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Evaluation of biocompatibility and bactericidal activity of hierarchically porous PLA-TiO2 nanocomposite films fabricated by breath-figure method
Materials Chemistry and Physics 230 (2019) 308–318
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Evaluation of biocompatibility and bactericidal activity of hierarchically porous PLA-TiO2 nanocomposite films fabricated by breath-figure method
T
A. Shebi, S. Lisa∗ Soft Materials Research Laboratory, Department of Chemistry, National Institute of Technology Calicut, Kerala, 673601, India
HIGHLIGHTS
GRAPHICAL ABSTRACT
nigrum leaves extract was used to • Piper synthesize the filler (carbon doped titania).
membranes are made • Nanocomposite porous through breath figure formation.
are effectively resistant • Membranes against staphylococcus aureus. membranes are pro• Cytocompatible posed for wound dressing application.
ARTICLE INFO
ABSTRACT
Keywords: Piper nigrum Carbon doped TiO2 Honeycomb structure Antimicrobial activity Biomedical applications
The present research work is focused on the green synthesis of rutile titanium dioxide (GST) nanoparticles using leaf extract of piper nigrum plant. The synthesis yielded rutile GST with a particle size of 6.45 ± 0.81 nm and a zeta potential value of −27.51 mV. Breath-figure method was implemented to fabricate honeycomb films and the porous honeycomb PLA/GST (H-PLA/GST) nanocomposites have shown tremendous improvement of antibacterial activity against S. aureus under visible light or in darkness. The highest antibacterial potentiality expressed in terms of zone of inhibition (ZOI) was exhibited by the H-PLA/3GST nanocomposites (28 mm in visible and no activity in dark). H-PLA/GST after cytocompatibility studies had seeded cells with flattened morphology, showing better cell adhesion.
1. Introduction Poly(lactic acid) (PLA), though a biodegradable and biocompatible aliphatic polyester with wide applications [1–7], has major drawbacks like poor thermal properties and brittleness which limits its employment in the field of electronics [8]. Thus methods like copolymerization, blending, addition of nanofillers like silica, graphene, metal oxides has been adopted to improve the properties of PLA [9,10]. Among the metal oxides, titanium dioxide (TiO2) has been widely used as a filler as it is non-toxic, environment friendly and its ease of synthesis [11,12]. TiO2 nanoparticles exist in three different phases’ viz., anatase, rutile and brookite [13–15]. The photocatalytic property of TiO2 causes enhanced bactericidal activity on exposure to light. The development of
∗
resistance against microbes is a major challenge in the biomedical area. The search for possible modification in PLA based systems using nanomaterials and medicinal plants for imparting antimicrobial properties [16] is a thrust area. The presence of carbon impurity even at trace levels in the crystal structure of TiO2 furnishes excess electrons or holes, which activates TiO2 even under visible light. The carbon doping is identifiable from the colour of the powder obtained, i.e., a colour change from normal white to grey/blackish colour depending on the percentage of carbon present in the structure of titania. The carbon defect in the structure acts as a sensitizer for visible light absorption and also lowers the band gap of titania. The carbon doped nanoparticles can be synthesized chemically and
Corresponding author. E-mail address: [email protected] (S. Lisa).
https://doi.org/10.1016/j.matchemphys.2019.03.045 Received 3 December 2018; Received in revised form 11 March 2019; Accepted 13 March 2019 Available online 17 March 2019 0254-0584/ © 2019 Elsevier B.V. All rights reserved.
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biologically, but the synthesis involving biocapping agent is non-toxic. The synthesis procedure consists of only one step, with limited reactants and can be easily scaled up for bulk production. Moreover, the eco-friendly synthesis offers compatibility for biomedical applications [17–19], which is our topic of interest. There are innumerous studies on the biosynthesis of TiO2 nanoparticles using extracts of Psidium guajava [20], Jatropha curcas L. [21], Aloe vera [22] and Eclipta prostrate [23]. The main advantage of using bio-extract is its dual role as stabilizing and hydrolysing agent during the synthesis of biocompatible nanoparticles. The present work focus on piper nigrum plant extract mediated sol-gel synthesis of TiO2 nanoparticles, in which carbonyl group present in the extract, not only functions as a biocapping agent but also contributes carbon source for the formation of carbon doped TiO2 (GST). The resultant GST shows excellent antibacterial activity even in visible light. Porous polymer films find application in the field of tissue engineering, as the well ordered pores helps to control the sequence of cell attachment. Breath figure (BF) technique had drawn attention of many researchers, even though other porosity generating techniques such as particle leaching, freeze-drying, gas foaming, electrospinning etc. were also extensively reported. On comparing with those techniques, BF technique controls the pore dimension to attain honeycomb structured films just by varying the humidity, air flow and solvent. Breath figure technique is a widely accepted method for the fabrication of porous films [24,25]. The formation of biofunctional porous polymer film is a subject of research, as the porosity favors the cellbiomaterial interaction, thereby improves cell attachment on the structured material surfaces. The principle behind the porosity generation is the water droplet condensation on the polymer solution containing fast evaporating solvent, followed by subsequent reorganization of droplets on the surface, which then evaporates to form porous polymer film [26]. Thus, combination of the idea of incorporation of green synthesized TiO2 nanoparticles as filler in the PLA matrix and breath figure method (formation of well-ordered pores on the matrix) for tissue engineering application is a novel, green and promising approach. In the present study, novel GST nanoparticles were synthesized in piper nigrum leaf extract medium using titanium tetrachloride as precursor. The piper nigrum extract with various functional groups selectively hydrolyse the precursor, and at the same time acts as an in-situ carbon source for the formation of carbon doped TiO2 and also takes the role of stabilizing agent for the titania particles. The selection of acidic precursors promotes hydrolysis, which restricts the possible condensation reaction. The activation of hydrolysis reaction results in the formation of stable rutile phase of titania [27]. From the observations, it is evident that the incorporation of GST on PLA matrix via breath figure formation has a prominent effect on the thermal, mechanical, antibacterial and morphological properties of resultant porous H-PLA/GST nanocomposites.
of the collected leaves were boiled for about 0.5 h in 200 mL distilled water to obtain the aqueous extract. The extract was filtered and was preserved at 4 °C. 2.3. Green synthesis of titanium dioxide nanoparticles by direct thermalhydrolysis sol–gel method 5 mL of metal precursor, TiCl4 was slowly added to 20 mL distilled water maintained in ice cold bath. The solution was then brought to room temperature with stirring for 0.5 h to homogenize the solution. The bath temperature was raised to 150 °C and maintained for the formation of nanoparticles. 25 mL of piper nigrum extract was added slowly to the hot solution while stirring. The pH was set to 4–5 to avoid the re-dissolving of formed titania particles and also for expelling the chloride ions. The carboxylic acids in the extract assure the acidic pH and effectively envelopes the so formed GST. The solution now turns to brownish white colloid solution, without precipitation (Fig. S1b). The solution was then centrifuged and the GST was washed thoroughly to make the sample chlorine free.
TiCl 4 + 2H2 O
TiO2 + 4H+ + 4Cl
Scheme 1. Acid hydrolysis of TiCl4. The control was prepared by adopting the same procedure as that of GST, but the piper nigrum leaf extract was replaced by distilled water (colloidal dispersion photograph and SEM images are shown in Fig. S1a and Fig. S2 respectively). 2.4. Fabrication of honeycomb H-PLA/GST nanocomposites-Breath figure method The PLA and H-PLA/GST nanocomposites were prepared as follows: (i) 2.5 wt% PLA (w/v) is made soluble in DCM (PLA + DCMt); (ii) varying amount of GST (1 wt%(w/w), 2 wt%(w/w), 3 wt%(w/w) and 4 wt%(w/w) were added separately to the PLA solution for preparing 4 different PLA/GST composites. Then, the mixtures were sonicated for 15 min and degassed under vacuum. The porosity was introduced in the films by maintaining 95% relative humidity and with an air flow rate of 50 mL min−1 to obtain H-PLA/GST composites [28]. 3. Characterization The morphology of synthesized and developed samples was analysed by field-emission scanning electron microscope (FE-SEM; Hitachi Su 66000) and transmission electron microscopy (TEM, JEM-2010). Energy-dispersive spectroscopy (EDS, OXFORD XMX N) was utilized to evaluate the particle size distribution and the elemental percentage of GST. Beckman coulter-Delsa nano C was used to measure the zeta potential of GST. The obtained phase of GST was confirmed by scanning the sample from 10° to 80° using powder X-ray diffraction (Rigaku
2. Materials and methods 2.1. Materials Semi crystalline PLLA pellets (PLA) (MW = 160,000, PDI = 1.45, glass transition temperature = 65.3 °C with a D-lactide content of 8%) and TiCl4 (99.98%) were supplied by Sigma-Aldrich (USA). The solvent, dichloromethane (CH2Cl2 or DCM, 99.0%) was purchased from Merck (Germany). Piper nigrum leaves were obtained from a rural area in Kerala. 2.2. Preparation of piper nigrum leaf aqueous extract The locally collected piper nigrum leaves were cut into small pieces and washed with the detergent tween 20 followed by double distilled water for several times. It is then dried at room temperature. About 40 g
Scheme 1. Structure of carboxylic acids isolated from piper nigrum. 309
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Miniflex 600) instrument. Fourier transform infrared spectrometer (JASCO 4700) in ATR-FTIR mode was implemented to detect the functional groups in H-PLA/GST membranes. UV spectrophotometer (UV-2600 SHIMADZU) recorded the absorption spectra of the samples from 200 to 800 nm and the mechanical strength was assessed by Schimadzu Autograph, AG-Xplus series (load = 10 N; crosshead speed = 10 mm/min). Thermogravimetric analyser (TGA, TA Instruments, Q 50) was used to evaluate the thermal properties of samples by scanning in a range of 0–750 °C with a heating rate of 10 °C/ min under nitrogen atmosphere. The water contact angles of the membranes were evaluated for the hydrophilicity by using a standard goniometer (DIGIDROP Hamburg, France).
culture was treated with 200 μL MTT (5 mg/mL dissolved in PBS) solution. After incubating the above content at 37 °C for 3 h, 300 μL of DMSO was added to each culture well and was again incubated at room temperature for 30 min to obtain a homogenous colour. The optical density (OD) of test (with sample) and control (without sample) at a wavelength of 540 nm was measured using a microplate reader. The growth inhibition % was obtained by following equation [33].
% Viability =
OD of test X 100 OD of control
(3)
4. Results & discussion
3.1. Water vapor transmission rate (WVTR)
4.1. Biosynthesized carbon doped TiO2 (GST) nanoparticles
Water vapor permeability of the fabricated films was investigated by evaluating the rate of water vapor permeating through it at definite time intervals. For the study, circular shaped nanocomposite films (diameter 1.5 cm) were placed on the glass vials filled with 5 mL water. The weight of the each vial (W0) was noted and was then kept in desiccators. The vials were weighed after regular intervals of time (Wt). WVTR was estimated as follows [29,30].
The biosynthesized GST was evaluated using various characterization techniques for the confirmation of formed particles.
WVTR =
4.1.1. FT-IR spectroscopy The FTIR spectrum of GST (Fig. 7), showed a vibrational band observed around 500 cm−1 which can be assigned to Ti-O of TiO2. The weak band observed at 2362 cm−1 indicates the H– C–H symmetric stretching of alkanes. The OH stretching of carboxylic acids in the piper nigrum extract exhibited a broad peak around 3000 cm−1. The bands at 1741 cm−1, 1621 cm−1 and 1370 cm−1 corresponds to C=O stretching of ketones, primary amines and C–H stretching in alkenes, respectively. These characteristic peaks show that the synthesized GST is stabilized by the proteins, terpenoids, flavanoids etc in piper nigrum leaves extract [34].
S X 24 A
where S is the slope (g/h), A (πr2) is the area of the membrane in m2 3.2. Antibacterial activity The bactericidal property of porous PLA and PLA/GST samples was determined against Staphylococcus aureus (S. aureus) in presence of dark and under visible light conditions by agar disc diffusion method. The test organism, S. aureus (gram positive) was inoculated into the sterilized petridishes containing 20 mL Muller Hinton Agar, in which five samples H-PLA and H-PLA/GST (1, 2, 3 and 4 wt%) discs were placed on the agar plates after swabbing. The petridish was sealed and exposed to visible light for 30 min (visible condition) and another petridish with five similar wells were kept in dark condition. The zone of inhibition formed around the wells, after incubation of the loaded petriplates at 37 °C for 24 h, was measured in millimetres [31].
4.1.2. Phase identification The crystallinity and phase purity of the GST were confirmed by Xray diffraction analysis (Fig. 1). The XRD patterns of GST were observed at 27.27°, 35.86°, 41.08°, 54.23°,62.8° and 69.06° which corresponds to rutile phase of TiO2 [35]. XRD patterns with distinct diffraction peaks at 27.27°, 35.86°, 41.08°, 54.23°, 56.29°, 62.8° and 69.06° could be indexed to the planes (110), (101), (111), (211), (220), (002) and (301) respectively. The average crystallite size of GST synthesized was determined to be 5.9 ± 1.50 nm. The controlled crystallite size might be due to the carbon doping onto TiO2, in which an oxygen atom gets substituted by a smaller carbon. This substitution results in a minor shift of diffraction peaks compared to that of commercial TiO2 samples. The diffraction pattern coincides with previous reports suggesting the higher purity of GST. The
3.3. Hemolysis test For hemolytic assay [32], initially, 5 mL of anticoagulated blood was diluted with 0.9% saline. The required volume of normal saline was incubated for about half an hour, from which 10 mL solution was taken in five different standard tubes. The porous H-PLA/GST was cut into 6 mm circular discs and immersed in the standard tubes, to which 0.2 mL diluted blood was added and were incubated for 1 h. 0.2 mL of diluted blood was diluted with saline to get negative control and positive control was obtained by required dilution of 0.2 mL blood with distilled water. After incubation and centrifugation, the supernatant solution was subjected to absorbance measurements at 541 nm. Four parallel measurements were taken for average and standard deviation. The hemolysis percentage (HP) was calculated as follows:
HP =
OD of test sample OD of( Ve)control 100 OD of( + Ve)control OD of( Ve)control
(2)
where OD stands for optical density. 3.4. Cytotoxicity According to ISO10993-5, the cytotoxicity of H-PLA/GST on L-929 mouse fibroblast cells was studied by direct microscopic observation as well as MTT assay. Cell images were captured after 5 days of treatment. Initially, the culture was sterilized with 1 x PBS solution and each mL
Fig. 1. XRD pattern of GST. 310
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Fig. 2. (a) TEM image for GST of 20 nm magnification, (inset) Particle distribution pattern for GST (b) SAED pattern of GST.
sharpening of the peaks gives a clear indication that the particles are of nanodimensions [36].
very well correlated with XRD spectrum [38]. The results of FESEM (Fig. 3a) revealed that the GST nanoparticles were spherical in shape with smoother surfaces and their average particle size is around 6.76 ± 1.41 nm. The size of GST from FESEM and TEM analysis were found to be complementary to each other. Further, the elements present in GST were figured out using EDS spectrum (Fig. 4). The spectrum elucidates the presence of carbon, Ti and O on the GST lattice with atomic % as 22.94, 13.92 and 63.13, respectively. The presence of C can only result from the in-situ conversion of carbohydrates or other groups in the plant extract. The complete conversion of the precursor (TiCl4) and the removal of byproduct were confirmed by the absence of Cl in the spectra. Additionally, the proteins and carbohydrates from the extract were found to evenly stabilize the particles, which is clear from the presence of C along with Ti and O and also from the nanodimension of particles seen in morphological studies [39].
4.1.3. Particle size analyser A size distribution analysis for determining the average size of synthesized TiO2 dispersed in aqueous solution was carried out using nanoparticle size analyser. In Fig. S3, the plot shows that the particles size was ranging from 20 to 70 nm, with an average size of 40 nm. This result gives a preliminary confirmation for the nano-dimension of green synthesized TiO2 (GST) particles. The zeta-potential value of GST was found to be −27.51 mV (Fig. S4). The doped carbon in GST traps excess electrons, resulting in a more negative charged surface. The high negative zeta potential value of GST shows high stability of its colloidal dispersion. Further morphological studies have to be done for the justification of size and shape of the GST [37]. 4.1.4. Morphological studies TEM analysis was carried out to investigate the morphology and size of GST (Fig. 2a) and thus confirmed the particles to be in the nanoregime with a spherical shape having an average particle size of 6.45 ± 0.81 nm. This is surprisingly in good agreement with the average particle size obtained from the XRD pattern. On the other hand, the selected area electron diffraction (SAED) pattern of GST with brighter spot and intense rings were evaluated for the crystalline and amorphous nature of GST (Fig. 2b). The SAED pattern were analysed to obtain the d-spacing, which resulted in indexing of lattice planes (110), (101), (111), (211), (002) and (301) that can be
4.1.5. Determination of band gap The optical band gap of GST at room temperature was recorded using UV-visible instrument. The absorption edge from the UV-visible spectra (Fig. 5a) of GST was found to be at 417 nm, which is a preliminary indication of synthesized titania. The absorption data were subjected to Kubelka–Munk transformation to estimate the optical band gap of the material. The optical absorption coefficient (ά) for the semiconducting material is expressed by the equation,
Fig. 3. (a) SEM image (onset) Physical colour (b) Particle size distribution histogram and log normal fitting of carbon doped TiO2 (GST). 311
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accommodation of carbon atom by replacing one of the highly electronegative oxygen atoms in TiO2 lattice, indicating the formation of Ti3+. The high resolution XPS spectra of O1s (Fig. 6d) showed two peaks at 529.8 eV and 531.5 eV corresponding to Ti-O-Ti bonds and C-O-Ti bonds, respectively [43,44]. The peaks at binding energies of 284.45 eV, 285.79 eV and 288.31 eV in C1s spectrum (Fig. 6c) can be assigned to C=C, C-O and Ti-O-C/O-Ti-C, respectively [45]. Thus the chemical composition of GST is confirmed with its structure as carbon doped (yellow colored as shown in Fig. 3a (onset)). 4.2. H-PLA/GST nanocomposites The obtained GST particles in different weight % compositions (1 wt %, 2 wt%, 3 wt%, and 4 wt%) were mixed with high molecular weight PLA in an organic solvent, DCM and were dried in the humid atmosphere with a constant air flow to obtain four different nanocomposites and their characterization were detailed. The codes for the samples were tabulated in Table 1.
Fig. 4. EDS spectra for GST and corresponding elemental composition (onset).
=
1
A (h
Eg ) n h
4.2.1. FT-IR analysis FT-IR spectra of PLA, pure GST nanopowders, together with spectrum of H-PLA/GST composite are shown in Fig. 7. For the composite, a broad band is seen around 3000-3500 cm−1, which is due to the presence of hydroxyl groups present on the surface of titanium dioxide nanoparticles, absorbed water as well as the OH stretching for the functional groups in the extract. The peak in the range 2852–2921 cm−1 could be attributed to the vibration of -CH2 bond in PLA molecules [46]. The characteristic peak of Ti-O (400–600 cm−1) is retained in the composite indicating the incorporation of GST in PLA matrix.
(4)
where hυ is an energy of the photon, Eg is the band gap energy, A is a constant and n = 1/2 for allowed direct transition, n = 2 for allowed indirect transition, which varies according to the nature of transition. It was previously reported that titania have an indirect band gap associated with allowed transition [40], so n = 2 was assumed for GST. The Tauc's plot assisted for determining the optical band gap of GST by extrapolating straight portion of the plot (άhυ)1/2 versus hυ as shown in Fig. 5b. The estimated value for the optical band gap (Eg) was 2.95 eV, claiming the semiconducting nature of GST. The doping of carbon on TiO2 narrows the band gap of TiO2 as well as improves the visible light absorption as reported [41].
4.2.2. Porosity evaluation The morphological studies on the nanocomposites were carried out to understand the pore formation in the films. The SEM image (Fig. 8) obtained for the composites shows that the membrane is hierarchically porous in nature. This could be attributed to the breath forming principle where DCM acts as the solvent under a relative humidity (RH) of 95%, resulting in porous structure. Pores were not obtained for pure PLA (as shown in Fig. S5a), but the addition of GST accelerates the condensation of moisture from the humid environment and also disperses the condensed water droplets uniformly.
4.1.6. X-ray photoelectron spectroscopy (XPS) The technique was used for confirming the surface chemical composition of the material. Fig. 6a proves the presence of Ti and O, as well as the doped carbon in the crystal lattice of GST. The binding energy of commercial TiO2 nanoparticles could be 458.7 eV [42]. But Fig. 6b depicts a shift in the two typical peaks of Ti 2p spectrum from that of reported XPS spectrum for undoped commercial TiO2. The shift in peaks to 458.21 eV (Ti 2p3/2) and 463.97 eV (Ti 2p1/2) can be attributed to the
Fig. 5. (a) UV-visible spectra for GST (b) the Tauc's plot for GST. 312
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Fig. 6. (a) XPS survey spectrum of GST (b), (c) and (d) high resolution XPS spectra (Ti 2p, C 1s and O 1s) of GST.
The thermal, antimicrobial, and mechanical properties of the HPLA/GST nanocomposites can thus be effectively improved by the strong interaction between nanoparticles and the polymer matrix. Uniform dispersion of GST in the composite is observed, when the amount was less than or equal to 3 wt%. Some of the GST nanoparticles seems to be agglomerated due to the strong interaction among them at 4 wt% (Fig. S5b) [47,48]. The pore size distribution of the four composite membranes was shown in Fig. S6. The mean pore sizes were noticed as 3.2 ± 2.5 μm, 8.5 ± 3.7 μm, 10 ± 0.2 μm and 17 ± 7 μm for H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST, respectively. The uniformity of pores can be attributed to the effective dispersion of GST in the PLA matrix [49–51].
heat energy and have higher thermal stability. Dispersed GST filler in PLA matrix retards the PLA degradation effectively by blocking the movement of free radicals/volatile degradation product, resulting in a good thermal stability for polymer composites [52,53]. 4.2.4. Mechanical properties The tensile properties of pure PLA and H-PLA/GST nanocomposites are listed in Fig. 9. The tensile strength and % elongation increased significantly with increment in GST loading upto 3% and was found to decline on further addition. The reduction in tensile properties can be attributed to the aggregation of individual or undispersed TiO2 nanoparticles. The tensile strength of nanocomposites with 0 wt%, 1 wt%, 2 wt%, 3 wt% and 4 wt % GST are about 39.32 ± 1.98%, 43.94 ± 1.91%, 50.02 ± 2.19%, 63.81 ± 2.63% and 43.05 ± 2.13%, respectively. The tensile strength for the composites does not show much variation, but elongation at break had pronounced effect with GST loading. All samples, except for H-PLA/2GST & H-PLA/3GST showed a lower elongation at break, Fig. 9. The elongations of nanocomposites with 0 wt%, 1 wt%, 2 wt%, 3 wt% and 4 wt% GST are about 2.18 ± 0.32%, 3.31 ± 0.42%, 8.61 ± 0.51%, 10.93 ± 0.41% and 4.09 ± 0.25%, respectively, implying that the toughness or rigidity can be improved on appropriate loading of reinforced nanofiller like GST. Even though the nanocomposites are porous, the reinforcement effect of nanofiller might be responsible for the enhancement in mechanical property [54–56].
4.2.3. Thermal studies The improvement in thermal stability of PLA can be achieved by nanoparticles addition as fillers. These GST filler particles possess higher thermal stability due to higher interaction of doped carbon with TiO2, which in turn is directly imparted to the polymer on its addition (Fig. S7). Table 2 displays the thermal degradation at 5% weight loss (T0.05), and 10% weight loss (T0.10), the maximum degradation temperature (Tmax) and also the char formation at 500 °C. Here, T0.05 and T0.10 was recorded to study the effect of GST loading on the thermal stability of the H-PLA/GST nanocomposites. The thermal stability of PLA/GST was found to increase with the addition of GST up to 4%. This is because of the fact that TiO2 absorbs 313
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Table 2 TGA results of PLA and H-PLA/GST porous film. Sample
T0.05 (°C)
T0.10 (°C)
Tmax (°C)
Char formation at 500 °C
Pristine PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
298.74 304.61 307.40 318.47 325.14
315.94 318.20 320.33 327.80 332.34
339.53 339.80 341.21 343.26 346.36
2.61 3.63 4.31 5.45 6.85
4.2.5. Surface wetting properties of the composites It has been known from previous reports that the surface topography and chemistry have high impact on wettability of a material. The honeycomb pores are familiar for its hydrophobicity by forming air pockets between the droplet and the film surface. But more interestingly, the contact angle for the nanocomposites was found to be lower, confirming the hydrophilic nature [57]. The contact angles of PLA, HPLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites were noticed at 91 ± 3.5°, 78.2 ± 2.8°, 73.5 ± 2.6°, 69.2 ± 2.4°, and 65.5 ± 2.8°, respectively (Fig. S8). The pores were formed after the evaporation of water and thus GST act as pore wall in the films. The GST, being hydrophilic and its presence on the surface as well as on the pore walls induced the spreading of water droplets, causing lower contact angles for the composites. Fig. 7. FTIR spectra of PLA, GST and H-PLA/3GST.
4.2.6. Water vapor transmission rate (WVTR) studies The WVTR of H-PLA/1GST (1%), H-PLA/2GST (2%), H-PLA/3GST (3%) and H-PLA/4GST (4%) honeycomb films was studied at 37.8 ± 0.5 °C under 80% relative humidity, Fig. S9. The water vapor permeability for pristine PLA film was found to be 111 ± 32 gm2.24 h. The WVTR values increased on increasing filler contents up to 3% (up to 2365 ± 64 g/m2.24 h), which may be due to the presence of uniform pores on the substrate surface. On increasing filler content to 4 wt %, the uniform distribution of filler content was lost which may block the pores, resulting in lower WVTR for H-PLA/4GST [58].
Table 1 The formulations of samples with sample codes. Sample code
Sample description
PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
2.5% PLA 99% PLA + 98% PLA + 97% PLA + 96% PLA +
1% 2% 3% 4%
n-GST n-GST n-GST n-GST
Fig. 8. SEM image of Honeycomb film of (a) H-PLA/1GST (b) H-PLA/2GST (c) H-PLA/3GST (d) H-PLA/4GST and (onset) corresponding zoomed area. 314
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Fig. 9. (a) Tensile strength and (b) % of elongation of H-PLA/GST nanocomposites.
4.2.7. Hemolysis The nanocomposites H-PLA/3GST was studied for its compatibility with blood so as to function as a medical dressing material. The degree of hemolysis from the assay shows the extent of damage of erythrocytes. Absorbance in the test for the samples exposed to PLA, H-PLA/ 1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST were 0.036 ± 0.0012, 0.041 ± 0.0022, 0.058 ± 0.0034, 0.073 ± 0.0031 and 0.085 ± 0.0049, respectively. The hemolysis rate obtained for PLA, H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST were 0.24 ± 0.08, 0.53 ± 0.12, 1.53 ± 0.24, 2.423 ± 0.31 and 3.132 ± 0.27, respectively (Fig. 10), which was far below the threshold value of 5%. The hemolytic study stipulates the application of the fabricated composites as wound dressing materials [59,60].
while PLA/4GST nanocomposites with lower nano sized GST content than H-PLA/3GST, fluctuate from the observed trend in inhibition zone against S. aureus as illustrated in Table 3. The lowering of zone diameter with higher GST concentration can be attributed to the agglomeration effect of GST particles and an irregular morphology. The bactericidal property of the samples depends on morphology, size, surface area etc. The nano sized particles with more surface area shows good bactericidal property [61–63]. The maximum zone diameter was for H-PLA/3GST against S. aureus bacteria in visible conditions, as presented in Fig. 11. H-PLA/3GST shows enhanced antibacterial activity even after 30 min of visible light irradiation, when compared to other H-PLA/GST nanocomposites. This may be due to the uniform distribution of GST particles or band gap narrowing or excellent antimicrobial activity of piper nigrum [64–66]. The striking reasons for the action of GST in the present studied PLA matrix as an effective photocatalyst, could be due to the capability of the material to absorb or reflect visible light [67]. The colour of GST particles synthesized was not purely white, but they had a greyish shade for the particles (shown in Fig. 3a (onset)). It was reported that the carbon doped TiO2 can narrow the band gap and promote the electronic excitation between the bands [68]. The role of carbon in improving the reduction of rate of recombination of electrons/holes by capture of electrons, makes the photocatalyst highly reactive [69]. Even though there are numerous discussions on this area, the actual mechanism still remains unknown. The antibacterial action of GST in the presence of visible light was thus suggested as represented in Scheme 2. The reactive hydroxyl radicals thus generated react with cell membranes, DNA, and thus cause the cell death in bacteria. The GST on exposure to visible light creates photo induced electrons and holes. The electrons reduces the dissolved oxygen to superoxide anion (O−•) and the generated holes on the surface react with water forms hydroxyl radicals (OH•) and oxidative radicals (HO2•). These radicals destroy the cell membrane, causing deformation in cell structure leading to ultimate cell death. This increased availability of the GST particles at the surface, enhance the bactericidal activity as discussed earlier. This reveals that the porous and nanoparticle incorporated film have the ability to induce the death of approaching bacteria.
4.2.8. Antimicrobial activity The inhibition zone around the disks on the agar plates shows the antibacterial activity of PLA, H-PLA/1GST, H-PLA/2GST, H-PLA/3GST, and H-PLA/4GST against S. aureus, which was demonstrated under dark and visible light to confirm their enhanced photocatalytic effect owing to the increased surface area of nanocomposites (Fig. 11). The inhibition zone diameters for H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites were 18, 22, 28 and 24 mm respectively,
4.2.9. Cyto-toxicity For the purpose of wound dressing application, the fabricated PLA, H- PLA/GST films, was tested for its cytocompatibility using mouse fibroblast L929 cells for specific time intervals i.e., 24, 72 and 120 h, respectively. The morphology of the substrates is a known parameter
Fig. 10. Hemolysis percentage of H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites. 315
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Fig. 11. Antibacterial activity of H-PLA/GST against S. aureus bacteria (a) dark condition (b) visible irradiation. Table 3 Antibacterial activity of different concentration of GST on H-PLA/GST nanocomposites under dark and visible condition. Sample
Zone of inhibition in visible condition (mm)
Zone of inhibition under dark condition (mm)
PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
Nil 18 22 28 24
Nil Nil Nil Nil Nil
Fig. 12. Optical image of mouse fibroblasts L929 on (a) H-PLA/1GST (b) HPLA/2GST (c) H-PLA/3GST and (d) H-PLA/4GST nanocomposites after 5 days.
Scheme 2. Schematic representation of generation of reactive oxygen species (ROS) in n-GST.
for the cell-substrate adhesion [70,71]. Fig. 12 shows confocal laser scanning microscopy (CLSM) images of fibroblast L929 cells cultured on porous H-PLA/GST films and control substrate (Fig. S10). The CLSM shows that the cells seeded on H-PLA/ 1GST and H-PLA/4GST films with non-uniform pores had retained the spherical morphology with few irregular protrusions even after 120 h. But at the same time, H-PLA/3GST films with the uniform pore size (Fig. 12c) had seeded cells with more flattened morphology, indicating improved cell adhesion and spreading. The studies validate better adhesion with the seeded fibroblast L929 cells for H-PLA/3GST films than H-PLA/1GST, H-PLA/2GST and H-PLA/4GST. MTT cell viability assay was carried out to evaluate the cell proliferation of the nanocomposites (Fig. 13).
Fig. 13. Cell viability on the H-PLA/1GST, H-PLA/2GST, H-PLA/3GST, and HPLA/4GST films represented by the cell numbers determined by MTT assay at day 1, 3 and 5.
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The cell viability of porous H-PLA/GST composites ascertained the fact that it is due to the presence of GST particles in the modified PLA films. The well known and accepted statement that ‘the pore size greatly influences the cell behaviour’ is the basic concept employed in the work. The pore size and pore uniformity modulation with different wt% of GST had significantly varied the cell behaviors such as adhesion, spreading or proliferation [32].
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5. Conclusion Hierarchically porous H-PLA/GST nanocomposites were successfully synthesized through a green method and used for wound dressing application for the first time. The phase analysis data obtained for GST coincides well with the standard JCPDS (File No. 99-101-095) data validating the rutile phase of synthesized GST nanoparticles. The addition of plant extract in the precursor solution inhibited the appearance of brookite and anatase phase. The functionality of synthesized GST, such as O-Ti and –OH vibrational bands, various functional groups present in proteins, polyols, alkaloids from leaf extract were analysed through FTIR spectrum. The spherical morphology as well as the average particle size of around 6.45 ± 0.81 nm were confirmed from TEM images. The SAED pattern and the XRD pattern were found to be complementary to each other. The presence of carbon (confirmed from Electron diffraction spectrum and X-ray photoelectron spectroscopy) at the interstitial sites of GST was found to cause synergetic effect in the enhancement of the desired properties. Further, antibacterial activity of GST as a function of concentration was tested against S. aureus. The plant extract/TiO2 composite showed tremendously improved antibacterial activity against S. aureus in visible conditions. The enhanced visible light absorption is assumed to be due to the narrowing of band gap as a result of carbon doping onto GST. The eco friendly and cost effective GST with its notable bioactivity can thus be recommended as an effective wound healing dressing material owing to its remarkable antimicrobial potential against S. aureus. Acknowledgements This research was financially supported by Ministry of Human Resource Development (MHRD), India. We would like to thank Dr. G. Unnikrishnan, R. Rarima, Jithin Raj and P.K. Adnan, Department of Chemistry, NIT Calicut for their help with XRD and DLS measurements. We extend our gratitude to Dr. Shibu and Krishnakumar (STIC, CUSAT) for their cooperation in recording TEM. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.matchemphys.2019.03.045. References [1] J.C. Middleton, A.J. Tipton, Synthetic biodegradable polymers as orthopaedic devices, Biomaterials 21 (2000) 2335–2346. [2] H. Eufinger, C. Rasche, J. Lehmbrock, M. Wehmoller, S. Weihe, I. Schmitz, C. Schiller, M. Epple, Performance of functionally graded implants of polylactides and calcium phosphate/calcium carbonate in an ovine model for computer assisted craniotomy and cranioplasty, Biomaterials 28 (2007) 475–485. [3] R.K. Kulkarni, K.C. Pani, C. Neuman, F. Leonard, Polylactic acid for surgical implants, Arch. Surg. 93 (1966) 839–843. [4] S.S. Ray, P. Maiti, M. Okamoto, K. Yamada, K. Ueda, New polylactide/layered silicate nanocomposites 1. Preparation, characterization and properties, Macromolecules 35 (2002) 3104–3110. [5] A. González, C.I.A. Igarzabal, Stereocomplex formation between enantiomeric poly (lactics), Food Hydrocoll 33 (2013) 289–296. [6] H. Yamane, Y. Furuhashi, N. Yoshie, Y. Kimura, Higher-order structures and mechanical properties of stereocomplex- type poly(lactic acid) melt spun fibers, Polymer 47 (2006) 5965–5972. [7] Y. Ikada, K. Jamshidi, H. Tsuji, S.H. Hyon, Stereocomplex formation between enantiomeric poly(lactics), Macromolecules 20 (1987) 904–906. [8] Y. Li, H. Shimizu, Toughening of polylactide by melt blending with a biodegradable
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Evaluation of biocompatibility and bactericidal activity of hierarchically porous PLA-TiO2 nanocomposite films fabricated by breath-figure method
T
A. Shebi, S. Lisa∗ Soft Materials Research Laboratory, Department of Chemistry, National Institute of Technology Calicut, Kerala, 673601, India
HIGHLIGHTS
GRAPHICAL ABSTRACT
nigrum leaves extract was used to • Piper synthesize the filler (carbon doped titania).
membranes are made • Nanocomposite porous through breath figure formation.
are effectively resistant • Membranes against staphylococcus aureus. membranes are pro• Cytocompatible posed for wound dressing application.
ARTICLE INFO
ABSTRACT
Keywords: Piper nigrum Carbon doped TiO2 Honeycomb structure Antimicrobial activity Biomedical applications
The present research work is focused on the green synthesis of rutile titanium dioxide (GST) nanoparticles using leaf extract of piper nigrum plant. The synthesis yielded rutile GST with a particle size of 6.45 ± 0.81 nm and a zeta potential value of −27.51 mV. Breath-figure method was implemented to fabricate honeycomb films and the porous honeycomb PLA/GST (H-PLA/GST) nanocomposites have shown tremendous improvement of antibacterial activity against S. aureus under visible light or in darkness. The highest antibacterial potentiality expressed in terms of zone of inhibition (ZOI) was exhibited by the H-PLA/3GST nanocomposites (28 mm in visible and no activity in dark). H-PLA/GST after cytocompatibility studies had seeded cells with flattened morphology, showing better cell adhesion.
1. Introduction Poly(lactic acid) (PLA), though a biodegradable and biocompatible aliphatic polyester with wide applications [1–7], has major drawbacks like poor thermal properties and brittleness which limits its employment in the field of electronics [8]. Thus methods like copolymerization, blending, addition of nanofillers like silica, graphene, metal oxides has been adopted to improve the properties of PLA [9,10]. Among the metal oxides, titanium dioxide (TiO2) has been widely used as a filler as it is non-toxic, environment friendly and its ease of synthesis [11,12]. TiO2 nanoparticles exist in three different phases’ viz., anatase, rutile and brookite [13–15]. The photocatalytic property of TiO2 causes enhanced bactericidal activity on exposure to light. The development of
∗
resistance against microbes is a major challenge in the biomedical area. The search for possible modification in PLA based systems using nanomaterials and medicinal plants for imparting antimicrobial properties [16] is a thrust area. The presence of carbon impurity even at trace levels in the crystal structure of TiO2 furnishes excess electrons or holes, which activates TiO2 even under visible light. The carbon doping is identifiable from the colour of the powder obtained, i.e., a colour change from normal white to grey/blackish colour depending on the percentage of carbon present in the structure of titania. The carbon defect in the structure acts as a sensitizer for visible light absorption and also lowers the band gap of titania. The carbon doped nanoparticles can be synthesized chemically and
Corresponding author. E-mail address: [email protected] (S. Lisa).
https://doi.org/10.1016/j.matchemphys.2019.03.045 Received 3 December 2018; Received in revised form 11 March 2019; Accepted 13 March 2019 Available online 17 March 2019 0254-0584/ © 2019 Elsevier B.V. All rights reserved.
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biologically, but the synthesis involving biocapping agent is non-toxic. The synthesis procedure consists of only one step, with limited reactants and can be easily scaled up for bulk production. Moreover, the eco-friendly synthesis offers compatibility for biomedical applications [17–19], which is our topic of interest. There are innumerous studies on the biosynthesis of TiO2 nanoparticles using extracts of Psidium guajava [20], Jatropha curcas L. [21], Aloe vera [22] and Eclipta prostrate [23]. The main advantage of using bio-extract is its dual role as stabilizing and hydrolysing agent during the synthesis of biocompatible nanoparticles. The present work focus on piper nigrum plant extract mediated sol-gel synthesis of TiO2 nanoparticles, in which carbonyl group present in the extract, not only functions as a biocapping agent but also contributes carbon source for the formation of carbon doped TiO2 (GST). The resultant GST shows excellent antibacterial activity even in visible light. Porous polymer films find application in the field of tissue engineering, as the well ordered pores helps to control the sequence of cell attachment. Breath figure (BF) technique had drawn attention of many researchers, even though other porosity generating techniques such as particle leaching, freeze-drying, gas foaming, electrospinning etc. were also extensively reported. On comparing with those techniques, BF technique controls the pore dimension to attain honeycomb structured films just by varying the humidity, air flow and solvent. Breath figure technique is a widely accepted method for the fabrication of porous films [24,25]. The formation of biofunctional porous polymer film is a subject of research, as the porosity favors the cellbiomaterial interaction, thereby improves cell attachment on the structured material surfaces. The principle behind the porosity generation is the water droplet condensation on the polymer solution containing fast evaporating solvent, followed by subsequent reorganization of droplets on the surface, which then evaporates to form porous polymer film [26]. Thus, combination of the idea of incorporation of green synthesized TiO2 nanoparticles as filler in the PLA matrix and breath figure method (formation of well-ordered pores on the matrix) for tissue engineering application is a novel, green and promising approach. In the present study, novel GST nanoparticles were synthesized in piper nigrum leaf extract medium using titanium tetrachloride as precursor. The piper nigrum extract with various functional groups selectively hydrolyse the precursor, and at the same time acts as an in-situ carbon source for the formation of carbon doped TiO2 and also takes the role of stabilizing agent for the titania particles. The selection of acidic precursors promotes hydrolysis, which restricts the possible condensation reaction. The activation of hydrolysis reaction results in the formation of stable rutile phase of titania [27]. From the observations, it is evident that the incorporation of GST on PLA matrix via breath figure formation has a prominent effect on the thermal, mechanical, antibacterial and morphological properties of resultant porous H-PLA/GST nanocomposites.
of the collected leaves were boiled for about 0.5 h in 200 mL distilled water to obtain the aqueous extract. The extract was filtered and was preserved at 4 °C. 2.3. Green synthesis of titanium dioxide nanoparticles by direct thermalhydrolysis sol–gel method 5 mL of metal precursor, TiCl4 was slowly added to 20 mL distilled water maintained in ice cold bath. The solution was then brought to room temperature with stirring for 0.5 h to homogenize the solution. The bath temperature was raised to 150 °C and maintained for the formation of nanoparticles. 25 mL of piper nigrum extract was added slowly to the hot solution while stirring. The pH was set to 4–5 to avoid the re-dissolving of formed titania particles and also for expelling the chloride ions. The carboxylic acids in the extract assure the acidic pH and effectively envelopes the so formed GST. The solution now turns to brownish white colloid solution, without precipitation (Fig. S1b). The solution was then centrifuged and the GST was washed thoroughly to make the sample chlorine free.
TiCl 4 + 2H2 O
TiO2 + 4H+ + 4Cl
Scheme 1. Acid hydrolysis of TiCl4. The control was prepared by adopting the same procedure as that of GST, but the piper nigrum leaf extract was replaced by distilled water (colloidal dispersion photograph and SEM images are shown in Fig. S1a and Fig. S2 respectively). 2.4. Fabrication of honeycomb H-PLA/GST nanocomposites-Breath figure method The PLA and H-PLA/GST nanocomposites were prepared as follows: (i) 2.5 wt% PLA (w/v) is made soluble in DCM (PLA + DCMt); (ii) varying amount of GST (1 wt%(w/w), 2 wt%(w/w), 3 wt%(w/w) and 4 wt%(w/w) were added separately to the PLA solution for preparing 4 different PLA/GST composites. Then, the mixtures were sonicated for 15 min and degassed under vacuum. The porosity was introduced in the films by maintaining 95% relative humidity and with an air flow rate of 50 mL min−1 to obtain H-PLA/GST composites [28]. 3. Characterization The morphology of synthesized and developed samples was analysed by field-emission scanning electron microscope (FE-SEM; Hitachi Su 66000) and transmission electron microscopy (TEM, JEM-2010). Energy-dispersive spectroscopy (EDS, OXFORD XMX N) was utilized to evaluate the particle size distribution and the elemental percentage of GST. Beckman coulter-Delsa nano C was used to measure the zeta potential of GST. The obtained phase of GST was confirmed by scanning the sample from 10° to 80° using powder X-ray diffraction (Rigaku
2. Materials and methods 2.1. Materials Semi crystalline PLLA pellets (PLA) (MW = 160,000, PDI = 1.45, glass transition temperature = 65.3 °C with a D-lactide content of 8%) and TiCl4 (99.98%) were supplied by Sigma-Aldrich (USA). The solvent, dichloromethane (CH2Cl2 or DCM, 99.0%) was purchased from Merck (Germany). Piper nigrum leaves were obtained from a rural area in Kerala. 2.2. Preparation of piper nigrum leaf aqueous extract The locally collected piper nigrum leaves were cut into small pieces and washed with the detergent tween 20 followed by double distilled water for several times. It is then dried at room temperature. About 40 g
Scheme 1. Structure of carboxylic acids isolated from piper nigrum. 309
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Miniflex 600) instrument. Fourier transform infrared spectrometer (JASCO 4700) in ATR-FTIR mode was implemented to detect the functional groups in H-PLA/GST membranes. UV spectrophotometer (UV-2600 SHIMADZU) recorded the absorption spectra of the samples from 200 to 800 nm and the mechanical strength was assessed by Schimadzu Autograph, AG-Xplus series (load = 10 N; crosshead speed = 10 mm/min). Thermogravimetric analyser (TGA, TA Instruments, Q 50) was used to evaluate the thermal properties of samples by scanning in a range of 0–750 °C with a heating rate of 10 °C/ min under nitrogen atmosphere. The water contact angles of the membranes were evaluated for the hydrophilicity by using a standard goniometer (DIGIDROP Hamburg, France).
culture was treated with 200 μL MTT (5 mg/mL dissolved in PBS) solution. After incubating the above content at 37 °C for 3 h, 300 μL of DMSO was added to each culture well and was again incubated at room temperature for 30 min to obtain a homogenous colour. The optical density (OD) of test (with sample) and control (without sample) at a wavelength of 540 nm was measured using a microplate reader. The growth inhibition % was obtained by following equation [33].
% Viability =
OD of test X 100 OD of control
(3)
4. Results & discussion
3.1. Water vapor transmission rate (WVTR)
4.1. Biosynthesized carbon doped TiO2 (GST) nanoparticles
Water vapor permeability of the fabricated films was investigated by evaluating the rate of water vapor permeating through it at definite time intervals. For the study, circular shaped nanocomposite films (diameter 1.5 cm) were placed on the glass vials filled with 5 mL water. The weight of the each vial (W0) was noted and was then kept in desiccators. The vials were weighed after regular intervals of time (Wt). WVTR was estimated as follows [29,30].
The biosynthesized GST was evaluated using various characterization techniques for the confirmation of formed particles.
WVTR =
4.1.1. FT-IR spectroscopy The FTIR spectrum of GST (Fig. 7), showed a vibrational band observed around 500 cm−1 which can be assigned to Ti-O of TiO2. The weak band observed at 2362 cm−1 indicates the H– C–H symmetric stretching of alkanes. The OH stretching of carboxylic acids in the piper nigrum extract exhibited a broad peak around 3000 cm−1. The bands at 1741 cm−1, 1621 cm−1 and 1370 cm−1 corresponds to C=O stretching of ketones, primary amines and C–H stretching in alkenes, respectively. These characteristic peaks show that the synthesized GST is stabilized by the proteins, terpenoids, flavanoids etc in piper nigrum leaves extract [34].
S X 24 A
where S is the slope (g/h), A (πr2) is the area of the membrane in m2 3.2. Antibacterial activity The bactericidal property of porous PLA and PLA/GST samples was determined against Staphylococcus aureus (S. aureus) in presence of dark and under visible light conditions by agar disc diffusion method. The test organism, S. aureus (gram positive) was inoculated into the sterilized petridishes containing 20 mL Muller Hinton Agar, in which five samples H-PLA and H-PLA/GST (1, 2, 3 and 4 wt%) discs were placed on the agar plates after swabbing. The petridish was sealed and exposed to visible light for 30 min (visible condition) and another petridish with five similar wells were kept in dark condition. The zone of inhibition formed around the wells, after incubation of the loaded petriplates at 37 °C for 24 h, was measured in millimetres [31].
4.1.2. Phase identification The crystallinity and phase purity of the GST were confirmed by Xray diffraction analysis (Fig. 1). The XRD patterns of GST were observed at 27.27°, 35.86°, 41.08°, 54.23°,62.8° and 69.06° which corresponds to rutile phase of TiO2 [35]. XRD patterns with distinct diffraction peaks at 27.27°, 35.86°, 41.08°, 54.23°, 56.29°, 62.8° and 69.06° could be indexed to the planes (110), (101), (111), (211), (220), (002) and (301) respectively. The average crystallite size of GST synthesized was determined to be 5.9 ± 1.50 nm. The controlled crystallite size might be due to the carbon doping onto TiO2, in which an oxygen atom gets substituted by a smaller carbon. This substitution results in a minor shift of diffraction peaks compared to that of commercial TiO2 samples. The diffraction pattern coincides with previous reports suggesting the higher purity of GST. The
3.3. Hemolysis test For hemolytic assay [32], initially, 5 mL of anticoagulated blood was diluted with 0.9% saline. The required volume of normal saline was incubated for about half an hour, from which 10 mL solution was taken in five different standard tubes. The porous H-PLA/GST was cut into 6 mm circular discs and immersed in the standard tubes, to which 0.2 mL diluted blood was added and were incubated for 1 h. 0.2 mL of diluted blood was diluted with saline to get negative control and positive control was obtained by required dilution of 0.2 mL blood with distilled water. After incubation and centrifugation, the supernatant solution was subjected to absorbance measurements at 541 nm. Four parallel measurements were taken for average and standard deviation. The hemolysis percentage (HP) was calculated as follows:
HP =
OD of test sample OD of( Ve)control 100 OD of( + Ve)control OD of( Ve)control
(2)
where OD stands for optical density. 3.4. Cytotoxicity According to ISO10993-5, the cytotoxicity of H-PLA/GST on L-929 mouse fibroblast cells was studied by direct microscopic observation as well as MTT assay. Cell images were captured after 5 days of treatment. Initially, the culture was sterilized with 1 x PBS solution and each mL
Fig. 1. XRD pattern of GST. 310
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Fig. 2. (a) TEM image for GST of 20 nm magnification, (inset) Particle distribution pattern for GST (b) SAED pattern of GST.
sharpening of the peaks gives a clear indication that the particles are of nanodimensions [36].
very well correlated with XRD spectrum [38]. The results of FESEM (Fig. 3a) revealed that the GST nanoparticles were spherical in shape with smoother surfaces and their average particle size is around 6.76 ± 1.41 nm. The size of GST from FESEM and TEM analysis were found to be complementary to each other. Further, the elements present in GST were figured out using EDS spectrum (Fig. 4). The spectrum elucidates the presence of carbon, Ti and O on the GST lattice with atomic % as 22.94, 13.92 and 63.13, respectively. The presence of C can only result from the in-situ conversion of carbohydrates or other groups in the plant extract. The complete conversion of the precursor (TiCl4) and the removal of byproduct were confirmed by the absence of Cl in the spectra. Additionally, the proteins and carbohydrates from the extract were found to evenly stabilize the particles, which is clear from the presence of C along with Ti and O and also from the nanodimension of particles seen in morphological studies [39].
4.1.3. Particle size analyser A size distribution analysis for determining the average size of synthesized TiO2 dispersed in aqueous solution was carried out using nanoparticle size analyser. In Fig. S3, the plot shows that the particles size was ranging from 20 to 70 nm, with an average size of 40 nm. This result gives a preliminary confirmation for the nano-dimension of green synthesized TiO2 (GST) particles. The zeta-potential value of GST was found to be −27.51 mV (Fig. S4). The doped carbon in GST traps excess electrons, resulting in a more negative charged surface. The high negative zeta potential value of GST shows high stability of its colloidal dispersion. Further morphological studies have to be done for the justification of size and shape of the GST [37]. 4.1.4. Morphological studies TEM analysis was carried out to investigate the morphology and size of GST (Fig. 2a) and thus confirmed the particles to be in the nanoregime with a spherical shape having an average particle size of 6.45 ± 0.81 nm. This is surprisingly in good agreement with the average particle size obtained from the XRD pattern. On the other hand, the selected area electron diffraction (SAED) pattern of GST with brighter spot and intense rings were evaluated for the crystalline and amorphous nature of GST (Fig. 2b). The SAED pattern were analysed to obtain the d-spacing, which resulted in indexing of lattice planes (110), (101), (111), (211), (002) and (301) that can be
4.1.5. Determination of band gap The optical band gap of GST at room temperature was recorded using UV-visible instrument. The absorption edge from the UV-visible spectra (Fig. 5a) of GST was found to be at 417 nm, which is a preliminary indication of synthesized titania. The absorption data were subjected to Kubelka–Munk transformation to estimate the optical band gap of the material. The optical absorption coefficient (ά) for the semiconducting material is expressed by the equation,
Fig. 3. (a) SEM image (onset) Physical colour (b) Particle size distribution histogram and log normal fitting of carbon doped TiO2 (GST). 311
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accommodation of carbon atom by replacing one of the highly electronegative oxygen atoms in TiO2 lattice, indicating the formation of Ti3+. The high resolution XPS spectra of O1s (Fig. 6d) showed two peaks at 529.8 eV and 531.5 eV corresponding to Ti-O-Ti bonds and C-O-Ti bonds, respectively [43,44]. The peaks at binding energies of 284.45 eV, 285.79 eV and 288.31 eV in C1s spectrum (Fig. 6c) can be assigned to C=C, C-O and Ti-O-C/O-Ti-C, respectively [45]. Thus the chemical composition of GST is confirmed with its structure as carbon doped (yellow colored as shown in Fig. 3a (onset)). 4.2. H-PLA/GST nanocomposites The obtained GST particles in different weight % compositions (1 wt %, 2 wt%, 3 wt%, and 4 wt%) were mixed with high molecular weight PLA in an organic solvent, DCM and were dried in the humid atmosphere with a constant air flow to obtain four different nanocomposites and their characterization were detailed. The codes for the samples were tabulated in Table 1.
Fig. 4. EDS spectra for GST and corresponding elemental composition (onset).
=
1
A (h
Eg ) n h
4.2.1. FT-IR analysis FT-IR spectra of PLA, pure GST nanopowders, together with spectrum of H-PLA/GST composite are shown in Fig. 7. For the composite, a broad band is seen around 3000-3500 cm−1, which is due to the presence of hydroxyl groups present on the surface of titanium dioxide nanoparticles, absorbed water as well as the OH stretching for the functional groups in the extract. The peak in the range 2852–2921 cm−1 could be attributed to the vibration of -CH2 bond in PLA molecules [46]. The characteristic peak of Ti-O (400–600 cm−1) is retained in the composite indicating the incorporation of GST in PLA matrix.
(4)
where hυ is an energy of the photon, Eg is the band gap energy, A is a constant and n = 1/2 for allowed direct transition, n = 2 for allowed indirect transition, which varies according to the nature of transition. It was previously reported that titania have an indirect band gap associated with allowed transition [40], so n = 2 was assumed for GST. The Tauc's plot assisted for determining the optical band gap of GST by extrapolating straight portion of the plot (άhυ)1/2 versus hυ as shown in Fig. 5b. The estimated value for the optical band gap (Eg) was 2.95 eV, claiming the semiconducting nature of GST. The doping of carbon on TiO2 narrows the band gap of TiO2 as well as improves the visible light absorption as reported [41].
4.2.2. Porosity evaluation The morphological studies on the nanocomposites were carried out to understand the pore formation in the films. The SEM image (Fig. 8) obtained for the composites shows that the membrane is hierarchically porous in nature. This could be attributed to the breath forming principle where DCM acts as the solvent under a relative humidity (RH) of 95%, resulting in porous structure. Pores were not obtained for pure PLA (as shown in Fig. S5a), but the addition of GST accelerates the condensation of moisture from the humid environment and also disperses the condensed water droplets uniformly.
4.1.6. X-ray photoelectron spectroscopy (XPS) The technique was used for confirming the surface chemical composition of the material. Fig. 6a proves the presence of Ti and O, as well as the doped carbon in the crystal lattice of GST. The binding energy of commercial TiO2 nanoparticles could be 458.7 eV [42]. But Fig. 6b depicts a shift in the two typical peaks of Ti 2p spectrum from that of reported XPS spectrum for undoped commercial TiO2. The shift in peaks to 458.21 eV (Ti 2p3/2) and 463.97 eV (Ti 2p1/2) can be attributed to the
Fig. 5. (a) UV-visible spectra for GST (b) the Tauc's plot for GST. 312
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Fig. 6. (a) XPS survey spectrum of GST (b), (c) and (d) high resolution XPS spectra (Ti 2p, C 1s and O 1s) of GST.
The thermal, antimicrobial, and mechanical properties of the HPLA/GST nanocomposites can thus be effectively improved by the strong interaction between nanoparticles and the polymer matrix. Uniform dispersion of GST in the composite is observed, when the amount was less than or equal to 3 wt%. Some of the GST nanoparticles seems to be agglomerated due to the strong interaction among them at 4 wt% (Fig. S5b) [47,48]. The pore size distribution of the four composite membranes was shown in Fig. S6. The mean pore sizes were noticed as 3.2 ± 2.5 μm, 8.5 ± 3.7 μm, 10 ± 0.2 μm and 17 ± 7 μm for H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST, respectively. The uniformity of pores can be attributed to the effective dispersion of GST in the PLA matrix [49–51].
heat energy and have higher thermal stability. Dispersed GST filler in PLA matrix retards the PLA degradation effectively by blocking the movement of free radicals/volatile degradation product, resulting in a good thermal stability for polymer composites [52,53]. 4.2.4. Mechanical properties The tensile properties of pure PLA and H-PLA/GST nanocomposites are listed in Fig. 9. The tensile strength and % elongation increased significantly with increment in GST loading upto 3% and was found to decline on further addition. The reduction in tensile properties can be attributed to the aggregation of individual or undispersed TiO2 nanoparticles. The tensile strength of nanocomposites with 0 wt%, 1 wt%, 2 wt%, 3 wt% and 4 wt % GST are about 39.32 ± 1.98%, 43.94 ± 1.91%, 50.02 ± 2.19%, 63.81 ± 2.63% and 43.05 ± 2.13%, respectively. The tensile strength for the composites does not show much variation, but elongation at break had pronounced effect with GST loading. All samples, except for H-PLA/2GST & H-PLA/3GST showed a lower elongation at break, Fig. 9. The elongations of nanocomposites with 0 wt%, 1 wt%, 2 wt%, 3 wt% and 4 wt% GST are about 2.18 ± 0.32%, 3.31 ± 0.42%, 8.61 ± 0.51%, 10.93 ± 0.41% and 4.09 ± 0.25%, respectively, implying that the toughness or rigidity can be improved on appropriate loading of reinforced nanofiller like GST. Even though the nanocomposites are porous, the reinforcement effect of nanofiller might be responsible for the enhancement in mechanical property [54–56].
4.2.3. Thermal studies The improvement in thermal stability of PLA can be achieved by nanoparticles addition as fillers. These GST filler particles possess higher thermal stability due to higher interaction of doped carbon with TiO2, which in turn is directly imparted to the polymer on its addition (Fig. S7). Table 2 displays the thermal degradation at 5% weight loss (T0.05), and 10% weight loss (T0.10), the maximum degradation temperature (Tmax) and also the char formation at 500 °C. Here, T0.05 and T0.10 was recorded to study the effect of GST loading on the thermal stability of the H-PLA/GST nanocomposites. The thermal stability of PLA/GST was found to increase with the addition of GST up to 4%. This is because of the fact that TiO2 absorbs 313
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Table 2 TGA results of PLA and H-PLA/GST porous film. Sample
T0.05 (°C)
T0.10 (°C)
Tmax (°C)
Char formation at 500 °C
Pristine PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
298.74 304.61 307.40 318.47 325.14
315.94 318.20 320.33 327.80 332.34
339.53 339.80 341.21 343.26 346.36
2.61 3.63 4.31 5.45 6.85
4.2.5. Surface wetting properties of the composites It has been known from previous reports that the surface topography and chemistry have high impact on wettability of a material. The honeycomb pores are familiar for its hydrophobicity by forming air pockets between the droplet and the film surface. But more interestingly, the contact angle for the nanocomposites was found to be lower, confirming the hydrophilic nature [57]. The contact angles of PLA, HPLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites were noticed at 91 ± 3.5°, 78.2 ± 2.8°, 73.5 ± 2.6°, 69.2 ± 2.4°, and 65.5 ± 2.8°, respectively (Fig. S8). The pores were formed after the evaporation of water and thus GST act as pore wall in the films. The GST, being hydrophilic and its presence on the surface as well as on the pore walls induced the spreading of water droplets, causing lower contact angles for the composites. Fig. 7. FTIR spectra of PLA, GST and H-PLA/3GST.
4.2.6. Water vapor transmission rate (WVTR) studies The WVTR of H-PLA/1GST (1%), H-PLA/2GST (2%), H-PLA/3GST (3%) and H-PLA/4GST (4%) honeycomb films was studied at 37.8 ± 0.5 °C under 80% relative humidity, Fig. S9. The water vapor permeability for pristine PLA film was found to be 111 ± 32 gm2.24 h. The WVTR values increased on increasing filler contents up to 3% (up to 2365 ± 64 g/m2.24 h), which may be due to the presence of uniform pores on the substrate surface. On increasing filler content to 4 wt %, the uniform distribution of filler content was lost which may block the pores, resulting in lower WVTR for H-PLA/4GST [58].
Table 1 The formulations of samples with sample codes. Sample code
Sample description
PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
2.5% PLA 99% PLA + 98% PLA + 97% PLA + 96% PLA +
1% 2% 3% 4%
n-GST n-GST n-GST n-GST
Fig. 8. SEM image of Honeycomb film of (a) H-PLA/1GST (b) H-PLA/2GST (c) H-PLA/3GST (d) H-PLA/4GST and (onset) corresponding zoomed area. 314
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Fig. 9. (a) Tensile strength and (b) % of elongation of H-PLA/GST nanocomposites.
4.2.7. Hemolysis The nanocomposites H-PLA/3GST was studied for its compatibility with blood so as to function as a medical dressing material. The degree of hemolysis from the assay shows the extent of damage of erythrocytes. Absorbance in the test for the samples exposed to PLA, H-PLA/ 1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST were 0.036 ± 0.0012, 0.041 ± 0.0022, 0.058 ± 0.0034, 0.073 ± 0.0031 and 0.085 ± 0.0049, respectively. The hemolysis rate obtained for PLA, H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST were 0.24 ± 0.08, 0.53 ± 0.12, 1.53 ± 0.24, 2.423 ± 0.31 and 3.132 ± 0.27, respectively (Fig. 10), which was far below the threshold value of 5%. The hemolytic study stipulates the application of the fabricated composites as wound dressing materials [59,60].
while PLA/4GST nanocomposites with lower nano sized GST content than H-PLA/3GST, fluctuate from the observed trend in inhibition zone against S. aureus as illustrated in Table 3. The lowering of zone diameter with higher GST concentration can be attributed to the agglomeration effect of GST particles and an irregular morphology. The bactericidal property of the samples depends on morphology, size, surface area etc. The nano sized particles with more surface area shows good bactericidal property [61–63]. The maximum zone diameter was for H-PLA/3GST against S. aureus bacteria in visible conditions, as presented in Fig. 11. H-PLA/3GST shows enhanced antibacterial activity even after 30 min of visible light irradiation, when compared to other H-PLA/GST nanocomposites. This may be due to the uniform distribution of GST particles or band gap narrowing or excellent antimicrobial activity of piper nigrum [64–66]. The striking reasons for the action of GST in the present studied PLA matrix as an effective photocatalyst, could be due to the capability of the material to absorb or reflect visible light [67]. The colour of GST particles synthesized was not purely white, but they had a greyish shade for the particles (shown in Fig. 3a (onset)). It was reported that the carbon doped TiO2 can narrow the band gap and promote the electronic excitation between the bands [68]. The role of carbon in improving the reduction of rate of recombination of electrons/holes by capture of electrons, makes the photocatalyst highly reactive [69]. Even though there are numerous discussions on this area, the actual mechanism still remains unknown. The antibacterial action of GST in the presence of visible light was thus suggested as represented in Scheme 2. The reactive hydroxyl radicals thus generated react with cell membranes, DNA, and thus cause the cell death in bacteria. The GST on exposure to visible light creates photo induced electrons and holes. The electrons reduces the dissolved oxygen to superoxide anion (O−•) and the generated holes on the surface react with water forms hydroxyl radicals (OH•) and oxidative radicals (HO2•). These radicals destroy the cell membrane, causing deformation in cell structure leading to ultimate cell death. This increased availability of the GST particles at the surface, enhance the bactericidal activity as discussed earlier. This reveals that the porous and nanoparticle incorporated film have the ability to induce the death of approaching bacteria.
4.2.8. Antimicrobial activity The inhibition zone around the disks on the agar plates shows the antibacterial activity of PLA, H-PLA/1GST, H-PLA/2GST, H-PLA/3GST, and H-PLA/4GST against S. aureus, which was demonstrated under dark and visible light to confirm their enhanced photocatalytic effect owing to the increased surface area of nanocomposites (Fig. 11). The inhibition zone diameters for H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites were 18, 22, 28 and 24 mm respectively,
4.2.9. Cyto-toxicity For the purpose of wound dressing application, the fabricated PLA, H- PLA/GST films, was tested for its cytocompatibility using mouse fibroblast L929 cells for specific time intervals i.e., 24, 72 and 120 h, respectively. The morphology of the substrates is a known parameter
Fig. 10. Hemolysis percentage of H-PLA/1GST, H-PLA/2GST, H-PLA/3GST and H-PLA/4GST nanocomposites. 315
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Fig. 11. Antibacterial activity of H-PLA/GST against S. aureus bacteria (a) dark condition (b) visible irradiation. Table 3 Antibacterial activity of different concentration of GST on H-PLA/GST nanocomposites under dark and visible condition. Sample
Zone of inhibition in visible condition (mm)
Zone of inhibition under dark condition (mm)
PLA H-PLA/1GST H-PLA/2GST H-PLA/3GST H-PLA/4GST
Nil 18 22 28 24
Nil Nil Nil Nil Nil
Fig. 12. Optical image of mouse fibroblasts L929 on (a) H-PLA/1GST (b) HPLA/2GST (c) H-PLA/3GST and (d) H-PLA/4GST nanocomposites after 5 days.
Scheme 2. Schematic representation of generation of reactive oxygen species (ROS) in n-GST.
for the cell-substrate adhesion [70,71]. Fig. 12 shows confocal laser scanning microscopy (CLSM) images of fibroblast L929 cells cultured on porous H-PLA/GST films and control substrate (Fig. S10). The CLSM shows that the cells seeded on H-PLA/ 1GST and H-PLA/4GST films with non-uniform pores had retained the spherical morphology with few irregular protrusions even after 120 h. But at the same time, H-PLA/3GST films with the uniform pore size (Fig. 12c) had seeded cells with more flattened morphology, indicating improved cell adhesion and spreading. The studies validate better adhesion with the seeded fibroblast L929 cells for H-PLA/3GST films than H-PLA/1GST, H-PLA/2GST and H-PLA/4GST. MTT cell viability assay was carried out to evaluate the cell proliferation of the nanocomposites (Fig. 13).
Fig. 13. Cell viability on the H-PLA/1GST, H-PLA/2GST, H-PLA/3GST, and HPLA/4GST films represented by the cell numbers determined by MTT assay at day 1, 3 and 5.
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The cell viability of porous H-PLA/GST composites ascertained the fact that it is due to the presence of GST particles in the modified PLA films. The well known and accepted statement that ‘the pore size greatly influences the cell behaviour’ is the basic concept employed in the work. The pore size and pore uniformity modulation with different wt% of GST had significantly varied the cell behaviors such as adhesion, spreading or proliferation [32].
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5. Conclusion Hierarchically porous H-PLA/GST nanocomposites were successfully synthesized through a green method and used for wound dressing application for the first time. The phase analysis data obtained for GST coincides well with the standard JCPDS (File No. 99-101-095) data validating the rutile phase of synthesized GST nanoparticles. The addition of plant extract in the precursor solution inhibited the appearance of brookite and anatase phase. The functionality of synthesized GST, such as O-Ti and –OH vibrational bands, various functional groups present in proteins, polyols, alkaloids from leaf extract were analysed through FTIR spectrum. The spherical morphology as well as the average particle size of around 6.45 ± 0.81 nm were confirmed from TEM images. The SAED pattern and the XRD pattern were found to be complementary to each other. The presence of carbon (confirmed from Electron diffraction spectrum and X-ray photoelectron spectroscopy) at the interstitial sites of GST was found to cause synergetic effect in the enhancement of the desired properties. Further, antibacterial activity of GST as a function of concentration was tested against S. aureus. The plant extract/TiO2 composite showed tremendously improved antibacterial activity against S. aureus in visible conditions. The enhanced visible light absorption is assumed to be due to the narrowing of band gap as a result of carbon doping onto GST. The eco friendly and cost effective GST with its notable bioactivity can thus be recommended as an effective wound healing dressing material owing to its remarkable antimicrobial potential against S. aureus. Acknowledgements This research was financially supported by Ministry of Human Resource Development (MHRD), India. We would like to thank Dr. G. Unnikrishnan, R. Rarima, Jithin Raj and P.K. Adnan, Department of Chemistry, NIT Calicut for their help with XRD and DLS measurements. We extend our gratitude to Dr. Shibu and Krishnakumar (STIC, CUSAT) for their cooperation in recording TEM. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.matchemphys.2019.03.045. References [1] J.C. Middleton, A.J. Tipton, Synthetic biodegradable polymers as orthopaedic devices, Biomaterials 21 (2000) 2335–2346. [2] H. Eufinger, C. Rasche, J. Lehmbrock, M. Wehmoller, S. Weihe, I. Schmitz, C. Schiller, M. Epple, Performance of functionally graded implants of polylactides and calcium phosphate/calcium carbonate in an ovine model for computer assisted craniotomy and cranioplasty, Biomaterials 28 (2007) 475–485. [3] R.K. Kulkarni, K.C. Pani, C. Neuman, F. Leonard, Polylactic acid for surgical implants, Arch. Surg. 93 (1966) 839–843. [4] S.S. Ray, P. Maiti, M. Okamoto, K. Yamada, K. Ueda, New polylactide/layered silicate nanocomposites 1. Preparation, characterization and properties, Macromolecules 35 (2002) 3104–3110. [5] A. González, C.I.A. Igarzabal, Stereocomplex formation between enantiomeric poly (lactics), Food Hydrocoll 33 (2013) 289–296. [6] H. Yamane, Y. Furuhashi, N. Yoshie, Y. Kimura, Higher-order structures and mechanical properties of stereocomplex- type poly(lactic acid) melt spun fibers, Polymer 47 (2006) 5965–5972. [7] Y. Ikada, K. Jamshidi, H. Tsuji, S.H. Hyon, Stereocomplex formation between enantiomeric poly(lactics), Macromolecules 20 (1987) 904–906. [8] Y. Li, H. Shimizu, Toughening of polylactide by melt blending with a biodegradable
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