Abstract
A novel route for obtaining ordered microporous polylactide films has been explored using zinc oxide and a Schiff base. The SEM images reveal that the water vapor condensation near the surface of a polylactide solution containing a synthesized Schiff base and zinc oxide results in well-dispersed water droplets, which upon subsequent evaporation can assist breath figure patterning. The generation of ordered micropores at the film surface is attributed to the interaction between the surface +ve charge of well-dispersed zinc oxide particles and the δ-bearing water droplets from moisture. The potential for biocompatible applications has been revealed for the membranes by the cell viability assay against mice fibroblast (L929) cells and the hemocompatibility assay. The findings suggest an efficient route for the development of porous biodegradable polylactide membranes for various applications, typically for wound dressing.
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Introduction
Polymer membranes play a vital role in various applications such as wastewater treatment [1], food and preservation [2] and biomedical engineering [3,4,5]. Interesting attempts have been made by researchers for the fabrication of porous polymer membranes. The reported methods for their fabrication include porogen leaching, gas foaming, phase separation, electrospinning and 3D printing [6,7,8,9,10,11,12,13]. Porogen leaching technique involves the blending of a water-soluble porogen such as sodium chloride or sodium citrate with a biodegradable polymer followed by solution casting. After drying, the water-soluble porogens are leached out, forming pores within the polymer membrane. The quality and quantity of leachable particles have direct influence on the pore size distribution [14, 15]. The gas foaming technique for porous membrane fabrication does not utilize organic solvents, but generally uses high-pressure carbon dioxide gas. The membranes are initially exposed to high-pressure gas, which is later lowered to cause thermodynamic instability followed by nucleation, within the matrix. The dissolved gas diffuses into the nuclei to create macropores. The porosity developed depends upon the amount of gas dissolved and the diffusion rate of the gas molecules through the pores [16, 17].
In many polymeric systems, phase separation occurs due to the thermodynamic demixing of the homogenous polymer solution on exposure to a non-solvent. The solvent is removed by freeze drying to obtain the polymer with a porous topography. Thermally induced, solid–liquid and liquid–liquid phase separation routes are categorized under this technique [18,19,20]. Electrospinning is an interesting technique to produce fibers with controlled diameter, ranging from micrometers to nanometers. Electrospun fibers are prominently used in biomedical fields as scaffolds, drug carriers, wound dressing materials and so on [21]. The solvent for dissolving the polymer can influence the surface morphology of the nanofiber. Bognitzki et al. reported PLLA fibers with uniform pore structures caused by the rapid phase separation of dichloromethane [22]. 3D printing is a technique that creates porous 3D structure on the polymer with a computer-aided design. This method has drawn lot of attention due to the wide applicability in biomedical field. Kim et al. evaluated the survival of hepatocytes on microporous 3D polymer scaffolds [23]. The scaffolds were found to possess micropores with diameter 45–150 μm. PLA scaffolds with pore size ranging from 38 to 150 μm were fabricated by Zeltinger et al. using the combination of 3D printing and salt leaching technique [24].
Among the various techniques for the fabrication of porous membranes with biocompatibility, solvent casting followed by breath figure (BF) formation is a facile approach that allows the control of different characteristics such as pore size and pore interconnection degrees. Hiroshi et al. [25] described the control of cell adhesion by microporous polymer films. They utilized polycaprolactone (PCL) as a substrate for obtaining honeycomb morphology with 5 μm pore size for growing endothelial cells. The size of the periodic microstructures in BFs falls within the range of hundreds of nanometers to tens of micrometers [26, 27]. BF films with pore size of 4.6 μm have been found to show enhanced cell adhesion and proliferation [28]. The inhibition of cancer cells and bacteria by BF films has been investigated by Ma et al. [29]. They attributed the suppression to the growth of cancer cells due to the hydrophobicity of the matrix and the presence of air in the pores.
Interesting investigations for biocompatible porous films have been carried out using polymers such as polylactide (PLA), polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA) [30,31,32]. PLA films, for a variety of applications, have been developed and characterized by different research groups [33,34,35].
The present work highlights the development of ordered microporous PLA films using zinc oxide (ZnO) and a Schiff base through breath figure formation. The utilization of a Schiff base for the effective dispersion of ZnO particles in PLA matrix and the subsequent microporous patterning has not yet been explored. The films developed through the work have been characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction technique (XRD), thermogravimetric analysis (TGA), differential scanning calorimeter (DSC) and UV–visible spectroscopy. The water vapor transmission rate, antibacterial activity and hemolytic activity of the films have also been examined in view of the possible utilization of the films for wound healing. The in vitro cytotoxicity of the films was evaluated by using mouse fibroblast cells.
Experimental
Materials
Benzil (98%) and 2-aminopyridine (99%) were procured from Alfa Aesar, England. Polylactide was supplied by Sigma-Aldrich, Germany, and ZnO was purchased from Loba Chemie, India. The solvents used were methanol, hexane, dichloromethane (Merck, India) and absolute ethanol (Hayman, UK). All the chemicals were used as received, without further purification.
Synthesis of Schiff base
The Schiff base, N-(1,2-diphenyl-2-(pyridin-2-ylimino)ethylidene)pyridin-2-amine (B), was synthesized (Fig. 1) as reported earlier [36]. In a typical synthesis step, 20 mL methanolic solution of 2-aminopyridine (2 mmol) was added dropwise to a methanolic solution of benzil (1 mmol) and was refluxed overnight till a pale yellow color appeared. The solvent was evaporated under reduced pressure. The precipitate, obtained after solvent evaporation under reduced pressure, was washed three times with cold methanol and recrystallized using absolute ethanol.
Synthesis route for N-(1,2-diphenyl-2-(pyridin-2-ylimino)ethylidene)pyridin-2-amine
Preparation of porous membranes
Porous membranes were developed through the formation of breath figures in the polylactide matrix with the Schiff base (B), prepared as above, and zinc oxide as modifiers. The membranes, designated as P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO, were prepared with ZnO/B ratios, viz. 1:1, 3:3, 5:5 wt%, with respect to the wt% of polylactide in dichloromethane, respectively. The polylactide and Schiff base (B) were directly dissolved in dichloromethane, while ZnO was dispersed in the solution by sonication. The solutions were casted into films in a petri dish at 80% relative humid atmosphere. After the solvent evaporation, the films were dried in vacuum oven for 8 h to remove the moisture content to attain surface patterned membranes.
Characterization
The structure of the synthesized Schiff base was confirmed by proton nuclear magnetic resonance (1H NMR) spectroscopy. The dried sample was dissolved in DMSO and was analyzed using a Bruker Avance III proton NMR (400 MHz) at 25 °C. The functionality of Schiff base and its interaction with PLA and ZnO were examined using a Fourier transform infrared (FTIR) spectrometer (Jasco 4700) in the region of 400–4000 cm−1.
The morphology and the pore diameter distribution were studied by a field emission scanning electron microscope (FE-SEM; Hitachi Su 66000) at an accelerating voltage of 5 kV. Each sample was coated with Au/Pd (5 nm) in a vacuum sputtering facility before making SEM observations. The diameter of the pores from different zones of SEM images was analyzed by using ImageJ analysis software (image J 1.50i).XRD patterns of the samples under investigation were recorded at room temperature using a Rigaku miniflex 600 benchtop at a scanning speed of 15°/min with CuKα (λ = 1.540 Å) as X-ray source and with a scanning range of 5°–90°.
The transmittance behavior of the membranes was examined by UV–visible spectroscopy, recorded over a range of 200–800 nm with a UV spectrophotometer (UV-2600 SHIMADZU). TGA analysis was performed with a Mettler Toledo thermal analyzer (TGA/TA Instruments, Q50). For this, approximately 5 mg of the samples was heated at a rate of 10 °C min−1 from room temperature to 700 °C under nitrogen atmosphere (flow rate 50 mL min−1). DSC scans of PLA and P-3B-3ZnO were done at 20 °C min−1 from 0 to 200 °C under a nitrogen flow of 50 mL min−1.
Porosity measurement
The porosity (%P) of the composite membranes was measured by using ImageJ analysis software as [37]:
Water vapor transmission rate (WVTR)
WVTR is a measure of the rate of water passing through a film for a fixed time period. The membranes were cut into circular shapes with 1.6 cm diameter and were placed on the top of four different glass vials containing 10 mL water. Constant humidity was maintained by placing them in a desiccator, and the weight of the vials was measured at regular intervals up to 24 h for each film. The WVTR, evaluated at 37.8 ± 0.5 °C under 80% relative humidity, was calculated using the equation [38]:
where ‘S’ is the slope (g/h) of the plot between change in weight in grams (g) and time in hours (h) and ‘A’ is the area (m2) of the membrane.
Antibacterial activity
The in vitro antibacterial activities of the samples against Escherichia coli (EC8099) were examined by the zone inhibition method [39]. Initially, the bacteria were cultivated in a nutrient agar for 24 h. Diluted bacteria suspensions were then spread on the agar plate. The circular-shaped films, both PLA and P-B-ZnO (6 mm), were placed on the agar plate containing lawn of Escherichia coli (E. Coli), followed by incubation at 37 °C for a day.
Hemocompatibility assay
The potential to use the membranes as a wound dressing material was verified by hemolysis assay. The anticoagulant blood was diluted with 0.9% saline and was stored as a stock solution. Circular membranes with 6 mm diameter were immersed in 8 mL of 0.9% saline and were incubated at 37 °C for about half an hour. 200 μL of the stock solution was added to every sample and also to distilled water (positive control or PC) and 0.9% saline (negative control or NC). Each solution was then incubated at 37 °C for an hour. The supernatant after centrifuging was checked for its absorbance at 541 nm. The percentage of hemolysis (HP) was calculated as [40]:
where OD represents the optical density or absorbance.
Cell viability studies
As per ISO10993-5, the cytotoxicity of the samples was evaluated using L-929 mouse fibroblast cell culture. The cells were seeded on a 48-well plate, and the films were introduced after incubation. The viable cells after the test were quantified by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [41]. During the assay, the cells reduce the tetrazolium salt (MTT) into purple-colored formazan in the presence of succinate dehydrogenase. The number of viable cells would be proportional to the intensity of purple color obtained. The phosphate-buffered saline (PBS) was used to wash the cell culture, to which 200 μL MTT/mL of culture was added, and was incubated for 3 h. 300 μL of dimethyl sulfoxide (DMSO) was added to each well, incubated further for 30 min and then centrifuged. The cells treated with MTT solution without the samples are set as control. The absorbance at 540 nm was measured spectrophotometrically. The % viability was calculated using Eq. (4),
where Atest is the absorbance of cell with sample after MTT assay and Acontrol is the absorbance of cell treated with MTT (without sample).
Results and discussion
Characterization of Schiff base
The Schiff base, N-(1,2-diphenyl-2-(pyridin-2-ylimino)ethylidene)pyridin-2-amine (B) was subjected to FTIR and proton NMR analyses for the confirmation of the successful condensation reaction between benzil and 2-aminopyridine.
Proton NMR analysis (1H NMR)
The compound ‘B’ shows 4 resonance signals [(Fig. 2a): 7.64 (t) ppm and 7.8 (m) ppm (–CH– of benzene ring); 7.9 (dd) ppm (–CH– of pyridine ring); and 8.24 (s) ppm (–CH– of pyridine ring, α C=N)].
a 1H NMR spectrum and b FTIR spectrum of Schiff base (B)
The intense peak at 3–4 ppm corresponds to DMSO-H2O, and that at 2–3 ppm denotes the chemical shift for the deuterated solvent, DMSO-d6.
Fourier transform infrared (FTIR) spectroscopy
The FTIR spectrum of the synthesised Schiff base (B) is given in Fig. 2b. The vibrational stretching frequencies at 1658 and 1675 cm−1 correspond to the azomethine (> C=N–) group in the Schiff base. The band at 3060 cm−1 is assigned to aromatic C–H stretching, and that at 1592 cm−1 corresponds to the C=C stretching. The C–N stretching frequency is indicated at 1324 cm−1.
The aromatic C–H bending frequency is indicated at 875, 795 and 718 cm−1, respectively. The bands corresponding to the azomethine group confirm the effective condensation of benzil with 2-aminopyridine.
Investigation of microporous composite films
The synthesized Schiff base (B) was used as a dispersing agent for ZnO particles in the polylactide membrane. Figure 3 shows the macrostructure of the PLA matrix containing the Schiff base and ZnO. The introduction of B has been observed to improve the flexibility of PLA. The interaction between the polylactide chains and the Schiff base additive is dependent on the coordination between the nitrogen atom in Schiff base and the carbonyl group in polylactide chain [42], weakening the interaction between polymer chains. This subsequently facilitates the uniform distribution of ZnO in the matrix. Our experiments showed that the Schiff base could easily disperse ZnO particles in PLA up to 5 wt%; however, even 1 wt% of ZnO dispersion was difficult in the absence of the former.
Schematic representation for the occupancy of modifier between the chains
The observed development of uniform porous structure within the matrix can be attributed to a breath figure patterning, caused by the excellent distribution of ZnO. The fast evaporation of dichloromethane (solvent used) from the polylactide/Schiff base/ZnO dispersion initially causes the reduction in air–solution interface temperature below atmospheric temperature. Subsequently, water droplets from the atmosphere condense onto the surface, which gets evenly aligned by the ZnO particles that can also penetrate through the polylactide matrix to create intense pores. Ordered microporous polymer films are formed after the complete evaporation of solvent followed by water droplets [43]. The coalescence of water droplets is prevented by surfactants, which could stabilize the droplets and forms, ordered pores on the surface of the polymer film [44].The fabricated composites were investigated for their morphology and properties.
Morphological analysis
The SEM images (Fig. 4a–d) show that the composites possess a porous structure, while polylactide alone is nonporous. This is attributed to the breath figure formation, in which dichloromethane acts as the solvent and moisture in the atmosphere acts as the non solvent.
Pore formation on PLA membrane: a PLA alone, b PLA/ZnO, c PLA/Schiff base, d PLA/Schiff base/ZnO (P-3B-3ZnO)
The dispersion of ZnO alone in the polylactide solution causes the formation of pores (Fig. 4b); however they are non-uniformly distributed as ZnO precipitation could occur after dispersion. The addition of Schiff base alone results in single layer pores, only on the surface, which cannot penetrate through the layers of the polymer matrix (Fig. 4c). The addition of ZnO particles with the aid of Schiff base results in the formation of interconnected pores within the membrane (Fig. 4d).
A threshold concentration of ZnO has been found to be ideal for the uniform patterning of pores in PLA. Figure 5 shows the SEM images of PLA, P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO. The P-1B-1ZnO does not contain sufficient ZnO to develop homogeneity. The P-5B-5ZnO system has been found to be overloaded with ZnO, as the uniformity in pore distribution can be disrupted by ZnO agglomeration.
SEM images of: a PLA, b P-1B-1ZnO, c P-3B-3ZnO, d P-5B-5ZnO
The homogeneity in ZnO dispersion can be observed in the SEM image displayed in Fig. S1. The smaller ZnO particles are predominantly seen in P-3B-3ZnO, while agglomerated particles are found on the surface of P-5B-5ZnO (Figure S1). A relatively, narrow distribution has been observed for the P-3B-3ZnO system compared to the other two compositions.
The zinc oxide particles possess a strong surface (+) charge which can form electrical bondages with the δ- in the water droplets [45]. The well-dispersed ZnO particles, with the aid of Schiff base, due to their smaller size effectively penetrate through the matrix along with moisture and the subsequent evaporation of moisture from the matrix results in the interesting architecture.
The distribution diagram (Fig. 6) shows that the average pore diameter of the composites P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO is 5.52±1.35, 6.36±1.36 and 3.88± 0.94 μm, respectively. The percentage of porosity of P-1B-IZnO, P-3B-3ZnO and P-5B-5ZnO composites is 52, 63 and 37%, respectively. The porosity was found to be least for P-5B-5ZnO due to the higher filler concentration. The introduction of porosity on the surface of a biocompatible membrane can enhance cell adhesion, proliferation and differentiation [46].
Histograms for pore size distribution in PLA composites; a P-1B-1ZnO, b P-3B-3ZnO and c P-5B-5ZnO
Attenuated total reflectance Fourier transform (ATR-FTIR) spectroscopy
The IR spectra for neat PLA and the membranes (P-1B-1ZnO, P-3B-3ZnO, P-5B-5ZnO) are displayed in Fig. 7. It is observed that all the membranes possess the characteristic frequencies, viz. 2994 and 2944 cm−1 (symmetric and asymmetric vibrations of > C–H stretching), 1747 cm−1 (–C=O stretch), 1453 and 1381 cm−1 (symmetric and asymmetric C–H bending vibrations) and 1180 and 1079 cm−1 (–C–O–C stretch), corresponding to the fundamental features of PLA.
ATR-FTIR spectra: a PLA, b P-1B-1ZnO, c P-3B-3ZnO, d P-5B-5ZnO
The higher –C=O peak intensity for P-1B-1ZnO clearly highlights the lower interaction of ZnO with the carbonyl group of the polymer. The decreased intensity of –C=O stretching frequency for P-3B-3ZnO is definitely due to the better interaction of ZnO particles with the –C=O group of ester in PLA. The re-appearance of a maximum intense peak at 1747 cm−1 for P-5B-5ZnO is the manifestation of ZnO agglomeration after the threshold concentration.
Additionally, the IR spectra highlight the improvement in the crystallinity of the composites with the dispersion of ZnO particles. The two bands at 865 and 755 cm−1 correspond to the amorphous (*A) and crystalline (*C) phase of PLA, respectively [47,48,49]. It is clear from the spectra that the crystalline nature of PLA has been increased with the addition of ZnO (observed from the increased intensity of crystalline peak and the decreased intensity of amorphous peak). The negligible crystalline peak intensity at 755 cm−1 for P-1B-1ZnO could be attributed to the non availability of sufficient amount of ZnO as an additive compared to the P-3B-3ZnO and P-5B-5ZnO systems.
X-ray diffraction analysis
The enhancement in crystallinity with the Schiff base-assisted incorporation of ZnO into polylactide has been confirmed through XRD analysis as shown in Fig. 8; with characteristic peaks at 2θ = 31.8°, 34.4°, 36.3° for P-3B-3ZnO and P-5B-5ZnO and two extra peaks at 2θ = 56.5°, 68° for P-3B-3ZnO. The highly intense peak at 36.3° corresponds to the wurtzite structure of ZnO. These observations are in good agreement with JCPDS: 80-0075 of wurtzite ZnO. The increase in crystalline peak intensity can be attributed to the increase in number of diffracting particles in the respective planes. As observed from FTIR spectrum, the composites P-3B-3ZnO and P-5B-5ZnO are dominantly crystalline in nature due to the presence of sufficient ZnO particles within the matrix.
XRD patterns of PLA and PLA composites
It is observed that PLA–ZnO film prepared without the Schiff base and that with a lower ZnO concentration do not show any crystallinity. The absence of crystalline peaks for PLA–ZnO and P-1B-1ZnO (with very low ZnO concentration) might be due to the entrapment of ZnO particles within the matrix, without appropriate dispersion. The average sizes of ZnO fillers (Table 2) were estimated according to Debye–Scherrer equation [50]:
where λ is the wavelength of X rays and K is a dimensionless constant; β—the width of the XRD peak at half maximum; θ—the diffraction angle; and D—the size of the crystalline domains (K = 0.9, λ = 0.154 nm, β = 0.043 rad). The indexed XRD data are given in Table 1.
An enhancement in crystallinity with the incorporation of ZnO particles in a polyester matrix has earlier been reported [51].
UV–visible spectroscopy
Transmission of UV and visible light through PLA films, incorporated with ZnO particles and the Schiff base, recorded at a range of 200–800 nm is presented in Fig. 9. PLA alone shows no UV transmittance in the lower range of UV (at UV-C); however, after 230 nm (UV-A and UV-B) its transmittance increases significantly.
UV blocking property and transparency of PLA and PLA composites
Interestingly, the PLA and the composites have been found to be highly transparent, i.e., about 99% in the visible region (400–800 nm). The percent transmittance (%T) of the pure PLA films in UV range has been found to be decreased with the addition of ZnO. The neat PLA film exhibits transparency with a transmittance value (at 300 nm) of 71.59%, while the P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO composites show very low transmittance values, i.e., 2.16, 0 and 11.24%, respectively. The UV cutoff in the transmittance spectrum of PLA is seen to be around 200–230 nm, while that for composites is found to be in the range of 200–370 nm.
The Schiff base-assisted uniform distribution of ZnO particles in the polylactide has been observed to trigger excellent UV shielding property for the latter. The P-3B-3ZnO is found to block the entire wavelengths in UV region. This can be attributed to the intrinsic UV-screening features of ZnO particles coupled with the multiple reflections of UV rays in the interior cavities of the porous membrane.
For utilizing PLA films as wound dressing materials, one may require additives those are resistant to UV rays. The ZnO particles are found to play a critical role in the UV shielding property of membranes [52]. Evenly distributed ZnO particles in the PLA matrix hinder or scatter the light falling on it. The blocking of UV-B and UV-C rays is critical in wound healing, as they can damage nucleic acids or proteins and may lead to cell death [53].
Thermal analysis
Thermal stability of pure PLA, P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO is shown in Fig. 10. The TGA curve of PLA shows only one-step degradation attributed to the loss of ester groups from the polymer [54]. The incorporation of ZnO particles into the PLA reduces the thermal stability of the polymer, which is directly related to the amount of ZnO particles [50, 55]. The catalytic effect of ZnO could be the reason for this observation [56].
TGA curves of PLA and PLA composites
The thermal decomposition of neat polymer begins (T5wt%) at 308.23 °C and reaches the maximum (Tpeak) at 342.89 °C as shown in Table 2. According to the thermogram, the composites begin to degrade almost 100 °C below than that of unreinforced PLA. The residue has been found to be increased with an increase in the content of ZnO particles. Even though there is a reduction in the thermal stability of PLA with the incorporation of ZnO, all the membranes with ZnO have been found to be stable up to 200 °C, which is sufficient for many of their practical applications, typically for wound healing materials. The effect of filler addition on the Tg value of PLA has been further evaluated by differential scanning calorimetry (DSC) and is discussed in the supporting information (Section 1.2, Figure S2).
Water vapor transmission rate
The water vapor transmission rate (WVTR) of PLA, P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO films is shown in Fig. 11. The WVTR is related to the slope (plot of change in weight: ∆W and change in time: ∆t) (Fig. S3) and area of the membrane.
WVTR for the films monitored in 1 day
It is seen that the WVTR values increase up to 2101 ± 35 g/m2· 24 h, upon the addition of 3% ZnO into the membrane. This is definitely associated with the microporous structure with interconnected micropores. However, a further increase in ZnO causes a reduction in WVTR to 1865 ± 20 g/m2·24 h because of the slow disintegration of the uniform porous structure, after the threshold value, attributed to the ZnO agglomeration. The decreased porosity for P-5B-5ZnO due to the higher concentration of additives within the same polymer concentration has been found to be the contributing factor for the diminished WVTR.
For effective wound healing, a moist atmosphere is essential and dehydration should be minimum. The dressing with a material possessing an optimal WVTR, in the range of 2101 ± 35 g/m2·24 h, can enhance the cell proliferation [57].
Antibacterial activity
The bactericidal activity of polylactide and its composites (Fig. 12) was checked against E. coli bacteria. All the samples except P-3B-3ZnO have been found to be inert toward the bacteria. It has been observed that the composite P-3B-3ZnO possesses the highest inhibition zone with a diameter of 39.34 mm, with E. coli. The lowest activity of P-1B-1ZnO might be due to the non-availability of ZnO on its surface for effective contact with bacterial cell wall and its rupture, while ZnO agglomeration in P-5B-5ZnO (Fig. S1) causes a reduced activity for it against the bacteria.
Resistance study of PLA and PLA composites against E. coli
Hemocompatibility assay
A major issue to be evaluated for materials to act as wound healers is their biocompatibility, particularly hemocompatibility. The hemolytic tendency of the films was investigated and was quantified by noting the absorbance at 541 nm (Fig. 13a). It was observed that the spectra of all the samples were closer to the negative (0.9% saline) control and were far less than positive control (water) spectrum. The test was carried out to demonstrate the non-toxicity of Schiff base and ZnO-incorporated PLA on direct contact with blood. The absorbance at 541 nm is directly related to the hemoglobin concentration. The released free hemoglobin from the blood has been found to be very low as attested by the very low absorbance obtained for the samples.
Hemocompatibility analysis a UV–visible spectrum and b hemolytic percentage histogram of PLA and PLA composites
Further, the hemolytic percentage was calculated for a comparative evaluation of samples with the positive and negative controls. The hemolytic percentage (Fig. 13b) of PLA, P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO was found to be 0.2 ± 0.05, 0.66 ± 0.17, 1.6 ± 0.22 and 1.93 ± 0.29%, respectively. The allowed hemolytic percentage is 5% [40]. The assay recommends the non-hemolytic nature of all the samples under study.
Cell viability studies
An in vitro cytotoxicity study was carried out on the membranes for evaluating the harmful effect of materials released by the membrane in the cell cultured medium. The base polymer PLA is known to be biocompatible, and the cytotoxic effect of Schiff base and ZnO-incorporated membrane has not been reported yet. Figure 14 shows the phase contrast optical microscope images of the cells seeded on the membranes, P-1B-1ZnO (Fig. 14b), P-3B-3ZnO (Fig. 14c) and P-5B-5ZnO (Fig. 14d) after 5 days. The MTT assay results (Fig. 15) show that the membranes, P-1B-1ZnO and P-3B-3ZnO possess relatively good cell viability in comparison with the control. A toxicating effect has been observed for P-5B-5ZnO, which might be due to the cell damage caused by the agglomerated ZnO particles on the surface of the membranes [58].
Phase contrast optical microscope images of the cells with: a control, b P-1B-1ZnO, c P-3B-3ZnO, d P-5B-5ZnO
Cell viability of P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO after 1 day, 3 days and 5 days
The variation in the properties of ZnO such as size, morphology, purity would influence the cytotoxicity. The electrons in ZnO particles can chemisorb oxygen to form O2− ions and generate hydrogen peroxide (H2O2) [59].
The presence of H2O2 is known to induce cell proliferation. It is also known that a higher content of H2O2 can adversely affect the healing process due to cell toxication [60]. Thus, it follows that the composition P-3B-3ZnO would be ideal for wound healing applications.
Conclusions
A Schiff base viz., N-(1,2-diphenyl-2-(pyridin-2-ylimino)ethylidene)pyridin-2-amine was synthesized and was innovatively utilized as a ZnO dispersing agent as well as a hydrophilic filler in a PLA membrane, to generate uniform pores. The observed development of interconnected pores in the membranes loaded with ZnO and Schiff base has been attributed to breath figure formation. The average pore diameter of the composites with different concentrations of Schiff base and ZnO, i.e., for P-1B-1ZnO, P-3B-3ZnO and P-5B-5ZnO, has been found to be 5.52 ± 1.35, 6.36 ± 1.36 and 3.88 ± 0.94 μm, respectively. The crystallinity introduced upon the incorporation of ZnO particles into the PLA matrix has been confirmed by both ATR-FTIR and XRD studies. The percentage of transmittance in the UV region for the composites was found to be lower than that for the pure PLA membrane, highlighting the UV shielding property inducted by ZnO and the newly generated porous topography. The values of water vapor transmission rate (WVTR) for the composites (1984 ± 31 g/m2·24 h, 2101 ± 35 g/m2·24 h, 1865 ± 20 g/m2·24 h) have been found to be ideal for wound dressing applications. The adhesion and proliferation of mice fibroblast cells on the membranes were monitored to evaluate the cytocompatibility. The composite, P-3B-3ZnO with excellent UV light screening, optimal WVTR, less hemolytic nature, antibacterial property and cytocompatibility have been proposed as an ideal system for wound dressing applications.
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Acknowledgements
R.R. acknowledges MHRD, India, for a research fellowship. The authors thankfully acknowledge the help by Dr. A. Sujith and Mr. Manaf O., Materials Research Laboratory, NITC.
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Rarima, R., Asaletha, R. & Unnikrishnan, G. Schiff base-assisted surface patterning of polylactide–zinc oxide films: generation, characterization and biocompatibility evaluation. J Mater Sci 53, 9943–9957 (2018). https://doi.org/10.1007/s10853-018-2338-9
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DOI: https://doi.org/10.1007/s10853-018-2338-9