Ionic Liquid Treated Bacterial Cellulose Sheets as Prospective Biodegradable Implant Materials

Ionic Liquid Treated Bacterial Cellulose Sheets as Prospective Biodegradable Implant Materials | 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 Ionic Liquid Treated Bacterial Cellulose Sheets as Prospective Biodegradable Implant Materials Muniba Munir, Sadaf Nosheen, Nawshad Muhammad, Maliha Uroos, Waleed Mustafa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3223070/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jan, 2024 Read the published version in Cellulose → Version 1 posted 4 You are reading this latest preprint version Abstract The purpose of this research was to create BC membranes / sheets which can be degraded by the enzymes in body fluids on implantation for soft and hard tissue regeneration. Bacterial Cellulose has been explored for its use in hard and soft tissue regeneration such as bone, dental, wound, hernia, dura mater, skin, nerve, cornea, and blood vessels. The limiting factor in the use of BC as biomedical implant material is that it is practically non-biodegradable in vitro and in vivo. However, reactive hydroxyl groups on BC allow a variety of chemical modifications which can be beneficial for the development of smart degradable biomedical materials. The use of Ionic Liquids (ILs) is the greener and non-toxic alternative to the chemical treatment for the degradation of BC. The ILs affect the degradability of BC by interacting with the functional groups and decreasing its crystallinity. Two non-toxic and biocompatible ILs i.e Pyridinium hydrogen sulfate (Py-HSO4) and 1-butyl-3-methyl imidazolium hydrogen sulfate (BMIM-HSO4) were used in the current study. The biodegradation of BC using these ILs has not been studied previously for biomedical implants. The characterizations of the IL treated BC were done using XRD, FTIR analysis, SEM, contact angle studies, degradation assay, drug delivery, and in vitro biocompatibility. SEM results suggest a clear change in the morphology of the BC nano fibers after treatment with ionic liquids. Furthermore, significant degradation was observed over 28 days where BC (Py-HSO4) degraded by 36% and BC (BMIM-HSO4) treated had degraded by 56%. Additionally, the IL treated BC could carry antibacterial drugs and showed potential for their sustained release. The modified membranes supported cell attachment and proliferation and were non-toxic and highly biocompatible. These results suggest that BC pellicles / sheets treated with ILs can be used as a degradable implant material for tissue engineering, regeneration, and drug delivery for various regenerative biomedical applications. Bacterial cellulose Ionic liquids degradable biomaterials tissue regeneration drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Bacterial cellulose (BC) is an economical natural polymer that is chemically pure and free of lignin and hemicellulose found in plant cellulose. It is produced as an extracellular, gelatinous matrix by acetic acid-producing bacteria such as Acetobacter xylinum [ 1 ]. Microbial BC is produced in the form of a membrane at the air-medium interface [ 2 , 3 ]. The unique structural, physicochemical, mechanical, and biological properties, such as purity, high degree of polymerization, three-dimensional (3D) fibrous structure makes it an attractive polymer for biomedical applications [ 1 , 4 – 8 ]. Its naturally synthesized network of interconnected nanofibrils with a high surface area and porosity leads to high liquid-absorption and retention [ 6 ]. High Young's modulus of the BC sheets exists due to its super-molecular structure that preserves the biological fibrils and binds them securely with hydrogen bonds [ 9 ]. These characteristics make it a next-generation natural polymer candidate for biomedical applications [ 10 ]. The extensive use of BC in medical science as a tissue replacement has resulted in numerous biomedical materials, some of which are commercially available [ 11 ]. Its non-cytotoxic and fibrous nature allows cell attachment, proliferation, migration, and differentiation [ 12 ]. These characteristics enable fast wound healing and re-epithelialization of the damaged tissues [ 13 ]. Furthermore, the remarkable hydrophilicity of BC allows it to maintain a moist environment at the wound site while maintaining an optimal temperature of 25.3°C – 37.3°C at the wound site [ 14 ]. Additionally, the fibrous morphology also allows adequate gas exchange, blood and exudate absorption, low tissue adhesion, and thermal insulation [ 15 ]. Based on all above factors, BC can be used as a biomaterial for hard tissue engineering (bone and dental), wound dressing, skin regeneration, dura-mater, nerve regeneration, hernioplasty, soft tissue reconstruction, cornea and blood vessel formation [ 11 ]. In addition, BC can also be utilized for the delivery of anti-inflammatory and antibacterial drugs with increased efficiency [ 16 – 18 ]. Despite having tremendous properties BC has a disadvantage of being non-biodegradable in vitro and in vivo [ 19 ]. Ideally the biodegradable biomaterial implants should not require a second surgery to be removed, because, these can be degraded into non-toxic substances through hydrolytic routes and excreted from the body [ 20 – 23 ]. The main factors that affect the biodegradation of BC in physiological environment are crystallinity, molecular weight, hydrophilicity and modification strategy. The biodegradation of materials in vivo involves hydrolysis, enzymatic degradation, oxidation and physical mechanism [ 11 ]. In order to enhance the biosorption of BC inside the living body, buffers, polymers, proteins and solvents have been used. Therefore, to induce degradability into BC, several studies have been carried out previously. For example in a prior study the dispersed ribbons from bacterial cellulose have been digested in a mixture of cloned Ce17A [CBH] I) and Ce16A (CBHII) in the presence of beta glucosidase enzyme into soluble sugars [ 24 ]. Similarly, periodate oxidation was used to prepare a biodegradable BC scaffold for tissue engineering [ 25 ]. Another technique used was LiCl/DMAc solvent system to form degradable BC. Although it is an effective technique but is expensive and only doable in lab settings [ 26 ]. Moreover, these methods completely degrade the cellulose content and dismember the sheet form. Therefore, the use of less toxic and cost-effective alternative methods to keep the sheet form intact needed to be explored. For this purpose, Ionic liquids (ILs) could be great substitutes for the chemical dissolution of BC. The ILs are the liquid salts which are entirely made up of ions and have a melting point below 100°C [ 27 ]. Due to their unique thermal, physical, chemical, and biological qualities, they are possible candidates for a clean, efficient, and eco-friendly replacement for volatile organic solvents [ 28 , 29 ]. The advantages of ILs in comparison to mineral acids and bases are that ILs are less viscous, non-volatile, soluble, thermally stable and non-corrosive [ 30 ]. The ILs tend to decrease the crystallinity of BC by interacting with its functional groups to make BC degradable. Therefore, to form degradable BC we used BMIM-HSO 4 and Py-HSO 4 synthesized in our lab. These ILs were selected from two distinct groups of ILs synthesized in our lab based on their non-toxicity in vitro in our prior cell culture study. For effective bacterial cellulose breakdown anions with strong hydrogen bond acceptance and cations with strong acidic protons should be chosen to design the ILs [ 31 ]. Pretreatment with ILs can lead to in vivo BC degradation into low molecular weight by-products on implantation [ 31 ]. Due to this degradable behavior of bacterial cellulose, it can contribute to the controlled drug release and a wide range of tissue engineering applications. Research Methodologies 3.1 Synthesis of Bacterial Cellulose Bacterial cellulose was synthesized in Hestrin-Schramm culture medium (HS) using 2% glucose w/v, 0.5% tryptone w/v, and 0.5% yeast extract w/v. For pre-culture, the bacteria and yeast co cultured scoby was used. The culture thus produced was further mixed in the HS media and was allowed to grow under static conditions for 2–4 days to make bacterial cellulose pellicles at 28°C in IL beaker. The produced BC from the medium was isolated in sheet form and purified by boiling in a 0.4% percent NaOH solution for 40 min at 100°C and then heated twice for 40 min at 100°C with distilled water and were air-dried [ 32 ]. 3.2 Modification of bacterial cellulose by ionic liquids The dried BC was suspended in two types of ionic liquids (1 –butyl- 3 -methylimidazolium hydrogen sulfate BMIM-HSO 4 and Pyridinium hydrogen sulphate Py-HSO 4 . The three IL concentrations were selected from prior optimization of BC membranes with both types of ionic liquids at 25%, 75% and 100% concentrations. The treatment time was 5 min, 10 min, 15 min, 20 min, 1.5 hrs, 2.5 hrs, and 3 hrs at 100℃. The subsequent findings demonstrated that the treatment of BC in IL concentration of 75% and temp 100°C for 20 min were ideal to make BC sheets having optimal and controllable degradation rate. Therefore, all BC sheets for this study were treated with 75% of each IL at 100°C for 20 mins. The treated BC membranes were then thoroughly washed with distilled water thrice till all unconjugated components of the ILs were removed. Hence two variants of IL treated BC were obtained which were named as BC (BMIM-HSO 4 ), BC (Py-HSO 4 ). Characterization of BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) Characterization of BC treated with ionic liquids was done by SEM analysis, FTIR spectroscopy, drug release profile, swelling test, tensile measurements, degradation assay, bacterial studies, and in vitro cell viability tests. 3.3 FTIR spectroscopy FTIR spectroscopic analysis was carried out to map the functional groups and chemical make-up of the BC (BMIM-HSO 4 ), BC (Py-HSO 4 ). The absorbance spectra of all samples were obtained using FTIR spectroscope (thermos Nicolet scientific 6700 USA) in Attenuated total reflection mode. Spectra of absorbance were captured at wavenumbers between 500 and 4000cm − 1 with 128 scans and 8 cm − 1 resolutions. 3.4 Scanning Electron Microscopy (SEM) The shape and structure of the BC (BMIM-HSO 4 ) and BC (Py-HSO 4 ) was determined in comparison to control BC using scanning electron microscope (TESCAN vega3 LMU). SEM analysis was performed at a 15 kv accelerating voltage range. Prior to imaging, each sample was gold coated after mounting on aluminum stubs for improved conductivity. 3.5 X- Ray-Diffraction The BC (Control) and BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) were scanned using an XRD analyzer (Rigaku) at room temperature with Cu Kα radiation. A scanning range of 0° to 90° was covered at a rate of 0.02° per step. 3.6 Contact Angle Measurements An optical microscope (Nikon) with a Sony camera adaptor (CMA-D2) and a 458 right-angle prism were used to measure contact angles, and the images were taken and uploaded to a computer for analysis using a video capture card (ATI). All procedures used purified water (18.2 MV cm). The contact angle between a water droplet of 2µl size and the substrate surface was measured for both the non-treated and ILs treated materials. Just before measuring the angle, 0.5 µl of solvent was added to the initial droplet to approximate the increasing contact angle approach. The angle between the tangent of the droplet and the substrate was determined after this image was shot on a computer. At least 600 measurements were taken per sample automatically to take the mean. 3.7 Swelling Assays Dry BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) and BC (control) were used to determine the swelling ratio. Membranes were weighed before immersion in Phosphate buffered saline for 24 hrs at ambient temp in 6 well plate. Samples were retrieved from the media at various time points (1, 2, 3, 4, 5, 24 hrs), The filter paper was used to properly blot away the excess solvent, and the membranes were weighed and submerged once again. Results were repeated for each sample, and the average ± SD was used. SR % = (W wet - W dry ) / W dry × 100 (1) Equation 1 was used to compute the swelling of the membranes. 3.8 In vitro Degradation Analysis The degradation of BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) and BC (Control) was studied by immersion in a PBS solution of pH 7.4 at 37℃. The studies were carried out for up to 28 days. The change in weight was measured by using a weighing balance (OHAUS®) for each membrane. The weight was calculated in percentage weight reduction. All samples were tested thrice, and the results were presented as the average ± SD. % weight loss = (W dry - W wet )/ W dry × 100 (2) Equation 2 was used to calculate the degradation of membranes: 3.9 Drug Release Analysis To ascertain the drug release from BC (BMIM-HSO 4 ) and BC (Py-HSO 4 ) two synthetic drugs chloramphenicol (CAP) and metronidazole (MNZ) were used. In vitro drug release was investigated using 6ml PBS (pH 7.4) where membranes were incubated for different time intervals at 37°C and 50 rpm. After, 1, 2, 3, 4, 5, 6, and 24, 48 and 72 hrs, 3 ml of the PBS was taken out and substituted with 3 ml of new PBS. CAP was measured spectrophotometrically at 278 nm and MNZ at 320 nm after each time interval. To ascertain the unknown drug concentrations discharged from samples, standard curves were employed. 3.10 Antibacterial Activity Evaluation Two strains, Staphylococcus aureus 6538TM ( S. aureus ) and Escherichia coli ATCC R 8739TM ( E. coli ) were used for the antibacterial testing. The plates were streaked with a loopful inoculum from the glycerol stocks, and incubated at 37°C overnight. Then 150 mL of broth was infused with a single colony into a 500-ml Erlenmeyer flask, creating the culture incubated at 37℃. The culture was then diluted to 0.5 MacFarland concentration, which is equivalent to 1.5 108 CFU/mL and has an optical density (OD) of 0.132 at 600 nm. The assay was carried out by diluting the inoculum with nutritional broth at a ratio of 1:500 until the right cell concentration was obtained. The swelling-diffusion technique was used to incorporate CAP/ MNZ into the BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) membranes. The 1 cm 2 oven-dried membranes were submerged in CAP (0.5%) or MNZ (1%) stock solutions for 8 hrs. To remove the unabsorbed CAP/ MNZ, membranes were taken out of the CAP/ MNZ stock solution (CAP/ MNZ residual) and submerged in 1 mL of distilled water for 10 seconds (CAP/ MNZ washing sol.). 3.11 In Vitro Biocompatibility of BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) In this research, fibroblast cells NIH3T3 were used for all cell studies. These cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (10%) and penicillin-streptomycin solution (1%) in a humidified incubator with 5% CO 2 at 37°C. The cells were sub-cultured every two to three days till the required number was achieved. 50000 cells were seeded on the surface of each (BMIM-HSO 4 ) and BC (Py-HSO 4 ) sheet sample and cultured for 24hrs in a 24 well tissue culture plate. The BC sheet samples were taken out of the medium after 24 hrs of incubation, and the cells were washed with PBS then filled with 500 µl of Alamar Blue solution (10%), and then allowed to incubate in the dark for 4 hrs. To take absorbance readings 100 µl of the Alamar blue solution (in 8 replicates) was aliquoted into a 96 well plate in an Absorbance plate reader (Bio-Tek Instruments, Inc.) at 570 nm. Results and Discussions 4.1 FTIR analysis FTIR spectra of pure ILs PY-HSO 4 & BMIM-HSO 4 and IL treated BC sheets i.e BC (Py-HSO 4 ) & BC (BMIM-HSO 4 ) is shown in Fig. 1 . FTIR spectra of ILs showed that 800–950 range is attributed to S-O single bond and the range 950–1300 is responsible for S═O and 1300–1450 showed S-OH bonds (Fig. 1 A). All the distinguishing bands of bacterial cellulose have been identified in accordance with the literature. The broad band at 3340–3500 cm − 1 range shows functional group of bacterial cellulose and is ascribed to the OH stretching. Alkane (CH stretch) vibrations and asymmetric CH 2 stretching are responsible for the peak at 2900 cm − 1 . The peak at 2700–2800 cm − 1 depicts CH 2 symmetric stretching. The peak at 1640 cm − 1 is due to OH distortion. The peak at 1400–1430 cm − 1 is due to CH 2 deformation and the band at 1370 cm − 1 is ascribed to CH 3 deformation. At 1340 cm − 1 , IR displays OH deformation, and the bands responsible between 1320 cm − 1 and 1030 cm − 1 correspond to CO deformation [ 33 ]. ILs used to treat BC left no noticeable bands behind, showing that the treated cellulose had been thoroughly washed [ 23 ] as shown in Fig. 1 B and C. After being treated with both ILs, the peak at 1640 cm − 1 which is dramatically reduced has been linked to OH distortion. Similar to this, after being treated with two different types of ILs, the intensity of the band at 1340 that has been linked to OH deformation [ 34 ]. When compared to the original cellulose, IR spectra of BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) show no remarkable differences which is an indication of no chemical changes due to ILs treatment on BC [ 23 ]. 4.2 Scanning electron microscopy (SEM) The structural morphology of BC after treatment with two types of ILs (BMIM-HSO 4 , Py-HSO 4 ) at concentrations of 75% and temp of 100°C was examined using Scanning Electron Microscopy, as seen in Fig. 2 . The structure of BC (control) was smooth, with visible normal BC fibrous structure. Continuous nanofibers with a diameter of roughly 60 nm made up the BC membrane. The BC nanofibers displayed a smooth surface morphology and a three-dimensional porous network structure [44]. Microfibrils that are intertwined, securely fastened, and extremely robust constitute the surface morphology of BC (Fig. 2 ) [45]. Fiber networks structured erratically or arbitrarily in three dimensions were converted into bacterial cellulose films by bacteria during fermentation. BC web ribbons range in length from 1 to 9 m and produce an extensive network of hydrogen bonding in a complex reticulation pattern serving as a stabilizer for framework [ 35 ]. Following IL treatment, morphological alterations occurred in both BC (BMIM-HSO 4 ), BC (Py-HSO 4 ) membranes. The treatment of BC caused partial degradation in both cases. The fibrous structure of BC after treatment with BC (Py-HSO 4 ) resulted in partial melting of the fibers and they became merged into one another (Fig. 2 D, E, F). While after treatment with BMIM-HSO 4 , the fibrous structure of BC became thin and sowed some degradability (Fig. 2 G, H, I). Because BMIM-HSO 4 and Py-HSO 4 are solvents in their own capacity therefore they have the potential to break hydrogen bonds inside BC fibers, resulting in amorphous and fragile-looking particles and fibers [ 36 ]. The two ionic liquids were utilized in three different concentrations (25%, 75%,100%) to treat BC. The BC sheets were not degradable after three hours at a concentration of 25% at 100 o C. Similarly, no changes were seen using 75% at 100 o C for 5, 10, or 15 minutes, but after 20 minutes, BC became porous and thinner. However further exposure for longer time disintegrated the fibrous structure followed by dissolution. When BC was treated with 100% ILs for 5, 10, and 15 minutes, it became thin and partly disintegrated and further exposure after 15 minutes completely disintegrated and dissolved. Therefore, in comparison to other concentrations the 75% of ILs at 100°C was considered to be more suitable for developing degradable BC without disturbing its integrity. 4.3 XRD Analysis of BC and ILs treated BC Figure 3 displays the diffractogram of BC after treatment with BMIM-HSO 4 and Py-HSO 4 . Three intense peaks can be seen in the diffraction patterns of 22.7°, 14.4°, 16.6° which denote the planar crystals of (100), (010), and (110). BC has a cellulose I crystal structure that created a triclinic structure [ 36 ]. BC (Fig. 3 ) possesses a crystal structure indicative of cellulose type I and a high crystallinity (84–89%) [ 8 ]. After treatment with ionic liquid BMIM-HSO 4 , crystallinity of BC sheet was reduced at a concentration of 75% at 100℃. It demonstrates that crystallinity of BC was lost and relates to the morphology of the material that exhibits damage. After being treated with Py-HSO 4 , crystallinity of BC was lost at 75% conc. and 100℃ and the fibrous structure of BC became more distorted. After the treatments of two ILs, the crystallinity is disrupted. As a result of the reaction of ILs, the structure of BC becomes amorphous. 4.4 Contact Angle Measurements A precise metric for detecting changes in surface characteristics of polymer is wettability [ 37 ]. Variations in the contact angle of a liquid on the surface are used to quantify wettability. Untreated BC surfaces typically have contact angles below 90° because they are hydrophilic. The contact angle of the untreated BC was 57° (Fig. 4 A). It is clear that the ILs treatments had a significant impact on the contact angle reduction, increasing the hydrophilic properties of the material (Fig. 4 B & C). Py-HSO 4 treated BC showed contact angle of 46° and the contact angle of BC after treatment with BMIM-HSO 4 was 30°. Hydrophilic nature is attributed to more amorphous regions. 4.5 Swelling Studies of BC-ILs The swelling or water absorption of the IL treated BC is shown in Fig. 5 . All BC-ILs membranes quickly absorb water during the first three hours of the assay before gradually increasing for next 24 hrs. Bacterial cellulose shows a higher swelling ratio after being treated with ILs than it does without treatment. Initially the control BC membrane absorbed a significant amount of water (Fig. 5 ) within an hr the BC membrane attained swelling ratio of 421.6%, which subsequently slightly rose to 513% after 2 hrs and 586% after 3 hrs and after 4 hrs 651% and 696% 5 hrs. The total swelling ratio achieved after 24 hrs was 718%. After one hr, the swelling ratio of BC (Py-HSO 4 ) reached a value of 591% and then gradually increased to 743% after 2 hrs, and 816% after 3 hrs, 907% after 4 hrs and 951% after 5 hrs. BC (Py-HSO 4 ) reached its maximum swelling ratio of 996% after 24 hrs. BC (BMIM-HSO 4 ), exhibited significantly higher swelling ratio than BC (Control) and BC (Py-HSO 4 ). The swelling ratio BC (BMIM-HSO 4) reached 764% after one hour, and it gradually increased to 799% after 2 hrs, 968% after 3 hrs,1051% after 4 hrs and 1087% after 5 hrs and 1113% after 24 hrs (Fig. 5 ). Both BC (BMIM-HSO 4 ) and BC (PY-HSO 4 ) swell at a rate that is noticeably higher than BC (control), which is associated with the hydrophilic character of the ILs [ 38 ]. BC has a lower swelling ratio because of its increased crystallinity. The BC fiber structure is, however, degraded by ILs, and as a result, BC membranes treated with ionic liquids became amorphous. Therefore, BC treated with these ionic liquids has higher swelling rate. 4.6 In vitro Degradation Assays Figure 6 illustrates the in vitro degradation of BC and BC treated with ILs in PBS over various time periods (3, 7, 14, 21, 28 days) at 37℃. Weight loss percentages were determined according to the differences between dry weights of the treated BC. At all-time points, weight loss of BC (BMIM- HSO 4 ) was significantly higher than BC (Py-HSO 4 ). Higher hydrophilicity of BC (BMIM-HSO 4 ) as compared to BC (Py-HSO 4 ) may be the cause of the greater weight loss. After 28 days, BC (BMIM-HSO 4 ) showed a weight reduction of 56%, while BC (Py-HSO 4 ) had a weight loss of 36%. PBS can cause BC micro-fibrils to swell many folds after some time. The C-O bonds in the BC molecular chains weaken and eventually break under the influence of ions and molecules of water from the area with weak bonding force. Similar mechanism of BC degradation in PBS was also explained in literature [ 39 ].The degradation rate of BC continuously increased up to 14 days and then became constant after a weight reduction of approximately 14.3%. This BC breakdown in PBS is primarily caused by the significantly high swelling of BC fibers [ 39 ]. The treatment of bacterial cellulose membranes by Ionic liquids BMIM HSO 4 and PY-HSO 4 resulted in a significantly faster degradation over time (Fig. 6 ). The degradation of BC (Py-HSO 4 ) was 9%, 18%, 21.6%, 27%, and 36% after 3, 7, 14, 21 and 28 days respectively. However, there was a significantly higher degradation in the case of BC (BMIM-HSO 4 ) compared to the BC (Py-HSO 4 ). After 3 days the degradation was 12.25%, after 7 days it was 21%, after 14 days it was 35%, after 21 days it was 49.1% and after 28 days it was 56.1%. These values were 20.1% higher than the BC (Py-HSO 4 ) and were 41.8% higher than Control. 4.7 Drug Release Profile The UV-VIS absorbance values for CAP and MNZ were recorded at specific wavelengths of 280 nm and 320 nm respectively. These absorbance values (y) of CAP and MNZ were used to draw standard curve using the equation (y = mx + c) to find the value of unknown concentrations. Quantitative UV-VIS spectrophotometry was performed to study the drug release profile of CAP and MNZ. From the standard straight-line curve, the unknown concentration of drug released was determined. Figure 7 MNZ release profile of BC and ILs-BC(A), CAP release profile of BC and ILs-BC (B) The time-dependent drug release from BC membranes treated with Py-HSO 4 and BMIM-HSO 4 was investigated using PBS and UV-VIS spectroscopy in the 200–700 nm region. CAP and MNZ had lambda max of 320 nm and 278 nm, respectively. As illustrated in Fig. 7, the % cumulative drug release was used to study the release pattern over a three-day period. When compared to BC, membranes treated with ILs that were loaded with CAP and MNZ demonstrated sustained release. The chosen time period was 0.25, 0.5, 0.75, 1, 2, 3, 4, 24, 48, and 72 hrs in order to monitor the release profile. In the case of BC (Py-HSO 4 ) loaded with MNZ, the treated membranes first demonstrated burst release of 31% within first 4 hrs, and then sustained release of 48% for the next 72 hrs. The BC (BMIM-HSO 4 ) treated membranes showed 20% release after 4 hrs and after this time interval and sustained release of 37% after 72 hrs. Whereas, metronidazole loaded BC membranes showed 71% release within 72 hours (Fig. 7). CAP loaded BC (Py-HSO 4 ) had a cumulative drug release of 51.09% after 3 hrs and 79% after 72 hrs. In the case of BC (BMIM-HSO 4 ) treated membranes that had been loaded with CAP, the cumulative drug release was 37% after 3 hrs, and 72% after 70 hrs. On the contrary the BC (Control) loaded with CAP displayed burst release of 99.8% within 70 hrs (Fig. 7). Since, degradable membranes showed sustained release which is supportive of wound healing process. 4.8 Anti-Bacterial Studies The effectiveness of CAP-loaded BC-ILs membranes on S. aureus and E. coli was examined. The inhibitory zones difference between pure ionic liquids treated BC membranes and membranes containing CAP was measured (Fig. 8 ). The CAP-containing BC membranes treated with BMIM-HSO 4 and Py-HSO 4 both shown antibacterial activity against the two model bacteria with inhibitory zones of 35 mm. Similarly, MNZ loaded Py-HSO 4 and BMIM-HSO 4 treated BC showed inhibitory zones of 40 mm on E. coli as well as S. aureus . The positive controls that are CAP and MNZ showed inhibitory zones of 22 mm on E. coli and S. aureus . It can be concluded that the antimicrobial activity seen in disc diffusion experiments is due to CAP and MNZ loaded into BC-ILs sheets. The drugs have a known effect on both pathogens and here it is indicative of the loading efficiency and sustained release behavior of BC-ILs sheets. It can thus be envisaged that these IL treated BC sheets can be used in wound healing applications using any drug. 4.9 In Vitro Biocompatibility of BC-ILs membranes In vitro cytotoxicity studies were performed using fibroblast NIH3T3 cells to determine whether the BC-ILs membranes and drug loaded BC-ILs membranes are biocompatible. The results of cytotoxicity studies of BC (control) and BC-ILs sheets are shown in Fig. 9 . Cytocompatibility was measured after direct contact with the materials for 1, 3, and 7 days. BC is known for its biocompatibility, and the values as high as 95% from BC-ILs and drug-loaded BC sheets show that these are non-toxic and biocompatible too. All samples had 100% biocompatibility after the first day there was no statistical difference from the control (p > 0.05). BC (Py-HSO 4 ) and BC (BMIM-HSO 4 ) showed almost equal cell viability [ 40 ]. Furthermore, MNZ loaded BC (Py-HSO 4 ) and BC (BMIM-HSO 4 ) show higher cell viability as compared to CAP loaded sheets. After 7 days of direct contact with the samples, there wasn't any significant difference between the cytocompatibility of BC (Control), BC-ILs and drug loaded BC-ILs. According to ASTM standards a biocompatibility higher than 70% is considered acceptable for the implant biomaterials. Conclusions Bacterial cellulose sheets are non-degradable biomaterials, which limits its use as a regenerative implant material. In this research BC pellicles / sheets were made biodegradable by treating with ILs. Degradability of BC treated with ILs BMIM-HSO 4 and Py-HSO 4 have been confirmed by SEM, FTIR, XRD & degradation studies. FTIR confirmed that there was no chemical change after treatment with ILs and no residue remained after washing. SEM analysis showed that BC fibers became thinner and had been partially broken. Moreover, BC treated with ILs became more hydrophilic and developed sustained release profile of drugs that were loaded on them. Furthermore, in vitro biocompatibility test confirmed that the IL treated BC was biocompatible and non-cytotoxic. Our results conclude that the BC sheets became not only biodegradable but also more hydrophilic and efficient drug carriers after treatment with ILs. Hence these BC-ILs can be used for tissue regenerative and wound healing applications, as degradable implants, drug carriers and tissue engineered scaffolds. Further in vivo studies can be helpful in establishing the effectiveness as implant material. Declarations Acknowledgements The authors would like to acknowledge the support of HEC under NRPU-7787 and Ministry of Science and Technology, Government of Pakistan for a developmental grant titled "Establishment of Center for Advance Technologies in Biomedical Material" under its knowledge economy initiative. We also extebd our sincere appreciation to the researchers supporting project RSPD2023R755 KSU,Ryadh, Saudi Arabia. Authors would also like to thank Dr Aqif Anwar Chaudhry and Dr. Zulifqar Ali for support during the project. Ethics approval and consent to participate Not Applicable Consent for publication All authors consent for publication of the submitted manuscript Availability of data and material Not Applicable Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding The project was funded by Higher Edication Commission, Pakistant under Grant number HEC-NRPU-7787 Author contributions All authors contributed to the study conception and design specially the corresponding author Dr Faiza Sharif. Material preparation, data collection and analysis were performed by Muniba Muneer and Sadaf Nosheen. The first draft of the manuscript was written by Muniba Muneer and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Ullah, M.W., et al., Innovative production of bio-cellulose using a cell-free system derived from a single cell line. Carbohydrate Polymers, 2015. 132: p. 286-294. Khan, S., et al., Preparation and structural characterization of surface modified microporous bacterial cellulose scaffolds: A potential material for skin regeneration applications in vitro and in vivo. International Journal of Biological Macromolecules, 2018. 117: p. 1200-1210. Khalid, A., et al., Bacterial cellulose–TiO 2 nanocomposites promote healing and tissue regeneration in burn mice model. 2017. 7(75): p. 47662-47668. Ul-Islam, M.J.C.p.d., Comparative synthesis and characterization of bio-cellulose from local waste and cheap resources. 2019. 25(34): p. 3664-3671. Shah, N., et al., Overview of bacterial cellulose composites: A multipurpose advanced material. Carbohydrate Polymers, 2013. 98(2): p. 1585-1598. Ul-Islam, M., et al., Comparative study of plant and bacterial cellulose pellicles regenerated from dissolved states. International Journal of Biological Macromolecules, 2019. 137: p. 247-252. Ul-Islam, M., T. Khan, and J.K. Park, Water holding and release properties of bacterial cellulose obtained by in situ and ex situ modification. Carbohydrate Polymers, 2012. 88(2): p. 596-603. Czaja, W., D. Romanovicz, and R.m. Brown, Structural investigations of microbial cellulose produced in stationary and agitated culture. Cellulose, 2004. 11(3): p. 403-411. Yamanaka, S., et al., The structure and mechanical properties of sheets prepared from bacterial cellulose. Journal of Materials Science, 1989. 24(9): p. 3141-3145. Swingler, S., et al., Recent Advances and Applications of Bacterial Cellulose in Biomedicine. Polymers 2021, 13, 412 , 2021, s Note: MDPI stays neutral with regard to jurisdictional claims in published …. Torgbo, S. and P. Sukyai, Biodegradation and thermal stability of bacterial cellulose as biomaterial: The relevance in biomedical applications. Polymer Degradation and Stability, 2020. 179: p. 109232. Torres, F.G., S. Commeaux, and O.P. Troncoso, Biocompatibility of Bacterial Cellulose Based Biomaterials. Journal of Functional Biomaterials, 2012. 3(4). Shpichka, A., et al., Skin tissue regeneration for burn injury. Stem Cell Research & Therapy, 2019. 10(1): p. 94. Rahman, M.M. and A.N. Netravali, Aligned Bacterial Cellulose Arrays as “Green” Nanofibers for Composite Materials. ACS Macro Letters, 2016. 5(9): p. 1070-1074. Sajjad, W., et al., Development of modified montmorillonite-bacterial cellulose nanocomposites as a novel substitute for burn skin and tissue regeneration. Carbohydr Polym, 2019. 206: p. 548-556. Shao, W., et al., Tetracycline hydrochloride loaded regenerated cellulose composite membranes with controlled release and efficient antibacterial performance. 2016. 6(4): p. 3068-3073. Alkhatib, Y., et al., Controlled extended octenidine release from a bacterial nanocellulose/Poloxamer hybrid system. European Journal of Pharmaceutics and Biopharmaceutics, 2017. 112: p. 164-176. Gupta, A., et al., Synthesis of Silver Nanoparticles Using Curcumin-Cyclodextrins Loaded into Bacterial Cellulose-Based Hydrogels for Wound Dressing Applications. Biomacromolecules, 2020. 21(5): p. 1802-1811. Manoukian, O.S., et al., Biomaterials for Tissue Engineering and Regenerative Medicine , in Encyclopedia of Biomedical Engineering , R. Narayan, Editor 2019, Elsevier: Oxford. p. 462-482. Ginjupalli, K., et al., Poly (α-hydroxy acid) based polymers: A review on material and degradation aspects. 2017. 144: p. 520-535. Hasnain, M.S., et al., Biodegradable polymer matrix nanocomposites for bone tissue engineering , in Applications of nanocomposite materials in orthopedics 2019, Elsevier. p. 1-37. Ulery, B.D., L.S. Nair, and C.T.J.J.o.p.s.P.B.p.p. Laurencin, Biomedical applications of biodegradable polymers. 2011. 49(12): p. 832-864. Amarasekara, A.S. and B. Wiredu, Degradation of Cellulose in Dilute Aqueous Solutions of Acidic Ionic Liquid 1-(1-Propylsulfonic)-3-methylimidazolium Chloride, and p-Toluenesulfonic Acid at Moderate Temperatures and Pressures. Industrial & Engineering Chemistry Research, 2011. 50(21): p. 12276-12280. Boisset, C., et al., Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. 2000. 66(4): p. 1444-1452. Li, J., et al., Preparation and characterization of 2, 3-dialdehyde bacterial cellulose for potential biodegradable tissue engineering scaffolds. 2009. 29(5): p. 1635-1642. Lima, G.d.M., et al., Characterisation of bacterial cellulose partly acetylated by dimethylacetamide/lithium chloride. Materials Science and Engineering: C, 2011. 31(2): p. 190-197. Hallett, J.P. and T. Welton, Room-Temperature Ionic Liquids: Solvents for Synthesis and Catalysis. 2. Chemical Reviews, 2011. 111(5): p. 3508-3576. Singh, S.K. and A.W. Savoy, Ionic liquids synthesis and applications: An overview. Journal of Molecular Liquids, 2020. 297: p. 112038. Singh, S.K., Solubility of lignin and chitin in ionic liquids and their biomedical applications. International Journal of Biological Macromolecules, 2019. 132: p. 265-277. Welton, T.J.C.r., Room-temperature ionic liquids. Solvents for synthesis and catalysis. 1999. 99(8): p. 2071-2084. Isik, M.S.H.M.D.I.L., C.M. Cellulose: Dissolution, and M. Preparation of New Cellulosic International Journal of Molecular Sciences, 2014. 15, 11922-11940 DOI: 10.3390/ijms150711922. Kiziltas, E.E., A. Kiziltas, and D.J.J.C.P. Gardner, Synthesis of bacterial cellulose using hot water extracted wood sugars. 2015. 124: p. 131-138. Costa, L.M.M., G.M. de Olyveira, and P.J.J.o.B. Basmaji, Bacterial cellulose towards functional green composites materials. 2011. 5(2): p. 167-172. Spiridon, I., C.-A. Teacă, and R. Bodîrlău, Structural changes evidenced by FTIR spectroscopy in cellulosic materials after pre-treatment with ionic liquid and enzymatic hydrolysis. BioResources, 2011. 6: p. 400-413. Suryanto, H., et al., The Mechanical Strength and Morphology of Bacterial Cellulose Films: The Effect of NaOH Concentration. IOP Conference Series Materials Science and Engineering (Online), 2019. 515(1): p. 7. Suryanto, H., et al. Morphology and structure of bacterial cellulose film after ionic liquid treatment . in Journal of Physics: Conference Series . 2020. IOP Publishing. Adamson, A.W. and A.P. Gast, Physical chemistry of surfaces . Vol. 150. 1967: Interscience publishers New York. Morais, E.S., et al., Anti-inflammatory and antioxidant nanostructured cellulose membranes loaded with phenolic-based ionic liquids for cutaneous application. Carbohydrate Polymers, 2019. 206: p. 187-197. Chen, Y., et al., In Vitro Structural Changes of Nano-Bacterial Cellulose Immersed in Phosphate Buffer Solution. Journal of Biomimetics, Biomaterials, and Tissue Engineering, 2011. 10. Ventura, S.P.M., et al., Imidazolium and Pyridinium Ionic Liquids from Mandelic Acid Derivatives: Synthesis and Bacteria and Algae Toxicity Evaluation. ACS Sustainable Chemistry & Engineering, 2013. 1(4): p. 393-402. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2024 Read the published version in Cellulose → Version 1 posted Editorial decision: Major revision 13 Aug, 2023 Submission checks completed at journal 12 Aug, 2023 Editor assigned by journal 12 Aug, 2023 First submitted to journal 01 Aug, 2023 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3223070","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":225910584,"identity":"06315e7b-3c3e-427b-b734-0b66c821a708","order_by":0,"name":"Muniba Munir","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muniba","middleName":"","lastName":"Munir","suffix":""},{"id":225910585,"identity":"e3a529d0-3f81-4be3-b213-dd648f38e37a","order_by":1,"name":"Sadaf Nosheen","email":"","orcid":"","institution":"Government College University, Lahore","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sadaf","middleName":"","lastName":"Nosheen","suffix":""},{"id":225910586,"identity":"22348358-daeb-4d09-ac8e-dc24ef247a37","order_by":2,"name":"Nawshad Muhammad","email":"","orcid":"","institution":"Khyber Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nawshad","middleName":"","lastName":"Muhammad","suffix":""},{"id":225910587,"identity":"eb241efb-c8c0-4b97-99ea-bf67574b0298","order_by":3,"name":"Maliha Uroos","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maliha","middleName":"","lastName":"Uroos","suffix":""},{"id":225910588,"identity":"8e2dce73-c8e5-46d6-974f-31d1a14a8e13","order_by":4,"name":"Waleed Mustafa","email":"","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Waleed","middleName":"","lastName":"Mustafa","suffix":""},{"id":225910589,"identity":"d3e5ea0d-7641-4bc1-8275-7e78ed488153","order_by":5,"name":"Rawaiz Khan","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rawaiz","middleName":"","lastName":"Khan","suffix":""},{"id":225910590,"identity":"d3634232-0c7d-475c-a1aa-8e698df18f70","order_by":6,"name":"Rong Wang","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Wang","suffix":""},{"id":225910591,"identity":"66b2da96-5a3a-4849-984d-10724151eb39","order_by":7,"name":"Faiza Sharif","email":"data:image/png;base64,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","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Faiza","middleName":"","lastName":"Sharif","suffix":""}],"badges":[],"createdAt":"2023-08-01 06:44:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3223070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3223070/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10570-024-05741-y","type":"published","date":"2024-01-30T15:00:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41719723,"identity":"fd032263-b90f-4161-8100-f74c369c1bf1","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":361768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) FTIR spectra of Py-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026amp; BMIM-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4, \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(B and C)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eFTIR spectra of BC (Control), BC (Py-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) \u0026amp; BC (BMIM-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/d635fe0e8b64c72f045c675b.jpg"},{"id":41719724,"identity":"3d143ba4-d59b-4276-b499-65c90e473bcd","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":943385,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of BC membranes, (A-C) BC (Control), (D-F) BC(BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), \u0026amp; (G-I) BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/3822f3ea4fea5f41734f606f.jpg"},{"id":41721659,"identity":"ca401515-c0cb-45f5-abd1-1bfba9a97e1e","added_by":"auto","created_at":"2023-08-17 17:42:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":149470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD of BC, BC (BMIM-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4)\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026amp; BC (Py-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/2c33b55832a93019e71366e7.jpg"},{"id":41719726,"identity":"491cd8d3-bde6-4c92-a993-04fed175ae23","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175810,"visible":true,"origin":"","legend":"\u003cp\u003eContact angle of BC, (B) Contact angle of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) \u0026amp; (C) BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/9dcd19a95decaa08d48653fa.png"},{"id":41722349,"identity":"e038a21b-faef-48c1-91ff-15b5b47aa4e6","added_by":"auto","created_at":"2023-08-17 17:50:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":209481,"visible":true,"origin":"","legend":"\u003cp\u003eSwelling analysis of BC, BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) \u0026amp; BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/1e50f0f74fc4c2b8232f372a.jpg"},{"id":41719733,"identity":"6ec281ae-c6e2-4950-b29b-b62183433605","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":205420,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation of BC-Control, BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) \u0026amp; BC-(PY HSO\u003csub\u003e4\u003c/sub\u003e)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/66eb0383aabbec55dc7c82b0.jpg"},{"id":41719727,"identity":"995832e2-cf1f-4ec4-8cae-1033f0148b17","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71830,"visible":true,"origin":"","legend":"\u003cp\u003eMNZ release profile of BC and ILs-BC(A), CAP release profile of BC and ILs-BC (B)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/6e75fa654fc3960b6735dd2e.png"},{"id":41721661,"identity":"d0db107b-7ce7-4abf-a3d3-1910dd127e59","added_by":"auto","created_at":"2023-08-17 17:42:35","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":202574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial activity of ILs treated drug loaded BC (A) CAP loaded BC-ILs membranes, (B) MNZ loaded BC-ILs membranes\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/d9fdcdf8e3f28101ef749275.jpg"},{"id":41719730,"identity":"e947b357-b76f-4253-b54a-a088d198258e","added_by":"auto","created_at":"2023-08-17 17:34:35","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":80987,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of BC, BC-ILs \u0026amp; drugs loaded BC-ILs\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/fadfeab76b2709a1a344a1c0.png"},{"id":50673808,"identity":"70c6ff14-fa99-4bad-8a34-c0c15278260a","added_by":"auto","created_at":"2024-02-05 15:06:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1393420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/4ee2b5c1-28b6-494e-ab18-c4b6dde755f3.pdf"},{"id":41721658,"identity":"dfc5bab5-b43d-45ea-aed1-f660ec63193e","added_by":"auto","created_at":"2023-08-17 17:42:35","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2068453,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3223070/v1/1d7159313803c027b4e8b57f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ionic Liquid Treated Bacterial Cellulose Sheets as Prospective Biodegradable Implant Materials","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacterial cellulose (BC) is an economical natural polymer that is chemically pure and free of lignin and hemicellulose found in plant cellulose. It is produced as an extracellular, gelatinous matrix by acetic acid-producing bacteria such as \u003cem\u003eAcetobacter xylinum\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Microbial BC is produced in the form of a membrane at the air-medium interface [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The unique structural, physicochemical, mechanical, and biological properties, such as purity, high degree of polymerization, three-dimensional (3D) fibrous structure makes it an attractive polymer for biomedical applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its naturally synthesized network of interconnected nanofibrils with a high surface area and porosity leads to high liquid-absorption and retention [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. High Young's modulus of the BC sheets exists due to its super-molecular structure that preserves the biological fibrils and binds them securely with hydrogen bonds [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These characteristics make it a next-generation natural polymer candidate for biomedical applications [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe extensive use of BC in medical science as a tissue replacement has resulted in numerous biomedical materials, some of which are commercially available [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Its non-cytotoxic and fibrous nature allows cell attachment, proliferation, migration, and differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These characteristics enable fast wound healing and re-epithelialization of the damaged tissues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, the remarkable hydrophilicity of BC allows it to maintain a moist environment at the wound site while maintaining an optimal temperature of 25.3\u0026deg;C \u0026ndash; 37.3\u0026deg;C at the wound site [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, the fibrous morphology also allows adequate gas exchange, blood and exudate absorption, low tissue adhesion, and thermal insulation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on all above factors, BC can be used as a biomaterial for hard tissue engineering (bone and dental), wound dressing, skin regeneration, dura-mater, nerve regeneration, hernioplasty, soft tissue reconstruction, cornea and blood vessel formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, BC can also be utilized for the delivery of anti-inflammatory and antibacterial drugs with increased efficiency [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite having tremendous properties BC has a disadvantage of being non-biodegradable \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Ideally the biodegradable biomaterial implants should not require a second surgery to be removed, because, these can be degraded into non-toxic substances through hydrolytic routes and excreted from the body [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The main factors that affect the biodegradation of BC in physiological environment are crystallinity, molecular weight, hydrophilicity and modification strategy. The biodegradation of materials \u003cem\u003ein vivo\u003c/em\u003e involves hydrolysis, enzymatic degradation, oxidation and physical mechanism [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In order to enhance the biosorption of BC inside the living body, buffers, polymers, proteins and solvents have been used. Therefore, to induce degradability into BC, several studies have been carried out previously. For example in a prior study the dispersed ribbons from bacterial cellulose have been digested in a mixture of cloned Ce17A [CBH] I) and Ce16A (CBHII) in the presence of beta glucosidase enzyme into soluble sugars [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, periodate oxidation was used to prepare a biodegradable BC scaffold for tissue engineering [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Another technique used was LiCl/DMAc solvent system to form degradable BC. Although it is an effective technique but is expensive and only doable in lab settings [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, these methods completely degrade the cellulose content and dismember the sheet form. Therefore, the use of less toxic and cost-effective alternative methods to keep the sheet form intact needed to be explored. For this purpose, Ionic liquids (ILs) could be great substitutes for the chemical dissolution of BC.\u003c/p\u003e \u003cp\u003eThe ILs are the liquid salts which are entirely made up of ions and have a melting point below 100\u0026deg;C [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Due to their unique thermal, physical, chemical, and biological qualities, they are possible candidates for a clean, efficient, and eco-friendly replacement for volatile organic solvents [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The advantages of ILs in comparison to mineral acids and bases are that ILs are less viscous, non-volatile, soluble, thermally stable and non-corrosive [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The ILs tend to decrease the crystallinity of BC by interacting with its functional groups to make BC degradable. Therefore, to form degradable BC we used BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Py-HSO\u003csub\u003e4\u003c/sub\u003e synthesized in our lab. These ILs were selected from two distinct groups of ILs synthesized in our lab based on their non-toxicity \u003cem\u003ein vitro\u003c/em\u003e in our prior cell culture study. For effective bacterial cellulose breakdown anions with strong hydrogen bond acceptance and cations with strong acidic protons should be chosen to design the ILs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Pretreatment with ILs can lead to \u003cem\u003ein vivo\u003c/em\u003e BC degradation into low molecular weight by-products on implantation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Due to this degradable behavior of bacterial cellulose, it can contribute to the controlled drug release and a wide range of tissue engineering applications.\u003c/p\u003e"},{"header":"Research Methodologies","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Synthesis of Bacterial Cellulose\u003c/h2\u003e\n\u003cp\u003eBacterial cellulose was synthesized in Hestrin-Schramm culture medium (HS) using 2% glucose w/v, 0.5% tryptone w/v, and 0.5% yeast extract w/v. For pre-culture, the bacteria and yeast co cultured scoby was used. The culture thus produced was further mixed in the HS media and was allowed to grow under static conditions for 2\u0026ndash;4 days to make bacterial cellulose pellicles at 28\u0026deg;C in IL beaker. The produced BC from the medium was isolated in sheet form and purified by boiling in a 0.4% percent NaOH solution for 40 min at 100\u0026deg;C and then heated twice for 40 min at 100\u0026deg;C with distilled water and were air-dried [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Modification of bacterial cellulose by ionic liquids\u003c/h2\u003e\n\u003cp\u003eThe dried BC was suspended in two types of ionic liquids (1 \u0026ndash;butyl- 3 -methylimidazolium hydrogen sulfate BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Pyridinium hydrogen sulphate Py-HSO\u003csub\u003e4\u003c/sub\u003e. The three IL concentrations were selected from prior optimization of BC membranes with both types of ionic liquids at 25%, 75% and 100% concentrations. The treatment time was 5 min, 10 min, 15 min, 20 min, 1.5 hrs, 2.5 hrs, and 3 hrs at 100℃. The subsequent findings demonstrated that the treatment of BC in IL concentration of 75% and temp 100\u0026deg;C for 20 min were ideal to make BC sheets having optimal and controllable degradation rate. Therefore, all BC sheets for this study were treated with 75% of each IL at 100\u0026deg;C for 20 mins. The treated BC membranes were then thoroughly washed with distilled water thrice till all unconjugated components of the ILs were removed. Hence two variants of IL treated BC were obtained which were named as BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of BC (BMIM-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e), BC (Py-HSO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of BC treated with ionic liquids was done by SEM analysis, FTIR spectroscopy, drug release profile, swelling test, tensile measurements, degradation assay, bacterial studies, and \u003cem\u003ein vitro\u003c/em\u003e cell viability tests.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 FTIR spectroscopy\u003c/h2\u003e\n\u003cp\u003eFTIR spectroscopic analysis was carried out to map the functional groups and chemical make-up of the BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e). The absorbance spectra of all samples were obtained using FTIR spectroscope (thermos Nicolet scientific 6700 USA) in Attenuated total reflection mode. Spectra of absorbance were captured at wavenumbers between 500 and 4000cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 128 scans and 8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resolutions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\n\u003cp\u003eThe shape and structure of the BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) was determined in comparison to control BC using scanning electron microscope (TESCAN vega3 LMU). SEM analysis was performed at a 15 kv accelerating voltage range. Prior to imaging, each sample was gold coated after mounting on aluminum stubs for improved conductivity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 X- Ray-Diffraction\u003c/h2\u003e\n\u003cp\u003eThe BC (Control) and BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) were scanned using an XRD analyzer (Rigaku) at room temperature with Cu K\u0026alpha; radiation. A scanning range of 0\u0026deg; to 90\u0026deg; was covered at a rate of 0.02\u0026deg; per step.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Contact Angle Measurements\u003c/h2\u003e\n\u003cp\u003eAn optical microscope (Nikon) with a Sony camera adaptor (CMA-D2) and a 458 right-angle prism were used to measure contact angles, and the images were taken and uploaded to a computer for analysis using a video capture card (ATI). All procedures used purified water (18.2 MV cm). The contact angle between a water droplet of 2\u0026micro;l size and the substrate surface was measured for both the non-treated and ILs treated materials. Just before measuring the angle, 0.5 \u0026micro;l of solvent was added to the initial droplet to approximate the increasing contact angle approach. The angle between the tangent of the droplet and the substrate was determined after this image was shot on a computer. At least 600 measurements were taken per sample automatically to take the mean.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 Swelling Assays\u003c/h2\u003e\n\u003cp\u003eDry BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (control) were used to determine the swelling ratio. Membranes were weighed before immersion in Phosphate buffered saline for 24 hrs at ambient temp in 6 well plate. Samples were retrieved from the media at various time points (1, 2, 3, 4, 5, 24 hrs), The filter paper was used to properly blot away the excess solvent, and the membranes were weighed and submerged once again. Results were repeated for each sample, and the average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD was used.\u003c/p\u003e\n\u003cp\u003eSR % = (W\u003csub\u003ewet\u003c/sub\u003e - W\u003csub\u003edry\u003c/sub\u003e) / W\u003csub\u003edry\u003c/sub\u003e\u0026times; 100 (1)\u003c/p\u003e\n\u003cp\u003eEquation 1 was used to compute the swelling of the membranes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e3.8 \u003cem\u003eIn vitro\u003c/em\u003e Degradation Analysis\u003c/h2\u003e\n\u003cp\u003eThe degradation of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (Control) was studied by immersion in a PBS solution of pH 7.4 at 37℃. The studies were carried out for up to 28 days. The change in weight was measured by using a weighing balance (OHAUS\u0026reg;) for each membrane. The weight was calculated in percentage weight reduction. All samples were tested thrice, and the results were presented as the average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\n\u003cp\u003e% weight loss = (W\u003csub\u003edry\u003c/sub\u003e- W\u003csub\u003ewet\u003c/sub\u003e)/ W\u003csub\u003edry\u003c/sub\u003e\u0026times; 100 (2)\u003c/p\u003e\n\u003cp\u003eEquation 2 was used to calculate the degradation of membranes:\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.9 Drug Release Analysis\u003c/h2\u003e\n\u003cp\u003eTo ascertain the drug release from BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) two synthetic drugs chloramphenicol (CAP) and metronidazole (MNZ) were used. \u003cem\u003eIn vitro\u003c/em\u003e drug release was investigated using 6ml PBS (pH 7.4) where membranes were incubated for different time intervals at 37\u0026deg;C and 50 rpm. After, 1, 2, 3, 4, 5, 6, and 24, 48 and 72 hrs, 3 ml of the PBS was taken out and substituted with 3 ml of new PBS. CAP was measured spectrophotometrically at 278 nm and MNZ at 320 nm after each time interval. To ascertain the unknown drug concentrations discharged from samples, standard curves were employed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.10 Antibacterial Activity Evaluation\u003c/h2\u003e\n\u003cp\u003eTwo strains, Staphylococcus aureus 6538TM (\u003cem\u003eS. aureus\u003c/em\u003e) and Escherichia coli ATCC R 8739TM (\u003cem\u003eE. coli\u003c/em\u003e) were used for the antibacterial testing. The plates were streaked with a loopful inoculum from the glycerol stocks, and incubated at 37\u0026deg;C overnight. Then 150 mL of broth was infused with a single colony into a 500-ml Erlenmeyer flask, creating the culture incubated at 37℃. The culture was then diluted to 0.5 MacFarland concentration, which is equivalent to 1.5 108 CFU/mL and has an optical density (OD) of 0.132 at 600 nm. The assay was carried out by diluting the inoculum with nutritional broth at a ratio of 1:500 until the right cell concentration was obtained. The swelling-diffusion technique was used to incorporate CAP/ MNZ into the BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) membranes. The 1 cm\u003csup\u003e2\u003c/sup\u003e oven-dried membranes were submerged in CAP (0.5%) or MNZ (1%) stock solutions for 8 hrs. To remove the unabsorbed CAP/ MNZ, membranes were taken out of the CAP/ MNZ stock solution (CAP/ MNZ residual) and submerged in 1 mL of distilled water for 10 seconds (CAP/ MNZ washing sol.).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.11 \u003cem\u003eIn Vitro\u003c/em\u003e Biocompatibility of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e)\u003c/h2\u003e\n\u003cp\u003eIn this research, fibroblast cells NIH3T3 were used for all cell studies. These cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (10%) and penicillin-streptomycin solution (1%) in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. The cells were sub-cultured every two to three days till the required number was achieved. 50000 cells were seeded on the surface of each (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) sheet sample and cultured for 24hrs in a 24 well tissue culture plate. The BC sheet samples were taken out of the medium after 24 hrs of incubation, and the cells were washed with PBS then filled with 500 \u0026micro;l of Alamar Blue solution (10%), and then allowed to incubate in the dark for 4 hrs. To take absorbance readings 100 \u0026micro;l of the Alamar blue solution (in 8 replicates) was aliquoted into a 96 well plate in an Absorbance plate reader (Bio-Tek Instruments, Inc.) at 570 nm.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussions","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e4.1 FTIR analysis\u003c/h2\u003e\n\u003cp\u003eFTIR spectra of pure ILs PY-HSO\u003csub\u003e4\u003c/sub\u003e \u0026amp; BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and IL treated BC sheets i.e BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) \u0026amp; BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. FTIR spectra of ILs showed that 800\u0026ndash;950 range is attributed to S-O single bond and the range 950\u0026ndash;1300 is responsible for S═O and 1300\u0026ndash;1450 showed S-OH bonds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eAll the distinguishing bands of bacterial cellulose have been identified in accordance with the literature. The broad band at 3340\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range shows functional group of bacterial cellulose and is ascribed to the OH stretching. Alkane (CH stretch) vibrations and asymmetric CH\u003csub\u003e2\u003c/sub\u003e stretching are responsible for the peak at 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The peak at 2700\u0026ndash;2800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e depicts CH\u003csub\u003e2\u003c/sub\u003e symmetric stretching. The peak at 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is due to OH distortion. The peak at 1400\u0026ndash;1430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is due to CH\u003csub\u003e2\u003c/sub\u003e deformation and the band at 1370 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is ascribed to CH\u003csub\u003e3\u003c/sub\u003e deformation. At 1340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, IR displays OH deformation, and the bands responsible between 1320 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to CO deformation [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eILs used to treat BC left no noticeable bands behind, showing that the treated cellulose had been thoroughly washed [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and C. After being treated with both ILs, the peak at 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which is dramatically reduced has been linked to OH distortion. Similar to this, after being treated with two different types of ILs, the intensity of the band at 1340 that has been linked to OH deformation [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. When compared to the original cellulose, IR spectra of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) show no remarkable differences which is an indication of no chemical changes due to ILs treatment on BC [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e4.2 Scanning electron microscopy (SEM)\u003c/h2\u003e\n\u003cp\u003eThe structural morphology of BC after treatment with two types of ILs (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e, Py-HSO\u003csub\u003e4\u003c/sub\u003e) at concentrations of 75% and temp of 100\u0026deg;C was examined using Scanning Electron Microscopy, as seen in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The structure of BC (control) was smooth, with visible normal BC fibrous structure. Continuous nanofibers with a diameter of roughly 60 nm made up the BC membrane. The BC nanofibers displayed a smooth surface morphology and a three-dimensional porous network structure [44]. Microfibrils that are intertwined, securely fastened, and extremely robust constitute the surface morphology of BC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) [45].\u003c/p\u003e\n\u003cp\u003eFiber networks structured erratically or arbitrarily in three dimensions were converted into bacterial cellulose films by bacteria during fermentation. BC web ribbons range in length from 1 to 9 m and produce an extensive network of hydrogen bonding in a complex reticulation pattern serving as a stabilizer for framework [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Following IL treatment, morphological alterations occurred in both BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) membranes. The treatment of BC caused partial degradation in both cases. The fibrous structure of BC after treatment with BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) resulted in partial melting of the fibers and they became merged into one another (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, E, F). While after treatment with BMIM-HSO\u003csub\u003e4\u003c/sub\u003e, the fibrous structure of BC became thin and sowed some degradability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG, H, I). Because BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Py-HSO\u003csub\u003e4\u003c/sub\u003e are solvents in their own capacity therefore they have the potential to break hydrogen bonds inside BC fibers, resulting in amorphous and fragile-looking particles and fibers [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe two ionic liquids were utilized in three different concentrations (25%, 75%,100%) to treat BC. The BC sheets were not degradable after three hours at a concentration of 25% at 100\u003csup\u003eo\u003c/sup\u003eC. Similarly, no changes were seen using 75% at 100\u003csup\u003eo\u003c/sup\u003eC for 5, 10, or 15 minutes, but after 20 minutes, BC became porous and thinner. However further exposure for longer time disintegrated the fibrous structure followed by dissolution. When BC was treated with 100% ILs for 5, 10, and 15 minutes, it became thin and partly disintegrated and further exposure after 15 minutes completely disintegrated and dissolved. Therefore, in comparison to other concentrations the 75% of ILs at 100\u0026deg;C was considered to be more suitable for developing degradable BC without disturbing its integrity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e4.3 XRD Analysis of BC and ILs treated BC\u003c/h2\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e displays the diffractogram of BC after treatment with BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Py-HSO\u003csub\u003e4\u003c/sub\u003e. Three intense peaks can be seen in the diffraction patterns of 22.7\u0026deg;, 14.4\u0026deg;, 16.6\u0026deg; which denote the planar crystals of (100), (010), and (110). BC has a cellulose I crystal structure that created a triclinic structure [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. BC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) possesses a crystal structure indicative of cellulose type I and a high crystallinity (84\u0026ndash;89%) [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. After treatment with ionic liquid BMIM-HSO\u003csub\u003e4\u003c/sub\u003e, crystallinity of BC sheet was reduced at a concentration of 75% at 100℃. It demonstrates that crystallinity of BC was lost and relates to the morphology of the material that exhibits damage. After being treated with Py-HSO\u003csub\u003e4\u003c/sub\u003e, crystallinity of BC was lost at 75% conc. and 100℃ and the fibrous structure of BC became more distorted. After the treatments of two ILs, the crystallinity is disrupted. As a result of the reaction of ILs, the structure of BC becomes amorphous.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e4.4 Contact Angle Measurements\u003c/h2\u003e\n\u003cp\u003eA precise metric for detecting changes in surface characteristics of polymer is wettability [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Variations in the contact angle of a liquid on the surface are used to quantify wettability. Untreated BC surfaces typically have contact angles below 90\u0026deg; because they are hydrophilic. The contact angle of the untreated BC was 57\u0026deg; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). It is clear that the ILs treatments had a significant impact on the contact angle reduction, increasing the hydrophilic properties of the material (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; C). Py-HSO\u003csub\u003e4\u003c/sub\u003e treated BC showed contact angle of 46\u0026deg; and the contact angle of BC after treatment with BMIM-HSO\u003csub\u003e4\u003c/sub\u003e was 30\u0026deg;. Hydrophilic nature is attributed to more amorphous regions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e4.5 Swelling Studies of BC-ILs\u003c/h2\u003e\n\u003cp\u003eThe swelling or water absorption of the IL treated BC is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. All BC-ILs membranes quickly absorb water during the first three hours of the assay before gradually increasing for next 24 hrs. Bacterial cellulose shows a higher swelling ratio after being treated with ILs than it does without treatment. Initially the control BC membrane absorbed a significant amount of water (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) within an hr the BC membrane attained swelling ratio of 421.6%, which subsequently slightly rose to 513% after 2 hrs and 586% after 3 hrs and after 4 hrs 651% and 696% 5 hrs. The total swelling ratio achieved after 24 hrs was 718%. After one hr, the swelling ratio of BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) reached a value of 591% and then gradually increased to 743% after 2 hrs, and 816% after 3 hrs, 907% after 4 hrs and 951% after 5 hrs. BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) reached its maximum swelling ratio of 996% after 24 hrs. BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e), exhibited significantly higher swelling ratio than BC (Control) and BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e). The swelling ratio BC (BMIM-HSO\u003csub\u003e4)\u003c/sub\u003e reached 764% after one hour, and it gradually increased to 799% after 2 hrs, 968% after 3 hrs,1051% after 4 hrs and 1087% after 5 hrs and 1113% after 24 hrs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Both BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (PY-HSO\u003csub\u003e4\u003c/sub\u003e) swell at a rate that is noticeably higher than BC (control), which is associated with the hydrophilic character of the ILs [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. BC has a lower swelling ratio because of its increased crystallinity. The BC fiber structure is, however, degraded by ILs, and as a result, BC membranes treated with ionic liquids became amorphous. Therefore, BC treated with these ionic liquids has higher swelling rate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e4.6 \u003cem\u003eIn vitro\u003c/em\u003e Degradation Assays\u003c/h2\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the \u003cem\u003ein vitro\u003c/em\u003e degradation of BC and BC treated with ILs in PBS over various time periods (3, 7, 14, 21, 28 days) at 37℃. Weight loss percentages were determined according to the differences between dry weights of the treated BC. At all-time points, weight loss of BC (BMIM- HSO\u003csub\u003e4\u003c/sub\u003e) was significantly higher than BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e). Higher hydrophilicity of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) as compared to BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) may be the cause of the greater weight loss. After 28 days, BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) showed a weight reduction of 56%, while BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) had a weight loss of 36%.\u003c/p\u003e\n\u003cp\u003ePBS can cause BC micro-fibrils to swell many folds after some time. The C-O bonds in the BC molecular chains weaken and eventually break under the influence of ions and molecules of water from the area with weak bonding force. Similar mechanism of BC degradation in PBS was also explained in literature [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].The degradation rate of BC continuously increased up to 14 days and then became constant after a weight reduction of approximately 14.3%. This BC breakdown in PBS is primarily caused by the significantly high swelling of BC fibers [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. The treatment of bacterial cellulose membranes by Ionic liquids BMIM HSO\u003csub\u003e4\u003c/sub\u003e and PY-HSO\u003csub\u003e4\u003c/sub\u003e resulted in a significantly faster degradation over time (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The degradation of BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) was 9%, 18%, 21.6%, 27%, and 36% after 3, 7, 14, 21 and 28 days respectively. However, there was a significantly higher degradation in the case of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) compared to the BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e). After 3 days the degradation was 12.25%, after 7 days it was 21%, after 14 days it was 35%, after 21 days it was 49.1% and after 28 days it was 56.1%. These values were 20.1% higher than the BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) and were 41.8% higher than Control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e4.7 Drug Release Profile\u003c/h2\u003e\n\u003cp\u003eThe UV-VIS absorbance values for CAP and MNZ were recorded at specific wavelengths of 280 nm and 320 nm respectively. These absorbance values (y) of CAP and MNZ were used to draw standard curve using the equation (y\u0026thinsp;=\u0026thinsp;mx\u0026thinsp;+\u0026thinsp;c) to find the value of unknown concentrations. Quantitative UV-VIS spectrophotometry was performed to study the drug release profile of CAP and MNZ. From the standard straight-line curve, the unknown concentration of drug released was determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure\u0026nbsp;7 MNZ release profile of BC and ILs-BC(A), CAP release profile of BC and ILs-BC (B)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe time-dependent drug release from BC membranes treated with Py-HSO\u003csub\u003e4\u003c/sub\u003e and BMIM-HSO\u003csub\u003e4\u003c/sub\u003e was investigated using PBS and UV-VIS spectroscopy in the 200\u0026ndash;700 nm region. CAP and MNZ had lambda max of 320 nm and 278 nm, respectively. As illustrated in Fig.\u0026nbsp;7, the % cumulative drug release was used to study the release pattern over a three-day period. When compared to BC, membranes treated with ILs that were loaded with CAP and MNZ demonstrated sustained release. The chosen time period was 0.25, 0.5, 0.75, 1, 2, 3, 4, 24, 48, and 72 hrs in order to monitor the release profile. In the case of BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) loaded with MNZ, the treated membranes first demonstrated burst release of 31% within first 4 hrs, and then sustained release of 48% for the next 72 hrs. The BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) treated membranes showed 20% release after 4 hrs and after this time interval and sustained release of 37% after 72 hrs. Whereas, metronidazole loaded BC membranes showed 71% release within 72 hours (Fig.\u0026nbsp;7). CAP loaded BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) had a cumulative drug release of 51.09% after 3 hrs and 79% after 72 hrs. In the case of BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) treated membranes that had been loaded with CAP, the cumulative drug release was 37% after 3 hrs, and 72% after 70 hrs. On the contrary the BC (Control) loaded with CAP displayed burst release of 99.8% within 70 hrs (Fig.\u0026nbsp;7). Since, degradable membranes showed sustained release which is supportive of wound healing process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e4.8 Anti-Bacterial Studies\u003c/h2\u003e\n\u003cp\u003eThe effectiveness of CAP-loaded BC-ILs membranes on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e was examined. The inhibitory zones difference between pure ionic liquids treated BC membranes and membranes containing CAP was measured (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The CAP-containing BC membranes treated with BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Py-HSO\u003csub\u003e4\u003c/sub\u003e both shown antibacterial activity against the two model bacteria with inhibitory zones of 35 mm. Similarly, MNZ loaded Py-HSO\u003csub\u003e4\u003c/sub\u003e and BMIM-HSO\u003csub\u003e4\u003c/sub\u003e treated BC showed inhibitory zones of 40 mm on \u003cem\u003eE. coli\u003c/em\u003e as well as \u003cem\u003eS. aureus\u003c/em\u003e. The positive controls that are CAP and MNZ showed inhibitory zones of 22 mm on \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIt can be concluded that the antimicrobial activity seen in disc diffusion experiments is due to CAP and MNZ loaded into BC-ILs sheets. The drugs have a known effect on both pathogens and here it is indicative of the loading efficiency and sustained release behavior of BC-ILs sheets. It can thus be envisaged that these IL treated BC sheets can be used in wound healing applications using any drug.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e4.9 In Vitro Biocompatibility of BC-ILs membranes\u003c/h2\u003e\n\u003cp\u003eIn vitro cytotoxicity studies were performed using fibroblast NIH3T3 cells to determine whether the BC-ILs membranes and drug loaded BC-ILs membranes are biocompatible. The results of cytotoxicity studies of BC (control) and BC-ILs sheets are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. Cytocompatibility was measured after direct contact with the materials for 1, 3, and 7 days. BC is known for its biocompatibility, and the values as high as 95% from BC-ILs and drug-loaded BC sheets show that these are non-toxic and biocompatible too. All samples had 100% biocompatibility after the first day there was no statistical difference from the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) showed almost equal cell viability [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, MNZ loaded BC (Py-HSO\u003csub\u003e4\u003c/sub\u003e) and BC (BMIM-HSO\u003csub\u003e4\u003c/sub\u003e) show higher cell viability as compared to CAP loaded sheets. After 7 days of direct contact with the samples, there wasn't any significant difference between the cytocompatibility of BC (Control), BC-ILs and drug loaded BC-ILs. According to ASTM standards a biocompatibility higher than 70% is considered acceptable for the implant biomaterials.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBacterial cellulose sheets are non-degradable biomaterials, which limits its use as a regenerative implant material. In this research BC pellicles / sheets were made biodegradable by treating with ILs. Degradability of BC treated with ILs BMIM-HSO\u003csub\u003e4\u003c/sub\u003e and Py-HSO\u003csub\u003e4\u003c/sub\u003e have been confirmed by SEM, FTIR, XRD \u0026amp; degradation studies. FTIR confirmed that there was no chemical change after treatment with ILs and no residue remained after washing. SEM analysis showed that BC fibers became thinner and had been partially broken. Moreover, BC treated with ILs became more hydrophilic and developed sustained release profile of drugs that were loaded on them. Furthermore, \u003cem\u003ein vitro\u003c/em\u003e biocompatibility test confirmed that the IL treated BC was biocompatible and non-cytotoxic. Our results conclude that the BC sheets became not only biodegradable but also more hydrophilic and efficient drug carriers after treatment with ILs. Hence these BC-ILs can be used for tissue regenerative and wound healing applications, as degradable implants, drug carriers and tissue engineered scaffolds. Further \u003cem\u003ein vivo\u003c/em\u003e studies can be helpful in establishing the effectiveness as implant material.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the support of HEC under NRPU-7787 and Ministry of Science and Technology, Government of Pakistan for a developmental grant titled \u0026quot;Establishment of Center for Advance Technologies in Biomedical Material\u0026quot; under its knowledge economy initiative. We also extebd our sincere appreciation to the researchers supporting project RSPD2023R755 KSU,Ryadh, Saudi Arabia. Authors would also like to thank Dr Aqif Anwar Chaudhry and Dr. Zulifqar Ali for support during the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent for publication of the submitted manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was funded by Higher Edication Commission, Pakistant under Grant number HEC-NRPU-7787\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design specially the corresponding author Dr Faiza Sharif. Material preparation, data collection and analysis were performed by Muniba Muneer and Sadaf Nosheen. The first draft of the manuscript was written by Muniba Muneer and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eUllah, M.W., et al., \u003cem\u003eInnovative production of bio-cellulose using a cell-free system derived from a single cell line.\u003c/em\u003e Carbohydrate Polymers, 2015. 132: p. 286-294.\u003c/li\u003e\n\u003cli\u003eKhan, S., et al., \u003cem\u003ePreparation and structural characterization of surface modified microporous bacterial cellulose scaffolds: A potential material for skin regeneration applications in vitro and in vivo.\u003c/em\u003e International Journal of Biological Macromolecules, 2018. 117: p. 1200-1210.\u003c/li\u003e\n\u003cli\u003eKhalid, A., et al., \u003cem\u003eBacterial cellulose\u0026ndash;TiO 2 nanocomposites promote healing and tissue regeneration in burn mice model.\u003c/em\u003e 2017. 7(75): p. 47662-47668.\u003c/li\u003e\n\u003cli\u003eUl-Islam, M.J.C.p.d., \u003cem\u003eComparative synthesis and characterization of bio-cellulose from local waste and cheap resources.\u003c/em\u003e 2019. 25(34): p. 3664-3671.\u003c/li\u003e\n\u003cli\u003eShah, N., et al., \u003cem\u003eOverview of bacterial cellulose composites: A multipurpose advanced material.\u003c/em\u003e Carbohydrate Polymers, 2013. 98(2): p. 1585-1598.\u003c/li\u003e\n\u003cli\u003eUl-Islam, M., et al., \u003cem\u003eComparative study of plant and bacterial cellulose pellicles regenerated from dissolved states.\u003c/em\u003e International Journal of Biological Macromolecules, 2019. 137: p. 247-252.\u003c/li\u003e\n\u003cli\u003eUl-Islam, M., T. Khan, and J.K. Park, \u003cem\u003eWater holding and release properties of bacterial cellulose obtained by in situ and ex situ modification.\u003c/em\u003e Carbohydrate Polymers, 2012. 88(2): p. 596-603.\u003c/li\u003e\n\u003cli\u003eCzaja, W., D. Romanovicz, and R.m. Brown, \u003cem\u003eStructural investigations of microbial cellulose produced in stationary and agitated culture.\u003c/em\u003e Cellulose, 2004. 11(3): p. 403-411.\u003c/li\u003e\n\u003cli\u003eYamanaka, S., et al., \u003cem\u003eThe structure and mechanical properties of sheets prepared from bacterial cellulose.\u003c/em\u003e Journal of Materials Science, 1989. 24(9): p. 3141-3145.\u003c/li\u003e\n\u003cli\u003eSwingler, S., et al., \u003cem\u003eRecent Advances and Applications of Bacterial Cellulose in Biomedicine. Polymers 2021, 13, 412\u003c/em\u003e, 2021, s Note: MDPI stays neutral with regard to jurisdictional claims in published \u0026hellip;.\u003c/li\u003e\n\u003cli\u003eTorgbo, S. and P. Sukyai, \u003cem\u003eBiodegradation and thermal stability of bacterial cellulose as biomaterial: The relevance in biomedical applications.\u003c/em\u003e Polymer Degradation and Stability, 2020. 179: p. 109232.\u003c/li\u003e\n\u003cli\u003eTorres, F.G., S. Commeaux, and O.P. Troncoso, \u003cem\u003eBiocompatibility of Bacterial Cellulose Based Biomaterials.\u003c/em\u003e Journal of Functional Biomaterials, 2012. 3(4).\u003c/li\u003e\n\u003cli\u003eShpichka, A., et al., \u003cem\u003eSkin tissue regeneration for burn injury.\u003c/em\u003e Stem Cell Research \u0026amp; Therapy, 2019. 10(1): p. 94.\u003c/li\u003e\n\u003cli\u003eRahman, M.M. and A.N. Netravali, \u003cem\u003eAligned Bacterial Cellulose Arrays as \u0026ldquo;Green\u0026rdquo; Nanofibers for Composite Materials.\u003c/em\u003e ACS Macro Letters, 2016. 5(9): p. 1070-1074.\u003c/li\u003e\n\u003cli\u003eSajjad, W., et al., \u003cem\u003eDevelopment of modified montmorillonite-bacterial cellulose nanocomposites as a novel substitute for burn skin and tissue regeneration.\u003c/em\u003e Carbohydr Polym, 2019. 206: p. 548-556.\u003c/li\u003e\n\u003cli\u003eShao, W., et al., \u003cem\u003eTetracycline hydrochloride loaded regenerated cellulose composite membranes with controlled release and efficient antibacterial performance.\u003c/em\u003e 2016. 6(4): p. 3068-3073.\u003c/li\u003e\n\u003cli\u003eAlkhatib, Y., et al., \u003cem\u003eControlled extended octenidine release from a bacterial nanocellulose/Poloxamer hybrid system.\u003c/em\u003e European Journal of Pharmaceutics and Biopharmaceutics, 2017. 112: p. 164-176.\u003c/li\u003e\n\u003cli\u003eGupta, A., et al., \u003cem\u003eSynthesis of Silver Nanoparticles Using Curcumin-Cyclodextrins Loaded into Bacterial Cellulose-Based Hydrogels for Wound Dressing Applications.\u003c/em\u003e Biomacromolecules, 2020. 21(5): p. 1802-1811.\u003c/li\u003e\n\u003cli\u003eManoukian, O.S., et al., \u003cem\u003eBiomaterials for Tissue Engineering and Regenerative Medicine\u003c/em\u003e, in \u003cem\u003eEncyclopedia of Biomedical Engineering\u003c/em\u003e, R. Narayan, Editor 2019, Elsevier: Oxford. p. 462-482.\u003c/li\u003e\n\u003cli\u003eGinjupalli, K., et al., \u003cem\u003ePoly (\u0026alpha;-hydroxy acid) based polymers: A review on material and degradation aspects.\u003c/em\u003e 2017. 144: p. 520-535.\u003c/li\u003e\n\u003cli\u003eHasnain, M.S., et al., \u003cem\u003eBiodegradable polymer matrix nanocomposites for bone tissue engineering\u003c/em\u003e, in \u003cem\u003eApplications of nanocomposite materials in orthopedics\u003c/em\u003e2019, Elsevier. p. 1-37.\u003c/li\u003e\n\u003cli\u003eUlery, B.D., L.S. Nair, and C.T.J.J.o.p.s.P.B.p.p. Laurencin, \u003cem\u003eBiomedical applications of biodegradable polymers.\u003c/em\u003e 2011. 49(12): p. 832-864.\u003c/li\u003e\n\u003cli\u003eAmarasekara, A.S. and B. Wiredu, \u003cem\u003eDegradation of Cellulose in Dilute Aqueous Solutions of Acidic Ionic Liquid 1-(1-Propylsulfonic)-3-methylimidazolium Chloride, and p-Toluenesulfonic Acid at Moderate Temperatures and Pressures.\u003c/em\u003e Industrial \u0026amp; Engineering Chemistry Research, 2011. 50(21): p. 12276-12280.\u003c/li\u003e\n\u003cli\u003eBoisset, C., et al., \u003cem\u003eImaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A.\u003c/em\u003e 2000. 66(4): p. 1444-1452.\u003c/li\u003e\n\u003cli\u003eLi, J., et al., \u003cem\u003ePreparation and characterization of 2, 3-dialdehyde bacterial cellulose for potential biodegradable tissue engineering scaffolds.\u003c/em\u003e 2009. 29(5): p. 1635-1642.\u003c/li\u003e\n\u003cli\u003eLima, G.d.M., et al., \u003cem\u003eCharacterisation of bacterial cellulose partly acetylated by dimethylacetamide/lithium chloride.\u003c/em\u003e Materials Science and Engineering: C, 2011. 31(2): p. 190-197.\u003c/li\u003e\n\u003cli\u003eHallett, J.P. and T. Welton, \u003cem\u003eRoom-Temperature Ionic Liquids: Solvents for Synthesis and Catalysis. 2.\u003c/em\u003e Chemical Reviews, 2011. 111(5): p. 3508-3576.\u003c/li\u003e\n\u003cli\u003eSingh, S.K. and A.W. Savoy, \u003cem\u003eIonic liquids synthesis and applications: An overview.\u003c/em\u003e Journal of Molecular Liquids, 2020. 297: p. 112038.\u003c/li\u003e\n\u003cli\u003eSingh, S.K., \u003cem\u003eSolubility of lignin and chitin in ionic liquids and their biomedical applications.\u003c/em\u003e International Journal of Biological Macromolecules, 2019. 132: p. 265-277.\u003c/li\u003e\n\u003cli\u003eWelton, T.J.C.r., \u003cem\u003eRoom-temperature ionic liquids. Solvents for synthesis and catalysis.\u003c/em\u003e 1999. 99(8): p. 2071-2084.\u003c/li\u003e\n\u003cli\u003eIsik, M.S.H.M.D.I.L., C.M. Cellulose: Dissolution, and M. Preparation of New Cellulosic International Journal of Molecular Sciences, 2014. 15, 11922-11940 DOI: 10.3390/ijms150711922.\u003c/li\u003e\n\u003cli\u003eKiziltas, E.E., A. Kiziltas, and D.J.J.C.P. Gardner, \u003cem\u003eSynthesis of bacterial cellulose using hot water extracted wood sugars.\u003c/em\u003e 2015. 124: p. 131-138.\u003c/li\u003e\n\u003cli\u003eCosta, L.M.M., G.M. de Olyveira, and P.J.J.o.B. Basmaji, \u003cem\u003eBacterial cellulose towards functional green composites materials.\u003c/em\u003e 2011. 5(2): p. 167-172.\u003c/li\u003e\n\u003cli\u003eSpiridon, I., C.-A. Teacă, and R. Bod\u0026icirc;rlău, \u003cem\u003eStructural changes evidenced by FTIR spectroscopy in cellulosic materials after pre-treatment with ionic liquid and enzymatic hydrolysis.\u003c/em\u003e BioResources, 2011. 6: p. 400-413.\u003c/li\u003e\n\u003cli\u003eSuryanto, H., et al., \u003cem\u003eThe Mechanical Strength and Morphology of Bacterial Cellulose Films: The Effect of NaOH Concentration.\u003c/em\u003e IOP Conference Series Materials Science and Engineering (Online), 2019. 515(1): p. 7.\u003c/li\u003e\n\u003cli\u003eSuryanto, H., et al. \u003cem\u003eMorphology and structure of bacterial cellulose film after ionic liquid treatment\u003c/em\u003e. in \u003cem\u003eJournal of Physics: Conference Series\u003c/em\u003e. 2020. IOP Publishing.\u003c/li\u003e\n\u003cli\u003eAdamson, A.W. and A.P. Gast, \u003cem\u003ePhysical chemistry of surfaces\u003c/em\u003e. Vol. 150. 1967: Interscience publishers New York.\u003c/li\u003e\n\u003cli\u003eMorais, E.S., et al., \u003cem\u003eAnti-inflammatory and antioxidant nanostructured cellulose membranes loaded with phenolic-based ionic liquids for cutaneous application.\u003c/em\u003e Carbohydrate Polymers, 2019. 206: p. 187-197.\u003c/li\u003e\n\u003cli\u003eChen, Y., et al., \u003cem\u003eIn Vitro Structural Changes of Nano-Bacterial Cellulose Immersed in Phosphate Buffer Solution.\u003c/em\u003e Journal of Biomimetics, Biomaterials, and Tissue Engineering, 2011. 10.\u003c/li\u003e\n\u003cli\u003eVentura, S.P.M., et al., \u003cem\u003eImidazolium and Pyridinium Ionic Liquids from Mandelic Acid Derivatives: Synthesis and Bacteria and Algae Toxicity Evaluation.\u003c/em\u003e ACS Sustainable Chemistry \u0026amp; Engineering, 2013. 1(4): p. 393-402.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacterial cellulose, Ionic liquids, degradable biomaterials, tissue regeneration, drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-3223070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3223070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe purpose of this research was to create BC membranes / sheets which can be degraded by the enzymes in body fluids on implantation for soft and hard tissue regeneration. Bacterial Cellulose has been explored for its use in hard and soft tissue regeneration such as bone, dental, wound, hernia, dura mater, skin, nerve, cornea, and blood vessels. The limiting factor in the use of BC as biomedical implant material is that it is practically non-biodegradable in vitro and in vivo. However, reactive hydroxyl groups on BC allow a variety of chemical modifications which can be beneficial for the development of smart degradable biomedical materials. The use of Ionic Liquids (ILs) is the greener and non-toxic alternative to the chemical treatment for the degradation of BC. The ILs affect the degradability of BC by interacting with the functional groups and decreasing its crystallinity.\u003c/p\u003e\n\u003cp\u003eTwo non-toxic and biocompatible ILs i.e Pyridinium hydrogen sulfate (Py-HSO4) and 1-butyl-3-methyl imidazolium hydrogen sulfate (BMIM-HSO4) were used in the current study. The biodegradation of BC using these ILs has not been studied previously for biomedical implants. The characterizations of the IL treated BC were done using XRD, FTIR analysis, SEM, contact angle studies, degradation assay, drug delivery, and in vitro biocompatibility. SEM results suggest a clear change in the morphology of the BC nano fibers after treatment with ionic liquids. Furthermore, significant degradation was observed over 28 days where BC (Py-HSO4) degraded by 36% and BC (BMIM-HSO4) treated had degraded by 56%. Additionally, the IL treated BC could carry antibacterial drugs and showed potential for their sustained release. The modified membranes supported cell attachment and proliferation and were non-toxic and highly biocompatible. These results suggest that BC pellicles / sheets treated with ILs can be used as a degradable implant material for tissue engineering, regeneration, and drug delivery for various regenerative biomedical applications.\u003c/p\u003e","manuscriptTitle":"Ionic Liquid Treated Bacterial Cellulose Sheets as Prospective Biodegradable Implant Materials","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-17 17:34:30","doi":"10.21203/rs.3.rs-3223070/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-08-13T20:45:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-12T07:13:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-12T07:13:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellulose","date":"2023-08-01T06:30:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fb4f044a-deba-45b3-8a34-07307c949d0b","owner":[],"postedDate":"August 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-05T15:02:46+00:00","versionOfRecord":{"articleIdentity":"rs-3223070","link":"https://doi.org/10.1007/s10570-024-05741-y","journal":{"identity":"cellulose","isVorOnly":false,"title":"Cellulose"},"publishedOn":"2024-01-30 15:00:50","publishedOnDateReadable":"January 30th, 2024"},"versionCreatedAt":"2023-08-17 17:34:30","video":"","vorDoi":"10.1007/s10570-024-05741-y","vorDoiUrl":"https://doi.org/10.1007/s10570-024-05741-y","workflowStages":[]},"version":"v1","identity":"rs-3223070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3223070","identity":"rs-3223070","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}