM) was used to characterize spherical morphology and roughness
of CLP surfaces before and after protein coating and enzymatic cross-linking to evaluate aggregation
between NPs after the treatments. For the imaging, 10 µL of CLP dispersion was pipetted on a freshly
cleaved mica sheet and dried overnight at ambient temperature. All samples were imaged in tapping
mode in ambient air using a Multimode 8 AFM equipped with a Nanoscope V controller from Bruker
Corporation, Santa Barbara, CA, USA). NCHV-A probes with a fundamental resonance frequency of
320–370 kHz, a nominal spring constant of 40 N m−1 , and a tip radius below 10 nm were used for
imaging. At least three sample areas were imaged from the same mica sheet without further processing
of the images except flattening using Nanoscope Analysis 8.15 software from Bruker (USA).
Furthermore, AFM was used to measure adhesion energies between Col IV and lignin, Col IV
and gelatin as well as Col IV and casein. For the force measurements the tip less silicon cantilever
(CSC38/No Al coating, MicroMasch, Tallinn, Estonia) with a normal spring constant of 40 N m−1
was used to study the interactions. Prior to force measurements, the nominal spring constant was
determined analyzing the thermal vibration spectra using Sader method [53]. The biomaterial-coated
probe was prepared with same method as previously [54]. Adsorption of Col IV onto the colloidal
probe was performed in several steps. First, the collagen solution (1 mg mL−1 ) was placed in ice-filled
beaker and thawed by sonication (2 × 10 min). Then, the glass probe was surface modified with 5 vol-%
3-aminopropyl triethoxysilane (APTES) dissolved in ethanol to improve physical adsorption of protein
on the glass probe (45 min). Unreacted APTES was rinsed with ethanol and dried. The APTES-modified
probe was glued with an optical adhesive (Norland Products, Inc., Cranbury, New Jersey, USA) on the
free-end of the cantilever with 3D micromanipulator following UV curing (15 min) at the wavelength
of 365 nm. After gluing, the colloidal probes modified with APTES were mounted on metallic disc
facilitated with double-side tape and few drops of collagen solution were spin-coated (40 s) at 1000 rpm.
The collagen-coated probes were dried overnight and rinsed with Milli-Q water before use. The neat
glass probe was used a reference.
AFM force measurements were performed using a Multimode 8 AFM NanoScope V controller
coupled with a Pico Force (PF) scanner from Bruker (USA) in a liquid mode. The colloidal probe was
mounted on the liquid cell and subsequently inserted into the AFM head. Few drops of PBS buffer
(pH 7.4) were injected onto sample film and equilibrated (10 min) before the force measurements.
The rate of the approach and retraction of the colloidal probe towards the surface was 2 µm s−1 . At least
three random locations were probed to ensure the homogeneity of the film surface. The deflection
sensitivity was determined from a freshly cleaved mica surface. The recorded data were converted to
the profiles of normalized force and the separation distances, where D = 0 was adjusted to be at the
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maximum applied load [55]. The measured force profiles were compared to the DLVO theory [56,57].
and the adhesion energy was calculated through the integration over the adhesion area. For the
proteins (Col IV), the Hamaker constant for calculation of van der Waals forces was 7.5 × 10−21 J [58].
2.13. TEM
FEI Tecnai 12 (Hope, CA, USA) operating at 120 kV was used to obtain transmission electron
microscopy (TEM) images from the CLP dispersions. For the imaging, 3 µL of the sample was applied
on a carbon film supported grid and incubated (2 min). The excess of the solvent was removed by
blotting the side of the grid onto paper. Imaging was performed in the brightfield mode with slight
under focus.
2.14. Sample Preparation
Chamois specimens washed with acetone and dried with filter paper were cut to narrow strips
(3.5 cm × 1.0 cm) following stabilization in the standard conditions (25 ◦ C, 50% humidity). The area
used for adhesion was 1 cm−2 . In addition to aqueous NP dispersions (CLP, β-casein and CLP coated
with β-casein) in 1 mg mL−1 concentration, Tgase (100 nkat cm−2 ) was used for curing β-casein
coated CLPs joints. Furthermore, NPs (1 mg mL−1 ) were dispersed in diluted water-soluble adhesive
(10 mg mL−1 ) to study the effect of the NPs on the adhesion in the agglutinative formulation. Lignin
dissolved in THF (1 mg mL−1 ) and diluted adhesive formulation (H2 O:THF, 99:1, v:v) in 10 mg mL−1
concentration were used as references. After sticking the specimens with NP dispersions (50 µL and
100 µL), the samples where kept under a metal plate (ca. 200 g) in the standard conditions for 3 days
prior to tensile testing (MTS400, MTS Systems Corporation, Eden Prairie, MN, USA).
Tiny and bilayer protein coated CLPs (mass ratio 1 g g−1 lignin) were linked on the CNF surfaces
using esterification reaction between the carboxylic acid groups with hydroxyls of CNF [49,59]. For the
analysis, two drops of modified CLP dispersions were coated (4000 rpm, 1 min) on the CNF surface
using a spin-coater from Laurell Technologies Corp., (North Wales, PA, USA). Heated up to 105 ◦ C
(10 min) following 5 min treatment at 155 ◦ C. To remove unbound particles, CNF surfaces were rinsed
with H2 O and dried under nitrogen flow.
3. Results and Discussion
3.1. Tailoring CLP Surfaces with Proteins
Proteins adsorb on lignin surface. The extent of the interactions depends on the physicochemical
properties of the biomolecule resulting from the three-dimensional (3D) structure and amino acid
composition of the protein [42,50]. To show potential to exploit actual by-product from industry,
purified β-casein, previously used in wood [60] and food [21] adhesives was used a model protein for
the surface functionalization of CLPs.
3.1.1. β-Casein
Adsorption of β-casein on lignin surface was studied at pH 7.4 using QCM-D (Figure S1A). Gelatin,
serum proteins and PL, commonly used to coat tissues to improve cell adhesion [61], were studied
for comparison. Positively charged gelatin (47 kDa, pI 7.0–9.0) at pH 7.4 adsorbed better on lignin
surface than smaller negatively charged β-casein (24 kDa, pI 4.6–5.1). Adsorption of serum proteins
and polypeptide (PL) were weaker. The increase in dissipation was considerably higher at similar
frequency values for the β-casein coating compared to other proteins, indicating that the coating is
softer and contains more water (Figure S1B).
For the coating of individual CLPs with β-casein, protein adsorption on lignin surface was
examined at pH range 3.0–8.5 (Figure 1). It was the highest at pH 3.0 due to positive charge of β-casein
in the acidic reaction conditions. A similar adsorbed mass was observed at pH 8.5, but in this case
negatively charged β-casein formed particles (ca. 300 nm) that adsorbed on the lignin surface together
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value when β
—
with the polymeric protein. This is further confirmed when comparing the increase in dissipation
(Figure S1C). The dissipation is much higher for layers adsorbed at alkaline pH compared to pH 3.5,
indicating that these layers are more loosely bound and containπ–π
more water due to the nanoparticulate
morphology. The formation of β-casein NPs depends on the pH, time, mixing, protein and salt
concentration [20].
Figure 1. Adsorption
Adsorption of
of β
β-casein on lignin thin film at different pH observed using QCM-D.
Hence, pH 3.0 was selected for the coating CLPs with β-casein for further experiments. Figure 2a
value when β
shows the zeta potential of the CLPs varying from negative (ca. −25 mV) to positive (ca. 25 mV)
—
value when β-casein concentration increased. During the coating, CLPs aggregated when the surface
charge of the particles was close to zero (CLP—protein ratio ca. 0.01), as shown in Figure 2b. On the
other hand, once clearly positively charged, the protein-coated CLP dispersions were stable for weeks.
π–π
Large-scale all atom MD simulations [62] have shown that aromatic residues contribute significantly
to the protein adsorption on hydrophobic surfaces via strong π–π stacking interactions between
p2-carbons. The basic residues such as arginine and lysine play equally strong role for the adsorption.
The effect of proline residues has been demonstrated recently [42].
In Figure 2 are shown representative TEM images from single CLPs (Figure 2c) including
β-casein coated CLPs (Figure 2d) confirming that after protein adsorption, and enzymatic cross-linking
(Figure 2e) CLPs remain individual spherical NPs and the aggregation of the particles is minor.
During sample preparation on the carbon grid, some of the particles moved close to each other due
to water evaporation during drying. The corresponding AFM images are shown in Figure 2f–h and
Figure S2A–D. When CLPs were coated with β-casein, very small protein particles could be detected
from the background of TEM (Figure 2d) and AFM (Figure S2B,D) images, not visible in the references
(Figure 2f,i–k and Figure S2A,C). Apparently, some of these particles adsorbed on CLP surface along
with polymeric β-casein since several protruding points (ca. 40 nm) could be imaged from the CLP
surfaces by AFM (Figure
S2B,D).
Adsorption
of β
(a)
(b)
Figure 2. Cont.
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Figure 2. Coating
Coating CLPs
CLPswith
withβ β-casein (pH 3.0) evidenced using zeta potential (a) and DLC
), ββ-casein
(b) measurements as a function of time. TEM images measured from unmodified CLPs (c),
and β-casein
β
coated CLPs (d) ) and
coated CLPs cross-linked with Tgase (e). In (f,g,h) are shown
representative AFM images from enzymatically stabilized CLPs and in (i,j,k) are presented the
corresponding references.
3.1.2. Poly(L-glutamic acid)
c) including β
Feasibility to coat CLP surfaces using selected proteins to maximize specific interactions with the
substrate such as CNF surface was evaluated. Thus, negatively charged CLPs were first coated with
positively charged PL following modification with PGA containing large number of carboxylic acid
–
groups for the esterification reaction with hydroxyls on nanocellulose surface via fast heat treatment.
–D.
When
CLPs
were
coated
with
β
It was hypothesized that tiny CLPs below 100 nm in size coat single CNF fibrils better than larger
particles since the typical width for the nanocellulose fibrils is ca. 5–20 nm and the length several
–
micrometers. The average molecular masses of enzymatically polymerized and cross-linked lignin
surface along with polymeric β
used for tiny CLP preparation are shown in Figure 3 and the characterization using FTIR spectroscopy
in Tables S1–S3. In general, the changes in the FTIR spectra between the references and laccase treated
samples were minor due to heterogeneous cross-linking reactions and residual moisture in the samples
slightly interfering the interpretation of the spectra.
The appearance of the CLP dispersions at pH 6.0 are shown in Figure S3. The average particle
sizes for CLPs prepared from enzymatically oxidized lignin were below 100 nm (Table 1). For the
references, the particle size was half of that obtained according to the method of Lievonen at al. [25].
In both cases, the zeta potentials were on the same order of magnitude as previously described [25].
The increased molecular weight, higher hydrophobicity of the polymerized and cross-linked lignin
–
as well as lower concentration enabled tight packing of enzymatically oxidized
lignin fast mixing
promoting tiny NP CLP formation. In the laccase-catalyzed reactions, the cross-links are formed
in lignin via different radical reactions. Enzymatic initiation of the radicalization starts from the
phenolic hydroxyl groups–of lignin following condensation of the free radicals to covalent chemical
bonds [37,38,46,47,63]. The representative AFM images of the different CLPs (Figure S4) verify the
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spherical and smooth surface structure of the NPs stable for several weeks (Table S4), as evident from
the TEM images (Figure S5). Solid lignin NPs 10–30 nm in size can be produced using mechanical
shearing [32] and are also potential modifiers for CNF surfaces.
Figure 3. Average molecular masses and standard deviations of the enzymatically oxidized and
polymerized lignins. Molecular masses were analyzed also for the CLPs prepared from the
enzymatically treated lignins. The smaller values of dissolved CLPs are most likely due to slower
solubility of CLPs in alkaline reaction conditions than powdered lignin.
Table 1. Characterization of CLPs prepared from laccase-treated lignin.
Sample
Average Size (nm)
Zeta Potential (mV)
PDI
pH 6.0
Reference
MaL-treatment
ThL-treatment
131 ± 1
75 ± 1
82 ± 2
−30 ± 1
−25 ± 1
−33 ± 1
0.27
0.40
0.40
pH 8.0
Reference
MaL-treatment
ThL-treatment
125 ± 1
65 ± 1
-
−22 ± 1
−30 ± 1
-
0.25
0.25
-
(-) Not determined. The enzyme is not reactive at alkaline reaction conditions.
–
Bilayer protein coated tiny CLPs (ca. 131 nm) are shown in Figure S6. Significant increase in
the average particle size from ca. 200 nm to ca. 400 nm confirmed coating of the NPs with small
PGA (15–50 kDa). When using larger PGA (150–300 kDa), the effect on the average particle size and
extent of the surface coating was minor. Apparently, shorter polymer chain is more desirable for
− the both cases, zeta potential values
the modification of CLP surfaces over large ones. However, in
decreased from ca. 40 mV to 30 mV, verifying dual coating −of CLPs. In Figure S7A,B are shown
−
adsorption of PL peptide on the intact CLP surface following adsorption of low and high molecular
weight PGA confirming the above conclusions.
−22 ± 1
In the body, NP-specific protein coronas are formed on hard
NPs in minutes comprising
−30inorganic
±1
hundreds of proteins. Adsorption of serum proteins on lignin (Figure S1A,B) show that CLP surfaces
could be tailored accordingly to increase cellular interaction. Compared to hard NPs, soft CLPs are
presumably safer since undesirable penetration of the elastic NPs through the biological membranes
is minor. Low cytotoxicity [31,64] and resistance for the enzymatic hydrolysis increase potential to
–
exploit CLPs in value-added applications in medicine and cosmetics [2,20].
–
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3.2. Stability of β-Casein Coated CLP
3.2.1. Effect of Enzymatic Cross-Linking
Tailoring CLP surfaces with β-casein allows further modification of the particles with cross-linking
enzymes [65]. In vitro studies have shown that Tgase can cross-link proteins in a gel in minutes [17,48].
Hence, to improve stability of β-casein coated CLPs at physiological pHs, for example in stomach
(pH 1.0–3.0), duodenum (pH 4.8–8.2) and blood (pH 7.4), Tgase was used to cross-link the β-casein
coating. Figure S8A shows the increase in the average particle size of CLPs as a function of enzyme
dosage. Particles cross-linked with high enzyme dosage above 20 nkat g−1 aggregated immediately
because covalent bonds were also formed between the individual particles. The zeta potential values
(Figure S8B) decreased after 24-h incubation and, after seven-day treatment, extensive cross-linking of
the NPs was detected by eye. Reasonable enzyme dosage for the cross-linking only β-casein coating
was found to be 15 nkat g−1 . Prior to pH stability studies of the enzymatically cross-linked particles,
Tgase activity was removed from the dispersions using ultra-centrifugation (Figure S9). After the
treatment, some of the unbound β-casein adsorbed on CLP surfaces increasing the average particle
size by ca. 40 nm. Then, the particles were stable for several weeks.
Elasticity of the enzymatically cross-linked β-casein coating was studied by QCM-D
(Figure S10A,B). After adsorption of β-casein on lignin film until a stable baseline was observed
(Figure S1), Tgase (2 min) was injected into the chamber following cross-linking (10 min) of the
protein film. When using the low enzyme dosage (5 nkat), loosely bound β-casein was washed away.
However, at the same time, the cross-linking reaction proceeded to a certain extent since a sharp
decrease in dissipation was detected due to the formation of elastic networked protein coating. Instead,
when using higher enzyme dosages in the measuring cell (25 and 50 nkat), first a small amount of
unbound β-casein was removed (tiny increase in the frequency signal). After that, a sharp drop in the
frequency was detected due to enzyme adsorption on the β-casein surface. The cross-linking reaction
proceeded as in the case of low enzyme dosage. During the enzymatic reaction (10 min), the frequency
increased slightly, verifying an accompanied loss of small molecule reaction product (-NH3 + ) [66] as
well as decreased water binding capacity of the cross-linked protein coating. Tgase injection following
stabilization of the cross-linking reaction was repeated three times until stable baseline was observed.
Figure 2c–h shows representative TEM and AFM images of β-casein coated CLPs cross-linked with
Tgase. Compared to CLPs coated with β-casein (Figure S2B,D) the surfaces of cross-linked particles
were smoother. From the corresponding TEM images (Figure 2d,c), it is evident that also small protein
particles in the background were cross-linked to larger particles. Regarding the applicability of the
enzymatically stabilized protein coated CLP dispersions, polymerization of free β-casein to nanosized
particles [65] improves homogeneity and stability of the dispersion.
3.2.2. Effect of pH
Understanding dispersion properties is essential for the exploitation CLPs in adhesive
formulations. CLPs are stable in wide pH range [25]. However, due to better electrostatic stability in
alkaline conditions, the reactivity of CLPs is much higher than in acidic conditions [39,63]. For medical,
cosmetic and food applications, it is pivotal that tailored CLPs are stable in physiological conditions.
Figure 4a,b shows the average particle sizes and zeta potential values for β-casein coated CLPs
at pH 3.0 and 7.4 as a function of time. In acidic dispersion, NPs started to aggregate after four-day
incubation, which was evident also from the more positive zeta potential values. After 25 days,
protein-coated particles precipitated. At pH 3.0, β-casein was positively charged, but CLP surface was
nearly neutral, promoting aggregation of the coated CLPs. At slightly alkaline dispersion (pH 7.4),
the stability of β-casein coated CLPs was excellent. The average particle sizes and zeta potential values
remained nearly unchanged for 25 days.
β
7.4), the stability
of β
Nanomaterials
2018, 8, 1001
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(a)
(b)
ilityofofβ-casein
β
Figure 4. Stability
coated and enzymatically cross-linked CLPs at (a) pH 3.0 and (b) pH
7.4 evidenced using average particle size and zeta potential measurements. In both cases, the protein
coating following enzymatic cross-linking was performed at 3.0. Enzyme activity was removed using
ultracentrifugation following redispersion of the particles at above pH.
To
ofthe
theβ β-casein coated CLPs at pH 3.0, the optimized Tgase dosage
Toimprove
improve stability of
(15− nkat g−1 ) was used to cross-link the surfaces of the particles. The stability of cross-linked protein
coated CLPs was excellent compared to the non-cross-linked particles (Figure 4a). The average particle
sizes remained nearly unchanged for 25 days. Instead, the zeta potential values became slightly more
positive due to some instability of CLPs, as explained above. Instead, at pH 7.4 (Figure 4b), the zeta
potential values were stable for weeks, but the average particle sizes increased during the first day
after enzyme inhibition and solvent exchange. Then, the cross-linked protein coated CLPs were stable
for weeks.
3.3. Adhesive Interactions
AFM force measurement was used to compare strength of the interactions between lignin and
model proteins (Figure 5). Col IV is the main component of the skin and, therefore, understanding the
interactions of Col IV with other proteins and lignin could be exploited in wound healing and other
biomedical applications.
Long-range attractive interactions were detected when Col IV surface was brought into contact
with casein and gelatin (Figure 5a), while no meaningful adhesions were detected when the neat glass
surface was used (Figure 5b). The force interactions between Col IV and lignin were three times larger
than the ones between Col IV and model proteins (Figure 5c) with adhesion energy being 0.0102 ±
0.0006 nJ m−1 . Interestingly, the adhesion energy between Col IV and casein as well as Col IV and
gelatin was nearly identical at pH 7.4, evident also from the overlapping retraction force profiles
(Figure 5a). At high salt concentration, the repulsions detected from the approach force profiles was
much longer-ranged than predicted by DLVO theory for pure electrostatic double-layer repulsion
(Figure 5d). This reveals that steric repulsions dominate [67]. Based on the approach force profiles
separation distance between Col IV and gelatin pair is nearly twofold compared to that of Col IV and
casein, while Col IV and lignin was placed in between. Even though casein show lower adhesion
energy with Col IV, it is an attractive protein to coat CLPs compared to poorly soluble gelatin having
high tendency to aggregate, as recently demonstrated [42]. Thus, purified β-casein containing many
reactive sites for enzymatic cross-linking and stabilization is an excellent coat protein for CLPs enabling
also fast curing enzymatic means [65].
Nanomaterials 2018, 8, 1001
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Figure 5. AFM force profiles. Interaction between (a) Col IV probe and other proteins and lignin.
(b) Interaction of the model surfaces with a neat glass probe. (c) Corresponding histograms of mean
adhesion energy of above interactions including standard deviation. (d) DLVO fitting of the data
presented in (b). The gray dashed line represents the DLVO fitting for PBS (150 mM) at pH 7.4. All force
profiles were normalized with the radius of the probe.
3.4. Applications
Chamois specimens and CNF were used model soft matrixes to demonstrate effect of tailored
CLPs to adhere soft materials. Furthermore, enzymatic and chemical heat treatment were exploited for
−
fast covalent curing of the agglutinations.
3.4.1. Agglutination of Chamois Specimens with CLPs
Figure 6a show the chamois leather specimen including CLP dispersion used for adhesion. Tensile
testing was used to measure the strength of adhesive joints until break down. The representative
stress–strain curves for various NP formulations are shown in Figure 6b,c.
tendency to aggregate, as recently demonstrated [42]. Thus, purified β
(a)
(b)
(c)
Figure 6. Tensile testing. (a) Two chamois specimens to be adhered with CLP dispersion (above).
–
Adhered strips subjected
to a controlled tension until failure (below). (b) Representative stress–strain
–
–
curves for the CLPs used for adhesion including the corresponding references. (c) Representative
●
,●β
stress–strain curves for the same samples as in (b) dispersed in water-soluble adhesive. CLPs (blue),
, ● CLPs coated with β
, ● commercial adhesive
, ● polymeric
β-casein NPs (red), CLPs coated with β-casein (purple), commercial adhesive (black), polymeric
lignin dissolved in THF (grey).
Nanomaterials 2018, 8, 1001
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Figure 7a shows the effect of different NPs for adhering protein matrix that was significantly
better than that of lignin and commercial water-soluble adhesive. When the number of NPs doubled,
the agglutination of the specimens was more than twofold stronger, which was evident from the
measured stress values. The differences between the type of the soft material, i.e., lignin or protein,
used for the NP preparation was apparent when high NP concentration was used. Adhesive property of
β-casein NPs on protein matrix was the highest and for CLPs only half of that. Effect of β-casein coated
CLPs on the adhesion was between these two cases showing potential as a method to prepare functional
low-cost β-casein NPs from lignin via self-assembly. The tensile strain (Figure 7b) measured from the
corresponding samples followed the same order as the stress values. However, the differences between
the samples were smaller. When β-casein coated CLPs were used for adhering, the strain values were
rather similar between two different quantities. In the case of large number of NPs, the repeatability was
poor, presumably due to instability of the β-casein coating at pH 3.0, as explained above. Regarding
to the references, lignin powder dissolved in THF and diluted adhesive, the agglutination of the
specimens was minor compared to that of NPs. Due to rough, hairy surface of chamois, the NPs
spread, adsorbed and penetrated on the soft material better than the thick adhesive formulation,
efficiently dissipating energy and retarding fracture of the joints under stress.
–
Figure 7. Comparison between tensile stress–strain
histograms. (a,b) Different CLPs including the
references at two quantities. (c,d) Effect of Tgase catalyzed cross-linking on the adhesion. (e,f) Various
NPs including the references dispersed in diluted water-soluble adhesive.
train values
Figure 7c,d shows the increased stress and strain
values when
whenββ-casein coated CLPs were
covalently linked to chamois specimens using low Tgase dosage (100 nkat). Increasing the enzyme
activity, the strength of the adhesion could be increased [47,62]. These results suggest that protein
coated CLPs could also be linked to biological matrixes using enzymes enabling fast curing in moist
environments essential for the medical applications. In such seals, CLPs remain single nanoparticles
Nanomaterials 2018, 8, 1001
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retaining their physicochemical properties since the covalent linkages are formed via protein coating.
It is also plausible that cross-links were formed between amine groups in lysine side-chains and the
acyl group derived from the carboxylic acid groups present in CLPs due to side reactions of Tgase [17].
If CLPs are coated with tissue specific proteins, the potential rejection reactions could be diminished
during the wound healing, yielding small scars important for cosmetic applications. Accordingly, it is
presupposed that CLP formulations could be exploited for the preparation of edible coatings for foods.
Furthermore, to study potential to use CLPs as additive in adhesive formulations, NPs and the
references were dispersed in the water-soluble adhesive (Figure 7e,f). Unexpectedly, the stress value
was the largest (ca. 5%) for the dispersion containing lignin powder compared to that of diluted
adhesive. Mixing protein NPs in the adhesive formulation decreased stress value ca. 3%, and for
the unmodified CLPs ca. 7%. In the case of tensile strain measurement, the results were opposite.
For CLPs, β-casein NPs and β-casein coated CLPs, the tensile strain improved ca. 5%, 12% and 10%,
respectively. For lignin powder, the elasticity of the adhesive joint degreased ca. 5% compared to the
diluted adhesive.
Figure 8 shows histograms of Young’s modulus for various NPs. The values doubled for the
specimens adhered with high number of NPs regardless of the type of the polymer used for the
adhesion. Instead, when the NPs were dispersed in the adhesive formulation, the differences between
the raw
raw materials
materialsbecame
becamevisible.
visible.In
Inthe
thecase
caseof
ofCLPs,
CLPs,Young’s
Young’smodulus
modulusincreased
increased(ca.
(ca.70
70kPa),
kPa),but
but,
the
for the protein-based NPs it degreased (ca. 43 kPa), showing more elastic agglutination due to softer
structure of the particles and improved compatibility with the substrate. In the adhesive formulation,
theYoung’s
Young’s modulus
modulus was
was the
thehighest
highestfor
forthe
thepolymeric
polymericlignin
ligninshowing
showingmuch
muchstronger
strongeradhesive
adhesivejoint
joint,
the
likely due to varying chemical reactions and interactions between the adhesive components and the
substrate. Furthermore, interactions between lignin and protein matrix differ from the ones between
CLPs and the substrate. In aqueous dispersion, hydrophobic groups are buried inside the CLPs’
’
hydrophilic groups locating on the surfaces of NPs. Furthermore, the instability of lignin complicated
the analyses. The results obtained in the adhesive formulation should be critically considered. Young’s
modulusmodulus
determined
for the diluted adhesive was between that of CLPs and lignin powder.
Young’s
determine
(a)
(b)
) CLPs,
casein and
Figure 8.Young’s
Young’smodulus
modulusfor
for (a)
CLPs,ββ-casein
andCLPs
CLPscoated
coatedwith
withββ-casein using two quantities.
(b) Corresponding histograms for the NPs dispersed in diluted adhesive including the references.
These results show that, when using NP prepared from soft natural matter (technical lignin,
proteins and their combinations), it is possible to obtain strong elastic agglutinations between soft
surfaces, as evidenced recently [24,68,69]. The type of biopolymer affects significantly the strength,
–
Nanomaterials 2018, 8, 1001
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flexibility and elongation of the joint. Studies with soybean-based adhesives containing polymeric
lignin pointed out that protein–lignin ratio is the most critical parameter affecting the adhesive
interactions [70,71]. In textiles, enzymatic cross-linking has been used to strengthen lignin-containing
adhesives [72]. Apparently, exploitation of technical lignin in nanoparticulate morphology in stable
dispersion for adhering is an interesting approach for many lignin applications reviewed [73].
3.4.2. Grafting CLPs on CNF Surfaces
Feasibility to coat CNF with varying sized CLPs was also investigated. Figure 9a shows
nanocellulose fibrils spin coated with several layers of tiny CLPs (ca. 75 nm). Covalently linked
CLPs clearly follow the fibrous morphology of nanocellulose, changing the surface properties of the
hydrophilic polymer to that of more hydrophobic lignin surface. Linking of the NPs was obtained via
esterification reaction between carboxylic acid groups of lignin and hydroxyl groups of CNF surface via
heat treatment [49]. After washing with H2 O for the removal of unbound CLPs, the fibers accumulated
to some extent, however, CLPs remaining linked on the CNF surfaces due–to covalent –linkages. Hence,
it is proposed that CNF modified with CLPs could be excellent bionanomaterials for tissue repairing,
having improved stability in body fluids, culture media and resistance against enzymatic hydrolysis.
(a)
(b)
(c)
Figure 9. CNF surfaces coated with CLPs: (a) Tiny CLPs; (b) PL and PGA coated CLPs; and (c) reference.
It was also shown that CNF surfaces (Figure 9b) could be coated accordingly with protein
coated CLPs using heat treatment. To increase the reactivity between CNF and protein coated CLPs,
nanoparticles were modified first with positively charged PL and then with negatively charged PGA
containing large number of carboxylic acid groups. CLPs coated only with PL could not be linked
on the slightly negative CNF surface to the same extent. In that case, surface modification of CNF,
e.g., via carboxylation to increase negative charges on the surface, is a prerequisite. After the heat
treatment, the average particle sizes of the modified CLPs clearly decreased (Figure S6A and Figure 9b)
due to the formation of covalent linkages between carboxylic acids in PGA and hydroxyls in CLP.
After washing with water, most of the bilayer coated CLPs remained on the CNF surface. Feasibility
for the bilayer protein coating of CLPs was demonstrated also using QCM-D under identical reaction
conditions (Figure S7A,B). PL peptide was first adsorbed to CNF film following adsorption of CLPs
yielding intact lignin nanoparticulate surface. Then, PL and PGA (15–50 and 50–100 kDa) were
adsorbed on the surface that was finally washed with water. As shown in Figures S6 and S7, it was
evident that small PGA adsorbed on CLP surface better than large PGA.
Current polymeric tissue adhesives are often unstable in physiological pH requiring complex
in vivo analytical systems for the control of the polymerization and cross-linking reactions.
Furthermore, they are often toxic [6,69]. Coating fibers with specific CLPs could be an effective way to
improve mechanical strength. Adhesives based on nanobridging via hard inorganic NPs [16] as well
as colloidal mesoporous silica (CMS) particles [68] have been proposed as alternatives for traditional
Nanomaterials 2018, 8, 1001
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medical adhesives. Since the adhesion energy is proportional to the surface area of NPs, enzymatically
cross-linked CLPs [39] with tailored functionalities could be potential additives for medical adhesives
following enzymatic or thermal treatment for fast curing shown above. Additionally, porous structure
of CLPs enables quicker decomposition in biological media than inorganic NPs preventing undesirable
accumulation in the body. Due to strong autofluorescence of lignin, it is an attractive raw material
enabling sensitive real time detection crucial for the development of image-guided procedures for
clinical applications.
4. Conclusions
Development of green technologies [74,75] for the preparation of bio(nano)materials [76] from the
forest process side-streams such as adhesives and coatings [77,78] is increasing constantly. Different
CLPs prepared and modified to adhere chamois and to modify CNF surface could be potential additives
for various formulations to be exploited for wound sealing, edible coatings and fiber modification for
textiles to improve adhesion, hydrophobicity, antimicrobial and antioxidative properties of the coatings.
Since the cross-linking methods are fast and feasible in the moist environment, clinical fluorescence
imaging of aromatic CLPs is possible. Furthermore, it was concluded that, when using tissue specific
proteins, e.g., hydrolyzed from collagen, sericins extracted from silk and caseins fractionated from
the dairy side-streams, compatibility of the NPs with the substrates could be enhanced. Compared to
NPs prepared solely from proteins, the costs of the raw materials are remarkably lower. Apparently,
these results pave the way for the exploitation of technical lignin in multiple forms.
Supplementary Materials: The electronic supplementary data associated with this article is available online
at https://www.mdpi.com/2079-4991/8/12/1001/s1. Figure S1: Adsorption of proteins on lignin thin films
analyzed by QCM-D, Figure S2: AFM images of β-casein coated CLPs, Figure S3: CLP dispersions prepared from
laccases treated lignins, Figure S4: AFM height images from CLPs prepared from laccase treated lignin at pH 6,
Figure S5: TEM images from CLPs prepared from enzymatically treated lignin one month after preparation, Figure
S6: Average particle size and zeta potential of CLPs as a function of PL−CLP mass ratio, Figure S7: Adsorption of
PL, PGA and CLPs on slightly negatively charged CNF analyzed by QCM-D, Figure S8: β-Casein coating and
enzymatic stabilization of the particles with Tgase, Figure S9: Variation of stabilized CLPs in size after removal of
enzyme activity using ultracentrifugation, Figure S10: Elasticity of enzymatically cross-linked β-casein coating,
Tables S1–S3: Characterization of laccase treated lignins by FTIR, Table S4: Effect of time on the average particle
size, zeta potential and polydispersity (PDI) of CLPs prepared from different lignins at starting concentration
0.5 g L−1 .
Author Contributions: M.-L.M. supervised the work including writing of the final version of the manuscript.
Furthermore, she was responsible for the adsorption, coating and stabilization studies, TEM imaging as well
as planning of the application studies. G.R. prepared and characterized CLP dispersions using different
physicochemical methods such as AFM, SEC and FTIR spectroscopy. R.W.N.N. and T.L. carried out the AFM
retraction force measurements. A.H. carried out the adhesion experiments with various NPs and chamois
specimens using tensile testing. O.N. purified and characterized enzymes used in the study at VTT. J.J.V.-D.
conducted the AFM imaging. M.A.K. and M.Ö. provided facilities, scientific discussion and guidance for the
study. All authors contributed to the production of the manuscript.
Funding: This research was funded by the Academy of Finland (TaBioMat, Tailored biomass derived
self-assembling building blocks for bionanomaterial applications, grant number 276696). Furthermore, this work has
received funding from the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020
research and innovation programme under grant agreement No 720303 (Zelcor project).
Acknowledgments: MSc students Anni Pyysing, Helena Båtsman, Teemu Kemppainen, Ari Ruotsalainen,
Mustafa Çan, Andrey Vinogradov (Aalto University, JOIN-E3000 Life science technologies project course, coated
lignin nanoparticles for biomaterial applications, Finland) and MSc student Zhenxing Yan (Aalto University,
Finland) are acknowledged for the laboratory assistance.
Conflicts of Interest: There are no conflicts to declare.
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