A New Dual Functional PHB-grafted Lignin Copolymer: Synthesis

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A New Dual Functional PHB-grafted Lignin Copolymer: Synthesis, Mechanical Properties and Biocompatibility Studies Dan Kai, Kangyi Zhang, Sing Shy Liow, and Xian Jun Loh ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00445 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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ACS Applied Bio Materials

A New Dual Functional PHB-grafted Lignin Copolymer: Synthesis, Mechanical Properties and Biocompatibility Studies

Dan Kai1*, Kangyi Zhang1, Sing Shy Liow1, Xian Jun Loh1,2,3*

1Institute

of Materials Research and Engineering (IMRE), A*STAR, 2 Fusionopolis Way, #08-

03 Innovis, Singapore 138634 2Department

of Materials Science and Engineering, National University of Singapore, 9

Engineering Drive 1, Singapore 117576 3Singapore

Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751

* Corresponding

Author

E-mail: [email protected], [email protected]

1 ACS Paragon Plus Environment

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Abstract Poly(3-hydroxybutyrate) (PHB) is a sustainable and biodegradable biopolymer from bacteria, but its brittle nature greatly limits its applications. In this study, we developed a lignin-PHB copolymer to enhance the mechanical properties of PHB. β-Butyrolactone was grafted onto lignin core by using solvent free ring-opening polymerization (ROP). Then different amounts of lignin-PHB copolymers were blended into PHB and then engineered into nanofibers via electrospinning. The composite nanofibers with lignin-PHB copolymer exhibit much stronger mechanical properties than pure PHB fibers. Composite nanofibers with 2% lignin copolymer demonstrate the best mechanical performance with tensile strength increasing from 1.45 ± 0.36 MPa to 5.61 ± 0.63 MPa, Young’s modulus increasing from 54.7 ± 1.2 MPa to 84.6 ± 10.0 MPa, and elongation increasing from 9.6 ± 2.2 % to 93.5 ± 7.6%. Moreover, PHB/lignin nanofibers demonstrate tuneable antioxidant activity, allowing the neutralization of excess free radicals in our body. Animal studies also demonstrate that the PHB/lignin nanofibers are nonirritant and biocompatible. Hence, these new PHB/lignin nanofibers hold great potential for biomedical applications.

Keywords:

Poly(3-hydroxybutyrate),

solvent-free

polymerization,

electrospinning,

mechanical reinforcement, plasticizer, antioxidant, biocompatibility


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Introduction Growing concerns over the consumption of fossil fuels and associated environmental issues have spurred the development of sustainable materials from renewable resources. Among biopolymers, polyhydroxyalkanoate (PHA) is a class of sustainable aliphatic polyesters produced by various microorganisms for energy storage purposes 1-2. Poly(3-hydroxybutyrate) (PHB), the most common member of the PHA family, is biodegradable, biocompatible and thermoplastic 3. For biomedical applications, PHB has been used as tissue engineering scaffolds for various tissues, including bone, skin, nerve and even myocardium 4-6. PHB also has applications in the agricultural and packaging sectors. However, industrial and commercial applications of PHB are limited mainly by its poor mechanical properties. The brittle nature of PHB is due to intrinsic large spherulites and large extent of secondary crystallization 7. To improve

mechanical

properties,

many

copolymerization, blending and annealing

methods 8-9.

have

been

employed

including

Mechanical strength and stiffness of such

modified PHB materials are enhanced, but the brittleness remains. The deformation capacity of most of these materials is still below 10% 10. Lignin is the second most abundant natural polymer on earth and a by-product of the paper milling industry. Approximately 70 million tons of lignin are produced annually, but less than 2% is used in value-added applications. Lignin, with its advantageous properties, is a promising alternative to existing fossil fuel-based materials. Lignin presents high stiffness, decent antibacterial, anti-oxidative and UV-blocking properties

11-13.

Lignin has become one of the

most popular reinforcement agents for polymer composites due to its native function of mechanical support in plants 14-15. Lignin has been used as fillers to modify thermal, mechanical and rheological performances of PHB materials

9, 16-18.

Most work focused on the effect of

lignin on thermal and crystallization properties of PHB, while the aspect of mechanical reinforcement is largely unexplored. In certain cases, lignin fillers can increase the stiffness 3 ACS Paragon Plus Environment


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and strength of the composites, but decrease its deformation capacity. Phase separation and interfacial incompatibility between unmodified lignin and PHB are the main reasons for this unfavorable outcome. Grafting polymers onto lignin could enhance the interfacial adhesion between lignin fillers and PHB matrix for mechanical improvement. In this study, we synthesized lignin-PHB via the solvent free ring-opening polymerization (ROP) of β-butyrolactone onto alkali lignin. Lignin-PHB copolymer was blended with PHB and electrospun into composite fibers. Electrospinning is an attractive technique to produce ultrafine polymer fibers with diameters in the micro/nanoscale. Electrospun fibers with high surface area to volume ratio and high porosity have been utilized in multiple applications, including water filtration, cosmetic masks, energy storage, biosensors and tissue engineering scaffolds 19-21. Herein, different amounts of lignin-PHB copolymer were incorporated into PHB fibers. The antioxidant activity, topographical and mechanical properties of the composite fibers were investigated. Furthermore, cells were cultured on the fibers to evaluate their potential for bone tissue engineering applications.

Materials and methods Synthesis of lignin-PHB copolymer Alkali lignin was dried to remove moisture before experiment. Lignin (Mn = 5 kDa, 2 g), βbutyrolactone (8 g) and tin(II) 2-ethylhexanoate (0.2 ml as catalyst) were mixed and reacted at 130°C for 24 hours under N2. After the reaction, chloroform was added to dissolve the obtained copolymer. After stirring for 2 hours, the solution was centrifuged to remove the unreacted lignin. Lignin-PHB was obtained by precipitating the supernatant into hexane.


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Lignin-PHB copolymer was then characterized by 1H NMR (Bruker 400 MHz) by dissolving in deuterated chloroform. Waters® gel permeation chromatography (GPC) was used to characterize the molecular weight and polydispersity index of the copolymer.

Scheme 1 Polymerization method (solvent-free ring-opening) to synthesize lignin-PHB copolymer from kraft lignin and β-butyrolactone (monomer). Tin(II) 2-ethylhexanoate was used as a catalyst.

Electrospinning of PHB/lignin-PHB nanofibers Natural PHB was purchased from Sigma (Product No. 363502) and purified by precipitation in hexane before use. The apparent Mn and Mw of the purified PHB were 250 and 760 kDa respectively (by GPC with PMMA standards). Electrospinning solutions were prepared by dissolving PHB and lignin-PHB

copolymers into 1,1,1,3,3,3-hexafluoro-2-propanol with a total concentration of 5 wt%. The mass ratios of the two materials were 99:1, 98:2, 95:5, 90:10 and 80:20. The flow rate was set at 0.8 ml/hour, and 10 kV of voltage was applied on blunt 22 G needles. Pure PHB fibers, as a control, were fabricated under the same parameters.

Characterization of electrospun fibers



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The morphology, mechanical and thermal behaviors properties of the electrospun fibers were characterized using SEM, uniaxial tensile testing machine, DSC and TGA. The parameters were based on our previous work 22. Cold crystallization temperature (Tcc), cold crystallization enthalpy change (ΔHcc, negative), glass transition temperature (Tg), melting temperature (Tm) and melting enthalpy change (ΔHm, positive) were determined from the DSC curves. The crystallinity (Xc) was calculated using the following equation (1)

Xc = (ΔHm + ΔHcc) / ΔHm0 × 100%

ΔHm0 is 146 J/g for 100% crystalline PHB 23. The antioxidant activity of the PHB/lignin-PHB fibers was evaluated by 1,1-diphenyl-2picrylhydrazyl (DPPH) assay by soaking 20 mg fibers into 10 ml DPPH solution (60 μM in methanol) 24.

Fluorescent staining of fibroblasts on nanofibers NIH/3T3 fibroblasts (ATCC® CRL-1658TM) were cultured on various PHB / lignin-PHB nanofibers for 8 days. Cells were grown according to manufacturer’s protocol on 24-well plates with a seeding density of 5000 cells per well. Cells were then fixed with 2.5% glutaraldehyde before permeabilization with 0.1% Triton X-100. DAPI (Sigma-Aldrich) and fluorescent phalloidin (MoBiTec Molecular Technology) were used to stain cell nuclei and actin filaments respectively. The DAPI stock solution (300 nM) was mixed with PBS at a ratio of 1:20,000. Phalloidin stock solution (6.6 µM) was diluted with PBS at a ratio of 1:22. Next, 200µl of each type of working solutions were added to each nanofiber sample. After washing steps and mounting, the cells were visualized with a Leica DMi8 inverted microscope fitted with the appropriate filters. 6 ACS Paragon Plus Environment

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In vivo study PHB/lignin-PHB fibers (5 mm diameter with 0.3 mm thickness) were subcutaneously implanted into the backs of female Sprague-Dawley rats to assess in vivo biocompatibility for up to 35 days. PHB fibers were used as a reference biomaterial for control surgeries. The animals were randomly assigned to 5 post-implantation time-points (days 7, 14, 21, 28 and 35), with 5 animals per time-point. Each animal received 8 implants (4 of each biomaterial) and were housed in 2 or 3 animal cages for the duration of the study. All animals were closely monitored for clinical signs, site-specific skin reaction and body weight. Animals were sacrificed on their respective time-points. Intact biomaterials and their surrounding tissue measuring 1.5 x 1.5 cm were harvested for histopathological studies. The experiments were conducted in accordance to IACUC guidelines. Skin tissues were fixed with 10% formalin, dehydrated and embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT).

Results and discussion Synthesis and characterization of lignin-PHB copolymer Lignin polymerization is a good approach to modify its surface properties in order to overcome its drawbacks. To date, quite a few polymerization methods have been established for graft polymerization on lignin 25-33. Chung et al. prepared lignin-lactide copolymers with a solventfree ROP by using triazabicyclodecene as an organocatalyst

34.

In our study, lignin-PHB

copolymer was firstly synthesized by solvent-free ROP (Scheme 1). Figure 1 shows the representative NMR spectrum of lignin-PHB. Lignin showed its characteristic chemical shifts 7 ACS Paragon Plus Environment


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at 3.80 ppm (methoxyl groups) and 6.80 ppm (phenyl rings). Characteristic resonances of PHB were observed at 1.29 ppm, 2.57 ppm and 5.27 ppm corresponding to the protons of –CH3, CH2 and –CH. Peak g at 4.21 ppm presents the end group of PHB chain. Based on the ratio of peak d and g (the integration area of peak d divided by the area of peak g), the average PHB chain length in the copolymer could be calculated as n = 8.8. Fourier-transform infrared (FTIR) was also carried out to characterize the chemical structure of the lignin copolymer. Besides the absorption of lignin (supporting information Figure S2), the characteristic peaks for PHB were also observed, such as 2980 cm-1 corresponding to symmetric CH2 stretching, 1740 cm-1 to carbonyl stretching and 1270 cm-1 to C-O stretching.

Figure 1 NMR spectrum of lignin-PHB in CDCl3


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Analyzed by GPC, and the Mn and Mw of the copolymer were 71.9 kDa and 84.4 kDa respectively. The DSC curve (supporting information Figure S3) shows that the copolymer is semi-crystalline with Tg of 3 °C, Tm of 181 °C and ΔHm of 7.0 J/g. The very low ΔHm suggests that the lignin-PHB has a hyperbranched structure. Unlike the highly-crystalline linear PHB, lignin-PHB has multiple but short PHB chains. These short PHB chains tend to form irregular, entangle coils (amorphous regions), instead of growing into an ordered and aligned molecular structure (crystalline regions). All above results indicate that lignin was successfully grafted with PHB. The TGA curve (supporting information Figure S4) shows that the copolymer underwent 5% weight loss (thermal decomposition temperature, Td) at 195 °C, and that a residual of 18.2% remained at 500 °C. Based on the residual of unmodified lignin at 500 °C (~58%), the mass percent of lignin in lignin-PHB copolymer was 31.4%. In our study, we have tried solvent-free ring-opening polymerization with different types of lignin from different sources. The polymerization also works for lignosulfonates and some kraft lignin from China, but the reaction parameters need to be tuned accordingly. Interestingly, we also found that the solvent-free approach did not work for the kraft lignin provided by TCI chemicals, but this softwood lignin can be polymerized within a solvent system. The reason is still unclear.



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Figure 2 SEM images of electrospun (A) PHB, (B)PHB1LP, (C) PHB2LP, (D) PHB5LP, (E) PHB10LP and (F) PHB20LP nanofibers. The scale bar = 5 μm.

Fiber morphology As shown in Figure 2, all the PHB/lignin-PHB nanofibers displayed uniform and randomlyorientated nanostructure without any bead. Diameters of each fiber sample are summarized in Table 1. Pure PHB displayed the largest fiber diameter of 951 ± 155 nm. The addition of ligninPHB copolymer in small amounts (< 5%) slightly reduced the fiber diameter, and higher amounts of the copolymer (> 10%) significantly decreased the fiber diameter down to 413 ± 68 nm for PHB10LP. The lignin-PHB copolymer with high surface charge density could enhance the repulsive force on Taylor cone during electrospinning, therefore generating thinner nanofibers.

Table 1 Material properties of PHB/lignin-PHB nanofibers Fibers

PHB : ligninPHB

Fiber diameter (nm)

Tensile strength (MPa)

Y modulus (MPa)

Elongation (%)


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PHB

100:0

951 ± 155

1.45 ± 0.36

54.7 ± 1.2

9.6 ± 2.2

PHB1LP

99:1

724 ± 193*

4.37 ± 0.80*

106.7 ± 16.1*

47.2 ± 16.7*

PHB2LP

98:2

900 ± 206

5.61 ± 0.63*

84.6 ± 10.0*

93.5 ± 7.6*

PHB5LP

95:5

903 ± 200

4.89 ± 0.49*

77.8 ± 5.4*

44.7 ± 7.0*

PHB10LP

90:10

413 ± 68*

2.76 ± 0.42*

58.8 ± 13.0

25.3 ± 6.6*

PHB20LP

80:20

653 ± 154*

3.64 ± 1.42*

86.4 ± 14.3*

16.5 ± 3.4

* Significantly different from corresponding parameters of PHB fibers (p < 0.05)

Mechanical behavior The mechanical behavior of PHB/lignin-PHB nanofibers was determined by tensile testing (Table 1 and Figure 3). Pure PHB nanofibers display weak mechanical properties (tensile strength of 1.45 ± 0.36 MPa; Young’s modulus of 54.7 ± 1.2 MPa and elongation-at-break of 9.6 ± 2.2 %) due to its brittle nature with intrinsic large spherulites. The weak strength and brittleness of PHB nanofibers greatly limit its applications. Interestingly, the composite nanofibers with lignin-PHB copolymer exhibits much stronger mechanical properties. PHB2LP with only 2% lignin filler demonstrates better mechanical performance: the tensile strength increased to 5.61 ± 0.63 MPa (3.9 times of PHB), Young’s modulus increased to 84.6 ± 10.0 MPa (1.5 times of PHB), and most importantly the elongation increased to 93.5 ± 7.6% (9.7 times of PHB). The addition of extra lignin-PHB causes weakening of mechanical properties. 10% lignin copolymer in PHB nanofibers reduced the tensile strength to 2.76 ± 0.42 MPa, Young’s modulus to 58.8 ± 13.0 MPa, and elongation to 25.3 ± 6.6%. Notably, these values are still higher than those of pure PHB nanofibers. It is noteworthy that the fiber diameter decreased with the addition of lignin-PHB. It has been reported that tensile strength and modulus increases with a decrease in fiber diameter, due to better molecular orientation and crystallinity among smaller fiber diameters

35.

However, compared to pure PHB fibers,

PHB2LP and PHB5LP showed similar fiber diameters, but their mechanical properties were 11 ACS Paragon Plus Environment


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significantly higher. This indicates that the reinforcement effect was due to lignin-PHB instead of the change of fiber diameter. Lignin has been blended into PHB to modify the thermal properties, degradation behavior and processing stability of the composites

8, 17, 36.

Blending two brittle materials to achieve

mechanical reinforcement is remarkably interesting. Previous reports of incorporating unmodified lignin in thermoplastic led to a deterioration in mechanical properties 37. Angelini et al. reported that 30% of lignin-rich residue (LRR) in PHB composites decreased the tensile strength from 29.6 MPa to 11.6 MP and elongation-at-break from 1.54 % to 0.55 %. The addition of LRR also reduced the flexural and impact properties of the PHB composites

36.

Camargo et al. found that adding lignin (from sugarcane bagasse) into poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) reduced the tensile strength, elongation-at-break and flexural strength of the composite (lignin/PHBV in 50/50) 38. Lignin molecule is relatively polar due to its numerous hydroxyl groups, presenting poor compatibility or immiscibility with non-polar or weak-polar polymer matrix. This results in poor stress transfer between the phases 39. Hence, our approach requires the use of modified lignin. Chemical modification can effectively modify lignin properties and improve its compatibility with other polymers. Lignin copolymers can demonstrate good miscibility in host polymer matrix (such as PP, PCL, PLA), leading to positive reinforcement results 14, 34, 40. In our study, PHB chains were grafted onto the surface of lignin core to improve its compatibility with the PHB matrix. These PHB chains can form tough interfacial binding with PHB matrix to reduce crack formation. Mangeon et al. reported that PHB-grafted carbon nanotube could act as reinforcing agent for improving the tensile strength (11% incremental) and elongation-at-break (89% incremental) of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofibers 41.


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Figure 3 Typical tensile stress–strain curves of electrospun PHB and PHB/lignin-PHB nanofibers.

Thermal behavior The thermal behavior of PHB and PHB/lignin-PHB nanofibers was analyzed by TGA and DSC. TGA profiles (supporting information Figure S5 and Table 2) reveal that pure PHB nanofibers exhibit the thermal decomposition temperature (Td) of 235 ºC, while the addition of ligninPHB do not remarkably influence the Td of the composite nanofibers (~240 ºC).



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Figure 4 DSC results of electrospun PHB/lignin-PHB nanofibers. A is the first heating run and B is the second heating run.

Table 2 Thermal properties of PHB/lignin-PHB nanofibers Fibers

Td a (°C)

1st heating run

2st heating run

Tm b (°C)

ΔHm b (J/g)

Xc c (%)

Tg b (°C)

Tcc b (°C)

ΔHcc b (J/g)

Tm b (°C)

ΔHm b (J/g)

Xc (%)

PHB

235

170

91.2

62.5

3.2

NA

NA

150

86.9

59.5

PHB1LP

249

172

91.0

63.0

9.3

NA

NA

152

84.5

58.4

PHB2LP

235

172

90.1

63.0

2.1

51

-19.68

156

85.8

46.2

PHB5LP

238

170

85.9

61.9

1.9

51

-12.87

152

81.2

49.3

PHB10L P

241

169

78.6

59.8

-2.3

49

-27.7

146

71.4

33.3

PHB20L P

230

165

73.3

62.8

-1.0

46

-38.1

147

69.7

27.1

a)T , d

thermal decomposition temperature (5% weight loss). b) determined by DSC. NA: not applicable

DSC analysis was used to investigate the thermal behavior of the nanofibers (Table 2 and Figure 4). All samples show similar Tm (~170 °C) and Xc (62%), but no obvious Tg and Tcc were observed in the 1st heating circle (Figure 4A). It is well known that electrospinning (fast quenching) is able to form high crystallinity by creating ordered alignment of polymer-chain. The process can also introduce a higher level of chain entanglement in polymer structure 42. This results in good mechanical properties of the nanofibers. 14 ACS Paragon Plus Environment

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In the 2nd heating cycle of DSC (Figure 4B), pure PHB exhibit the properties of a classic semicrystalline polymer with 3.2°C for Tg, 150 °C for Tm and 59.5% for Xc. PHB1LP shows similar melting behavior as PHB nanofibers (Tm = 152 °C; Xc = 58.4%), but with higher Tg of 9.3°C. No cold crystallization temperature is observed for both nanofibers, indicating that 1% of lignin-PHB do not influence the crystal formation of PHB. The increased Tg and good crystallization of PHB1LP may contribute to its improved mechanical properties. All the PHB/lignin-PHB nanofibers display single Tg, indicating that the lignin-PHB copolymer is miscible with PHB matrix without phase separation. In addition, DSC results show that the incorporation of more lignin-PHB (> 2%) decreases the Tg, generates cold crystallization peak and reduces the crystallization of the resulting nanofibers. In such case, the lignin-PHB has disordered the molecular packing, reduced crystallization of PHB matrix and increased its amorphous portion. Less ordered packing of PHB polymer chains may also contribute to the weaker mechanical properties of these nanofibers containing high amounts of lignin copolymers. Similar results are reported by other researchers. Doherty et al. found that Tm, ΔHm and Xc of PHB/lignin composites decreases with increase in lignin content (soda lignin extracted from bagasse), and Xc decreases to 21% with 60% of lignin in the composite

18.

McDonald et al. prepared the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/lignin composites via in situ reactive extrusion, and they found that the Xc of the composites decreases from 64.3% for pristine PHBV to 52.4% for the sample grafted with dicumyl peroxide 39. Later, the same group reported that the addition of esterified lignin (30%) into PHBV slightly decreases the degree of crystallinity 37.

Antioxidant properties of PHB/lignin-PHB nanofibers



Lignin is well-known for its antioxidant activity

43-47.

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Lignin possesses decent antioxidant

activity due to its high content of phenolic moieties. However, the ROP of β-butyrolactone would likely react with some aliphatic and phenolic hydroxyl groups on lignin, resulting in decreased antioxidant activity as compared to unmodified lignin. However, we found that lignin-PHB possesses good antioxidant properties. As shown in Figure 5, the antioxidant activity of pure PHB nanofiber is quite low. PHB only shows 18.2 ± 9.1% of free radical inhibition after 48 hours of incubation, remarkably lower than all lignin-PHB fibers. A higher amount of lignin-PHB copolymer in nanofibers results in higher inhibition capability. PHB20LP nanofiber with the highest lignin content (20 %) displays the highest antioxidant activity, whereby its DPPH scavenging percentage attained 98.8 ± 0.9 % in 4 hours. Our results indicate that the phenolic groups have partially reacted with β-butyrolactone, and therefore even after grafting of PHB, the copolymers still display the inherent antioxidant properties from lignin. These antioxidant nanofibers have healthcare applications such as biomaterials for reducing tissue damage caused by oxidative stress.


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Figure 5 Antioxidant activity of PHB/lignin-PHB nanofibers (DPPH assay).

Biocompatibility of nanofibers in vitro Spindle-like morphology of the fibroblasts can be observed across all the PHB and PHB-lignin nanofibers (Figure 6). This elongated shape is characteristic of NIH/3T3 fibroblasts. Cell nuclei are stained blue while the actin filaments are stained green. The tight mesh of cells after 8 days of culture indicates very high confluency and good biocompatibility. Hence, these nanofibers have potential biomedical applications such as in tissue engineering.



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A

B

C

D

E

F

Figure 6 NIH-3T3 fibroblasts stained with DAPI and phalloidin after 8 days of culture on various nanofibers. (A) PHB (B) PHB1LP (C) PHB2LP (D) PHB5LP (E) PHB10LP and (F) PHB20LP nanofibers (Scale bar = 75 μm) In vivo test To further assess the biocompatibility of lignin-PHB nanofibers, samples were subcutaneously implanted into the rat models. The PHB2LP nanofibers were selected for the vivo study, due to its outstanding mechanical properties and good antioxidant activity. PHB nanofibers were used as control. Histological evaluation was performed up to 35 days after the surgery. H&E staining results are shown in Figure 7A and B. The combined scores for polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, neo-vascularisation, fibrosis and fatty infiltrate were calculated (supporting information Figure S6). The overall conclusion for all 5 time points is that the PHB2LP nanofibers are non-irritant. This conclusion is based on the 0.4 mean difference between the PHB2LP score (27.6 mean over all time points) and the PHB score (27.2 mean over all time points). The mean score per group is shown per time point in Figure 7A. In this analysis, the general conclusion holds true for each time point. (On day 35th the difference observed is 1.75 while on day 7 it is -0.75). 18 ACS Paragon Plus Environment

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Figure 7 (A) Integrated histopathology score per time point from H&E staining. (B) H&E staining images of the implanted nanofibers at different time points. (C) Combined MT staining score per time point. The collagen-rich area (blue) over total scanned area is represented as percent. (D) MT staining images of the implanted nanofibers at different time points. *** presents implanted nanofibers. Scale bar: 200 µm. MT staining results are shown in Figure 7C and D. In this stain, a three-dye protocol selectively stains muscle, erythrocytes and cytoplasm as red, collagen as blue, and cell nuclei as brown. The area of blue colour (collagen) was calculated as a percentage of total area. The data compiled for all 20 animals over 5 time points show a gradual increase in collagen formation in PHB2LP as compared to the PHB group (Figure 7C). From day 7 to 35, an increase in collagen-positive area from 79.34% to 82.95% can be seen in the PHB2LP group while for PHB group, the collagen-positive area increases 70.66% to 79.25%. This may be attributed to ongoing fibrosis in the implant area of PHB2LP post-implantation. The overall difference between PHB2LP and PHB group indicates that lignin-PHB is also non-irritant.

Conclusion In this study, we report a lignin-based functional filler to improve the mechanical performance of brittle PHB. The lignin-PHB copolymer was synthesized via solvent-free ROP. Only 2% of



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such copolymer is required to increase tensile strength by ~4 times, stiffness by 1.5 times and elongation by ~10 times as compared to pure PHB. The lignin/PHB nanofibers display tuneable antioxidant activity and good biocompatibility both in vitro and in vivo, indicating that ligninPHB filler can broaden the applications of PHB in the biomedical fields. For example, such lignin-based nanofibers can be used as antioxidative biomaterials to protect human body from oxidative stress, such as promoting cartilage tissue regeneration and neutralizing free radicals generated by osteoarthritis. These lignin polymers can also be applied in personal care or cosmetic products to protect our skin from UV radiation and environmental pollutants. Overall, our study has widened the approaches for lignin valorization.

Acknowledgements The authors gratefully acknowledge the financial support from the Institute of Materials Research and Engineering (IMRE) under the Agency of Science, Technology and Research (A*STAR).

Supporting Information Experimental details, Structure of raw lignin; FTIR, DSC and TGA curves of lignin-PHB copolymer; TGA and density of PHB/lignin-PHB nanofibers; Individual histopathology scores per time point for animal study.

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A New Dual Functional PHB-grafted Lignin Copolymer: Synthesis

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