Biofunctionalization of Textile Materials.1. Biofunctionalization of Poly(Propylene) (PP) Nonwovens Fabrics by Alafosfalin

: This paper presents the method of obtaining poly(propylene) (PP) nonwoven fabrics with antimicrobial properties, using Alafosfalin as the nonwoven modifying agent. Alafosfalin, namely L-alanyl-L-1-aminoethylphosphonic acid, presents representative P-terminal phosphonodipeptide, which possesses a strong, broad spectrum of antimicrobial properties. The analysis of these biofunctionalized nonwoven fabrics processed by the melt-blown technique, included: scanning electron microscopy (SEM), UV / Vis transmittance, FTIR spectrometry, and air permeability. The nonwovens were subjected to microbial activity tests against colonies of Gram-positive ( Staphylococcus aureus ) and Gram-negative ( Escherichia coli ) bacteria. Results indicate that the described nonwovens can be successfully used as an antibacterial material. guanidine hydrochloride; Gc—gentamycine chloride; g—grafted; H—heparin NIPAAm- N -isopropyl acrylamide; NVP— N -Vinyl-2-pyrrolidone; Oct—octenidine; PLLA—poly(L-lactide); PP—polypropylene nonwoven; aPTT—activated partial thromboplastin time.


Introduction
Poly(propylene) nonwoven (PP nonwoven) fabric is a thermoplastic, small density material with excellent comprehensive performance, mildew resistant, with a great development potential as medical materials [1]. Thus, PP nonwoven fabrics are applied as containers, suture material and surgical meshes, oxygenator membranes [2], or wound dressings [3,4]. In order to expand the functions of PP nonwoven fabrics to medical requirements, it is necessary to modify and process it. In this regard, biocompatible hybrids carrying and releasing bioactive drugs in a controlled and sustained manner may significantly improve the efficacy of wound therapies compared with current treatments. Antibacterial polypropylene nonwoven is often used as a hybrid material combining the PP nonwoven with other antibacterial components.
The recent, more representative examples of antibacterial PP nonwoven are listed in Table 1.
Bioactivity assessments on the chitosan-immobilized surfaces were anticipated by activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration.
Aminophosphonate compounds of special biological importance are presented in Table 2. Aminophosphonate compounds of special biological importance are presented in Table 2.
Alafosfalin was selected from a range of related peptides on the basis of its activity, low toxicity in mammals, pharmacokinetic properties and stability to peptidases [40,[44][45][46][47]. Alaphosphin proved active against majority of Serratiamarcescens strains [48], and also in vitro potentiation of cephalosporins against urinary tract bacteria [49] possessing potential usefulness in human medicine for the treatment of urinary tract of bacterial infections.
Despite the large range of applications of aminophosphonate compounds, there is only a Blanchard and Graves paper on their usage as cellulose hybrids [50].
As a part of our research program directed on the synthesis of biologically active functionalized phosphonates [26,27,[51][52][53] and their grafting on polymer matrix [54] we present the preparation and biological exploration of a polymer hybrid, based on polypropylene (PP) nonwoven fabric modified on surface by representative P-terminal phosphonopeptide-Alafosfalin (Alaphosphin).

Materials and Methods
The most widespread, first AA P isolated in natural sources [18,[34][35][36] Glu γP(Me) (phosphinothricin, PPT) Aminophosphonate compounds of special biological importance are presented in Table 2. Ala-Ala P selectively inhibited peptidoglycan biosynthesis in both Gram-negative and Gram-positive bacteria.
Alafosfalin was selected from a range of related peptides on the basis of its activity, low toxicity in mammals, pharmacokinetic properties and stability to peptidases [40,[44][45][46][47]. Alaphosphin proved active against majority of Serratiamarcescens strains [48], and also in vitro potentiation of cephalosporins against urinary tract bacteria [49] possessing potential usefulness in human medicine for the treatment of urinary tract of bacterial infections.
Despite the large range of applications of aminophosphonate compounds, there is only a Blanchard and Graves paper on their usage as cellulose hybrids [50].
As a part of our research program directed on the synthesis of biologically active functionalized phosphonates [26,27,[51][52][53] and their grafting on polymer matrix [54] we present the preparation and biological exploration of a polymer hybrid, based on polypropylene (PP) nonwoven fabric modified on surface by representative P-terminal phosphonopeptide-Alafosfalin (Alaphosphin).

Materials and Methods
A naturally occurring broad-spectrum systemic herbicide produced by several species of Streptomyces; inhibiting glutamine synthetase [18,37] PMG (PhosphonoMethyl-Glycine; Glyphosate) Ala-Ala P selectively inhibited peptidoglycan biosynthesis in both Gram-negative and Gram-positive bacteria.
Alafosfalin was selected from a range of related peptides on the basis of its activity, low toxicity in mammals, pharmacokinetic properties and stability to peptidases [40,[44][45][46][47]. Alaphosphin proved active against majority of Serratiamarcescens strains [48], and also in vitro potentiation of cephalosporins against urinary tract bacteria [49] possessing potential usefulness in human medicine for the treatment of urinary tract of bacterial infections.
Despite the large range of applications of aminophosphonate compounds, there is only a Blanchard and Graves paper on their usage as cellulose hybrids [50].
As a part of our research program directed on the synthesis of biologically active functionalized phosphonates [26,27,[51][52][53] and their grafting on polymer matrix [54] we present the preparation and biological exploration of a polymer hybrid, based on polypropylene (PP) nonwoven fabric modified on surface by representative P-terminal phosphonopeptide-Alafosfalin (Alaphosphin).
Alafosfalin was selected from a range of related peptides on the basis of its activity, low toxicity in mammals, pharmacokinetic properties and stability to peptidases [40,[44][45][46][47]. Alaphosphin proved active against majority of Serratiamarcescens strains [48], and also in vitro potentiation of cephalosporins against urinary tract bacteria [49] possessing potential usefulness in human medicine for the treatment of urinary tract of bacterial infections.
Despite the large range of applications of aminophosphonate compounds, there is only a Blanchard and Graves paper on their usage as cellulose hybrids [50].
As a part of our research program directed on the synthesis of biologically active functionalized phosphonates [26,27,[51][52][53] and their grafting on polymer matrix [54] we present the preparation and biological exploration of a polymer hybrid, based on polypropylene (PP) nonwoven fabric modified on surface by representative P-terminal phosphonopeptide-Alafosfalin (Alaphosphin).
Alafosfalin was selected from a range of related peptides on the basis of its activity, low toxicity in mammals, pharmacokinetic properties and stability to peptidases [40,[44][45][46][47]. Alaphosphin proved active against majority of Serratiamarcescens strains [48], and also in vitro potentiation of cephalosporins against urinary tract bacteria [49] possessing potential usefulness in human medicine for the treatment of urinary tract of bacterial infections.
Despite the large range of applications of aminophosphonate compounds, there is only a Blanchard and Graves paper on their usage as cellulose hybrids [50].
As a part of our research program directed on the synthesis of biologically active functionalized phosphonates [26,27,[51][52][53] and their grafting on polymer matrix [54] we present the preparation and biological exploration of a polymer hybrid, based on polypropylene (PP) nonwoven fabric modified on surface by representative P-terminal phosphonopeptide-Alafosfalin (Alaphosphin).

Polymers
Polypropylene (PP) produced by Borealis (Vienna, Austria) type HL612 FB, MFR = 1200 g/10 min, in the form of granulates was used for fabrication of nonwoven samples.

Nonwoven Fabrics
Nonwovens were fabricated by the melt-blown technique using a one-screw laboratory extruder (Axon, Nyvang, Sweden) with a head with 30 holes of 0.35 mm diameter each, compressed air heater and collecting drum. Processing parameters for fabrication of PP nonwoven are presented in Table 3.

Dip-Coating of Nonwoven Fabric and Modification of Phosphonodipeptide
The coating pastes of homogeneous dispersion and appropriate viscosity (about 60-70 dPas) were prepared based on styrene-acrylic resin, wetting agent as well as thickening agent and water.
Thickening agent ensures viscosity and appropriate grip of product, wetting agent prevents the formation of agglomerates in coating paste and uniformity in coating dispersion, and styrene-acrylic resin combines all ingredients of coating paste with nonwoven fabric. The applied ratio of component composition has main influence on the mechanical properties of the textile material.
Alafosfalin powder was added into the paste in 5 variants of concentrations: 0.005%, 0.01%, 0.05%, 0.1%, and 0.15% and then mixed for 10 min. The nonwoven samples were impregnated with the paste, squeezed and dried for 3 h at 50 • C (to constant weight). The increase of sample dry mass after modification was 5%. Component composition of used pastes is listed in Table 4. Table 4. Component composition of used pastes (%).

Scanning Electron Microscopy
The microscope analysis (SEM) was performed on a TESCAN VEGA 3 scanning electron microscope (Brno, Czech Republic). The SEM microscopic examination of the surface topography was carried out in a high vacuum using the energy of the probe beam 20 ekV. The surface of each preparation was

ATR-FTIR
Changes of the chemical structure of polypropylene surface products were assessed using ATR-FTIR spectroscopy. The study was performed in the range of 400-4000 cm −1 using a spectrometer Jasco 4200 series with ATR attachment (Pike Gladi ATR, Fitchburg, WI, USA).

UV-Vis Analysis
Changes of the physical properties as transmittance (%T) of nonwoven polypropylene fabric before and after phosphonodipeptide incorporation were assessed using a double beam Jasco V-550 UV/Vis spectrophotometer (Tokyo, Japan) with integrating sphere attachment in the range: 200-800 nm.

Filtration Parameters
Air permeability was determined for one layer of nonwoven sample and the test based on EN ISO 9237:1998 standard [55]. An FX 3300 TEXTEST AG permeability tester (Schwerzenbach, Switzerland) was used. Air at a pressure of 100 and 200 Pa was passed through a fabric area of 20 cm 2 diameter for testing. An average of 10 values was taken to be the final value of the sample.

Tensile Testing
Tensile testing of nonwoven fabrics was carried out in accordance with the EN ISO 10319:2015-08 standard [56]. A Tinius Olsen H50KS tester (Horsham, PA, USA) was used. Stretching speed was 20 mm/min.

Antimicrobial Activity
The antibacterial activity of the resulting nonwoven fabrics was tested according to PN-EN ISO 20645:2006 [57] (textile fabrics-determination of antibacterial activity-agar diffusion plate test).
Gram-negative bacteria E. coli (ATCC 25922) and Gram-positive bacteria S. aureus (ATCC 6538) were used as model bacteria in this study.
Antibacterial activity of PP/Ala-AlaP hybrids was tested by the agar diffusion method using Muller Hinton medium agar. The test was initiated by pouring each agar onto sterilized Petri dishes and allowing it to solidify. The surface of the growth media was inoculated by overnight broth cultures of bacteria (0.4 × 10 8 CFU mL −1 ). Samples of sterile PP discs (10 mm) were charged with coating pastes with various amounts of Ala-Ala P ( Table 5) and then discs with hybrids Ala-Ala P /PP were placed onto the inoculated agar and incubated at 37 • C for 24 h. The diameter of the clear zone around the sample was measured as an indication of growth inhibition. All tests were carried out in duplicate. Simultaneously, the same tests were carried out as control samples with samples of unmodified polypropylene nonwoven.

Results and Discussion
The analysis of the biofunctionalized nonwoven fabrics covered: scanning electron microscopy (SEM), UV/Vis transmittance, ATR-FTIR spectrometry, and also technical parameters, including filtration parameters and tensile properties (durability for stretching and relative elongation). For samples with the highest charge with Ala-Ala P , an antimicrobial activity against representative Gram-positive and Gram-negative bacteria was determined.

Scanning Electron Microscopy
Scanning electron microscopy SEM spectra of polypropylene nonwoven (PP) and polypropylene nonwoven charged with phosphonodipeptide modifier (alafosfalin paste concentrations 0.15%) are Coatings 2019, 9, 412 6 of 12 presented in Figure 2. SEM spectra of polypropylene nonwoven present smooth surface of unmodified polymer fibers. The modification of PP by a surface deposition of Ala-Ala P lead to a rough surface of the textile, covered with dots/smudges of the modifier on PP fibers, accompanied by formation of more loose texture of the modified nonwoven.
samples with the highest charge with Ala-Ala P , an antimicrobial activity against representative Gram-positive and Gram-negative bacteria was determined.

Scanning Electron Microscopy
Scanning electron microscopy SEM spectra of polypropylene nonwoven (PP) and polypropylene nonwoven charged with phosphonodipeptide modifier (alafosfalin paste concentrations 0.15%) are presented in Figure 2. SEM spectra of polypropylene nonwoven present smooth surface of unmodified polymer fibers. The modification of PP by a surface deposition of Ala-Ala P lead to a rough surface of the textile, covered with dots/smudges of the modifier on PP fibers, accompanied by formation of more loose texture of the modified nonwoven.

ATR-FTIR Spectra
Comparison of ATR-FTIR spectra of Ala-Ala P , polypropylene nonwoven (PP), and polypropylene nonwoven charged with Ala-Ala P (Ala-Ala P /PP) is presented on Figure 3.
Characteristic FTIR signals of Ala-Ala P , polypropylene (PP) and Ala-Ala P -polypropylene nonwoven hybrid (PP/Ala-Ala P ) are summarized in Table 5.

ATR-FTIR Spectra
Comparison of ATR-FTIR spectra of Ala-Ala P , polypropylene nonwoven (PP), and polypropylene nonwoven charged with Ala-Ala P (Ala-Ala P /PP) is presented on Figure 3. Characteristic FTIR signals of Ala-Ala P , polypropylene (PP) and Ala-Ala P -polypropylene nonwoven hybrid (PP/Ala-Ala P ) are summarized in Table 5.

ATR-FTIR Spectra
Comparison of ATR-FTIR spectra of Ala-Ala P , polypropylene nonwoven (PP), and polypropylene nonwoven charged with Ala-Ala P (Ala-Ala P /PP) is presented on Figure 3. Characteristic FTIR signals of Ala-Ala P , polypropylene (PP) and Ala-Ala P -polypropylene nonwoven hybrid (PP/Ala-Ala P ) are summarized in Table 5.

UV/Vis Transmittance Spectra
Comparison of transmittance spectra (%T) in the range λ = 200-800 nm of polypropylene nonwoven samples without (PP) and with L-Ala-Ala P modifier (PP-MOD-Ala-Ala P paste concentrations 0.15%) is given in Figure 4. Recorded UV/Vis transmittance spectra (%T) of samples after phosphonodipeptide modification revealed changes in macrostructure of nonwoven after modification expressed by the depress of transmittance ability in the range λ = 200-800 nm. All polypropylene modified nonwoven samples with different L-Ala-Ala P contents had the similar spectral characteristics and transmittance.

Vibrations
Legend: ν-stretching vibration, δ-deformation, ρb-bending deformation, γ-twisting vibrations, CA-the carbon atom of the amide bond, NA-the nitrogen atom of the amide bond, Cs-the carbon atom of the spacer group between alanine and phosphonate group, CCH 3 -the carbon atom of the methyl group, CCOOH-the carbon atom of the carbonyl group, Ala-part of N-terminal alanine, and S-shoulder.

UV/Vis Transmittance Spectra
Comparison of transmittance spectra (%T) in the range λ = 200-800 nm of polypropylene nonwoven samples without (PP) and with L-Ala-Ala P modifier (PP-MOD-Ala-Ala P paste concentrations 0.15%) is given in Figure 4. Recorded UV/Vis transmittance spectra (%T) of samples after phosphonodipeptide modification revealed changes in macrostructure of nonwoven after modification expressed by the depress of transmittance ability in the range λ = 200-800 nm. All polypropylene modified nonwoven samples with different L-Ala-Ala P contents had the similar spectral characteristics and transmittance.
Legend: ν-stretching vibration, δ-deformation, ρb-bending deformation, γ-twisting vibrations, CA-the carbon atom of the amide bond, NA-the nitrogen atom of the amide bond, Cs-the carbon atom of the spacer group between alanine and phosphonate group, CCH3-the carbon atom of the methyl group, CCOOH-the carbon atom of the carbonyl group, Ala-part of N-terminal alanine, and S-shoulder.

UV/Vis Transmittance Spectra
Comparison of transmittance spectra (%T) in the range λ = 200-800 nm of polypropylene nonwoven samples without (PP) and with L-Ala-Ala P modifier (PP-MOD-Ala-Ala P paste concentrations 0.15%) is given in Figure 4. Recorded UV/Vis transmittance spectra (%T) of samples after phosphonodipeptide modification revealed changes in macrostructure of nonwoven after modification expressed by the depress of transmittance ability in the range λ = 200-800 nm. All polypropylene modified nonwoven samples with different L-Ala-Ala P contents had the similar spectral characteristics and transmittance.

Technical Parameters
Filtration parameters expressed by the air permeability were detected for clean polypropylene nonwoven and nonwovens with addition of Alafosfalin. All these results indicated that phosphonodipeptide modification decreased filtration properties of nonwovens (Table 6). Modified nonwoven samples with different L-Ala-Ala P contents had an approximately similar result of filtration properties. The content of coating pastes L-Ala-Ala P applied on the polypropylene nonwoven samples does not affect filtration properties. The results of tensile strength testing, including durability for stretching (kN/m) and relative elongation at maximum load (%) of PP nonwoven fabrics and PP/Ala-Ala P hybrids are listed in Table 7. The results revealed an increase of these parameters for PP loaded with the paste in comparison with PP, namely distinct in tensile strength (0.32 to 0.40 (kN/m)) and slow in relative elongations (35.0% to 38.7%), respectively.

Antimicrobial Activity
The nonwovens prepared were subjected to antimicrobial activity tests against E. coli (ATCC 25922) and S. aureus (ATCC 6538) [60,61] (Table 8). Results of these studies gave new Ala-Ala P biofunctionalized materials which provide antimicrobial protection against different bacterial microorganisms (Gram-negative and Gram-positive). Table 8. Results of tests on the antibacterial activity of L-Ala-Ala P modified nonwovens. 0.15 14 14 Surface of applied disks with 1 cm diameter equals 0.785 cm 2 Concentration of inoculum (bacterial suspension) amount of live bacteria CFU/mL = 0.4 × 10 8 Amounts of 0.01% Ala-Ala P coating paste concentration sapplied on polypropylene nonwovens provide antimicrobial properties for E. coli and no less than 0.05% phosphonodipeptide modification of PP nonwoven showed antibacterial activity against S. aureus (Table 8). At the same time, applied surface modification of the nonwovens with phosphonopeptide had no effect on mechanical properties of the modified textile.

Conclusions
The development of new products is closely related to progress in many areas of science, especially in chemistry and biochemistry. The interdisciplinary study focused on functionalization of textile polymers by an insertion (or coating) of bioactive compounds into/onto the surface of their fibers which can be an interesting direction of the textile product engineering development.
These new, described nonwovens can be successfully used as an antibacterial material. An important attribute of the presented solution is the simple process of implementation, low production costs and good efficiency of antimicrobial properties.
Apart from the antibacterial activity, such products also show acceptable air filtration ability and tensile strength, independently from the amounts of applied phosphonodipeptide surface concentrations.
The introduction of bioactive aminophosphonateclass (PCN compounds) into the structure of textiles is a novelty in the scientific literature, both in the modification of textiles and in the title compounds itself. Alafosfalin and other phosphonodipeptides compounds are characterized by uncomplicated synthesis and safety of use, for the above reasons there are large possibilities for wider use in the material and textile manufacturing industry.
Author Contributions: M.H.K. designed the research study and contributed to the data interpretation and to the manuscript drafting and revisions, analyzed the data and contributed to writing the manuscript. Z.M. analyzed the data and participated in the publication preparation. A.W. analyzed the data and participated in the publication preparation and J.S.-L. analyzed the data and participated in the publication preparation.