Ahmed Salama

Peter Hesemann

Synthesis and characterization of chitosan-N-guanidinium/silica ionic hybrids as templates for calcium phosphate mineralization

Keywords:

HAL is

Graphical Abstract

Introduction.

Calcium phosphate with controlled morphologies and hierarchical structure recently triggered extensive research in the area of biomaterials [START_REF] Schweizer | Polymer-controlled, bio-inspired calcium phosphate mineralization from aqueous solution[END_REF][START_REF] Salama | Cellulose/calcium phosphate hybrids: New materials for biomedical and environmental applications[END_REF]. Organic matrix mediated biomineralization using templates such as collagen and amino acids were found to modulate efficiently calcium phosphate mineralization in living organisms [START_REF] Liu | Intrafibrillar collagen mineralization produced by biomimetic hierarchical nanoapatite assembly[END_REF].

Moreover, anionic non-collagenous proteins are reported to induce hydroxyapatite deposition within collagen fibers [START_REF] Hu | Development of Biomimetic Scaffolds with Both Intrafibrillar and Extrafibrillar Mineralization[END_REF]. Due to the limitations and high cost of noncollagenous proteins, several attempts to mimic these proteins via the use anionic polymers like poly(acrylic acid) and poly(aspartic acid) were reported [START_REF] Ogiwara | Calcium Phosphate Mineralization in Cellulose Derivative/Poly(acrylic acid) Composites Having a Chiral Nematic Mesomorphic Structure[END_REF][START_REF] Salama | Carboxymethyl cellulose-g-poly (acrylic acid)/calcium phosphate composite as a multifunctional hydrogel material[END_REF]. Recently, it was shown that functionalized polysaccharides with appropriate biocompatibility and biodegradability can exert a strong influence on the external morphology and crystalline structure of inorganic mineralization [START_REF] Schweizer | Polymer-controlled, bio-inspired calcium phosphate mineralization from aqueous solution[END_REF][START_REF] Coleman | Hydroxyapatite mineralization in the presence of anionic polymers[END_REF][START_REF] Salama | Ionic liquid-assisted formation of cellulose/calcium phosphate hybrid materials[END_REF].

Chitosan, i.e. deacetylated chitin, is a linear polysaccharide composed of glucosamine and N-acetylglucosamine units linked by β (1-4) glycosidic bonds. Chitosan is among the most studied polysaccharides and can chemically be modified to obtain derivatives with improved biological properties. The presence of primary amine groups as well as primary and secondary hydroxyl groups allows its derivatization and the introduction of functional groups via covalent or ionic bonding with silica precursors [START_REF] Connell | Chemical characterisation and fabrication of chitosan-silica hybrid scaffolds with 3-glycidoxypropyl trimethoxysilane[END_REF][START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF][START_REF] Salama | New N-guanidinium chitosan/silica ionic microhybrids as efficient adsorbent for dye removal from waste water[END_REF]. Due to their biocompatibility, biodegrability, anti-bacterial activity and cellular compatibility, chitosan derivatives are promising candidates for applications in the biomedical field [START_REF] Thakur | Recent advances in graft copolymerization and applications of chitosan: A Review[END_REF]. However, in the area of biomineralization, chitosan-based frameworks showed low efficiency for controlled nucleation and growth of inorganic materials, especially calcium phosphate, because of their smooth surface [START_REF] Malafaya | Bilayered chitosan-based scaffolds for osteochondral tissue engineering: Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor[END_REF][START_REF] Wang | Construction of a fluorescent nanostructured chitosan-hydroxyapatite scaffold by nanocrystallon induced biomimetic mineralization and its cell biocompatibility[END_REF].

Only few studies have been reported which evaluate the potential of chitosan as template for controlling the biomineralization processes. For example, Wang et. al.

prepared specific hierarchical structures from chitosan/calcium phosphate nanocomplexes through ionotropic gelation using tripolyphosphate as both cross linking agent and phosphate precursor. The porous texture of the prepared hybrid material results in a high efficiency in dye adsorption and enzymatic catalysis [START_REF] Wang | Facile one-pot preparation of chitosan/calcium pyrophosphate hybrid Micro flowers[END_REF].

Another trial was carried out by using genipin as crosslinking reagent to access hydroxyapatite coated genipin-chitosan scaffolds. The results showed that the mineralized hydroxyapatite nanocrystals mechanically reinforce the produced materials. Moreover, the biomimetically synthesized hydroxyapatite influences the cellular morphology and the cytoskeleton organization of rat bone marrow-derived mesenchymal stem cells [START_REF] Wang | Construction of a fluorescent nanostructured chitosan-hydroxyapatite scaffold by nanocrystallon induced biomimetic mineralization and its cell biocompatibility[END_REF].

The guanidinium moiety is an important active unit for many biological receptors especially those for RNA [START_REF] Calnan | Argininemediated RNA recognition: the arginine fork[END_REF]. It can be found in many drugs with numerous therapeutic applications and biological activities such as antidiabetic [START_REF] Hu | Synthesis, characterization and antibacterial activity of guanidinylated chitosan[END_REF] and broadspectrum antimicrobial drugs [START_REF] Sang | Synthesis and preservative application of quaternized carboxymethyl chitosan containing guanidine groups[END_REF]. Moreover, the guanidinium group can form ionic interaction with a wide range of anionic functional groups like carboxylate and phosphate as well as hydrogen bonding [START_REF] Sasaki | Specific, multiple-point binding of ATP and AMP to a guanidinium-functionalized monolayer[END_REF]. Several trials have been carried out to prepare guanidinium functionalized chitosan via the reaction between chitosan and arginine [START_REF] Zhang | Preparation of arginine modified PEIconjugated chitosan copolymer for DNA delivery[END_REF] or through direct guanylation between chitosan and cyanamide [START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF][START_REF] Salama | New N-guanidinium chitosan/silica ionic microhybrids as efficient adsorbent for dye removal from waste water[END_REF].

The sol-gel technique has been widely used to produce functional hybrid materials because it has allowed overcoming the difficulty of using the classical silica glassbased biomaterials for manipulating the internal microstructure of the matrix as well as avoiding high temperature [START_REF] Iafisco | Silica gel template for calcium phosphates crystallization[END_REF]. A huge number of organic functional groups can be incorporated within silica matrices via the sol-gel process, ranging from active pharmaceutical ingredients to polysaccharides [START_REF] Salama | Carboxymethyl cellulose/silica hybrids as templates for calcium phosphate biomimetic mineralization[END_REF] and synthetic polymers [START_REF] Pirzada | Hybrid Carbon Silica Nano fi bers through Sol -Gel Electrospinning[END_REF] up to biological entities, like peptides [START_REF] Jedlicka | Surface analysis by X-ray photoelectron spectroscopy of sol-gel silica modified with covalently bound peptides[END_REF], with the aim of providing enhanced functionality to the resulting biomaterials.

In this article, we report new chitosan/silica hybrid materials for calcium phosphate mineralization. Firstly, the guanylation of neat chitosan was carried out in acidic aqueous solution using scandium(ӀӀӀ) triflate as a catalyst. Then, this cationic Nguanidinium chitosan acetate was treated with two anionic silica precursors to form new N-guanidinium chitosan/silica ionic hybrids via sol-gel chemistry. The formation of these supramolecular hybrids consisting of N-guanidinium chitosan acetate and hybrid silica result from a high affinity between these two components which is based on ionic interactions between the cationic guanidinium groups of chitosan and the anionic functions of the silica precursors. Finally, we investigated the activity of the two ionic hybrids toward calcium phosphate mineralization from simulated bidy fluid (SBF) solutions. This strategy towards three-dimensional ionic templates increases the density of functional groups that act as nucleation sites toward inorganic mineralization. The chemical composition, morphology, internal crystal structure and content of the biomimetically mineralized calcium phosphate was investigated as a function of the quantity of sulfonate or carboxylate groups in intermediate ionic hybrids.

Materials and methods.

Materials

Medium molecular weight chitosan (degree of deacetylation, 75-85%), acetic acid, cyanamide, scandium(III) triflate were obtained from Sigma-Aldrich. Carboxyethylsilanetriol, sodium salt; 25% in water and 3-(trihydroxysilyl)-1propansulfonic acid, 30-35% in water were supplied from ABCR GmbH. All chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared using deionized water.

Syntheses

N-guanidinium chitosan acetate (GC)

. 10 ml of a 2% solution of chitosan in 2% acetic acid was mixed with 84 mg cyanamide (0.002 mol) and 30 mg (60 nmol) of scandium(III) triflate. The solution was stirred at 100°C for 48 h and the resulting mixture was precipitated and washed repeatedly with acetone. The guanuylated material GC was obtained as a white powder.

GC/silica containing sulfonic acid and carboxylate groups ionic hybrids (CGSS and CGSC)

. N-guanidinium chitosan/silica containing sulfonic and carboxylic acid ionic hybrid were synthesized in two steps. In the first step, to a solution of material GC (0.4 g) in 100 ml of deionized water 2 equivalents of 3-(trihydroxysilyl)-1-propanesulfonic acid or carboxyethylsilanetriol, sodium salt were added under vigorous stirring. Then, 0.02 M HCl solution (10 mL) was slowly added into the alkoxide solution and the mixture was heated at 60 °C for 48 h. Finally, the resulting white suspension was filtered. The precipitates of CGSS and CGSC were thoroughly washed with deionized water until neutral pH and finally washed with methanol followed by freeze-drying for 24 h.

Calcium phosphate biomineralization.

Biomimetic calcium phosphate mineralization was carried out as described in our previous study [START_REF] Salama | Chitosan based hydrogel assisted spongelike calcium phosphate mineralization for in-vitro BSA release[END_REF]. The ionic hybrids CGSC and CGSS were soaked in doubly concentrated simulated body fluid (2 x SBF) to enhance the biomimetic calcium phosphate nucleation. The biomimetic mineralization process continued for 7 days after soaking the ionic hybrids in 100 mL 2 x SBF with continuous renewed every 24 h. The mineralized CGSC and CGSS hybrids were washed with water then dried in a freeze-drier.

Characterization methods.

FT-IR Spectroscopy.

The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of materials were obtained using a Thermo Nicolet FT-IR Avatar 320 with a diamond crystal Spectra were recorded from 500 to 4000 cm -1 .

2.4.2.

Solid state NMR spectroscopy. 29 Si-NMR spectra were recorded on a Varian VNMRS 400 MHz spectrometer using a two-channel probe with 3.2 mm ZrO 2 rotors.

Results and discussion

Methodology

In a first step, N-guanidinium chitosan acetate (GC) was synthesized through a direct guanylation reaction between chitosan and cyanamide in the presence of scandium (III) triflate as reported in our previous studies [START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF][START_REF] Salama | New N-guanidinium chitosan/silica ionic microhybrids as efficient adsorbent for dye removal from waste water[END_REF]. The two different anionic silica precursors, carboxyethylsilanetriol sodium salt and 3-(trihydroxysilyl)-1propansulfonic acid, have been used to form GC-silica hybrid composites via a solgel reaction. Ionic interactions between GC and the silica precursors ensured high affinity between both components and led to the formation of N-guanidinium-chitosan acetate/silica containing sulfonate groups (CGSS) and N-guanidinium-chitosan acetate/silica containing carboxylate groups (CGSC). The bioactivity of the two hybrid materials was finally investigated toward biomimetic calcium phosphate mineralization, thus yielding the mineralized hybrid materials containing calcium phosphate CGSS-CP and CGSC-CP. All materials were characterized to monitor the chemical and morphological modifications induced by i) the sol-gel reaction and ii) the calcium phosphate mineralization process.

IR spectroscopic analyses

The FT-IR spectra of chitosan and N-guanidinium-chitosan together with those of the CGSC and CGSS ionic hybrids and mineralized hybrids are shown in figure 1. The spectra of chitosan and CG are virtually similar and show the same characteristic bands of the chitosan skeleton. The broad band at 3450 cm -1 can be attributed to NH, OH symmetric stretching vibration and inter-and intra-molecular hydrogen bonds of chitosan molecules. The weak band at 2921 cm -1 is ascribed to the C-H stretching vibrations of chitosan and the characteristic peaks at 1656 and 1580 cm -1 are due to the C=O stretching (amide I) and -NH stretching ( amide II), respectively. However, the increase of the intensity of the adsorption band at 1630 cm -1 observed in the spectrum of CG suggests the presence of guanidinium moiety as shown in figure

1 [START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF].

The spectrum of GC After the sol-gel reaction with the two silica hybrid precursors show strong absorption bands between 1100 and 1000 cm -1 which can be assigned to the siloxane networks (Si-O-Si) [START_REF] Han | Sol-gel-derived organicinorganic hybrid materials[END_REF]. The presence of these typical silica peaks in the spectra of two ionic hybrids (GCSS and GCSC) confirms the formation of a silica network around the N-guanidinium-chitosan acetate polymer chains. Furthermore, GCSS and GCSC ionic hybrids showed a broad band at 3200-3500 cm -1 that can be assigned to the silanol groups, localized on the surface of the silica hybrid material due to the incomplete condensation reaction under acid-catalyzed conditions. This incomplete condensation in the silica is beneficial for improving the compatibility between silica and N-guanidinium-chitosan acetate through hydrogen bonding. After calcium phosphate mineralization by immersing the two ionic hybrids in simulated body fluid (SBF) solution, the two mineralized hybrids exhibited the characteristic bands of phosphate group vibration modes. The peaks at 1033 and 1180 cm -1 (P-O ν 3 mode), 569 cm -1 (P-O ν 4 mode), and 904 cm -1 (P-O ν 1 mode) are assigned to CGSC-CP. In addition, nearly the same vibration modes were also found in the spectrum of the sulfonate sample CGSS-CP. These results confirm that a mineralization process took place.

Solid state NMR spectroscopy

The solid state NMR spectroscopic analysis allows monitoring the chemical modification of the materials. Here, we can get information about the chemical modification of chitosan via guanylation, resulting in the formation of the material GC, and, in a second time, the formation of the chitosan/silica hybrid composites CGSC and CGSS.

The spectra of neat chitosan and GC are given in the supporting information. As already reported in our previeous work [START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF], the appearance of a signal at a chemical shift of =160 ppm in the 13 C CP-MAS spectrum of GC reflects the presence of the newly formed guanidinium groups. Furthermore, a shoulder at around 180 ppm can be attributed to acetate counter anions, as the guanylation reaction was performed in the presence of acetic acid. In this way, 13 C CP-MAS solid state NMR spectroscopy gives clear evidence for the formation of the chitosan supported guanidinium acetate groups.

X-ray diffraction

SEM and EDX

We performed SEM and EDX measurements in order to monitor the morphological and chemical modifications of the materials during the subsequent synthesis steps.

Figure 5 shows the SEM images of the prepared materials. N-guanidinium chitosan (GC) shows a relatively homogeneous surface without specific morphology. Nguanidinium-chitosan acetate/silica containing sulfonate groups (GCSS) consists of separated particles of flax seeds-like shape with an average diameter of about 500 nm.

However, N-guanidinium-chitosan acetate/silica containing carboxylate groups (GCSC) showed soft morphology without distinct particle shape that may reflect the hydrogen bond formation between N-guanidinium-chitosan acetate and silica containing carboxylic groups.

CGSC and CGSS

In addition, SEM analyses were carried out to investigate the effect of anionic groups (carboxylate or sulfonate) in the N-guanidinium chitosan acetate/silica hybrids on the morphology of mineralized calcium phosphate. Figure 6 shows SEM images and EDXs spectra of GCSC-CP and GCSS-CP. SEM images show that the two materials display different morphologies. The mineralized calcium phosphate with carboxylate containing groups (GCSC-CP) show a sponge-like morphology with micrometer sized globular aggregates composed of hierarchical nanoplates. The length of these nanoplates is less than 300 nm. On the other side, the material GCSS-CP, is composed of calcium phosphate layers and nanospheres without a well-defined morphology. As the mineralization occurred under similar condition in terms of pH and time, these results indicate that nature of the functional ionic group plays an important role in the mineralization process and led to materials with different morphologies and textures. As the intermediate GCSC and GCSS chitosan/silica hybrid composites contain a high amount of charged species, strong ionic interactions with both calcium and phosphate ions during the biomineralization process can be assumed. The effect of functional groups of the polymeric materials on the morphology of the mineralized calcium phosphate was already reported in the literature [START_REF] Bleek | New developments in polymer-controlled, bioinspired calcium phosphate mineralization from aqueous solution[END_REF]. For example, Mai et al. reported that poly(ethylene oxide) and poly(3sulfopropyl methacrylate) block copolymers initiate the formation of well-defined hydroxyapatite spheres. The strong effect of this copolymer is probably due to the formation of auto-assembled phases the highly charged block copolymer [START_REF] Lkel | Poly(ethylene oxide)bpoly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and bio film inhibition[END_REF].

SEM combined with energy-dispersive X-ray spectroscopy (EDX) allows mapping of the elements that are forming these structures, as shown in figure 6. EDX spectra show that the CGSS-CP and CGSC-CP phases contain carbon, oxygen, nitrogen, sodium, silicon, phosphorus, and calcium. The presence of carbon, nitrogen and oxygen is due to the presence of N-guanidinium-chitosan acetate. Silicon originates from silica network. The homogenous distribution of silicon suggests true organic/inorganic hybrid nature of the material. The other elements are due to the formation of the calcium phosphate phases. As the mineralization was carried out in SBF, some other elements besides calcium, phosphorus, and oxygen have also been incorporated. EDX thus shows that the grown calcium phosphate is closely related to natural bone in the sense that the apatite contains a variety of elements that are also found in bone such as Na, k and Mg. The Ca/P molar ratios are of 1.48 and 1.44 for CGSC-CP and CGSS-CP. This value is lower than the theoretical value for pure hydroxyapatite (1.67) [START_REF] Schweizer | Polymer-controlled, bio-inspired calcium phosphate mineralization from aqueous solution[END_REF]. The XRD results and also Ca/P ratio from EDXs measurements suggest that the mineralized calcium phosphate is mainly constituted of hydroxyapatite. However, the deviation of Ca/P ratio from that of pure hydroxyl apatite can be attributed to the formation of additional amorphous calcium phosphate or other crystalline calcium phosphate phases as already reported in the literature [START_REF] Schweizer | Polymer-controlled, bio-inspired calcium phosphate mineralization from aqueous solution[END_REF][START_REF] Salama | Regenerated cellulose/wool blend enhanced biomimetic hydroxyapatite mineralization[END_REF].

TEM analysis

TEM images of the materials are given in figure 7. These micrographs further confirm the hierarchical structure of the sponge like architecture of GCSC-CP which consists of plate-shaped nanoparticles. Precipitation of calcium-phosphate salt preferentially occurred on the carboxylate containing hybrid because carboxylic groups acted as Ca 2+ -binding site [START_REF] Nardecchia | In situ precipitation of amorphous calcium phosphate and ciprofloxacin crystals during the formation of chitosan hydrogels and its application for drug delivery purposes[END_REF]. On the other side, TEM micrographs of GCSS-CP showed the presence of granular-like aggregates that resembled those observed in aqueous solutions.

Thermogravimetric analyses

It has been reported that silica content increases the thermal stability of chitosanbased hybrids [START_REF] Metin | Efficient removal of acidic dye using low-cost biocomposite beads[END_REF]. Figure 8 shows the TGA thermograms of all reported hybrid materials compared between all the hybrids in the term of inorganic content and thermal stability. At a first glance, the residual mass of the GCSS and GCSC materials is relatively similar (16.2% vs. 17.7%). This result indicates a similar content of anionic silica hybrid material within the two materials. However, after calcium phosphate mineralization, we observed important differences between the two resulting materials GCSS-CP and GCSC-CP, with residual masses of 29.3 % of the first one and 39.7 % for the second one. These results indicate that the calcium phosphate content of the N-guanidinium-chitosan acetate/silica containing carboxylate groups hybrid after 7 days incubation was significantly higher than the one obtained with N-guanidinium-chitosan acetate/silica containing sulfonate groups. We attribute these differences to the chemical nature of the intermediate chitosan-silica hybrid composites. It appears that the presence of carboxylate groups in GCSC not only affects the morphology of nucleated calcium phosphate, but also resulted in a significant increase of the amount of formed calcium phosphate. The retardation effect of sulfonate groups in the GCSS may be the reason for this behavior. Indeed, a significant retardation in calcium phosphate precipitation has already been reported using the charged polymers, and this in particular compared to polymer-free conditions [START_REF] Lkel | Poly(ethylene oxide)bpoly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and bio film inhibition[END_REF].

Conclusion

Novel ionic hybrids were successfully prepared and investigated to enhance the biomimetic mineralization of calcium phosphate. Sulfonate and carboxylatecontaining chitosan/silica hybrid composites were synthesized from anionic trialcoxysilylated precursors and N-guanidinium-chitosan acetate. The ionic hybrids showed a strong effect on the mineralization of calcium phosphate. The nature of the anionic groups (carboxylate or sulfonate) not only affects the morphology of the formed calcium phosphate, but also resulted in a significant difference of the amount of formed calcium phosphate between the two materials. Our results show that Nguanidinium-chitosan acetate/silica hybrid composite containing carboxylate groups is more efficient to induce heterogeneous nucleation and growth of hydroxyapatite.

Overall, our data suggest that N-guanidinium-chitosan acetate/silica containing

13 C

 13 Cross Polarization Magic Angle Spinning (CP/MAS) was used to obtain a high signal-to-noise ratio with 5 ms contact time and 5 s recycling delay.2.4.3. Thermogravimetric Analysis.TGA measurements were carried out with a NETZSCH STA 409 PC instrument. All materials were burned under air flux between 25 and 900 °C at a heating rate of 5°C/min.2.4.4. SEM Analysis.The surface morphology of N-guanidinium chitosan and the Nguanidinium chitosan silica microhybrid was determined by using a Hitachi S-4800 scanning electron microscope.

Figure 1 :

 1 Figure 1: FT-IR spectra of chitosan (C), GC, GCSC, GCSS, GCSC-CP and GCSS-CP.

The 13 C

 13 CP-MAS solid state NMR spectra of the CGSC and CGSS ionic hybrids are given in figure 2. In the spectrum of CGSC, new signals in the range of 13-32 ppm and at 185 ppm indicate the presence of the organosilylated carboxylate precursor and can be attributed to the carbon centers of the ethyl chain (C15/C16) and the carboxylate groups (C14), respectively. On the other side, the 13 C CP-MAS solid state NMR spectra of the sulfonate ionic hybrid CGSS shows new signals resonances in the range of 13-19 ppm characteristic for the propyl chain of the silylated sulfonate precursor (C12/C13/C14). Finally, the disappearance of the signal at around 180 ppm reflects the substitution of acetate anions via anion exchange involving the silylated carboxylate or sulfonate compounds.

Figure 2 .Figure 3 :

 23 Figure 2. 13 C NMR of CGSS (C) and CGSC (D)

Figure 4

 4 Figure4shows X-ray diffractograms of chitosan and N-guanidinium chitosan acetate GC (left), CGSS-CP and CGSC-CP (right). Neat chitosan shows the typical reflection at 2θ = 20.1° which can be attributed to the crystalline structure of the chitosan chains[START_REF] Salama | Synthesis of N-Guanidinium-Chitosan/Silica Hybrid Composites: Efficient Adsorbents for Anionic Pollutants[END_REF]. In the diffractogram of GC, this peak can also be observed. A new diffraction ray at 2θ = 26.2 and other broad rays at 34.9° and 39.1° reflect the formation of a new crystalline structure for the new guanidinium side chains in this latter material. The increase of the crystallinity of chitosan by derivatization, i.e. the introduction of new side chains on the chitosan backbone, has already been reported in in literature[START_REF] Kurita | Chemoselective protection of the amino groups of chitosan by controlled phthaloylation: Facile preparation of a precursor useful for chemical modifications[END_REF].The X-ray diffractograms of the mineralized N-guanidinium chitosan/silica hybrids CGSS-CP and CGSC-CP are more complex. They show diffraction peaks which assigned to the nucleated hydroxyapatite. These peaks are centered at 2θ = 31.9°, 39°, 46.7°, and 49.7°, corresponding to (002), (211), (112), (300), and (222) planes of hydroxyapatite, respectively[START_REF] Salama | New sustainable hybrid material as adsorbent for dye removal from aqueous solutions[END_REF]. These results indicate that the nucleated calcium phosphate phases in CGSC and CGSS are constituted of similar crystalline phases and mainly contain hydroxyapatite.

Figure 4 :

 4 Figure 4: XRD patterns of chitosan (C), N-guanidinium chitosan acetate (GC), mineralized N-guanidinium-chitosan acetate/silica containing sulfonate groups (GCSS-CP) and mineralized N-guanidinium-chitosan acetate/silica containing carboxylate groups (GCSC-CP).

Figure 5 :

 5 Figure 5: SEM images of N-guanidinium-chitosan acetate (GC), and the materials

Figure 6 :

 6 Figure 6: SEM images and EDXs of GCSC-CP and GCSS-CP and elemental maps for CGSC-CP.

Figure 7 :

 7 Figure 7: TEM images of CGSC-CP (left) and CGSS-CP (right)

Figure 8 :

 8 Figure 8: Thermogravimetric analyses of CGSC, CGSS, CGSC-CP and CGSS-CP.

ACKNOWLEDGMENT

This work was financially supported by the Embassy of France in Egypt -Institut Français d'Egypte (IFE) and Science & Technology Development Fund (STDF) in Egypt for the financial support.

anionic groups are interesting materials for applications in the biomaterials field, in particular for biomineralization processes.

Notes:

The authors declare no competing financial interest


Ahmed Salama

Mohamed Hasanin

Peter Hesemann

Synthesis and antimicrobial properties of new chitosan derivatives containing guanidinium groups

Keywords:

Highlights

 New chitosan derivatives bearing guanidinium functions were prepared and characterized.  The four derivatives displayed improved antimicrobial activity in comparison with neat chitosan.  Biosourced and renewable chitosan derivatives could be applied as antimicrobial surfaces.

1-Introduction

The antimicrobial resistance toward conventional antibiotics can often lead to serious diseases in humans [START_REF] Sahariah | Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms[END_REF]. Because of the increased emergence of antibiotic resistant microbial, different innovative techniques to combat bacteria have been established. A novel method uses natural and biodegradable polymers as antimicrobials for the prevention the growth of microorganisms. Among them, polysaccharides have recently emerged as promising candidates for the development of functional biomaterials due to their low cost, non-toxicity, biodegradability and their virtually unlimited availability [START_REF] Salama | Cellulose/calcium phosphate hybrids: New materials for biomedical and environmental applications[END_REF][START_REF] Salama | Soy protein hydrolysate grafted cellulose nanofibrils with bioactive signals for bone repair and regeneration[END_REF]. Polysaccharides with antimicrobial properties were proposed as potential antibiotic and microbial resistant materials for biomedical applications [START_REF] Salama | Dicarboxylic cellulose decorated with silver nanoparticles as sustainable antibacterial nanocomposite material[END_REF].

In this context, chitin, a biopolymer derived from the exoskeleton of insects and crustaceans, can be chemically converted to its partially deacetylated form, called chitosan [START_REF] Hassan | International Journal of Biological Macromolecules New chitosan / silica / zinc oxide nanocomposite as adsorbent for dye removal[END_REF]. The chemical structure of chitosan permits a variety of chemical modifications to synthesize novel derivatives containing functional groups, such as cationic or other hydrophilic and hydrophobic moieties [START_REF] Holappa | Synthesis and Characterization of Chitosan N -Betainates J o u r n a l P r e -p r o o f Having Various Degrees of Substitution[END_REF][START_REF] Sahariah | Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N -Alkyl and N , N -Dialkyl Chitosan Derivatives[END_REF]. Neat chitosan is insoluble in aqueous medium. In acidic medium, the amino groups are converted to ammonium cations. Moreover, these amino groups in chitosan increase the hydrophilicity and improve its water solubility in acidic aqueous solutions. Chitosan has been reported as a promising sustainable material for different applications such as adsorption [START_REF] Monier | Preparation of ruthenium (III) ion-imprinted beads based on 2-pyridylthiourea modified chitosan[END_REF], biomineralization [START_REF] Salama | Chitosan based hydrogel assisted spongelike calcium phosphate mineralization for in-vitro BSA release[END_REF] and drug delivery [START_REF] Salama | Preparation of polyelectrolyte/calcium phosphate hybrids for drug delivery application[END_REF]. Hence, chitosan is a highly versatile biopolymer J o u r n a l P r e -p r o o f allowing a variety of chemical modifications. It is noteworthy that chitosan displays weak inherent antimicrobial activity which disappeared with many microbial strains.

Native chitosan offered limited ability for killing microorganisms, where the lone pair of electrons on amino groups is controlled via unique structure. For this reason, many researchers tried to improve the antimicrobial activity of chitosan via chemical modifications [START_REF] Sahariah | Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N -Alkyl and N , N -Dialkyl Chitosan Derivatives[END_REF]Shehabeldine & Hasanin, 2019). The effect of the cationic charge on the antimicrobial activity of chitosan derivatives has been matter of various studies. The cationic charges are responsible for binding of chitosan to anionic components in the bacterial membrane through electrostatic attractions followed by the rupture of the cell membrane permeability control. Its ability to inhibit microbial growth is observed only in acidic medium, where the polymer is carrying positive charges (Salama & Hesemann, 2018a). Furthermore, the antimicrobial properties of chitosan can be increased not only through functionalization with functional groups such as cationic alkylammonium, guanidinium, anionic carboxylate functions, or thiol-containing groups but also with more hydrophobic substituents, such as alkyl, phenyl and benzyl groups [START_REF] Sahariah | Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N -Alkyl and N , N -Dialkyl Chitosan Derivatives[END_REF][START_REF] Sahariah | Antimicrobial Chitosan and Chitosan Derivatives: A Review of the Structure-Activity Relationship[END_REF].

As already mentioned, the elaboration of chitosan with enhanced antibacterial activity via derivatization with cationic moieties attracted considerable attention. For example, Rúnarsson et al. reported that the presence of ammonium or pyridinium groups on the chitosan chains improved antibacterial activity compared to neat chitosan [START_REF] Vidar Rúnarsson | Antibacterial activity of N-quaternary chitosan derivatives: Synthesis, characterization and structure activity relationship (SAR) investigations[END_REF]. Moreover, surface-quaternized derivatives of chitosan containing high charge density together with the presence of hydrophobic benzyl groups inhibited the growth of different bacteria compared with derivatives containing propyl groups [START_REF] Wiarachai | Surfacequaternized chitosan particles as an alternative and effective organic antibacterial material[END_REF].

The guandine/guanidinium entity is an important functional group in biochemistry as biological receptors especially for RNA. Due to its high pKa value, guanidine exclusively exists in aqueous solution in its protonated form, i.e. the guanidinium cation. The distinctive property of the guanidinium cation is the ability to interact with various anionic functional groups via the formation of ion pairs combined with strong hydrogen bonding. The introduction of guanidinium groups into chitosan via reaction between chitosan and arginine was studied by [START_REF] Zhang | Preparation of arginine modified PEI-conjugated chitosan copolymer for DNA delivery[END_REF]. Hu et al. investigated the introduction of the guanidine moiety on the chitosan backbone via reaction with aminoiminomethanesulfonic acid (Hu et al., J o u r n a l P r e -p r o o f 2007). In our previous work, the formation of N-guanidinium chitosan acetate was achieved through direct guanylation of chitosan with cyanamide in presence of scandium (III) [START_REF] Salama | Synthesis and characterization of N -guanidinium chitosan / silica ionic hybrids as templates for calcium phosphate mineralization[END_REF]. We used this material for the formation of microhybrids via a sol gel technique involving 3-glycidoxypropyl trimethoxysilane.

The resulting material was used as new adsorbent for water purification from organic dyes [START_REF] Salama | Synthesis of N-Guanidinium-chitosan/silica hybrid composites: Efficient adsorbents for anionic pollutants[END_REF]. Another study focused on the formation of ionic composite materials via the sol-gel reaction with 3-(trihydroxysilyl)-1propanesulphonic acid. The resulting ionic microhybrids were also successfully used as new adsorbents for water purification (Salama & Hesemann, 2018a).

In this study, we investigated the impact of guanidinium groups on the biological activity of guanylated chitosan derivatives. Besides, guanylated chitosan obtained via scandium(III) catalyzed guanylation reaction (vide supra), we synthesized new guanylated chitosan materials by reacting chitosan with carbodiimides in ionic liquid media. The resulting guanylated chitosan derivatives were characterized using various techniques. The impact of the guanidinium moieties and the counter anion in the study of antibacterial activity was the main objective of this study. The antimicrobial profile was monitored via the estimation of minimal inhibitory concentration (MIC) and time required for killing, to evaluate the bioavailability and antimicrobial efficiency of the materials.

Experimental part

Materials

Medium molecular weight chitosan (degree of deacetylation, 75-85%), cyanamide, N,N′-dicyclohexylcarbodiimide, N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and scandium(III) triflate were obtained from Sigma-Aldrich. 1-Butyl-3-methylimidazolium chloride (BMIM Cl) was purchased from io-li-tec. All other chemicals of analytical grade and used without further purification. All aqueous solutions were prepared using deionized water. All reagents and microbial media were of analytical grade and used without any previous treatment.

Syntheses

Synthesis of N-guanidinium chitosan acetate (B) and N-guanidinium chitosan chloride (C).

J o u r n a l P r e -p r o o f Synthesis of materials B and C was performed by preparing 10 ml of a 2% solution of chitosan (A) in 2% acetic acid, or 2% hydrochloric acid, in case of material B and C, respectively. These solutions were mixed with 0.084 g cyanamide (2 mmol) and 0.03 g (60 nmol) of scandium(ӀӀӀ) triflate. The solution was stirred at 100°C for 48 h, the resulting mixture was precipitated and washed several times with acetone. Material B from acetic acid and material C isolated from hydrochloric acid were freeze-dried to obtain the pure products B and C as a light brown solid.

Synthesis of N-guanidinium chitosan (N,N′-dicyclohexyl) chloride (D) and Nguanidinium chitosan (N-(3-dimethylaminopropyl)-N'-ethyl hydrochloride) chloride (E)

Chitosan (0.1 g) was dissolved in BMIM Cl (10 g) at 90 o C under magnetic stirring for 2 days. The reaction of chitosan with N,N′-dicyclohexylcarbodiimide or N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.2 mmole) was allowed to proceed at 100 o C for 2 days. The products were isolated by precipitation in 450 mL methanol. The polymers were isolated by filtration and washed three times with 50 mL aliquots of methanol. The collected chitosan derivatives (D and E) were freeze-dried and separated as brown solids.

Characterization methods.

Elemental analysis. EA was done with an Elemental Vario Micro Cube apparatus.

Solid state NMR spectroscopy. 13 C Cross Polarization Magic Angle Spinning (CP/MAS) was used to obtain a high signal-to-noise ratio with 5 ms contact time and 5 s recycling delay.

FT-IR Spectroscopy. The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of materials were obtained using a Thermo Nicolet FT-IR Avatar 320 with a diamond crystal. Spectra were recorded from 500 to 4000 cm -1 . Thermogravimetric Analysis. TGA measurements were carried out with a NETZSCH STA 409 PC instrument. All materials were burned under air between 25 and 800 °C at a heating rate of 5 °C/min.

Antimicrobial studies

The antimicrobial studies were carried out to evaluate the antimicrobial behavior of the new chitosan derivatives. Turbidimetric method was used to judge the antimicrobial activity according to the procedure described by J. Balouiri et. al. [START_REF] Balouiri | Methods for in vitro evaluating antimicrobial activity: A review[END_REF]. The minimal inhibitory concentration (MIC) was J o u r n a l P r e -p r o o f carried out according to our previous work (M. S. [START_REF] Hasanin | New potential green, bioactive and antimicrobial nanocomposites based on cellulose and amino acid[END_REF]. The serial dilution method was used from 100 mg/ml to 0 mg/ml as a negative control.

Cultures of the following microorganisms were used in the tests: (i) Gram-negative bacteria: Escherichia coli (NCTC-10416) and Pseudomonas aeruginosa (NCID-9016); (ii) Gram-positive bacteria: Staphylococcus aureus (NCTC-7447) and Bacillus subtilis (NCID-3610); (iii) unicellular fungi: namely, Candida albicans (NCCLS 11) using nutrient broth medium. One colony of each microbial strain was suspended in a physiological saline solution (NaCl 0.9% in distilled water at pH 6.5). Mueller Hinton broth medium was inoculated by the above-mentioned bacterial strains and incubated individually at 37 °C for 24 h. After the incubation process, the turbidity was measured with V-630 UV-vis spectrophotometer (Jasco, Japan) at a wavelength of 530 nm, and the above concentrations were used to determine the MIC values under the same incubation conditions. The inhibition percentage was calculated according to equation

(1). 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒-𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 (1)
Data are expressed as mean ± standard deviation (SD) values. The data analysis was done using the Origin lab 9 professional edition program to carry out these statistical tests.

For determination the time required for killing, the assayed microorganisms strains were subjected to cultivation in the concentration of 400 cells which refers to the logarithmic growth phase. The prepared samples were diluted to 106 colony forming units (CFU) in milliliter for each microorganism strain (CFU/mL). MIC values of each microbial strain were multiplied by four times and used at initial concentration in Mueller Hinton broth media. Aliquots (100 mL) were collected from each treatment after 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h of incubation at 37 °C and subsequently serially diluted in phosphate-buffered saline (PBS). Microorganisms were then transferred to Mueller Hinton agar plates and incubated at 37 °C for 24 h before determining the viable results of CFU/mL [START_REF] Eid | Arylthiazole antibiotics targeting intracellular methicillinresistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis[END_REF].

Results and discussion

J o u r n a l P r e -p r o o f

Synthesis of the chitosan derivatives.

Chemical modifications of chitosan are expected to enhance its antimicrobial activity toward bacteria. Various functional groups such as quaternary ammonium groups containing alkyl chains were generated on the chitosan chains to enhance its antimicrobial activity. These studies showed that the quaternary ammonium groups and the presence of long alkyl chains effectively control the efficacy against bacteria [START_REF] Sahariah | Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms[END_REF]. The formation of guanidinium functions by reacting primary amines and cyanamide has already been reported. However, these reactions often require harsh conditions and were sometimes accompanied by the formation of secondary products which are difficult to eliminate, e.g. pyrazole. Short and Darby reported the formation of guanidinium salts via the reaction of ammonium starting materials and cyanamide at elevated temperatures and in the presence of concentrated hydrochloric acid [START_REF] Short | Sympathetic nervous system blocking agents. 3. Derivatives of benzylguanidine[END_REF]. More recently, Tsubokura et al. reported a new protocol requiring milder reaction conditions. This reaction strategy involved the use of scandium(ӀӀӀ) triflate as Lewis acid catalyst [START_REF] Tsubokura | Direct guanylation of amino groups by cyanamide in water: Catalytic generation and activation of unsubstituted carbodiimide by scandium(iii) triflate[END_REF]. On the other side, carbodiimides have attracted increasing attention for preparing complexes with a substituted propiolamidine group or a substituted guanidinate groups [START_REF] J O U R N A L P R E -P R O O F Cao | Activation of Carbodiimide and Transformation with Amine to Guanidinate Group by Ln(OAr) 3 (THF) 2 (Ln: Lanthanide and Yttrium) and Ln(OAr) 3 (THF) 2 as a Novel Precatalyst for Addition of Amines to Carbodiimides: Influence of Aryloxide Group[END_REF]. Here, we used these two protocols for preparing new chitosan supported guanidinium groups as shown in scheme 1.

Scheme 1 illustrates the synthesis of the four N-guanidinium chitosan derivatives B -E through the reaction of chitosan A with cyanimide (for synthesis materials B, C) and carbodiimides (for synthesis materials D, E). The conversion of the amino groups to guanidinium functions was monitored via elemental analysis, FT-IR and 13 C CP-MAS-NMR spectroscopy as well as by TGA.

The results of elemental analysis measurements for materials B, C, D and E are given in table 1. The results indicate significant increase of the nitrogen content from 7.7 for neat chitosan to 11.06, 12.33, and 10.47 for materials B, C and E, respectively.

However, the nitrogen content doesn't increase in material D may be due to the presence of two carbon rich moieties, cyclohexyl rings. These results together with the decreased values observed for carbon give a first indication for the successful formation of guanidinium moieties supported on chitosan.

J o u r n a l P r e -p r o o f

FT-IR study

The functional groups of chitosan and its derivatives were confirmed by means of FTIR spectroscopy. In the spectrum of material B, an additional signal at 160 ppm can be assigned to the carbon centers of the newly formed guanidinium groups (C9). Furthermore, the signal at 180 ppm can be ascribed to acetate counter ions. Hence, 13 C CP-MAS solid state NMR spectroscopy confirms the synthesis of chitosan containing guanidinium groups.

The spectrum of material C, N-guanidinium chitosan chloride, is given in figure 2b.

Similarly to the spectrum of material B, a new signal at 160 ppm indicates the formation of guanidinium groups. The higher intensity of this signal indicates a higher guanylation degree in this material; the guanylation reactions seems to occur more efficiently in the presence of hydrochloric acid.

TGA

The thermogravimetric analysis (TGA) curves for chitosan A and its derivatives B -E over the 30-800°C range, are displayed in figure 3. In all materials, the weight losses can be separated into three main temperature regions, designed as regions I, II and III.

From room temperature to 120°C (region I), the weight loss ranging from 0.4 to 8.1%, may be attributed to the evaporation of physically adsorbed water. The amount of show only slight differences which confirm that the thermal stability of chitosan derivatives is close to the thermal behavior of neat chitosan. These results also show that the crystal structure of neat chitosan has not been completely destroyed via dissolution and regeneration even in acidic aqueous solutions or in ionic liquids.

Finally, at temperatures above 300°C, we observed a complete decomposition of the materials. The third weight loss, region III (300 -700°C), recorded high stability for N-guanidinium chitosan chloride comparing with neat chitosan and other derivatives.

For example, the weight loss% reached 81.9, 77.5, 70.8, 84.8, 81.5% for the materials A, B, C, D and E, respectively, at 550 o C.

Antimicrobial activity

The antimicrobial activity of the materials could be evaluated through various protocols depending on the solubility performance of the assayed samples. During this study, the antimicrobial activity of chitosan and its new derivatives was estimated through two techniques, the minimal inhibitory concentration (MIC) and the time required for killing. The MIC test was estimated through turbidimetric technique using different concentrations, from 100 to zero mg/ml (The results were presented in tables S1 to S5). Figure 4 Further studies displayed that the MIC of chitosan films for gram-negative was almost 200 mg/ml and for gram-positive was 40 mg/ml [START_REF] Takahashi | Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree[END_REF]. Another studies showed that MIC values of chitosan for most gram negative J o u r n a l P r e -p r o o f bacteria was around 100 to 10 mg/ml [START_REF] Helander | Chitosan disrupts the barrier properties of the outer membrane of Gramnegative bacteria[END_REF][START_REF] Tsai | Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation[END_REF]. Moreover, several studies described the creation of various active groups on the chitosan backbone to support and extend its antimicrobial activity [START_REF] Avadi | Diethylmethyl chitosan as an antimicrobial agent: Synthesis, characterization and antibacterial effects[END_REF]Shehabeldine & Hasanin, 2019).

It is well known that the chitosan antimicrobial activity arises from the action of the two electron lone pairs presented in the amino groups. These groups react with microorganism cell wall via complexion reaction which produced choppy microorganism wall (Shehabeldine & Hasanin, 2019). However, the antimicrobial activity of the novel materials (B to E) increased significantly compared to chitosan which can be attributed to the formed guanidinium groups. These groups are cationic and can enhance the permeability of cytoplasmic membrane via adsorption and bonding to intracellular constituents and kills the organisms. Moreover, the cell membrane disorganization and pore formation disrupt the cells permeability mechanisms. Also, the guanidine compounds effect on DNA and cellular proteins of bacteria [START_REF] Zhou | Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine J o u r n a l P r e -p r o o f[END_REF].

More specifically, N-guanidinium chitosan acetate B exhibited the highest antimicrobial activity as shown in figure 4. This material showed the lowest MICs value (less than 3.125 mg/ml) with all assayed organisms which refers to its effective broad spectrum antimicrobial activity. This high value of reactivity arises from the presence of anionic acetate counter ion which also possesses inherited antimicrobial activity [START_REF] Cacic | Synthesis and Antimicrobial Activity of Some Derivatives on the Basis (7-hydroxy-2-oxo-2Hchromen-4-yl)-acetic Acid Hydrazide[END_REF][START_REF] Chung | Durable Press and Antimicrobial Finishing of Cotton Fabrics with a Citric Acid and Chitosan Treatment[END_REF]. In this context, the samples C, D and E showed intermediate antimicrobial activity against all assayed microorganisms which is still higher compared to chitosan. The novel derivatives recorded the following order: C > E > D. However, material C has high antimicrobial reactivity comparing the other two materials, D and E. This observation may be due to the delocalization of the positive charge on guanidinium groups via the alky chains in the materials D and E. Moreover, the free mobility of Cl - atom in material C improved its antimicrobial activity [START_REF] Kim | Comparison of the Antimicrobial Effects of Chlorine, Silver Ion, and Tobramycin on Biofilm[END_REF]. Material D, therefore, exhibited the lowest antimicrobial activity among the investigated samples. These results may be due to special structure of material D which contains two bulky cyclohexyl groups. These groups act as a barrier for interactions between the new derivative and the cell wall [START_REF] Malík | The Structure-Antimicrobial Activity Relationships of a Promising Class of the Compounds Containing the N-Arylpiperazine Scaffold[END_REF]. Using different bioassay will be required for complete picture of the antimicrobial action of the novel materials [START_REF] Houghton | Uses and abuses of in vitro tests in ethnopharmacology: Visualizing an elephant[END_REF].

Conclusion

New chitosan derivatives bearing guanidinium functions were synthesized by direct reaction between chitosan and either cyanamide in aqueous medium or carbodiimides in ionic liquid media. The new derivatives exhibited high broad spectrum antimicrobial activity. Among the new materials, N-guanidinium chitosan acetate showed the highest antimicrobial activity with high rate of killing according to the time required

  Figure 1 a/b shows the FT-IR spectroscopy of materials A, B, C, D and E. The spectrum of neat chitosan (A) shows a broad band at around 3450 cm -1 which can be attributed to N-H and O-H symmetric stretching vibration and inter-and intra-molecular hydrogen bonds. The weak band at 2902 cm -1 is ascribed to the C-H stretch vibrations of chitosan. Moreover, the characteristic peaks at 1656 and 1580 cm -1 were due to the C=O stretching (amide I) and -NH stretching (amide II), respectively. All chitosan derivatives spectra showed the characteristic bands of chitosan skeleton. Besides, increasing the intensity of the adsorption band at 1630 cm - 1 was observed in the spectrum of all derivatives B to E which suggests the generation of guanidinium moiety. The intensity of the band at 3450 cm -1 was decreased in case of materials D and E, comparing with neat chitosan, indicating a decrease in the density of guanidinium and hydroxyl groups arising from the presence of new alkyl and cyclohexyl branches. Moreover, the weak band at 2921 cm -1 , ascribed to the CH stretch vibrations of chitosan A, significantly increased in the charts D and E. This increase refers to the increasing in the CH stretch vibration which arises from the formation of N-guanidinium chitosan (N, N′-dicyclohexyl) chloride D and Nguanidinium chitosan (N-(3-Dimethylaminopropyl)-N'-ethyl hydrochloride) chloride E. 3.3. 13 C CP-MAS solid state NMR The characterization of the materials via 13 C CP-MAS solid state NMR spectroscopy is a highly efficient tool to follow the derivatization and chemical evolution of the materials via the different guanylation reactions. We therefore performed 13 C CP-MAS solid state NMR experiments for the parent chitosan A and the new derivatives B -E. The spectra are given in the figure 2a/b. The spectra of chitosan A principally shows signals in the area of 55-110 ppm, due to the anhydroglucose units. More specifically, the signals at 55 ppm can be attributed to the CH2 (C6), whereas the signal at 70 ppm is due to a superposition of the C2, C3, and C5 carbon centers. The signals of the C1 and C4 carbon are shifted to lower field (80 and 104 ppm) due to strong electron withdrawing effect of neighboring oxygen atoms. Finally, the signals of low intensity J o u r n a l P r e -p r o o f observed at 25 and 175 ppm are due to residual acetyl groups and reflect the partial deacetylation of chitosan.

Figure 2 d

 2 Figure 2 d/e shows the 13 C CP-MAS solid state NMR for N-guanidinium chitosan (N, N′-dicyclohexyl) acetate D and N-guanidinium chitosan (N-(3-Dimethylaminopropyl)-N'-ethyl hydrochloride E. In both spectra, additional signals, in the region between 20 -45 ppm, indicate the presence of aliphatic carbon centers, originate from the grafting of the carbodiimides on the amine groups of the chitosan chains. Furthermore, the signals of the carbons center of the guanidinium groups around 160 ppm can also be seen in both spectra.

J o u r

  n a l P r e -p r o o f physically adsorbed water is significantly higher in chitosan derivatives containing acetate anions compared to N-guanidinium chitosan chloride. This difference may be due to the higher hydrophilicity of chitosan derivatives containing acetate anion with the same compound containing chloride anions. In region II (120 -300°C), a sharp weight loss indicates the thermal degradation of chitosan scaffolds. The degradation temperature at 80% weight loss follows the ascending order: A (279) ~ B (278) ˂ D (258) ˂ C (250) ˂ E (242), showing that the guanylated chitosan materials in general show a lower thermal stability compared to the parent neat chitosan. The thermograms

  displays the statistical analysis of the MICs results for the all materials (A, B, C, and D). Chitosan A displays low antimicrobial activity in comparison with the new derivatives, especially with Gram negative bacteria. The MICs values of chitosan recorded 25 mg/mL for Escherichia coli (NCTC-10416), Staphylococcus aureus (NCTC-7447), Bacillus subtilis (NCID-3610), Candida albicans (NCCLS 11) and 50 mg/mL for Pseudomonas aeruginosa (NCID-9016).

J o u r

  n a l P r e -p r o o f Consequently, the time required for killing was calculated considering chitosan as reference sample and N-guanidinium chitosan acetate B as the highest antimicrobial derivative and DMSO as blank. Figure 5 shows the surviving of assayed microorganisms against time during treatment with chitosan, material B and DMSO. The native chitosan displays the long time for treatment with Escherichia coli (NCTC-10416), Pseudomonas aeruginosa (NCID-9016), Staphylococcus aureus (NCTC-7447), Bacillus subtilis (NCID-3610) and Candida albicans (NCCLS 11) by 16, 20, 20, 18 and 18 hours, respectively. The rate of killing in case of material B was reduced to 12, 10, 12, 14, and 12 hours for Escherichia coli (NCTC-10416), Pseudomonas aeruginosa (NCID-9016), Staphylococcus aureus (NCTC-7447), Bacillus subtilis (NCID-3610) and Candida albicans (NCCLS 11). Material B showed a synergetic effect arising from both guanidinium groups and anionic acetate which enhanced the rate of microbial cells destruction (M. Hasanin, El-Henawy, Eisa, El-Saied, & Sameeh, 2019; M. S.[START_REF] Hasanin | New potential green, bioactive and antimicrobial nanocomposites based on cellulose and amino acid[END_REF]. Overall, the novel derivatives have excellent antimicrobial activity with high rate of microorganisms killing. Therefore, the new derivatives from B -E can be considered as broad spectrum antimicrobial agents.

Figure 1 :

 1 Figure 1: FT-IR study of chitosan and its derivatives B, C, D and E

J

  Figure 2d/e: 13 C CP-MAS solid state NMR for N-guanidinium chitosan (derivatives D and E).

  r n a l P r e -p r o o f

Figure 3 :Figure 4 :

 34 Figure 3: TGA of chitosan A and its derivatives B, C, D and E (under air flux, heating ramp 5°C/min)

Figure 5 :

 5 Figure 5: Time required for killing (E. coli, P. aeruginosa, S. aureus, B. subtilis and C. albicans.) for A and C.

Table 1 :

 1 Elemental analysis of chitosan (A) and its derivatives (B, C, D and E).

	Samples	C[%]	H[%]	N[%]
	A	43.06±0.02	6.73±0.07	7.7±0.13
	B	41.34±0.42	6.48±0.21	11.06±0.07
	C	34.0±0.3	6.21±0.24	12.33±0.17
	D	39.31±0.23	6.84±0.28	7.06±0.17
	E	36.57±0.24	8.24±0.13	10.47±0.01

ACKNOWLEDGMENT

This work was financially supported by the Embassy of France in Egypt -Institut Français d'Egypte (IFE) and Science & Technology Development Fund (STDF) in Egypt.

Author Statement

Ahmed Salama and Peter Hesemann developed the polymer synthesis and designed the experiments. They shared equally in data curation, funding acquisition, investigation, project administration, resources, software and writing. Mohamed Hasanin assayed the antimicrobial study. All authors commented on the manuscript and supported data analysis.

Notes:

The authors declare no competing financial interest