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Article Application of FTIR Method for the Assessment of Immobilization of Active Substances in the Matrix of Biomedical Materials

Dorota Kowalczuk 1,* and Monika Pitucha 2 1 Chair and Department of Medicinal , Faculty of Pharmacy with Division of Medical Analytics, the Medical University of Lublin, 20-090 Lublin, Poland 2 Independent Radiopharmacy Unit, Faculty of Pharmacy with Division of Medical Analytics, the Medical University of Lublin, 20-093 Lublin, Poland * Correspondence: [email protected]; Tel.: +48-81-448-7380

 Received: 17 August 2019; Accepted: 11 September 2019; Published: 13 September 2019 

Abstract: Background: The purpose of the study was to demonstrate the usefulness of the (FTIR) method for the evaluation of the modification process of biomaterials with the participation of active substances. Methods: Modified catheter samples were prepared by activating the matrix with an acid, iodine, or bromine, and then immobilizing the active . To carry out the modification process, the Fourier transform infrared-attenuated total reflectance (FTIR-ATR) method was used. Results: FTIR analysis indicated the presence of the immobilized substances in the catheter matrix and site-specific reactions. Conclusion: We surmise that the infrared spectroscopic technique is an ideal tool for the assessment of the drug immobilization and the changes occurring in the course of the modification process.

Keywords: FTIR analysis; biomaterial characterization; biomaterial modification; immobilization

1. Introduction Fourier transform infrared spectroscopy (FTIR) is a universal analytical tool for evaluation of a wide range of materials [1,2], especially for identification of unknown materials [3]. The FTIR technique has been used to identify pure substances [4], mixtures [5], impurities [4,6], and compositions of various materials [7,8]. It has also been helpful in the elucidation of different processes concerning plenty of compounds or materials. Papers available in the specialist literature report application of infrared spectroscopy for the analysis of structural changes [9] as well as for monitoring production (bioproduction) processes [10–12]. From the viewpoint of pharmaceutical sciences, the study of substance content in formulations, the study of substance release from formulations, and other kinetic processes, including substance penetration or distribution, are important applications of mid infrared spectroscopy [13,14]. FTIR spectroscopy has also been helpful in performing more advanced analyses aimed at the characterization of artistic materials [14] and disease diagnosis [15,16]. With regard to its medical applications, the use of FTIR for structure characterization based on the linkage of the peptides is of great importance as well [16]. FTIR spectroscopy is a pivotal technique for characterization of polymeric and biopolymeric materials [17,18]. The FTIR technology has proved to be useful in identification and characterization of homopolymers, copolymers, or composite, and it has been successfully applied in the evaluation of polymerization process, characterization of the polymer structure, polymer surface, , and polymer modification [17,19,20]. More common applications of FTIR methodology include: Quality verification of materials; analysis of thin films and coatings [21]; decomposition of and other materials, often through thermogravimetry

Materials 2019, 12, 2972; doi:10.3390/ma12182972 www.mdpi.com/journal/materials Materials 2019, 12, 2972 2 of 13 combined with FTIR [22,23] and ; of materials to identify contaminants; monitoring of emissions; and failure analysis [24–26]. The speed of FTIR analysis makes it particularly useful in screening applications, while its sensitivity allows many advanced research applications. Thus, infrared spectroscopy is a valuable technique for analysis of pharmaceuticals, polymers and plastics, food, environment, and falsified materials, including medicines [27–29]. Advance techniques of FTIR are as follows: Fourier transform infrared-attenuated total reflectance (FTIR-ATR), Fourier transform infrared-photo acoustic spectroscopy (FTIR-PAS), Fourier transform infrared imaging spectroscopy (FTIR combined with an optical , FTIR-), Fourier transform infrared microspectroscopy, and Fourier transform infrared nanospectroscopy (nano-FTIR spectroscopy), which achieves nanoscale-level spatial resolution by combining IR spectroscopy and scattering-type near-field scanning microscopy [1,2,9,10,29,30]. Infrared nanospectroscopy opens up new possibilities for chemical and structural analysis and quality control in various fields, ranging from materials’ sciences to biomedicine [30]. Over the past years, much attention has been focused on modification of biomaterials and their potential applications in medical implants or other biomedical devices. The advanced FTIR technologies are also considered very helpful in assessing material changes [31–38]. Therefore, it is not surprising that the universality of FTIR methods has often been reported by researchers [31,32,34]. Infrared spectroscopy has been widely used for characterization of materials (e.g., latex, silicone, polyurethane) subjected to different physicochemical treatment when better hydrophilicity, lubricity, haemocompatibility, biocompatibility, or antibacterial activity were aimed at [31–35]. Evaluation of the modification progress relied on comparing the surface spectra of the untreated and treated materials. Registration of the material sample spectra after each subsequent stage of modification enabled identification of even the smallest changes without the infrared spectra library searching [31,32,35]. A variety of material modifications related to the use of physical (plasma, or UV radiation, electron beam irradiation), chemical (e.g., ozone oxidation, enzymatic oxidation, surface graft polymerization, site-specific reactions), or physicochemical treatment were monitored by means of infrared spectroscopic instrumentation [31–38]. A qualitative FTIR analysis is often the first step in the analytical process of a material. Fourier transform infrared spectroscopic method, however, provides a quantitative analysis as well. What is more, FTIR is a relatively cheap technique that might permit direct semi-quantitative measurements of various organic entities, including the adsorbed/immobilized compounds. As a result of using a standard of known concentrations, such measurements become quantitative [39,40]. The advantages of the FTIR quantitative procedure include direct evaluation of the adsorbed/immobilized amount as well as exact measurement of compounds that are hard to quantify, since they do not absorb UV-visible light, or they require expensive analytical procedures [39]. The FTIR spectroscopy combined with chemometric analysis provides very rapid, accurate, and generic approaches to the characterization not only of chemical compounds and biomolecules, but also potential biomarkers and microorganisms, including microbial pathogens [41]. IR spectrochemical analysis in combination with the chemometric technique (for example partial least square discriminant analysis, partial least square regression) can also be applied as a new diagnostic tool in microbiological and medical diagnostics [42,43]. Vibrational spectroscopy, like FTIR and , plays an important role in assessing the covalent and non-covalent functionalization of biomaterials obtained through the incorporation of various functionalities at the material matrix or surface, including immobilization of active compounds [44,45]. These complementary methods can be used for the discrimination between covalent and non-covalent immobilizations. The appearance of new functional groups in the FTIR spectra of functionalized materials may testify to covalent modifications, while the shift of spectral bands characteristic of the material or immobilized may indicate non-covalent interactions (Van der Waals interactions, bonds) [17,19,46,47]. Materials 2019, 12, 2972 3 of 13

In the previous studies, we used FTIR-ATR method for analysis of the multistep modification of biomaterials with the covalent immobilized molecules [46,47]. Continuing the modification research, we have developed a two-step method of non-covalent immobilization of the active substance in the material matrix. The aim of this study was to present some examples of biomaterial modification in order to point out the importance of FTIR spectroscopy in the evaluation of the presence of an active substance introduced to a biomaterial matrix, site-specific changes, as well as to elucidate the mechanism of immobilization.

2. Materials and Methods All chemicals used for the modification process of biomaterials were purchased from Sigma-Aldrich (Poznan, Poland), and POCH (Polish Chemical Reagents, Gliwice, Poland) companies. The modification process of biomaterials was performed using the selected patented procedures [48,49], adapted for the presented research.

2.1. Preparation of P-2-CA-Treated Catheter Samples The samples were prepared by immobilization of pyrrole-2-carbaldehyde (P-2-CA, from Sigma- Aldrich Sp. z o.o., Poznan, Poland), on commercial polyurethane intravenous catheters (BD Becton Dickinson, Franklin Lakes, NJ, USA). The catheters were cut into 1 cm long fragments and activated in an acid solution under gentle stirring at 50 ◦C. Then the samples were removed, rinsed several times with distilled water, and dried. In the next stage, the samples were placed in Eppendorf tubes, 1 immersed in 1 mL of 2 mg mL− solution of pyrrole-2-carbaldehyde in , and incubated in closed tubes for 30 min, at 60 ◦C with stirring at 100 rpm, and then in open tubes to evaporate the solvent. Next, the catheters were removed, rinsed with water, and left to dry overnight.

2.2. Preparation of Sparfloxacin-Treated Catheter Samples The samples were prepared by immobilization of sparfloxacin obtained from Sigma-Aldrich, on commercial latex catheters (Rusch, Germany). The catheters were cut into 0.5 cm long fragments and activated with 2% methanolic iodine solution for 1 h under gentle stirring at 40 ◦C. Next, the samples were removed, rinsed with distilled water, and dried. In the next stage, the samples were placed in 1 Eppendorf tubes, immersed in 1 mL of 2 mg mL− solution of sparfloxacin and incubated in closed tubes for 12 h, at 60 ◦C with stirring at 100 rpm. Then the catheters were removed, rinsed with water, and left to dry overnight.

2.3. Preparation of Sethacridine-Treated Catheter Samples The samples were prepared by immobilization of ethacridine lactate obtained from Sigma-Aldrich, on commercial polyurethane intravenous catheters (BD Becton Dickinson). The catheters were cut into 1 cm long fragments and activated with 1% methanolic bromine solution for 1 h under gentle stirring at 40 ◦C. Next, the samples were removed, rinsed with distilled water, and dried. In the next stage, 1 the samples were placed in Eppendorf tubes, immersed in 1 mL of 2 mg mL− solution of ethacridine and incubated in closed tubes for 1 h at 60 ◦C with stirring at 100 rpm, and then in open tubes about 5 h. Then the catheters were removed, rinsed with water, and left to dry overnight.

2.4. Fourier Transform Infrared Spectroscopy (FTIR) Characterization FTIR with transmittance mode was used to characterize the presence of specific chemical groups in the tested catheter samples. FTIR measurements were carried out on a Nicolet 6700 spectrometer (Thermo Fisher Scientific Inc., Warsaw, Poland) equipped with a deuterated triglycine sulfate detector (DTGS/KBr) and a versatile Attenuated Total Reflectance (ATR) sampling accessory with a diamond 1 1 crystal plate. Spectra were recorded in the spectral range of 4000–600 cm− at 4 cm− spectral resolution, Materials 2019, 12, 2972 4 of 13 Materials 2019, 12, x FOR PEER REVIEW 4 of 13

2spectral sampleresolution, gain, and 322 samplesample/ backgroundgain, and 32 scans sample/background using OMNIC 8.1 scans computer using software OMNIC (Thermo 8.1 computer Fisher Scientificsoftware (Thermo Inc.). Fisher Scientific Inc.).

3. Results and Discussion We have been doing doing research research into into immobilization immobilization of of active active substances substances on onthe the surface surface (or (orin the in thematrix) matrix) of biomaterials of biomaterials for formany many years. years. In Inour our wo work,rk, in inorder order to to assess assess the the effectiveness effectiveness of the modificationmodification process, we have used mainly FourierFourier transform infrared spectroscopyspectroscopy (FTIR). For this purpose, the FTIR spectra of the chemically treatedtreated and untreated samples were collected in the mid-infrared regionregion using using attenuated attenuated total total reflection reflection (ATR) (ATR) sampling sampling mode mode and and then then compared. compared. In this In paper,this paper, we present we present the selected the selected results results of the of evaluation the evaluation of the of modified the modified biomedical biomedical materials. materials.

3.1. Surface Characterization of the Porphyrin-Coated Catheter Bacteria thatthat formform aa heterogeneousheterogeneous and and organized organized biofilm biofilm structure structure show show increased increased resistance resistance to antibioticsto antibiotics and and they they are thereforeare therefore difficult difficult to eradicate to eradicate by means by means of a conventional of a conventional antibiotic antibiotic therapy. However,therapy. However, promising promising results have results been have obtained been with obtained the use with of athe photodynamic use of a photodynamic therapy (PDT) therapy with cationic(PDT) with porphyrins cationic porphyrins as a photosensitizer. as a photosensitizer. Studies have Studies shown have that shown the positive that the chargepositive of charge cationic of porphyrinscationic porphyrins determines determines the activity the againstactivity Gram-positive against Gram-positive and Gram-negative and Gram-negative bacteria. Itbacteria. has been It provedhas been that proved PDT is that highly PDT useful is highly in eliminating useful in bacterial, eliminating fungal, bacterial, protozoan, fungal, and viralprotozoan, infections and [50 viral–52]. PDTinfections is a promising [50–52]. PDT technology is a promising for the treatmenttechnology of for infectious the treatment diseases of causedinfectious by diseases antibiotic-resistant caused by bacteriaantibiotic-resistant [52]. It can bacteria be used [52]. especially It can forbe theused eradication especially offor biofilms the eradication from the of surface biofilms of catheters,from the prostheses,surface of catheters, drains [53 prostheses,], and for the drains treatment [53], and of the for localized the treatment infections of the [54 localized]. Thus, photodynamicinfections [54]. antimicrobialThus, photodynamic chemotherapy antimicrobial (PACT) chemotherapy may be an alternative (PACT) to may the conventional be an alternative antibiotic to the therapy conventional [50,52]. Inantibiotic PACT, thetherapy combination [50,52]. ofIn aPACT, photosensitizer the combination (light absorbing of a photosensitizer molecule) and (light light, absorbing in the presence molecule) of oxygen,and light, results in the in presence the formation of oxygen, of cytotoxic results free in the radicals formation (reactive of oxygencytotoxic species) free radicals that lead (reactive to the selectiveoxygen species) destruction that oflead microbial to the selective cells [55]. destruction Taking into of account microbial the cells effectiveness [55]. Taking of PACT, into account we covered the aeffectiveness catheter with of aPACT, porphyrin we covered photosensitizer a catheter with with a view a porphyrin to prevention photosensitizer of biofilm formation with a view on the to catheterprevention surface. of biofilm The catheter formation was on modified the catheter by activating surface. The its surface catheter with was a modified solution ofby nitric activating acid and its thensurface immobilization with a solution of pyrrole-2-carbaldehyde. of nitric acid and then Inimmobilization the acidic environment of pyrrole-2-carbaldehyde. and in the presence In of the air, theacidic tetrapyrrolic environment chain and was in formedthe presence [56,57] of on air, the catheterthe tetrapyrrolic surface (Scheme chain wa1):s formed [56,57] on the catheter surface (Scheme 1): H

NH N H +2H+, temp.,air 4 H H H (Scheme 1) N - 4H2O H O N NH 1H-pyrrole-2-carbaldehyde Molecular Formula:

C5H5NO H Porphyrin Molecular Formula:

C20H14N4

Scheme 1. Scheme 1. SynthesisSynthesis of porphyrin.porphyrin. In the course of time, the surface of the catheter became reddish-violet suggesting the formation In the course of time, the surface of the catheter became reddish-violet suggesting the formation of porphyrin (Figure1). of porphyrin (Figure 1).

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FigureFigure 1.1. Catheter sample untreated ((Aa)) and and porphyrin-coated porphyrin-coated ( (bB).). Figure 1. Catheter sample untreated (a) and porphyrin-coated (b). The FTIR analysis (Figure 22)) ofof thethe modifiedmodified cathetercatheter surfacesurface confirmedconfirmed ourour visual observations. The untreatedThe FTIR analysis polyurethane (Figure catheter 2) of the (Sample modified 1) exhibited catheter surface a typical confirmed distribution our of visual absorption observations. bands 1 forThe this untreated type of polyurethanematerial. The The cathetervibrational vibrational (Sample band at 1) 3330 exhibited cm −1 correspondsacorresponds typical distribution to to N-H N-H stretching stretchingof absorption vibrations. vibrations. bands 1 −1 Thefor this peakspeaks type in inof the material.the range range from The from 3000 vibrational to3000 2800 to band cm 2800− atare cm3330 connected−1 cmare connectedcorresponds with C-H with asymmetrical to N-H C-H stretching asymmetrical and symmetrical vibrations. and −1 1 symmetricalstretchingThe peaks vibrations. in stretching the range The vibrations. doublefrom 3000 peakThe todouble in the2800 region peak cm in ofare the 1680–1740 connectedregion of cm 1680–1740 −with(C= C-HO stretching cm asymmetrical−1 (C=O vibrations), stretching and 1 −1 vibrations),thesymmetrical bands above thestretching bands 1500 cm vibrations.above− (probably 1500 The cm N-Hdouble−1 (probably bending peak in vibrationsN-H the regionbending and of C-Nvibrations1680–1740 stretching cmand vibrations),(C=OC-N stretching and −1 1 vibrations),the strong bands andthe thebands in thestrong regionabove bands of1500 1300–1000 in cmthe region(probably cm− of(asymmetrical 1300–1000 N-H bending cm and−1 (asymmetricalvibrations symmetrical and O-C-Oand C-N symmetrical stretching −1 O-C-Ovibrations)vibrations), stretching areand associated the vibrations) strong with bands are the associated bondsin the ofregion wi urethaneth theof 1300–1000 bonds group of (Scheme urethane cm (asymmetrical2): group (Scheme and 2):symmetrical O-C-O stretching vibrations) are associated with the bonds of urethane group (Scheme 2): O NH NH O O C NH R NH C O C R C O O O O SchemeScheme 2. 2. OrganicOrganic unit unit typical typical of a urethane polymer.polymer. Scheme 2. Organic unit typical of a urethane polymer. Similar assessmentsassessments of of polyurethane polyurethane based-materials based-materials have have been presentedbeen presented earlier [earlier58,59]. Besides,[58,59]. Similar assessments of polyurethane based-materials have been presented earlier [58,59]. Besides,the available the publicationsavailable publications [59,60] report [59,60] that thereport band that at 1702 the cm band1 is at assigned 1702 cm to the−1 is hydrogen-bonded assigned to the Besides, the available publications [59,60] report that the band− at 1702 cm−1 is assigned to the hydrogen-bondedC=O (hydrogen bonding C=O (hydrogen between C =bondingO and N-H) between of urethane C=O and linkage, N-H) while of urethane the peak linkage, at about 1720while cm the1 hydrogen-bonded C=O (hydrogen bonding between C=O and N-H) of urethane linkage, while the− peakis related at about to free 1720 C= cmO group.−1 is related to free C=O group. peak at about 1720 cm−1 is related to free C=O group. In thethe casecase of of porphyrin porphyrin FTIR FTIR spectrum, spectrum, the main the bandsmain canbands be assignedcan be assigned according according the published the In the case of porphyrin FTIR spectrum, the main bands can be assigned according the publisheddata. The bandsdata. The at about bands 3300 at about cm 1 3300and 960cm−1 cm and1 960are relatedcm−1 are to related N-H bond to N-H stretching bond stretching and bending and published data. The bands at about− 3300 cm−1 and− 960 cm−1 are related to N-H bond stretching and bendingfrequencies frequencies of the pyrrole of the ring pyrrole (Scheme ring1), (Scheme the zone 1), of the 3100–2900 zone of cm 3100–29001 is assigned cm−1 is to assigned C-H bond to of C-H the bending frequencies of the pyrrole ring (Scheme 1), the zone of 3100–2900− cm−1 is assigned to C-H bondporphyrin of the ring porphyrin (Scheme ring1), the (Scheme region 1), of 1500–1680the region cmof 1500–16801, and the cm band−1, and at about the band 1350 at cm about1 are 1350 assigned cm−1 bond of the porphyrin ring (Scheme 1), the region of 1500–1680− cm−1, and the band at about− 1350 cm−1 areto C assigned=C stretching to C=C and stretching C=N stretching and C=N vibrations, stretching respectively, vibrations, while respectively, the band while at about the 760 band cm at1 aboutrefers are assigned to C=C stretching and C=N stretching vibrations, respectively, while the band at− about 760to the cm C-H−1 refers bond to bending the C-H vibrationsbond bending [61]. vibrations [61]. 760 cm−1 refers to the C-H bond bending vibrations [61]. The FTIR spectra of the unmodifiedunmodified and modifiedmodified catheter are similar. However, several new porphyrin-specificThe FTIR spectra peaks of theappear unmodified in the spectrum and modified of the catheter modified are catheter, similar. especiallyHowever, atseveral 16501 cmnew−1 porphyrin-specific peaks appear in the spectrum of the modified catheter, especially at 1650 cm− (C=C−1 (C=Cporphyrin-specific stretching vibrations), peaks appear 13501 in cm the−1 (C=Nspectrum stretching of the modifiedvibrations), catheter, and1 760 especially cm−1 (=C-H at 1650 bending cm stretching vibrations), 1350 cm− (C=N− stretching1 vibrations), and 760 cm− (=C-H bending−1 vibrations) vibrations)(C=C stretching vibrations), 1350 cm (C=N stretching vibrations), and 760 cm (=C-H bending vibrations)

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FigureFigure 2.2. FourierFourier transform transform infrared infrared spectroscopy spectroscopy (FTIR) (FTI spectraR) spectra of the of untreated the untreated polyurethane polyurethane catheter Figure 2. Fourier transform infrared spectroscopy (FTIR) spectra of the untreated polyurethane catheter(Sample ( 1Sample) and P-2-CA-treated 1) and P-2-CA-treated catheter catheter with the with formation the formation of porphyrin of porphyrin (Sample ( 2Sample). 2). catheter (Sample 1) and P-2-CA-treated catheter with the formation of porphyrin (Sample 2). 3.2. Surface Characterization of the Catheter Coated with a Chemotherapeutic 3.2.3.2. Surface Surface Characterizatio Characterizationn of of the the Catheter Catheter Coated Coated with with a a Chemotherapeutic Chemotherapeutic In order to restrict the catheter-associated infections, it is recommended that the conventional In order to restrict the catheter-associated infections, it is recommended that the conventional antibioticIn order therapy to restrict should the be applied,catheter-associated and the catheters infections, should it beis impregnatedrecommended with that silver the orconventional antibiotics. antibiotic therapy should be applied, and the catheters should be impregnated with silver or Modificationantibiotic therapy of biomaterials should be isapplied, the subject and ofthe many catheters scientific should studies. be impregnated One of the with strategies silver or is antibiotics. Modification of biomaterials is the subject of many scientific studies. One of the strategies incorporationantibiotics. Modification of an antibacterial of biomater substanceials is the into subject the matrix of many of the scientific biomaterial studies. (not One only of tothe its strategies surface) is incorporation of an antibacterial substance into the matrix of the biomaterial (not only to its fromis incorporation which the substance of an antibacterial could be gradually substance released into the (drug-eluting matrix of the biomaterials). biomaterial We(not performed only to its a surface) from which the substance could be gradually released (drug-eluting biomaterials). We two-stepsurface) modificationfrom which bythe activating substance the could latex be catheter gradually with thereleased iodine (drug-eluting solution and thenbiomaterials). incorporating We performed a two-step modification by activating the latex catheter with the iodine solution and then theperformed antimicrobial a two-step molecule modification from the by fluoroquinolone activating the group-sparfloxacinlatex catheter with the (Scheme iodine3): solution and then incorporatingincorporating the the antimicrobial antimicrobial molecule molecule from from th thee fluoroquinolone fluoroquinolone group-sparfloxacin group-sparfloxacin (Scheme (Scheme 3): 3): CH3 CH3 HFN HFN N N N N H3C H3C F COOH F COOH NH2 O NH2 O Scheme 3. Structure of sparfloxacin. Scheme 3. StructureStructure of sparfloxacin.sparfloxacin.

ThisThisThis group groupgroup of ofof antibiotics antibiotics was was selected selected with with regard regard to to a a broad broad spectrum spectrum of of antimicrobial antimicrobial activity, activity, goodgoodgood penetration penetration properties, properties,properties, and and hence hencehence their theirtheir efficacy eefficacyfficacy against againstagainst the thethe “young”“young” and andand “old” “old”“old” biofilm biofilmbiofilm andand long-timelong-timelong-time persistence persistencepersistence in inin the thethe biofilm biofilmbiofilm [62]. [[62].62]. TheTheThe presence presence of of sparfloxacin sparfloxacinsparfloxacin on onon the the surface surface of of the the biomaterialbiomaterial was was confirmed confirmedconfirmed by by assessing assessing the the cathetercathetercatheter surface surfacesurface at at each each stage stage of of it itsitss modification modificationmodification using using thethe FTIRFTIR method.method. TheTheThe FTIR FTIR spectrumspectrum ofof sparfloxacinsparfloxacinsparfloxacin showsshowsshows manymamanyny absorptionabsorptionabsorption bandsbandsbands (Figure(Figure(Figure3 ). 3).3). TheTheThe mostmostmost 1 1 characteristiccharacteristic bands bands can be assigned to:to: Primary amineamine ((ννN-HN-H inin NHNH2)) atat 34603460 cmcm−1 and 3340 cmcm−1,, characteristic bands can be assigned to: Primary amine (νN-H in NH2 2) at 3460 cm−−1 and 3340 cm−−1, 1 1 carbonylcarbonyl ( νC=O)C=O) in in COOH at 1714 cmcm−1,, carbonyl carbonyl ( νC=O)C=O) in in 4-quinolone 4-quinolone ring at 1639 cmcm−1,, aromatic aromatic carbonyl (νC=O) in COOH at 1714 cm−−1, carbonyl (νC=O) in 4-quinolone ring at 1639 cm−−1, aromatic 1 ringring (νC=C)C=C) in the regionregion ofof 1585–14001585–1400 cm cm−1(with (with N-HN-H in NHin NH), C-N2), C-N bonding bonding (νC-N (νC-N in C-NH in C-NH), C-O2), ring (νC=C) in the region of 1585–1400 cm− −1 (with N-H in 2NH2), C-N bonding (νC-N in C-NH2 2), ν 1 −1 C-ObondingC-O bondingbonding ( C-O ((νν inC-OC-O COOH) inin COOH)COOH) in the in rangein thethe ofrangerange 1350–1260 ofof 1350–12601350–1260 cm− ,and cmcm C-F−1,and,and stretching C-FC-F stretchingstretching vibrations vibrationsvibrations in the range inin thethe of 1 −1 range1250–1100range of of 1250–1100 1250–1100 cm− [46 ].cm cm−1[46]. [46].

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FigureFigure 3. 3. FTIRFTIR spectrum spectrum of of sparfloxacin. sparfloxacin. Figure 3. FTIR spectrum of sparfloxacin. TheThe spectrum ofof thethe iodine-activated iodine-activated latex latex catheter cathet (Figureer (Figure4, Sample 4, Sample 1) shows 1) shows the the absorbance bands The spectrum of the iodine-activated latex catheter (Figure 4, Sample 1) shows the absorbance1 bandscharacteristic characteristic of latex: of C-Hlatex: stretching C-H stretching vibrations vibrations (CH3 and (CH CH3 and groups) CH ingroups) the range in the of 3000–2850range of 3000– cm− , 1 2850bandsC=C cm stretching characteristic−1, C=C vibrationsstretching of latex: in vibrations theC-H range stretching ofin 1680–1600the vibrations rangecm of − (CH1680–1600, C-H3 and blending CH cm groups)−1, vibrations, C-H inblending the in-plane range vibrations, of at 3000– about −1 1 1 −1 in-plane28501450–1350 cm at, cmaboutC=C− andstretching 1450–1350 out of plane vibrationscm−1 atand about outin 840theof plane cmrange− [at54 ofabout], as1680–1600 well 840as cm the −cm1 [54], bands, C-H as that well blending appeared as the bandsvibrations, under that the −1 1 1 −1 appearedin-planeinfluence at ofunder about iodine the1450–1350 at aboutinfluence 1550 cm of cm and iodine− , out 1050 ofat cm planeabout− , probably at1550 about cm as 840−1 a, result 1050cm ofcm[54], oxidation−1, asprobably well inas the theas double abands result bondthat of −1 −1 oxidationappearedregion [63 in].under the double the influence bond region of iodine [63]. at about 1550 cm , 1050 cm , probably as a result of oxidationInIn thethe in FTIR FTIRthe double spectrum spectrum bond of the ofregion modifiedthe [63].modified material materi (activatedal (activated with iodine with and iodine treated and with treated sparfloxacin) with sparfloxacin)thereIn are the bands FTIR there characteristic spectrum are bands ofof characteristic sparfloxacin,the modified of which sparfloxacin,materi definitelyal (activated which confirms definitelywith its presence:iodine confirms and The treated intenseits presence: bandswith 1 1 Thesparfloxacin)at 3419 intense cm− bands andthere 3295 areat cm3419bands− associatedcm characteristic−1 and 3295 with N-Hcmof sparfloxacin,−1 asymmetricassociated whichwith and symmetricN-H definitely asymmetric stretching confirms and vibrations, its symmetric presence: the 1 −1 1 −1 stretchingThepeaks intense at 1717 vibrations, bands cm− atand the 3419 1631peaks cm cm at −and 1717corresponding 3295 cm− 1 cmand 1631associated to νcmC=−1 Ocorresponding with in COOH N-H andasymmetric to to νC=OνC= Oin and inCOOH4-quinolone symmetric and to −1 −1 νstretchingring,C=O respectively,in 4-quinolone vibrations, and ring,the the peaks respectively, drug at peaks 1717 andcm overlapping the and drug 1631 peaks with cm the correspondingoverlapping bands of thewith to matrix νtheC=O bands correspondingin COOH of the matrixand to to correspondingνstretchingC=O in 4-quinolone vibrations to stretching ring, of the respectively, vibrations aromatic ring of and the and the aromatic C-O,drug C-N,peaks ring C-F andoverlapping bonds C-O, C-N, of sparfloxacinwith C-F the bonds bands moleculeof ofsparfloxacin the matrix in the 1 moleculecorrespondingrange of 1450–1100in the to range stretching cm of− 1450–1100. vibrations cm− of1. the aromatic ring and C-O, C-N, C-F bonds of sparfloxacin molecule in the range of 1450–1100 cm−1.

Figure 4. FTIR spectra of the iodine-activated latex catheter (Sample 1) and sparfloxacin-treated Figure 4. FTIR spectra of the iodine-activated latex catheter (Sample 1) and sparfloxacin-treated catheterFigure 4. (Sample FTIR spectra 2). of the iodine-activated latex catheter (Sample 1) and sparfloxacin-treated cathetercatheter ( (SampleSample 2 2).). 3.3. Surface Characterization of the Catheter Coated with an Antiseptic 3.3. Surface Characterization of the Catheter Coated with an Antiseptic Another strategy for the protection of the biomaterial against the formation of a bacterial biofilmAnother is the introductionstrategy for ofthe an protection antiseptic intoof the its matrixbiomaterial instead against of an antibiotic.the formation of a bacterial biofilm is the introduction of an antiseptic into its matrix instead of an antibiotic.

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3.3. Surface Characterization of the Catheter Coated with an Antiseptic Another strategy for the protection of the biomaterial against the formation of a bacterial biofilm isMaterials the introduction 2019, 12, x FOR of PEER an antiseptic REVIEW into its matrix instead of an antibiotic. 8 of 13 Materials 2019, 12, x FOR PEER REVIEW 8 of 13 Thus, in a similar manner as before, polyurethane-based catheters were subjected to a two-stage Thus, in a similar manner as before, polyurethane-based catheters were subjected to a two-stage modification.Thus, in a Initially,similar manner the polyurethane as before, polyurethane material was-based activated catheters using were a bromine subjected solution to a two-stage and then treatedmodification. with ethacridine Initially, the lactate polyurethane (Scheme4 ):material was activated using a bromine solution and then modification.treated with ethacridine Initially, the lactate polyurethane (Scheme 4):material was activated using a bromine solution and then treated with ethacridine lactate (Scheme 4): NH2 NH2 OCH - 3 HO O- OCH3 OH O + H2N N+ H3C O 2NH NH 3CH O H SchemeScheme 4. 4. StructureStructure of ethacridine lactate.lactate. Scheme 4. Structure of ethacridine lactate. The intense fluorescence of the catheter under UV light at 366 nm, similar to the fluorescence of The intense fluorescence of the catheter under UV light at 366 nm, similar to the fluorescence of ethacridine solution, may indicate the presence of ethacridine in the catheter matrix (Figure5). ethacridineThe intense solution, fluorescenc may indicatee of the the catheter presence under of ethacridine UV light at in 366 the nm, catheter similar matrix to the (Figure fluorescence 5). of

Figure 5. Ethacridine solution (a), the untreated catheter (b) and the ethacridine-treated catheter (c). FigureFigure 5.5. EthacridineEthacridine solutionsolution ((Aa), the untreated catheter (Bb) and the ethacridine-treated cathetercatheter ((Cc).). The above observations confirmed the FTIR analysis of the modified catheter. The FTIR spectra The above observations confirmed the FTIR analysis of the modified catheter. The FTIR spectra obtainedThe abovefor the observations bromine-activated confirmed catheter the FTIRand the analys ethacridine-treatedis of the modified catheter catheter. are The shown FTIR in spectra Figure obtained for the bromine-activated catheter and the ethacridine-treated catheter are shown in Figure6. obtained6. for the bromine-activated catheter and the ethacridine-treated catheter are shown in Figure The FTIR spectrum of ethacridine lactate [64] exhibits the bands in the region of 3500–3100 cm 1 6. The FTIR spectrum of ethacridine lactate [64] exhibits the bands in the region of 3500–3100 cm−−1 which are assigned to N-H asymmetric and symmetric stretching vibrations of primary aromatic−1 whichThe are FTIR assigned spectrum to N-H of ethacridine asymmetric lactate and [64]symmetric exhibits stretching the bands vibrations in the region of primaryof 3500–3100 aromatic cm amine and hydrogen-bonded N-H stretching vibrations. The appearance of these bands in the FTIR whichamine andare assignedhydrogen-bonded to N-H asymmetric N-H stretching and vibrations.symmetric Thestretching appearance vibrations of these of bandsprimary in aromaticthe FTIR spectra of the modified catheter located at about 3450, 3320, and 3200 cm 1, indicates the presence aminespectra and of the hydrogen-bonded modified catheter N-H located stretching at about vibrations. 3450, 3320, The and appearance 3200 cm− 1of−, indicates these bands the presencein the FTIR of of ethacridine in the catheter matrix. Alongside this, the appearance of− a1 relatively strong band at spectraethacridine of the in modifiedthe catheter catheter matrix. located Alongside at about this, 3450, the appearance 3320, and 3200 of a cmrelatively, indicates strong the band presence at 1630 of 1630 cm 1 corresponding to C=N stretching vibrations of the acridine ring can provide further evidence ethacridinecm−1 corresponding− in the catheter to C=N matrix. stretching Alongside vibrations this, of the the appearance acridine ring of cana relatively provide strong further band evidence at 1630 of of the−1 incorporation of ethacridine into the catheter matrix. cmthe incorporation corresponding of toethacridine C=N stretching into the vibrations catheter ofmatrix. the acridine ring can provide further evidence of the incorporation of ethacridine into the catheter matrix.

Materials 2019, 12, 2972 9 of 13 Materials 2019, 12, x FOR PEER REVIEW 9 of 13

FigureFigure 6.6. FTIR spectrum of ethacridine lactate lactate ( (aa);); FTIR FTIR spectra spectra of of the the bromine-treated bromine-treated catheter catheter (green(green line) line) andand thethe ethacridine-treatedethacridine-treated cathetercatheter (red line) ( b).

InIn thethe casecase ofof immobilization of of sparfloxacin sparfloxacin and and ethacridine, ethacridine, no no vibrational vibrational bands bands indicating indicating a acovalent covalent binding binding of of the the drug drug with with the the formation formation of of aa new new moiety moiety were were observed. observed. Contrary Contrary to to the the previousprevious studies,studies, whenwhen sparfloxacinsparfloxacin was covalently bound bound on on a a catheter catheter coated coated with with a aheparin heparin layer, layer, wewe observed observed the the appearance appearance of of an an intense intense vibration vibration band band belonging belonging to a newto a functionalnew group formed asformed a result as of a linkingresult of the linking amino the group amino of thegroup drug of with the drug the carbonyl with the group carbonyl of the group oxidized of the heparin oxidized [46 ]. heparinAn increase[46]. in the band intensity or shifting to lower values compared to pure substancesAn increase (shift ofin 10–40the band units) intensity could suggestor shifting Van to der lower Waals wavenumber type interactions values orcompared hydrogen to bondspure betweensubstances the (shift introduced of 10–40 drug units) and could matrix suggest structure. Van The der substances Waals type have interact probablyions or been hydrogen incorporated bonds to thebetween matrix the by introduced a process of drug activator-enhanced and matrix structure. diffusion The and substances/or by the have changes probably in the been polymer incorporated structure madeto the by matrix the activating by a process agent of which activator-enhanced were observed diffusion in the spectrum and/or ofbypolyurethane the changes in polymer the polymer treated withstructure iodine. made by the activating agent which were observed in the spectrum of polyurethane polymer treated with iodine.

4. Conclusion

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4. Conclusions To sum up, our studies clearly indicate that the FTIR method with attenuated total reflection (ATR) is an important, indispensable, and effective tool for the assessment of the immobilization of active substances in the matrix of biomedical materials. Moreover, the fact that FIR-ATR is a relatively inexpensive, rapid, simple, and nondestructive technique, without sample pretreatment was of great importance in selecting the most appropriate experimental technique for the presented analyses. Three examples have been presented in order to illustrate the significant role of the combination of library searching focused on identification of characteristic spectral bands of the polymer, with the analysis of the registered spectra focused on identification of the functional groups of active compounds introduced to polymer matrix Obtained modified biomaterials containing the immobilized, pharmacologically active substance are suggestive of potential applications in medicine to prevent the formation of bacterial biofilms that commonly trigger infections.

Author Contributions: D.K. created the concept, designed the study, and coordinated FTIR analyses. The resources, review, and funding acquisition were performed by M.P. Both authors read and approved the final manuscript. Funding: This research was funded by the Polish Ministry of Scientific Research and Information Technology, grant number N N405 385037. Acknowledgments: The authors thank students Agata Gladysz and Jakub Krol (at Chair and Department of , the Medical University of Lublin) for preparing modified biomaterials. Conflicts of Interest: The authors declare no conflict of interest

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