Poly(lactic acid)/Zinc/Alginate Complex Material: Preparation and Antimicrobial Properties

The aim of this study was to investigate an antimicrobial and degradable composite material consisting of melt-blown poly(lactic acid) nonwoven fabrics, alginate, and zinc. This paper describes the method of preparation and the characterization of the physicochemical and antimicrobial properties of the new fibrous composite material. The procedure consists of fabrication of nonwoven fabric and two steps of dip-coating modification: (1) impregnation of nonwoven samples in the solution of alginic sodium salt and (2) immersion in a solution of zinc (II) chloride. The characterization and analysis of new material included scanning electron microscopy (SEM), specific surface area (SSA), and total/average pore volume (BET). The polylactide/alginate/Zn fibrous composite were subjected to microbial activity tests against colonies of Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli) bacterial strains, and the following fungal strains: Aspergillus niger van Tieghem and Chaetomium globosum. These results lay a technical foundation for the development and potential application of new composite as an antibacterial/antifungal material in biomedical areas.


Introduction
Wound healing is a dynamic and complex process (phases: hemostasis, inflammation, proliferation, and maturation) affected by several factors, which needs an appropriate surrounding to achieve accelerated healing [1]. Modern wound healing dressing should exhibit non-toxic and non-allergenic properties, be capable of maintaining high humidity at the wound site while removing excess exudate, antibacterial characteristics or at least impermeability to bacteria, enable gaseous exchange, and be cost effective [2][3][4][5]. These requirements are partly fulfilled by the physico-chemical properties of PLA (biocompatibility, biodegradability, mechanical strength) [6][7][8][9][10][11][12][13][14][15] and to a much larger extent by PLA-composites equipped with a wide spectrum of antimicrobial agents, including inorganic microbials [16,17].
Since an effective antibacterial PLA composite should exhibit prolonged antibacterial activity, a stable surface deposition/attachment of zinc to PLA presents a major problem.
As part of our investigations focused on the functionalization of textile materials [125][126][127][128][129][130][131], we propose the use of an alginate film covering the PLA matrix (PLA-ALG), which, after adding copper salts, was cross-linked to form an outer space coating with strongly bound copper ions (Figure 1). Such a PLA-Alg-Zn (2+) composite slowly releases zinc ions, ensuring its long-lasting antibacterial activity ( Figure 2).   The poly(lactic acid) melt-blown nonwoven fabrics with a basis weight of 250 g/m 2 were produced by using a one-screw laboratory extruder (Axon, Limmared, Sweden). The extruder head has 30 holes with a 0.25 mm die orifice diameter each. The polylactide (PLA) granulate for melt blowing was dried at 80 • C to constant weight. The process parameters of melt blowing are given in Table 1.

Modification-Dip-Coting
The PLA melt-blown nonwoven fabrics samples were modified by dip-coting, a twostep method: (1) impregnation in the solution of alginic acid sodium salt and (2) immersion in the solution of zinc (II) chloride. Samples of PLA nonwoven fabric were impregnated in a homogeneously dispersed polysaccharide solution (0.5%) for 1 min (sample: PLA-Alg-Na (+) ), then each sample was immediately transferred into two different aqueous solutions of zinc (II) chloride and re-immersed for 1 min (sample PLA-Alg-Zn (2+) -1 re-immersed in 5% ZnCl 2 solution and sample PLA-Alg-Zn (2+) -2 re-immersed in 10% ZnCl 2 solution). Then, the samples were squeezed and dried for 5 h at 50 • C to constant weight. The modifier components are given in Table 2.

Morphological and Structural Characterization-Scanning Electron Microscopy
Examination of the microscopic structures was carried out using a HITACHI S-4700 scanning electron microscope equipped with a Thermo NORAN EDS X-ray microanalyzer.
The topographic analysis of the tested samples was carried out in low vacuum with a beam energy of 10 kV and at magnifications of 800× and 1600×.

Morphological and Structural Characterization-Specific Surface Area
The specific surface area was determined by the Brunauer, Emmet, and Teller method (BET). Measurements were carried out on an Autosorb-1 apparatus (Quantachrome Instruments, Boynton Beach, FL, USA), using nitrogen as a sorption agent and an adsorption isotherm at 77 K. In each experiment, approximately 1-2 g of a given sample were weighed and used. Prior to the analysis, the samples were dried in 105 • C for 24 h and degassed at room temperature. Measurements were made in duplicate, and the results were presented as a mean value.

Morphological and Structural Characterization-Contact Angle and Wettability
Surface wettability was determined by static measurements of the water wetting angle. The wetting angle was measured by the "sitting droplet" method using a drop shape analysis (DSA) system DSA 10 Mk2 (Kruss GmbH, Hamburg, Germany). Water drops of 0.25 µL were applied to each clean and dry sample. The apparent contact angle was calculated as the average of 10 measurements [132].

Chemical Characterization-Atomic Absorption Spectrometry with Flame Excitation
Determination of the zinc content in PLA-Alg-Zn (2+) composites was assessed by the FAAS method. The previous sample was mineralized ( Figure 3) using a single-module Magnum II microwave mineralizer from Ertec (Wroclaw, Poland), in similar way as was described earlier [133].

Morphological and Structural Characterization-Scanning Electron Microscopy
Examination of the microscopic structures was carried out using a HITACHI S-4700 scanning electron microscope equipped with a Thermo NORAN EDS X-ray microanalyzer. The topographic analysis of the tested samples was carried out in low vacuum with a beam energy of 10 kV and at magnifications of 800× and 1600×.

Morphological and Structural Characterization-Specific Surface Area
The specific surface area was determined by the Brunauer, Emmet, and Teller method (BET). Measurements were carried out on an Autosorb-1 apparatus (Quantachrome Instruments, Boynton Beach, FL, USA), using nitrogen as a sorption agent and an adsorption isotherm at 77 K. In each experiment, approximately 1-2 g of a given sample were weighed and used. Prior to the analysis, the samples were dried in 105 °C for 24 h and degassed at room temperature. Measurements were made in duplicate, and the results were presented as a mean value.

Morphological and Structural Characterization-Contact Angle and Wettability
Surface wettability was determined by static measurements of the water wetting angle. The wetting angle was measured by the "sitting droplet" method using a drop shape analysis (DSA) system DSA 10 Mk2 (Kruss GmbH, Hamburg, Germany). Water drops of 0.25 µ L were applied to each clean and dry sample. The apparent contact angle was calculated as the average of 10 measurements [132].

Chemical Characterization-Atomic Absorption Spectrometry with Flame Excitation
Determination of the zinc content in PLA-Alg-Zn (2+) composites was assessed by the FAAS method. The previous sample was mineralized ( Figure 3) using a single-module Magnum II microwave mineralizer from Ertec (Wroclaw, Poland), in similar way as was described earlier [133]. Determination of the zinc (II) ions was performed by atomic absorption spectrometry with flame excitation using a Thermo Scientific Thermo Solar M6 (LabWrench, Midland, ON, Canada) spectrometer equipped in similar way as was described earlier [133,134]. Measurements were made in triplicate and the results were presented as a mean value. Determination of the zinc (II) ions was performed by atomic absorption spectrometry with flame excitation using a Thermo Scientific Thermo Solar M6 (LabWrench, Midland, ON, Canada) spectrometer equipped in similar way as was described earlier [133,134]. Measurements were made in triplicate and the results were presented as a mean value.

Antimicrobial Activity
The antibacterial activity of PLA-Alg-Zn (2+) material was assessed according to standard PN-EN ISO 20645:2006 [135] against a representative colony of Gram-negative and Gram-positive bacteria (Escherchia coli/Staphylococcus aureus). The antifungal activity of composites was tested according to PN-EN 14119:2005 [136] against an Aspergillus niger van Tieghem (ATCC 6275) and Chaetomium globosum (ATCC 6205). All tests (modified samples and unmodified PLA) were carried out in duplicate.

Scanning Electron Microscopy
The presented SEM images illustrate changes in the morphology of the fiber surface of the investigated samples. These changes are the result of modification of PLA nonwoven fabric with sodium salt solution of alginic acid and ZnCl 2 .
The SEM image of the unmodified nonwoven fabric presents a mesh of randomly oriented fibers with interconnected pores and a comparatively smooth surface (Figure 4). Modification of the nonwoven fabric with sodium alginate solution (PLA→PLA-ALG (−) Na (+) ) induced the formation of a film on the fiber surface, during which the PLA fibrous structure serves as a matrix for a deposited alginate, presumably by the formation of hydrogen bonds between the HO group of alginate and the carbonyls of the carboester functions of PLA. This modification slightly affected the overall morphology of the formed composite surface, maintaining the fibrous structure of the PLA→PLA-ALG (−) Na (+) composites but with more irregular shapes and less visible pores. Additionally, agglomerates of sodium alginate (ALG-Na (+) ) can be seen on single fibers of PLA-ALG -Na (+) ( Figure 5). Further modification of the temporary composite PLA-ALG (−) Na (+) with ZnCl 2 , (PLA-ALG (−) Na (+) →PLA-ALG-Zn (2+) ) led to immense changes in its morphology, probably due to the formation of interchain complexes PLA 1 -ALG 1 . Therefore, the fibrous structure of the resulting composite was largely deformed with an increase of surface roughness, with multiple zinc agglomerates (Figure 6), resembling a polymer film, with integrated PLA fibers. Figures 7-9 present example of the EDS spectra for PLA, PLA-Alg-Na (+) , and PLA-Alg-Zn (2+) (10%) samples (EDS data are presented as a plot of the peak intensity versus energy (keV)). Table 3 shows the chemical composition of the tested materials, obtained by quantitative analysis using EDS X-ray microanalysis. The table presents the averaged results obtained from five measurement points. The antibacterial activity of PLA-Alg-Zn (2+) material was assessed according to standard PN-EN ISO 20645:2006 [135] against a representative colony of Gram-negative and Gram-positive bacteria (Escherchia coli/Staphylococcus aureus). The antifungal activity of composites was tested according to PN-EN 14119:2005 [136] against an Aspergillus niger van Tieghem (ATCC 6275) and Chaetomium globosum (ATCC 6205). All tests (modified samples and unmodified PLA) were carried out in duplicate.

Scanning Electron Microscopy
The presented SEM images illustrate changes in the morphology of the fiber surface of the investigated samples. These changes are the result of modification of PLA nonwoven fabric with sodium salt solution of alginic acid and ZnCl2.
The SEM image of the unmodified nonwoven fabric presents a mesh of randomly oriented fibers with interconnected pores and a comparatively smooth surface (Figure 4). Modification of the nonwoven fabric with sodium alginate solution (PLA→PLA-ALG (−) Na (+) ) induced the formation of a film on the fiber surface, during which the PLA fibrous structure serves as a matrix for a deposited alginate, presumably by the formation of hydrogen bonds between the HO group of alginate and the carbonyls of the carboester functions of PLA. This modification slightly affected the overall morphology of the formed composite surface, maintaining the fibrous structure of the PLA→PLA-ALG (−) Na (+) composites but with more irregular shapes and less visible pores. Additionally, agglomerates of sodium alginate (ALG-Na (+) ) can be seen on single fibers of PLA-ALG -Na (+) ( Figure 5). Further modification of the temporary composite PLA-ALG (-) Na (+) with ZnCl2, (PLA-ALG (−) Na (+) →PLA-ALG-Zn (2+) ) led to immense changes in its morphology, probably due to the formation of interchain complexes PLA 1 -ALG 1 -Zn (2+) -ALG 2 -PLA 2 (PLA 1 -ALG 1(−) Na (+) →PLA 1 -ALG 1 -Zn (2+) →PLA 1 -ALG 1 -Zn (2+) -ALG 2 -PLA 2 ). Therefore, the fibrous structure of the resulting composite was largely deformed with an increase of surface roughness, with multiple zinc agglomerates (Figure 6), resembling a polymer film, with integrated PLA fibers.   Figures 7-9 present example of the EDS spectra for PLA, PLA-Alg-Na (+) , and PLA-Alg-Zn (2+) (10%) samples (EDS data are presented as a plot of the peak intensity versus energy (keV)). Table 3 shows the chemical composition of the tested materials, obtained by quantitative analysis using EDS X-ray microanalysis. The table presents the averaged results obtained from five measurement points.  Figures 7-9 present example of the EDS spectra for PLA, PLA-Alg-Na (+) , and PLA-Alg-Zn (2+) (10%) samples (EDS data are presented as a plot of the peak intensity versus energy (keV)). Table 3 shows the chemical composition of the tested materials, obtained by quantitative analysis using EDS X-ray microanalysis. The table presents the averaged results obtained from five measurement points.          When analyzing the spectrum in Figure 7, it can be observed that the sample contains elements characteristic for polylactide, that is, carbon and oxygen. Whereas the PLA- ALG-Na (+) sample, because of the modification with sodium alginate, is characterized by the presence of an additional peak, derived from Na atoms (Figure 8). Additionally, modification of PLA-ALG-Na (+) nonwoven fabric with zinc (II) chloride contributed to the presence of further peaks, originating from zinc and chloride. The presence of chloride is confirmed by the content of NaCl in the samples.
Quantitative EDS analysis showed that modification of PLA nonwoven fabric with sodium alginate did not significantly change the carbon and oxygen contents of the samples. Slight decreases in the content of these elements were observed: about 0.28 at. % and about 1.38 at. % for carbon and oxygen, respectively. Additionally, small concentrations of sodium (1.65 at %) were noted for the PLA-ALG-Na (+) sample. The EDS results for the samples modified with ZnCl 2 show that the content of Zn and Cl in the samples increased with the increasing chloride concentration. However, because it showed the best antibacterial and antifungal properties, the results are presented for the PLA-ALG-Zn (2+) sample (10%). We can observe that because of the rapid cross-linking of the sample surface and the formation of a kind of film on the surface, the sodium content increased twice, compared to the PLA-ALG-Na (+) sample. On the other hand, the high content of zinc (14.22%) confirms that the agglomerates shown in the photos are agglomerates of a divalent metal ( Figure 9). Moreover, it can be seen that when the samples were modified with ZnCl 2 , the carbon and oxygen content decreased significantly (by 23.39 at. % and 11.26 at. %, respectively). A high value of standard deviation indicates an irregular distribution of the chemical components.

Contact Angle and Wettability
The analysis of the contact angle enabled an assessment of the wettability of the surfaces of the tested materials. The results demonstrated that PLA and PLA-ALG Zn (2+) nonwoven fabrics were characterized by a hydrophobic character of the surface, for which  (Figure 10a,c). In contrast, the modification with sodium alginate changed the character of the material surface, which became hydrophilic with an average contact angle of 45.67 • (Figure 10b).

Contact Angle and Wettability
The analysis of the contact angle enabled an assessment of the wettability of the surfaces of the tested materials. The results demonstrated that PLA and PLA-ALG Zn (2+) nonwoven fabrics were characterized by a hydrophobic character of the surface, for which the average contact angle was 122.79° and 114.98°, respectively (Figure 10a,c). In contrast, the modification with sodium alginate changed the character of the material surface, which became hydrophilic with an average contact angle of 45.67° (Figure 10b). As shown in Figures 10 and 11, modification of PLA nonwoven fabric with sodium alginate significantly changed the nature of the nonwoven fabric surface from hydrophobic to hydrophilic. This is due to the presence of the Na + ions contained in alginate, which make it soluble in water. On the other hand, another modification with ZnCl2 resulted in substitution of sodium ions with divalent metal ions (Zn 2+ ), thanks to which the crosslinking process occurred on the surface and alginate covering fibers became insoluble in water. This changed the nature of the nonwoven surface from hydrophilic to hydrophobic again. However, it is characterized by lower surface hydrophobicity than the PLA nonwoven fabric, due to the Na + ions present on the surface in small amounts, which was confirmed by SEM-EDS analysis. As shown in Figures 10 and 11, modification of PLA nonwoven fabric with sodium alginate significantly changed the nature of the nonwoven fabric surface from hydrophobic to hydrophilic. This is due to the presence of the Na + ions contained in alginate, which make it soluble in water. On the other hand, another modification with ZnCl 2 resulted in substitution of sodium ions with divalent metal ions (Zn 2+ ), thanks to which the crosslinking process occurred on the surface and alginate covering fibers became insoluble in water. This changed the nature of the nonwoven surface from hydrophilic to hydrophobic again. However, it is characterized by lower surface hydrophobicity than the PLA nonwoven fabric, due to the Na + ions present on the surface in small amounts, which was confirmed by SEM-EDS analysis.
Antibiotics 2021, 10, x FOR PEER REVIEW 10 of 19 Figure 11. The static water contact angle of PLA and its composites.

Flame Atomic Absorption Spectrometry
Determination of the metal concentration in the PLA-Alg-Zn (2+) samples was assessed by the FAAS spectrometry, and the results are shown in Table 5.

Flame Atomic Absorption Spectrometry
Determination of the metal concentration in the PLA-Alg-Zn (2+) samples was assessed by the FAAS spectrometry, and the results are shown in Table 5. Table 5. Zinc concentration in PLA-Alg-Zn (2+) samples.

Sample Zn Concentration [g/kg]
PLA 0.003 PLA-Alg-Na (+) 0.003 PLA-Alg-Zn (2+) -1 11.55 PLA-Alg-Zn (2+) -2 39.71 The results of the determination of the zinc concentration in PLA-Alg-Zn (2+) samples show that the metal content in complex materials depends on the variant of applied modifier dip-coating solution and type of solution of Zinc (II) chloride ( Table 5). The higher concentration of the used zinc (II) chloride solutions (10%) resulted in the higher content of the metal on PLA-Alg-Zn (2+) -2 material (39.71 g/kg), and the lower concentration of zinc (II) chloride (5%) gave a relatively lower content of Zn in the sample (PLA-Alg-Zn (2+) -1-11.55 g/kg). Additionally, the FAAS spectrometry measurements also indicate that the distribution of zinc in the material is quite uniform (approximately 7.5%).
Control samples as unmodified PLA and modified by alginic acid sodium salt material (PLA-Alg-Na (+) ) exhibited strong growth of bacterial and fungal colonies covering the entire surface of the samples placed on Petri dishes (Figures 12a,c and 13a,c). Poly(lactic acid) material functionalized with a zinc/alginate complex, regardless of the metal concentration in PLA-Alg-Zn (2+) composites in the range from 11 to 39 [g/kg] (Table 5), showed an inhibitory effect against S. aureus bacteria and fungus species expressed by zones of inhibition and no visible growth on/under the samples (Tables 6 and 7). The increase of the zinc concentration in PLA-Alg-Zn (2+) composites caused an increase of the antimicrobial properties of the material. The PLA-Alg-Zn (2+) -2 (39 [g/kg] of Zn) sample showed antimicrobial properties for all variants of the tested microorganisms (Figures 12b,d and 13b,d). The results obtained in accordance with the EN-ISO 20645:2006 and EN 14119:2005 standards confirm the antimicrobial protection of PLA-Alg-Zn (2+) complexes against various microorganisms [135,136].

Conclusions
In this study, we developed and characterized a complex material consisting of alginic acid sodium salt, poly(lactide), and zinc (II) chloride. PLA-ALG-Zn (2+) composite was fabricated from biodegradable PLA polymer by the melt-blown method, and then the obtained fibrous material was modified by dip-coating, using alginic acid sodium salt and zinc (II) chloride. Structural characterization of the new material was achieved by scanning electron microscopy (SEM), and determination of the specific surface area and wettability. The chemical compositions of the PLA-Alg-Zn (2+) composites were identified using energy-dispersive X-ray spectroscopy EDS (C, O, Zn surface analysis) and atomic absorption spectrometry with flame excitation (Zn content in bulk). The obtained complex material exhibited an antimicrobial in vitro action against representative bacteria: Escherichia coli and Staphylococcus aureus, and fungus species: Chaetomium globosum and Aspergillus niger. From the point of view of our previous work, these results are promising regarding the material's applicability in different fields of materials science in the medical or healthcare industry.

Conclusions
In this study, we developed and characterized a complex material consisting of alginic acid sodium salt, poly(lactide), and zinc (II) chloride. PLA-ALG-Zn (2+) composite was fabricated from biodegradable PLA polymer by the melt-blown method, and then the obtained fibrous material was modified by dip-coating, using alginic acid sodium salt and zinc (II) chloride. Structural characterization of the new material was achieved by scanning electron microscopy (SEM), and determination of the specific surface area and wettability. The chemical compositions of the PLA-Alg-Zn (2+) composites were identified using energydispersive X-ray spectroscopy EDS (C, O, Zn surface analysis) and atomic absorption spectrometry with flame excitation (Zn content in bulk). The obtained complex material exhibited an antimicrobial in vitro action against representative bacteria: Escherichia coli and Staphylococcus aureus, and fungus species: Chaetomium globosum and Aspergillus niger. From the point of view of our previous work, these results are promising regarding the material's applicability in different fields of materials science in the medical or healthcare industry.