Low and medium molecular weight chitosan matrices with ibuprofen as a potential base for soft capsules for oral use: evaluation of physicochemical properties and molecular structure

Low and medium molecular weight chitosan matrices with ibuprofen as a potential base for soft capsules for oral use: evaluation of physicochemical properties and molecular structure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Low and medium molecular weight chitosan matrices with ibuprofen as a potential base for soft capsules for oral use: evaluation of physicochemical properties and molecular structure Barbara Gieroba, Paulina Kazimierczak, Grzegorz Kalisz, Vladyslav Vivcharenko, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7416784/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Chitosan, which is a biocompatible and biodegradable natural polysaccharide, has been considered as a potential biopolymer-based carrier that can be applied in drug release systems, such as capsules for ibuprofen to overcome physiological barriers. In this study, we have examined three different variants of chitosan matrices for oral administration of ibuprofen (IBU) with various molecular weights (MW) or concentrations - 2% (w/v) and 4% (w/v) low MW, and 2% (w/v) medium MW. Molecular arrangement and physicochemical properties were determined before and after drug release from matrices with two powerful spectroscopic techniques - Raman spectroscopy and Diffuse Reflectance Infrared Fourier Transform DRIFT spectroscopy. It was proved that IBU was successfully incorporated into all studied chitosan matrices, however, the burst and highest drug release was demonstrated for the 2% low MW sample, where more amino and hydroxyl groups were observed interacting with the cationic IBU. It has been shown that chitosan can be an effective drug carrier, improving their pharmacokinetic and pharmacodynamic parameters. chitosan physicochemical properties matrix drug release system ibuprofen natural polysaccharide polymers molecular weight Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Chitosan (CS), poly[β-(1→4)-linked-2-amino-2-deoxy-D-glucose] (Figure 1), is a natural linear polysaccharide obtained by the partial or complete alkaline deacetylation of chitin. It consists of D-glucosamine and N -acetyl-D-glucosamine units, linked by β-1,4 glycosidic bonds (Muxika et al. 2017). Chitosan is not a singular polymer with a defined structure but a series of molecules with variations in size, composition, and monomer distribution (Aranaz et al. 2021). The molar percentage between two glucosamine monomeric units is an indicator of the degree of deacetylation (DDA) (Verlee et al. 2017). It ranges from 0 (chitin) to 100 (entirely deacetylated chitin) (Pérez-Álvarez et al. 2018). This parameter is considered an important factor that influences numerous crucial physicochemical and biological properties of chitosan and its derivatives, like hydrophilicity, crystallinity, tensile strength, elastic modulus, moisture content, degradation, and finally, protein adsorption and cell response (Yuan et al. 2011; Mathaba and Daramola 2020). DDA is influenced by the temperature, time of reaction, alkali concentration, particle size, and density (Sivashankari and Prabaharan 2016; Pellis et al. 2022). Generally, chitosan with a higher DDA is characterized by better biological features than poorly deacetylated ones because of the higher concentration of the amino (NH 2 ) groups in relation to the N-acetyl glucosamine moieties (Kasaai 2009; Pérez-Álvarez et al. 2018). Chitosan with a greater DDA is characterized by an elongated conformation with more flexible chains, and those with decreased DDA often take on a spiral shape, mainly because of the low charge density in the polymeric chain (Morris et al. 2009). Moreover, along with the increase of DDA during processing, the molecular weight (MW) of chitosan decreases; MW also depends on the source of material (Harish Prashanth et al. 2002). Most commercial chitosans have MW of 50–2000 kDa, with an average DDA of 50–100% (usually 80–90%). Based on the MW, chitosan can be divided into low molecular weight (1000 kDa) (Gonçalves et al. 2021). MW determines not only the fundamental properties of molecules, such as conformation, viscosity, solubility, and cytotoxicity, but in the case of chitosan, additionally the ability to create different forms and their strengths, and even the releasing rates of the substances carried by the molecules (Zhang et al. 2017). MW and DDA may also influence the crystallinity of the chitosan films, which in turn affects mechanical properties, especially tensile strength (Hoekstra et al. 1998), and determines the water sorption ability. Less crystalline chitosan films (low-ordered non-crystalline and amorphous forms) will absorb more water vapor and, inversely, in high crystalline forms (Saito et al. 2000). Chitosan is insoluble in neutral or alkaline solvents and easily soluble in many aqueous solutions of organic and inorganic acids (Ogawa et al. 2004). In water solutions, chitosan has high viscosity and forms a quasi-globular conformation, stabilized by inter- and intramolecular hydrogen bonds between hydroxyl and amino groups (Morris et al. 2009). Moreover, its (bio)chemical reactivity is quite high due to free primary amino groups distributed evenly in the molecular chain (Ogawa et al. 2004). Chitosan has gel-forming ability at low pHs (Dimida et al. 2017). When it is exposed to an acidic environment, the amino groups in the polymer chains protonate, creating NH 3 + moieties. As a consequence, it becomes cationic, allowing it to interplay with different types of molecules (Lizardi-Mendoza et al. 2016). In practice, chitosan occurs in solution as a poly-cationic form; thereby, it is able to react with negatively charged molecules, like anions and poly-anions, creating ionic complexes (Dimida et al. 2017). This positive charge is also considered responsible for the antimicrobial properties of chitosan through the interaction with the negatively charged cell membranes of bacteria (Yılmaz Atay and Çelik 2017). Due to its solubility in acidic aqueous media, it can be manufactured in various forms, i.e., hydrogels, films, nanofibers, or even pastes (Shi et al. 2006). However, there are some reports on its formulation of non-dissolved powdery and flakes (Ardila et al. 2017). Chitosan possesses exquisite biological properties, such as biodegradability, non-toxicity, mucoadhesiveness, hemocompatibility, and low immunogenicity, and exhibits antioxidant, anticancer, and antibacterial activities (Zhao et al. 2018). Therefore, it is widely employed in the biotechnological industry in diverse cosmetic and pharmaceutical formulations and in creating gene, protein, drug, and vaccine delivery systems(Khademi et al. 2018; Barbosa et al. 2020; Lima et al. 2021; Thareja et al. 2021). It is also utilized in biomedical applications, mainly in the field of wound dressings, tissue engineering, and implant coatings (Shi et al. 2006). Its non-medical usages are food processing and packaging, water clarification, environmental remediation, waste management, and sewage disposal, separators, and agricultural materials production (Lavertu et al. 2012). Chitosan is often used for the drug release systems of non-steroidal drugs, usually in the form of cross-linked films or tablets to accommodate the selected drug inside the polymeric material (Vieira et al. 2013). One of these drugs is ibuprofen (IBU), belonging to a group of medicines called Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and is used extensively for the management of acute pain (Motov et al. 2019). It is a low-soluble crystal powder in class II in the biopharmaceutics classification system (BCS). IBU is characterized by a short half-life (2 hours) after oral administration, leading to the need for increasing and multiplying the doses and, in consequence, resulting in missed medication aliquots (and associated with poor patient compliance) and severe side effects. Additional issues arising from oral administration are gastric irritation and hepatic first-pass metabolism (Bushra and Aslam 2010; Mahmood et al. 2021). Thus, there is a need to overcome these disadvantages and extend the bioactive effect of IBU, where the drug release is performed through a matrix, such as chitosan, helping to reduce a series of undesirable adverse effects (Vieira et al. 2013). This research aimed to investigate chitosan matrices with ibuprofen as a potential base for soft capsules for oral use. The particular aim of this study was to evaluate the physicochemical features and molecular arrangements of chitosan films consisting of molecules with various preparation methods (low - 2 % (w/v) and 4 % (w/v) in 1% acetic acid, and medium - 2 % (w/v) in 1% acetic acid) with and without ibuprofen (or after drug release). Due to problems with chitosan solubility during sample preparation, 4 % (w/v) chitosan medium molecular weight was excluded from this study. The specimens without IBU were control samples. These properties were determined by combining several analytical techniques - drug release studies, Raman spectroscopy (RS), and diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. All matrices were tested before the IBU release test (prior to placing them in PBS) and after the drug release test. Specifically, assessing these structural, chemical, and physical characteristics will help comprehend the possible applications of the chitosan matrices in the biomedical field, especially as a drug delivery and release system. The following variants were examined: 2% chitosan low molecular weight - 2CS_L 2% chitosan low molecular weight soaked in PBS - 2CS_L_R 2% chitosan low molecular weight + ibuprofen - 2CS_L/IBU 2% chitosan low molecular weight + ibuprofen after release test - 2CS_L/IBU_R 4% chitosan low molecular weight - 4CS_L 4% chitosan low molecular weight soaked in PBS - 4CS_L_R 4% chitosan low molecular weight + ibuprofen - 4CS_L/IBU 4% chitosan low molecular weight + ibuprofen after release test - 4CS_L/IBU_R 2% chitosan medium molecular weight - 2CS_M 2% chitosan medium molecular weight soaked in PBS - 2CS_ M_R 2% chitosan medium molecular weight + ibuprofen - 2CS_ M/IBU 2% chitosan medium molecular weight + ibuprofen after release test - 2CS_ M/IBU_R The control samples without the drug were soaked in PBS in parallel during the release test, just like the matrices with IBU. 2. Materials and Methods 2.1. Samples preparation Three types of chitosan matrices made of 2% ( w/v ) chitosan with low molecular weight (50–190 kDa MW, Sigma-Aldrich Chemicals, Warsaw, Poland; sample marked as 2CS_L), 4% ( w/v ) chitosan with low molecular weight (50–190 kDa MW; sample marked as 4CS_L), and 2% ( w/v ) chitosan with medium molecular weight (217 kDa MW, kindly obtained from National Marine Fisheries Research Institute, Gdynia, Poland; sample marked as 2CS_M) were fabricated. Each chitosan matrix was additionally loaded with 240 µg of ibuprofen (Sigma-Aldrich Chemicals, Warsaw, Poland; samples marked as 2CS_L/IBU, 4CS_L/IBU, 2CS_M/IBU, respectively). In brief, chitosan matrices were prepared by dissolving appropriate amounts of chitosan in 1% ( v/v ) CH 3 COOH (Avantor Performance Materials, Gliwice, Poland). To obtain the homogenous distribution of ibuprofen within chitosan matrices, dissolved chitosan was mixed with ibuprofen solution on a magnetic stirrer. Next, 600 µL of obtained gels were spread in a thin layer on the glass coverslips, soaked in 1% ( w/v ) NaOH (Avantor Performance Materials, Gliwice, Poland) for 5 min, then rinsed with deionized water, and left to air dry. The thickness of the dried samples was measured using an electronic micrometer with an accuracy of 0.001 mm (Schut Geometrical Metrology, Groningen, The Netherlands). The thickness of the chitosan films was 90 µm ± 9.6 µm. 2.2. Assessment of ibuprofen release To evaluate the ibuprofen release, the ibuprofen-loaded matrices (2CS_L/IBU, 4CS_L/IBU, 2CS_M/IBU) were submerged in 10 mL of phosphate-buffered saline (PBS) solution (pH 7.4, Sigma Aldrich-Chemicals, Warsaw, Poland) and incubated at 37 °C. Control CS matrices (without drug) were also soaked in PBS and served as control matrices during spectroscopic analyses. At defined time intervals, 0.5 mL samples were collected to evaluate ibuprofen concentration. Fresh PBS was added to maintain the original volume of the PBS solution during the test. Ibuprofen concentration was assessed by measuring the absorbance values at the wavelength of 225 nm using a UV-spectrophotometer (Genesys 6 UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA). A calibration curve was done for known concentrations of ibuprofen solutions, which were prepared in PBS in the 12.5 - 400 μg/mL range. The ibuprofen release profile from the chitosan matrices was expressed as the percentage of released drug at defined time intervals in a cumulative graph. Results obtained in this study were presented as mean values ± standard deviation (n ≥ 3). Statistical analysis was performed by using one-way ANOVA followed by Tukey's test with statistical significance at p < 0.05. (GraphPad Prism 8.0.0 Software, GraphPad Software Inc., La Jolla, CA, USA). 2.3. Raman spectroscopy The Raman spectra were recorded in the 100-4000 cm −1 range using a Witec alpha300R (WITec, Gmbh, Ulm, Germany) with 532 nm excitation laser wavelength and registered in the ControlSIX software (WITec Gmbh, Ulm, Germany). All spectra were obtained with an integration time of 0.5 s with 9 mW laser power and 10 accumulations, applied through x100 objective lens (Zeiss, Germany). The collected light through an objective lens was directed via a 100 mm diameter silica fiber, acting as a confocal pinhole to a spectrograph (UHTS300S, WITec Gmbh, Ulm, Germany) with a 600 g/mm grating and charge-coupled device (CCD) camera. The results are presented as an average of 10 spectra for every sample after processing (baseline correction and smoothing with nine points Savitzky-Golay algorithm) with ProjectSIX (WITec Gmbh, Ulm, Germany). 2.4. Diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTs) Spectra were recorded with a FTIR Nicolet iS50 spectrometer (Thermo Scientific, Waltham, MA, USA) in diffuse reflectance mode using a continuous flow Harrick Praying Mantis chamber. Typically, 100 scans were collected at a resolution of 1 cm -1 . Prior to the measurement, each sample was purged in He flow (45 ml min -1 ) for 30 min. DRIFTS technique was used to study structural properties and the surface chemistry of bare and Ibuprofen-containing samples with a minimum of sample preparation steps to avoid any possible influence on the analysis. Typically, the sample was placed on a flat surface inside a chamber. The IR radiation was directed onto the sample via two ZnS windows, and the light reflected by diffuse scattering was collected with an external spherical mirror (outside the cell). The DRIFT spectra are presented in the Kubelka–Munk function. 3. Results 3.1. Assessment of ibuprofen release profile The percentage of drug release with time from the IBU-loaded chitosan matrices is shown in Figure 2. The 2CS_L/IBU showed a high initial burst release of ibuprofen, reaching the plateau effect within the first 0.5 h (35.16 ± 4.99% of the drug was released). Further ibuprofen release was not detectable for this sample. The 4CS_L/IBU showed a similar profile of ibuprofen release; however, it reached a plateau after 2 h of the experiment (32.16 ± 9.14% of the drug was released). In turn, the 2CS_M/IBU showed burst release of the ibuprofen within the first 2 h followed by a subsequent slow, sustained release until reaching the plateau effect after 48 h of the test (49.95 ± 9.48% of the drug was released). 3.2. Raman spectroscopy Raman analysis of samples placed on coverslips was performed. High magnification was chosen for confocal measurements of polymers in all mentioned variants. Raman spectra of native forms were recorded and presented in Figure 3A, and the assignments of bands are shown in Table 1. The characteristic chitosan bands were identified according to the literature (Zając et al. 2015; Biniaś et al. 2016; Gieroba et al. 2020). Recorded spectra of 2CS_L and 4CS_L have a similar course with the most distinct changes comparing low to medium MW. The most affected ranges are 2880-3000 and 300-500 cm -1 , with an upshift from 900 to 929 cm -1 and the disappearance of bands at 3310 and 360 cm -1 . It suggested the modifications of stretching of CH and CH 2 groups as CH 2 and CH 3 stretching was more pronounced in 2CS_M, and C-C-C by forming more interlacing bonds between secondary carbon atoms of chitosan during chemically induced gel forming. Additionally, 4CS_L, compared to the 2 CS_L spectrum, does not present bands at 363 and 442 cm -1 assigned to ring out-of-lane vibrations, indicating the higher density of the resulting polymer by C-C-C and C-O-C linking. Table 1. Assignments to Raman shifts recorded in studied native samples (Movasaghi et al. 2007; Zając et al. 2015; Gieroba et al. 2020; Camerlingo et al. 2022) Raman shift (cm -1 ) Assignments 2CS_L 4CS_L 2CS_M 3312 3310 - ν s NH 2 and N-H, ν O-H 2932 2932 2933 ν CH 3 2890 2888 2890 ν CH 2 ν CH 1461 1463 1463 δ as CH, τ CH 2 (in-plane) CH and OH scissoring, CH 2 wagging 1417 - 1417 CH 3 and CH scissoring 1377 1378 1384 δ CH δ COH 1266 1266 1263 δ-CH (in-plane), CH 2 OH 1113 - 1120 C-O-C (ether) - 1092 - C-C stretch of glucosamine rings, ν s (C–O–C) 929 - 929 δ -CH (out-of-plane) 900 900 900 β-glycosidic bonds 442 - - τ HCC (out-of-plane) COC scissoring OH and ring out-of-plane vibration 363 - - δ C-C-C ν – stretching, δ – deformation, τ – bending, s – symmetric, as – antisymmetric In the initial evaluation, more distinct changes were observed between low and medium MW chitosan in the mentioned native samples before enrichment with IBU. To acquire background information on PBS's potential influence in drug release experiments, native samples were exposed to the continuous fluid flow for an equal amount of time in the same conditions as IBU-enriched samples. The data from those samples is shown in Figure 3C. Indicated bands in the native sample were also observed in PBS-treated materials without significant shifts or differences in spectra. Subsequently, thin films enriched with IBU matrices before and after drug release performed in PBS were analyzed, presented in Figures 3B, and 3D, respectively. Additionally, the pure ibuprofen spectrum was recorded and presented in Figure 5 that presents the Raman spectrum of ibuprofen (IBU) added to polymer matrices with described characteristic bands. The IBU spectrum (Figure 5) exhibits characteristic bands at 2992, 1703, and 1229 cm -1 , attributed to the OH stretching mode, the asymmetrical wagging vibrations from the carbonyl group (C-O), and C=O stretching, respectively (Guan et al. 2024). Other typical bands for IBU are the asymmetric alkyl stretching bands at 2956 cm −1 , carbonyl stretching bands at 1722 cm −1 , CH–CO strain at 1420 cm −1 and deformation of CH 2 at 780 cm −1 (Pereira et al. 2020). IBU addition resulted in new bands appearing at 1416 cm -1 in 2CS_L_IBU and 4CS_L_IBU or 1424 cm -1 in 2CS_M_IBU, assigned to CH, CH 3 bending of IBU and deformation of hydroxyl and amino groups, by the formation of non-covalent bonds (Zhang et al. 2011; Azueta-Aguayo et al. 2022). The characteristic band for crystalline ibuprofen at 1608 was not observed in spectra, nor was the band at 1342 cm -1 (Kazarian and Martirosyan 2002; Krukiewicz et al. 2020). In the range 1000-1500 cm -1 , the spectra are similar, but interestingly 2CS_M presents the broadening of the band to lower Raman shifts with the highest Raman intensity at 1093 cm -1 . It can be assigned to CO stretching and COH bending, providing information that IBU was incorporated in all polymer matrices and that the bond is strongest in 2CS_M. An additional band at 3373 cm -1 suggests that OH, NH, and NH 2 groups were affected by intramolecular and intermolecular hydrogen bonding in medium molecular weight samples. β-glycosidic bonds were not affected by the incorporation of IBU in all samples, thus it does not change the chitosan backbone, rather interacting with free hydroxyl and methyl groups (Jia et al. 2020). Interestingly, the measurements of films after release in PBS (Figure 3D) do not change Raman spectra compared to those presented in Figure 3C, proving that changes in the structure of chitosan gel are not reversible in a water environment and after IBU release. For a better understanding of changes and their influence on individual CS matrices, the spectra grouped to 2CS_L, 4CS_L, and 2CS_M were presented concerning IBU enrichment in Figure 4. In the 2CS_L group (Figure 4A), the addition of IBU leads to a slight shift of 1267 to 1266, near 3300 cm -1 and the disappearance of bands in region 300-500 cm -1 . The highest level of similarity appears to be in 2CS_L_R and 2CS_L/IBU_R, indicating a marginal influence on PBS flow during release. In the 4CS_L group (Figure 4B), a full spectra comparison of native and experimental samples was similar to the 2CS_L, with a decrease in intensity for CH 2 and CH 3 . The addition of ibuprofen resulted in shifts from 3310 to 3308 and from 1092 to 1116 cm -1 the disappearance of the 1463 cm -1 band, and a significant lowering of a band at 3310 cm -1 assigned to hydroxyl group stretching. At low Raman shifts similar modifications of C-C ring vibrations were observed in the drug consisting of matrices. The most interesting changes were observed in the 2CS_M group (Figure 4C), where with higher MW, changes in matrices were more significant. The native 2CS_M polymer shares bands at 900, 1120, and 1378-1380 cm -1 across all spectra; however, significant differences were also observed. 2CS_M displays the highest intensity at 2933 cm -1 with an altered spectrum in the range of 2800-3000 cm -1 , but does not display bands over 3300 cm -1 , indicating modification in CH and CH 2 groups. 3.3. DRIFT spectroscopy The DRIFT spectra of polymer samples are represented in Fig. 6. For all studied samples, three main regions can be identified in the frequency ranges between (1) 3600-3000 cm -1 , characteristic for OH stretching modes of the different types of hydrate water molecules; (2) 3100-2800 cm -1 , characteristic for ν C-H and ν N-H bands, CH and NH stretching vibrational modes, with corresponding (3) 1700–1200 cm −1 (NH and CH deformation modes, CO and CC stretching modes) with characteristic modes for carbohydrate ring (920-750 cm −1 ). The most detailed analysis of DRIFT spectra of samples (Figure 5) can be assigned as presented in Tables 2 and 3. Table 2. Assignments of DRIFT spectra of the native CS and CS+IBU samples Literature (Meyer et al. 1993; Chopin and Whalen 1993; Bouchard and Douek 1993; Armaroli et al. 2004; Kalisz et al. 2021; Katan et al. 2022; Xu et al. 2023) Functional groups Wavenumber 2CS_M 2CS_L 4CS_L O-H stretching 3600-3300 3493 3513 3512 C-H stretching 3100-2800 2990 2942 2908 2990 2939 2897 2990 2939 2909 O–H stretching of water molecules Absorbed water 1650-1633 1679 1682 1678 C=O stretching N-H bending 1600-1588 1614 1615 1598 CH 2 bending 1464-1405 1432 1435 1433 CH 3 bending 1395-1355 1389 1389 1394w C-N stretching 1317-1320 1326 1326 1326w C-OH stretching 1264-1260 1264 1231vw 1267 1231 1273w C-O-C stretching 1165-1162 1166 1168 1167 C-O stretching 1200 – 800 1126 1047 997 1127 1047 998 1126 1047s 998 C-O-C ring vibration of carbohydrate (β-glycosidic bond) 920 –758 904 906 906 852 Out-of-plane O-H bending 681 681 681 Table 3. CS+IBU samples assignments (Meyer et al. 1993; Chopin and Whalen 1993; Bouchard and Douek 1993; Armaroli et al. 2004; Kalisz et al. 2021; Katan et al. 2022; Xu et al. 2023) Functional groups Wavenumber 2CS_M/IBU 2CS_L/IBU 4CS_L/IBU O-H stretching 3600 –3300 3467 3507 3500 C-H stretching 3100-2800 2927 2939 2936 O–H stretching of water molecules Absorbed water 1650-1633 1681 1681 1681 C=O stretching N-H bending 1600-1588 1616 1613 1609 CH2 bending 1464-1405 1435 1434 1435 CH 3 bending 1395-1355 1394 1391 1390 C-N stretching 1317-1320 1339 1330 1327w C-OH stretching 1264-1260 1271 1230w 1273w C-O-C stretching 1165-1162 1155 1167 1166 C-O stretching 1200 – 800 1079 975 1127 1047 1003 1127 1046 1000 C-O-C ring vibration of carbohydrate (β-glycosidic bond) 920 –758 802 906 906 Out-of-plane O-H bending 670 736 736 4. Discussion Spectroscopy (ATR FT-IR, DRIFT, XPS, and Raman spectroscopies) includes several methods of identifying and classifying compounds based on the vibrations of their bonds that are used to determine and demonstrate the differences in the molecular structure of studied compounds (Pirutin et al. 2023). Based on the measured spectra and assigned proper functional groups and vibrations, it is possible to detect chemical interactions between chitosan and ibuprofen in the process of creating polymer matrices and then changes in matrices after drug release (van Haaren et al. 2023). Spectroscopic methods are versatile tools in pharmaceutics and biopharmaceutics, with a wide field of applications ranging from characterization of drug formulations in both qualitative and quantitative analyses, like: study drug release from semisolid formulations in a non-invasive way; measuring drug diffusion from appropriate pharmaceutical systems such as polymers, gels, films, liposomes, etc.; investigate drug penetration into relevant acceptor systems as well as reveal the mechanism of drug release; characterize interactions between drugs and (semi)synthetic, and native macromolecules (Dole et al. 2011; Song et al. 2020). Fourier Transform Infrared Spectroscopy (FT-IR, a complementary method to Raman spectroscopy) is one of the techniques allowing for the characterization of chitosan-based on different bands corresponding to the −NH 2 group, which can be allocated to the symmetrical COO− gather extending vibration. At the same time, Raman analysis, depending on the inelastic diffusing of photons, is utilized to obtain a material's structural fingerprint. The research on the chemical structure of chitosan exposes a six-membered ring containing oxygen. The prominent bands are present at 471 cm −1 (bending vibration of C-C-O bonds) and 895 cm −1 , 1146 cm −1 (stretching vibration of C-C-O bonds) (Sudatta et al. 2020). Moreover, the presence of free -NH 2 and -OH groups (3600-3000 cm -1 ) in CS's backbone enables chemical modifications by joining different molecules with biological and pharmacological activity, enhancing its properties for more biomedical applications (Bhattacharjee et al. 2023). Our study found that the chitosan–ibuprofen matrices showed a grain morphological structure, and the introduction of ibuprofen changed the molecular arrangement of chitosan, attributing probably to the electrostatic and hydrophobic interactions between the drug and the polymer. It is considered the most likely IBU interacted with CS with its aromatic ring and hydrophilic carboxylic groups, changing surface hydrophobicity. At the same time, cationic CS contains a non-polar region and ammonium groups providing hydrogen bonding capacity and possessing high affinity for oppositely charged molecules of IBU (Mahmood et al. 2021). Consequently, the surface becomes much less hydrophilic. IBU is a molecule of inherently low polarity, which results in a lack of anchoring sites required for further organization; therefore, chitosan matrices may facilitate the close packing and the structural ordering of IBU (Mani et al. 2023). The kinetics of drug release profiles confirmed that in low MW CS samples, IBU is more tightly packed than in medium MW CS, which manifests in the burst release of the drug. Spectroscopically, less C-O groups were detected in the 2CS_M/IBU sample, which may mean better binding to the matrix and, consequently smaller and slower release. For all CS/IBU conjugates, the spectral data confirmed the formation of amide I and II bonds between the carboxyl group of IBU and the amino group of the CS chain through amide bonds, improving water solubility, and proving the occurrence of grafting reaction (Guan et al. 2024). The IBU/CS aerogels were also prepared for transdermal administration by Chen Li et al. This bioformulation exhibited nanopore morphology structure, and the addition of IBU changed the crystallinity of CS connected with the electrostatic and hydrophobic interplay between the drug and the polymer. Moreover, the IBU/CS aerogels possessed an excellent swelling ratio and higher thermal stability, ensuring controlled drug release (Li et al. 2021). As chitosan is widely used in the pharmaceutical industry, its use with NSAIDs, particularly ibuprofen, for potential treatment was studied from a clinical perspective and assumed as promising (Pooresmaeil and Namazi 2021). As an over-the-counter medication, it is commonly used for pain relief by inhibiting cyclooxygenase (COX), which is involved in the production of prostaglandins. The reduction of its activity leads to a decrease in this production, thus decreasing inflammation (Rainsford 2009). It has a good safety profile, but the main adverse drug reactions are related to gastrointestinal, hepato-renal, and cardiovascular risks related to the most common oral use of the compound (Rainsford 2009; Lanas et al. 2010; Collaboration Coxib and traditional NSAID Trialists’ (CNT) 2013). To avoid adverse side effects, studies have looked at both modifications of drug action and alternative methods of drug delivery. Topical applications (gels, creams, sprays, etc.) were investigated concerning musculoskeletal pain. However, absorption was decreased compared to oral IBU, which may differ 300 times in plasma concentration (Tegeder et al. 1999). Moreover, topical formulations have a drawback of costs, as topical NSAIDs are more expensive in therapy. However, a chronic application was not studied in this perspective (Manoukian et al. 2017). To address this issue, the modifications of drug carriers might be beneficial for patients. Already studied formulations tend to use biocompatible, degradable polymers as vehicles. The examples can be porous materials (e.g., vaterite investigated with cyclodextrins and doxorubicin). However, as concluded by Campbell et al., it can be influenced by matrix content incubation temperature, ionic strength, pH, drug concentration, etc. which results in different performance of the resulting capsules (Campbell et al. 2022). Diego-Taboada et al. have studied ibuprofen release from sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores in simulated gastric (SGF) and gastrointestinal fluid (GIT). This experiment indicated that an encapsulated material can be released rapidly in high yield from SEC triggered by pH. A potential application for targeted release into the GIT was demonstrated by 88 ± 1% of the drug retained in SGF after 45 minutes and 85 ± 2% released after 5 min in buffer (PBS; pH 7.4) (Diego-Taboada et al. 2013). Previous research has shown that the drug release from ibuprofen-loaded biomaterials as tablets is pH sensitive. The chitosan carrier may allow more rapid IBU release in stomach fluids at pH 1.2 due to the easier favorable protonation of the amino groups in this acidic condition, opposite to the behavior that occurs at pH 7.4, that of for example PBS, where an increase of the rate of CS swelling occurs. In an acidic environment of the stomach, the chitosan-based drug carrier will dissolve very fast, releasing whole amount of the immobilized IBU and providing immediate therapeutic effect (Vieira et al. 2013). Based on this research, we can expect faster and greater IBU release at low pH mimicking gastric fluid conditions than at pH 7.4 as it was conducted in this study. The materials developed by dos S. Pereira et al. consisting of IBU, CS, and silver nanoparticles (AgNPs) presented a lower swelling rate at neutral pH because, in the GIT, the amine groups are deprotonated (dehydronated), and the material is characterized by a negatively-charged surface that electrostatically repels the molecules of IBU, leading to its greater release. Additionally, the hybrid compound exhibited greater drug release capacity and bactericidal activity (Pereira et al. 2020). 5. Conclusions Within this study, it was proven that chitosan matrices may be a promising base for the production of oral ibuprofen-loaded capsules. The effective incorporation of ibuprofen into chitosan matrices was evidenced by Raman spectroscopy by the disappearance of the bands at 1608 and 1342 cm -1 characteristic for crystalline IBU, and the appearance of new bands at 1416 and 1424 cm -1 assigned to CH, CH 3 bending of IBU and deformation of hydroxyl and amino groups, by the formation of non-covalent bonds between CS and IBU. Among all tested samples, 2CS_L/IBU appeared to be the most promising drug carrier since it revealed the burst release of the ibuprofen within just half an hour. Importantly, it may be assumed that in the acidic environment of the gastric juice, the drug release will be significantly faster, providing excellent therapeutic effect after oral administration. This research demonstrated that spectroscopic methods can be a powerful tools in the investigation of polymer/drug interactions and their relevance to controlled drug release processes. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Acknowledgments The research was funded by the Ministry of Science and Higher Education in Poland through the statutory activity of the Medical University of Lublin (DS 630 project). Additionally, it received partial support from the Ministry of Science and Higher Education in Poland under the PBmb 170 project. We would also like to acknowledge the financial support provided by the Foundation for Polish Science through the Reintegration grant (grant number POIR.04.04.00-00-4398/17-00, REINTEGRATION/2017-4/14). CRediT authorship contribution statement Conceptualization: ASB, BG and AP, Funding acquisition: ASB and AP, Investigation: BG, GK, PK, VV, MK, ISP, OM, LN, RN, Methodology: BG, PK, VV, GK, OM, MK, Project administration: AP, ASB, Resources: ASB, AP, ISP, Supervision: ASB, Visualization ISP, VV, GK, Writing – original draft: BG, GK, OM, PK, VV Writing – review and editing: AP, ISP, ASB Data statement Data available on request from the authors. A preprint has previously been published Barbara Gieroba et al. 2024 (https://doi.org/10.2139/ssrn.4995823). References Aranaz I, Alcántara AR, Civera MC, et al (2021) Chitosan: An Overview of Its Properties and Applications. Polymers 13:3256. https://doi.org/10.3390/polym13193256 Ardila N, Daigle F, Heuzey M-CC, Ajji A (2017) Antibacterial Activity of Neat Chitosan Powder and Flakes. Molecules 22:100. https://doi.org/10.3390/molecules22010100 Armaroli T, Bécue T, Gautier S (2004) Diffuse Reflection Infrared Spectroscopy (DRIFTS): Application to the in situ Analysis of Catalysts. Oil & Gas Science and Technology-Rev IFP 59:215–237 Azueta-Aguayo PH, Chuc-Gamboa MG, Aguilar-Pérez FJ, et al (2022) Effects of Neutralization on the Physicochemical, Mechanical, and Biological Properties of Ammonium-Hydroxide-Crosslinked Chitosan Scaffolds. International Journal of Molecular Sciences 23:14822. https://doi.org/10.3390/ijms232314822 Barbosa FC, Silva MC da, Silva HN da, et al (2020) Progress in the Development of Chitosan Based Insulin Delivery Systems: A Systematic Literature Review. Polymers 12:2499. https://doi.org/10.3390/polym12112499 Bhattacharjee S, Goswami S, Das S, et al (2023) pH-responsive, stable, and biocompatible functional nanogels based on chitosan (CS)/poly methacrylic acid (PMAA) polymers: Synthesis and characterization. Materials Today Communications 36:106541. https://doi.org/10.1016/j.mtcomm.2023.106541 Biniaś D, Biniaś W, Janicki J (2016) Application of Raman spectroscopy for evaluation of chemical changes in dibutyrylchitin fibres. Fibres and Textiles in Eastern Europe 24:27–38. https://doi.org/10.5604/12303666.1207842 Bouchard J, Douek M (1993) Structural and Concentration Effects on the Diffuse Reflectance Ftir Spectra of Cellulose, Lignin and Pulp. Journal of Wood Chemistry and Technology 13:481–499. https://doi.org/10.1080/02773819308020530 Bushra R, Aslam N (2010) An overview of clinical pharmacology of Ibuprofen. Oman medical journal 25:155–161. https://doi.org/10.5001/OMJ.2010.49 Camerlingo C, Portaccio M, D’Apuzzo F, et al (2022) μ-FTIR, μ-Raman, and SERS Analysis of Amide I Spectral Region in Oral Biofluid Samples during Orthodontic Treatment. Sensors 22:7874. https://doi.org/10.3390/s22207874 Campbell J, Ferreira AM, Bowker L, et al (2022) Dextran and Its Derivatives: Biopolymer Additives for the Modulation of Vaterite CaCO 3 Crystal Morphology and Adhesion to Cells. Advanced Materials Interfaces 9:. https://doi.org/10.1002/admi.202201196 Chopin T, Whalen E (1993) A new and rapid method for carrageenan identification by FT IR diffuse reflectance spectroscopy directly on dried, ground algal material. Carbohydrate Research 246:51–59. https://doi.org/10.1016/0008-6215(93)84023-Y Collaboration Coxib and traditional NSAID Trialists’ (CNT) (2013) Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. The Lancet 382:769–779. https://doi.org/10.1016/S0140-6736(13)60900-9 Diego-Taboada A, Maillet L, Banoub JH, et al (2013) Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking. J Mater Chem B 1:707–713. https://doi.org/10.1039/C2TB00228K Dimida S, Barca A, Cancelli N, et al (2017) Effects of Genipin Concentration on Cross-Linked Chitosan Scaffolds for Bone Tissue Engineering: Structural Characterization and Evidence of Biocompatibility Features. International Journal of Polymer Science 2017:8410750. https://doi.org/10.1155/2017/8410750 Dole MN, Patel PA, Sawant S, Shedpure PS (2011) ADVANCE APPLICATIONS OF FOURIER TRANSFORM INFRARED SPECTROSCOPY Review Article Gieroba B, Sroka-Bartnicka A, Kazimierczak P, et al (2020) Spectroscopic studies on the temperature-dependent molecular arrangements in hybrid chitosan/1,3-β-D-glucan polymeric matrices. International Journal of Biological Macromolecules 159:911–921. https://doi.org/10.1016/j.ijbiomac.2020.05.155 Gonçalves C, Ferreira N, Lourenço L (2021) Production of Low Molecular Weight Chitosan and Chitooligosaccharides (COS): A Review. Polymers 2021, Vol 13, Page 2466 13:2466. https://doi.org/10.3390/POLYM13152466 Guan X, Yao H, Wu J (2024) Photocrosslinkable hydrogel of ibuprofen‐chitosan methacrylate modulates inflammatory response. Journal of Biomedical Materials Research Part A 112:2001–2017. https://doi.org/10.1002/jbm.a.37758 Harish Prashanth KV, Kittur FS, Tharanathan RN (2002) Solid state structure of chitosan prepared under different N-deacetylating conditions. Carbohydrate Polymers 50:27–33. https://doi.org/10.1016/S0144-8617(01)00371-X Hoekstra A, Struszczyk H, Kivekäs O (1998) Percutaneous microcrystalline chitosan application for sealing arterial puncture sites. Biomaterials 19:1467–1471. https://doi.org/10.1016/S0142-9612(98)00060-X Jia S, Pan H, Lin Q, et al (2020) Study on the preparation and mechanism of chitosan-based nano-mesoporous carbons by hydrothermal method. Nanotechnology 31:365604. https://doi.org/10.1088/1361-6528/ab9575 Kalisz G, Przekora A, Kazimierczak P, et al (2021) Physicochemical changes of the chitosan/β-1,3-glucan/hydroxyapatite biocomposite caused by mesenchymal stem cells cultured on its surface in vitro. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251:119439. https://doi.org/10.1016/j.saa.2021.119439 Kasaai MR (2009) Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review. Journal of agricultural and food chemistry 57:1667–1676. https://doi.org/10.1021/JF803001M Katan T, Kargl R, Mohan T, et al (2022) Solid Phase Peptide Synthesis on Chitosan Thin Films. Biomacromolecules 23:731–742. https://doi.org/10.1021/ACS.BIOMAC.1C01155/ASSET/IMAGES/LARGE/BM1C01155_0008.JPEG Kazarian SG, Martirosyan GG (2002) Spectroscopy of polymer/drug formulations processed with supercritical fluids: in situ ATR–IR and Raman study of impregnation of ibuprofen into PVP. International Journal of Pharmaceutics 232:81–90. https://doi.org/10.1016/S0378-5173(01)00905-X Khademi F, Derakhshan M, Yousefi-Avarvand A, Tafaghodi M (2018) Potential of polymeric particles as future vaccine delivery systems/adjuvants for parenteral and non-parenteral immunization against tuberculosis: A systematic review. Iranian journal of basic medical sciences 21:116–123. https://doi.org/10.22038/IJBMS.2017.22059.5648 Krukiewicz K, Kowalik A, Turczyn R, Biggs MJP (2020) In vitro attenuation of astrocyte activation and neuroinflammation through ibuprofen-doping of poly(3,4-ethylenedioxypyrrole) formulations. Bioelectrochemistry 134:107528. https://doi.org/10.1016/j.bioelechem.2020.107528 Lanas A, Tornero J, Zamorano JL (2010) Assessment of gastrointestinal and cardiovascular risk in patients with osteoarthritis who require NSAIDs: the LOGICA study. Annals of the Rheumatic Diseases 69:1453–1458. https://doi.org/10.1136/ard.2009.123166 Lavertu M, Darras V, Buschmann MD (2012) Kinetics and efficiency of chitosan reacetylation. Carbohydrate Polymers 87:1192–1198. https://doi.org/10.1016/j.carbpol.2011.08.096 Li C, Wang K, Xie D (2021) Green Fabrication and Release Mechanisms of pH-Sensitive Chitosan–Ibuprofen Aerogels for Controlled Transdermal Delivery of Ibuprofen. Frontiers in Chemistry 9:. https://doi.org/10.3389/fchem.2021.767923 Lima BV, Oliveira MJ, Barbosa MA, et al (2021) Immunomodulatory potential of chitosan-based materials for cancer therapy: a systematic review of in vitro , in vivo and clinical studies. Biomaterials Science 9:3209–3227. https://doi.org/10.1039/D0BM01984D Lizardi-Mendoza J, Argüelles Monal WM, Goycoolea Valencia FM (2016) Chemical Characteristics and Functional Properties of Chitosan. Chitosan in the Preservation of Agricultural Commodities 3–31. https://doi.org/10.1016/B978-0-12-802735-6.00001-X Mahmood S, Almurisi SH, Al-Japairai K, et al (2021) Ibuprofen-Loaded Chitosan–Lipid Nanoconjugate Hydrogel with Gum Arabic: Green Synthesis, Characterisation, In Vitro Kinetics Mechanistic Release Study and PGE2 Production Test. Gels 7:254. https://doi.org/10.3390/gels7040254 Mani R, Peltonen L, Strachan CJ, et al (2023) Nonclassical Crystallization and Core–Shell Structure Formation of Ibuprofen from Binary Solvent Solutions. Crystal Growth & Design 23:236–245. https://doi.org/10.1021/acs.cgd.2c00971 Manoukian MAC, Migdal CW, Tembhekar AR, et al (2017) Topical Administration of Ibuprofen for Injured Athletes: Considerations, Formulations, and Comparison to Oral Delivery. Sports Medicine - Open 3:36. https://doi.org/10.1186/s40798-017-0103-2 Mathaba M, Daramola MO (2020) Effect of Chitosan’s Degree of Deacetylation on the Performance of PES Membrane Infused with Chitosan during AMD Treatment. Membranes 2020, Vol 10, Page 52 10:52. https://doi.org/10.3390/MEMBRANES10030052 Meyer U, Gilbert C, Kokot S (1993) Application of DRIFT Spectroscopy and Chemometrics for the Comparison of Cotton Fabrics. Applied Spectroscopy, Vol 47, Issue 6, pp 741-748 47:741–748 Morris GA, Castile J, Smith A, et al (2009) Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach. Carbohydrate Polymers 76:616–621. https://doi.org/10.1016/J.CARBPOL.2008.11.025 Motov S, Masoudi A, Drapkin J, et al (2019) Comparison of Oral Ibuprofen at Three Single-Dose Regimens for Treating Acute Pain in the Emergency Department: A Randomized Controlled Trial. Annals of emergency medicine 74:530–537. https://doi.org/10.1016/J.ANNEMERGMED.2019.05.037 Movasaghi Z, Rehman S, Rehman IU (2007) Raman Spectroscopy of Biological Tissues. Applied Spectroscopy Reviews 42:493–541. https://doi.org/10.1080/05704920701551530 Muxika A, Etxabide A, Uranga J, et al (2017) Chitosan as a bioactive polymer: Processing, properties and applications. International journal of biological macromolecules 105:1358–1368. https://doi.org/10.1016/J.IJBIOMAC.2017.07.087 Ogawa K, Yui T, Okuyama K (2004) Three D structures of chitosan. International Journal of Biological Macromolecules 34:1–8. https://doi.org/10.1016/j.ijbiomac.2003.11.002 Pellis A, Guebitz GM, Nyanhongo GS (2022) Chitosan: Sources, Processing and Modification Techniques. Gels 2022, Vol 8, Page 393 8:393. https://doi.org/10.3390/GELS8070393 Pereira AK dos S, Reis DT, Barbosa KM, et al (2020) Antibacterial effects and ibuprofen release potential using chitosan microspheres loaded with silver nanoparticles. Carbohydrate Research 488:107891. https://doi.org/10.1016/j.carres.2019.107891 Pérez-Álvarez L, Ruiz-Rubio L, Vilas-Vilela JL (2018) Determining the Deacetylation Degree of Chitosan: Opportunities to Learn Instrumental Techniques. Journal of Chemical Education 95:1022–1028. https://doi.org/10.1021/ACS.JCHEMED.7B00902 Pirutin SK, Jia S, Yusipovich AI, et al (2023) Vibrational Spectroscopy as a Tool for Bioanalytical and Biomonitoring Studies. International Journal of Molecular Sciences 24:6947. https://doi.org/10.3390/ijms24086947 Pooresmaeil M, Namazi H (2021) Developments on carboxymethyl starch-based smart systems as promising drug carriers: A review. Carbohydrate Polymers 258:117654. https://doi.org/10.1016/j.carbpol.2021.117654 Rainsford KD (2009) Ibuprofen: pharmacology, efficacy and safety. Inflammopharmacology 17:275–342. https://doi.org/10.1007/s10787-009-0016-x Saito Y, Okano T, Gaill F, et al (2000) Structural data on the intra-crystalline swelling of beta-chitin. International journal of biological macromolecules 28:81–88. https://doi.org/10.1016/S0141-8130(00)00147-1 Shi C, Zhu Y, Ran X, et al (2006) Therapeutic potential of chitosan and its derivatives in regenerative medicine. The Journal of surgical research 133:185–192. https://doi.org/10.1016/J.JSS.2005.12.013 Sivashankari PR, Prabaharan M (2016) Deacetylation modification techniques of chitin and chitosan. Chitosan Based Biomaterials: Fundamentals: Volume 1 1:117–133. https://doi.org/10.1016/B978-0-08-100230-8.00005-4 Song Y, Cong Y, Wang B, Zhang N (2020) Applications of Fourier transform infrared spectroscopy to pharmaceutical preparations. Expert Opinion on Drug Delivery 17:551–571. https://doi.org/10.1080/17425247.2020.1737671 Sudatta BP, Sugumar V, Varma R, Nigariga P (2020) Extraction, characterization and antimicrobial activity of chitosan from pen shell, Pinna bicolor. International Journal of Biological Macromolecules 163:423–430. https://doi.org/10.1016/j.ijbiomac.2020.06.291 Tegeder I, Muthselbach U, Lotsch J, et al (1999) Application of microdialysis for the determination of muscle and subcutaneous tissue concentrations after oral and topical ibuprofen administration. Clinical Pharmacology & Therapeutics 65:357–368. https://doi.org/10.1016/S0009-9236(99)70128-1 Thareja A, Hughes H, Alvarez-Lorenzo C, et al (2021) Penetration Enhancers for Topical Drug Delivery to the Ocular Posterior Segment—A Systematic Review. Pharmaceutics 13:276. https://doi.org/10.3390/pharmaceutics13020276 van Haaren C, De Bock M, Kazarian SG (2023) Advances in ATR-FTIR Spectroscopic Imaging for the Analysis of Tablet Dissolution and Drug Release. Molecules 28:4705. https://doi.org/10.3390/molecules28124705 Verlee A, Mincke S, Stevens CV (2017) Recent developments in antibacterial and antifungal chitosan and its derivatives. Carbohydrate polymers 164:268–283. https://doi.org/10.1016/J.CARBPOL.2017.02.001 Vieira AP, Badshah S, Airoldi C (2013) Ibuprofen-loaded chitosan and chemically modified chitosans—Release features from tablet and film forms. International Journal of Biological Macromolecules 52:107–115. https://doi.org/10.1016/j.ijbiomac.2012.09.013 Xu C, Xu Y, Zhong D, et al (2023) Zr4+ and glutaraldehyde cross-linked polyethyleneimine functionalized chitosan composite: Synthesis, characterization, Cr(VI) adsorption performance, mechanism and regeneration. International journal of biological macromolecules 239:. https://doi.org/10.1016/J.IJBIOMAC.2023.124266 Yılmaz Atay H, Çelik E (2017) Investigations of antibacterial activity of chitosan in the polymeric composite coatings. Progress in Organic Coatings PB:194–200. https://doi.org/10.1016/J.PORGCOAT.2016.10.013 Yuan Y, Chesnutt BM, Haggard WO, Bumgardner JD (2011) Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures. Materials 2011, Vol 4, Pages 1399-1416 4:1399–1416. https://doi.org/10.3390/MA4081399 Zając A, Hanuza J, Wandas M, Dymińska L (2015) Determination of N-acetylation degree in chitosan using Raman spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134:114–120. https://doi.org/10.1016/j.saa.2014.06.071 Zhang K, Peschel D, Helm J, et al (2011) FT Raman investigation of novel chitosan sulfates exhibiting osteogenic capacity. Carbohydrate Polymers 83:60–65. https://doi.org/10.1016/j.carbpol.2010.07.021 Zhang M, Ma Y, Wang Z, et al (2017) Optimizing molecular weight of octyl chitosan as drug carrier for improving tumor therapeutic efficacy. Oncotarget 8:64237. https://doi.org/10.18632/ONCOTARGET.19452 Zhao D, Yu S, Sun B, et al (2018) Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers 2018, Vol 10, Page 462 10:462. https://doi.org/10.3390/POLYM10040462 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 08 May, 2026 Reviews received at journal 04 May, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers invited by journal 10 Apr, 2026 Editor assigned by journal 24 Aug, 2025 Submission checks completed at journal 22 Aug, 2025 First submitted to journal 20 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7416784","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621660333,"identity":"b2f5831f-abc3-4724-a76d-3f17abed0812","order_by":0,"name":"Barbara Gieroba","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACAwYGNjADRDIzMNgwMLCD2RZEa0kDkSC2BGEtDBAthwlrMWc/++zBzz12dn3svQ8fF9ScT1zbzGP2gHEHbi2WPenmhj3PkpPbeI4bG884djtx22EecwPGM3gcdiCNTYLnAHMym0QamzQPG1iLmQRjGx4t55+xSf45UJ/MJv8MqOXfOSK03AAZfuCwHZsEG5s0b9sBwlosZzxjN5Y5cDyBjSeN2XhmX7LxtsNs5QaJePxizp/G9vDNgWp7+fZjjI8LvtnJbjvevO3Bxx02OLXAQGIDPi5WYI/KZSRCyygYBaNgFIwYAAAVHk3dyrM5egAAAABJRU5ErkJggg==","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":true,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Gieroba","suffix":""},{"id":621660334,"identity":"6c76b90a-c48e-4859-b641-464337b62c37","order_by":1,"name":"Paulina Kazimierczak","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Paulina","middleName":"","lastName":"Kazimierczak","suffix":""},{"id":621660335,"identity":"2c5d6a4c-8a67-4476-834d-511908fee4da","order_by":2,"name":"Grzegorz Kalisz","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Grzegorz","middleName":"","lastName":"Kalisz","suffix":""},{"id":621660336,"identity":"cf764a1c-d2cf-4164-acba-85c12d1beeee","order_by":3,"name":"Vladyslav Vivcharenko","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Vladyslav","middleName":"","lastName":"Vivcharenko","suffix":""},{"id":621660337,"identity":"dfba97b6-610b-4ab6-ba94-046485510f50","order_by":4,"name":"Olena Mozgova","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Olena","middleName":"","lastName":"Mozgova","suffix":""},{"id":621660338,"identity":"39648920-7bbc-4375-baa1-d76383cbd175","order_by":5,"name":"Maryna Khalavka","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Maryna","middleName":"","lastName":"Khalavka","suffix":""},{"id":621660339,"identity":"54e1212a-e5b2-41ba-b695-39bfa550f4dd","order_by":6,"name":"Liudmyla Nosach","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Liudmyla","middleName":"","lastName":"Nosach","suffix":""},{"id":621660340,"identity":"be36439a-0858-473f-9198-ff4b8ab1339b","order_by":7,"name":"Izabela S. Pięta","email":"","orcid":"","institution":"Polish Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"S.","lastName":"Pięta","suffix":""},{"id":621660341,"identity":"d4e1cfd4-c3c2-47ab-8bc8-0317c0b8ecb8","order_by":8,"name":"Agata Przekora","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Przekora","suffix":""},{"id":621660342,"identity":"d661545e-6a58-4a51-9bd8-9f6d0b64e6a0","order_by":9,"name":"Anna Sroka-Bartnicka","email":"","orcid":"","institution":"Medical University of Lublin","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Sroka-Bartnicka","suffix":""}],"badges":[],"createdAt":"2025-08-20 11:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7416784/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7416784/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107482868,"identity":"48e3f962-5148-45d7-9eba-1fae9f5232d7","added_by":"auto","created_at":"2026-04-22 02:25:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33058,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of chitosan.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/65b645a773a3f9e14cc976b3.png"},{"id":107481974,"identity":"bfbef141-ed86-49ff-823f-f923e0873877","added_by":"auto","created_at":"2026-04-22 02:21:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85874,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of ibuprofen release from ibuprofen-loaded chitosan samples (\u003csup\u003e*\u003c/sup\u003estatistically significant results compared to 4CS_L/IBU, \u003csup\u003e#\u003c/sup\u003estatistically significant results compared to 2CS_L/IBU, P \u0026lt; 0.05, one-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/f160f24a5ab15389a343653a.png"},{"id":107199849,"identity":"cf7f542e-68f1-4a3a-acb1-e3b582f7027e","added_by":"auto","created_at":"2026-04-18 03:22:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":224519,"visible":true,"origin":"","legend":"\u003cp\u003eRaman spectra of (A) native polymers, (B) polymers treated with PBS, (C) IBU-enriched, and (D) IBU-enriched after release.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/7a6570568830957de283e100.png"},{"id":107199851,"identity":"68c9e767-d62e-4d19-9503-30fc88d34b74","added_by":"auto","created_at":"2026-04-18 03:22:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192835,"visible":true,"origin":"","legend":"\u003cp\u003eRaman spectra of (A) 2% CS low MW, (B) 4% CS low MW, (C) 2% medium MW, each group with and without ibuprofen.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/c160b9ccdc7c1196714ac103.png"},{"id":107483786,"identity":"2d91fe45-e656-467a-b770-226bb99f3b69","added_by":"auto","created_at":"2026-04-22 02:29:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96884,"visible":true,"origin":"","legend":"\u003cp\u003eRaman spectrum of IBU added to polymer matrices\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/ff555785d3a198016897913a.png"},{"id":107199853,"identity":"41e2f781-10ff-4b70-b9e0-0d418bee48a5","added_by":"auto","created_at":"2026-04-18 03:22:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":286766,"visible":true,"origin":"","legend":"\u003cp\u003eDRIFT spectra of the (A) CS native samples and (B, C) CS+IBU samples in regions (B) O-H stretching and (C) C-O stretching.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/071fe5d0e4c8577a7da89554.png"},{"id":107486205,"identity":"127c0f4d-0ad9-4fb9-9467-33a3a4e2ec90","added_by":"auto","created_at":"2026-04-22 02:37:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1465410,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7416784/v1/a4c04a27-8f01-4735-8d65-66f4cf27f070.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low and medium molecular weight chitosan matrices with ibuprofen as a potential base for soft capsules for oral use: evaluation of physicochemical properties and molecular structure","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChitosan (CS), poly[\u0026beta;-(1\u0026rarr;4)-linked-2-amino-2-deoxy-D-glucose] (Figure 1), is a natural linear polysaccharide obtained by the partial or complete alkaline deacetylation of chitin. It consists of D-glucosamine and \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosamine units, linked by \u0026beta;-1,4 glycosidic bonds (Muxika et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChitosan is not a singular polymer with a defined structure but a series of molecules with variations in size, composition, and monomer distribution (Aranaz et al. 2021). The molar percentage between two glucosamine monomeric units is an indicator of the degree of deacetylation (DDA) (Verlee et al. 2017). It ranges from 0 (chitin) to 100 (entirely deacetylated chitin) (P\u0026eacute;rez-\u0026Aacute;lvarez et al. 2018). This parameter is considered an important factor that influences numerous crucial physicochemical and biological properties of chitosan and its derivatives, like hydrophilicity, crystallinity, tensile strength, elastic modulus, moisture content, degradation, and finally, protein adsorption and cell response (Yuan et al. 2011; Mathaba and Daramola 2020). DDA is influenced by the temperature, time of reaction, alkali concentration, particle size, and density (Sivashankari and Prabaharan 2016; Pellis et al. 2022). Generally, chitosan with a higher DDA is characterized by better biological features than poorly deacetylated ones because of the higher concentration of the amino (NH\u003csub\u003e2\u003c/sub\u003e) groups in relation to the N-acetyl glucosamine moieties (Kasaai 2009; P\u0026eacute;rez-\u0026Aacute;lvarez et al. 2018). Chitosan with a greater DDA is characterized by an elongated conformation with more flexible chains, and those with decreased DDA often take on a spiral shape, mainly because of the low charge density in the polymeric chain (Morris et al. 2009). Moreover, along with the increase of DDA during processing, the molecular weight (MW) of chitosan decreases; MW also depends on the source of material (Harish Prashanth et al. 2002). Most commercial chitosans have MW of 50\u0026ndash;2000 kDa, with an average DDA of 50\u0026ndash;100% (usually 80\u0026ndash;90%). Based on the MW, chitosan can be divided into low molecular weight (\u0026lt;100 kDa), medium molecular weight (range from 100 to 1000 kDa), and high molecular weight (\u0026gt;1000 kDa) (Gon\u0026ccedil;alves et al. 2021). MW determines not only the fundamental properties of molecules, such as conformation, viscosity, solubility, and cytotoxicity, but in the case of chitosan, additionally the ability to create different forms and their strengths, and even the releasing rates of the substances carried by the molecules (Zhang et al. 2017). MW and DDA may also influence the crystallinity of the chitosan films, which in turn affects mechanical properties, especially tensile strength (Hoekstra et al. 1998), and determines the water sorption ability. Less crystalline chitosan films (low-ordered non-crystalline and amorphous forms) will absorb more water vapor and, inversely, in high crystalline forms (Saito et al. 2000).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChitosan is insoluble in neutral or alkaline solvents and easily soluble in many aqueous solutions of organic and inorganic acids (Ogawa et al. 2004). In water solutions, chitosan has high viscosity and forms a quasi-globular conformation, stabilized by inter- and intramolecular hydrogen bonds between hydroxyl and amino groups (Morris et al. 2009). Moreover, its (bio)chemical reactivity is quite high due to free primary amino groups distributed evenly in the molecular chain (Ogawa et al. 2004). Chitosan has gel-forming ability at low pHs (Dimida et al. 2017). When it is exposed to an acidic environment, the amino groups in the polymer chains protonate, creating NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e moieties. As a consequence, it becomes cationic, allowing it to interplay with different types of molecules (Lizardi-Mendoza et al. 2016). In practice, chitosan occurs in solution as a poly-cationic form; thereby, it is able to react with negatively charged molecules, like anions and poly-anions, creating ionic complexes (Dimida et al. 2017). This positive charge is also considered responsible for the antimicrobial properties of chitosan through the interaction with the negatively charged cell membranes of bacteria (Yılmaz Atay and \u0026Ccedil;elik 2017). Due to its solubility in acidic aqueous media, it can be manufactured in various forms, i.e., hydrogels, films, nanofibers, or even pastes (Shi et al. 2006). However, there are some reports on its \u0026nbsp;formulation of non-dissolved powdery and flakes (Ardila et al. 2017).\u003c/p\u003e\n\u003cp\u003eChitosan possesses exquisite biological properties, such as biodegradability, non-toxicity, mucoadhesiveness, hemocompatibility, and low immunogenicity, and exhibits antioxidant, anticancer, and antibacterial activities (Zhao et al. 2018). Therefore, it is widely employed in the biotechnological industry in diverse cosmetic and pharmaceutical formulations and in creating gene, protein, drug, and vaccine delivery systems(Khademi et al. 2018; Barbosa et al. 2020; Lima et al. 2021; Thareja et al. 2021). It is also utilized in biomedical applications, mainly in the field of wound dressings, tissue engineering, and implant coatings (Shi et al. 2006). Its non-medical usages are food processing and packaging, water clarification, environmental remediation, waste management, and sewage disposal, separators, and agricultural materials production (Lavertu et al. 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChitosan is often used for the drug release systems of non-steroidal drugs, usually in the form of cross-linked films or tablets to accommodate the selected drug inside the polymeric material (Vieira et al. 2013). One of these drugs is ibuprofen (IBU), belonging to a group of medicines called Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and is used extensively for the management of acute pain (Motov et al. 2019). It is a low-soluble crystal powder in class II in the biopharmaceutics classification system (BCS). IBU is characterized by a short half-life (2 hours) after oral administration, leading to the need for increasing and multiplying the doses and, in consequence, resulting in missed medication aliquots (and associated with poor patient compliance) and severe side effects. Additional issues arising from oral administration are gastric irritation and hepatic first-pass metabolism (Bushra and Aslam 2010; Mahmood et al. 2021). Thus, there is a need to overcome these disadvantages and extend the bioactive effect of IBU, where the drug release is performed through a matrix, such as chitosan, helping to reduce a series of undesirable adverse effects (Vieira et al. 2013).\u003c/p\u003e\n\u003cp\u003eThis research aimed to investigate chitosan matrices with ibuprofen as a potential base for soft capsules for oral use. The particular aim of this study was to evaluate the physicochemical features and molecular arrangements of chitosan films consisting of molecules with various preparation methods (low - 2 % (w/v) and 4 % (w/v) in 1% acetic acid, and medium - 2 % (w/v) in 1% acetic acid) with and without ibuprofen (or after drug release). Due to problems with chitosan solubility during sample preparation, 4 % (w/v) chitosan medium molecular weight was excluded from this study. The specimens without IBU were control samples. These properties were determined by combining several analytical techniques - drug release studies, Raman spectroscopy (RS), and diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. All matrices were tested before the IBU release test (prior to placing them in PBS) and after the drug release test. Specifically, assessing these structural, chemical, and physical characteristics will help comprehend the possible applications of the chitosan matrices in the biomedical field, especially as a drug delivery and release system.\u003c/p\u003e\n\u003cp\u003eThe following variants were examined:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e2% chitosan low molecular weight - \u003cstrong\u003e2CS_L\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan low molecular weight soaked in PBS - \u003cstrong\u003e2CS_L_R\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan low molecular weight + ibuprofen - \u003cstrong\u003e2CS_L/IBU\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan low molecular weight + ibuprofen after release test - \u003cstrong\u003e2CS_L/IBU_R\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e4% chitosan low molecular weight - \u003cstrong\u003e4CS_L\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e4% chitosan low molecular weight soaked in PBS - \u003cstrong\u003e4CS_L_R\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e4% chitosan low molecular weight + ibuprofen - \u003cstrong\u003e4CS_L/IBU\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e4% chitosan low molecular weight + ibuprofen after release test - \u003cstrong\u003e4CS_L/IBU_R\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan medium molecular weight - \u003cstrong\u003e2CS_M\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan medium molecular weight soaked in PBS - \u003cstrong\u003e2CS_\u003c/strong\u003e\u003cstrong\u003eM_R\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan medium molecular weight + ibuprofen - \u003cstrong\u003e2CS_\u003c/strong\u003e\u003cstrong\u003eM/IBU\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e2% chitosan medium molecular weight + ibuprofen after release test - \u003cstrong\u003e2CS_\u003c/strong\u003e\u003cstrong\u003eM/IBU_R\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe control samples without the drug were soaked in PBS in parallel during the release test, just like the matrices with IBU.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Samples preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree types of chitosan matrices made of 2% (\u003cem\u003ew/v\u003c/em\u003e) chitosan with low molecular weight (50\u0026ndash;190 kDa MW, Sigma-Aldrich Chemicals, Warsaw, Poland; sample marked as 2CS_L), 4% (\u003cem\u003ew/v\u003c/em\u003e) chitosan with low molecular weight (50\u0026ndash;190 kDa MW; sample marked as 4CS_L), and 2% (\u003cem\u003ew/v\u003c/em\u003e) chitosan with medium molecular weight (217 kDa MW, kindly obtained from National Marine Fisheries Research Institute, Gdynia, Poland; sample marked as 2CS_M) were fabricated. Each chitosan matrix was additionally loaded with 240 \u0026micro;g of ibuprofen (Sigma-Aldrich Chemicals, Warsaw, Poland; samples marked as 2CS_L/IBU, 4CS_L/IBU, 2CS_M/IBU, respectively). In brief, chitosan matrices were prepared by dissolving appropriate amounts of chitosan in 1% (\u003cem\u003ev/v\u003c/em\u003e) CH\u003csub\u003e3\u003c/sub\u003eCOOH (Avantor Performance Materials, Gliwice, Poland). To obtain the homogenous distribution of ibuprofen within chitosan matrices, dissolved chitosan was mixed with ibuprofen solution on a magnetic stirrer. Next, 600 \u0026micro;L of obtained gels were spread in a thin layer on the glass coverslips, soaked in 1% (\u003cem\u003ew/v\u003c/em\u003e) NaOH (Avantor Performance Materials, Gliwice, Poland) for 5 min, then rinsed with deionized water, and left to air dry. The thickness of the dried samples was measured using an electronic micrometer with an accuracy of 0.001 mm (Schut Geometrical Metrology, Groningen, The Netherlands). The thickness of the chitosan films was 90 \u0026micro;m \u0026plusmn; 9.6 \u0026micro;m.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Assessment of ibuprofen release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the ibuprofen release, the ibuprofen-loaded matrices (2CS_L/IBU, 4CS_L/IBU, 2CS_M/IBU) were submerged in 10\u0026thinsp;mL of phosphate-buffered saline (PBS) solution (pH 7.4, Sigma Aldrich-Chemicals, Warsaw, Poland) and incubated at 37 \u0026deg;C. Control CS matrices (without drug) were also soaked in PBS and served as control matrices during spectroscopic analyses. At defined time intervals, 0.5 mL samples were collected to evaluate ibuprofen concentration. Fresh PBS was added to maintain the original volume of the PBS solution during the test. Ibuprofen concentration was assessed by measuring the absorbance values at the wavelength of 225 nm using a UV-spectrophotometer (Genesys 6 UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA). A calibration curve was done for known concentrations of ibuprofen solutions, which were prepared in PBS in the 12.5 - 400 \u0026mu;g/mL range. The ibuprofen release profile from the chitosan matrices was expressed as the percentage of released drug at defined time intervals in a cumulative graph. Results obtained in this study were presented as mean values \u0026plusmn; standard deviation (n \u0026ge; 3). Statistical analysis was performed by using one-way ANOVA followed by Tukey\u0026apos;s test with statistical significance at p \u0026lt; 0.05. (GraphPad Prism 8.0.0 Software, GraphPad Software Inc., La Jolla, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Raman spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Raman spectra were recorded in the 100-4000 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e range using a Witec alpha300R (WITec, Gmbh, Ulm, Germany) with 532 nm excitation laser wavelength and registered in the ControlSIX software (WITec Gmbh, Ulm, Germany). All spectra were obtained with an integration time of 0.5 s with 9 mW laser power and 10 accumulations, applied through x100 objective lens (Zeiss, Germany). The collected light through an objective lens was directed via a 100 mm diameter silica fiber, acting as a confocal pinhole to a spectrograph (UHTS300S, WITec Gmbh, Ulm, Germany) with a 600 g/mm grating and charge-coupled device (CCD) camera. The results are presented as an average of 10 spectra for every sample after processing (baseline correction and smoothing with nine points Savitzky-Golay algorithm) with ProjectSIX (WITec Gmbh, Ulm, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpectra were recorded with a FTIR Nicolet iS50 spectrometer (Thermo Scientific, Waltham, MA, USA) in diffuse reflectance mode using a continuous flow Harrick Praying Mantis chamber. Typically, 100 scans were collected at a resolution of 1 cm\u003csup\u003e-1\u003c/sup\u003e. Prior to the measurement, each sample was purged in He flow (45 ml min\u003csup\u003e-1\u003c/sup\u003e) for 30 min. DRIFTS technique was used to study structural properties and the surface chemistry of bare and Ibuprofen-containing samples with a minimum of sample preparation steps to avoid any possible influence on the analysis. Typically, the sample was placed on a flat surface inside a chamber. The IR radiation was directed onto the sample via two ZnS windows, and the light reflected by diffuse scattering was collected with an external spherical mirror (outside the cell). The DRIFT spectra are presented in the Kubelka\u0026ndash;Munk function.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Assessment of ibuprofen release profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage of drug release with time from the IBU-loaded chitosan matrices is shown in Figure 2. The 2CS_L/IBU showed a high initial burst release of ibuprofen, reaching the plateau effect within the first 0.5 h (35.16 \u0026plusmn; 4.99% of the drug was released). Further ibuprofen release was not detectable for this sample. The 4CS_L/IBU showed a similar profile of ibuprofen release; however, it reached a plateau after 2 h of the experiment (32.16 \u0026plusmn; 9.14% of the drug was released). In turn, the 2CS_M/IBU showed burst release of the ibuprofen within the first 2 h followed by a subsequent slow, sustained release until reaching the plateau effect after 48 h of the test (49.95 \u0026plusmn; 9.48% of the drug was released).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Raman spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaman analysis of samples placed on coverslips was performed. High magnification was chosen for confocal measurements of polymers in all mentioned variants. Raman spectra of native forms were recorded and presented in Figure 3A, and the assignments of bands are shown in Table 1. The characteristic chitosan bands were identified according to the literature (Zając et al. 2015; Biniaś et al. 2016; Gieroba et al. 2020). Recorded spectra of 2CS_L and 4CS_L have a similar course with the most distinct changes comparing low to medium MW. The most affected ranges are 2880-3000 and 300-500 cm\u003csup\u003e-1\u003c/sup\u003e, with an upshift from 900 to 929 cm\u003csup\u003e-1\u003c/sup\u003e and the disappearance of bands at 3310 and 360 cm\u003csup\u003e-1\u003c/sup\u003e. It suggested the modifications of stretching of CH and CH\u003csub\u003e2\u003c/sub\u003e groups as CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e stretching was more pronounced in 2CS_M, and C-C-C by forming more interlacing bonds between secondary carbon atoms of chitosan during chemically induced gel forming. Additionally, 4CS_L, compared to the 2 CS_L spectrum, does not present bands at 363 and 442 cm\u003csup\u003e-1\u003c/sup\u003e assigned to ring out-of-lane vibrations, indicating the higher density of the resulting polymer by C-C-C and C-O-C linking.\u003c/p\u003e\n\u003cp\u003eTable 1. Assignments to Raman shifts recorded in studied native samples (Movasaghi et al. 2007; Zając et al. 2015; Gieroba et al. 2020; Camerlingo et al. 2022)\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRaman shift (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssignments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4CS_L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_M\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e3312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e3310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nu;\u003csub\u003es\u003c/sub\u003e NH\u003csub\u003e2\u003c/sub\u003e and N-H,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nu; O-H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nu; CH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e2890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nu; CH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nu; CH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026delta;\u003csub\u003eas\u003c/sub\u003e CH,\u003c/p\u003e\n \u003cp\u003e\u0026tau; CH\u003csub\u003e2\u003c/sub\u003e (in-plane)\u003c/p\u003e\n \u003cp\u003eCH and OH scissoring,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e wagging\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1417\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1417\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e and CH scissoring\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1377\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026delta; CH\u003c/p\u003e\n \u003cp\u003e\u0026delta; COH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026delta;-CH (in-plane), CH\u003csub\u003e2\u003c/sub\u003eOH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003eC-O-C (ether)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e1092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003eC-C stretch of glucosamine rings,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nu;\u003csub\u003es\u003c/sub\u003e (C\u0026ndash;O\u0026ndash;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026delta; -CH (out-of-plane)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026beta;-glycosidic bonds\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026tau; HCC (out-of-plane)\u003c/p\u003e\n \u003cp\u003eCOC scissoring\u003c/p\u003e\n \u003cp\u003eOH and ring out-of-plane vibration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e363\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026delta; C-C-C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u0026nu; \u0026ndash; stretching, \u0026delta; \u0026ndash; deformation, \u0026tau; \u0026ndash; bending, \u003csub\u003es\u003c/sub\u003e \u0026ndash; symmetric, \u003csub\u003eas\u003c/sub\u003e \u0026ndash; antisymmetric\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn the initial evaluation, more distinct changes were observed between low and medium MW chitosan in the mentioned native samples before enrichment with IBU. To acquire background information on PBS\u0026apos;s potential influence in drug release experiments, native samples were exposed to the continuous fluid flow for an equal amount of time in the same conditions as IBU-enriched samples. The data from those samples is shown in Figure 3C. Indicated bands in the native sample were also observed in PBS-treated materials without significant shifts or differences in spectra.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, thin films enriched with IBU matrices before and after drug release performed in PBS were analyzed, presented in Figures 3B, and 3D, respectively. Additionally, the pure ibuprofen spectrum was recorded and presented in Figure 5 that presents the Raman spectrum of ibuprofen (IBU) added to polymer matrices with described characteristic bands.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe IBU spectrum (Figure 5) exhibits characteristic bands at 2992, 1703, and 1229 cm\u003csup\u003e-1\u003c/sup\u003e, attributed to the OH stretching mode, the asymmetrical wagging vibrations from the carbonyl group (C-O), and C=O stretching, respectively (Guan et al. 2024). Other typical bands for IBU are the asymmetric alkyl stretching bands at 2956 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e,\u0026nbsp;carbonyl stretching bands at 1722 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, CH\u0026ndash;CO strain at 1420 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and deformation of CH\u003csub\u003e2\u003c/sub\u003e at 780 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (Pereira et al. 2020). IBU addition resulted in new bands appearing at 1416 cm\u003csup\u003e-1\u003c/sup\u003e in 2CS_L_IBU and 4CS_L_IBU or 1424 cm\u003csup\u003e-1\u003c/sup\u003e in 2CS_M_IBU, assigned to CH, CH\u003csub\u003e3\u003c/sub\u003e bending of IBU and deformation of hydroxyl and amino groups, by the formation of non-covalent bonds (Zhang et al. 2011; Azueta-Aguayo et al. 2022). The characteristic band for crystalline ibuprofen at 1608 was not observed in spectra, nor was the band at 1342 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e(Kazarian and Martirosyan 2002; Krukiewicz et al. 2020). In the range 1000-1500 cm\u003csup\u003e-1\u003c/sup\u003e, the spectra are similar, but interestingly 2CS_M presents the broadening of the band to lower Raman shifts with the highest Raman intensity at 1093 cm\u003csup\u003e-1\u003c/sup\u003e. It can be assigned to CO stretching and COH bending, providing information that IBU was incorporated in all polymer matrices and that the bond is strongest in 2CS_M. An additional band at 3373 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003esuggests that OH, NH, and NH\u003csub\u003e2\u003c/sub\u003e groups were affected by intramolecular and intermolecular hydrogen bonding in medium molecular weight samples. \u0026beta;-glycosidic bonds were not affected by the incorporation of IBU in all samples, thus it does not change the chitosan backbone, rather interacting with free hydroxyl and methyl groups (Jia et al. 2020). Interestingly, the measurements of films after release in PBS (Figure 3D) do not change Raman spectra compared to those presented in Figure 3C, proving that changes in the structure of chitosan gel are not reversible in a water environment and after IBU release.\u003c/p\u003e\n\u003cp\u003eFor a better understanding of changes and their influence on individual CS matrices, the spectra grouped to 2CS_L, 4CS_L, and 2CS_M were presented concerning IBU enrichment in Figure 4. In the 2CS_L group (Figure 4A), the addition of IBU leads to a slight shift of 1267 to 1266, near 3300 cm\u003csup\u003e-1\u003c/sup\u003e and the disappearance of bands in region 300-500 cm\u003csup\u003e-1\u003c/sup\u003e. The highest level of similarity appears to be in 2CS_L_R and 2CS_L/IBU_R, indicating a marginal influence on PBS flow during release.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the 4CS_L group (Figure 4B), a full spectra comparison of native and experimental samples was similar to the 2CS_L, with a decrease in intensity for CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e. The addition of ibuprofen resulted in shifts from 3310 to 3308 and from 1092 to 1116 cm\u003csup\u003e-1\u003c/sup\u003e the disappearance of the 1463 cm\u003csup\u003e-1\u003c/sup\u003e band, and a significant lowering of a band at 3310 cm\u003csup\u003e-1\u003c/sup\u003e assigned to hydroxyl group stretching. At low Raman shifts similar modifications of C-C ring vibrations were observed in the drug consisting of matrices.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe most interesting changes were observed in the 2CS_M group (Figure 4C), where with higher MW, changes in matrices were more significant. The native 2CS_M polymer shares bands at 900, 1120, and 1378-1380 cm\u003csup\u003e-1\u003c/sup\u003e across all spectra; however, significant differences were also observed. 2CS_M displays the highest intensity at 2933 cm\u003csup\u003e-1\u003c/sup\u003e with an altered spectrum in the range of 2800-3000 cm\u003csup\u003e-1\u003c/sup\u003e, but does not display bands over 3300 cm\u003csup\u003e-1\u003c/sup\u003e, indicating modification in CH and CH\u003csub\u003e2\u003c/sub\u003e groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. DRIFT spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DRIFT spectra of polymer samples are represented in Fig. 6. For all studied samples, three main regions can be identified in the frequency ranges between (1) 3600-3000 cm\u003csup\u003e-1\u003c/sup\u003e, characteristic for OH stretching modes of the different types of hydrate water molecules; (2) 3100-2800 cm\u003csup\u003e-1\u003c/sup\u003e, characteristic for \u0026nu;\u003csub\u003eC-H\u003c/sub\u003e and \u0026nu;\u003csub\u003eN-H\u003c/sub\u003e bands, CH and NH stretching vibrational modes, with corresponding (3) 1700\u0026ndash;1200 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (NH and CH deformation modes, CO and CC stretching modes) with characteristic modes for carbohydrate ring (920-750 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e). The most detailed analysis of DRIFT spectra of samples (Figure 5) can be assigned as presented in Tables 2 and 3.\u003c/p\u003e\n\u003cp\u003eTable 2. Assignments of DRIFT spectra of the native CS \u0026nbsp;and CS+IBU samples Literature (Meyer et al. 1993; Chopin and Whalen 1993; Bouchard and Douek 1993; Armaroli et al. 2004; Kalisz et al. 2021; Katan et al. 2022; Xu et al. 2023)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunctional groups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWavenumber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_M\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4CS_L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eO-H stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3600-3300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3512\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-H stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3100-2800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2990\u003c/p\u003e\n \u003cp\u003e2942\u003c/p\u003e\n \u003cp\u003e2908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2990\u003c/p\u003e\n \u003cp\u003e2939\u003c/p\u003e\n \u003cp\u003e2897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2990\u003c/p\u003e\n \u003cp\u003e2939\u003c/p\u003e\n \u003cp\u003e2909\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eO\u0026ndash;H stretching of water molecules\u003c/p\u003e\n \u003cp\u003eAbsorbed water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1650-1633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1678\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC=O stretching\u003c/p\u003e\n \u003cp\u003eN-H bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1600-1588\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1598\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1464-1405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1433\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1395-1355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1394w\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-N stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1317-1320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1326w\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-OH stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1264-1260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1264\u003c/p\u003e\n \u003cp\u003e1231vw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1267\u003c/p\u003e\n \u003cp\u003e1231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1273w\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-O-C stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1165-1162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1167\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-O stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1200 \u0026ndash; 800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1126\u003c/p\u003e\n \u003cp\u003e1047\u003c/p\u003e\n \u003cp\u003e997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1127\u003c/p\u003e\n \u003cp\u003e1047\u003c/p\u003e\n \u003cp\u003e998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1126\u003c/p\u003e\n \u003cp\u003e1047s\u003c/p\u003e\n \u003cp\u003e998\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eC-O-C ring vibration of carbohydrate (\u0026beta;-glycosidic bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e920 \u0026ndash;758\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e906\u003c/p\u003e\n \u003cp\u003e852\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOut-of-plane O-H bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e681\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 3. CS+IBU samples assignments (Meyer et al. 1993; Chopin and Whalen 1993; Bouchard and Douek 1993; Armaroli et al. 2004; Kalisz et al. 2021; Katan et al. 2022; Xu et al. 2023)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"605\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunctional groups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWavenumber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_M/IBU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2CS_L/IBU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4CS_L/IBU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eO-H stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3600 \u0026ndash;3300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e3467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-H stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3100-2800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2936\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eO\u0026ndash;H stretching of water molecules\u003c/p\u003e\n \u003cp\u003eAbsorbed water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1650-1633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1681\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC=O stretching\u003c/p\u003e\n \u003cp\u003eN-H bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1600-1588\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1609\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eCH2 bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1464-1405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1395-1355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-N stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1317-1320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1327w\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-OH stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1264-1260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1230w\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1273w\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-O-C stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1165-1162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1166\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-O stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1200 \u0026ndash; 800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1079\u003c/p\u003e\n \u003cp\u003e975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1127\u003c/p\u003e\n \u003cp\u003e1047\u003c/p\u003e\n \u003cp\u003e1003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e1127\u003c/p\u003e\n \u003cp\u003e1046\u003c/p\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eC-O-C ring vibration of carbohydrate (\u0026beta;-glycosidic bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e920 \u0026ndash;758\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e906\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eOut-of-plane O-H bending\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e670\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e736\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e736\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSpectroscopy (ATR FT-IR, DRIFT, XPS, and Raman spectroscopies) includes several methods of identifying and classifying compounds based on the vibrations of their bonds that are used to determine and demonstrate the differences in the molecular structure of studied compounds (Pirutin et al. 2023). Based on the measured spectra and assigned proper functional groups and vibrations, it is possible to detect chemical interactions between chitosan and ibuprofen in the process of creating polymer matrices and then changes in matrices after drug release (van Haaren et al. 2023). Spectroscopic methods are versatile tools in pharmaceutics and biopharmaceutics, with a wide field of applications ranging from characterization of drug formulations in both qualitative and quantitative analyses, like:\u003c/p\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003estudy drug release from semisolid formulations in a non-invasive way;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003emeasuring drug diffusion from appropriate pharmaceutical systems such as polymers, gels, films, liposomes, etc.;\u003c/li\u003e\n \u003cli\u003einvestigate drug penetration into relevant acceptor systems as well as reveal the mechanism of drug release;\u003c/li\u003e\n \u003cli\u003echaracterize interactions between drugs and (semi)synthetic, and native macromolecules (Dole et al. 2011; Song et al. 2020).\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eFourier Transform Infrared Spectroscopy (FT-IR, a complementary method to Raman spectroscopy) is one of the techniques allowing for the characterization of chitosan-based on different bands corresponding to the \u0026minus;NH\u003csub\u003e2\u003c/sub\u003e group, which can be allocated to the symmetrical COO\u0026minus; gather extending vibration. At the same time, Raman analysis, depending on the inelastic diffusing of photons, is utilized to obtain a material\u0026apos;s structural fingerprint. The research on the chemical structure of chitosan exposes a six-membered ring containing oxygen. The prominent bands are present at 471 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (bending vibration of C-C-O bonds) and 895 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, 1146 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (stretching vibration of C-C-O bonds) (Sudatta et al. 2020). Moreover, the presence of free -NH\u003csub\u003e2\u003c/sub\u003e and -OH groups (3600-3000 cm\u003csup\u003e-1\u003c/sup\u003e) in CS\u0026apos;s backbone enables chemical modifications by joining different molecules with biological and pharmacological activity, enhancing its properties for more biomedical applications (Bhattacharjee et al. 2023).\u003c/p\u003e\n\u003cp\u003eOur study found that the chitosan\u0026ndash;ibuprofen matrices showed a grain morphological structure, and the introduction of ibuprofen changed the molecular arrangement of chitosan, attributing probably to the electrostatic and hydrophobic interactions between the drug and the polymer. It is considered the most likely IBU interacted with CS with its aromatic ring and hydrophilic carboxylic groups, changing surface hydrophobicity. At the same time, cationic CS contains a non-polar region and ammonium groups providing hydrogen bonding capacity and possessing high affinity for oppositely charged molecules of IBU (Mahmood et al. 2021). Consequently, the surface becomes much less hydrophilic. IBU is a molecule of inherently low polarity, which results in a lack of anchoring sites required for further organization; therefore, chitosan matrices may facilitate the close packing and the structural ordering of IBU (Mani et al. 2023). The kinetics of drug release profiles confirmed that in low MW CS samples, IBU is more tightly packed than in medium MW CS, which manifests in the burst release of the drug. Spectroscopically, less C-O groups were detected in the 2CS_M/IBU sample, which may mean better binding to the matrix and, consequently smaller and slower release. For all CS/IBU conjugates, the spectral data confirmed the formation of amide I and II bonds between the carboxyl group of IBU and the amino group of the CS chain through amide bonds, improving water solubility, and proving the occurrence of grafting reaction (Guan et al. 2024). The IBU/CS aerogels were also prepared for transdermal administration by Chen Li et al. This bioformulation exhibited nanopore morphology structure, and the addition of IBU changed the crystallinity of CS connected with the electrostatic and hydrophobic interplay between the drug and the polymer. Moreover, the IBU/CS aerogels possessed an excellent swelling ratio and higher thermal stability, ensuring controlled drug release (Li et al. 2021).\u003c/p\u003e\n\u003cp\u003eAs chitosan is widely used in the pharmaceutical industry, its use with NSAIDs, particularly ibuprofen, for potential treatment was studied from a clinical perspective and assumed as promising (Pooresmaeil and Namazi 2021). As an over-the-counter medication, it is commonly used for pain relief by inhibiting cyclooxygenase (COX), which is involved in the production of prostaglandins. The reduction of its activity leads to a decrease in this production, thus decreasing inflammation (Rainsford 2009). It has a good safety profile, but the main adverse drug reactions are related to gastrointestinal, hepato-renal, and cardiovascular risks related to the most common oral use of the compound (Rainsford 2009; Lanas et al. 2010; Collaboration Coxib and traditional NSAID Trialists\u0026rsquo; (CNT) 2013). To avoid adverse side effects, studies have looked at both modifications of drug action and alternative methods of drug delivery. Topical applications (gels, creams, sprays, etc.) were investigated concerning musculoskeletal pain. However, absorption was decreased compared to oral IBU, which may differ 300 times in plasma concentration (Tegeder et al. 1999). Moreover, topical formulations have a drawback of costs, as topical NSAIDs are more expensive in therapy. However, a chronic application was not studied in this perspective (Manoukian et al. 2017). To address this issue, the modifications of drug carriers might be beneficial for patients. Already studied formulations tend to use biocompatible, degradable polymers as vehicles. The examples can be porous materials (e.g., vaterite investigated with cyclodextrins and doxorubicin). However, as concluded by Campbell et al., it can be influenced by matrix content incubation temperature, ionic strength, pH, drug concentration, etc. which results in different performance of the resulting capsules (Campbell et al. 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiego-Taboada et al. have studied ibuprofen release from sporopollenin exine capsules (SEC) extracted from \u003cem\u003eLycopodium clavatum\u003c/em\u003e spores in simulated gastric (SGF) and gastrointestinal fluid (GIT). This experiment indicated that an encapsulated material can be released rapidly in high yield from SEC triggered by pH. A potential application for targeted release into the GIT was demonstrated by 88 \u0026plusmn; 1% of the drug retained in SGF after 45 minutes and 85 \u0026plusmn; 2% released after 5 min in buffer (PBS; pH 7.4) (Diego-Taboada et al. 2013). Previous research has shown that the drug release from ibuprofen-loaded biomaterials as tablets is pH sensitive. The chitosan carrier may allow more rapid IBU release in stomach fluids at pH 1.2 due to the easier favorable protonation of the amino groups in this acidic condition, opposite to the behavior that occurs at pH 7.4, that of for example PBS, where an increase of the rate of CS swelling occurs. In an acidic environment of the stomach, the chitosan-based drug carrier will dissolve very fast, releasing whole amount of the immobilized IBU and providing immediate therapeutic effect (Vieira et al. 2013). Based on this research, we can expect faster and greater IBU release at low pH mimicking gastric fluid conditions than at pH 7.4 as it was conducted in this study. The materials developed by dos S. Pereira et al. consisting of IBU, CS, and silver nanoparticles (AgNPs) presented a lower swelling rate at neutral pH because, in the GIT, the amine groups are deprotonated (dehydronated), and the material is characterized by a negatively-charged surface that electrostatically repels the molecules of IBU, leading to its greater release. Additionally, the hybrid compound exhibited greater drug release capacity and bactericidal activity (Pereira et al. 2020).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWithin this study, it was proven that chitosan matrices may be a promising base for the production of oral ibuprofen-loaded capsules. The effective incorporation of ibuprofen into chitosan matrices was evidenced by Raman spectroscopy by the disappearance of the bands at 1608 and 1342 cm\u003csup\u003e-1\u003c/sup\u003e characteristic for crystalline IBU, and the appearance of new bands at 1416 and 1424 cm\u003csup\u003e-1\u003c/sup\u003e assigned to CH, CH\u003csub\u003e3\u003c/sub\u003e bending of IBU and deformation of hydroxyl and amino groups, by the formation of non-covalent bonds between CS and IBU. Among all tested samples, 2CS_L/IBU appeared to be the most promising drug carrier since it revealed the burst release of the ibuprofen within just half an hour. Importantly, it may be assumed that in the acidic environment of the gastric juice, the drug release will be significantly faster, providing excellent therapeutic effect after oral administration. This research demonstrated that spectroscopic methods can be a powerful tools in the investigation of polymer/drug interactions and their relevance to controlled drug release processes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by the Ministry of Science and Higher Education in Poland through the statutory activity of the Medical University of Lublin (DS 630 project). Additionally, it received partial support from the Ministry of Science and Higher Education in Poland under the PBmb 170 project. We would also like to acknowledge the financial support provided by the Foundation for Polish Science through the Reintegration grant (grant number POIR.04.04.00-00-4398/17-00, REINTEGRATION/2017-4/14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: ASB, BG and AP, Funding acquisition: ASB and AP, Investigation: BG, GK, PK, VV, MK, ISP, OM, LN, RN, Methodology: BG, PK, VV, GK, OM, MK, Project administration: AP, ASB, Resources: ASB, AP, ISP, Supervision: ASB, Visualization ISP, VV, GK, Writing \u0026ndash; original draft: BG, GK, OM, PK, VV Writing \u0026ndash; review and editing: AP, ISP, ASB\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request from the authors. A preprint has previously been published Barbara Gieroba et al. 2024 (https://doi.org/10.2139/ssrn.4995823).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAranaz I, Alc\u0026aacute;ntara AR, Civera MC, et al (2021) Chitosan: An Overview of Its Properties and Applications. Polymers 13:3256. https://doi.org/10.3390/polym13193256\u003c/li\u003e\n\u003cli\u003eArdila N, Daigle F, Heuzey M-CC, Ajji A (2017) Antibacterial Activity of Neat Chitosan Powder and Flakes. Molecules 22:100. https://doi.org/10.3390/molecules22010100\u003c/li\u003e\n\u003cli\u003eArmaroli T, B\u0026eacute;cue T, Gautier S (2004) Diffuse Reflection Infrared Spectroscopy (DRIFTS): Application to the in situ Analysis of Catalysts. Oil \u0026amp; Gas Science and Technology-Rev IFP 59:215\u0026ndash;237\u003c/li\u003e\n\u003cli\u003eAzueta-Aguayo PH, Chuc-Gamboa MG, Aguilar-P\u0026eacute;rez FJ, et al (2022) Effects of Neutralization on the Physicochemical, Mechanical, and Biological Properties of Ammonium-Hydroxide-Crosslinked Chitosan Scaffolds. International Journal of Molecular Sciences 23:14822. https://doi.org/10.3390/ijms232314822\u003c/li\u003e\n\u003cli\u003eBarbosa FC, Silva MC da, Silva HN da, et al (2020) Progress in the Development of Chitosan Based Insulin Delivery Systems: A Systematic Literature Review. Polymers 12:2499. https://doi.org/10.3390/polym12112499\u003c/li\u003e\n\u003cli\u003eBhattacharjee S, Goswami S, Das S, et al (2023) pH-responsive, stable, and biocompatible functional nanogels based on chitosan (CS)/poly methacrylic acid (PMAA) polymers: Synthesis and characterization. Materials Today Communications 36:106541. https://doi.org/10.1016/j.mtcomm.2023.106541\u003c/li\u003e\n\u003cli\u003eBiniaś D, Biniaś W, Janicki J (2016) Application of Raman spectroscopy for evaluation of chemical changes in dibutyrylchitin fibres. Fibres and Textiles in Eastern Europe 24:27\u0026ndash;38. https://doi.org/10.5604/12303666.1207842\u003c/li\u003e\n\u003cli\u003eBouchard J, Douek M (1993) Structural and Concentration Effects on the Diffuse Reflectance Ftir Spectra of Cellulose, Lignin and Pulp. Journal of Wood Chemistry and Technology 13:481\u0026ndash;499. https://doi.org/10.1080/02773819308020530\u003c/li\u003e\n\u003cli\u003eBushra R, Aslam N (2010) An overview of clinical pharmacology of Ibuprofen. Oman medical journal 25:155\u0026ndash;161. https://doi.org/10.5001/OMJ.2010.49\u003c/li\u003e\n\u003cli\u003eCamerlingo C, Portaccio M, D\u0026rsquo;Apuzzo F, et al (2022) \u0026mu;-FTIR, \u0026mu;-Raman, and SERS Analysis of Amide I Spectral Region in Oral Biofluid Samples during Orthodontic Treatment. Sensors 22:7874. https://doi.org/10.3390/s22207874\u003c/li\u003e\n\u003cli\u003eCampbell J, Ferreira AM, Bowker L, et al (2022) Dextran and Its Derivatives: Biopolymer Additives for the Modulation of Vaterite CaCO 3 Crystal Morphology and Adhesion to Cells. Advanced Materials Interfaces 9:. https://doi.org/10.1002/admi.202201196\u003c/li\u003e\n\u003cli\u003eChopin T, Whalen E (1993) A new and rapid method for carrageenan identification by FT IR diffuse reflectance spectroscopy directly on dried, ground algal material. Carbohydrate Research 246:51\u0026ndash;59. https://doi.org/10.1016/0008-6215(93)84023-Y\u003c/li\u003e\n\u003cli\u003eCollaboration Coxib and traditional NSAID Trialists\u0026rsquo; (CNT) (2013) Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. The Lancet 382:769\u0026ndash;779. https://doi.org/10.1016/S0140-6736(13)60900-9\u003c/li\u003e\n\u003cli\u003eDiego-Taboada A, Maillet L, Banoub JH, et al (2013) Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking. J Mater Chem B 1:707\u0026ndash;713. https://doi.org/10.1039/C2TB00228K\u003c/li\u003e\n\u003cli\u003eDimida S, Barca A, Cancelli N, et al (2017) Effects of Genipin Concentration on Cross-Linked Chitosan Scaffolds for Bone Tissue Engineering: Structural Characterization and Evidence of Biocompatibility Features. International Journal of Polymer Science 2017:8410750. https://doi.org/10.1155/2017/8410750\u003c/li\u003e\n\u003cli\u003eDole MN, Patel PA, Sawant S, Shedpure PS (2011) ADVANCE APPLICATIONS OF FOURIER TRANSFORM INFRARED SPECTROSCOPY Review Article\u003c/li\u003e\n\u003cli\u003eGieroba B, Sroka-Bartnicka A, Kazimierczak P, et al (2020) Spectroscopic studies on the temperature-dependent molecular arrangements in hybrid chitosan/1,3-\u0026beta;-D-glucan polymeric matrices. International Journal of Biological Macromolecules 159:911\u0026ndash;921. https://doi.org/10.1016/j.ijbiomac.2020.05.155\u003c/li\u003e\n\u003cli\u003eGon\u0026ccedil;alves C, Ferreira N, Louren\u0026ccedil;o L (2021) Production of Low Molecular Weight Chitosan and Chitooligosaccharides (COS): A Review. Polymers 2021, Vol 13, Page 2466 13:2466. https://doi.org/10.3390/POLYM13152466\u003c/li\u003e\n\u003cli\u003eGuan X, Yao H, Wu J (2024) Photocrosslinkable hydrogel of ibuprofen‐chitosan methacrylate modulates inflammatory response. Journal of Biomedical Materials Research Part A 112:2001\u0026ndash;2017. https://doi.org/10.1002/jbm.a.37758\u003c/li\u003e\n\u003cli\u003eHarish Prashanth KV, Kittur FS, Tharanathan RN (2002) Solid state structure of chitosan prepared under different N-deacetylating conditions. Carbohydrate Polymers 50:27\u0026ndash;33. https://doi.org/10.1016/S0144-8617(01)00371-X\u003c/li\u003e\n\u003cli\u003eHoekstra A, Struszczyk H, Kivek\u0026auml;s O (1998) Percutaneous microcrystalline chitosan application for sealing arterial puncture sites. Biomaterials 19:1467\u0026ndash;1471. https://doi.org/10.1016/S0142-9612(98)00060-X\u003c/li\u003e\n\u003cli\u003eJia S, Pan H, Lin Q, et al (2020) Study on the preparation and mechanism of chitosan-based nano-mesoporous carbons by hydrothermal method. Nanotechnology 31:365604. https://doi.org/10.1088/1361-6528/ab9575\u003c/li\u003e\n\u003cli\u003eKalisz G, Przekora A, Kazimierczak P, et al (2021) Physicochemical changes of the chitosan/\u0026beta;-1,3-glucan/hydroxyapatite biocomposite caused by mesenchymal stem cells cultured on its surface in vitro. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 251:119439. https://doi.org/10.1016/j.saa.2021.119439\u003c/li\u003e\n\u003cli\u003eKasaai MR (2009) Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review. Journal of agricultural and food chemistry 57:1667\u0026ndash;1676. https://doi.org/10.1021/JF803001M\u003c/li\u003e\n\u003cli\u003eKatan T, Kargl R, Mohan T, et al (2022) Solid Phase Peptide Synthesis on Chitosan Thin Films. Biomacromolecules 23:731\u0026ndash;742. https://doi.org/10.1021/ACS.BIOMAC.1C01155/ASSET/IMAGES/LARGE/BM1C01155_0008.JPEG\u003c/li\u003e\n\u003cli\u003eKazarian SG, Martirosyan GG (2002) Spectroscopy of polymer/drug formulations processed with supercritical fluids: in situ ATR\u0026ndash;IR and Raman study of impregnation of ibuprofen into PVP. International Journal of Pharmaceutics 232:81\u0026ndash;90. https://doi.org/10.1016/S0378-5173(01)00905-X\u003c/li\u003e\n\u003cli\u003eKhademi F, Derakhshan M, Yousefi-Avarvand A, Tafaghodi M (2018) Potential of polymeric particles as future vaccine delivery systems/adjuvants for parenteral and non-parenteral immunization against tuberculosis: A systematic review. Iranian journal of basic medical sciences 21:116\u0026ndash;123. https://doi.org/10.22038/IJBMS.2017.22059.5648\u003c/li\u003e\n\u003cli\u003eKrukiewicz K, Kowalik A, Turczyn R, Biggs MJP (2020) In vitro attenuation of astrocyte activation and neuroinflammation through ibuprofen-doping of poly(3,4-ethylenedioxypyrrole) formulations. Bioelectrochemistry 134:107528. https://doi.org/10.1016/j.bioelechem.2020.107528\u003c/li\u003e\n\u003cli\u003eLanas A, Tornero J, Zamorano JL (2010) Assessment of gastrointestinal and cardiovascular risk in patients with osteoarthritis who require NSAIDs: the LOGICA study. Annals of the Rheumatic Diseases 69:1453\u0026ndash;1458. https://doi.org/10.1136/ard.2009.123166\u003c/li\u003e\n\u003cli\u003eLavertu M, Darras V, Buschmann MD (2012) Kinetics and efficiency of chitosan reacetylation. Carbohydrate Polymers 87:1192\u0026ndash;1198. https://doi.org/10.1016/j.carbpol.2011.08.096\u003c/li\u003e\n\u003cli\u003eLi C, Wang K, Xie D (2021) Green Fabrication and Release Mechanisms of pH-Sensitive Chitosan\u0026ndash;Ibuprofen Aerogels for Controlled Transdermal Delivery of Ibuprofen. Frontiers in Chemistry 9:. https://doi.org/10.3389/fchem.2021.767923\u003c/li\u003e\n\u003cli\u003eLima BV, Oliveira MJ, Barbosa MA, et al (2021) Immunomodulatory potential of chitosan-based materials for cancer therapy: a systematic review of in vitro , in vivo and clinical studies. Biomaterials Science 9:3209\u0026ndash;3227. https://doi.org/10.1039/D0BM01984D\u003c/li\u003e\n\u003cli\u003eLizardi-Mendoza J, Arg\u0026uuml;elles Monal WM, Goycoolea Valencia FM (2016) Chemical Characteristics and Functional Properties of Chitosan. Chitosan in the Preservation of Agricultural Commodities 3\u0026ndash;31. https://doi.org/10.1016/B978-0-12-802735-6.00001-X\u003c/li\u003e\n\u003cli\u003eMahmood S, Almurisi SH, Al-Japairai K, et al (2021) Ibuprofen-Loaded Chitosan\u0026ndash;Lipid Nanoconjugate Hydrogel with Gum Arabic: Green Synthesis, Characterisation, In Vitro Kinetics Mechanistic Release Study and PGE2 Production Test. Gels 7:254. https://doi.org/10.3390/gels7040254\u003c/li\u003e\n\u003cli\u003eMani R, Peltonen L, Strachan CJ, et al (2023) Nonclassical Crystallization and Core\u0026ndash;Shell Structure Formation of Ibuprofen from Binary Solvent Solutions. Crystal Growth \u0026amp; Design 23:236\u0026ndash;245. https://doi.org/10.1021/acs.cgd.2c00971\u003c/li\u003e\n\u003cli\u003eManoukian MAC, Migdal CW, Tembhekar AR, et al (2017) Topical Administration of Ibuprofen for Injured Athletes: Considerations, Formulations, and Comparison to Oral Delivery. Sports Medicine - Open 3:36. https://doi.org/10.1186/s40798-017-0103-2\u003c/li\u003e\n\u003cli\u003eMathaba M, Daramola MO (2020) Effect of Chitosan\u0026rsquo;s Degree of Deacetylation on the Performance of PES Membrane Infused with Chitosan during AMD Treatment. Membranes 2020, Vol 10, Page 52 10:52. https://doi.org/10.3390/MEMBRANES10030052\u003c/li\u003e\n\u003cli\u003eMeyer U, Gilbert C, Kokot S (1993) Application of DRIFT Spectroscopy and Chemometrics for the Comparison of Cotton Fabrics. Applied Spectroscopy, Vol 47, Issue 6, pp 741-748 47:741\u0026ndash;748\u003c/li\u003e\n\u003cli\u003eMorris GA, Castile J, Smith A, et al (2009) Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach. Carbohydrate Polymers 76:616\u0026ndash;621. https://doi.org/10.1016/J.CARBPOL.2008.11.025\u003c/li\u003e\n\u003cli\u003eMotov S, Masoudi A, Drapkin J, et al (2019) Comparison of Oral Ibuprofen at Three Single-Dose Regimens for Treating Acute Pain in the Emergency Department: A Randomized Controlled Trial. Annals of emergency medicine 74:530\u0026ndash;537. https://doi.org/10.1016/J.ANNEMERGMED.2019.05.037\u003c/li\u003e\n\u003cli\u003eMovasaghi Z, Rehman S, Rehman IU (2007) Raman Spectroscopy of Biological Tissues. Applied Spectroscopy Reviews 42:493\u0026ndash;541. https://doi.org/10.1080/05704920701551530\u003c/li\u003e\n\u003cli\u003eMuxika A, Etxabide A, Uranga J, et al (2017) Chitosan as a bioactive polymer: Processing, properties and applications. International journal of biological macromolecules 105:1358\u0026ndash;1368. https://doi.org/10.1016/J.IJBIOMAC.2017.07.087\u003c/li\u003e\n\u003cli\u003eOgawa K, Yui T, Okuyama K (2004) Three D structures of chitosan. International Journal of Biological Macromolecules 34:1\u0026ndash;8. https://doi.org/10.1016/j.ijbiomac.2003.11.002\u003c/li\u003e\n\u003cli\u003ePellis A, Guebitz GM, Nyanhongo GS (2022) Chitosan: Sources, Processing and Modification Techniques. Gels 2022, Vol 8, Page 393 8:393. https://doi.org/10.3390/GELS8070393\u003c/li\u003e\n\u003cli\u003ePereira AK dos S, Reis DT, Barbosa KM, et al (2020) Antibacterial effects and ibuprofen release potential using chitosan microspheres loaded with silver nanoparticles. Carbohydrate Research 488:107891. https://doi.org/10.1016/j.carres.2019.107891\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-\u0026Aacute;lvarez L, Ruiz-Rubio L, Vilas-Vilela JL (2018) Determining the Deacetylation Degree of Chitosan: Opportunities to Learn Instrumental Techniques. Journal of Chemical Education 95:1022\u0026ndash;1028. https://doi.org/10.1021/ACS.JCHEMED.7B00902\u003c/li\u003e\n\u003cli\u003ePirutin SK, Jia S, Yusipovich AI, et al (2023) Vibrational Spectroscopy as a Tool for Bioanalytical and Biomonitoring Studies. International Journal of Molecular Sciences 24:6947. https://doi.org/10.3390/ijms24086947\u003c/li\u003e\n\u003cli\u003ePooresmaeil M, Namazi H (2021) Developments on carboxymethyl starch-based smart systems as promising drug carriers: A review. Carbohydrate Polymers 258:117654. https://doi.org/10.1016/j.carbpol.2021.117654\u003c/li\u003e\n\u003cli\u003eRainsford KD (2009) Ibuprofen: pharmacology, efficacy and safety. Inflammopharmacology 17:275\u0026ndash;342. https://doi.org/10.1007/s10787-009-0016-x\u003c/li\u003e\n\u003cli\u003eSaito Y, Okano T, Gaill F, et al (2000) Structural data on the intra-crystalline swelling of beta-chitin. International journal of biological macromolecules 28:81\u0026ndash;88. https://doi.org/10.1016/S0141-8130(00)00147-1\u003c/li\u003e\n\u003cli\u003eShi C, Zhu Y, Ran X, et al (2006) Therapeutic potential of chitosan and its derivatives in regenerative medicine. The Journal of surgical research 133:185\u0026ndash;192. https://doi.org/10.1016/J.JSS.2005.12.013\u003c/li\u003e\n\u003cli\u003eSivashankari PR, Prabaharan M (2016) Deacetylation modification techniques of chitin and chitosan. Chitosan Based Biomaterials: Fundamentals: Volume 1 1:117\u0026ndash;133. https://doi.org/10.1016/B978-0-08-100230-8.00005-4\u003c/li\u003e\n\u003cli\u003eSong Y, Cong Y, Wang B, Zhang N (2020) Applications of Fourier transform infrared spectroscopy to pharmaceutical preparations. Expert Opinion on Drug Delivery 17:551\u0026ndash;571. https://doi.org/10.1080/17425247.2020.1737671\u003c/li\u003e\n\u003cli\u003eSudatta BP, Sugumar V, Varma R, Nigariga P (2020) Extraction, characterization and antimicrobial activity of chitosan from pen shell, Pinna bicolor. International Journal of Biological Macromolecules 163:423\u0026ndash;430. https://doi.org/10.1016/j.ijbiomac.2020.06.291\u003c/li\u003e\n\u003cli\u003eTegeder I, Muthselbach U, Lotsch J, et al (1999) Application of microdialysis for the determination of muscle and subcutaneous tissue concentrations after oral and topical ibuprofen administration. Clinical Pharmacology \u0026amp; Therapeutics 65:357\u0026ndash;368. https://doi.org/10.1016/S0009-9236(99)70128-1\u003c/li\u003e\n\u003cli\u003eThareja A, Hughes H, Alvarez-Lorenzo C, et al (2021) Penetration Enhancers for Topical Drug Delivery to the Ocular Posterior Segment\u0026mdash;A Systematic Review. Pharmaceutics 13:276. https://doi.org/10.3390/pharmaceutics13020276\u003c/li\u003e\n\u003cli\u003evan Haaren C, De Bock M, Kazarian SG (2023) Advances in ATR-FTIR Spectroscopic Imaging for the Analysis of Tablet Dissolution and Drug Release. Molecules 28:4705. https://doi.org/10.3390/molecules28124705\u003c/li\u003e\n\u003cli\u003eVerlee A, Mincke S, Stevens CV (2017) Recent developments in antibacterial and antifungal chitosan and its derivatives. Carbohydrate polymers 164:268\u0026ndash;283. https://doi.org/10.1016/J.CARBPOL.2017.02.001\u003c/li\u003e\n\u003cli\u003eVieira AP, Badshah S, Airoldi C (2013) Ibuprofen-loaded chitosan and chemically modified chitosans\u0026mdash;Release features from tablet and film forms. International Journal of Biological Macromolecules 52:107\u0026ndash;115. https://doi.org/10.1016/j.ijbiomac.2012.09.013\u003c/li\u003e\n\u003cli\u003eXu C, Xu Y, Zhong D, et al (2023) Zr4+ and glutaraldehyde cross-linked polyethyleneimine functionalized chitosan composite: Synthesis, characterization, Cr(VI) adsorption performance, mechanism and regeneration. International journal of biological macromolecules 239:. https://doi.org/10.1016/J.IJBIOMAC.2023.124266\u003c/li\u003e\n\u003cli\u003eYılmaz Atay H, \u0026Ccedil;elik E (2017) Investigations of antibacterial activity of chitosan in the polymeric composite coatings. Progress in Organic Coatings PB:194\u0026ndash;200. https://doi.org/10.1016/J.PORGCOAT.2016.10.013\u003c/li\u003e\n\u003cli\u003eYuan Y, Chesnutt BM, Haggard WO, Bumgardner JD (2011) Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures. Materials 2011, Vol 4, Pages 1399-1416 4:1399\u0026ndash;1416. https://doi.org/10.3390/MA4081399\u003c/li\u003e\n\u003cli\u003eZając A, Hanuza J, Wandas M, Dymińska L (2015) Determination of N-acetylation degree in chitosan using Raman spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134:114\u0026ndash;120. https://doi.org/10.1016/j.saa.2014.06.071\u003c/li\u003e\n\u003cli\u003eZhang K, Peschel D, Helm J, et al (2011) FT Raman investigation of novel chitosan sulfates exhibiting osteogenic capacity. Carbohydrate Polymers 83:60\u0026ndash;65. https://doi.org/10.1016/j.carbpol.2010.07.021\u003c/li\u003e\n\u003cli\u003eZhang M, Ma Y, Wang Z, et al (2017) Optimizing molecular weight of octyl chitosan as drug carrier for improving tumor therapeutic efficacy. Oncotarget 8:64237. https://doi.org/10.18632/ONCOTARGET.19452\u003c/li\u003e\n\u003cli\u003eZhao D, Yu S, Sun B, et al (2018) Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers 2018, Vol 10, Page 462 10:462. https://doi.org/10.3390/POLYM10040462\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chitosan, physicochemical properties, matrix drug release system, ibuprofen, natural polysaccharide polymers, molecular weight","lastPublishedDoi":"10.21203/rs.3.rs-7416784/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7416784/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Chitosan, which is a biocompatible and biodegradable natural polysaccharide, has been considered as a potential biopolymer-based carrier that can be applied in drug release systems, such as capsules for ibuprofen to overcome physiological barriers. In this study, we have examined three different variants of chitosan matrices for oral administration of ibuprofen (IBU) with various molecular weights (MW) or concentrations - 2% (w/v) and 4% (w/v) low MW, and 2% (w/v) medium MW. Molecular arrangement and physicochemical properties were determined before and after drug release from matrices with two powerful spectroscopic techniques - Raman spectroscopy and Diffuse Reflectance Infrared Fourier Transform DRIFT spectroscopy. It was proved that IBU was successfully incorporated into all studied chitosan matrices, however, the burst and highest drug release was demonstrated for the 2% low MW sample, where more amino and hydroxyl groups were observed interacting with the cationic IBU. It has been shown that chitosan can be an effective drug carrier, improving their pharmacokinetic and pharmacodynamic parameters.","manuscriptTitle":"Low and medium molecular weight chitosan matrices with ibuprofen as a potential base for soft capsules for oral use: evaluation of physicochemical properties and molecular structure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-18 03:22:44","doi":"10.21203/rs.3.rs-7416784/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"83964510402468672502274795040003278734","date":"2026-05-08T07:17:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T09:34:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140796478335321086047246991037579830784","date":"2026-04-10T17:38:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-10T07:17:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-24T14:50:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-22T12:46:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Polymer Bulletin","date":"2025-08-20T11:03:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d1152fd2-85e8-4b96-b9ad-e488e9140f0e","owner":[],"postedDate":"April 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"83964510402468672502274795040003278734","date":"2026-05-08T07:17:21+00:00","index":23,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T09:34:57+00:00","index":17,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-18T03:22:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-18 03:22:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7416784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7416784","identity":"rs-7416784","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}