Novel acryloylated and methacryloylated nanocellulose derivatives with improved mucoadhesive properties

In this work, three nanocellulose derivatives are synthesized with the aim of preparing new mucoadhesive materials. Nanocellulose is reacted with glycidyl methacrylate in dimethylsulphoxide, and with acryloyl and methacryloyl chloride in dimethylacetamide in the presence of 4-(N,N-dimethylamino)pyridine as a catalyst. These reactions are carried out under heterogeneous conditions, and the reaction products are characterized using various spectroscopic techniques, X-ray diﬀraction, atomic force microscopy, and thermogravimetric analysis. The Fourier-transform infrared spectra showed all the characteristic absorption bands typical for cellulose and also new peaks at 1720 cm − 1 for the carbonyl group (C ═ O) and 1639, 812 cm − 1 for the double bond (C ═ C). It is established that the crystal structure of the nanocellulose is slightly changed with derivatisation and the thermal stability of these derivatives increased. Mucoadhesive properties of nanocellulose and its derivatives is evaluated using the tensile test, rotating basket method, and ﬂuorescence ﬂow-through method. The retention of these polymers is evaluated on sheep oral mucosal tissue ex vivo using artiﬁcial saliva. Test results demonstrated that the new derivatives of nanocellulose have improved mucoadhesive properties compared to the parent nanocellulose.


Introduction
Cellulose is the most abundant natural and renewable biopolymer on earth.[13] NC can be extracted from different cellulose sources (wood, cotton, kenaf, bamboo, sisal, etc.) [14][15][16][17] using chemical, physical, enzymatic methods or their combination. [18,19]ellulose has three hydroxyl groups in its elementary structure, and the abundant hydroxyl functional groups in its macromolecules allow a wide range of functionalization via chemical reactions, leading to the development of various materials with tunable properties.Cellulose derivatives (carboxymethyl cellulose (CMC), methyl cellulose (MC), and hydroxypropyl methyl cellulose (HPMC)) are widely used for drug delivery in pharmacy, biomedicine and are considered as the first generation (nonspecific) of mucoadhesive polymers. [20]Some of them (for example, CMC) forms hydrogen bonds with mucosal membranes, and others (MC, HPMC) spread onto mucus and form the interpenetration layer with mucus gel through diffusion. [21]However, their adhesion to mucous membranes is often insufficiently strong.There are some strategies to enhance the mucoadhesive properties of conventional polymers.For example, the introduction of thiol groups into a polymer backbone allows for covalent anchoring to the mucus layer through the formation of disulfide bonds. [22]urthermore, the acryloylate, [23] methacryloylate, [24] boronate, [25] maleimide [26,27] and aldehyde [28] groups improve mucoadhesive properties of polymers.The acryloyl group has a reactive double bond that can form a covalent linkage with the free thiol groups of cysteine residues found in mucin glycoproteins via thiol-ene click reactions occurring under physiological conditions.NC as well as microcrystalline cellulose (MCC) can be used as an inert filler, binder, and drug delivery vehicles in pharmaceutics, [9,29] and it can be adapted for the design of mucoadhesive formulations. [30]The conjugation of the above functional groups to the NC backbone will enhance its mucoadhesive capabilities.Although the NC unit cell has three active hydroxyl groups, these hydroxyl groups play a major role in forming hydrogen bonds in the formation of fibrils, and numerous intramacromolecular hydrogen bonds limit the solubility of cellulose in many solvents.So, the majority of reactions used for the synthesis of cellulose derivatives (CMC, MC, and HPMC) usually are conducted in heterogeneous conditions.
In the present study, we synthesized acryloylated nanocellulose (NC-ACh) and methacryloylated nanocellulose (NC-MACh) derivatives and investigated their stricture and mucoadhesive properties using freshly excised sheep oral mucosa.To the best of our knowledge, this is the first study reporting the enhancement of mucoadhesive properties of NC using these strategies.

Results and Discussion
Three different approaches have been undertaken to introduce unsaturated groups into the structure of NC using reactions with glycidyl methacrylate, methacryloyl chloride and acryloyl chloride as shown in Figure 1.
For the esterification of the sterically hindered hydroxyl group electron-rich pyridines such as DMAP provide supe-rior levels of catalytic turnover. [39,40]There are two reaction routes that explain chemical modifications of natural and synthetic polymers through the use of the GMA: transesterification and epoxide ring-opening mechanisms depending on reaction conditions. [41]n the reactions of NC with acryloyl and methacryloyl chlorides, DMAP plays the role of a nucleophilic agent that promotes the formation of the (meta)acryloylpyridinium salt intermediate. [42]This intermediate can be attacked by hydroxyl groups of cellulose more easily, and DMAP is eliminated for the next catalytic cycle.
The H 1 NMR spectra of NC and its resulting derivatives (Figure 2) show the distinctive peaks typical for cellulose at  3.0-4.2ppm. [43,44]With the cellulose derivatives, the new peaks at 2.25-2.75ppm and additional peaks at 6.25 ppm are observed responsible for the protons that belong to unsaturated C═C groups of GMA, ACh, and MACh.Additionally new peak at 2.7 ppm is observed in all three derivatives due to the protons of ═CH 2 group.Furthermore, a new peak appears in NCC-GMA conjugate at 2.3 ppm due to the protons of C─CH 2 -group.
FT-IR spectra show characteristic absorption bands for NC (Figure 3) at 3400 cm -1 , which are related to the stretching vibrations of O-H, at 1420, 1335, 1202, 1075-1060 cm −1 corresponding to the bending vibrations of ─CH─, ─CH 2 ─, ─OH, ─CO, the stretching vibrations of C─O and the pyranose rings. [14]The appearance of new bands in the FTIR spectra of NC derivatives at 1721 cm −1 is attributed to the C═O stretching    frequency of conjugated ester groups, [45] and at 1650, 813 cm −1 indicate a double C═C bond. [33,46]The degrees of substitution were calculated using FTIR spectra by subtracting the spectra and integrating the absorption bands of the respective groups.
The physicochemical characteristics of the synthesized derivatives are presented in Table 1.
The results of UV spectroscopic studies show (Figure 4) that there are three absorbance peaks at ≈196, 226, and 290 nm associated with the C═O of the carboxyl groups (at 240, 290 nm) and the carboxyl groups (at 190-210 nm).As can be seen from the UV spectra of NC derivatives, electronic transitions -* and n-* of С═О and С═С conjugated bonds are observed, which differ in intensity, in relation to the selection rules for electronic transitions.In the region of 190-230 nm, absorption bands observed are associated with -* transitions of electrons and a bathochromic shift towards long wavelengths occurs with an increase in the number of conjugated bonds in the chain of macromolecules.The absorption bands at 260-290 nm are associated with n-* electronic transitions of conjugated bonds and carbonyl groups.UV spectroscopic studies have shown that there is a shift of absorption bands to the long wavelength region, which is associated with conjugated bonds.The absorption band at 350 nm is associated with n- * electronic transitions of C═O groups, but due to the number of these groups, according to Woodward's law, [47] they are shifted to the long wavelength region.
To confirm the presence of unsaturated C═C groups qualitatively a reaction with potassium permanganate was used in aqueous suspensions of NC derivatives.Mixing these derivatives with potassium permanganate solutions results in color changes from purple to brown, which confirms the successful modification of  cellulose. [34]The use of unmodified NC as a control does not result in the color changes.
The crystal index of NC derivatives was changed and they had 88% for NC, 82% for NC-GMA, 73% for NC-MACh and 63% for ACh.The modification process of NC partially disrupted the cellulose crystal structure. [48]The interplanar distances (d) of NC derivatives crystals have also increased (Table 2).
This also confirms the interplanar distances (d), whose values increase in the functionalized NC sample.The results of Xray diffraction analysis show that the functionalization process affects the size of NC crystallites anisotropically, since the size of crystallites increases in one direction but does not change in another direction, which may be due to the accessibility of the surface of NC crystallites for the modifying reagent (GMA, ACh, and MACh).The chemical modification is supposed to begin at the surface of cellulose crystallites and then gradually move anisotropically into deeper layers of the crystalline structure.The use of a smaller size of the modifying reagent results in a greater degree of substitution and a lower degree of sample crystallinity.
The TGA thermograms of NC and its derivatives (Figure 6) showed that the weight loss for all samples proceeds in three stages.The first weight loss event upon heating to 100 °C (5-9%) is possibly related to the removal of moisture. [48,49]he second stage is probably due to the decomposition process related to the thermal oxidation of cellulose at 200-300 °C.Although cellulose derivatives usually have a lower decomposition temperature than the parent cellulose, [33,50] in our case, NC derivatives have a higher degradation temperature than NC.It is likely due to the presence of unsaturated C═C bonds in NC derivatives.An increase in temperature leads to the polymerization of these unsaturated bonds and the formation of cross-linked structures, whose degradation temperature will be higher. [51]Samples with a higher degree of substitution of NC have greater decomposition temperatures.
The AFM study showed that NC particles have a needle-like shape with a width of 20-80 nm and a length of 180-600 nm (Figure 7).
The size and shape of NC particles are substantially altered following the modification, resulting in the formation of  elongated ellipsoid particles that range in size from 200 to 350 nm.It appears that this is because the side groups of cellulose macromolecules grow in size during modification, which causes crystallites to enlarge in a direction perpendicular to the axis of macromolecules.

Retention on Sheep Oral Mucosa
The retention studies on different mucosal surfaces were described in previous publications. [26,52]In our work we carried   out experiments on freshly excised sheep oral mucosa, irrigated with artificial saliva.Fluorescent images of these samples are presented in Figure 8.
The superior mucoadhesive behavior of all NC derivatives as shown in Figure 9 is likely due to the presence unsaturated (meth)acrylate groups that can form covalent bonds with thiols of mucin present on the mucosal surface. [53,54]

Detachment from Sheep Oral Mucosa
In this method we studied the detachment of the synthesized NC derivatives from sheep oral mucosa.The force of detachment or adhesive strength is the force required to overcome the adhesive bonds between the polymer materials and mucosa, while the total work of adhesion is the area under the force-distance curves. [37]he results showed that NC-GMA, NC-MACh, and NC-Ach were 2-6 times more mucoadhesive compared with the unmodified NC (Figure 10).The NC-ACh sample has the highest mucoadhesiveness that is related to its degree of substitution.Overall, the adhesive force of the polymers correlated well with their work of adhesion values as NC-MACh and NC-ACh exhibited greater force of detachment and work of adhesion relative to the neat NC.

Rotating Cylinder Method to Evaluate Mucoadhesive Properties
In order to evaluate the binding to the mucosa as well as the cohesiveness of the tablets, an appropriate method has been used as described by the Bernkop-Schnurch group earlier. [38]In this method the tablets were attached to the mucosal tissues secured on a rotating cylinder immersed into artificial saliva solution at 37 °C.The detachment or desintegration time of these tablets was recorded visually.The results of mucoadhesion studies performed using the rotating cylinder method are shown in Figure 11.
The adhesion time of the NC-ACh tablets was 9.0-fold greater, and the adhesion time of the NC-MACh tablets was 6.0-fold greater than the adhesion time of the unmodified NC.It can be explained by the formation of covalent bonds between the acryloyl groups and the thiol groups of the mucus.
All mucoadhesion studies showed that the new NC derivatives have good mucoadhesive properties and they are significantly more adhesive compared with the unmodified NC.Based on the mucoadhesiveness, all samples could be arranged in the following order: NC-ACh>NC-MACh>NC-GMA>NC.This suggests that the modified NC derivatives could be exploited further for transmucosal drug delivery due to their superior mucoadhesive features.

Conclusion
In this study glycidyl methacrylate, acrylate, and methacrylate groups were grafted onto NC through the heterogeneous reaction using dimethyl aminopyridine as a catalyst to synthesize novel mucoadhesive polymers. 1H NMR, FTIR and permanganate analysis confirmed the successful synthesis of the NC derivatives.X-ray, FTIR and TGA analysis showed that that the smaller the size of the modifying reagent, the higher the degree of substitution and the lower the degree of crystallinity.The synthesized novel NC derivatives have greater mucoadhesive properties on the sheep oral mucosa than the parent NC.New NC derivatives with enhanced mucoadhesive properties could be of interest for application in transmucosal drug delivery.
These mucoadhesive NC derivatives may be used in the future to develop formulations with various active pharmaceutical Macromol.Biosci.2024, 2400183

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ingredients.To the best of our knowledge, this is the first study reporting the chemical modification of NC with the aim to enhance its mucoadhesive properties.
Synthesis of Nanocellulose Derivatives-Synthesis of Glycydylmethacrylated Nanocellulose (NC-GMA): NC-GMA was synthesized by reacting NC with glycydylmethacrylate using a published method with a slight modification. [32]Briefly, NC (10.0 g) was suspended in DMSO (250 mL) in a 0.5 L round-bottom flask equipped with a cover reflux condenser under a nitrogen atmosphere.The suspension was stirred during 1 h at 100 °C to activate NC.Then the temperature of the suspension was decreased to 40 °C and DMAP (0.2 g), hydroquinone (2.0 mL of 2% solution in ethanol) and GMA (0.95 mL) were added.The suspension was stirred at room temperature for 72 h, after which the reaction was terminated by adding an equimolar amount of concentrated HCl to neutralize DMAP.Then the suspension was centrifugated and the product was separated.The product was dispersed in deionized water, and purified by dialysis against 4.5 L deionized water with 6 water changes over 72 h.The final product was freeze-dried using HetoPowerDry LL3000 Freeze Dryer (Thermo Scientific, UK).
Synthesis of Acryloylated Nanocellulose (NC-ACh) and Methacryloylated Nanocellulose (NC-MACh): NC-ACh and NC-MACh were synthesized by reacting NC with acryloyl chloride and methacryloyl chloride, respectively, using a reported protocol with some modifications. [33]Briefly, NC (10.0 g) was suspended in DMAc (250 mL) and stirred at 100 °C for 1 h in 0.5 L round-bottom flask equipped with a cover reflux condenser.The slurry was then cooled to 40 °C, and DMAP (0.2 g), hydroquinone (2.0 mL of 2%solution in ethanol) and either AСh (0.75 mL) or MACh (0.9 mL) were added.The suspension was stirred at room temperature for 72 h under a nitrogen atmosphere, after which the reaction was terminated by adding an equimolar amount of concentrated HCl to neutralize DMAP.After the suspension was centrifugated and the products were separated these were dispersed in deionized water and transferred to a dialysis tube and extensively dialyzed for 72 h against deionized water at 25 °C.The final products were lyophilized using HetoPowerDry LL3000 Freeze Dryer (Thermo Scientific, UK).
Characterization of Nanocellulose Derivatives-1H Nuclear Magnetic Resonance Spectroscopy (1H NMR): Solutions of NC derivatives (0.3% w v −1 ) were prepared in TFA and allowed to be dissolved overnight at room temperature.The 1 H NMR spectra were recorded using 400 MHz Ultrashield Plus B-ACS 60 spectrometer (Bruker, UK).
Fourier Transform Infrared (FT-IR) Analysis: FT-IR spectra of dry samples were recorded with an Inventio-S IR Fourier (Bruker, Germany) using an attenuated total reflectance technique.The spectra were obtained in the range 4000-500 cm −1 at room temperature, with a resolution of 4 cm −1 and 16 scans.

X-Ray Diffractometry:
The influence of the functionality of samples on the NC crystallinity was evaluated using XRD Miniflex 600 (Rigaku, Japan) with monochromatic CuK radiation isolated by a nickel filter with a wavelength of 1.5418 Å at 40 kV and the current strength of 15 mA.Solid samples of NC, NC-GMA, NC-ACh, and NC-MACh were examined in the form of a powder, scanning from 2°to 70°with a scan step of 0.02°, generating characteristic diffractograms at the rate of 2.5 scans min −1 .The data processing of experimental diffraction patterns, peak deconvolution, describing the peaks used by Miller indices, peak shape, and the basis for the amorphous contribution were conducted using SmartLab Studio II software.
Atomic Force Microscopy (AFM): Morphological studies of NC and its derivatives were performed by using AFM Agilent 5500 (Agilent, USA).The silicon cantilevers with a stiffness of 9.5 N m −2 were used and the frequency was 262 kHz.The AFM scan area (x -y -z) was 3.0 -3.0 -1 μm.
Thermogravimetric Analysis (TGA): Thermal analysis of the dry samples was carried out with TG-DSC/DTA synchronous thermal analyzer STA PT1600 (Linseis, Germany) by heating ≈20 mg of each sample in an air atmosphere at a heating rate of 10 °C min −1 from 25 °to 900 °C.The samples were loaded in aluminium pans.
UV Spectroscopy: UV spectra of NC and its derivatives were recorded with a Specord 210 UV-spectrophotometer (Analytic Jena, Germany) by using quartz cells 1 cm in diameter and 1 nm slit; the scanning range of measurement was 190-1000 nm, a scanning speed was 5 nm s −1 .The spectra were recorded using 1% aqueous solutions of the samples.
Permanganate Test to Qualify Unsaturated C═C groups: The quality analysis of unsaturated groups in NC derivatives was carried out using previously published method with slight modifications. [34]Briefly, 0.1 g NC derivative was suspended in 20 mL deionized water and left stirring for 2 h.To this suspension 3 mL of 0.1 N potassium permanganate was added, followed with the addition of 4 mL of 0.1 N oxalic acid from a microburette and the suspension was stirred for 15 min.This resulted in a change of solution color from purple to brown, which was used as a titration endpoint.The presence of small quantities of unsaturated bonds in the NC derivatives resulted in reduction of some MnO 4 − ions to Mn 2+ , which act as a catalyst and speed up the reaction of permanganate ions with oxalic acid added subsequently.
Ex Vivo Sheep Mucoadhesion Studies-Preparation of Artificial Saliva Fluid: Artificial saliva fluid used to wash a mucosal surface was prepared as reported previously. [35]It was composed of NaCl (0.43 g), CaCl 2 (0.22 g), KCl (0.75 g), NaHCO 3 (0.20 g) and KH 2 PO 4 (0.9 g) dissolved in 1 L of deionized water, pH 6.75 and the solution was kept at 37 °C throughout the experiments.
Fluorescent Labelling of Nanocellulose Derivatives: NC and its derivatives were labelled with fluorescein isothiocyanate (FITC) using a slightly modified reported procedure. [36]Briefly, 1 g of NC derivative (NC-GMA, NC-ACh, NC-MACh) was suspended in 100 mL DMSO and the mixture was stirred for 30 min at 90 °C under nitrogen atmosphere until it was homogeneously mixed.0.01 g of fluorescein isothiocyanate was added and stirred for 10 min and 0.3% of dibutyltin dilaurate was added subsequently.The mixture was further stirred for 4 h at 90 °C.The product was washed with acetone 3 times and the precipitate was dialyzed in deionized water for 3 days.The dialyzed product was centrifuged and separated from liquid.The product was then freeze-dried.
Retention on Sheep Oral Mucosa: Fluorescence microscopy (MZ10F microscope; Leica Microsystems, Milton Keynes, UK), coupled to an "ET GFP" filter camera (Zeiss Imager A1/AxioCamMRm camera, 1296 × 966 pixels, 0.8 × magnification) was used to investigate the mucosal retention of fluorescein sodium in the presence of the polymeric carriers based on a slightly modified protocol reported by Kolawole et al. [37] Freshly excised sheep oral tissue stored on ice was used in this study within 24 h of procurement.The mucosal side of the oral tissue was preserved during excision of the required section (≈1.5 × 2.5 cm) and rinsed with artificial saliva solution (∼3 mL) prior to blank tissue imaging.The mucosal tissue was placed on a 75 mm × 25 mm glass slide and maintained in an incubator at 37 °C during saliva wash-out.Initially, fluorescence images of mucosal tissues were recorded for each sample as

Figure 1 .
Figure 1.Reaction scheme for the synthesis of NC-GMA, NC-ACh, and NC-MACh.

Figure 9 .
Figure 9. Mucosal retention of FITC-labeled NC-GMA, NC-MACh, and NC-Ach on sheep oral tissue; FITC-dextran served as a negative control and FITC-NC (unmodified NC) as a positive control.Result presented as mean ± standard deviation, n = 3, *depicts statistically significant differences between samples (p < 0.05).

Table 1 .
Synthetic yield, physical properties and degrees of substitution (determined using FTIR spectroscopy) of NC derivatives.

Table 2 .
Structural parameters of NC and NC derivatives.