# **Modification and Processing of Biodegradable Polymers**

Edited by Krzysztof Moraczewski Printed Edition of the Special Issue Published in *Materials*

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## **Modification and Processing of Biodegradable Polymers**

## **Modification and Processing of Biodegradable Polymers**

Editor

**Krzysztof Moraczewski**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Krzysztof Moraczewski Department of Materials Engineering Kazimierz Wielki University Bydgoszcz Poland

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Materials* (ISSN 1996-1944) (available at: www.mdpi.com/journal/materials/special issues/modification process biodegradable polym).

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 Currently, he is the Dean of the Faculty of Materials Engineering. He is the author or co-author of 100 scientific publications. He conducts research in the field of materials engineering, which is one of the dynamically developing areas of polymer science and methods of their modification. In his scientific work, he deals with issues related to the modification and metallization of the surface layer of polymers and processes related to the aging of polymer materials.

## *Article* **Characterization of a Sandwich PLGA-Gallic Acid-PLGA Coating on Mg Alloy ZK60 for Bioresorbable Coronary Artery Stents**

#### **Li-Han Lin 1 , Hung-Pang Lee 2 and Ming-Long Yeh 1,3,\***

Received: 8 November 2020; Accepted: 2 December 2020; Published: 4 December 2020

**Abstract:** Absorbable magnesium stents have become alternatives for treating restenosis owing to their better mechanical properties than those of bioabsorbable polymer stents. However, without modification, magnesium alloys cannot provide the proper degradation rate required to match the vascular reform speed. Gallic acid is a phenolic acid with attractive biological functions, including anti-inflammation, promotion of endothelial cell proliferation, and inhibition of smooth muscle cell growth. Thus, in the present work, a small-molecule eluting coating is designed using a sandwich-like configuration with a gallic acid layer enclosed between poly (d,l-lactide-co-glycolide) layers. This coating was deposited on ZK60 substrate, a magnesium alloy that is used to fabricate bioresorbable coronary artery stents. Electrochemical analysis showed that the corrosion rate of the specimen was ~2000 times lower than that of the bare counterpart. The released gallic acid molecules from sandwich coating inhibit oxidation by capturing free radicals, selectively promote the proliferation of endothelial cells, and inhibit smooth muscle cell growth. In a cell migration assay, sandwich coating delayed wound closure in smooth muscle cells. The sandwich coating not only improved the corrosion resistance but also promoted endothelialization, and it thus has great potential for the development of functional vascular stents that prevent late-stent restenosis.

**Keywords:** magnesium alloy; cardiovascular stents; callic acid; dip coating; endothelialization; anticorrosion

#### **1. Introduction**

According to reports from the Global Health Organization in 2016, cardiovascular diseases (CVDs), the prevalence of which increased by 15% in the past decade, are the leading cause of death (44%) among non-communicable diseases [1]. In clinical settings, a percutaneous coronary intervention (PCI) combined with balloon angioplasty and stent implantation is the gold standard for treating stenotic arteries. Currently, dual antiplatelet therapy is suggested for at least 12 months to alleviate mural thrombosis caused by PCI [2]. The traditional materials used in cardiovascular stents are 316 L stainless steels, cobalt-chromium alloys, and nickel-titanium alloys [3]. However, the permanent installation of devices inside the human body causes a chronic inflammatory local reaction and long-term endothelial dysfunction [4]. Biodegradable magnesium (Mg) and its alloys are ideal candidates for stent platforms because they have less neointimal formation and provide long-term inhibition of the growth of smooth muscle cells (SMCs), unlike stainless steel [5,6]. The Mg alloys also have twice the tensile strength of

unmodified biodegradable poly-l-lactide (PLLA), which leads to flexible stenting deployment and better radial force [7–9].

To properly use a magnesium alloy as a biodegradable stent platform for physiological applications, the corrosion rate must be controlled. To date, the surfaces of degradable magnesium alloys have been modified in several ways, such as alkaline heat treatment (AHT), the sol-gel process, chemical conversion, and micro-arc oxidation (MAO) [10,11]. Although ceramic MAO processing may provide a consolidated surface [12–14] for clinical applications, a prior study showed that this process did not improve corrosion resistance after three months [15]. In contrast, a sol-gel coating can greatly improve the corrosion resistance with chemical modifications [16]. Polymetric processes create a physical barrier [17] that enhances the corrosion resistance of Mg-stents, and the barrier also serves as an absorbable drug-loading system [18,19]. A polymer coating of a specific thickness can prevent complete degradation of Mg alloys for periods of 1 to 12 months [20].

Phenolic molecules, such as gallic acids (GA), exert and induce specific levels of selective viability in human endothelial cells (ECs) and SMCs [21–24]. Furthermore, these extracted molecules have anti-oxidation characteristics that inhibit inflammation. Since cellular antioxidants prevent the formation of oxidized low-density lipoprotein (LDL) and antiplatelet activation, this is a remarkable method by which to treat atherosclerosis [25]. Although several articles have indicated that the phenolic conversion coating on a Mg-alloy is non-toxic and anti-corrosive, chemical conversion may compromise stent integrity through the phenolic-Mg conversion process [26–29].

Mg-Zn alloys are well-known for their good corrosion resistance and physiological safety [30]. The ZK60 Mg-6Zn-0.5Zr alloy has been used to make vascular stents due to its better biocompatibility than those of other Mg alloys [31]. In addition, ZK60 has a high tensile strength (315 MPa) that prolongs the radial force of Mg stents. Nevertheless, the rapid degradation rate of bare ZK60 represents a shortcoming of the alloy for its use to fabricate bioresorbable coronary artery stents [15]. In this paper, a coating comprising of GA sandwiched between two layers of Poly (d,l-lactide-co-glycolide) (PLGA), a novel sandwich coating, on Mg alloy ZK60 concept was proposed. It can serve not only as a protective barrier that increases corrosion resistance but also as a reservoir for controlling GA release. The surface properties of coated-Mg stent and corrosion behavior of a Mg alloy were determined. Furthermore, cell viability and cell migration tests were performed to investigate the cell regulation of released GA in endothelialization as well as the effects on the growth of smooth muscle cells.

#### **2. Materials and Methods**

#### *2.1. Materials and Specimen Preparation*

In this study, extruded commercial ZK60 alloy bars (Zn 5.48 wt.%, Zr 0.42 wt.% and balance Mg) comprised the starting material for the substrate [32]. PLGA in molar ratio of 85/15 and GA with purity above 97.5% were purchased from Sigma-Aldrich (St. Louis, MO, USA; Figure 1a). ZK60 disks cut from the bars were reduced to 12 mm in diameter and 5 mm in thickness (Figure 1c). These specimens were ground with silicone carbide sandpaper (150–5000 mesh) and polished. Before being dried in a stream of air, all specimens were rinsed ultrasonically with acetone, ethanol, and distilled water for 5 min, respectively. Mg-stent was machined by an INTAI Technology and CHONG HUAI laser (Taichung, Taiwan) for further evaluation.

#### *2.2. Sandwich Coating Films Preparation*

Mirror-polished ZK60 specimens were soaked in an alkaline solution composed of 20 wt.% NaOH. The solution was stirred at 240 rpm and then heated for 90 min at 60 ◦C to equilibrate the specimens. After 90 min, the specimens were drawn out of the alkaline solution and rinsed with deionized water. The rinsed specimens were incubated at 80 ◦C for 30 min until they were dried. Subsequently, heat treatment at 120 ◦C for another 30 min was conducted to stabilize the oxide film. To fabricate the sol-gel PLGA coating layer, PLGA was dissolved at a concentration of 4 wt.% in 10 mL of chloroform. The PLGA film thickness depended on the drawing speed of 3 mm/s, and the film was dried in a stream of air to form a uniform dip-coating surface. Subsequently, different coating layers were prepared with or without GA solution. The samples were dipped into GA solution at a concentration of 1 wt.% and 10 mL acetone. Finally, the samples were ultrasonically rinsed with ethanol and air-dried. The coating steps were conducted on the Mg-stent for surface morphology observation.

#### *2.3. Characterization of the Surface, the Cross-Section Structure, and the Elements Content*

The crystallinity of ZK60 was analyzed using thin film X-ray diffraction (TF-XRD Bruker D8 Discover, Brucker, Karlsruhe, Germany) with Cu-Kα radiation. Diffraction patterns were acquired at 2θ values of 20–80◦ . The surface morphology and element distributions of the coating films were studied by scanning electron microscopy (SEM JSM-6700F, JEOL, Tokyo, Japan) under 10 kV acceleration voltage and energy dispersive spectrometry (EDS JSM-6700F, JEOL, Tokyo, Japan) under 0.2 keV acquisition energy, respectively. The structures of the films were recorded with a Fourier transform-infrared (FT-IR) spectrophotometer (FTIR-4600, Jasco, Tokyo, Japan) at a transmitter ratio (T%) and infra spectra resolution of 4 cm−1 . The spectra were collected in the range of 600–4000 cm−1 .

#### *2.4. Electrochemical Corrosion and Hydrogen Evolution Tests*

The electrochemical and hydrogen evolution tests were performed in revised simulated body fluid (r-SBF) solution (per liter, 0.072 g of NaSO4, 0.182 g of K2HPO4, 0.225 g of KCl, 0.310 g of MgCl2 6H2O, 0.736 g of NaHCO3, 0.923 g of CaCl2, 2.036 g Na2CO3, 5.403 g of NaCl, and 11.928 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic avid (HEPES) dissolved in deionized water), respectively. In the electrochemical corrosion tests, a PARSTAT 2273 electrochemistry workstation (PARSTAT 2273, AMETEK, Berwyn, PA, USA) was operated at a scanning rate of 1 mV s−1 at −2.0 V to 1.0 V with a step height of 2.5 mV [12]. A reference saturated calomel electrode (SCE KCl) combined with a conventional three-electrode electrode cell and a platinum plate was used for the electrochemical analysis. The area of the working electrode exposed to the electrolyte was controlled by a polylactic acid (PLA) holder to within 1 cm2 . Each sample was tested for once and immersed in r-SBF solution at least three hours [12]. In hydrogen evolution test, all samples (n = 3) were freshly prepared and then placed in the r-SBF solution for 48 h at a pH of 7.4 and a temperature of 37 ◦C. The equipment then recorded the total volume of the hydrogen released from the magnesium alloys [32].

#### *2.5. Cytocompatibility Evaluation*

The cytocompatibility tests examined cytotoxicity, cell proliferation, and migration. A human umbilical vein cell line (EA. hy926) and a rat aortic smooth muscle cell line (RASMC) were provided by Wen-Tai Chiu (National Cheng Kung University, Tainan, Taiwan). The growth behaviors of the EA. hy926 and RASMC represented the cardiovascular ECs and SMCs, respectively. Before cell culturing, the sterile samples were immersed in free fetal bovine serum Dulbecco's Modified Eagle Medium (FBS DMEM) (approximately 1.25 cm2 /mL in DMEM) for 24-h extraction. The resulting filtered (0.2 µm filter) medium was then diluted to a 15 mL volume. The cells were seeded separately at a density of 4000 cells/well in 96-well plates. The 90% DMEM with 10% dimethyl sulfoxide (DMSO) was used as the positive control, and 90% DMEM with 10% FBS was used as the negative control. The culture medium was replaced with 90% extracted medium with 10% FBS overnight. Before collection of the optical density (OD) values at 450 nm, cell counting kit-8 (CCK8) solution was added into each well and the mixture was incubated for 2 h. Briefly, the cell proliferation was recorded on days 1, 4, and 7. Three replications were conducted and then the results were calculated using Equation (1) below.

Relative growth rate % = [(OD test − OD positive)/(OD negative − OD positive)] × 100% (1)

#### *2.6. Hemolysis Tests Materials* **2020**, *13*, x FOR PEER REVIEW 4 of 15

The hemolysis tests were performed according to the ISO 10993-4 standard for biomaterials [33]. Sodium citrate (4 wt.%) to the fresh blood samples in the ratio of 1:9 was taken 30 min before the tests. All specimens were immersed in centrifuge tubes containing 10 mL of normal saline and incubated for 30 min at 37 ◦C. Deionized water was used as the positive control, and normal saline was used as the negative control. After 30 min of incubating, 0.2 mL of the diluted blood, prepared with normal saline at a volume ratio of 4:5, was added into the centrifuge tubes, and all the tubes were incubated for 60 min at 37 ◦C. After 60 min, the tubes were centrifuged for 5 min at 2500 rpm, and the supernatant was collected and carefully transferred to a 96-well plate for spectroscopic measurement. The hemolysis data, read by ELISA, were calculated using Equation (2) below and were based on the average of three replications: The hemolysis tests were performed according to the ISO 10993-4 standard for biomaterials [33]. Sodium citrate (4 wt.%) to the fresh blood samples in the ratio of 1:9 was taken 30 min before the tests. All specimens were immersed in centrifuge tubes containing 10 mL of normal saline and incubated for 30 min at 37 °C. Deionized water was used as the positive control, and normal saline was used as the negative control. After 30 min of incubating, 0.2 mL of the diluted blood, prepared with normal saline at a volume ratio of 4:5, was added into the centrifuge tubes, and all the tubes were incubated for 60 min at 37 °C. After 60 min, the tubes were centrifuged for 5 min at 2500 rpm, and the supernatant was collected and carefully transferred to a 96-well plate for spectroscopic measurement. The hemolysis data, read by ELISA, were calculated using Equation (2) below and were based on the average of three replications:

> Hemolysis % = [(OD test − OD negative)/(OD positive − OD negative)] × 100% (2) Hemolysis % = [(OD test − OD negative)/(OD positive − OD negative)] × 100% (2)

(**a**)

(**b**)

(**c**)

**Figure 1.** Composition of ZK60 (**a**) [34], the steps in the dip-coating procedure (**b**), and FT-IR spectra of GA, PLGA, and the sandwich coating (**c**). **Figure 1.** Composition of ZK60 (**a**) [34], the steps in the dip-coating procedure (**b**), and FT-IR spectra of GA, PLGA, and the sandwich coating (**c**).

#### *2.7. Free Radical Activity Tests*

The free radical activity tests, measured diphenyl-2-picrylhydrazyl (DPPH), were performed according to application on bleached teeth and several antioxidant tests on magnesium alloys [34]. The DPPH powder was diluted in 0.2 mmol/mL DPPH solution with 70% ethanol. All specimens were immersed in 2 mL of DPPH solution and incubated at 37 ◦C for 1 h in the dark. Similarly, DPPH solution was used as the control group. Then the absorbance of DPPH data, read by microplate reader, were calculated using Equation (3) below based on the average of three replications:

$$\text{Inhibition } \%= \text{[(OD control - OD test)(OD control)]} \times 100\% \tag{3}$$

#### *2.8. Statistical Analysis*

In this study, all quantitative results are expressed as standard deviation (SD) unless indicated. Each assay was performed in at least three replicated tests, as described above. Measured experimental results from GraphPad Prism software (Prism 9.0, GraphPad, San Diego, CA, USA) were analyzed by a non-parametric test (Kruskal-Wallis Test) combined with Uncorrected Dunn's multiple comparisons test (each comparison stands alone), and a *p*-value < 0.05 was considered significant.

#### **3. Results**

#### *3.1. Modification of the ZK60 Surface*

An as-extruded ZK60 disk scanned by XRD indicated three peaks at 32.2◦ , 34.5◦ , and 36.8◦ , corresponding to Mg in Appendix A (Figure A1). As-extruded ZK60 cut disks were modified with AHT to develop a layer of magnesium hydroxide as Equation (4) below. The Mg(OH)2 layer was then treated at high temperature to form a rough MgO layer as Equation (5) following by the PLGA dip coating. These chemical reactions produced a less-active ZK60 surface and a compact void-free oxide for the dip-coating process. The void-free structure prevented inner bulk erosion on the polymer layer and external pitting corrosion on the highly active magnesium alloy. In comparison to the bare ZK60, the MgO coating had higher corrosion resistance to delay magnesium ions from bursting out in Appendix A (Figure A2). The sandwich coating steps were schematized and are depicted in order in Figure 1b. As illustrated in that figure, the bare ZK60 substrate was first treated with AHT and then coated with sandwich coating layers. Since hydrophilic GA was unable to dissolve in the oil phase, the hydrophobic PLGA was able to remain compact in the sandwich layer coating process.

The FT-IR analysis confirmed the encapsulation of GA in the polymer sandwich layers (Figure 1c). The wavenumbers of the functional group in GA were sourced from a published FT-IR article [35]. The presence of the benzene signal at 1482 cm−1 allowed a decisive characterization of the phenolic GA compound even though PLGA and GA shared several similar functional groups. The GA group had benzene vibration peaks at 1482 cm−1 , and PLGA did not, while the interference of C=O (1750 cm−1 ) and C-O (1250–1100 cm−1 ) vibration peaks caused a broad benzene peak of the sandwich coating from 1600 cm−1 to 1300 cm−1 . Also, the O-H peaks, representing tri-hydroxyl groups at 3683 cm−1 , provided substantial evidence that the sandwich coating immobilized the GA in the sandwich structure.

$$\rm Mg^{2+}(aq) + 2OH^{-}(aq) \rightleftharpoons Mg(OH)\_{2}(s) \tag{4}$$

$$\text{Mg(OH)}\_{2}\text{(s)} \xrightarrow{\Delta} \text{MgO(s)} + \text{H}\_{2}\text{O(g)}\uparrow\tag{5}$$

#### *3.2. E*ff*ects of PLGA Dip-Coating and Phenolic Layer on the Coating Morphology*

The SEM images of the sandwich coating showed that the AHT-modified ZK60 surface became more uniform after a series of dip-coating processes (Figure 2). Initially, the morphology of the MgO after AHT was rough and coarse. After the PLGA dip-coating, a non-homogeneous microstructure formed on the MgO surface. After the second PLGA dip-coating, the top PLGA film layer smoothed the rough GA layer in the form of a sandwiched-layer structure. In addition, the SEM images indicated that PLGA and sandwich coatings had film thicknesses of 1.1 ± 0.4 µm and 2.1 ± 0.3 µm, respectively, in the cross-section view. The thickness of the coating's cross-sections was proportionate with the number of PLGA layers. formed on the MgO surface. After the second PLGA dip-coating, the top PLGA film layer smoothed the rough GA layer in the form of a sandwiched-layer structure. In addition, the SEM images indicated that PLGA and sandwich coatings had film thicknesses of 1.1 ± 0.4 µm and 2.1 ± 0.3 µm, respectively, in the cross-section view. The thickness of the coating's cross-sections was proportionate with the number of PLGA layers.

**Figure 2.** SEM images of the coating steps: MgO, PLGA-MgO, GA/PLGA-MgO, and Sandwich-MgO, the cross-section images of PLGA-MgO (1.1 ± 0.4 µm) and Sandwich-MgO (2.1 ± 0.3 µm). **Figure 2.** SEM images of the coating steps: MgO, PLGA-MgO, GA/PLGA-MgO, and Sandwich-MgO, the cross-section images of PLGA-MgO (1.1 ± 0.4 µm) and Sandwich-MgO (2.1 ± 0.3 µm).

EDS detection confirmed that the sandwich layers covered the bare ZK60, as shown in the elemental distribution in Figure 3a. In the MgO layer, magnesium and oxygen accounted for 36.9% and 63.1%, respectively, while in the final sandwich coating film, Mg and oxygen (O) accounted for 7.2% and 23.4%, respectively, with the balance being carbon (C). The decreases in the contents of Mg and O with the layers verified that the PLGA coating protected the MgO surface. Furthermore, the rising C contents of GA and PLGA indicated bare Mg substrate was covered by the coating. Next, an EDS line scan was applied to analyze the elemental changes in the sandwich coating (Figure 3b,c). Based on the changes in the ratios of the element weights, the highest content of Mg was in the ZK60 region. Due to the rich oxygen contents of MgO, GA, and PLGA, the increasing O and declining Mg contents indicated the MgO layer and sandwich coating. Furthermore, the rising carbon signal originated from the mounting epoxy resin. The film thickness observed in the SEM images was consistent with the EDS analysis measured from the first Mg–O crossing point to the second Mg–C crossing point in the PLGA and sandwich coating films. EDS detection confirmed that the sandwich layers covered the bare ZK60, as shown in the elemental distribution in Figure 3a. In the MgO layer, magnesium and oxygen accounted for 36.9% and 63.1%, respectively, while in the final sandwich coating film, Mg and oxygen (O) accounted for 7.2% and 23.4%, respectively, with the balance being carbon (C). The decreases in the contents of Mg and O with the layers verified that the PLGA coating protected the MgO surface. Furthermore, the rising C contents of GA and PLGA indicated bare Mg substrate was covered by the coating. Next, an EDS line scan was applied to analyze the elemental changes in the sandwich coating (Figure 3b,c). Based on the changes in the ratios of the element weights, the highest content of Mg was in the ZK60 region. Due to the rich oxygen contents of MgO, GA, and PLGA, the increasing O and declining Mg contents indicated the MgO layer and sandwich coating. Furthermore, the rising carbon signal originated from the mounting epoxy resin. The film thickness observed in the SEM images was consistent with the EDS analysis measured from the first Mg–O crossing point to the second Mg–C crossing point in the PLGA and sandwich coating films.

To evaluate the dip-coating effect on the ZK60 stent platform, the coating steps mentioned above were repeated on the ZK60 stent prototype. Although the ZK60 stent developed irregular corrosive struts without the coating, the coated Mg-stent retained its stent integrity, and a uniform polymer coating formed on its surface (Figure 4).

(**a**)

with the number of PLGA layers.

formed on the MgO surface. After the second PLGA dip-coating, the top PLGA film layer smoothed the rough GA layer in the form of a sandwiched-layer structure. In addition, the SEM images indicated that PLGA and sandwich coatings had film thicknesses of 1.1 ± 0.4 µm and 2.1 ± 0.3 µm, respectively, in the cross-section view. The thickness of the coating's cross-sections was proportionate

**Figure 2.** SEM images of the coating steps: MgO, PLGA-MgO, GA/PLGA-MgO, and Sandwich-MgO,

EDS detection confirmed that the sandwich layers covered the bare ZK60, as shown in the elemental distribution in Figure 3a. In the MgO layer, magnesium and oxygen accounted for 36.9% and 63.1%, respectively, while in the final sandwich coating film, Mg and oxygen (O) accounted for 7.2% and 23.4%, respectively, with the balance being carbon (C). The decreases in the contents of Mg and O with the layers verified that the PLGA coating protected the MgO surface. Furthermore, the rising C contents of GA and PLGA indicated bare Mg substrate was covered by the coating. Next, an EDS line scan was applied to analyze the elemental changes in the sandwich coating (Figure 3b,c). Based on the changes in the ratios of the element weights, the highest content of Mg was in the ZK60 region. Due to the rich oxygen contents of MgO, GA, and PLGA, the increasing O and declining Mg contents indicated the MgO layer and sandwich coating. Furthermore, the rising carbon signal originated from the mounting epoxy resin. The film thickness observed in the SEM images was

the cross-section images of PLGA-MgO (1.1 ± 0.4 µm) and Sandwich-MgO (2.1 ± 0.3 µm).

**Figure 3.** EDS composition analysis of (**a**) different layers and cross-sections of the (**b**) PLGA, and (**c**) sandwich coating films. **Figure 3.** EDS composition analysis of (**a**) different layers and cross-sections of the (**b**) PLGA, and (**c**) sandwich coating films. struts without the coating, the coated Mg-stent retained its stent integrity, and a uniform polymer coating formed on its surface (Figure 4).

**Figure 4.** Mg-stent prototype, and the SEM images of the bare strut and sandwich-coated strut. **Figure 4.** Mg-stent prototype, and the SEM images of the bare strut and sandwich-coated strut.

#### *3.3. Anti-Corrosion Behavior 3.3. Anti-Corrosion Behavior*

better corrosion resistance.

better corrosion resistance.

**Figure 4.** Mg-stent prototype, and the SEM images of the bare strut and sandwich-coated strut. *3.3. Anti-Corrosion Behavior*  The hydrogen evolution was recorded for 48 h. The results indicated the highest H2 volume for the untreated ZK60, and the lowest, for sandwich coating (Figure 5a). The electrochemical analysis showed results comparable to those for hydrogen release. The lowest corrosion density of the sandwich coating, acquired from the potentiodynamic polarization curves, suggested that the sandwich coating improved the corrosion resistance (Figure 5b). The unmodified ZK60 had the lowest corrosion potential, −1.6 V, and the highest corrosion current density, 20.51 µA/cm2, which corresponded to the H2 release, indicating that the bare surfaces did suffer a severe corrosion attack 120 *µm* 120 *µm* 1 *mm* The hydrogen evolution was recorded for 48 h. The results indicated the highest H2 volume for the untreated ZK60, and the lowest, for sandwich coating (Figure 5a). The electrochemical analysis showed results comparable to those for hydrogen release. The lowest corrosion density of the sandwich coating, acquired from the potentiodynamic polarization curves, suggested that the sandwich coating improved the corrosion resistance (Figure 5b). The unmodified ZK60 had the lowest corrosion potential, −1.6 V, and the highest corrosion current density, 20.51 µA/cm2, which corresponded to the H2 release, indicating that the bare surfaces did suffer a severe corrosion attack in the physiological environment (Table 1). Furthermore, the PLGA had a higher corrosion potential, −0.4 V, and a lower corrosion current density, 1.79 µA/cm2, and the sandwich coating also exhibited a slightly increased corrosion potential of −0.2 V and a decreased corrosion current density of 0.01 µA/cm2 than those of the bare ZK60. These findings suggested a significant difference between PLGA and sandwich coating in terms of electrochemical performance. This sandwich structure provided a The hydrogen evolution was recorded for 48 h. The results indicated the highest H2 volume for the untreated ZK60, and the lowest, for sandwich coating (Figure 5a). The electrochemical analysis showed results comparable to those for hydrogen release. The lowest corrosion density of the sandwich coating, acquired from the potentiodynamic polarization curves, suggested that the sandwich coating improved the corrosion resistance (Figure 5b). The unmodified ZK60 had the lowest corrosion potential, −1.6 V, and the highest corrosion current density, 20.51 µA/cm2 , which corresponded to the H2 release, indicating that the bare surfaces did suffer a severe corrosion attack in the physiological environment (Table 1). Furthermore, the PLGA had a higher corrosion potential, −0.4 V, and a lower corrosion current density, 1.79 µA/cm2 , and the sandwich coating also exhibited a slightly increased corrosion potential of −0.2 V and a decreased corrosion current density of 0.01 µA/cm2 than those of the bare ZK60. These findings suggested a significant difference between PLGA and sandwich coating in terms of electrochemical performance. This sandwich structure provided a better corrosion resistance.

in the physiological environment (Table 1). Furthermore, the PLGA had a higher corrosion potential, −0.4 V, and a lower corrosion current density, 1.79 µA/cm2, and the sandwich coating also exhibited a slightly increased corrosion potential of −0.2 V and a decreased corrosion current density of 0.01

**Figure 5.** Corrosion resistance tests: (**a**) Hydrogen evolution curves of ZK60, PLGA coating and sandwich coating and (**b**) Potentiodynamic polarization curves of ZK60, PLGA coating, and sandwich coating in SBF solution. **Figure 5.** Corrosion resistance tests: (**a**) Hydrogen evolution curves of ZK60, PLGA coating and sandwich coating and (**b**) Potentiodynamic polarization curves of ZK60, PLGA coating, and sandwich coating in SBF solution.

**Table 1.** Fitting results for the potentiodynamic polarization curves related to Figure 5b. The inhibition efficiency was calculated as "η % = [1 − (Icorr. sample/Icorr. Bare ZK60)] × 100%". **Table 1.** Fitting results for the potentiodynamic polarization curves related to Figure 5b. The inhibition efficiency was calculated as "η% = [1 − (Icorr. sample/Icorr. Bare ZK60)] × 100%".

#### *3.4. Effect of Phenolic Molecules on ECs and SMCs in TERMS of Cell Viability and Hemolysis 3.4. E*ff*ect of Phenolic Molecules on ECs and SMCs in TERMS of Cell Viability and Hemolysis*

The sandwich coating layers promoted EC proliferation but inhibited the growth of SMCs (Figure 6). Changes in GA gradient resulted in varying EC vitality, demonstrating the relationship between EC tolerance and GA toxicity in Appendix A (Figure A3). The outcome demonstrated that the GA 1 wt.% group had better viability than did the other two groups, while the highlyconcentrated GA suggested that direct over-exposure could cause apoptosis due to the high antioxidant activity. After comparison to different groups, including bare (ZK60), PLGA and sandwich coating, at 1, 4, and 7 days, sandwich coating showed no toxicity to ECs (Figure 6c). The sandwich coating layers promoted EC proliferation but inhibited the growth of SMCs (Figure 6). Changes in GA gradient resulted in varying EC vitality, demonstrating the relationship between EC tolerance and GA toxicity in Appendix A (Figure A3). The outcome demonstrated that the GA 1 wt.% group had better viability than did the other two groups, while the highly-concentrated GA suggested that direct over-exposure could cause apoptosis due to the high antioxidant activity. After comparison to different groups, including bare (ZK60), PLGA and sandwich coating, at 1, 4, and 7 days, sandwich coating showed no toxicity to ECs (Figure 6c).

Based on the cell viability of the ECs and SMCs, there were two notable points: the non-toxic growth rate of sandwich coating towards ECs, and the selective suppression of SMCs (Figure 6a,b). In comparison to bare ZK60, the PLGA and sandwich coatings exhibited significant viability in ECs. The SMCs exhibited proliferation in the PLGA group, while sandwich coating only had 90% viability after four days. Even though the proliferation of the ECs declined on Day 4, the decline could have been due to the high content of GA released in a static environment. The efficiency of GA, while not significant in the SMCs, was moderate in strength. These results indicated that the ECs had a robust proliferation growth rate of 150% compared to the SMC group, and sandwich coating inhibited SMC over-growth. None of the groups exhibited toxicity in the comparison tests; thus, the four-day test was sufficient to observe the growth trend, while a seven-day test would have soon reached 100% coverage and lost the trend in our relative growth study. Based on the cell viability of the ECs and SMCs, there were two notable points: the non-toxic growth rate of sandwich coating towards ECs, and the selective suppression of SMCs (Figure 6a,b). In comparison to bare ZK60, the PLGA and sandwich coatings exhibited significant viability in ECs. The SMCs exhibited proliferation in the PLGA group, while sandwich coating only had 90% viability after four days. Even though the proliferation of the ECs declined on Day 4, the decline could have been due to the high content of GA released in a static environment. The efficiency of GA, while not significant in the SMCs, was moderate in strength. These results indicated that the ECs had a robust proliferation growth rate of 150% compared to the SMC group, and sandwich coating inhibited SMC over-growth. None of the groups exhibited toxicity in the comparison tests; thus, the four-day test was sufficient to observe the growth trend, while a seven-day test would have soon reached 100% coverage and lost the trend in our relative growth study.

Sandwich coating presented a hemolysis ratio below 5%, corresponding to the clinical bio-safety standard (Figure 6d). The bare ZK60 had a high hemolysis ratio of more than 43%, while both sandwich coating and PLGA showed lower hemolysis rates of 2.1% and 3.8%, respectively. Overall, the sandwich demonstrated a promising ability to regulate ECs and SMCs with excellent hemocompatibility. Sandwich coating presented a hemolysis ratio below 5%, corresponding to the clinical bio-safety standard (Figure 6d). The bare ZK60 had a high hemolysis ratio of more than 43%, while both sandwich coating and PLGA showed lower hemolysis rates of 2.1% and 3.8%, respectively. Overall, the sandwich demonstrated a promising ability to regulate ECs and SMCs with excellent hemocompatibility.

**Figure 6.** The cell viability evaluation of (**a**) ECs and (**b**) SMCs on day 1, day 4, (**c**) coatings versus EC proliferation (\* *p* < 0.05 and \*\* *p* < 0.01, mean ± SD, N = 3) and (**d**) the hemolysis test. **Figure 6.** The cell viability evaluation of (**a**) ECs and (**b**) SMCs on day 1, day 4, (**c**) coatings versus EC proliferation (\* *p* < 0.05 and \*\* *p* < 0.01, mean ± SD, N = 3) and (**d**) the hemolysis test.

#### *3.5. Anti-Oxidation 3.5. Anti-Oxidation*

The linear regression retrieved from a gradient GA content test defined a standard reference of GA released weight and concentration (Figure 7a). The sandwich coating film triggered GA weight release at 1, 2, 3, and 6 h, with a reduced speed after three hours (Figure 7b). The free radical scavenging analysis showed that the bare, PLGA, and sandwich coatings could eliminate oxidant stress with antioxidant capacities of approximately 28%, 36%, and 63%, respectively. These data verified that the GA released from the sandwich coating film promoted scavenging of free radicals and protected the vascular tissue due to its significant anti-oxidative ability, suggesting that the sandwich coating is a promising material for vascular stents (Figure 7c). The linear regression retrieved from a gradient GA content test defined a standard reference of GA released weight and concentration (Figure 7a). The sandwich coating film triggered GA weight release at 1, 2, 3, and 6 h, with a reduced speed after three hours (Figure 7b). The free radical scavenging analysis showed that the bare, PLGA, and sandwich coatings could eliminate oxidant stress with antioxidant capacities of approximately 28%, 36%, and 63%, respectively. These data verified that the GA released from the sandwich coating film promoted scavenging of free radicals and protected the vascular tissue due to its significant anti-oxidative ability, suggesting that the sandwich coating is a promising material for vascular stents (Figure 7c).

#### *3.6. Cell Migration*

(**a**) (**b**) The sandwiched-layer structure ensured that the GA had an anti-proliferative effect on the SMCs and a slight influence on EC migration (Figure 8). Re-endothelialization at the lesion site is a particularly important standard after PCI and requires healthy EC proliferation, migration, and spreading. Additionally, ideal regulation of SMCs and ECs could prevent penetration from SMCs as well as late-stent restenosis. As the previous viability results showed, GA has a specific sensitivity to SMCs and ECs at GA−1wt.% (~4 µg/mL) in the sandwich-structured film. The results indicated a robust EC migration without a significant difference in migration length among all groups (Figure 8a). In contrast, the SMCs had a larger migration distance in the PLGA group than in the other groups, while the sandwich and control groups were alike (Figure 8b).

*3.5. Anti-Oxidation*

(**a**) (**b**)

(**c**) (**d**) **Figure 6.** The cell viability evaluation of (**a**) ECs and (**b**) SMCs on day 1, day 4, (**c**) coatings versus EC

*Materials* **2020**, *13*, x FOR PEER REVIEW 10 of 15

The linear regression retrieved from a gradient GA content test defined a standard reference of GA released weight and concentration (Figure 7a). The sandwich coating film triggered GA weight release at 1, 2, 3, and 6 h, with a reduced speed after three hours (Figure 7b). The free radical scavenging analysis showed that the bare, PLGA, and sandwich coatings could eliminate oxidant stress with antioxidant capacities of approximately 28%, 36%, and 63%, respectively. These data verified that the GA released from the sandwich coating film promoted scavenging of free radicals

proliferation (\* *p* < 0.05 and \*\* *p* < 0.01, mean ± SD, N = 3) and (**d**) the hemolysis test.

sandwich coating is a promising material for vascular stents (Figure 7c).

**Figure 7.** The anti-oxidation analysis of (**a**) the DPPH/GA efficiency curve, (**b**) the sandwich coating film release GA curve and (**c**) free radical scavenging activity (\*\*\* *p* < 0.001, mean ± SD, N = 6). **Figure 7.** The anti-oxidation analysis of (**a**) the DPPH/GA efficiency curve, (**b**) the sandwich coating film release GA curve and (**c**) free radical scavenging activity (\*\*\* *p* < 0.001, mean ± SD, N = 6). for SMCs. The results were consistent with the cytocompatibility test, indicating that sandwich coating preserved the viability of ECs and inhibited the SMCs with the release of GA.

coating preserved the viability of ECs and inhibited the SMCs with the release of GA. (**a**) **Figure 8.** *Cont.*

(**a**)

**Figure 8.** Cell migration of (**a**) EC, (**b**) SMC at 0, 24, and 48 h, and the fitting results for (**c**) EC and (**d**) SMC migration coverage (mean ± SEM, N = 3). **Figure 8.** Cell migration of (**a**) EC, (**b**) SMC at 0, 24, and 48 h, and the fitting results for (**c**) EC and (**d**) SMC migration coverage (mean ± SEM, N = 3).

**4. Discussion**  One of the goals of surface modification of ZK60 is to enhance its corrosion resistance in simulated body fluid and hence improve its potential for use in fabricating bioresorbable coronary artery stents. Mg(OH)2 forms a passive layer on the magnesium surface through AHT [36]. This process protects the Mg surface from ion attack and prevents the accelerated degradation of ZK60 magnesium, which is supported by the polarization test in Figure A2. Based on the limitations in our lab, discussing the LA:GA ratio of PLGA and the coating techniques was beyond the scope of the An overall analysis of the ECs and SMCs after 48 h showed 81% and 42% migration from sides to the middle wound closure, respectively, in the control group. In the PLGA group, both the ECs and SMCs exhibited migration of 90% and 55%, respectively (Figure 8c,d). In the bare group, the ECs exhibited mild movement of 68%, and the SMCs showed no significant difference. Furthermore, the sandwich coating, similarly to the control group, displayed migration rates of 82% for ECs and 38% for SMCs. The results were consistent with the cytocompatibility test, indicating that sandwich coating preserved the viability of ECs and inhibited the SMCs with the release of GA.

#### study.  Discussion**

degradation of the PLGA (50:50) occurred after 102 days, whereas only about 60% of the PLGA (85:15) degraded within the same period [37]. Therefore, we simply discuss the weight percentage of PLGA (based on the 85:15 ratio) for its long-term degradation process and chose the best group as the control group in this study. An oxide-passive layer on the Mg-disk led to a coarse surface until the second PLGA layer was deposited on it to fill the cracks (Figure 2), yet this passive layer prevented the initial hydrogen evolution from the ZK60 surface and prevented the acid degradation products of the PLGA layer from penetrating directly into the ZK60 substrate. This phenomenon explains why the NaOH passive layer is vital to make PLGA layer more completed and concrete on the coated surface. Although dip-coating might not be ideal for stent coating due to the complicated geometry, this technique can be simply conducted and qualifies for further bench testing, such as cytocompatibility and immersion test. One of the goals of surface modification of ZK60 is to enhance its corrosion resistance in simulated body fluid and hence improve its potential for use in fabricating bioresorbable coronary artery stents. Mg(OH)2 forms a passive layer on the magnesium surface through AHT [36]. This process protects the Mg surface from ion attack and prevents the accelerated degradation of ZK60 magnesium, which is supported by the polarization test in Figure A2. Based on the limitations in our lab, discussing the LA:GA ratio of PLGA and the coating techniques was beyond the scope of the study. A comprehensive degradation study on PLGA (50:50) and (85:15) revealed that complete degradation of the PLGA (50:50) occurred after 102 days, whereas only about 60% of the PLGA (85:15) degraded within the same period [37]. Therefore, we simply discuss the weight percentage of PLGA (based on the 85:15 ratio) for its long-term degradation process and chose the best group as the control group in this study.

An oxide-passive layer on the Mg-disk led to a coarse surface until the second PLGA layer was deposited on it to fill the cracks (Figure 2), yet this passive layer prevented the initial hydrogen evolution from the ZK60 surface and prevented the acid degradation products of the PLGA layer from penetrating directly into the ZK60 substrate. This phenomenon explains why the NaOH passive layer is vital to make PLGA layer more completed and concrete on the coated surface. Although dip-coating might not be ideal for stent coating due to the complicated geometry, this technique can be simply conducted and qualifies for further bench testing, such as cytocompatibility and immersion test.

Many studies have investigated the use of phenolic molecules to regulate endothelialization based on their specific properties on SMCs and ECs [28,29]. These applications not only prevent the over-growth of SMCs from invading the EC wall but also assist in the formation of a uniform endothelial layer in the vascular system. Further, the goal of drug elution changed from anti-immunity to anti-proliferation since sirolimus (SR) replaced paclitaxel (PTX) as the main drug system for drug-eluting stent [38]. We believe our sandwiched GA concept can bring a novel direction to apply small-molecule on eluting stent instead of traditional SR or PTX. In this paper, the healing process can be nearly completed in 48 h, so a four-day viability test is consistent with the endothelial cell healing process (Figures 6 and 8). Endothelial cells are unique in their growth pattern, preferring to grow in flat structures, so the extreme growth rate without an appropriate dynamic environment, a laminar blood flow, tends to cause apoptosis in limited living space [39,40]. Nevertheless, the opposite behavior between ECs and SMCs in this study suggests that the PLGA films promoted the proliferation of SMCs, while sandwich coating notably delayed them with similar behavior to that of the control group. Many recent clinical trials have revealed that polymer-based stents [18] do not mitigate late-stent restenosis, the SMC migration results may explain the deficiencies related to the use of polymers and offer a possible solution.

The anti-inflammatory effect is another concern related to cardiovascular stents. Phenolic molecules exhibit extraordinary, inherent anti-oxidative abilities, thus helping to scavenge reactive oxygen species and protect vascular tissue. In addition, free radical scavenging decreases oxidants and inhibits the atherogenesis initiated by oxidation of LDL. These antioxidant mechanisms prevent late myocardial infarction and in-stent restenosis after PCI. Although the drug delivery strategy is beyond the scope of our study, the interaction of free-radical capture and the GA delivery trend are what we are interested in. Recently, a free radical scavenging analysis completed by DPPH was conducted to evaluate the antioxidant capacity of the stent platform [34]. Phenolic molecules, such as ECGC and TA [28–31], have been discussed in similar studies, yet the GA application under PLGA eluting stents has not been sufficiently investigated. In our previous study, a phenolic-modified ceramic coating on ZK60 was proven to be efficient on osteo-like cell activity [12]. Due to its ability to capture free radicals and cell selectivity, GA can potentially be applied in the modified polymer coating. However, the GA content is hard to characterize in the host because of the circulation system, which is also correlated to the cell tolerance and apoptosis occurring in a static environment.

Under the limitations of a coating strategy for the complicated geometry of stents and the in vivo environment, only the surface modification and the interaction of materials and cells can be discussed within our scope. To optimize the scale of this research, other coating techniques, such as spray coating, and the in vivo environment will be discussed in the near future.

#### **5. Conclusions**

PLGA dip-coatings with gallic acid (GA) were prepared on a ZK60 surface in a sandwiched-layer structure. The a close-packed sandwiched layer showed enhanced corrosion resistance and a homogeneous film surface. An in vitro assay demonstrated that the sandwich coating behaved selectively with bioactivity on ECs, significant suppression of SMC over-proliferation, anti-hemolysis ability, and anti-oxidation effects compared to PLGA. This simple technique with a sandwiched-layer structure is a promising surface treatment for a commercialized stent platform for treating atherosclerosis and preventing late-stent restenosis.

**Author Contributions:** Conceptualization, L.-H.L. and H.-P.L.; methodology, L.-H.L.; software, L.-H.L.; validation, L.-H.L.; formal analysis, L.-H.L.; investigation, L.-H.L.; resources, L.-H.L.; data curation, L.-H.L.; writing—original draft preparation, L.-H.L.; writing—review and editing, H.-P.L. and M.-L.Y.; visualization, L.-H.L. and H.-P.L.; supervision, M.-L.Y.; project administration, M.-L.Y.; funding acquisition, M.-L.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by Ministry of Science and Technology in Taiwan through Grants MOST-106-3114-E-006-012 and Medical Device Innovation Center (MDIC), National Cheng Kung University (NCKU) from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MoE) in Taiwan. *Materials* **2020**, *13*, x FOR PEER REVIEW 13 of 15 **Acknowledgments:** This research was supported by the Nano Biomedical and Tissue Engineering LAB, *Materials* **2020**, *13*, x FOR PEER REVIEW 13 of 15 **Acknowledgments:** This research was supported by the Nano Biomedical and Tissue Engineering LAB,

**Acknowledgments:** This research was supported by the Nano Biomedical and Tissue Engineering LAB, Department of BME, National Cheng Kung University. We thank INTAI Technology and CHONG HUAI Laser for providing the Laser technique of Mg-stents. Department of BME, National Cheng Kung University. We thank INTAI Technology and CHONG HUAI Laser for providing the Laser technique of Mg-stents. Department of BME, National Cheng Kung University. We thank INTAI Technology and CHONG HUAI Laser for providing the Laser technique of Mg-stents.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. **Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. **Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Appendix A Appendix A Appendix A**

**Figure A1.** XRD patterns of as-extruded ZK60. **Figure A1.** XRD patterns of as-extruded ZK60.

**Figure A2.** The MG63 attachment morphology of bare ZK60 and MgO and the potentiodynamic polarization curves of bare ZK60 and MgO. **Figure A2.** The MG63 attachment morphology of bare ZK60 and MgO and the potentiodynamic polarization curves of bare ZK60 and MgO.

**Figure A2.** The MG63 attachment morphology of bare ZK60 and MgO and the potentiodynamic

polarization curves of bare ZK60 and MgO.

SD, N = 3).

**References**

SD, N = 3).

**References**

**Figure A3.** The cytocompatibility evaluation of GA content versus the relative growth rate (mean ±

**Figure A3.** The cytocompatibility evaluation of GA content versus the relative growth rate (mean ±

1. Letchumanan, I.; Arshad, M.M.; Gopinath, S.C. Nanodiagnostic Attainments and Clinical Perspectives on

2. Stone, G.W.; Aronow, H.D. Long-term Care After Percutaneous Coronary Intervention: Focus on the Role

1. Letchumanan, I.; Arshad, M.M.; Gopinath, S.C. Nanodiagnostic Attainments and Clinical Perspectives on

2. Stone, G.W.; Aronow, H.D. Long-term Care After Percutaneous Coronary Intervention: Focus on the Role

C-Reactive Protein: Cardiovascular Disease Risks Assessment. *Curr. Med. Chem.* **2020**, *27*, 1.

C-Reactive Protein: Cardiovascular Disease Risks Assessment. *Curr. Med. Chem.* **2020**, *27*, 1.

of Antiplatelet Therapy. *Mayo Clin. Proc.* **2006**, *81*, 641–652.

of Antiplatelet Therapy. *Mayo Clin. Proc.* **2006**, *81*, 641–652.

polarization curves of bare ZK60 and MgO.

for providing the Laser technique of Mg-stents.

publish the results.

**Appendix A**

**Acknowledgments:** This research was supported by the Nano Biomedical and Tissue Engineering LAB, Department of BME, National Cheng Kung University. We thank INTAI Technology and CHONG HUAI Laser

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to

**Figure A1.** XRD patterns of as-extruded ZK60.

**Figure A3.** The cytocompatibility evaluation of GA content versus the relative growth rate (mean ± SD, N = 3). **Figure A3.** The cytocompatibility evaluation of GA content versus the relative growth rate (mean ± SD, N = 3).

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## *Article* **Antibacterial Films Based on Polylactide with the Addition of Quercetin and Poly(Ethylene Glycol)**

**Ewa Olewnik-Kruszkowska 1,\* , Magdalena Gierszewska 1 , Agnieszka Richert 2 , Sylwia Grabska-Zieli ´nska 1 , Anna Rudawska 3 and Mohamed Bouaziz 4**

> 1 Faculty of Chemistry, Chair of Physical Chemistry and Physicochemistry of Polymers, Nicolaus Copernicus University in Toru ´n, Gagarin 7 Street, 87-100 Toru ´n, Poland<EMAIL_ADDRESS>(M.G<EMAIL_ADDRESS>(S.G.-Z.)

> 2 Faculty of Biological and Veterinary Sciences, Chair of Genetics, Nicolaus Copernicus University in Toru ´n, Lwowska 1 Street, 87-100 Toru ´n, Poland<EMAIL_ADDRESS>

**Abstract:** A series of new films with antibacterial properties has been obtained by means of solvent casting method. Biodegradable materials including polylactide (PLA), quercetin (Q) acting as an antibacterial compound and polyethylene glycol (PEG) acting as a plasticizer have been used in the process. The effect of quercetin as well as the amount of PEG on the structural, thermal, mechanical and antibacterial properties of the obtained materials has been determined. It was found that an addition of quercetin significantly influences thermal stability. It should be stressed that samples containing the studied flavonoid are characterized by a higher Young modulus and elongation at break than materials consisting only of PLA and PEG. Moreover, the introduction of 1% of quercetin grants antibacterial properties to the new materials. Recorded results showed that the amount of plasticizer did not influence the antibacterial properties; it does, however, cause changes in physicochemical properties of the obtained materials. These results prove that quercetin could be used as an antibacterial compound and simultaneously improve mechanical and thermal properties of polylactide-based films.

**Keywords:** polylactide; quercetin; antibacterial properties; food packaging

#### **1. Introduction**

In recent years, polymers have become one of the most common materials used for packaging, replacing materials such as glass, metal, paper and wood. It is estimated that about 40% of industrial packaging and about 50% of consumables packaging is made of polymers. Such a large interest in plastics as packaging materials is due to their many beneficial properties, such as low permeability and high mechanical strength [1–3].

Packaging materials, in particular those intended for food, have to meet a significant number of requirements. Most of all they have to ensure effective protection of the product against harmful factors (such as oxygen, UV radiation, moisture, bacteria and fungi) during food transport, storage and shelf life. For this reason materials with an additional function of so called active packaging are becoming more popular. The most popular include polymers modified with various substances characterized by such properties as absorption of moisture, oxygen or carbon dioxide. The group of active agents being introduced into the polymer matrix also includes substances with biocidal properties. The main benefit of using food packaging materials with antibacterial properties is the extension of products' shelf life and protection against bacteria and fungi, which can be hazardous to human health [2,4–6].

**Citation:** Olewnik-Kruszkowska, E.; Gierszewska, M.; Richert, A.; Grabska-Zieli ´nska, S.; Rudawska, A.; Bouaziz, M. Antibacterial Films Based on Polylactide with the Addition of Quercetin and Poly(Ethylene Glycol). *Materials* **2021**, *14*, 1643. https:// doi.org/10.3390/ma14071643

Academic Editor: John T. Kiwi

Received: 25 February 2021 Accepted: 24 March 2021 Published: 27 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Currently, materials utilized in the production of packaging consist mainly of polyethylene, poly(ethylene terephthalate), polypropylene, poly(vinyl chloride) and polystyrene. Unfortunately, the use of polymer materials leads to the accumulation of huge amounts of waste that are harmful to the natural environment. This is due to their high durability and inherent resistance to biodegradation. Plastic waste accounts for about 30% of the weight of all the waste in the world. The growing problem of waste has recently resulted in an increased popularity and interest in biodegradable polymers. It seems to be very likely that in the immediate future they may completely replace traditional polymer materials. One of the polymers that will in all probability become an alternative to the currently used polymers of petrochemical origin is polylactide (PLA) [7,8]. It has to be stressed that polylactide is an aliphatic biodegradable polyester that can be obtained from renewable raw materials of plant origin [9]. The most popular materials used in an aim to manufacture lactic acid include corn, rice, barley, wheat or cassava [10].

In order to obtain antibacterial packaging materials based on polylactide, different compounds such as nanosilver, nisin, polyhexamethylene guanidine derivatives, cinnamon or tea tree essential oil [11–16] have been used. Among different active substances, flavonoids constitute a group of very interesting compounds characterized by varied antibacterial properties.

Molecular structure of flavonoids is characterized by two aromatic rings, which are connected by a three-carbon bridge. In the group of flavonoids we can distinguish: flavones, isoflavones, flavonols, flavanones, flavanols and anthocyanins. Flavonoids can, for example, be found in leaves, flowers, plant seeds, fruit (e.g., in citrus fruits, blueberries, berries, grapes, chokeberry), vegetables (e.g., in peppers, onions, tomatoes, broccoli) and in coffee and cocoa beans. It has to be noted that they possess many beneficial properties. They are characterized by anti-inflammatory, antiviral, antiatherosclerosis, antiallergic and anti-cancer properties. In addition, they perform a protective function, for instance deterring insects and inhibiting fungi, a common hazard to plants in which flavonoids can be found. The presence of these compounds also hinders the adverse effects of ultraviolet radiation [17–19].

One of the most popular flavonoids is quercetin—3,30 ,40 ,5,7-pentahydroxylflavone (Figure 1). It can be found in many types of fruit, vegetables, leaves, seeds and grains. Quercetin is also present in medicinal botanicals, including Ginkgo biloba and in varieties of honey from different plant sources. Recently scientists focused on its biological and antioxidative characteristics [20–23]. Moreover, it should also be mentioned that the flavonoid in question was used in the formulation of packaging films consisting mainly of ethylene-vinyl alcohol copolymer (EVOH) [24]. It was also scrutinized as an antioxidant and environmentally-friendly colored indicator of ageing time in a topas cyclo-olefin copolymer (ethylene-norbornene) [25]. Additional research devoted to the possible applications of quercetin has been described in the work of Kost et al. [26], where the fibers based on polylactide and β-cyclodextrin loaded with quercetin were used as dressing materials characterized with antibacterial properties. Other possible applications of the quercetin have been presented in the work of Hao et al. [27], where quercetin was encapsulated using chitosan-coated nanoliposomes. *Materials* **2021**, *14*, x FOR PEER REVIEW 3 of 22

In the present work polylactide-based films with an addition of poly(ethylene glycol)

Polylactide, type 2002D with melt flow rate 5–7 g/10 min (2.16 kg; 190 °C), with average molecular weight of 155,500 Da and content of monomeric units D and L equal to

In an aim to analyze antibacterial properties of the obtained materials, two bacterial reference strains were used in the study: Escherichia coli (ATCC 8739) and Staphylococcus

In aim to obtain all of the PLA-based films, the solvent evaporation technique were applied. In the first stage, 1.5 g of pure and dry polylactide was dissolved in 35 mL of chloroform by vigorous mixing at room temperature. In the second stage, an appropriate amount of PEG (5% or 10% *w*/*w* of PLA, 0.075 g or 0.15 g of PEG, respectively) was added. Quercetin was dissolved in 15 mL of acetone and the mixture was then introduced into the solution containing PLA and PEG. The resulting solutions were cast onto clean glass plates, 145 mm in diameter and dried at ambient temperature for 48 h. Designations and

containing a composition [g/L]: tryptone peptone—15, phyton peptone—5, sodium chlo-

ride—5, agar—agar—15 was acquired from Oxoid (Hampshire, UK).

compositions of the obtained materials are presented in Table 1.

**Sample Quercetin Content 1**

**Table 1.** Compositions of the obtained materials (L-polylactide; P-PEG, Q-quercetin).

LP5 - 5 LP10 - 10 LP5Q1 1 5 LP10Q1 1 10 LP5Q2 2 5 LP10Q2 2 10

, St. Cloud, MN, USA). Moreover, an agar medium

**[wt.%] PEG Content 1**

(Min-

**[wt.%]**

3.5% and 96.5%, respectively was delivered in the form of pellets by Nature Works®

netonka, MN, USA). Quercetin, an antibacterial compound, was purchased from Sigma-Aldrich (Steinheim, Germany). Poly(ethylene glycol) with an average molecular weight of 1500 (Sigma-Aldrich, Steinheim, Germany) was applied as the plasticizing agent. Acetone and chloroform were purchased from Avantor Performance Materials Poland S.A.

acting as a plasticizer and quercetin used as a biocidal agent are suggested as new anti-

PLA films, it has to be stressed that they present an enormous potential for application in

**Figure 1.** The structure of quercetin. **Figure 1.** The structure of quercetin.

**2. Materials and Methods**

the food industry.

(Gliwice, Poland).

1 Relative to PLA mass.

aureus (ATCC 6538P) (Microbiologics®

*2.2. Formation of PLA-Based Films*

*2.1. Materials*

In the present work polylactide-based films with an addition of poly(ethylene glycol) acting as a plasticizer and quercetin used as a biocidal agent are suggested as new antibacterial packaging materials. Taking into account the properties of quercetin-infused PLA films, it has to be stressed that they present an enormous potential for application in the food industry.

#### **2. Materials and Methods**

#### *2.1. Materials*

Polylactide, type 2002D with melt flow rate 5–7 g/10 min (2.16 kg; 190 ◦C), with average molecular weight of 155,500 Da and content of monomeric units D and L equal to 3.5% and 96.5%, respectively was delivered in the form of pellets by Nature Works® (Minnetonka, MN, USA). Quercetin, an antibacterial compound, was purchased from Sigma-Aldrich (Steinheim, Germany). Poly(ethylene glycol) with an average molecular weight of 1500 (Sigma-Aldrich, Steinheim, Germany) was applied as the plasticizing agent. Acetone and chloroform were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

In an aim to analyze antibacterial properties of the obtained materials, two bacterial reference strains were used in the study: Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 6538P) (Microbiologics®, St. Cloud, MN, USA). Moreover, an agar medium containing a composition [g/L]: tryptone peptone—15, phyton peptone—5, sodium chloride—5, agar—agar—15 was acquired from Oxoid (Hampshire, UK).

#### *2.2. Formation of PLA-Based Films*

In aim to obtain all of the PLA-based films, the solvent evaporation technique were applied. In the first stage, 1.5 g of pure and dry polylactide was dissolved in 35 mL of chloroform by vigorous mixing at room temperature. In the second stage, an appropriate amount of PEG (5% or 10% *w*/*w* of PLA, 0.075 g or 0.15 g of PEG, respectively) was added. Quercetin was dissolved in 15 mL of acetone and the mixture was then introduced into the solution containing PLA and PEG. The resulting solutions were cast onto clean glass plates, 145 mm in diameter and dried at ambient temperature for 48 h. Designations and compositions of the obtained materials are presented in Table 1.

**Table 1.** Compositions of the obtained materials (L-polylactide; P-PEG, Q-quercetin).

1 Relative to PLA mass.

#### *2.3. Fourier Transform Infrared Analysis Analysis*

The Fourier transform infrared analysis of all studied materials was performed by means of a Nicolet iS10 (Thermo Fisher Scientific, Waltham, MA, USA). The spectra were recorded in the frequency range of 500–4000 cm−1 at a resolution of 4 cm−1 and scanned 64 times. The spectrum of quercetin in KBr disc form was obtained in the same conditions. All spectra were analyzed using the OMNIC 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.4. Scanning Electron Microscopy (SEM)*

Changes in the structure of the PLA-based films caused by an addition of PEG and quercetin were analyzed using a scanning electron microscope (Quanta 3D FEG, FEI Company, Hillsboro, OR, USA). In an aim to obtain high quality images, the samples were covered with a thin layer of gold. Images of all samples were taken at 5000× magnification.

#### *2.5. Atomic Force Microscopy (AFM)*

The surface pictures of the PLA-based films were obtained by means of an AFM microscope with a scanning probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, CA, USA). Analyses were performed in the tapping mode, in air, at room temperature. Using a scan area of 5 × 5 µm and Nanoscope software (Veeco Metrology, Inc. Santa Barbara, CA, USA), the roughness parameters such as the root mean square (*Rq*) and arithmetical mean deviation of the assessed profile (*Ra*) were calculated.

#### *2.6. Thermogravimetric Analysis*

Thermogravimetric (TG) analyses of the PLA films with an addition of PEG and quercetin were performed on Simultaneous TGA-DTA Thermal Analysis type SDT 2960 (TA Instruments, London, UK). All measurements were carried out at a heating rate of 10 ◦C/min under air flow from room temperature to 600 ◦C.

#### *2.7. Differential Scanning Calorimetry Method*

Thermal analyses were carried out on DSC (Polymer Laboratories, Epsom, UK). Experiments were performed under nitrogen screening. The thermal response in the obtained materials was investigated at the temperatures ranging from 25 ◦C to 180 ◦C (1st heating cycle) and with a heating rate of 10 ◦C/min. The PL5 software version v5.40 (Polymer Laboratories, Epsom, UK) was applied in an aim to establish the detailed information extracted from the DSC data. The degree of crystallinity (*Xm*) was established by applying the following Equation (1) [28,29]:

$$X\_m = \frac{\Delta H\_m}{\Delta H^0 \cdot X\_{PLA}} \cdot 100\% \tag{1}$$

where ∆*Hm* is the measured heat of fusion of sample, ∆*H*0 is the heat of fusion of a 100% crystalline polylactide and ∆*H*0 = 109 mJ/mg, *XPLA* is the mass fraction of polylactide.

#### *2.8. Mechanical Properties*

The mechanical properties of the PLA-based films with and without an addition of quercetin were analyzed by means of the Instron 1193 machine (Instron Corp., Canton, OH, USA) test according to the PN-EN ISO 527-1, -3 standard [30,31]. The crosshead speed was 20 mm/min with an applied 100 N force. In the case of each type of the studied materials, at least five samples were analyzed. The obtained results allowed to establish the Young's modulus (*E*), elongation at break (*ε*) and tensile stress (*σm*).

#### *2.9. Transparency*

Transparency of the obtained materials was established based on the method presented in work of [32]. Absorbance of the polymeric films with and without quercetin at 600 nm (*A*600) was measured by means of UV spectrophotometer (Ruili Analytical Instrument Company, Beijing, China). The analysed films were placed directly in a spectrophotometer test cell while an empty cell was used as the blank. The transparency (*T*) of the obtained materials was calculated according to the following Equation (2):

$$T = \frac{A\_{600}}{d} \ \left[ \mu m^{-1} \right] \tag{2}$$

where *d* is the film thickness [mm]. It should be noted that a higher transparency value is equivalent to lower transparency.

#### *2.10. Colour Measurement*

The changes in the color of the obtained films caused by the introduction of quercetin and PEG were studied by means of a MICRO-COLOR II LCM 6 (Dr Lange, Berlin, Germany) colorimeter. The CIE L\*a\*b\* system was applied in aim to calculate the colour difference (∆*E*) of materials. The following Equations (3) and (4) were used in order to establish the total values of ∆*E* and color intensity (*C*), respectively.

$$
\Delta E = \sqrt{(L - L^\*)^2 + (a - a^\*)^2 + (b - b^\*)^2},
\tag{3}
$$

$$\mathcal{C} = \sqrt{{(a^\*)}^2 + {(b^\*)}^2},\tag{4}$$

where *L* is the component describing lightness, a represents the colors ranging from green (−*a*) to red (*+a*), while *b* represents a parameter change from blue (−*b*). The color of the control film (in this case a LP5 film), was expressed as *L\** (lightness), *a* ∗ (redness/greenness) and *b* ∗ (yellowness/blueness) values [14]. In an attempt to obtain reliable data at least five measurements were performed for each of the samples, then average values were calculated. Moreover, based on the obtained results yellowness index (*YI*) was established using the Equation (5) described in the work of Pathare et al. [33]:

$$
\gamma Y = \frac{142.86 \cdot b^\*}{L^\*}. \tag{5}
$$

#### *2.11. Analysis of Antibacterial Properties*

Antibacterial properties were determined according to the ISO 20645:2006 standard: "Flat textile products. Determination of antibacterial activity. Diffusion method on an agar plate" [34]. Agar medium consisting of [g/L]: tryptone peptone—15, phyton peptone—5, sodium chloride—5, agar—agar—15 was poured onto each petri dish and allowed to gel. The medium was then infused with a bacterial culture at a concentration of 1.5 × 108 cfu/mL (0.5 McFarlanda). Centrally tested samples and control samples in the shape of a circle with a diameter of 25 ± 5 mm (four replicates) were placed on the dishes prepared in this way. Plates were incubated for 20 h at 37 ± 1 ◦C. After the end of the incubation time, the presence or absence of zones inhibiting the growth of microorganisms was determined. The width of the braking zone (*H*), i.e., the zone without bacteria near the edge of the sample, was calculated using the following Formula (6):

$$H = D - \frac{d}{2} \text{ [mm]},\tag{6}$$

where:

*H*—braking zone width [mm],

*D*—total diameter of the working sample and width of the braking zone [mm],

*d*—diameter of the working sample [mm].

Stereoscopic microscope SZX 12 (Olympus, Tokyo, Japan) was used in an aim to establish the size of inhibition zones of bacterial growth as well as the extent of microorganism proliferation in contact of the studied films with agar. The appearance of agar plates after the studied materials had been removed was recorded by means of a SCAN® 1200 colony counter (Interscience, Saint-Nom-la-Bretèche, France). The scale shown in Table 2 was used to assess the potency of antibacterial properties.

**Table 2.** Antibacterial effect of antibacterial treatment (ISO 20645, 2006) [34].

(a) Bacterial growth on the medium under the working sample. (b) The dynamometer range should only be partially taken into account in the calculations. An increase in the braking zone may be due to excess of active substance or unevenness of the substance in the article. (c) Lack of growth with a simultaneous lack of braking zone can be considered a good effect. A braking zone may not be possible due to limited diffusion. (d) "Almost as good as lack of growth"—an indication of limited efficiency. (e) Limited bacterial growth density means both the number of colonies and the diameter of the colonies.

#### **3. Results and Discussion**

#### *3.1. FTIR Analysis*

FTIR spectroscopy was used in an aim to establish the structure of the obtained materials with an addition of quercetin and different amounts of PEG. The FTIR spectrum of quercetin is shown in Figure 2, where the bands of characteristic groups can be observed. According to the literature [35,36] particular bands were assigned to the characteristic groups present in the quercetin structure. A broad band with the maximum at 3127 cm−1 belongs to the hydroxyl groups (phenolic -O-H stretching). The band at 1653 cm−1 indicates the presence of stretching vibrations of the -C=O group, while the bands at 1614 cm−1 , 1567 cm−1 and 1513 cm−1 belong to the C=C stretching bonds in the aromatic rings of quercetin [36,37]. In the spectrum of the neat quercetin an intense band at 1401 cm−1 is visible and can be ascribed to deformation vibrations of -OH groups. Additionally, in the discussed spectral bands, the maximum values of 1316 cm−1 and 1247 cm−1 correspond to the deformation vibrations of the -C-OH group. Other vibrations at 1167 cm−1 and 1092 cm−1 can be assigned to the anti-symmetrical and symmetrical stretching vibration of the -C-O-C group. The band observed at 997 cm−1 indicates the deformation vibrations of the -OH group, while the band at 933 cm−1 relates to the stretching vibrations of the -C-O. Other characteristic bands in the range between 884 cm−1 and 808 cm−1 correspond to the deformative vibrations of the -CH groups [35,37]. In Figures 3 and 4, the spectra of the materials consisting of PLA, PEG with or without addition of quercetin are depicted. The chemical structure of polylactide films is well known. It was also described in our previous publications [38]. Based on our study, it can be clearly seen that the bands at 3657 cm−1 and 3502 cm−1 correspond to -OH groups at the end of PLA chains. Absorption bands at 2996 cm−1 and 2947 cm−1 belong to the symmetrical and asymmetrical stretching vibrations of the -CH3 group, while the band at 1763 cm−1 relates to the characteristic -C=O carbonyl group. Moreover, symmetrical stretching vibrations of -C-O-C were recorded at 1207 cm−1 and at 1127 cm−1 . The bands describing the stretching vibration of C-COO and the deformation vibration of CO can be seen at 873 cm−1 and 756 cm−1 .

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**Figure 2.** The FTIR spectrum of quercetin. **Figure 2.** The FTIR spectrum of quercetin. **Figure 2.** The FTIR spectrum of quercetin.

**Figure 3.** The FTIR spectra of materials consisting of PLA, PEG 5 wt.% with an addition of querce-**Figure 3.** The FTIR spectra of materials consisting of PLA, PEG 5 wt.% with an addition of quercetin.

**Figure 3.** The FTIR spectra of materials consisting of PLA, PEG 5 wt.% with an addition of querce-

tin.

tin.

**Figure 4.** The FTIR spectra of materials consisting of PLA, PEG 10 wt.% with an addition of quer-**Figure 4.** The FTIR spectra of materials consisting of PLA, PEG 10 wt.% with an addition of quercetin.

cetin. In the case of films based on PLA and PEG the typical vibrations caused by the -CH2 group at 2878 cm−1 can be observed. Moreover, a peak at 3446 cm−1 for poly(ethylene glycol), which corresponds to the terminal hydroxyl group, has been recognized. Furthermore, it can be clearly noticed that the intensity of the band at 2878 cm−1 increases with In the case of films based on PLA and PEG the typical vibrations caused by the -CH2 group at 2878 cm−1 can be observed. Moreover, a peak at 3446 cm−1 for poly(ethylene glycol), which corresponds to the terminal hydroxyl group, has been recognized. Furthermore, it can be clearly noticed that the intensity of the band at 2878 cm−1 increases with the increase in the amount of PEG.

the increase in the amount of PEG. Introduction of quercetin into the PLA-PEG systems reveals a broad band at 3300 cm-1 which can be assigned to the -OH groups present in the structure of quercetin. Moreover, new bans at 1655 cm−1, 1615 cm−1 and 1600 cm−1 corresponding respectively to stretching vibrations of the -C=O group and the C=C stretching bonds in the aromatic rings have been recognized. It has to be stressed that the intensities of the described new Introduction of quercetin into the PLA-PEG systems reveals a broad band at 3300 cm−1 which can be assigned to the -OH groups present in the structure of quercetin. Moreover, new bans at 1655 cm−1 , 1615 cm−1 and 1600 cm−1 corresponding respectively to stretching vibrations of the -C=O group and the C=C stretching bonds in the aromatic rings have been recognized. It has to be stressed that the intensities of the described new bands increased with the quantity of quercetin in the obtained films [37].

bands increased with the quantity of quercetin in the obtained films [37]. The spectra of the studied films after the addition of quercetin present certain changes in the intensity of bands at 915 cm−1 and 955 cm−1 belong to the amorphous and crystalline phases of PLA, respectively [39,40]. Increase in intensity of band at 915 cm−1 The spectra of the studied films after the addition of quercetin present certain changes in the intensity of bands at 915 cm−1 and 955 cm−1 belong to the amorphous and crystalline phases of PLA, respectively [39,40]. Increase in intensity of band at 915 cm−1 indicates that quercetin influences the crystallinity of the obtained materials.

#### indicates that quercetin influences the crystallinity of the obtained materials. *3.2. SEM and AFM Analyses*

*3.2. SEM and AFM Analyses* Topography and surface morphology constitute factors which are taken into account when the potential application of materials is discussed. They are extremely important in the case of polymeric films used as food packaging as well as films with medical applications. In the present work we focused on the PLA-based materials with an addition of quercetin and poly(ethylene glycol). Obtained materials were characterized by antibacterial properties. In order to analyze their surface morphology atomic force microscopy and scanning electron microscopy were applied. The SEM images presented in Figure 5 reveal that samples of polylactide with an addition of 5 wt.% or 10 wt.% of poly(ethylene glycol), which plays the role of a plasticizer, were characterized by a smooth and flat surface, without any cracks or fissures. The same observation was made in the work of Jasim Ahmed at al. [41] where PLA-based films with an addition of 20 wt.% of PEG were studied. Topography and surface morphology constitute factors which are taken into account when the potential application of materials is discussed. They are extremely important in the case of polymeric films used as food packaging as well as films with medical applications. In the present work we focused on the PLA-based materials with an addition of quercetin and poly(ethylene glycol). Obtained materials were characterized by antibacterial properties. In order to analyze their surface morphology atomic force microscopy and scanning electron microscopy were applied. The SEM images presented in Figure 5 reveal that samples of polylactide with an addition of 5 wt.% or 10 wt.% of poly(ethylene glycol), which plays the role of a plasticizer, were characterized by a smooth and flat surface, without any cracks or fissures. The same observation was made in the work of Jasim Ahmed at al. [41] where PLA-based films with an addition of 20 wt.% of PEG were studied. In the mentioned work, SEM photographs depicted good dispersion of PEG into the polylactide matrix, indicating that the obtained materials form homogeneous

blends. Incorporation of quercetin into the PLA-PEG blends significantly influences the morphology of the obtained polymeric films It has to be taken into account, however, that the surfaces of films with a higher amount of PEG (10 wt.%) are characterized by larger convexities and deeper concavities of various shapes, evenly distributed throughout the surface in comparison with materials containing 5 wt.% of plasticizer. ylactide matrix, indicating that the obtained materials form homogeneous blends. Incorporation of quercetin into the PLA-PEG blends significantly influences the morphology of the obtained polymeric films It has to be taken into account, however, that the surfaces of films with a higher amount of PEG (10 wt.%) are characterized by larger convexities and deeper concavities of various shapes, evenly distributed throughout the surface in comparison with materials containing 5 wt.% of plasticizer.

In the mentioned work, SEM photographs depicted good dispersion of PEG into the pol-

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In an aim to establish the size and roughness of the studied surface, the AFM technique was applied. It is well known that roughness constitutes one of the factors which can significantly affect the antibacterial properties of the obtained materials. In Figure 6, three

dimensional pictures of surfaces of PLA-based films are presented. It can be observed that the size of cavities formed on the surface of the obtained polymeric films containing quercetin, with an addition of 10 wt.% of PEG, are significantly enlarged. The diameter of the cavities in the case of LP5Q1 and LP5Q2 samples equaled 1.9 µm and 2.0 µm, respectively, while in the case of LP10Q1 and LP10Q2 materials the fissures measured 2.5 µm and 2.8 µm. Based on the presented images (Figure 6), the minimum depth of the formed cavities was also calculated. In the case of materials containing 5 wt.% of PEG (LP5Q1 and LP5Q2) the values of cavities' depth equaled 57 nm and 75 nm, while in the case of films with an addition of 10 wt.% of the plasticizer (LP10Q1 and LP10Q2) the depth of cavities increased to an extent of 60 nm and 80 nm, respectively. The results presented above confirm that the diameter of the formed fissures increases with the increase in the amount of PEG, while the depth of observed cavities increases with the increase in the amount of quercetin. three dimensional pictures of surfaces of PLA-based films are presented. It can be observed that the size of cavities formed on the surface of the obtained polymeric films containing quercetin, with an addition of 10 wt.% of PEG, are significantly enlarged. The diameter of the cavities in the case of LP5Q1 and LP5Q2 samples equaled 1.9 µm and 2.0 µm, respectively, while in the case of LP10Q1 and LP10Q2 materials the fissures measured 2.5 µm and 2.8 µm. Based on the presented images (Figure 6), the minimum depth of the formed cavities was also calculated. In the case of materials containing 5 wt.% of PEG (LP5Q1 and LP5Q2) the values of cavities' depth equaled 57 nm and 75 nm, while in the case of films with an addition of 10 wt.% of the plasticizer (LP10Q1 and LP10Q2) the depth of cavities increased to an extent of 60 nm and 80 nm, respectively. The results presented above confirm that the diameter of the formed fissures increases with the increase in the amount of PEG, while the depth of observed cavities increases with the increase in the amount of quercetin.

In an aim to establish the size and roughness of the studied surface, the AFM technique was applied. It is well known that roughness constitutes one of the factors which can significantly affect the antibacterial properties of the obtained materials. In Figure 6,

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**Figure 6.** 3D AFM images of obtained materials. **Figure 6.** 3D AFM images of obtained materials.

Based on the obtained results the roughness parameters such as *Rq* (mean square deviation of surface roughness), *Ra* values (mean arithmetic deviation of the profile from the mean line) and *Rmax* (maximum distance between the highest and lowest point of the recorded image) have been discussed (Table 3) [16].

**Table 3.** Roughness parameters of PLA and PLA-based films with and addition of quercetin and poly(ethylene glycol). LP5Q1 32.2 ± 0.5 22.0 ± 0.4 232.0 ± 2.0 LP5Q2 34.1 ± 0.6 26.8 ± 0.5 237.0 ± 2.0

Based on the obtained results the roughness parameters such as *Rq* (mean square deviation of surface roughness), *Ra* values (mean arithmetic deviation of the profile from the mean line) and *Rmax* (maximum distance between the highest and lowest point of the rec-

**Table 3.** Roughness parameters of PLA and PLA-based films with and addition of quercetin and

**Sample** *Rq* **[nm]** *Ra* **[nm]** *Rmax* **[nm]** LP5 1.4 ± 0.1 1.0 ± 0.1 10.5 ± 0.5 LP10 3.8 ± 0.2 3.1 ± 0.2 23.1 ± 1.1

It needs to be stressed that the values of all of the analyzed roughness parameters significantly depend on the amount of PEG. Obtained results suggest a dependence between the increase of the amount of PEG and the roughness parameters discussed above which are characterized by significantly higher values. Taking into account the amount of quercetin used, values of *Ra*, *Rq* and *Rmax* reveal that the same tendency as in the case of PEG. The introduction of 2 wt.% of quercetin, however, leads only to a slight increase in the values of roughness parameters in comparison with the change of values caused by the plasticizer. which are characterized by significantly higher values. Taking into account the amount of quercetin used, values of *Ra*, *Rq* and *Rmax* reveal that the same tendency as in the case of PEG. The introduction of 2 wt.% of quercetin, however, leads only to a slight increase in the values of roughness parameters in comparison with the change of values caused by the plasticizer. *3.3. Determination of Thermal Properties of Polylactide-Based Materials*

#### *3.3. Determination of Thermal Properties of Polylactide-Based Materials* In an aim to determine the effect of different amounts of plasticizer and quercetin on

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orded image) have been discussed (Table 3) [16].

poly(ethylene glycol).

In an aim to determine the effect of different amounts of plasticizer and quercetin on thermal stability of polylactide-based films, thermogravimetric analysis was conducted. Based on the obtained results, thermogravimetric (TG) curves for the samples LP5, LP5Q1, LP5Q2 are shown in Figure 7, while the TG curves relating to the samples LP10, LP10Q1 and LP10Q2 are depicted in Figure 8. thermal stability of polylactide-based films, thermogravimetric analysis was conducted. Based on the obtained results, thermogravimetric (TG) curves for the samples LP5, LP5Q1, LP5Q2 are shown in Figure 7, while the TG curves relating to the samples LP10, LP10Q1 and LP10Q2 are depicted in Figure 8.

**Figure 7.** Thermogravimetric curves for polylactide-based materials with 5 wt.% of PEG. **Figure 7.** Thermogravimetric curves for polylactide-based materials with 5 wt.% of PEG.

**Figure 8.** Thermogravimetric curves for polylactide-based materials with 10 wt.% of PEG. **Figure 8.** Thermogravimetric curves for polylactide-based materials with 10 wt.% of PEG.

It was previously discussed that the plasticizer as well as the biocidal agent can significantly affect the thermal stability of the polylactide-based films [16]. In the prevailing number of publications the temperatures at 5% mass loss are analyzed. In the work of Rhim et al. [42] and Tarach at al. [16], however, it was indicated that in the case of these types of materials, the mass loss of about 5% of the initial mass is connected with the evaporation of the solvent residue. As a result, the values of temperatures at 10%, 30% and 50% mass loss of all studied materials (signified as T10%, T30% and T50%, respectively) were selected and presented in Table 4. Taking into account the amount of the plasticizer used, it can be observed that with an increase in the amount of PEG the thermal It was previously discussed that the plasticizer as well as the biocidal agent can significantly affect the thermal stability of the polylactide-based films [16]. In the prevailing number of publications the temperatures at 5% mass loss are analyzed. In the work of Rhim et al. [42] and Tarach at al. [16], however, it was indicated that in the case of these types of materials, the mass loss of about 5% of the initial mass is connected with the evaporation of the solvent residue. As a result, the values of temperatures at 10%, 30% and 50% mass loss of all studied materials (signified as T10%, T30% and T50%, respectively) were selected and presented in Table 4. Taking into account the amount of the plasticizer used, it can be observed that with an increase in the amount of PEG the thermal stability of the PLA-PEG samples decreases. According to the work of Phaechamud [43], as well as Pielichowski [44], the observed reduction in thermal stability is the result of PEG decomposition, which occurs at a significantly lower temperature compared to polylactide.

stability of the PLA-PEG samples decreases. According to the work of Phaechamud [43],

as well as Pielichowski [44], the observed reduction in thermal stability is the result of **Table 4.** TG data for obtained materials with and without addition of quercetin.

LP5 289.5 ± 0.4 340.1 ± 1.5 353.7 ± 1.5 LP10 287.2 ± 0.5 335.6 ± 1.3 350.9 ± 1.4 LP5Q1 322.6 ± 0.7 347.9 ± 1.0 358.5 ± 1.4 LP5Q2 329.8 ± 1.2 348.3 ± 1.0 358.3 ± 1.6 LP10Q1 320.0 ± 1.2 340.8 ± 1.3 352.4 ± 1.7 LP10Q2 325.5 ± 1.1 345.1 ± 1.3 356.1 ± 1.7 The incorporation of quercetin into the materials consisting of PLA and PEG, contributed to an increase in thermal stability of the obtained films. In thermograms presented in Figures 7 and 8 relating to the materials with the addition of 5 wt.% of PEG and 10 wt.% of plasticizer, respectively, a significant increase in thermal stability with the increase in an amount of biocidal agent can be observed. In the case of samples LP5Q1 and LP5Q2, the temperature for a 10% mass loss, compared to the LP5 sample, increased by 33.1 ◦C and 40.3 ◦C, respectively. The same tendency was observed in the case of samples based on PLA with 10 wt.% of PEG and containing quercetin. The 10% loss of mass in films containing

The incorporation of quercetin into the materials consisting of PLA and PEG, con-

10 wt.% of plasticizer, respectively, a significant increase in thermal stability with the increase in an amount of biocidal agent can be observed. In the case of samples LP5Q1 and LP5Q2, the temperature for a 10% mass loss, compared to the LP5 sample, increased by 33.1 °C and 40.3 °C, respectively. The same tendency was observed in the case of samples based on PLA with 10 wt.% of PEG and containing quercetin. The 10% loss of mass in

10 wt.% of PEG and modified with 1 and 2 wt.% of quercetin occurred at the temperature 32.8 ◦C and 38.3 ◦C higher than in the case of the PL10 material. The obtained results indicate that the most significant changes in thermal stability of the obtained materials can be observed after the addition of 1 wt.% of quercetin. The abovementioned results reveal that after incorporation of 2 wt.% of biocidal agent into the PL5 as well as the PL10 systems, the values of T10%, T30% and T50% also increase. The changes between 1 wt.% and 2 wt.% of quercetin, however, are not as significant as after addition of 1 wt.% of quercetin. It should be mentioned that an improvement of thermal stability of the obtained materials after the addition of quercetin was also observed in the work of Samper et al. [45]. In the mentioned work an addition of 0.25 wt.% of quercetin resulted in a remarkably higher stability of polypropylene (PP) in comparison with non-stabilized PP. According to the literature [37,45] this phenomenon can be attributed to the structure of the phenolic compound used in the procedure, especially to the number of hydroxyl groups. Moreover, it has been proved that in the case of quercetin there is an optimum amount which provides the best thermal stability. Further introduction of this substance beyond the optimum point does not provide additional benefits in terms of structural stabilization. Summarizing, based on the analysis of the above mentioned data (Table 4, Figures 7 and 8), it is reasonable to assume that the incorporation of quercetin significantly influences thermal stability of obtained materials. Obtained results are consistent with the data presented by the researchers mentioned above. the PL10 systems, the values of T10%, T30% and T50% also increase. The changes between 1 wt.% and 2 wt.% of quercetin, however, are not as significant as after addition of 1 wt.% of quercetin. It should be mentioned that an improvement of thermal stability of the obtained materials after the addition of quercetin was also observed in the work of Samper et al. [45]. In the mentioned work an addition of 0.25 wt.% of quercetin resulted in a remarkably higher stability of polypropylene (PP) in comparison with non-stabilized PP. According to the literature [37,45] this phenomenon can be attributed to the structure of the phenolic compound used in the procedure, especially to the number of hydroxyl groups. Moreover, it has been proved that in the case of quercetin there is an optimum amount which provides the best thermal stability. Further introduction of this substance beyond the optimum point does not provide additional benefits in terms of structural stabilization. Summarizing, based on the analysis of the above mentioned data (Table 4, Figures 7 and 8), it is reasonable to assume that the incorporation of quercetin significantly influences thermal stability of obtained materials. Obtained results are consistent with the data presented by the researchers mentioned above. In order to determine changes in thermal parameters of the studied materials, caused

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films containing 10 wt.% of PEG and modified with 1 and 2 wt.% of quercetin occurred at the temperature 32.8 °C and 38.3 °C higher than in the case of the PL10 material. The obtained results indicate that the most significant changes in thermal stability of the obtained materials can be observed after the addition of 1 wt.% of quercetin. The abovementioned results reveal that after incorporation of 2 wt.% of biocidal agent into the PL5 as well as

In order to determine changes in thermal parameters of the studied materials, caused by the addition of a plasticizer and quercetin, differential scanning calorimetry has been carried out (Figures 9 and 10). by the addition of a plasticizer and quercetin, differential scanning calorimetry has been carried out (Figures 9 and 10).

**Figure 9.** DSC thermograms of LP5 with and without an addition of quercetin. **Figure 9.** DSC thermograms of LP5 with and without an addition of quercetin.

**Figure 10.** DSC thermograms of sample LP10 with and without an addition of quercetin. **Figure 10.** DSC thermograms of sample LP10 with and without an addition of quercetin.

The obtained parameters of the glass transition temperature (*Tg*), crystallization temperature (*Tc*), crystallization enthalpy (Δ*Hc*), melting point (*Tm*), melting enthalpy (*ΔHm*) The obtained parameters of the glass transition temperature (*Tg*), crystallization temperature (*Tc*), crystallization enthalpy (∆*Hc*), melting point (*Tm*), melting enthalpy (∆*Hm*) and the calculated values of the crystallization degree (χ) were presented in Table 5.

and the calculated values of the crystallization degree (χ) were presented in Table 5. **Table 5.** DSC parameters for the all studied PLA-based materials.

LP5Q2 58.6 ± 0.4 119.6 ± 0.8 15.4 ± 0.1 152.3 ± 0.2 21.0 ± 0.3 19.7 LP10 49.5 ± 0.1 95.5 ± 0.5 17.9 ± 0.2 153.5 ± 0.3 26.6 ± 0.5 27.1 LP10Q1 54.0 ± 0.2 106.5 ± 0.6 15.0 ± 0.2 146.2/152.6 ± 0.2/0.3 24.8 ± 0.4 25.6 LP10Q2 54.8 ± 0.2 108.9 ± 0.6 12.8 ± 0.1 146.7/152.8 ± 0.3/0.3 16.6 ± 0.2 17.3 Obtaining a homogeneous blend consisting of PLA and PEG depends to a large extent on the molecular mass of PEG. Information available in literature [46,47] indicates that blends of PLA with PEG1500 separate when the content of poly(ethylene glycol) exceeds 20 wt.%. For this reason it is reasonable to assume, that the obtained films present a homogeneous structure.

> Obtaining a homogeneous blend consisting of PLA and PEG depends to a large extent on the molecular mass of PEG. Information available in literature [46,47] indicates that blends of PLA with PEG1500 separate when the content of poly(ethylene glycol) exceeds 20 wt.%. For this reason it is reasonable to assume, that the obtained films present a homogeneous structure. Based on the obtained results it can be seen that the increase in the amount of PEG Based on the obtained results it can be seen that the increase in the amount of PEG causes a significant decrease in the glass transition temperature as well as crystallization temperature values. It was established by Piórkowska et al. [48] that the plasticizer molecules in the form of PEG significantly increase the free volume of the mixture, consequently allowing cooperative movements of macromolecules and, for this reason, decreasing the *Tg* value. Simultaneously, plasticization of PLA by means of PEG intensifies the crystallization of PLA and in this way lowers its *Tc*. Moreover, with the increase in PEG load, higher values of melting enthalpy and degree of crystallinity can be observed. The introduction of quercetin into the PLA-PEG systems results in an opposite effect. It has to

> causes a significant decrease in the glass transition temperature as well as crystallization temperature values. It was established by Piórkowska et al. [48] that the plasticizer mole-

> quently allowing cooperative movements of macromolecules and, for this reason, decreasing the *Tg* value. Simultaneously, plasticization of PLA by means of PEG intensifies the crystallization of PLA and in this way lowers its *Tc*. Moreover, with the increase in PEG load, higher values of melting enthalpy and degree of crystallinity can be observed. The introduction of quercetin into the PLA-PEG systems results in an opposite effect. It has to be stressed that the presence of quercetin leads to a shift in glass transition temperature and crystallization temperature to higher values. Simultaneously, it can be observed that the values of both enthalpies, crystallization and melting, increase with the rise in the quercetin content. Furthermore, it should be noted, that quercetin has a profound impact on the degree of crystallinity. The direct correlation between the amount of quercetin and

be stressed that the presence of quercetin leads to a shift in glass transition temperature and crystallization temperature to higher values. Simultaneously, it can be observed that the values of both enthalpies, crystallization and melting, increase with the rise in the quercetin content. Furthermore, it should be noted, that quercetin has a profound impact on the degree of crystallinity. The direct correlation between the amount of quercetin and the degree of crystallinity of the obtained materials is apparent. This finding is consistent with the FTIR results, where the intensity of the band assigned to the crystalline phase decreases with the increase in quercetin content in the PLA-based films.

Moreover, the tests reveal that in the case of materials containing 10% wt of PEG, incorporation of quercetin causes the formation of different crystalline PLA forms. This phenomenon was widely described by Tabi et al. [49]. The formation of two crystalline forms is not observed in the case of polymeric films consisting of PLA, 5% PEG and quercetin, which is well worth noting.

#### *3.4. Evaluation of Mechanical Properties*

It is a commonly recognized fact that mechanical properties of packaging materials are extremely important [16]. In order to establish the impact different amounts of plasticizer and biocidal agent in form of quercetin have on the mechanical properties, the Young's modulus (*E*), tensile strength (*σm*) and elongation at break (*ε*) were determined. The values of Young's modulus and tensile strength, are shown in Figure 11a,b, respectively. In the case of a sample with a higher PEG content (LP10) a significant decrease of the Young's modulus and tensile strength in comparison with LP5 sample has been observed. These observations are consistent with results obtained by other researches [50,51] in relation to similar composition. During the study it has been established that the decrease in tensile strength, as well in Young's modulus values, can be attributed to poor stress transfer between the PLA and PEG phases [52]. An introduction of quercetin into polylactide-PEG systems significantly improves the properties indicated above. The PLA-PEG samples containing 1 as well as 2 wt.% of quercetin was characterized by a remarkable reinforcement of their structure. The highest values of mechanical properties were obtained in relation to the LP5Q2 sample. The value of Young's modulus for this material was about 2060 MPa while the maximum tensile strength reached about 32 MPa. The elongation at break (*ε*) values are presented in Figure 11c.

It can be clearly seen that an increase in the elasticity is correlated with the increase in the PEG content. It is well known that this phenomenon is related to reduction of intermolecular friction between the polymer molecules caused by the plasticizer molecules. The addition of quercetin allowed obtaining films with higher values of elasticity compared to the LP5 and LP10 samples. The highest value of the elongation at break was recorded for LP10 filled with 2.0 wt.% of quercetin. Moreover, it has to be stressed that in the case of the LP10Q2 sample the values of Young's modulus and elongation at breaking point are twice as high as for the LP10 material. Taking into account the obtained results the plasticizing effect of quercetin could potentially seem surprising. The same tendency, however, was reported in literature [53,54]. In the work of Luzi et al. [55] it was established that an improvement of the mechanical properties can be related to the inter-molecular interactions between hydrophilic groups of the polymer matrix and the polyhydroxyl groups of quercetin. In the work of K. Rubini et al. [53], where the influence of quercetin on the mechanical properties of gelatin-based films was evaluated, the values of the Young's modulus, the stress at break and the deformation at break increased significantly up to a point in which the flavonoid concentration of 1.5 wt.% was achieved. This limitation was caused by the non-homogeneous distribution of quercetin in gelatin. The susceptibility to deformation was also observed in the work of M. Latos-Brozio and A. Masek [54] where mechanical properties of PLA impregnated with quercetin and other substances of plant origin were discussed. Based on the obtained results and indicated literature, it is reasonable to assume that an incorporation of quercetin into the PLA matrix leads to an increase in mechanical properties of the obtained polymeric films.

**Figure 11.** Mechanical properties of polylactide-based materials: (**a**) Young's modulus (E), (**b**) Tensile strength (*σm*), (**c**) Elongation at break (*ε*). **Figure 11.** Mechanical properties of polylactide-based materials: (**a**) Young's modulus (E), (**b**) Tensile strength (*σm*), (**c**) Elongation at break (*ε*).

#### *3.5. Thickness and Transparency of Studied Materials*

*3.5. Thickness and Transparency of Studied Materials* The values of thickness and transparency were established based on the absorbance measurements and presented in Table 6. The obtained results indicate that an increase in the amount of PEG significantly increases the thickness of LP10 film in comparison with the LP5 sample. These findings are consistent with the results presented in literature. Anuar et al. [56] have established that an introduction of PEG into the polylactide matrix caused a diffusion of the plasticizer between the PLA chains. As a result, an increase of free volume between polylactide chains as well as an increase of thickness can be observed. Incorporation of quercetin into the PLA-PEG systems slightly decreases the thickness of the obtained materials, which is quite unexpected. In most cases, the introduction of different types of substances, such as essential oils, into the polymer matrix results in an increase of material thickness [16,57,58]. In the case of quercetin the opposite effect has been observed. Presumably, this can be connected to the interactions between –COOH The values of thickness and transparency were established based on the absorbance measurements and presented in Table 6. The obtained results indicate that an increase in the amount of PEG significantly increases the thickness of LP10 film in comparison with the LP5 sample. These findings are consistent with the results presented in literature. Anuar et al. [56] have established that an introduction of PEG into the polylactide matrix caused a diffusion of the plasticizer between the PLA chains. As a result, an increase of free volume between polylactide chains as well as an increase of thickness can be observed. Incorporation of quercetin into the PLA-PEG systems slightly decreases the thickness of the obtained materials, which is quite unexpected. In most cases, the introduction of different types of substances, such as essential oils, into the polymer matrix results in an increase of material thickness [16,57,58]. In the case of quercetin the opposite effect has been observed. Presumably, this can be connected to the interactions between –COOH groups of PLA and -OH of quercetin.

groups of PLA and -OH of quercetin.

**Table 6.** Thickness and transparency values of studied materials.

Taking into account the results presented in Table 6, it was established that an addition of 10 wt.% of poly(ethylene glycol) significantly decreases the transparency in comparison with a polymeric film containing 5 wt.% of PEG. Observations indicated that the transparency of the LP5 films is twice as high as that of LP10. This is consistent with literature [58], where in the case of materials containing 20 wt.% of poly(ethylene glycol), the transparency value of the PLA-PEG system achieved a value of 2.63. Thus, it can be assumed the transparency of blends consisting of PLA and PEG significantly depends on the composition. An introduction of 1 wt.% as well as 2 wt.% of yellow quercetin significantly decreases transparency of the obtained materials. It should be stressed, however, that the decrease in transparency of films infused with the same amount of quercetin (LP5Q1, LP10Q1 as well as LP5Q2 and LP10Q2) has similar values regardless of the polymer compositions (LP5 and LP10). This phenomenon clearly indicates that in the case of studied materials transparency is mainly related to the amount of used quercetin rather than the applied PLA-PEG system. The obtained results suggest that polymeric films containing quercetin can be used as packaging materials, especially for products which are light sensitive. Summarizing, the application of materials consisting of PLA, PEG and quercetin will not allow for discoloration of products, loss of flavor or nutrients.

#### *3.6. Differences in Colour of Studied Materials*

In order to improve the product's appearance, which significantly influences the consumer's decision-making process in terms of purchase, the packaging materials are mixed with compounds which are able to change their color. In this paper the color of the polymeric films based on PLA with an addition of poly(ethylene glycol) as plasticizer and quercetin as antibacterial agent was analyzed. The values of measured parameters *L*, *a*, *b* and the calculated parameters such as total color difference (∆*E*), chroma (*C*) and yellowness index (*YI*) are listed in Table 7.

**Table 7.** Color (*L*, *a*, *b*, ∆*E*, *C*, *YI*) parameters of obtained PLA-based materials.

In the case of samples consisting of PLA and PEG, the color difference equals 0.9 and proves that an addition of a higher amount of plasticizer does not influence the color changes. According to literature [33] the values of ∆*E* lower than 3 indicate that only an experienced observer is able to notice the difference in color of the studied materials. The changes in color, however, were observed in the case of films comprising quercetin. The changes in color of the obtained materials were not intentional and were most likely caused by an addition of a yellow—quercetin which significantly influenced the appearance of the samples. It can be clearly seen that with an increasing amount of quercetin the PLA-based films seem to be darker. Moreover, the color shifts to yellow upon increasing the amount

of quercetin. The most significant color changes are observed after introducing 2 wt.% of an antibacterial agent into the LP5 and PL10 systems. In the case of the LP5Q2 sample ∆*E* equaled 31.9, while for the LP10Q2 film ∆*E* equaled 33.6. The same tendency was observed in the case of topas cyclo-olefin copolymer (ethylene-norbornene) with an addition of quercetin described in the work of A. Masek et al. [25].

The second color parameter analyzed in this work was chroma (*C*) which is strongly connected with colorfulness. It has to be stressed that the higher the chroma values, the higher is the color intensity of the analyzed materials indicated by observers [33]. It is not surprising, that in the case of obtained materials with an addition of quercetin the chroma value significantly increased. This phenomenon is connected with the yellow color which is assigned to the conjugation in the B-ring cinnamoyl system and is typical of flavonoids [59]. Similar observations can be made in the case of the yellowness index (*YI*) values. Significant differences in *b* and, as a result, in *C*, ∆*E* and *YI* values were established between samples with and without quercetin. Summarizing, the addition of quercetin into PLA-PEG systems significantly influences the color parameters of the obtained materials.

#### *3.7. Examination of Antibacterial Properties*

It is a widely recognized fact that improving the antibacterial properties of biodegradable packaging materials prevents the development of food-damaging pathogens and significantly reduces the volume of plastic waste, by providing extended shelf life. In order to ensure antibacterial properties of packaging materials, different compounds are introduced into the polymer matrix. Depending on the type of active additive, they can be divided into chemoactive and bioactive substances. The most chemoactive ingredients include iron, titanium and zinc. The characteristic feature of the compounds mentioned above is reactivity with oxygen. It should be stressed, however, that all of them can affect the chemical composition of the product as well as the interior environment of the package. In addition, they can cause adverse health effects and hinder the recycling process. The indisputable disadvantages of chemoactive compounds lead to a more extensive application of bioactive additives derived from natural sources, such as polyphenols, essential oils and other natural extracts [15–17]. These substances play an important role in active packaging. As antioxidants, they allow to reduce production costs and eliminate the risk related to food safety. In the present work, the disk-diffusion method was applied in order to establish the capacity to combat microorganisms characterizing the obtained materials imbued with quercetin. In Table 8 the results of antibacterial properties are presented. In the case of the LP5 and LP10 films assigned as control samples, the inhibition of *S. Aureus* and *E. coli* growth was not observed.

**Table 8.** Results of antibacterial activity of the obtained PLA-based materials.

1 in accordance with ISO 20645:2006 standard.

The same tendency was observed in literature [58,60]. Introduction of quercetin into the PLA-PEG systems significantly changed the antibacterial properties of the obtained materials (Figures S1 and S2). Moreover, it was established that quercetin creates a more significant obstacle for the microorganisms in the case of materials containing a higher amount of plasticizer (LP10Q2) (Figure 12). It is, therefore, reasonable to assume that the

mentioned above beneficial change resulted from an increase of free volume between PLA chains caused by PEG. The effect of poly(ethylene glycol) on the antibacterial properties was also evaluated in our previous publication [16]. sistent with the data published by Kost et al. [26]. Recent studies as well as the obtained results clearly indicate that quercetin has the potential to combat bacteria and for this reason the incorporation of quercetin contributed to an increase in antibacterial capacity of the obtained materials.

The same tendency was observed in literature [58,60]. Introduction of quercetin into the PLA-PEG systems significantly changed the antibacterial properties of the obtained materials (Figures S1 and S2). Moreover, it was established that quercetin creates a more significant obstacle for the microorganisms in the case of materials containing a higher amount of plasticizer (LP10Q2) (Figure 12). It is, therefore, reasonable to assume that the mentioned above beneficial change resulted from an increase of free volume between PLA chains caused by PEG. The effect of poly(ethylene glycol) on the antibacterial properties

In literature [61,62] quercetin is considered to be a biocidal agent. Ohemeng et al. [63] have established that quercetin is able to inhibit the *E. coli* DNA gyrase. Plaper et al. [64] showed that quercetin forms a bond with the GyrB subunit of *E. coli* DNA gyrase. Moreover, additional antibacterial mechanisms, triggered by quercetin, resulted in destabilizing of bacterial membrane functions were described by Mirzoeva et al. [65]. It should be stressed that the reported antibacterial activity of materials containing quercetin is con-

*Materials* **2021**, *14*, x FOR PEER REVIEW 19 of 22

was also evaluated in our previous publication [16].

**Figure 12.** Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with LP10Q2 material.

**Figure 12.** Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with LP10Q2 material. **4. Conclusions** In summary, the influence of quercetin on the physico-chemical and antibacterial properties of PLA based materials was examined. Quercetin was introduced into two polymer systems consisting of polylactide and poly(ethylene glycol) (5 and 10 wt.%, respectively). The obtained materials containing quercetin were characterized by a significant improvement in thermal and mechanical properties. Simultaneously, it was also proved that the observed changes are a result of the inter-molecular interactions between hydrophilic groups of polylactide and poly(ethylene glycol) with the polyhydroxyl groups of quercetin. Moreover, it should be stressed that the introduction of quercetin allows ob-In literature [61,62] quercetin is considered to be a biocidal agent. Ohemeng et al. [63] have established that quercetin is able to inhibit the *E. coli* DNA gyrase. Plaper et al. [64] showed that quercetin forms a bond with the GyrB subunit of *E. coli* DNA gyrase. Moreover, additional antibacterial mechanisms, triggered by quercetin, resulted in destabilizing of bacterial membrane functions were described by Mirzoeva et al. [65]. It should be stressed that the reported antibacterial activity of materials containing quercetin is consistent with the data published by Kost et al. [26]. Recent studies as well as the obtained results clearly indicate that quercetin has the potential to combat bacteria and for this reason the incorporation of quercetin contributed to an increase in antibacterial capacity of the obtained materials.

#### taining antibacterial polymeric films. Furthermore, it can be clearly noticed that the yellow **4. Conclusions**

color of quercetin influences the change in the color and transparency of the studied materials. The films consisting of PLA, PEG and quercetin constitute promising bactericidal materials which can be applied as packaging materials. **Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1, Figure S1: Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with samples containing 5% wt. of PEG, Figure S2: Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with samples containing 10% wt. of PEG. In summary, the influence of quercetin on the physico-chemical and antibacterial properties of PLA based materials was examined. Quercetin was introduced into two polymer systems consisting of polylactide and poly(ethylene glycol) (5 and 10 wt.%, respectively). The obtained materials containing quercetin were characterized by a significant improvement in thermal and mechanical properties. Simultaneously, it was also proved that the observed changes are a result of the inter-molecular interactions between hydrophilic groups of polylactide and poly(ethylene glycol) with the polyhydroxyl groups of quercetin. Moreover, it should be stressed that the introduction of quercetin allows obtaining antibacterial polymeric films. Furthermore, it can be clearly noticed that the yellow color of quercetin influences the change in the color and transparency of the studied materials. The films consisting of PLA, PEG and quercetin constitute promising bactericidal materials which can be applied as packaging materials.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ma14071643/s1, Figure S1: Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with samples containing 5% wt. of PEG, Figure S2: Photos of Bacteria (*E. coli* and *S. aureus*) growth in direct contact with samples containing 10% wt. of PEG.

**Author Contributions:** Conceptualization, E.O.-K.; methodology, E.O.-K. and A.R. (Agnieszka Richert); software, E.O.-K., M.G., S.G.-Z., M.B. and A.R. (Anna Rudawska); validation, E.O.-K., M.G., A.R. (Agnieszka Richert); formal analysis, E.O.-K., M.G., M.B. and S.G.-Z.; investigation, E.O.-K., M.G., A.R. (Agnieszka Richert) and A.R. (Anna Rudawska); writing—original draft preparation E.O.-K.; writing—review and editing, E.O.-K., M.G.; visualization, E.O.-K., M.G; supervision, E.O.-K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Nicolaus Copernicus University in Toru ´n.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Influence of Tea Tree Essential Oil and Poly(ethylene glycol) on Antibacterial and Physicochemical Properties of Polylactide-Based Films**

#### **Iwona Tarach 1 , Ewa Olewnik-Kruszkowska 1,\* , Agnieszka Richert 2 , Magdalena Gierszewska 1 and Anna Rudawska 3**

Received: 5 October 2020; Accepted: 1 November 2020; Published: 4 November 2020

**Abstract:** The aim of the study was to establish the influence of poly(ethylene glycol) (PEG) on the properties of potential biodegradable packaging materials with antibacterial properties, based on polylactide (PLA) and tea tree essential oil (TTO). The obtained polymeric films consisted of PLA, a natural biocide, and tea tree essential oil (5–20 wt. %) was prepared with or without an addition of 5 wt. % PEG. The PLA-based materials have been tested, taking into account their morphology, and their thermal, mechanical and antibacterial properties against Staphylococcus aureus and Escherichia coli. It was established that the introduction of a plasticizer into the PLA–TTO systems leads to an increase in tensile strength, resistance to deformation, as well an increased thermal stability, in comparison to films modified using only TTO. The incorporation of 5 wt. % PEG in the PLA solution containing 5 wt. % TTO allowed us to obtain a material exhibiting a satisfactory antibacterial effect on both groups of representative bacteria. The presented results indicated a beneficial effect of PEG on the antibacterial and functional properties of materials with the addition of TTO.

**Keywords:** polylactide; tea tree essential oil; poly(ethylene glycol); packaging material; antibacterial films

#### **1. Introduction**

The proliferation of bacteria contributes to the spread of diseases, as well as food spoilage [1,2]. This group of microorganisms includes mainly strains such as *Bacillus cereus*, *Listeria monocytogenes*, *Salmonella typhimurium* and *Staphylococcus aureus* [3,4]. Due to the possibility of the undesirable contact of microorganisms with food, effective and safe methods of combating pathogenic bacteria are being sought [5]. For this reason, the food industry is still looking for new, innovative solutions to combat microorganisms' proliferation in packaged products, and to extend their shelf-life [6].

The manufacturing of packaging with antibacterial properties relies on the direct incorporation of biologically active substances into the polymer matrix [7]. In particular, the application of natural antibacterial agents for this purpose is noteworthy [8]. Essential oils (EO) are natural, microbiologically active substances with an extensive spectrum of activity [9]. Due to the acceptance of PLA and EO by the American Food and Drug Administration as "Generally Recognized As Safe GRAS", a significant

development of studies of materials based on the above-mentioned compounds has taken place [10]. Ahmed et al. in their works described the results of detailed research devoted to films based on polylactide (PLA) with the addition of cinnamon oil [11,12]. The application of other essential oils, such as clove [4,13], garlic [9,12], as well as lemongrass, rosemary and bergamot essential oil [14], allowed the obtaining of PLA-based materials displaying antibacterial properties.