Cotton

Compatibilizing of cotton fabric with hydrophobic drug cover layer for anti-inflammatory performance with the implementation of ibuprofen

All used reagents, unless otherwise noted, were purchased from Merck KGaA, (Darmstadt, Germany). Raw cotton fabric (1/1 plain weave, thickness 0.33 mm, surface mass 120 g/m2, yarn density 18 tex (which means 18 g/1,000 m), yarn diameter 0.17 mm) was kindly provided by PTB (Pabianice, Poland). l,l-LA was purchased from Purac (99%, The Netherlands), recrystallized from dry 2-propanol and stored under reduced pressure. Trifluoromethanesulfonic acid (98%), methyl-β-cyclodextrin (MβCD, 99%, methylation degree per glucose unit: 1.6–2.0) was used without further purification. Tin(II) octoate (2-ethylhexanoate, Sn(Oct)2, Aldrich, 95%) was purified by two high-vacuum distillations at 140 °C and 3 × 10−1 mbar and stored under vacuum. 1,2-dichloromethane (DCM) was distilled over P2O5 and stored under reduced pressure. Tetrahyfrofurane (THF, analytical grade) was distilled over sodium pellets and stored under vacuum. Methanol (analytical grade) was used as received.

The main goal of the experiment was to investigate the influence of base material (cotton fabric) preparation on drug performance and its efficiency. Therefore, a wide range of samples was made, with different variants of the preparation process, as shown in Fig. 1. Before the polymerization stage, a raw cotton fabric was subjected to few pretreatments which included washing, bleaching, and mercerizing. After that, l,l-lactide was polymerized in three different ways, and then the samples were impregnated with an active layer by the exhausting method, which was widely discussed by Bonet29. Details of every stage are described in the following paragraphs, including chemicals used and modification routine. In the most extensive version of the process, a cotton sample was washed, bleached, mercerized, modified by lactide and impregnated with a carrier containing ibuprofen (sample B). In the shortest version, one of the samples (sample L) was only washed and impregnated with IBU dispersion (Table 1).

Figure 1
figure 1

General scheme of different preparations paths for cotton functionalising. Orange presents the pretreatment stage, green polymerization of lactide, and blue—implementation of ibuprofen.

Table 1 Detailed schedule of modification steps for cotton samples.

Characteristics of the cover layers containing IBU

Star-shaped polymers are characterized by a single branch point from which linear chains emerge. The functionality of this point is referred to as the number of arms leaving it (linear chains). Star-shaped polymers can be prepared with a convergent (“arm-first”, “arm-in”), divergent (“core-first”, “arm-out”), or grafting-onto approach30. Various star-shaped PLA structures can be prepared as summarized by Michalski et al.31. For this research, star-shaped PLLA was synthesized via ring opening polymerization method, with methylated β-cyclodextrin as an initiator and stannous octoate (Sn(Oct)2) as a catalyst, as described in the literature32,33,34. Briefly, the bulk polymerization was carried out under reduced pressure in a sealed glass at 130 °C. The Sn(Oct)2 (10 mg, 0.025 mmol) was used as a catalyst and MnβCD (0.46 mmol, 0.602 g) was used as initiators l,l-lactide (35.7 mmol, 5.15 g) polymerization. After polymerization, the prepared polymers (star-PLA was dissolved in DCM and purified by slow precipitation to cold methanol and dried for 24 h under reduced pressure. star-PLA possesses 12 wt.% of MnβCD in the polymer structure.

PEG-PLA copolymers are already known as a matrix for the preparation of drug delivery systems35,36,37,38. Some in vivo examinations were also conducted39,40. Moreover, the important feature of PLA-PEG copolymers is the increased surface wettability of materials made of them41. For the purposes of ibuprofen implementation, the PLA-PEG copolymer was synthesized according to the procedures described by Michalski et al.42. l,l-lactide (5.70 g, 40 mmol) was polymerized in a glass ampule under reduced pressure in tetrahydrofuran (THF) at 80A °C with stannous octoate Sn(Oct)2 as a catalyst (1 mL of a 0.25 mol L−1 solution in dry THF) and at the presence of mPEG-OH (0.25 g, 1.25 mmol) as an initiator42. Subsequently, after 24 h, PLA-PEG copolymers was precipitated to cold methanol and dried under vacuum.

Cotton pretreatment

Washing

The cotton fabric was washed in anionic detergent Roksol M7 (PCC Exol SA, Brzeg Dolny, Poland) with a ratio of 10 g of detergent to 1 L of water for 1 h. The temperature of the bath was 60 °C. Then, the material was rinsed with cold water, squeezed lightly, and left to dry freely at room temperature.

Mercerizing

Mercerizing is a process of modifying cotton fibers structure with sodium hydroxide solution, which results in some physical changes such as increased strength, water affinity, as well as, chemical reactivity43. In this case, mercerization allowed for the multiplication of active centers on the surface of cotton fabric, so the following polymerization of lactide would have an easier access to initiating OH groups. For this purpose, a 17% sodium hydroxide solution was prepared with the addition of Roksol M7 as a wetting agent with a ratio of 2 g to 1 L of solution. The modification was conducted at temperature of 21 °C, for 3 or 20 min. Some samples were also put under tension during the process (as seen in Table 1). After the set time passed, samples were neutralized in 3% acetic acid, rinsed, and dried for 24 h at room temperature and 60–65% relative humidity level.

Bleaching

According to Hausner43, the following recipe for bleaching was provided: samples were put in a heated up to 80 °C bath containing 30% hydrogen peroxide (H2O2, with a ratio of 30 mL/L), Roksol M7 (2 g/L), NaOH (1.5 g/L), and sodium silicate (8 g/L). After 1 h of bathing, samples were rinsed with cold water and left for drying for 24 h at room temperature and 60–65% relative humidity level.

Lactide polymerization on the cotton surface

To decrease the hydrophilic properties of cotton fabric surface and increase compatibility with enriched with IBU polylactide layer, grafting of l,l-lactide was performed in three different conditions, bulk polymerization with (1) or without the use of vacuum (2) with stannous (II) octoate Sn(Oct)2 as catalyst, and cationic polymerization (3) with triflic acid (trifluoromethanesulfonic acid, CF3SO3H). Before starting synthesis, l,l-lactide was crystallized from dry 2-propanol, and sublimed. Formerly pretreated (washed, mercerized, bleached) cotton samples were conditioned for 24 h at 130 °C to remove excessive moisture.

Approach 1

5 g of l,l-lactide and two cotton samples 2 × 2 cm were placed in the glass ampule and dried under reduced pressure within 3 h. After that, 10 mg of Sn(Oct)2 was added, and the glass vessel was sealed with a burner under a high vacuum. The ampule was kept in oven at 130 °C within 24 h until newly formed polymer was in solid state.

Approach 2

The 5 × 5 cm samples were immersed in a 20 mL solution of distilled dichloromethane (CH2Cl2) containing 25 mg of Sn(Oct)2 under an argon atmosphere and mixed at room temperature for 0.5 h. Then, dichloromethane was removed, and samples were dried in a vacuum for 1 h. The prepared sample of 5 g of l,l-lactide was thoroughly spread out to obtain a thin layer, which would gradually penetrate the structure of the fabric during melting. For this variant, during polymerization vacuum was not used and there were no changes to other conditions (130 °C and 24 h of time).

Approach 3

Two samples of cotton (2 × 2 cm) and 5 g (0.035 mol) of l,l-lactide were placed in a Schlenk flask. The flask was degassed under reduced pressure, filled with argon, and sealed with a silicone septum. Next, 10 mL of dried CH2Cl2 was added. After the complete dissolution of the lactide, 50 µL of triflic acid was introduced through the septum, and the polymerization was conducted at room temperature for 24 h. At the end of every procedure, samples were rinsed twice with dichloromethane to remove unbonded polymer, which was then precipitated into methanol for further examinations. In contrast, the samples dried freely at room temperature without additional purification.

Covering with the active layer

Ibuprofen was purchased from Pol-Aura (Morąg, Poland). In a 1-step active layer application, 0.2 g of IBU and 0.4 g of star-PLA were added to 100 mL of dichloromethane and stirred for 15 min to prepare the drug carrier solution. Then, the samples with a total mass of 2.2 g were put into the bath and stirred for 24 h at room temperature. The last 15 min, the temperature was raised to 50 °C. The same procedure was specified for the PLA-PEG. The ratio of polymer to IBU amount was again kept at 2:1. Whereas the 2-step application included firstly stirring the samples in 150 mL of dichloromethane with 0.2 g PLA-PEG at room temperature and then adding 0.2 g of IBU and 0.2 g of star-PLA dissolved simultaneously in 50 mL of dichloromethane and stirring for another 24 h. The last 15 min ran at the temperature of 50 °C, just as in the 1-step method. All samples were dried flat afterwards without any rinsing. The theoretical amount of loaded ibuprofen results from the proportion of the drug in correlation to the amount of cotton fabric used and is 1 mg/cm2.

Characterization of the modified cotton

The wetting test of cotton was performed to demonstrate the degree of hydrophobization of the cotton fabric surface after the polymerization of lactide. In this research, the static water contact angle was determined using the sessile drop method. Measurements were performed using the DSA100 goniometer by Kruss (Germany). The built-in camera photographed the droplet positioned on the surface of the fabric after 1 s, and the angle was determined and calculated automatically. The measurement done for every sample had 4 repetitions. Then, the average of the measurements and standard deviation were calculated. Fourier-transform infrared spectroscopy (ATR-FTIR) spectra were measured with a Nicolet 6700 spectrometer to observe structural changes on the surface of cotton. The examination was obtained by adding 64 scans at a 2 cm−1 resolution. The molecular weight of the synthesized PLA was determined by size-exclusion chromatography (SEC) using an Agilent Pump 1100 Series with 2× PLGel 5 microns MIXED-C columns and methylene chloride as an eluent.

To determine the release of ibuprofen from the dressings, 1 × 1 cm samples were cut and put into 20 mL of phosphate buffered saline (PBS 0.1 M, pH 7.4) at room temperature. The release profile of IBU in PBS was examined by recording UV–VIS spectra at 220 nm using the spectrophotometer Thermo Scientific™ Evolution 220 (USA) with Insight software. Samples were measured triplicate for every point, and the calculations were made to determine the average and standard deviation [SD] of the measurements. The calibration curve was prepared with R2 coefficient of 0.999, and the wavelength for the drug concentration measurements was 220 nm.

In vitro examinations

Cell culture

Reference L929 mouse fibroblasts (purchased in LGC Standards, Middlesex, UK) and human Hs68 skin fibroblasts (CRL-1635™, purchased in American Type Cell Cultures (ATCC), Rockville, MD, USA). Mouse L929 fibroblasts were cultured at 37 °C in a 5% CO2 in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and standard antibiotics: penicillin (100 U/mL) and streptomycin (100 µg/mL), while the Hs68 cell line was grown in high glucose RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (all cell culture components were from Biowest, Nuaillé, France). Cell cultures were supplemented with fresh medium two or three times weekly to keep them in the log phase. The confluent monolayer was treated with 0.25% trypsin–EDTA solution (Biowest, Nuaillé, France) to passage.

Direct contact cytotoxicity

Direct contact cytotoxicity assay of the tested materials, row cotton and A-L was examined using L929 and Hs68 cell lines (density of 2 × 105 cells/mL) according to the ISO norm 10993-5 (International Organization for Standardization, 2009; Biological evaluation of medical devices Part 5: tests for in vitro cytotoxicity), with a use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, as previously described44. Under aseptic conditions, each tested material was placed in an individual well of the non-adherent Nunclon Delta Surface 24-well culture plate (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) in four replicates each. During the 24 h incubation in 1 mL of culture medium at 34 °C and 5% CO2, the tested materials absorbed the liquid. Next, the tested materials, row cotton and A-L samples, were cut into small pieces and transferred to a 96-well cell culture plate containing previously prepared L929 and Hs68 cells. The cell cultures in medium alone, without the tested materials, were used as a positive control (PC) for cell viability, whereas cells treated with 0.03% H2O2 were used as a negative control (NC), i.e. 100% dead cells due to lysis, and additionally as a control—raw (unmodified) cotton. The absorbance was measured spectrophotometrically using a Multiskan EX plate reader (Thermo Scientific, Waltham, MA, USA) at 570 nm. MTT reduction was related to untreated cells (%) = (absorbance of treated cells/absorbance of untreated cells × 100%) × 100%.

Anti-inflammatory properties of tested materials—extracellular secretion of anti-inflammatory cytokines

To assess the inflammatory effect of the tested materials, raw cotton and A-L samples, the level of anti-inflammatory cytokines (IL-1β and IL-8) was evaluated. The Hs68 cells were cultured for 24 h with tested materials alone and with tested materials in combination with lipopolysaccharide (LPS) from E. coli (1 µg/mL) to assess the anti-inflammatory potential of extracts. The inflammatory process in cell lines was induced by the treatment with LPS from E. coli (1 µg/mL), while according to Batool (2018), ibuprofen alone (50 µg/mL) as well as in combination with LPS from E. coli (1 µg/mL) was used as a control of anti-inflammatory action. The level of cytokines IL-1β, and IL-8 concentration in cell culture supernatants was tested using commercial ELISA (Invivogen Houston, TX, USA) which were performed according to the manufacturer protocol (Invivogen Houston, TX, USA). The test’s sensitivity was 1.5 pg/mL for IL-B and 1.7 pg/mL for TNF α.

Statistical analyses

Data are presented as mean values ± standard deviation (SD). The differences between tested variables were assessed using Statistica 13 PL software (https://statistica.software.informer.com/13.3software, Krakow, Poland) with a nonparametric Mann–Whitney U test (statistical comparison among two groups) or the Kruskal–Wallis test (statistical comparison among different groups). The results were considered statistically significant when p < 0.05.


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