Digital light processing (DLP) 3D-fabricated antimicrobial hydrogel with a sustainable resin of methacrylated woody polysaccharides and hybrid silver-lignin nanospheres

Abstract

Lithography-based digital light processing (DLP) 3D printing has gained increasing interest in the fabrication of custom-designed hydrogel scaffolds. Current research development calls for the versatility of the bio-based resin formulations...

Full text

S1
Supporting Information
Digital light processing (DLP) 3D-fabricated antimicrobial hydrogel with a
sustainable resin of methacrylated woody polysaccharides and hybrid
silver-lignin nanospheres
Luyao Wang,a Qingbo Wang,a Anna Slita,b Oskar Backman,a Zahra Gounani,c Emil Rosqvist,d Jouko Peltonen,d
Stefan Willför,a Chunlin Xu,a Jessica M. Rosenholm,b and Xiaoju Wang *a,b
17 pages, 11 Figures, 9 Tables
a Laboratory of Natural Materials Technology, Faculty of Science and Engineering, Åbo
Akademi University, Henrikinkatu 2, Turku FI-20500, Finland
b Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi
University, Tykistökatu 6A, Turku FI-20520, Finland
c Physics, Åbo Akademi University, Henrikinkatu 2, Turku FI-20500, Finland
d Laboratory of Molecular Science and Engineering, Faculty of Science and Engineering, Åbo
Akademi University, Henrikinkatu 2, Turku FI-20500, Finland
*Corresponding author email address: Xiaoju.Wang@abo.fi
Electronic Supplementary Material (ESI) for Green Chemistry.
This journal is © The Royal Society of Chemistry 2022

S2
Characterizations
Transmission electron microscopy (TEM): The morphology of LNP and LNP@Ag were analyzed by a TEM
microscope (JEM-1400 PLUS, JEOL Ltd., Japan) in bright-field mode with an accelerating voltage of 80 kV. A
dispersion of LNP or LNP@Ag was prepared in distilled water at a concentration of 0.01 wt%, and 5 µL of the
dispersion was dropped onto a copper grid (200 mesh, TED PELLA INC. USA) coated with thin carbon film and
then incubating at ambient temperature for 3 min. The excess liquid was removed by blotting with filter paper
before loading in the TEM microscope. The average particle size of LNP and AgNPs was measured by imaging 150
- 200 particles from TEM images using Image J software.
Hydrodynamic diameter and ζ-potential analysis: The intensity weighted average hydrodynamic diameter (Z-
average), polydispersity index (PDI) by intensity, and surface charge (ζ-potential) of the as-synthesized LNP and
LNP@Ag were determined by using a Zetasizer Nano instrument (Malvern Instruments). The samples for dynamic
light scattering (DLS) and ζ-potential measurements were prepared in distilled water at a concentration of around
0.2 mg.mL-1 and analyzed at 25 °C. DLS measurement parameters were as follows: a Helium-Neon laser
wavelength of 632.8 nm; a scattering angle of 173o; the refractive index (RI) and viscosity of the dispersant
(distilled water) were set to be 1.324 and 0.887 x 10-3 pa.s, respectively; the RI and absorption value of the
LNP@Ag were set to be 1.595 and 0.200 respectively, which were the same as those of LNP. The Z-average, PDI,
and ζ-potential values were collected through three consecutive measurements, for which the mean result was
reported.
FTIR analysis: The infrared (IR) absorbance of the samples (i.e., LNP and LNP@Ag freeze-dried powders) were
measured with a Fourier-transform infrared spectroscopy (FTIR) (Nicolet iS50 FT-IR Spectrometer, ThermoFisher
ScientificTM) equipped with a standard sample compartment and a sample holder. All the samples for FTIR
measurements were prepared by uniformly mixing 200 mg of potassium bromide (KBr) with 2 mg of sample in
an agate mortar. Then the KBr/sample mixture was tableted and tested for FTIR analysis. Sixty-four scans for
each sample were taken with a resolution of 4 cm−1 ranging from 4000 to 400 cm−1.

S3
Fig. S1 Solubility of laccase-polymerized MeOH-s fraction (incubated with laccase for 4 hours) in THF/H2O (9:1,
v/v) and acetone/H2O (9:1, v/v). The image was taken after 1 day of dissolution.
Fig. S2 TEM image of L-i-PrOH-LNP@Ag aqueous dispersion. The inset was the photograph of the LNP-[Ag(NH3)2]+
aqueous dispersion over 4 hours reaction time.
Fig. S3 TEM images of MeOH-LNP@Ags used as a representative sample to show the deficient alkali resistance
of lignin without polymerization but possess the capacity to reduce Ag+. The preparation process for MeOH-
LNP@Ag was the same as LM-4-NP@Ag.

S4
XPS analysis of the LNP and LNP@Ag
The high-resolution XPS spectra of LNP and LNP@Ag are shown in Fig. S4. The detailed deconvolution of the
core-level regions of C 1s and O 1s, including the band binding energies and relative area percentages were listed
in Table S3. No significant binding energy shifts were found in the deconvoluted bands from C 1s and O 1s core-
level regions after the incorporation of AgNPs, whereas the band area did change. Briefly, the relative area
percentages of C2 (C-O/C-OH) and O2 (C-O-C/C-OH) in LM-4-NP@Ag decreased compared with the LM-4-NP
sample. Meanwhile, the relative area percentages of C3 (O-C-O/C=O), C4 (O-C=O), and O1 (C=O/C=O*-O)
concomitantly increased. These changes all indicate the oxidation of lignin hydroxyl groups to carbonyl/carboxyl
groups during the silver reduction.
Fig. S4 High-resolution XPS spectra over C 1s and O 1s core-level regions of the (a) (c) LNP and (b) (d) LNP@Ag
from laccase-polymerized MeOH-s lignin.

S5
Fig. S5 TEM images of LM-4-NPs@Ag that reacted with Ag(NH3)2NO3 (10 mg. mL-1) for (a) 4 hours and (b) 6 hours,
and their corresponding histogram (c-d) of particle diameter size distribution of AgNPs. Note: The TEM
magnification in (a) and (b) was 40,000×.
FTIR analysis of the LNP and LNP@Ag
To ascertain the oxidation of lignin by silver, the chemical composition of LM-4-NP and LM-4-NP@Ag were
analyzed by FTIR spectroscopy. All the characteristic absorptions related to the aromatic skeleton of lignin were
observed in LNP and LNP@Ag samples. In the carbonyl/carboxyl region of LM-4-NP@Ag, the peak at 1720 cm-1
belongs to the absorption of non-conjugated C=O stretching,1 and the band intensity at 1720 cm-1 was obviously
stronger than that of the original LM-4-NP. The C-H stretching vibration in aromatic methoxyl groups (-OCH3) and
in methyl and methylene groups of lignin side chains was observed at 2936 cm-1 and 2841 cm-1 in LM-4-NP, which
decreased in intensity in LM-4-NP@Ag. Furthermore, the sharp peak observed at 1215 cm-1 in LM-4-NP belongs
to the syringyl (S) ring breathing with C-O stretching,2 which shifted to a higher wavenumber in the FTIR spectrum
of L-M-4-LNP@Ag. These results confirm the oxidation of lignin with the formation of carbonyl/carboxyl and
quinone groups during the reaction with Ag(NH3)2NO3 solution. The above-mentioned changes were also
observed in the FTIR spectrum of LB-4-NP/LB-4-NP@Ag and LE-4-NP/LE-4-NP@Ag samples. However, neither the
peak position nor the peak intensity was changed for the LI-4-LNP sample after being incubated with
Ag(NH3)2NO3 solution for 4 hours, which is in agreement with the chemical inert property of laccase-treated i-
PrOH fraction.

S6
Fig. S6 FTIR spectra of the LNP and LNP@Ag prepared from (a) laccase-polymerized birch AL lignin, (b) laccase-
polymerized i-PrOH-s lignin, (c) laccase-polymerized EtOH-s lignin, and (d) laccase-polymerized MeOH-s lignin.

S7
Fig. S7 SEM image of LM-4-NP@Ag powder.
Fig. S8 TEM image of LM-4-NP@Ag showing the presence of lignin capping layer.

S8
Fig. S9 The CAD drawing of the crosshatch scaffolds.

S9
Fig. S10 Inhibition zone in disk diffusion test using GGMMA-based hydrogel (16 mm in diameter, 1 mm in
thickness) discs cast by UV405-LED.

S10
Mass balance analysis
Fig. S11 Mass balance analysis of the laccase-catalyzed lignin polymerization and LNP as well as LNP@Ag
preparation processes using LM-4-NP@Ag sample as a representative case.
Safety notes were addressed to the deployment of THF as the solvent for the LNP production and fresh
preparation of Ag(NH3)2NO3 and a recycle note was created for the precipitation of the residual Ag+ as AgCl at
the end of in situ AgNP generation on LNP.

S11
Table S1. DLS results of the LNPs prepared from birch-MeOH-s fraction and laccase-polymerized MeOH lignin in
aqueous media.
hydrodynamic diametera
dispersitya
LNPs
Z-average
(nm)
STDEV
PDI
STDEV
MeOH-NP
417.8
2.9
0.14
0.05
LM-1-NPb
290.1
5.0
0.15
0.03
LM-2-NP
242.1
1.3
0.12
0.04
LM-3-NP
216.1
2.5
0.12
0.01
LM-4-NP
167.0
2.6
0.11
0.01
LM-5-NP
143.5
1.0
0.24
0.02
LM-6-NP
117.9
1.9
0.28
0.02
athe results were obtained by calculating the average of three
consecutive measurements. bLM-x-NP, x denotes the reaction
time of lignin with laccase.
Table S2. UV-vis absorption bands and intensity of LM-4-NP@Ag dispersion in aqueous media.
lignin absorption
intensity
SPR bands
relative intensity
reaction
time
(min)
289 nm
295.8 nm
wavelength
(nm)
absorption
intensity
289 nm
/295.8 nm
SPR /289
nm
SPR / 295.8
nm
90
0.5518
0.5569
---a
---
0.9908
0
0
100
0.5408
0.5488
---
---
0.9853
0
0
120
0.5488
0.5573
---
---
0.9848
0
0
150
0.5511
0.5539
---
---
0.9950
0
0
160
0.5864
0.5866
360.8
0.4391
0.9998
0.7487
0.7486
170
0.5944
0.5968
365.8
0.4640
0.9959
0.7807
0.7775
180
0.5981
0.5975
375.4
0.4934
1.0010
0.8250
0.8258
190
0.6273
0.6300
400.8
0.5269
0.9957
0.8399
0.8363
200
0.6123
0.6102
410.6
0.5509
1.0034
0.8996
0.9027
220
0.6366
0.6316
418.6
0.6035
1.0080
0.9480
0.9556
230
0.6389
0.6377
420.2
0.6292
1.0019
0.9848
0.9867
240
0.6352
0.6315
424.0
0.6626
1.0058
1.0431
1.0492
250
0.6532
0.6503
425.8
0.6820
1.0045
1.0440
1.0487
260
0.6537
0.6480
426.2
0.7159
1.0088
1.0951
1.1048
270
0.6606
0.6575
427.4
0.7268
1.0046
1.1003
1.1054
280
0.6753
0.6675
428.4
0.7625
1.0116
1.1292
1.1423
290
0.6836
0.6798
429.2
0.7751
1.0056
1.1338
1.1401
310
0.6772
0.6683
430.8
0.7920
1.0133
1.1696
1.1852
320
0.6867
0.6816
430.8
0.8082
1.0074
1.1771
1.1858
330
0.6954
0.6852
430.8
0.8310
1.0114
1.1949
1.2127
340
0.6772
0.6696
430.2
0.8188
1.0149
1.2090
1.2228
350
0.6917
0.6817
431.4
0.8393
1.0147
1.2133
1.2311
360
0.7093
0.7043
431.4
0.8548
1.0071
1.2052
1.2137
a---, not detectable.

S12
Table S3. Binding energies, XPS spectral deconvolution constraints, and area percent of the deconvoluted
bands in each core-level region of the XPS spectra of LM-4-NP and LM-4-NP@Ag.
LM-4-NP
LM-4-NP@Ag
core-level
regions
bands
binding
energy (eV)
FWHM
(eV)
L/G Mix
(%)
Product
area
(%)
binding
energy (eV)
FWHM
(eV)
L/G Mix
(%)
Product
area
(%)
band
assignmentb
C1
284.80
1.49
30
48.3
284.86
1.50
30
45.4
C-C/C=C
C2
286.16
1.49
30
32.2
286.11
1.50
30
26.8
C-O/C-OH
C3
287.10
1.49
30
17.2
287.07
1.50
30
22.7
O-C-O/C=O
C 1s
C4
288.68
1.49
30
2.2
288.87
1.50
30
5.0
O-C=O
O1
531.73
1.70
30
11.8
531.99
1.70
30
27.0
C=O/C=O*-O
O 1s
O2
533.32
1.70
30
76.0
533.31
1.70
30
66.3
C-O-C/C-OH
O3
534.64
1.70
30
12.2
534.41
1.70
30
6.68
C-O
Ag 3d3/2
---a
---a
---a
---a
374.9
1.68
30
40.0
Ag 3d3/2
Ag 3d
Ag 3d5/2
---a
---a
---a
---a
368.8
1.68
30
60.0
Ag 3d5/2
a’---‘ denotes not detectable. bthe deconvoluted bands were assigned by comparing with the published literature.3,4

S13
The calculation of lattice constant (a0) and average crystallite size (d) of AgNP
The interplanar spacing (dhkl) and lattice constant (a0) of Ag cubic structure were calculated according to the
Bragg’s Law (equation (1)) and Miller indices (equation (2)), respectively.5 In brief, the lattice plane reflections
(hkl) = (111), (200), (220), (311) and (222), with the corresponding 2θ = 38.14o, 44.18o, 64.48o, 77.35o, and 81.47o,
were used for the calculation of Ag lattice parameter (Table S4). The average of the five calculated values of a0
was compared with the standard a0 (4.0857 Å) of Ag cubic structure in Table S4.5
(1)
𝑑ℎ𝑘𝑙 =𝑛𝜆 / 2𝑆𝑖𝑛𝜃
(2)
𝑎0=𝑑ℎ𝑘𝑙 ℎ2+𝑘2+𝑙2
dhkl is the interplanar spacing, Å
n is the order of the reflection (integer number), for first-order n=1
λ is the wavelength of the X-ray source, CuKa=1.54184 Å
θ is the Bragg angle in radians (rad)
a0 is the lattice constant, Å
The average crystallite size (d) of AgNP was calculated following the Scherrer formula (equation (3)),6,7 which use
the full width at half maximum (FWHM) of the major diffraction peak (111).
(3)
𝑑=𝐾𝜆 / 𝐵𝐶𝑜𝑠𝜃
d is the dimension of the crystallite, Å
K is the numerical shape constant, K = 0.89
λ is the wavelength of the X-ray source, CuKa=1.54184 Å
B is the corrected FWHM in radians (rad)
θ is the Bragg angle of the peak maximum in radians (rad)

S14
Table S4. The lattice constant (a0) of AgNPs calculated from XRD data using the Bragg’s Law and Miller indices
and compared with the standard a0 of Ag.
indices
Bragg angle θ
(rad)
d-spacing
(Å)
lattice constant
(Å)
average
a0 (Å)
standard a0
(JCPD card)
error
(%)
LM-4-NP@Ag-3
h
k
l
θ
Sinθ
dhkl
a0
a0
a0
a0
1
1
1
0.3328
0.3267
2.3597
4.0870
2
0
0
0.3855
0.3760
2.0503
4.1006
2
2
0
0.5627
0.5334
1.4453
4.0873
3
1
1
0.6750
0.6249
1.2337
4.0916
2
2
2
0.7110
0.6526
1.1813
4.0885
4.0910
4.0857
0.13
LM-4-NP@Ag-5
h
k
l
θ
Sinθ
dhkl
a0
a0
a0
a0
1
1
1
0.3326
0.3265
2.3612
4.0895
2
0
0
0.3853
0.3758
2.0514
4.1028
2
2
0
0.5626
0.5334
1.4453
4.0873
3
1
1
0.6750
0.6249
1.2337
4.0916
2
2
2
0.7110
0.6526
1.1813
4.0885
4.0920
4.0857
0.15
LM-4-NP@Ag-10
h
k
l
θ
Sinθ
dhkl
a0
a0
a0
a0
1
1
1
0.3328
0.3267
2.3597
4.0870
2
0
0
0.3854
0.3759
2.0509
4.1017
2
2
0
0.5630
0.5337
1.4445
4.0850
3
1
1
0.6757
0.6254
1.2327
4.0883
2
2
2
0.7112
0.6527
1.1811
4.0879
4.0900
4.0857
0.10
Table S5. Comparison of particle size of AgNPs measured from TEM and crystallite size (d) of Ag calculated from
XRD pattern using Scherrer formula.
XRD
TEM
samples
B (rad)
Cosθ
d (nm)
particle size (nm)
LM-4-NP@Ag-3
0.041
0.9451
3.5
8.1 ± 1.2
LM-4-NP@Ag-5
0.039
0.9452
3.7
16.1 ± 1.9
LM-4-NP@Ag-10
0.036
0.9451
4.0
20.7 ± 2.1
Table S6. The XPS results in atomic%.
sample
C 1s
O 1s
Ag 3d
LM-4-NP
77.9
22.1
0
LM-4-NP@Ag
75.1
21.5
3.4

S15
Table. S7 Mass-based metrics for evaluating "greenness" in LNP@Ag preparation.
mass input (mg)
product (mg)
yield (%)c
waste (mg)
mass-based metrics
samples
LNPa
Ag(HN3)2NO3b
reaction
time
(hours)
LNP@Ag
LNP@Ag
Ag(HN3)2NO3d
reaction mass
efficiencye (%)
E factorf
LM-4-
NP@Ag-3
16
12
4
17.4
109
7.4
62
0.42
LM-4-
NP@Ag-5
16
20
4
23.0
143
9.8
64
0.43
LM-4-
NP@Ag-10
16
40
4
24.2
150
28.6
43
1.18
LM-4-
NP@Ag-10*
16
40
6
23.0
144
29.8
41
1.29
a2 mL LNP dispersion; b4 mL Ag(NH3)2NO3 solution, ignore the weight of NH3.H2O; cyield (%) = 100 x m(LNP@Ag) (recovered by
centrifugation and freeze-drying) / m(LNP) fed initially;
dwaste Ag(NH3)2NO3 = mass input Ag(NH3)2NO3 – (mass of Ag+ reduced to metallic Ag), ignore the weight of NH3.H2O
ereaction mass efficiency = 100 x mass of product (LNP@Ag) / mass of total input
fE(nvironmental) factor = total waste/mass of product (LNP@Ag)
Table S8. Comparison of the antimicrobial activity of as-prepared GGMMA/LNP@Ag hydrogel with other
hydrogel or film that engaged lignin-capped AgNPs as the antimicrobial component.
antibacterial testing
bactericidal ratio (%)
materials
loading of
[lignin-Ag]-
based sample
(wt%)
testing
approach
testing
time (h)
P.
aerugin
osa
E.
coli
S.
aureus
S.
epidermid
is
L.
monocylo
genes
Ref.
GGMMA-LNP@Ag hydrogel
0.10
4
---a
99
99
---
---
this
work
polyurethane-[lignin-AgNP]
foam
0.12
24
99
---
90
---
---
8
poly(vinyl alcohol)-[lignin-
AgNP] hydrogel
14
24
---
99
99
---
---
9
poly(lactide)-[lignin-AgNP]
film
1.00
colony-
counting
method
3
---
100
---
---
99
10
polyacrylic acid-pectin-
[lignin-AgNP] hydrogel
> 0.08
24
---
97
---
98
---
11
agar-[lignin-AgNP] film
1.00
optical
density
3
---
99
---
---
37
(6 h, 99%)
12
a ‘---‘ the strain was not included in the antimicrobial experiment.

S16
Table S9. The yield of laccase-polymerized lignin from birch AL fractions and LNP as well as LNP@Ag yield
obtained from laccase-polymerized lignin.
laccase-catalyzed
polymerization
LNP preparation
LNP@Ag preparationc
lignin fractions
initial
weight
(mg)
isolated
weight
(mg)
yielda
(%)
initial
weight
(mg)
insoluble
fraction
(mg)
LNP
(mg)
yieldb
(%)
initial
weight
(mg)
LNP@Ag
obtained
(mg)
yieldd
(%)
birch AL
210
218
109
100
9
82
82
16
20
126
birch-i-PrOH-s
209
205
102
100
9
79
79
16
14
88
birch-EtOH-s
205
208
102
100
2
71
71
16
18
112
birch-MeOH-s
200
204
102
100
8
81
81
16
24
150
acalculated after 4 hours incubation with laccase, yield (%) = 100 x m(laccase-polymerized lignin) (recovered by acid precipitation, centrifugation,
and freeze-drying) / m(lignin fraction) fed initially
byield (%) = 100 x m(LNP) (recovered by centrifugation and freeze-drying) / m(laccase-polymerized lignin) fed initially
cthe LNP@Ag was prepared by impregnating LNP in Ag(NH3)2NO3 solution (10 mg.mL-1) for 4 hours
dyield (%) = 100 x m(LNP@Ag) (recovered by centrifugation and freeze-drying) / m(LNP) fed initially

S17
References
1 S. Piqueras, S. Füchtner, R. Rocha de Oliveira, A. Gómez-Sánchez, S. Jelavić, T. Keplinger, A. de Juan and
L. G. Thygesen, Front. Plant Sci., 2020, 10, 1–15.
2 C. G. Boeriu, D. Bravo, R. J. A. Gosselink and J. E. G. Van Dam, Ind. Crops Prod., 2004, 20, 205–218.
3 L. Zhang, H. Lu, J. Chu, J. Ma, Y. Fan, Z. Wang and Y. Ni, ACS Sustain. Chem. Eng., 2020, 8, 12655–12663.
4 Z. Tian, L. Zong, R. Niu, X. Wang, Y. Li and S. Ai, J. Appl. Polym. Sci, 2015, 132, 42057.
5 B. K. Mehta, M. Chhajlani and B. D. Shrivastava, J. Phys. Conf. Ser., 2017, 836, 012050.
6 K. He, N. Chen, C. Wang, L. Wei and J. Chen, Cryst. Res. Technol., 2018, 53, 1–6.
7 S. Chen, G. Wang, W. Sui, A. M. Parvez and C. Si, Green Chem., 2020, 22, 2879–2888.
8 A. G. Morena, I. Stefanov, K. Ivanova, S. Pérez-Rafael, M. Sánchez-Soto and T. Tzanov, Ind. Eng. Chem.
Res., 2020, 59, 4504–4514.
9 M. Li, X. Jiang, D. Wang, Z. Xu and M. Yang, Colloids Surf. B: Biointerfaces, 2019, 177, 370–376.
10 S. Shankar, J. W. Rhim and K. Won, Int. J. Biol. Macromol., 2018, 107, 1724–1731.
11 D. Gan, W. Xing, L. Jiang, J. Fang, C. Zhao, F. Ren, L. Fang, K. Wang and X. Lu, Nat. Commun., 2019, 10,
1–10.
12 S. Shankar and J. W. Rhim, Food Hydrocoll., 2017, 71, 76–84.