Engineering Poly(lactide)âLignin Nanofibers with Antioxidant Activity

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Engineering poly(lactide)-lignin nanofibers with antioxidant activity for biomedical application Dan Kai, Wei Ren, Lingling Tian, Pei Lin Chee, Ye Liu, Seeram Ramakrishna, and Xian Jun Loh ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00478 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Engineering poly(lactide)-lignin nanofibers with antioxidant activity for biomedical application Dan Kai1*†, Wei Ren1†, Lingling Tian2, Pei Lin Chee1, Ye Liu1, Seeram Ramakrishna2,3, Xian Jun Loh1,4,5*

1

Institute of Materials Research and Engineering (IMRE), A*STAR, 2 Fusionopolis Way.

Innovis, #08-03, 138634 Singapore 2

Centre for Nanofibers and Nanotechnology, Mechanical Engineering, National University of

Singapore, 2 Engineering Drive 3, 119260 Singapore 3

Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University,

Guangzhou 510632, China 4

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

Engineering Drive 1, Singapore 117576, Singapore 5

Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore

†

These authors contributed equally to this work.

E-mail: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

Abstract Biodegradable poly(lactic acid) (PLA)/lignin composites are considered to be promising renewable plastic materials towards a sustainable world. The addition of lignin to PLA may assist to combat the oxidative stress induced by PLA as biomaterials. In this study, PLAlignin copolymers with various contents of alkylated lignin (10 to 50 %) were synthesized by ring-opening polymerization. The molecular weight of such copolymers ranged from 28 to 75 kDa, while the PLA chain length varied from 5 to 38. These PLA-lignin copolymers were further blended with poly(L-lactide) (PLLA) and fabricated into nanofibrous composites by electrospinning. The PLLA/PLA-lignin nanofibers displayed uniform and bead-free nanostructures with fiber diameter of 350~500 nm, indicating the miscibility of PLLA and lignin copolymers in nanoscale. Unlike bulk materials, incorporation of PLA-lignin copolymers did not enhance the mechanical properties of the nanofibrous composites. Antioxidant assay showed that the lignin copolymers and PLLA/PLA-lignin nanofibers rendered excellent radical scavenging capacity for over 72 hours. Moreover, three different types of cells (PC12, human dermal fibroblasts and human mesenchymal stem cells) were cultured on the electrospun nanofibers to evaluate their biocompatibility. Lignin-containing nanofibers exhibited higher cell proliferation compared to neat PLLA nanofibers. PLLA/PLA-Lig20 nanofibers displayed the best biocompatibility as it achieved a balance between the antioxidant activities and the cytotoxicity. With excellent antioxidant activities and good biocompatibility, the PLLA/PLA-lignin electrospun nanofibers hold great potential to be used as biomedical materials for protecting cells from oxidative stress conditions.

Keywords: Lignin copolymer, Electrospinning, Oxidative stress, Sustainability, Cytotoxicity


Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction The overconsumption of fossil fuel resources and the resulting environmental issues have motivated research to focus on natural polymers and composites which are both renewable and biodegradable [1]. Lignin, as the second most abundant biopolymer on earth, has been considered as a promising alternative for existing fossil fuel resources. Although lignin is currently a waste product from papermaking and biorefinery industries, it exhibits some advanced properties, such as good stiffness, antimicrobial activity, antioxidant properties and ultraviolet (UV) radiation protection. As such, extensive studies have been carried out to investigate and explore the approaches of converting lignin into value-added products. Inspired by its native function as mechanical supporting material in plants, lignin has been used as a structural filler to reinforce the mechanical properties of polymeric composites [2]. It has also been blended into many different thermoplastics, including polypropylene (PP) [3, 4], polyethylene (PE) [5, 6] and polystyrene (PS) [7]. However, the poor miscibility of unmodified lignin always resulted in a negative influence on the mechanical properties of the composite materials. To counter the problem, graft polymerization was used to synthesize lignin-based copolymer and it was proven to be an effective approach to modify the surface of lignin and improve its compatibility with other polymers [8]. Poly(lactic acid) (PLA) is a renewable natural polymer derived from plant sources, such as potato and corn [9]. Owing to its abundant availability and established manufacturing procedures, PLA is believed to be one of the most promising environmentally friendly bioplastics towards sustainable world [10]. This green polyester has also obtained the approval from Food and Drug Administration (FDA) to be engineered into biomaterials (sutures or tissue engineering scaffolds) for various biomedical applications due to its good biodegradability and biocompatibility. Blending lignin into PLA seems to be a rational



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

strategy to achieve sustainable composites. Not only did it provide a mean to modify the physical properties of PLA, it also minimised the overall cost [11, 12]. To retain an acceptable level of material properties, only low content of lignin (< 20%) can be added into the system. As such, PLA/lignin blends often exhibit undesirable mechanical properties due to the aggregation of unmodified lignin in PLA matrix. To improve the dispersion of lignin in PLA, PLA-lignin copolymers have been synthesized. Studies showed that the incorporation of such copolymers enhanced the mechanical properties of the PLA/lignin films [13, 14]. Currently, all studies on the PLA-lignin composites are in bulk form. Compared to bulk materials, porous composites with higher surface area would be worthy to explore for developing new applications of lignin materials, including catalysts, adsorbents, energy storage, sensors and even biomedical materials. Moreover, incorporating lignin into PLA endowed the composites with extra benefits such as antioxidant property, which is important for packaging materials and biomaterials [15]. One of the major issues of using PLA as biomaterials is the induction of oxidative stress or reactive oxygen species (ROS), which can cause an inflammatory response and toxicity for the implantations [16]. PLA/lignin system with good antioxidant activity might be a promising solution for the issue. In this study, we synthesized a series of PLA-lignin copolymers (with different lignin%) via the ring-open polymerization of lactide onto selectively alkylated lignin. Subsequently, such copolymers were blended with poly(L-lactide) (PLLA) and then electrospun into composite non-woven fabrics. Electrospinning is a simple and versatile technique to fabricate uniform micro/nanofibers with controlled fiber diameters. The porous electrospun matrixes with nanoscale architecture have gained great interests in various applications, especially as tissue engineering (TE) scaffolds. Unlike film casting, PLLA and PLA-lignin copolymers were blended and engineered in nanoscale by electrospinning, and it would be interesting to investigate the performance of the resulting porous nanocomposites. Here we evaluated the 4 ACS Paragon Plus Environment

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


influence of the PLA-lignin copolymers on the topographical, mechanical properties and antioxidant activity of the resulting nanofibers. Furthermore, three different cell types - PC12, human dermal fibroblasts (HDFs) and human mesenchymal stem cells (MSCs) were cultured on the electrospun nanofibers to investigate the biocompatibility of PLA-lignin composites and to explore the potential of the lignin-based nanofibers for TE applications.

Materials and methods Materials All chemicals were purchased from Sigma-Aldrich Chemicals and used as received except where noted. Alkali lignin was purchased from TCI (Mn = 3,000 g/mol) America and dried at 105 °C overnight before use.

Scheme 1 Route to synthesize PLA-lignin copolymers

Synthesis of PLA-lignin copolymers PLA-lignin copolymers were synthesized in two steps as shown in Scheme 1 [17]. In the first step, alkylated lignin was obtained as follows: A solution of n-C12H25Br (40.0 g) in isopropyl alcohol (40 mL) was added to a mixture of alkali lignin (40.0 g) and K2CO3 (24.0 g) in H2O



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

(160 mL) in a 500 ml two-neck round bottomed flask. After refluxing with stirring for 60 hours at 130°C, the mixture was cooled down to room temperature with stirring, and then the precipitate was filtered and dried. The obtained alkylated lignin was washed with hexane to remove the unreacted alkyl bromide and then dried in vacuum oven at 50 °C for overnight. The lignin was modified with selective alkylation at the phenolic OH (determined by NMR, Figure S1) PLA was grafted onto the alkylated lignin via ring opening polymerization. In particular, a mixture of alkylated lignin (1.0 g), lactide and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 50 mg) in anhydrous dichloromethane (25 mL) was stirred at room temperature for 24 hours under N2 atmosphere. Then the reaction was quenched with acetic acid and followed by precipitation in MeOH (600 ml). The precipitate was filtered and dried in vacuum oven at 50 °C for 24 hours to yield PLA-lignin copolymers as an earth yellow solid. A series of PLAlignin copolymers were synthesized under similar conditions by varying the feeding ratio of lactide : lignin (Table 1).

Characterization of PLA-lignin copolymers PLA-lignin copolymers were characterized by 1H NMR (Bruker 400 MHz). Deuterated chloroform (CDCl3) was used as a solvent to dissolve synthesized materials. Molecular weight and polydispersity index of polymer samples were analysed by Waters gel permeation chromatography (GPC, a Shimadzu SCL-10A and LC-8A system equipped with two Phenogel 5 mm 50 and 1000 Å columns in series and a Shimadzu RID-10A refractive index detector) by using HPLC tetrahydrofuran as an eluent. The flow rate of tetrahydrofuran eluent was 1.0 mL/min at 25 °C. The number average molecular weights (Mn), weight average


Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecular weights (Mw) and polydispersity index (PDI, Mw/Mn) were determined with a calibration based on linear poly(methyl methacrylate) standards.

Electrospinning of PLLA/PLA-lignin nanofibers A mixture of PLLA and PLA-lignin copolymers was dissolved in 1,1,1,3,3,3-hexafluoro-2propanol. The mass ratio of PLLA and PLA-lignin copolymers was 2:1 and the total concentration of the solution was 6 % (w/v). After stirring for a day, the homogeneous solutions were loaded into 3 ml syringes with a blunt 25-gauge needle. Each solution was pumped out at 1 ml/hour with 12 kV of voltage applied on the needle. Fibers were electrospun onto an aluminium foil wrapped collector (15 cm away from the needle tip). After spinning, the obtained nanofibers were dried overnight in vacuum oven and used for characterization and cell culture assays. Neat PLLA nanofibers were fabricated under the same parameters as control.

Characterization of electrospun nanofibers The surface topographies of the electrospun nanofibers were characterized by using scanning electron microscopy (SEM, 6360LA, JEOL, Japan). Samples were sputter-coated with a thin layer of gold before imaging. Fiber diameter was determined from 50 random measurements per image by using Image J (National Institutes of Health, Bethesda, USA). The surface hydrophilic properties of the electrospun nanofibers were evaluated by sessile drop water contact angle measurement using a VCA Optima Surface Analysis system (AST products, Billerica, USA). Droplets of 0.5 µl deionized water were sprinkled onto the fibers and data was recorded after 12 seconds. Five samples were used for each composition. 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 30

The mechanical properties of the PLLA/PLA-lignin nanofibers were measured by using uniaxial tensile testing technique (Instron 5943, USA) with 10 N load capacity at a rate of 5 mm/min. Nanofibers were cut into a rectangular shape at 5 × 30 mm for testing, and the thicknesses of the samples were about 100 µm and the gauge length was 20 mm. At least 5 samples were prepared for each composition. Tensile strength, Young’s modulus and elongation at break were calculated based on the stress-strain curve of each sample. Thermal behaviors of the nanofibers were also investigated. Thermogravimetric analysis (TGA) was carried out on a thermogravimetric analyser (Q500, TA Instruments, USA). Samples were heated at 20 °C/min from room temperature to 800 °C in a dynamic nitrogen atmosphere (flow rate = 60 ml/min). Differential Scanning Calorimeter (DSC) thermal analysis was performed on a DSC (Q100, TA Instruments, USA) equipped with an autocool accessory and calibrated using indium. The following protocol was used for each sample: heating from -20 °C to +220 °C at 20 °C/min, holding at +220 °C from 5 min, cooling from +220 °C to -20 °C at 20 °C/min, and finally reheating from -20 to +220 °C at 20 °C/min. Melting temperatures were taken as peak maxima. Enthalpy change (∆Hm) of PLLA was determined from the endothermic melting peak. The crystallinity (Xc) was calculated using the equation Eq.1

Xc = ∆Hm/ ∆Hm0 × 100%

where ∆Hm is normalized based on the % mass of PLA segments in the fibers. ∆Hm0 is 139.5 J/g for 100% crystalline PLA.

The antioxidant activity of the PLLA/PLA-lignin nanofibers was evaluated by 1,1-diphenyl2-picrylhydrazyl (DPPH) assay [16, 18]. Nanofibers (on 15 mm cover slips) were placed in


Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24-well plate. A 60 µM DPPH solution in MeOH was prepared, and 2 ml of such solution was added into each well. DPPH free radical content was measured by monitoring the absorbance changes at 517 nm at each time point. All samples were prepared in triplicate. The antiradical activity was measured as %inhibition of free radicals by measuring the decrease in absorbance compared to control solutions.

In vitro cell study In this study, three different types of cells were cultured to evaluate the biocompatibility of the PLLA/PLA-lignin nanofibers. The nanofibers were collected on 15-mm round glass cover slips and sterilized under UV light for 2 hours. After sterilization, samples were placed in 24well plates and washed by 1× phosphate buffered saline (PBS) for 3 times. Then cells were seeded onto the nanofibers at a density of 5,000 cells/well. PC12 cells were cultured in DMEM (Sigma) supplemented with 10% horse serum, 5% fetal bovine serum and 1% antibiotic/antimycotic solution. HDFs and MSCs were cultured in DMEM (Gibco) + 10% fetal bovine serum + 1% antibiotic/antimycotic solution. Cells were incubated in a humidified incubator at 37 °C with 5% CO2 and the medium was changed every 2 days. Tissue culture plate (TCP) was used as positive control. To assess cell proliferation rate, alamarBlue assay was performed after 3, 6 and 9 days of culture. At each time point, each cell type/nanofiber substrate was incubated for 4 hours at 37 °C with fresh culture medium supplemented with 10 vol% alamarBlue. After incubation, 100 µL medium from each well was transferred to a 96-well black polystyrene microplates. The experiment was performed in triplicate. The absorbance was then measured using a microplate reader (Infinite M200, Tecan) at wavelength of 570 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

Statistical analysis All the data presented are expressed as mean ± standard deviation of the mean. Student’s ttest and one-way ANOVA were used, and differences between the groups are considered statistically significant at p < 0.05.

Ha

Me in PLA chain

Hb Me

OH O O

Ha

O O

Me

MeO

O

n

OMe

C12H 25

OMe OC 12H 25

Hb

Methoxyl groups

Aromatic ring

8.0 ppm (t1)

7.0

6.0

5.0

4.0

3.0

2.0

1.0

Figure 1 1H NMR (CDCl3) of PLA-lignin copolymer.

Results and discussion Synthesis and characterization of PLA-lignin copolymers


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Alkylated lignin was characterized by NMR (Supportive information Figure S1). Lignin showed its characteristic chemical shifts at 3.80 ppm corresponding to methide protons attached to oxygen atom and 6.80 ppm presenting the protons of the phenyl ring in lignin. The multiple peaks between 0.80-1.70 ppm were associated with the proton peaks of the alkyl chain of the alkylated lignin. The reason behind the specific alkylation of lignin was to improve its dissolvability in organic solvents and to enhance the reactivity of its left alkyl OH. It was also reported that alkylated lignin exhibited better miscibility with other polymers, such as polyolefins and polyesters [19-21]. After the alkylation, PLA was grafted onto alkylated lignin by ring-opening polymerization. Figure 1 shows 1H NMR spectra of the PLA-lignin copolymer. Characteristic peaks of PLA were observed at 4.30 and 5.20 ppm corresponding to the protons attached to the repeating carbon and terminal carbon, while the signals associated with the methoxyl groups of lignin (~3.8 ppm) was also found in the spectra. FTIR spectra of lignin, alkylated lignin and PLA-lignin copolymers are shown in supportive information Figure S2. Compared to lignin, the copolymers displays PLA characteristic stretching frequencies for –CH3 asymmetric, –CH3 symmetric, C=O, and C– O at 2995, 2946, 1746, and 1080 cm-1, respectively. These results confirmed that the PLA chains were successfully grafted onto the lignin core. The molecular weight of PLA-lignin copolymers were analysed by GPC. By varying the feed ratio of lactide to lignin in the graft polymerization, the Mn of the copolymers were tunable ranging from 28.5 kDa for PLA-Lig50 to 75.6 kDa for PLA-Lig10 and the repeat units of the grafted PLA chains increased from 5 for PLA-Lig50 to 38 for PLA-Lig10 (Table 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

Table 1 Molecular characterization of PLA-lignin copolymers Mn a

Mw a

[lactide] : [lignin]

(kDa)

(kDa)

PLA-Lig10

90:10

61.5

87.2

1.32

38

95:5

PLA-Lig20

80:20

49.0

57.2

1.35

26

94:6

PLA-Lig30

70:30

38.7

47.7

1.41

15

92:8

PLA-Lig40

60:40

28.5

35.9

1.23

10

89:11

PLA-Lig50

50:50

15.4

21.2

1.37

5

80:20

Feed ratio Copolymers

PDI a

Average length of PLA chain b

Mass ratio PLA : Lignin c

a)Determined by GPC. b) Determined by NMR. c) Determined by GPC based on the molecular weight of lignin (3000 g/mol)

Figure 2 SEM images of electrospun (A) PLLA, (B)PLLA/PLA-Lig10, (C) PLLA/PLA-Lig20, (D) PLLA/PLALig30, (E) PLLA/PLA-Lig40 and (F) PLLA/PLA-Lig50 nanofibers. The scale bar = 5 µm.

Electrospinning of PLLA/PLA-lignin nanofibers Morphology of PLLA/PLA-lignin nanofibers is shown in Figure 2 and fiber diameters are summarized in Table 2. All the fibers exhibited uniform and bead-free nanostructures, suggesting that PLLA and PLA-lignin copolymers were miscible in nanoscale. Neat PLLA 12 ACS Paragon Plus Environment

Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanofibers without any lignin copolymer showed the largest fiber diameter of 712 ± 63 nm. The incorporation of PLA-lignin copolymers into PLLA significantly reduced the fiber diameter. Most of the blending nanofibers displayed diameters in the range of 400 nm to 600 nm, while PLLA/PLA-Lig50 nanofibers showed the smallest fiber diameter of 350 ± 80 nm. It was also observed that copolymers with higher lignin% resulted in thinner nanofibers. This phenomenon may be attributed to the low molecular weight of the high-lignin% copolymer, which reduced the viscosity of the spinning solution [22, 23]. Similarly, it was reported that blending lignin-poly(methyl methacrylate) copolymers into PCL fibers significantly reduced the fiber diameters from 1058 ± 261 nm to 370 ± 100 nm [24]. It is well known that the morphology of nanofibers is highly dependent on electrospinning parameters. Choice of solvents affects the solution properties, which in turn influences the fiber properties, such as diameter and surface morphology. Various solvents, including chloroform, dichloromethane, 2,2,2-trifluoroethanol, ethanol, THF, dimethylformamide and acetone, have been used for electrospinning PLLA [25-27]. It was reported that changes in the solvent vapour pressure would affect the fiber diameter distribution and porosity. A solvent with high vapor pressure was found to result in a broad diameter distribution with a porous surface, while a solvent with lower vapor pressure was associated with smaller fiber diameter and higher crystallinity [25, 26]. The distance between the needle tip and the grounded collector is another important parameter that influences the fiber diameter and morphology [28, 29]. A suitable distance is essential for complete solvent evaporation prior to the fiber formation on the collector. Residual solvents caused by insufficient distance could result in non-ideal fibers that are wet or thicker than intended. In addition, the decrease in distance could also cause the morphology of the fibers to appear to be round instead of flat [30]. A short distance is only recommended when a highly volatile solvent is used in the spinning solution [31]. 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

Water contact angles of the PLLA/PLA-lignin nanofibers were investigated to evaluate the hydrophobicity of the nano-structural surface. As shown in Table 2 and Supportive information Figure S3, neat PLLA nanofibers exhibited the water contact angle of 144 ± 1 °, and the addition of PLA-lignin copolymers did not significantly influence the hydrophobicity of the nanofibers. In order to improve the solubility of such copolymers in organic solvents and to retain the hydrophobic characteristics of the fibers, the hydroxyls in lignin, which contribute to its hydrophilicity, were substituted with the alkyl and PLA chains.

Table 2 Material characterization of PLLA/PLA-lignin nanofibers Fibers

Fiber diameter (nm)

Tensile strength (MPa)

Y modulus (MPa)

Elongation at break (%)

Water contact angle (°)

PLLA

712 ± 63

3.49 ± 0.20

56.8 ± 1.6

49 ± 5

144 ± 1

PLLA/PLALig10

485 ± 38*

3.25 ± 0.17

55.2 ± 4.0

21 ± 5*

146 ± 1

PLLA/PLALig20

525 ± 38*

2.49 ± 0.14*

56.1 ± 8.0

19 ± 1*

147 ± 2

PLLA/PLALig30

480 ± 49*

2.55 ± 0.14*

54.3 ± 4.3

20 ± 1*

145 ± 3

PLLA/PLALig40

417 ± 52*

2.58 ± 0.21*

66.0 ± 1.4*

16 ± 4*

143 ± 2

PLLA/PLALig50

350 ± 80*

2.56 ± 0.07*

66.8 ± 2.8*

11 ± 2*

143 ± 2

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

Mechanical properties of PLLA/PLA-lignin nanofibers Tensile tests were carried out to evaluate the mechanical properties of the PLLA/PLA-lignin nanofibers, and results including tensile strength, Young’s modulus and elongation at break were summarized in Table 2. Typical stress–strain curves of the composite nanofibers were shown in Supportive information Figure S4. Neat PLLA nanofibers presented the highest 14 ACS Paragon Plus Environment

Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mechanical properties (tensile strength of 3.49 ± 0.20 MPa; Young’s modulus of 56.8 ± 1.6 MPa and elongation at break of 49 ± 5 %), while the composite nanofibers with PLA-lignin copolymer exhibited lower tensile strength and elongation. Among all the composite nanofibers, PLLA/PLA-Lig10 showed similar tensile strength (3.25 ± 0.17 MPa) and Young’s modulus (55.2 ± 4.0 MPa) to PLLA nanofibers, and PLLA/PLA-Lig40 and PLLA/PLA-Lig50 fibers displayed significantly higher Young’s modulus (66.0 ± 1.4 and 66.8 ± 2.8 MPa, respectively) than that of PLLA fibers. It was reported that blending lignin into PLLA reduced the mechanical properties of the composites, especially when the lignin content was beyond 20% [12]. Lignin modification is believed to be an effective approach to improve the compatibility between lignin and PLLA [2, 8, 32]. The composites of acetylated lignin and PLA (10% and 20% of lignin in PLA by extrusion) exhibited more than 2-time higher tensile strength and elongation at break than those of PLA/raw lignin composites [33]. Chung et al. synthesized PLA-lignin copolymers with a solvent free method, and they found that the incorporation of the copolymer with low lignin% (lactide : lignin = 9 : 1) into PLA matrix led to a small improvement in the tensile strength and elongation, but the copolymer with higher lignin% (lactide : lignin = 6 : 4) decreased the mechanical properties compared to neat PLA film [13]. To toughen PLA/lignin composite, a rubbery layer of poly(ε-caprolactone-co-lactide) was grafted onto the surface of lignin core followed by an outer segment of poly(D-lactide) (PDLA) [14]. The rubbery layer played an important role in mediating the interface and reducing the crack formation during deformation, while the outer PDLA segments were able to form strong interfacial interactions with PLLA matrix by stereocomplexation. Thereby, the unique copolymer fillers could enhance the mechanical properties of the PLLA/lignin composites, and the elongation at break and toughness were observed more than 5 times higher than neat PLLA. It is worthy to note that mechanical reinforcements were achieved by PLA-lignin copolymers as fillers, but 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

the content of such fillers was still low (< 15%). In our study, the content of PLA-lignin copolymers in nanofibers was 33.3%, which is much higher than those reported in the aforementioned studies. Our study further demonstrated that PLA-grafted lignin fillers might not improve the mechanical properties of a nanofibrous matrix even though they worked well in bulk materials or films. In bulk, a matrix mainly supports the strength of the whole material, while micro/nano-size fillers mildly influence the mechanical properties by modulating the crystallization or molecular orientation of the matrix. On the other hand, in the nanofibers, the strength of each fiber highly depends on the bonding between fillers and PLLA matrix or even between two filler particles, as the size of the copolymer fillers (several hundred nm) is comparable with fiber diameter [14].

Table 3 Thermal properties of PLLA/PLA-lignin nanofibers 1st heating run Fibers

Td (°C)

a

Residue a (%)

Tg b (°C)

Tm b (°C)

∆Hm b

(J/g) PLLA

2st heating run Xc c (%)

Tg b (°C)

Tcc b (°C)

Tm b (°C)

∆Hm b

(J/g)

Xc c (%)

311

1.9

68

185

60.8

65.3

63

128

175

37.1

39.8

PLLA/PLALig10

310

4.5

60

182

38.4

61.9

60

139

175

3.3

5.3

PLLA/PLALig20

302

6.6

59

181

38.2

61.5

60

143

176

3.7

6.0

PLLA/PLALig30

292

6.1

55

179

38.8

62.5

54

143

175

7.8

12.6

PLLA/PLALig40

290

9.8

59

181

39.5

63.6

61

133

176

6.1

9.8

PLLA/PLALig50

280

11.5

60

181

40.8

65.7

63

138

177

16.9

27.2

a) Td, thermal decomposition temperature, is defined as the temperature at which the mass of the sample is 5% less than its mass measured at 50 ºC. Residue is the mass% at 500 °C (determined by TGA) b) Tg (glass transition temperature), Tcc (cold crystallization temperature), Tm (melting temperature) and ∆Hm (enthalpy change) determined by DSC. c) Crystallinity (Xc) determined by the equation: X% = ∆Hm/ ∆Hm0 × 100% where ∆Hm0 is 93.1 J/g for 100% crystalline PLA.

Thermal properties of PLLA/PLA-lignin nanofibers


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The thermal properties of the PLLA and PLLA/PLA-lignin nanofibers were studied by using TGA and DSC. As shown in Table 3, pure PLLA nanofibers exhibited the thermal decomposition temperature (Td, 5% of weight loss) of 311 ºC with carbon residue of 1.9% at 500 ºC. Adding lignin copolymer into PLLA slightly decreased the Td of the resulting nanofibers, but increased the carbon residue. This could be attributed to the relative lower Td (270 ºC) and higher carbon residue (48.8% at 500 ºC) of the alkylated lignin. Among all the nanofibers, PLLA/PLA-Lig50 displayed the lowest Td of 280 ºC and the highest carbon residue of 11.5%. The other thermal parameters, such as glass transition temperature (Tg), cold crystallization temperature (Tcc), melting temperature (Tm), melt enthalpy (∆Hm) and crystallinity (Xc) were determined by DSC (Table 3). PLLA is a semi-crystalline polymer with a Tg of ~60 °C and Tm of ~180 °C, while PLA-lignin copolymers were amorphous polymers with Tg ranging from 49 ºC to 55 ºC (Supportive information Figure S5). In the first heating run of DSC, all the PLLA/PLA-lignin nanofibers showed similar Tcc, Tm and Xc, but slightly lower Tg compared to pure PLLA nanofibers. It was reported that electrospinning is able to enhance the crystallinity of the polymers by forming ordered inter-chain alignment and higher degree of chain entanglement in polymer structure [34]. In the second heating run of DSC, nanofibers exhibited similar Tg to the first heating run, in which blending the lignin copolymers into PLLA decreased its Tg. The single Tg confirmed the good miscibility of lignin-PLA and PLLA. As shown in Supportive information Figure S6, the incorporation of lignin copolymers affected the cold crystallinity and melt enthalpy of the PLLA. The cold crystallinity of the PLLA/PLA-lignin nanofibers almost disappeared after the addition of lignin copolymer. Although the nanofibers showed a similar Tm, the copolymers remarkably reduced the Xc of the nanofibers. It indicated that the addition of such lignin copolymers



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

disrupted the molecular packing and crystallization of PLLA matrix by increasing its amorphous region. In this study, we used racemic lactide for the preparation of PLA-lignin copolymers, which could possibly be the reason for the lower crystallization of the composites. The more amorphous PLLA/PLA-lignin nanofibers attained in this study might be suitable for biomaterial applications. Prior research has reported that the polymer crystallinity exerted a great influence over the cell growth, and an amorphous surface is more favourable for cell attachment and proliferation compared to a crystalline surface [35, 36]. In the future work, we will synthesize PLLA-lignin and PDLA-lignin copolymers to investigate their performances in PLLA matrices. The use of such homopolymers as additive might yield greater compatibilization and crystallization within PLLA.

Antioxidant activity of PLLA/PLA-lignin nanofibers Recently, lignin has been receiving increasing attention due to its antioxidant activity [4, 3740]. The antioxidant activities of PLA-lignin copolymers and lignin-based nanofibers were evaluated by DPPH assay. Here, we demonstrated that the copolymer with a higher content of lignin had a greater inhibition value, proposing that grafting lignin copolymer to PLA does not diminish/compromise the inherent antioxidant activities of lignin. (Supportive information Figure S7). These lignin copolymers were also introduced into PLLA and the composite nanofibers were found to exhibit good antioxidant activities. As shown in Figure 3, neat PLLA nanofibers displayed low antioxidant activities. Even after 72 hours, PLLA nanofibers only achieved 15.5 ± 6.2% free radical inhibition, significantly lower than all other lignin-containing nanofibers. Among all PLLA/PLA-lignin nanofibers, PLLA/PLALig50 exhibited the highest DPPH scavenging percentage at every time point, due to its 18 ACS Paragon Plus Environment

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


highest lignin content in the nanofibers. The inhibition% of such nanofibers reached 49.1 ± 3.7% at 72 hours. Lignin is a complex aromatic compound with plenty of hydroxyl and methoxy functional groups, and these functional groups would assist to donate hydrogen to terminate oxidation propagation reaction [41-43]. Lignin as natural radical scavenger could also be used for biomaterial applications. Implantation of biomaterials always induces inflammatory responses and oxidative stresses. Consequently, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are activated and ROS are generated in the body. Such pro-oxidant molecules will result in DNA damage and necrosis [16]. Hence, using such lignin-based nanofibers as biomaterials may locally reduce oxidative stress-related tissue damages or functional disorders.

Figure 3 Free radical inhibition (antioxidant activity) of PLLA/PLA-lignin nanofibers by DPPH assay. N ≥ 3, * Significantly different from inhibition% of PLLA fibers (p < 0.05).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

Biocompatibility of PLLA/PLA-lignin nanofibers It is critical to ensure the safety of lignin and its copolymers before applying them in any cosmetic products or biomedical materials. The biocompatibility of lignin is still debatable, as only few biological data reported on its possible cytotoxic effects. In our previous studies, we demonstrated that lignin copolymers, lignin-poly(ethylene glycol) methyl ether methacrylate (PEGMA) and lignin-poly(methyl methacrylate) (PMMA) had good biocompatibility with HDFs [24, 44]. Here, the biocompatibility of the PLLA/PLA-lignin nanofibers was evaluated by using rat PC12 cells, HDFs and human MSCs. As shown in Figure 4, the proliferation activities of all three cell lines on all the nanofibers were found to increase with culture time, proposing that such PLA-lignin copolymers had no cytotoxic effects on these cells. All three cell types exhibited low metabolic activities on neat PLLA nanofibers, probably due to the oxidative stress induced by the polyester itself. On the other hand, all lignin-containing nanofibers displayed higher cell proliferation values compared to PLLA nanofibers, indicating that the antioxidant activity of the biomass polymer may enhance the viability of the cells. PC12 cells are neural stem cells that are widely used as the model for neurotoxicological studies and nerve regeneration. As shown in Figure 4A, both PLLA/PLALig20 and PLLA/PLA-Lig50 exhibited higher cell proliferation of PC12 cells compared to other nanofibers. HDFs exist within the dermis layer of human skin. They play a critical role in generating connective tissue and wound healing. Such cells are also largely used as the model for studying skin irritation potential and cytotoxicity of materials. As shown in Figure 4B, HDFs displayed the highest cell proliferation rate on PLLA/PLA-Lig20 nanofibers. The human MSCs are multipotent stromal cells that are capable of differentiating into many different cell types, including bone cells, myocytes, nerve cells and chondrocytes. To date, MSCs have become “All-Star” stem cells for both cell therapy and tissue engineering. As shown in Figure 4C, higher cell proliferations of MSCs were found on PLLA/PLA-Lig20 and 20 ACS Paragon Plus Environment

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PLLA/PLA-Lig30 nanofibers. Recently, scientists have started to explore the potential of using lignin as biomedical materials. Lignin nanotubes were synthesized as carriers for gene delivery and concentrations of up to 90 mg/mL of these lignin nanotubes were found to be tolerated for human HeLa cells, which is 10 times higher than those for carbon nanotubes [45]. Lignin/hydroxyapatite composites were engineered for bone tissue engineering, and such lignin-containing biomaterials not only exhibited a good biocompatibility with MSCs in vitro [46], but also demonstrated osteoconductivity and osseointegration properties in vivo [47].

Figure 4 Proliferation of (A) rat PC12 cells, (B) human dermal fibroblasts (HDFs) and (C) human mesenchymal stem cells (MSCs) on PLLA and PLLA/PLA-lignin nanofibers, as determined by alamarBlue assay. #p < 0.05 compared to tissue culture place (TCP) at each time point; *p < 0.05 compared to PLLA nanofibers at each time point



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

In addition, it was not surprising that PLLA/PLA-Lig50 nanofibers exhibited relative low cell proliferation values. It has been reported that lignin could exert some negative influence on cell viability at very high concentrations. To investigate the skin irritation potential of different types of lignins, Ugartondo et al. cultured human keratinocytes and murine fibroblasts in lignin solutions [43]. They found that the cytotoxicity of lignins increased with culture period with IC50 values surpassing 400 µg/ml. This finding was supported by another independent study that was developing gelatin/lignin films for food packaging materials. The IC50 value (to murine fibroblasts) of a sulphur-free water-insoluble lignin was found to be 631 ± 92 µg/ml, which is remarkably higher (17 times) than its effective antioxidant concentration [48]. In our study, lignin has shown to possess good antioxidant properties but its applications in biomaterials are highly limited by its cytotoxicity at high concentration. Therefore, it is critical to find the optimal amount of lignin to be introduced into the material system to achieve a balance between the antioxidant activities and the cytotoxicity. The cell proliferation results (Figure 4) revealed that PLLA/PLA-Lig20 exhibited the best biocompatibility with all three cell types, suggesting that this could be the optimal amount of lignin to be used. Further studies will be carried out to investigate the antioxidant mechanism of lignin copolymers and the cell behaviour with such antioxidant materials.

Conclusion In this study, PLA-lignin copolymers were synthesized by ring-opening polymerization, and the molecular weight and PLA chain lengths were controllable by varying the feed ratio of lignin and lactide. Electrospinning of PLA-lignin copolymers and PLLA generated uniform and fine nanofibers, illustrating the good miscibility between the two materials in nanoscale. However, the incorporation of such copolymers decreased the mechanical properties of the


Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanofibrous composites, revealing that good fillers for bulk materials might not have the same effect in porous/nanofibrous materials. On the other side, PLLA/PLA-lignin nanofibers were demonstrated to have good antioxidant activities and biocompatibility for three different cell types, indicating such materials could be used as tissue engineering scaffolds for locally attenuating cellular oxidative stress.

Acknowledgement The authors would like to acknowledge the A*STAR Personal Care Programme Grant on “Polymer Bank for Personal Care Applications” in support of this work.

Supporting Information 1

H NMR (CDCl3) of alkylated lignin; FTIR spectra, DSC curves and free radical inhibition of

lignin and PLA-lignin copolymers; water contact angles, stress-strain curves and DSC curves of PLLA and PLLA/PLA-lignin nanofibers



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

Reference [1] Pandey MP, Kim CS. Lignin Depolymerization and Conversion: A Review of Thermochemical Methods. Chem Eng Technol 2011;34:29-41. [2] Thakur VK, Thakur MK, Raghavan P, Kessler MR. Progress in Green Polymer Composites from Lignin for Multifunctional Applications: A Review. ACS Sustain Chem Eng 2014;2:1072-92. [3] Xu X, He ZH, Lu SR, Guo D, Yu JH. Enhanced thermal and mechanical properties of lignin/polypropylene wood-plastic composite by using flexible segment-containing reactive compatibilizer. Macromol Res 2014;22:1084-9. [4] Pouteau C, Dole P, Cathala B, Averous L, Boquillon N. Antioxidant properties of lignin in polypropylene. Polym Degrad Stab 2003;81:9-18. [5] Sadeghifar H, Argyropoulos DS. Correlations of the Antioxidant Properties of Softwood Kraft Lignin Fractions with the Thermal Stability of Its Blends with Polyethylene. ACS Sustain Chem Eng 2015;3:349-56. [6] Hu L, Stevanovic T, Rodrigue D. Unmodified and Esterified Kraft Lignin-Filled Polyethylene Composites: Compatibilization by Free-Radical Grafting. J Appl Polym Sci 2015;132:8. [7] Pucciariello R, Villani V, Bonini C, D'Auria M, Vetere T. Physical properties of straw lignin-based polymer blends. Polymer 2004;45:4159-69. [8] Kai D, Tan MJ, Chee PL, Chua YK, Yap YL, Loh XJ. Towards lignin-based functional materials in a sustainable world. Green Chem 2016;18:1175-200. [9] Wang H, Sun XZ, Seib P. Mechanical properties of poly(lactic acid) and wheat starch blends with methylenediphenyl diisocyanate. J Appl Polym Sci 2002;84:1257-62.


Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[10] Aldana DS, Villa ED, Hernandez MD, Sanchez GG, Cruz QR, Gallardo SF, et al. Barrier Properties of Polylactic Acid in Cellulose Based Packages Using Montmorillonite as Filler. Polymers 2014;6:2386-403. [11] Ouyang WZ, Huang Y, Luo HJ, Wang DS. Poly(Lactic Acid) Blended with Cellulolytic Enzyme Lignin: Mechanical and Thermal Properties and Morphology Evaluation. J Polym Environ 2012;20:1-9. [12] Li JC, He Y, Inoue Y. Thermal and mechanical properties of biodegradable blends of poly(L-lactic acid) and lignin. Polym Int 2003;52:949-55. [13] Chung YL, Olsson JV, Li RJ, Frank CW, Waymouth RM, Billington SL, et al. A Renewable Lignin-Lactide Copolymer and Application in Biobased Composites. ACS Sustain Chem Eng 2013;1:1231-8. [14] Sun Y, Yang L, Lu X, He C. Biodegradable and renewable poly(lactide)-lignin composites: synthesis, interface and toughening mechanism. Journal of Materials Chemistry A 2015;3:3699-709. [15] Ugartondo V, Mitjans M, Vinardell MP. Applicability of lignins from different sources as antioxidants based on the protective effects on lipid peroxidation induced by oxygen radicals. Ind Crop Prod 2009;30:184-7. [16] van Lith R, Gregory EK, Yang J, Kibbe MR, Ameer GA. Engineering biodegradable polyester elastomers with antioxidant properties to attenuate oxidative stress in tissues. Biomaterials 2014;35:8113-22. [17] Ren W, Pan X, Wang G, Cheng W, Liu Y. Dodecylated lignin-g-PLA for effective toughening of PLA. Green Chem 2016 DOI: 10.1039/C6GC01341D. [18] Baheiraei N, Yeganeh H, Ai J, Gharibi R, Azami M, Faghihi F. Synthesis, characterization and antioxidant activity of a novel electroactive and biodegradable



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

polyurethane for cardiac tissue engineering application. Mater Sci Eng C-Mater Biol Appl 2014;44:24-37. [19] Li Y, Sarkanen S. Alkylated kraft lignin-based thermoplastic blends with aliphatic polyesters. Macromolecules 2002;35:9707-15. [20] Maldhure AV, Ekhe JD, Deenadayalan E. Mechanical properties of polypropylene blended with esterified and alkylated lignin. J Appl Polym Sci 2012;125:1701-12. [21] Chen F, Dai HH, Dong XL, Yang JT, Zhong MQ. Physical Properties of Lignin-Based Polypropylene Blends. Polym Compos 2011;32:1019-25. [22] Kai D, Jin G, Prabhakaran MP, Ramakrishna S. Electrospun synthetic and natural nanofibers for regenerative medicine and stem cell. Biotechnol J 2013;8:59-72. [23] Huang Z, Zhang Y, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites.

Compos

Sci Technol

2003;63:2223-53. [24] Kai D, Jiang S, Low ZW, Loh XJ. Engineering highly stretchable lignin-based electrospun nanofibers for potential biomedical applications. Journal of Materials Chemistry B 2015;3:6194-204. [25] Asran AS, Salama M, Popescu C, Michler GH. Solvent Influences the Morphology and Mechanical Properties of Electrospun Poly(L-lactic acid) Scaffold for Tissue Engineering Applications. In: Michler GH, Henning S, editors. Layered Nanostructures - Polymers with Improved Properties. Weinheim: Wiley-V C H Verlag Gmbh; 2010. p. 153-61. [26] Maleki H, Gharehaghaji AA, Moroni L, Dijkstra PJ. Influence of the solvent type on the morphology and mechanical properties of electrospun PLLA yarns. Biofabrication 2013;5:7. [27] Casasola R, Thomas NL, Trybala A, Georgiadou S. Electrospun poly lactic acid (PLA) fibres: Effect of different solvent systems on fibre morphology and diameter. Polymer 2014;55:4728-37.


Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[28] Pham QP, Sharma U, Mikos AG. Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Eng 2006;12:1197-211. [29] Kriegel C, Arrechi A, Kit K, McClements DJ, Weiss J. Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Crit Rev Food Sci Nutr 2008;48:77597. [30] Subbiah T, Bhat GS, Tock RW, Pararneswaran S, Ramkumar SS. Electrospinning of nanofibers. J Appl Polym Sci 2005;96:557-69. [31] Ashammakhi N, Ndreu A, Piras AM, Nikkola L, Sindelar T, Ylikauppila H, et al. Biodegradable nanomats produced by electrospinning: Expanding multifunctionality and potential for tissue engineering. J Nanosci Nanotechnol 2007;7:862-82. [32] Sen S, Patil S, Argyropoulos DS. Thermal properties of lignin in copolymers, blends, and composites: a review. Green Chem 2015;17:4862-87. [33] Gordobil O, Delucis R, Egues I, Labidi J. Kraft lignin as filler in PLA to improve ductility and thermal properties. Ind Crop Prod 2015;72:46-53. [34] Kai D, Prabhakaran MP, Chan BQY, Liow SS, Ramakrishna S, Xu F, et al. Elastic poly (ε-caprolactone)-polydimethylsiloxane copolymer fibers with shape memory effect for bone tissue engineering. Biomed Mater 2016;11:015007. [35] Washburn NR, Yamada KM, Simon CG, Kennedy SB, Amis EJ. High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation. Biomaterials 2004;25:1215-24. [36] Park A, Cima LG. In vitro cell response to differences in poly-L-lactide crystallinity. J Biomed Mater Res 1996;31:117-30. [37] Azadfar M, Gao AH, Bule MV, Chen SL. Structural characterization of lignin: A potential source of antioxidants guaiacol and 4-vinylguaiacol. Int J Biol Macromol 2015;75:58-66.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

[38] Morandim-Giannetti AA, Agnelli JAM, Lancas BZ, Magnabosco R, Casarin SA, Bettini SHP. Lignin as additive in polypropylene/coir composites: Thermal, mechanical and morphological properties. Carbohydr Polym 2012;87:2563-8. [39] Domenek S, Louaifi A, Guinault A, Baumberger S. Potential of Lignins as Antioxidant Additive in Active Biodegradable Packaging Materials. J Polym Environ 2013;21:692-701. [40] Acosta JLE, Chavez PIT, Ramrez-Wong B, Bello-Perez LA, Ros AV, Millan EC, et al. Mechanical, thermal, and antioxidant properties of composite films prepared from durum wheat starch and lignin. Starch-Starke 2015;67:502-11. [41] Ponomarenko J, Dizhbite T, Lauberts M, Volperts A, Dobele G, Telysheva G. Analytical pyrolysis - A tool for revealing of lignin structure-antioxidant activity relationship. J Anal Appl Pyrolysis 2015;113:360-9. [42] Dizhbite T, Telysheva G, Jurkjane V, Viesturs U. Characterization of the radical scavenging activity of lignins - natural antioxidants. Bioresour Technol 2004;95:309-17. [43] Ugartondo V, Mitjans M, Vinardell MP. Comparative antioxidant and cytotoxic effects of lignins from different sources. Bioresour Technol 2008;99:6683-7. [44] Kai D, Low ZW, Liow SS, Abdul Karim A, Ye H, Jin G, et al. Development of Lignin Supramolecular Hydrogels with Mechanically Responsive and Self-Healing Properties. ACS Sustain Chem Eng 2015;3:2160-9. [45] Ten E, Ling C, Wang Y, Srivastava A, Dempere LA, Vermerris W. Lignin Nanotubes As Vehicles for Gene Delivery into Human Cells. Biomacromolecules 2014;15:327-38. [46] Jankovic A, Erakovic S, Ristoscu C, Mihailescu N, Duta L, Visan A, et al. Structural and biological evaluation of lignin addition to simple and silver-doped hydroxyapatite thin films synthesized by matrix-assisted pulsed laser evaporation. J Mater Sci-Mater Med 2015;26:14.


Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[47] Valente FL, Santos LC, Sepulveda RV, Goncalves GP, Eleoterio RB, Reis ECC, et al. Hydroxyapatite-lignin composite as a metallic implant-bone tissue osseointegration improver: experimental study in dogs. Cienc Rural 2016;46:324-9. [48] Nunez-Flores R, Gimenez B, Fernandez-Martin F, Lopez-Caballero ME, Montero MP, Gomez-Guillen MC. Physical and functional characterization of active fish gelatin films incorporated with lignin. Food Hydrocolloids 2013;30:163-72.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 30

For Table of Contents Use Only

Manuscript title: "Engineering poly(lactide)-lignin nanofibers with antioxidant activity for biomedical application" Authors: Kai, Dan; Ren, Wei; Tian, Lingling; Chee, Pei Lin; Liu, Ye; Ramakrishna, Seeram; Loh, Xian Jun

PLLA/PLA-lignin nanofibers, with excellent antioxidant activities and biocompatibility, hold great potential to be used as biomaterials for protecting cells against oxidative stress conditions.


Engineering Poly(lactide)âLignin Nanofibers with Antioxidant Activity

Recommend Documents

Engineering Poly(lactide)âLignin Nanofibers with Antioxidant Activity