Physicochemical Characteristics of ChitosanâTiO2 Biomaterial. 1

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PHYSICOCHEMICAL CHARACTERISTICS OF CHITOSAN/TIO2 BIOMATERIAL PART I. STABILITY AND SWELLING PROPERTIES Agnieszka Ewa Wi#cek, Agata Gozdecka, and Ma#gorzata Jurak Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b04257 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Industrial & Engineering Chemistry Research

PHYSICOCHEMICAL

CHARACTERISTICS

OF

CHITOSAN/TIO2 BIOMATERIAL PART I. STABILITY AND SWELLING PROPERTIES

Agnieszka E. Wiącek*, Agata Gozdecka, Małgorzata Jurak

1) Department of Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, 20-031 Lublin, Poland

*Corresponding author: Agnieszka E. Wiącek Department of Interfacial Phenomena Faculty of Chemistry Maria Curie-Skłodowska University 20031 Lublin, Poland [email protected]

ACS Paragon Plus Environment

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Abstract The surface and electrokinetic properties of chitosan/TiO2 biomaterial have been investigated. Both carbohydrate polymer and titanium dioxide demonstrate unique physico-chemical properties appropriate for biotechnological applications. However, the changing stability of such systems restricts its usage. Improved stability can be controlled by the environmental parameters, process conditions or modifying TiO2 structure. Antibacterial chitosan was used because its protective barrier can retard ripening, water loss as well as destruction of products. The concentrations of compounds and solution pH were varied. Static and dynamic light scattering measurements make evident the relationship between the state of aggregation of the suspensions and the stability. The chitosan/TiO2 system is characterized by improved physicochemical properties in comparison with base TiO2 as an effect of electrosteric stability. Stable material was obtained with the precisely selected compounds ratio, proved by FTIR method, which can be developed due to valuable properties of individual components along with synergistic effects.

Keywords: chitosan; titanium dioxide; antibacterial; dynamic and static light scattering; zeta potential


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1. Introduction Among metal oxides, titanium dioxide is of special significance because of its applications, low cost, resistance to photo chemical and chemical erosion. Many different pharmacological, medical and cosmetic applications of titanium oxide and its various shape types: nanotubes, nanoparticles, nano rods, mesoporous and nanoporous containing materials are currently in use, most of them are of hydrophilic nature1-4. Recently, the nano-TiO2 composites have been examined for a skin substitute. These are photobacteriocides, deodorants and antiviral agents5. It is commonly known that hydrophilicity can benefit skin humidity and reduce the required healing period. On the other hand, such situation can facilitate bacterial growth, sometimes very dangerous for patients. Thus, it is desirable to use material which can accelerate wound healing and is characterized by high anti-infection activity and immune-enhancing effect. The chitosan-TiO2 systems might have a more beneficial effect than titanium dioxide alone and might be active as such wide-ranging nanomaterial. Wettability (hydrophilic-hydrophobic nature) of composite materials is of great significance. The very popular carbohydrate polymer, chitosan was selected because it is well known as a nontoxic anticancer agent and an immune enhancer. It is frequently used due to its biodegradability, adsorption properties and biocompatibility. Besides, chitosan can help control the nanoparticles size and dispersion due to its spreading out during the system preparation. This is a consequence of unique film forming chitosan characteristics, swellability in aqueous solution and gel forming properties. Lately, H. Su and co-authors6 prepared a chitosan-TiO2 composite film by incorporation of titanium dioxide nanopowder into chitosan. By SEM analysis the authors proved that the TiO2 powder was uniformly dispersed into the chitosan matrix. TiO2 addition led to increase of hydrophilicity, to better mechanical properties, and to decrease of visible light transmittance of the composite film. Besides, the chitosan-TiO2 film possessed efficient antimicrobial activity against food-borne pathogenic microbes: Escherichia coli, Staphylococcus aureus,


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Candida albicans, and Aspergillus niger. What’s more, in the case of composite the synergistic effect of properties (e.g. antibacterial) is possible6-10. B. Li and co-workers results indicated that chitosan/TiO2 nanocomposite (the ratio of 1:5) showed the strongest inhibition in growth of Xoo, rice bacterial pathogen (Xanthomonas oryzae pv. oryzae). Additionally, the antibacterial activity of such nanocomposite against is significantly higher than that of the two individual components (chitosan or titanium dioxide) under both light and dark conditions7. On the other hand, biocompatibility, antibacterial and immune properties depend on homogeneity and particle size of metal oxide system. Therefore intense physicochemical and rheological characteristics of such suspension is required because the dispersed oxide systems show a tendency to aggregate at natural pH, and that is why biopolymers are used to improve their dispersibility and suspension stability during production. Specifically, natural substances have been shown to produce changes in the dispersion behaviour of titanium dioxide through control of electrostatic and steric forces between the particles11-13. The interesting is the rheological

behavior

of

chitosan/titanium

dioxide

composite

described

by

Y. Tang and co-workers14. On the basis of the obtained results the authors showed that the increased TiO2 nanoparticle loadings decreased the viscosity and dynamic viscoelasticity of the as-prepared coatings, and improved the antibacterial activity and mechanical properties of surface-coated cellulosic paper. In addition, chitosan added to the titanium oxide can fulfill a number of other functions. For example, chitosan with high degree of deacetylation possesses lower affinity for enzymes in vitro, has a less porous structure and lower water-uptake capacity, which limits the rapidity of the degradation and may also manipulate the biopolymer immunoreactivity15. Chitosan as an effect of amino group in its molecule is positively charged and readily attaches to negatively charged surface, which enlarges its bioadhesive properties. Moreover, chitosan enhances the transport of polar drugs across the outer layer of skin


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surface16-18, accelerates the binding of fibroblast, simplifies tissue regeneration19-21 and enhances synthesis of extracellular matrix. Superior haemostatic effectiveness of chitosan through blood platelets activation and thrombin generation was also presented allowing chitosan-based system application in wound dressing15, 22. Moderately high solids concentrations are required in the preparation of chitosanbased system. The stability and homogeneity of the system is further compromised in such cases, and the formation of aggregates can decrease after modification of titania surface behaviour. A number of studies have been conducted on the physicochemical characteristics of unmodified titanium dioxide suspensions as a function of different factors, for example pH, ionic strength, temperature, particle size and volume fraction of solids1-4, 23, 24, 25-29. Regardless of numerous data concerning the chitosan or titanium dioxide applications singly in the cosmetics and pharmaceuticals, only sporadic studies have been contributed to physicochemical characteristics and stability of chitosan-titanium dioxide assemblies. In this paper the surface and electrokinetic properties of titanium dioxide suspensions are examined and deliberation is given to how chitosan modification can alter the size, electrokinetic potential, and stability of the system. The chitosan/titanium dioxide system can be a promising biomaterial with prospective application as for example in wound healing dressings, which not only has protection function but also can stimulate tissue healing being bioactive30. Increase of biocompatibility and immune response of hybrid formulation is undisputable, but a full characteristic is required. The adsorption of molecules on the titanium dioxide solid surface affects the van der Waals and/or steric interactions between the particles, and because chitosan molecules are charged, the electric double layer repulsion between them can also be modified. Another point of view of this research is choice of environmentally friendly materials. There is no doubt that the chitosan addition increases biodegradability of chitosan-TiO2


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systems and the combined products. In this paper the effects of pH and chitosan concentration on the physicochemical properties of titanium dioxide components are determined. The physicochemical characteristic of chitosan affects the biological properties which can advance or limit its practical use. The chitosan/TiO2 systems could be used as a suspension, a tablet (bead) for special purposes, a skin substitute or a special kind of dressing (film) as a removing smell agent, bactericide and antiseptic. What is important, its hydrophilicity can benefit skin humidity and reduce the required healing period however, this property can also facilitate dangerous bacterial growth. Therefore the analyses should clarify which factors can increase the chitosan/TiO2 suspension microstructure and stability for the purpose of skin protection, antibacterial (disinfecting) character and as an effect of biocompatibility. Our proposed strategy to preserve the properties of chitosan is physical crosslinking by small-size ions. We hope that titanium dioxide particles can satisfy these conditions because the hydrophilic character of titanium dioxide particles facilitates their miscibility with chitosan in the solution. To achieve this goal selection of precise composition of ingredients is necessary. On this basis we expect to obtain stable chitosan/TiO2 biomaterial with acceptable homogeneity of the components. More detailed experiments describing analogous systems but regarding wettability and widely understood biocompatibility are now conducted for better recognition of their behaviour31. New material obtained in this way could have more favourable properties than the compounds individually. We hope that the obtained chitosan/ TiO2 systems can be qualified as functional composites for wound and tissue repair owing to their unique anti-infection activity of both chitosan and TiO2 particles and additionally, human immunity enhancing agent as an effect of chitosan alone.

3. Materials and methods 3.1. Materials characteristics


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Carbohydrate polymer, chitosan (Acrōs Organics, ACRS34905) and TiO2 P-25 Evonik (early Degussa) were used. Polysaccharide biopolymer (molecular weight 100 000-300 000, degree of deacetylation equals 82 ±2%) was applied without purification. TiO2 is a fine-particulate, pure titanium dioxide (TiO2 99.9%) with hydrophilic character caused by hydroxyl groups on the surface. It is white powder with high specific surface area and unique combination of anatase and rutile crystal structure approximately 80:2032. X-ray diffraction confirmed such composition. Both crystal forms are tetragonal but with different dimensions of the elementary cell. At a temperature close to 300 °C, a slow conversion of anatase to the more stable rutile structure initiates. At temperatures higher than 600 °C, the conversion runs faster and additionally, a reduction of the specific surface is observed. Determined by us the specific surface area of P-25, using the BET thermal desorption of nitrogen method, is 48.3 m2/g ± 2 m2/g, density of ca. 4 g/cm³ consistent with the literature data33,

34

. The aggregates are several hundred nanometers in size, but the

minimal particles have a mean diameter of approximately 24.5 nm± 3 nm. For the treatment of the data, the refractive index of TiO2-P25 was set at n=2.57 (taking into account a proportion of anatase (n=2.561) and rutile (n=2.613) in the mixture. To remove impurities the cleaning procedure described by Preočanin and Kallay35 was used as in our previous paper36-39. After the cleaning procedure the particles were ground in an agate mortar to break aggregates and start from a well-dispersed sample, always with a similar initial macrostructure. The SEM images of the base TiO2 nanoparticles show that they are approximately in a spherical form and the main fractions of particles are in the range 10-30 nm (Figure1). Pharmaceutical and biomedical applications of nanoparticles require that these structures are characterized by a broadly defined biocompatibility goal. To achieve this goal the best way is to use an appropriate polymer coating (e.g. chitosan) which can modify the surface properties.


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The additional components, NaCl, NaOH, HCl and CH3COOH were the analytical grade reagents (POCh S.A., Poland). Water used for preparation of all suspensions was purified in the Milli-Q Plus 185 system from Millipore (USA) (the resistivity 18.2 MΩ cm).

3.2. Sample preparation Measurements using the static and dynamic light scattering methods were made for various concentrations of chitosan solution and different ratios of used ingredients to obtain optimal coating. The specified amounts of the chitosan solution in acetic acid (1g/1M CH3COOH) (0.04 mL; 0.08 mL; 0.2 mL; 0.4mL and 0.6 mL) for different tested concentrations were dispersed in 10–3 M NaCl (100 mL) solutions at natural pH. The suspensions were prepared by mixing 5mg of TiO2 powder with the chitosan solution. For some dispersion pH effect was checked and pH was adjusted to the values 3, 4.8, 6, 7 and 9 by adding a suitable amount of 0.1 M HCl or 0.1 M NaOH solutions. pH was measured before and after homogenization (5000 rpm for 3 min) and, if necessary, readjusted before measurements. Chitosan molecules interact with the P-25 surface via electrostatic interactions under the condition where titanium dioxide and the chitosan molecules are oppositely charged. After adding chitosan into the acetic acid solution to probe with the TiO2 solid, the following structure of chitosan/titanium dioxide composite is possible (Figure2), which can be proposed on the basis of FTIR measurements. On the other hand, it is known that chitosan is hygroscopic in nature, so it has a great capability of forming hydrogen bonding (with both hydroxyl and amino groups) with water or aqueous solution. From the DSC and DTG methods, it is common that on the chitosan surface oxygen is present largely in the form of O−, then again, all nitrogen amounts is located in the amine and amide groups40. The presence of absorbed water plays a considerable role especially in the solid chitosan-based system, affecting the flow properties and compressibility of the powders or


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tablets tensile strength. With the intention of hybrid biomaterial use in clearly defined applications, it is necessary to understand their structure, properties, function and relationship between them. Both hybrid compounds have good or excellent UV protecting characteristics and synergic effect is possible. The titanium dioxide can have a good UV shielding, the UV absorbance of chitosan alone is detectable but not as high as that of TiO2 and is dependent on the chitosan source. In the hybrid, the addition of higher concentration of titanium dioxide can reduce the transmittance of chitosan to UV and visible light. Detailed measurements are described in our next paper41. From a practical point of view the other very important properties of chitosan/titanium dioxide composite and both components isolated are their hydrophilicity and biodegradability. The literature reports that the hydrophilicity of chitosan film linearly decreases with the increase of concentration, but when titanium dioxide is added, hydrophilicity increases. Moreover, various functional groups of chitosan have a different susceptibility to degradation. TiO2 particles enhance the resistance of the -C-O-C- bond responsible for chain scission of chitosan due to thermal degradation42. In order to guarantee chitosan/titanium dioxide uniformity and proper functionality in the final product besides hydrophilicity, UV resistivity and thermal properties, the multimodal size distribution (polydispersity index by dynamic light scattering) should be clarified. We expect that the grain size of TiO2 will decrease using chitosan as a modifier as well as after homogenization resulting in a more uniform structure at a precisely selected chitosan concentration. This will be described in a detailed way in the next sections.

3.3 DLS method, electrophoresis and Turbiscan stability measurements The particle size and multimodal size distribution of the TiO2 suspensions and chitosan/TiO2 nanocomposite dispersed in an electrolyte were measured by the dynamic light scattering


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(DLS) method (also known as photon correlation spectroscopy, PCS or quasi-elastic light scattering, QELS) using a Zeta Pals-BiMass Zetameter (Brookhaven Instruments Corporation, USA) with a laser-light of the wavelength 670 nm at 25oC as a function of time 5, 30, 60, 120 min, 180 min and 24 h after the system preparation. Stability of titanium dioxide as a function of pH was checked for 1.25 x 10-3 M concentration of dispersion and the same after chitosan modification (0.6 x 10-5M). To cover uniformly and to separate the loosely agglomerated particles of TiO2, the samples were energetically stirred and homogenized just before measurements. The precise calculation of metal oxide particles coverage by chitosan molecules is not possible as an effect of a broad range of mean value of biopolymer molecular weight. The DLS method provides information on the size distribution of the dispersed particles as a result of analysis of the autocorrelation function of the laser light scattered by the particles during their Brownian motion. Zeta Pals-BiMass Zetameter was also applied for the analysis of the charge at the solid-liquid interface for both base and chitosan-modified titania particles. On the basis of the autocorrelation function oscillations, the electrophoretic mobility distribution can be achieved and the zeta potential can also be determined. The average results of five repeated runs were calculated. Smoluchowski approximation was applied for the zeta potential evaluation from the electrophoretic mobility data giving possibilities to compare the literature results33, 43. Equivalent measurements were conducted with the help of an optical analyzer Turbiscan Lab. Turbiscan can control the system behaviour by the use of a moving head equipped in a near-infrared light source with a transmission and backscatter detector. All probes were analysed vertically along the cuvette and the data were taken every 40 µm. Changes in the intensity of measured light depend mainly on the particle size and concentration and indicate particle migration in the sample (settling and/or creaming) as well as their stability/instability. The main rule of Turbiscan


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measurements is that the lowest value of TSI (Turbiscan Stability Index), the highest stability of the system is. The coefficient TSI is calculated with the special computer program Turbiscan Easy Soft using the following Equation:

where: Xi – average transmission (T) or backscattering (BS) of light for each minute of measurement, XT,BS – average X1, n – number of scans (repetitions of single measurements during the total time of the experiment). At all original figures from Turbiscan measurements on the right scale, each selected time are pointed by different colour (start of measurements 0 min˗pink; 15 min˗dark green; 30 min˗bottle green; 1h and 1.30 min˗different shades of green; 1.45 min˗light green and 2h˗red, end of measurements). If measured suspensions are stable these lines and colours are difficult to distinguish. X-axis means height of cuvette from 0 mm (bottom of cuvette) to 55 mm (top of cuvette). Fluctuation at the bottom and/or at the top of cuvette and extension of the band (each colour at figure visible as separate line) can mean start of destabilization process. Electrokinetic and Turbiscan measurements are complementary because the principles of used methods are different, but the sample characterisation is more complete. When the weight ratio of titanium dioxide/chitosan is increased in close proximity to the isoelectric point, the particle aggregation is much promoted so that the aggregates settle down, leaving an effectively dilute suspension between the turbiscan cell walls. The changes can be visible at the bottom and at the top of the cuvette. Incidentally, this problem is much less important in the case of electrophoretic mobility determination, since short measurement time in each sample (about 1-3 minutes), and low volume fractions of solids (10-4 or lower) suggest a secondary significance of settling.


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Besides in this method, measurements are done for one height of cuvette. All experiments for both methods were performed at room temperature 20 ± 1 °C.

3.4 Resistance to solvents and swelling measurements The resistance of chitosan and chitosan/TiO2 beads to different solvents used frequently in pharmacy and medicine was tested by a not complicated gravimetric method. Concisely, 0.1 g of chitosan or 0.1 g chitosan/TiO2 dry powder was dissolved in 0.1 M HCl (1 mL). After careful stirring two kinds of beads were formed which were immersed in 25 mL of solvents (Milli-Q water, ethanol or glycerol) at room temperature for 24 h. After that the beads were collected and dried up in a desiccator to a constant weight. Subsequently, to characterize the chitosan/TiO2 composites accurately the swelling behaviour of chitosan beads and chitosan/TiO2 beads were checked by the same following method. The beads of chitosan and chitosan with TiO2 were allowed to swell in redistilled water (Milli-Q plus) at room temperature. The swollen samples were periodically weighed (each 5 minutes) until no weight increase or bead dissolution was observed.

3.5 FTIR measurements To verify the influence of chitosan on TiO2 particles visible in the microscale, the FTIR-ATR spectra were taken with a FTIR Nicolet 8700, Thermo Scientific Inc. FTIR spectroscopy is a powerful tool to materials characterization. FTIR spectroscopy was chosen, because it is sensitive mainly to hetero-nuclear functional group vibrations and polar bonds, especially OH stretching in water.

4. Results and Discussion 4.1 Resistance to solvents and swelling properties of chitosan and chitosan/TiO2 beads


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After measurements of resistance to solvents, the alteration of mass was calculated for two kinds of beads (chitosan and chitosan/TiO2) using the following equation:

∆m% = (1- mt/mi) x 100

where mt is the mass of bead after the solvent treatment and mi is the initial mass of bead44, 45. This experiment demonstrated a non-linear relationship of the mass decrease for both kinds of beads in three different solvents. Generally, the mass loss varies from 3 to 85 %. It was observed that neither chitosan nor chitosan/TiO2 is soluble in glycerol but in water. Low compatibility between the carbohydrate polymer bead and the organic solvent molecules (e.g. glycerol) prevents the absorption process and is a very important property of chitosan and chitosan/TiO2 complex to be used in biotechnological applications (mass waste equal to 2.88 and 10.41%, respectively). When the beads are immersed in ethanol, the mass loss is more marked (similarly for chitosan and chitosan/TiO2, the measured mass loss was 83.5 and 84.9%, respectively). The effect of TiO2 content on the swelling degree of chitosan/TiO2 hybrid membranes in 90 wt% aqueous solution of ethanol at 80◦C was described by D. Yang and co-workers44. The authors stated that all of chitosan/TiO2 hybrid membranes exhibited higher swelling degree than pure chitosan membrane. In our case these properties were comparable. On the other hand, water molecules strongly interact with the carbohydrate polymer (or chitosan/titania dioxide) and could replace the hydrogen bonding between chitosan-TiO2 and their chains. The additional crosslinking effect between chitosan and TiO2 prevents moderately the mass loss and this process is slower. However, after 24 h of immersion both balls were disappeared beads (100% of mass loss).


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The swelling properties of chitosan were determined by the swelling ratio (Q, in percent) calculated from the equation:

Q = (ms - mo)/mo

where mo is the weight of primary sample at the initial time in the dry state and ms ̶ the weight of swollen sample at the specified time. During longer time of experiment the swelling ratio decreases as an effect of enhanced acidic character of the environment. The amine groups in the chitosan molecules are ionized (NH3+) in acidic solutions and cationic charges operate as repulsive forces between the polysaccharide molecules. As amine groups stabilize the D-glucosidic linkages cut by acids, parts of chitosan oligomers, especially not highly crosslinked, were slowly dissolved in the aqueous solution. Norranattrakul and co-workers45 described swelling behaviour of the chitosan film crosslinked with citric acid (CA) and mixed with 1 wt% TiO2. The sample was immersed in distilled water with a pH of 5.5 at the time interval of 1, 3, 5, and 7 days. It was observed that the swelling process reaches a stable equilibrium after 5 days. It was found that the swelling curve of non-crosslinked chitosan changed greatly as compared to that of the crosslinked films. The values of swelling degree for the modified system were in the range 0.85-1.5 depending mainly on the kind of crosslinking agent.The lowest degree of swelling was obtained for chitosan/TiO2 crosslinked with citric acid45. In our case hydrochloric acid was used for chitosan/TiO2 beads formation. The measurements showed swelling and slightly de-swelling behaviour of chitosan beads. Both bead types showed the high swelling ratio in the range 1.91-2.96, but moderately higher swelling ratios were observed for the primary chitosan (about 1.55 times) than for the chitosan/TiO2 beads, probably due to protonation of the amino groups of chitosan at acidic


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pH. As expected, the TiO2 particles slightly inhibit remarkable swelling behaviour of chitosan, but on the other hand, increase its stability.

4.2 FTIR-ATR measurements and thermal stability The linkage of titanium dioxide particles with chitosan molecules can offer a lot of benefits. Nanoparticles of TiO2 have no pronounced effect on the initial mechanical properties of chitosan, but have a significant effect on thermal stability of chitosan functional groups. On the other hand, biocompatibility of metal oxide is improved by the chitosan addition. FTIRATR measurements were done because this method is a effective tool to materials characterization sensitive to hetero-nuclear functional group vibrations and polar bonds and also to verify structure of the investigated biomaterial. Polysaccharides, like chitosan, absorb in the region 1000-3500 cm-1,

46

. The FTIR-

ATR spectra of the native untreated chitosan, TiO2 and the chitosan/TiO2 system are collected in Figure3. For untreated chitosan (Figure3a) a broad band due to the axial O-H and N-H stretching centered at 3353 cm-1 and C-H stretching bands registered in the range 2957-2875 cm-1 is detectable. The absorption centred at 1650 cm-1 attributable to the axial C=O stretching of the acetamido groups (named amide I), the one at 1589 cm-1 (angular deformation of N-H bonds of the amino groups), the bands in the range 1408-1453 cm-1 resulting from the coupling of C-N axial stretching and N-H angular deformation is visible. The band noticeable at 1375 cm-1 is assigned to the CH3 symmetrical deformation mode. Finally named finger print band, particularly useful for evidencing chitosan presence even for small coatings deposition are bands due to skeletal signals in the range 1150-900 cm-1. A strong signal at 1026-1067 cm-1 is assigned to the asymmetric stretching of C-O-C in glucose circle, the bands in the range 1151-872 cm-1 due to polysaccharide skeleton, including the glycosidic bonds (peak of (1-4) glucosidic band), C-O and C-O-C stretchings.


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FTIR spectra of anatase at about 650 and 400 cm-1 should be visible, band characteristic of the rutile phase can be observed at 587 cm-1. For TiO2 P25 combination of anatase and rutile crystal structure is approximately 80:20, so peak from anatase (397 cm-1) is the most noticeable (Figure3b, right side). The shift in the profiles to lower energies observed on the samples is associated with the modification of titania particles by chitosan molecules and/or with the new composite formation. On the basis of the other authors47-48 interpretation of the FTIR spectra for different chitosan products indicates that there is the primary -OH group and the NH2 group which are active in the chitosan molecules. Hence, in our case the TiO2/chitosan at composite spectrum (Figure3c) in the range attributed to these early mentioned groups, which was centered at about 3353 cm-1, the peak intensity greatly increased and was shifted to about 3302 cm-1 compared with that in the both control (chitosan alone, Figure3a) and (TiO2 alone, 3b). Shift can confirm the creation of new biomaterial regarding its possible structure presented in Figure2. The next change suggesting the creation of biomaterial is disappearing of band at 1589 cm-1 attributable to the angular deformation of N-H bonds of the amino groups and huge increase (three times more) of the peak intensity at 1576 cm-1. Also the most representative peak of chitosan alone (C-O-C, glucose circle) is little visible at composite spectrum, absorbance changed from about 0.9 to 0.3 (compare Figure3a and Figure3c). On the other hand significant increase of peak attributed to N-H bands of chitosan (1415 cm-1) is observable, which confirms the connections between chitosan group and Ti4+ (see Figure2). Additionally, the absorption of O-Ti-O for the chitosan-titanium dioxide systems appeared in the range 677-695 cm-1. The typical absorption in the wavenumber region below 1000 cm-1 shows the absorption for inorganic compounds. On the other hand, the inorganic particles are found to enhance the thermal stability of chitosan composition. Thermal degradation of the chitosan structure is a complex reaction


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involving two47 or even three degradation stages48, respectively. The first stage occurs at the temperature 30-165 oC and is assigned to the evaporation of the residual water and acetic acid loosely bound to the polymer. From our previous investigations40 this point is located in the range 85°C-86.2°C from the DSC and DTG measurements, respectively. The second stage attributed

to

the

chitosan

degradation

(deacetylation,

further

dehydratation

and

depolymerization) is observed in the range 180-365 oC and for the investigated chitosan it is close to 345 oC. The differences can be explained as a result of different molecular weights of chitosan46-49. Diab and co-workers48 detected the third stage at 470 oC with a subsequent weight loss of the chitosan sample. Thermal properties are important for complete system characterization but this is not the main goal of this paper because preparation of pharmaceutical carriers or biomedical devices with chitosan as a component does not usually involve heating above 100 oC 48-52. The inorganic particles are found to enhance the thermal stability of chitosan composition. As a consequence, fitting of exactly right proportion is very important in order to obtain chitosan/titanium dioxide micro- or nanoparticles of the desirable properties. Hence the procedure of selection of system compounds proportion will be described in the next section.

4.3 Characterization of surface and volume properties of titanium dioxide The transmission electron microscopy (TEM) image of the commercial titanium dioxide (without purification) shows that titanium dioxide particles exist in solution as small flocks. After milling, the particles are more homogeneous and their average size is c.a. 20 nm (Figure1). But in our dispersions there are usually values about 200 nm or more. The frequent properties of metal oxides and titania, are the same potential determining ions (H+ and OHions). Therefore, it is crucial to characterize the suspension at different pHs, especially at


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pHiep. The isoelectric point of base TiO2 was found at pH 6.1 (Figure4), in the literature range and according to our earlier published data (5.9 - 6.4)32,

33, 43

adsorption sites on the titanium dioxide surface30,

suggests more than one charge

32, 43

. The existence of different

generation mechanism. Mechanism is more complicated after chitosan modification, but bacteriological and immunological aspects are very important for medical, cosmetic and pharmaceutical application of such systems. For this reason, we chose a few values of pH: 3; 4.8; 6; 7 and 9 as the representative ones for intense and meaningful suspension characterization. For commercial oxide without modification at pH values 3 and 9 the particle size is lower (338.8 nm and 770.95 respectively) than those obtained from the TEM-images and those reported by the Evonik producer while for medium pH values are notably larger. This can indicate the existence of weakly interconnected aggregate structures. Near the TiO2 i.e.p., the values of effective diameters, standard deviation and also TSI are the highest (2305.7±110.4 nm and 11.5 respectively). The aggregate structure is important because different macroscopic properties can result from the breaking down of weak linkages between flocks and/or those creating (existing) much stronger bonds inside the aggregate43. In order to get complete characteristics of the processes occurring in the examined TiO2 and TiO2/chitosan nanocomposites and their stability, the electrokinetic and Turbiscan measurements were carried out. For a low concentrated oxide suspension no change could be observed during a long period of storage and visually there are stable. However, even light changes in their stability can be easily detected by the Turbiscan measurements. Correlations between the electrophoretic and turbiscan measurements are very useful for estimation of biomaterial stability because specificity of such measurements is different.


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Low values (below 2µm) of effective diameter (and standard error) and the lowest value of TSI below 10, almost three times lower than the others were obtained at pH4.8 (Table 1). On the basis of these results, such system appears to be the most appropriate due to its stability and for different skin applications (pH near skin pH). It seems logical that surface properties are in close relationship with the electrokinetic ones. It is hard to completely control the flocculation state and consequently, the behaviour of titanium dioxide dispersions by only regulating the system pH. A better control of sample stability can be achieved by pH regulation and biological modification, e.g. by the chitosan biopolymer. We propose that the mechanism by which pH affects such behaviour consists in the changes in the surface charge of titanium dioxide particles and chitosan molecules and as a result, the electrostatic attraction/repulsion between them. The relationship of zeta potential as a function of both the weight ratio of titania/chitosan and pH of suspension will be helpful for verify the mechanism of stability, which will be described in the next sections.

4.4 Characterization of chitosan-treated titanium dioxide particles (low concentration, series I) Despite the great potential of chitosan its poor stability during long time storage makes chitosan-based systems not available as final pharmaceutical products. On the other hand, titanium dioxide-based system stability can be increased after the chitosan addition. Because of the polar and hydrogen bonding interactions between chitosan and TiO2, the particles are readily dispersed in the suspension. The volume properties of titanium dioxide suspension are directly associated with the surface condition of the particles. Due to the hydrophilic character of titanium dioxide, water creates a closed film on the surface in which different substances can be simply carried away32, 33, 43.


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Regarding the carbohydrate polymer stabilization, the steric stabilization should usually be taken into account. The initial step of such kind of stabilization is the adsorption of the stabilizer onto the solid (particles) surface32,

33 43

. It is possible to combine charged

functional groups in the same stabilizer molecule to provide both steric and electrostatic stabilization30-33. In the suspensions with the chitosan biopolymer such situation takes place, as described for similar systems, e.g. chitosan-hyaluronate53, chitosan-iron oxide54 and chitosan-copper oxide55. On the basis of our former electrokinetic and rheological investigations [43], the most reproducible behaviour of titanium dioxide suspensions was obtained for the intermediate (5%) concentration of TiO2 particles. As it was similar in these measurements this solid concentration was selected for investigation of the carbohydrate polymer modification. Initially, the measurements of titanium dioxide properties after chitosan modification were conducted at natural pH (pH=4.5±0.75). A varying parameter was the chitosan concentration (series I and II). For the first series the amount of chitosan used was in the range 0.4 x 10-80.6 x 10-7M. Only minor effects were found on the particle size distribution and the zeta potential of TiO2 after treatment with the smallest concentration of chitosan solution (not presented here). This means that the effect of biopolymer adsorption in the case of dilute dispersions, if any, can only be associated with the electrostatic forces. All values of TSI obtained for the first series were low in the range 0.9-4.1. But the lowest value of TSI (0.9) and the most stable system were obtained for 0.8 x 10-8 M of chitosan concentration, which is visible as the minimal fluctuations of transmission of light (%) vs. time in Figure5. The rate of migration of titanium dioxide particles changes slightly during the first 15-30 minutes (violet and bottle green lines, right scale) in the range 5-20% (left scale).


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The pHiep of the uncoated (base commercial) TiO2 particles was determined by the microelectrophoretic measurements close to the value (pHiep~6.1). On the other hand, for the chitosan molecules at low pH the free amino groups are protonated causing electrostatic repulsions between the biopolymer chains and thus facilitate its solvation. The literature reports the chitosan pKa in the range 6.0-6.555,

56

, hence below this value the chitosan

molecules exhibit the cationic behaviour. Hence protonation from an acidic to neutral solution with a specifically pH-dependent charge density is possible. The presence of free hydroxyl and amino groups enables polysaccharide to interact with other substances by hydrogen and electrostatic bonding. Such behaviour was confirmed by FTIR measurements (Figure3). Chitosan undergoes strong hydrogen bonding and hydrophobic interactions which can affect physicochemical properties (viscosity, electrokinetic potential, water solubility). Moreover, it was found out that the coating of the solid particle surface by chitosan molecules not only shifts the isoelectric point but can also remove the distance of connection between two particles46-52. Therefore at natural pH after polysaccharide adsorption an evident move of the isoelectric point of titanium dioxide in the direction of acidic environment, about one unit, is observed (Figure4). For the chitosan concentration in the range 0.2-0.6 x 10-7M (series I) all values of zeta potential of titanium dioxide particles as a function of time are positive in the range 19-60 mV (Figure6) and the values tend to zero with increasing concentration of chitosan. After equilibrium fitting, usually through 2 or 3h for the studied probes, the correlation between the effective diameter and the zeta potential can be found. The higher absolute values of zeta potential the lower values of effective diameter of chitosan/titanium dioxide particles were detectable. The most stable values of zeta potential during the first 3 h were obtained for 0.2 x 107

M chitosan concentration, which was also proved by the low value of TSI (2.0), despite


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large values of effective diameter (even sometimes outside the measuring range of apparatus). Organic substances usually adsorb in patches or on preferred sites on the TiO2 particle surface rather than as a uniform monolayer1-10. Therefore, the electrostatic repulsive forces appearing on the solid particles could be also not identical affecting in nonuniform bond distribution. We suppose that fluctuation of values of zeta potential and diameter of particles, especially for higher chitosan concentration can be an effect of its no uniform coverage of TiO2 particles. However, it is important that in such a system with large organic molecules steric forces (steric stabilization) are also essential. Not high (the absolute mean value about 20 mV) but stable zeta potentials in cooperation with steric forces are responsible for stable values of transmission of light and stability of probe at all. An example of graph of stable suspension is shown in the Figure6. Combination of the two mechanisms described above, called jointly the electrosteric stabilization, is difficult to isolate in the electrokinetic measurements alone, but should be observed in other compatible techniques, for example the multimodal size distribution, rheological and also turbiscan measurements. With the increasing weight or volume fraction of particles, solid particles are closer to each other and the number of attached particles in the network is effectively enhanced. Consequently, the local bonding strength is also increased and the particles arrangement grows to be more uniform. Therefore for complete characteristic, electrokinetic behaviour should be determined more accurately for higher chitosan solution concentrations and invariable titanium dioxide amount vs. time. This type of studies is presented in the next paragraph.

4.5 Effect of higher chitosan concentration (series II) on the electrokinetic properties of the TiO2 particles


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Because steric stabilization and/or particle linkages may only occur in the concentrated systems, when the interparticle distance is comparable to the relatively small chain length of chitosan molecule, the second series was done in which the amount of chitosan used was in the range 0.4 x 10-6- 0.6 x 10-5M (Table 2, series II). A network of ionic bridges between the negatively charged TiO2 and the positively charged chitosan chains is formed. Among higher concentrations of polysaccharide solution the maximum value of suspension TSI was obtained for the value equal to 0.8 x 10-6 M, after that a slow decrease of TSI was noticeable. A further increase of chitosan concentration is not required since the comparable values of TSI below 10 for all probes (5.9-7.7) and similar values of zeta potential (15.37-18.23 mV) suggest parallel system stability as an effect of not only electrostatic forces but also steric repulsion. Values of zeta potential below 20 mV are not enough to electrostatic stabilization. Obtainment of stable suspensions, which was proved on the basis of low values of TSI, was possible thanks to steric stabilization of chitosan layer and/or creation of stable composite. However, for the highest concentration of biopolymer chitosan layer is thicker and probably polymer film creation on TiO2 particles and its swelling properties cause two or even three times higher values of suspension effective diameter (Table 2). Creation of stable biomaterial could be attributed to formation of a protective hydration layer around the titanium dioxide particles and also near the chitosan chains through the interchain hydrogen bonds. The addition of precise amount of chitosan solution to the TiO2 suspension can stabilize the zeta potential on the particles surface and prevent their aggregation over a storage period, e.g. one week. Changing the swelling ratio of chitosanbased formulations, as a consequence a prolonged and more controlled drug release profile can be attained. Lately not titanium but zinc ions have been used as agents which are able to improve the colloidal stability of chitosan polyelectrolyte complex. Such type of stabilization


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effect could be attributed to the arrangement of co-ordinate bonds that adjust the structure of the chitosan composites52. As a confirmation of the electrosteric stabilization mechanism, the zeta potential and mean diameter values obtained from the dynamic light scattering measurements were usually congruent with the Turbiscan measurements, especially with TSI (turbiscan stability index). However, finding the simple relationship between these parameters is difficult especially near the point of zero charge, because even the properties of base titanium dioxide suspensions exhibit the largest changes for pH near the isoelectric point, independent of components amount32, 44. The electrokinetic behaviour is pH-dependent, and together with the gravitational and hydrodynamic effects is expected to be in control. The relative balance of all strengths is affected by different factors, mostly the particle size, concentration, temperature and pH. At pH below pH pzc the surface of titanium dioxide is positively charged and adsorption of chitosan only by electrostatic forces rather does not occur. Furthermore, the creation of homogeneous layer of thin chitosan film on the titanium dioxide particles surface is also evidently pH-dependent. Hence, the performed measurements for the concentrated chitosanmodified titanium oxide suspensions vs. pH are presented in detail in the next section.

4.6 Effect of pH on the electrokinetic behavior of chitosan/TiO2 particles As was mentioned early we suppose that a better control of dispersion properties can be achieved by two simple method combination, first by pH regulation and second by the addition of chitosan biopolymer and as an effect enhanced biocompatibility. For that reason, the studies of the chitosan effect and characterization of the properties of carbohydrate polymer modified titanium oxide composition as a function of pH were another goal of this paper.


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In the pH range 5.5-6.5 (near the point of zero charge) the effective diameter (not presented here) and zeta potential curves of unmodified and chitosan-modified TiO2 particles (Figure4) were similar. Higher differences appeared for more acidic and more basic pH values. The maximum of effective diameter for the chitosan-TiO2 particles in the basic alkaline environment (pH 9, not presented) as well as large values in the acidic environment (pH 3, Figure7) were obtained. The minimum values were achieved at pH close to 4.8. Size diameter and multimodal size distribution for the chitosan-modified titanium dioxide suspensions are also strictly pH dependent (Figure7). On the other hand, one could expect reduction in the diameter with the addition of carbohydrate biopolymer as manifestation of steric stabilization. During measurements such a case was found for the titania suspensions with the positively charged chitosan molecules layer at pH 4.8 and very low zeta potential values was obtained. The achieved relationships prove the role of steric stabilization in the investigated suspensions with the chitosan modification (Figs. 7-8, Table 1). The steric stabilization mechanism is responsible for low viscosity and easy dispersibility. Reduction in viscosity is caused by the steric barrier whereas a close approach between the titanium dioxide particles is prevented due to the layer of carbohydrate biopolymer on the solid surface. The increased distance between the particles results in a reduction of the van der Waals forces, and therefore enhanced electrostatic repulsion between the particles. At all measured values of pH after chitosan addition not so high, satisfactory values of TSI for the chitosan/titanium dioxide system were obtained, lower than 10 units, even sometimes twice lower than for the unmodified titania (see Table 1). Enhanced electrostatic repulsion help to diminish the flocculated system organization. The addition of cationic polysaccharide induce usually more positive zeta potential for the titania particles, according to the adsorption preference of the opposite-charged molecules and hereby the surface charge can be regulated.


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In Figure4 after the chitosan film adsorption the shift of the i.e.p point to the acidic environment is observable. At pH 4.8 very low but stable values of zeta potential were obtained. Chitosan strongly interacts with titanium dioxide via the electrostatic interaction under the pH conditions where their molecules are oppositely charged because under weak and strong acidic conditions chitosan is a polycation. Understanding the interaction of titanium dioxide with polysaccharides or others (compounds of cell membranes) is an important step towards knowledge of the biological effects. Chitosan biopolymer is a watersoluble and bioadhesive substrate whose molecules readily bind to negatively charged surfaces such as biological membranes, for example mucosal membranes14-18. Purified quantities of chitosans are available for a lot of biomedical applications (e.g. nonviral gene delivery, infection of breast cancer cells with free nucleic acid, wound repairing) 56-58. The last case of applications is especially important to us and therefore the wettability and biocompatibility measurements are under evaluation, which will be described in a new article32. The original scans from Turbiscan for the titanium dioxide particles with and without chitosan modification are presented in Figure8. On the basis of the obtained relationships it can be stated that equilibrium is reached fast and fluctuations are rather small. For the soft stable suspension, after the first minutes the migration rates sharply decrease, but to move down all particles about 3-4 days are needed. The migration rates are the mean value estimated from the scans transported along 0-55 mm (bottom scale) of the calibrated cell. For stable titania suspensions (e.g. with efficient chitosan concentration) the migration rates are low (in the range 2 µm/min to 5 µm/min) and more compact relationships vs. time for a larger part of the system is observable (Figure8). It means that the chitosan surface coverage of oxide particles is similar, the stabilization of suspension is effective and formation of stable biomaterial is achievable. In the presence of long-chain molecules an


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effect of steric stabilization can play a significant role and additionally at higher concentration of chitosan the process of forming gel is also possible regarding its swelling properties. In our opinion for the titania-based systems the most favourable parameters of diameter, multimodal size distribution and zeta potential were obtained at pH=4.8, near skin pH (Figure7, 8). The values of pH for the human skin are in the range 4.5-6.2 and depend on age, gender and even lifestyle. At this pH a small positive charge but very stable as a function of time is acquired and this can make the composite bioadhesive. On the basis of our previous investigations for untreated TiO2, it was stated that the transition from the state of weak flocculation at low pH (pH 3) to the maximum flocculation at pH 6 (near the isoelectric point) and then back to a state of weak flocculation at high pH (pH 9) is possible43. But these investigations illustrate a general tendency while in the multicomponent oxide systems, for example after the chitosan addition the situation can be different. This concept was discussed in detail with the reference to the titanium dioxide suspensions of different particle sizes after the phospholipid (PC) modification33. A remarkable result was the increase of the yield stress when the PC coating is applied as compared to that of bare particles. The addition of the biosurfactant has a stabilizing effect through steric repulsion between the oxide particles, but it is strictly pH-dependent and depends on the surface coverage. In the investigated systems precise choice of pH can be also important stabilizing factor. The electrostatic favored adsorption results in sensitivity of titanium dioxide to pH adjustment, but electroneutral adsorption cannot be neglected being the dominant manner of adsorption of organic acids in solution59-61. The presence of chitosan in the pH range 6-9 also improved stability, because the turbiscan stability index (TSI) for the titanium dioxide suspension after the biological modification was lower than for base suspension (Table 1), it means that it will produce no or low aggregation with stable viscosity.


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Conclusions The aim of the present paper was to identify the behaviour of cationic polysaccharide chitosan in the titanium dioxide dispersion. The obtained results with the help of an optical analyzer, electrophoresis and by the dynamic light scattering method show significant influence of the chitosan on the measured quantities. The interactions between the biopolymer and the titanium dioxide particles as well as between the particles themselves depend on the charge state of the interface. The charge on both kinds of TiO2 surfaces can be controlled by pH, due to the acid/base reactions. Cationic biopolymer molecules adsorb to the TiO2 surface, driven by electrostatic interactions and steric effects, adsorption is strictly pH-dependent and creation of stable TiO2/chitosan biomaterial is possible. This process was observed on the basis of FTIR spectra, also during Turbiscan and zetameter measurements as a change of diameter, multimodal size distribution, electrophoretic mobility, stability index before and after the flocculation state changes. Up to now there have not been distinguished universal parameters of chitosan-titanium dioxide products which are essential to provide their maximal stability. In this case the chitosan-modified titanium dioxide particles are characterized by improved physicochemical properties in comparison to the base TiO2. The modification of titanium dioxide particles can be a result of the increase in structural packing (crosslinking) of chitosan, which is a consequence of the interactions between the chitosan and titanium dioxide particles. These interactions are associated with the presence of Ti4+ and hydroxyl and/or amino groups in chitosan. If intermolecular interactions are enough strong the chitosan/TiO2 biomaterial is creating. In our opinion, the obtained chitosan/titanium system with the precisely selected compounds ratio is a good candidate for the preparation of new biopolymer mixtures with high potential attractive properties (biocompatibility, biodegradability and non-toxicity). We


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hope that the hybrid materials will be elaborated due to valuable properties of individual components along with frequently synergistic effects and our investigations make a substantial progress in the scaling-up of titanium dioxide-chitosan applications (cosmetics, sunscreens, antibacterial materials, drug delivery systems and others). Acknowledgment The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Programme (contract no. POIG.02.01.00-06-024/09 Centre for Functional Nanomaterials).

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(32) Wiącek, A.E., Gozdecka, A., Physicochemical characteristics of chitosan/TiO2 composites part II. In aspect of wettability and biocompatibility, Carbohydr. Polym. 2017, in preparation (33) Chibowski, E., Hołysz, L., Terpiłowski, K., Wiącek, A.E., Influence of ionic surfactants and lecithin on stability of titanium dioxide in aqueous electrolyte solution, Croat. Chem. Acta, 2007, 80 (3-4), 395-403. (34) Le, Q.Ch, Ropers, M.H., Terrisse, H., Humbert, B., Interaction between phospholipids and titanium dioxide particles, Colloids Surf., B, 2014, 123, 150-157. (35) Preočanin, T., Kallay, N., Point of Zero Charge and Surface Charge Density of TiO2 in Aqueous Electrolyte Solution as Obtained by Potentiometric Mass Titration, Croat. Chem. Acta, 2006, 79, 95-106. (36) Wiącek, A.E., Changes in wetting properties of silica surface treated with DPPC in the presence of phospholipase A2 enzyme, Appl. Surf. Sci., 2010, 256 (24), 7672– 7677. (37) Wiącek, A.E., Investigations of DPPC effect on Al2O3 particles in the presence of (phospho)lipases by the zeta potential and effective diameter measurements, Appl. Surf. Sci., 2011, 257 (9), 4495-4504. (38) Wiącek, A.E., Changes in wetting properties of alumina surface treated with DPPC in the presence of phospholipase A2 enzyme, Colloids Surf., B, 2011, 87, 54-60. (39) Wiącek, A.E., The wetting and interfacial properties of alumina surface treated with dipalmitoylphosphatidylcholine and lipase enzyme, Powder Technol., 2011, 212, 332-339. (40) Terpiłowski, K., Wiącek, A.E., Jurak, M., Influence of PEEK nitrogen plasma treatment for wettability of deposited chitosan layers, Adv. Polym. Technol., 2016, doi: 10.1002/adv.21813 (41) Gozdecka, A., Wiącek, A. E., Effect of UV radiation and chitosan coating on the adsorption-photocatalytic activity of TiO2 particles, Carbohydr. polym., 2017 under revision (42) Książek, S., Mucha, M., Thermal stability of chitosan nanocomposites containing TiO2 and organomodified montmorillonite, Prog. Chem. Appl. Chitin Its Deriv., 2015, XX, 122-129. (43) Wiącek, A.E., Anitowska, E., Delgado, A.V., Hołysz, L., Chibowski, E., The electrokinetic and rheological behaviour of phosphatidylcholine-treated TiO2 suspensions, Colloids Surf., A, 2014, 440, 110-115.


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(44) Yang, D., Li, J., Jiang, Z., Lu, L., Chen, X., Chitosan/TiO2 nanocomposite pervaporation membranes for ethanol dehydration, Chem. Eng. Sci., 2009, 64, 3130 3137. (45) Norranattrakul, P., Siralertmukul, K., Nuisin, R., Fabrication of chitosan/titanium dioxide composites film for the photocatalytic degradation of dye, J. Met., Mater. Miner., 2013, 23 (2), 9-22. (46) Wiącek, A.E., Dul, K., Effect of surface modification on starch/biosurfactant wettability, Colloids Surf., A, 2015, 480, 351–359. (47) Lewandowska, K., Sionkowska, A., Kaczmarek, B., Furtos, G., Characterization of chitosan composites with various clays, Int. J. Biol. Macromol., 2014, 65, 534-541. (48) Diab, M.A., El-Sonbati, A.Z., Bader, D.M., Thermal stability and degradation of chitosan modified by benzophenone, Spectrochim. Acta, Part A, 2011, 79, 1057–1062. (49) de Britto, D., Campana-Filho, S. P., Kinetics of the thermal degradation of chitosan, Thermochim. Acta, 2007, 465, 73-82. (50) Corazzari, I., Nistico, R., Turci, F., Faga, M.G., Franzoso, F., Tabasso, S., Magnacca, G., Advanced physico-chemical characterization of chitosan by means of TGA coupled on-line with FTIR and GCMS: Thermal degradation and water adsorption capacity, Polym. Degrad. Stab. 2015, 112, 1-9. (51) Pieróg, M., Ostrowska-Czubenko, J., Gierszewska-Drużyńska, M., Thermal degradation of double crosslinked hydrogel chitosan membranes, Prog. Chem. Appl. Chitin Its Deriv., 2012, 17, 67-74. (52) Zawadzki, J., Kaczmarek, H., Thermal treatment of chitosan in various conditions, Carbohydr. Polym., 2010, 80 (2), 394-400. (53) Wu, D., Delair, T., Stabilization of chitosan/hyaluronate colloidal polyelectrolyte complex in physiological conditions, Carbohydr. Polym., 2015, 119, 149-158. (54) Konwar, A., Kalita, S., Kotoky, J., Chowdhury, D., Chitosan-Iron Oxide coated graphene oxide nanocomposite hydrogel: a robust and soft antimicrobial biofilm, ACS Appl. Mater. Interfaces, 2016, 8, 20625-20634. (55) Haldorai, Y., Shim, J.J., Multifunctional Chitosan-Copper Oxide Hybrid Material: Photocatalytic and Antibacterial Activities, Int. Journal of Photoenergy, 2013, 2013, 1-8 (56) Desbrieres, J., Autoassociative natural polymer derivatives: the alkylchitosans. Rheological behaviour and temperature stability, Polymer, 2004, 45, 3285-3295.


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Table’s captions Table 1 Turbiscan stability index (TSI) for TiO2 and TiO2/chitosan particles as a function of pH. Table 2 Effective diameter, zeta potential and Turbiscan stability index (TSI) of titanium dioxide as a function of chitosan concentration.


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Table 1 Turbiscan stability index (TSI) for the TiO2 and TiO2/chitosan particles as a function of pH.

TSI for TiO2/chitosan

pH of system

TSI for TiO2

3

9.5

4.6

4.8

3.8

6.3

6

11.5

5.5

7

11.0

4.2

9

10.6

5.7

particles

Table 2 Effective diameter, zeta potential and Turbiscan stability index (TSI) of titanium dioxide as a function of chitosan concentration

Chitosan

Turbiscan

Effective

concentration

stability index

diameter

Zeta potential

[mol/dm3]

TSI

0.4 x 10-6

7.6

579.8 ± 36.8

17.6 ± 0.6

0.8 x 10-6

7.7

1114.0 ± 63.3

17.0 ± 0.7

0.2 x 10-5

7.0

1610.0 ± 73.4

18.2 ± 1.0

0.4 x 10-5

6.7

3382.5 ± 360.1

16.3 ± 0.7

0.6 x 10-5

5.9

8323.3 ± 1044.6

15.4 ± 0.8


35



Page 36 of 45

Page 37 of 45

70 60 50

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 30 20 10 0 10

20

30

40

50

60

70

80

90

100

Diameter, nm Figure 1 Multimodal size distribution and SEM image (inside) of commercial titanium dioxide particles after milling

Figure 2 Possible structure of the chitosan/TiO2 biomaterial


1


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Figure 3 FTIR-ATR spectrum of a) base chitosan, b) base TiO2 c) TiO2/chitosan biomaterial


2

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35

Mean zeta potential [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

TiO2

25

TiO2+Chitosan

20 15 10 ...

5 0 -5 -10 -15 -20 3

4

5

6

pH

7

8

9

10

Figure 4 Relationship of zeta potential for base TiO2 and TiO2/chitosan suspension as a function of pH

Figure 5 Transmission of light for the TiO2/chitosan (0.8x10-8mol/dm3) particles as a function of time and height of cuvette


3


20000 -7

0.2 x 10 M -7 0.4 x 10 M -7 0.6 x 10 M

17500 15000

Effective diameter [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

12500 10000 7500 5000 2500 0 0

30

60

90

120

150

180

210

1360 1440

Time [min]


4

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65 60 55 50

Zeta potential [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45 -7

0.2 x 10 M -7 0.4 x 10 M -7 0.6 x 10 M

40 35 30 25 20 15 0

30

60

90

120

150

180

210

1360 1440

Time [min]

Figure 6 Effective diameter and zeta potential of titanium dioxide as a function of time for different chitosan solution concentrations (up). Model transmission of light for the TiO2/chitosan (0.2 x 10-7mol/dm3) particles as a function of time and height of cuvette (down)


5


12000

Effective diameter [nm]

10000

TiO2, pH = 3 TiO2, pH = 4.8 TiO2 + Chitosan, pH = 3 TiO2 + Chitosan, pH = 4.8

8000

6000

4000

2000

0 0

30

60

90

120

150

180

210

1360 1440

Time [min]

40 35 30

Zeta potential [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 45

25

TiO2, pH = 3 TiO2, pH = 4.8

20

TiO2 + Chitosan, pH = 3 TiO2 + Chitosan, pH = 4.8

15 10 5 0 -5 0

30

60

90

120

150

180

210

1360 1440

Time [min]

Figure 7 Effective diameter and zeta potential of titanium dioxide and titanium dioxide/chitosan as a function of time at acidic pH


6

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pH3

pH=4.8


7


Page 44 of 45

pH3

pH=4.8

Figure 8 Transmission of light for the TiO2 particles (up) and TiO2/chitosan particles (down) as a function of time and height of cuvette at pH 3 and pH 4.8, respectively


8

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Figure captions Figure 1 Multimodal size distribution and electron photograph (inside) of commercial titanium dioxide particles after milling Figure 2 Possible structure of the chitosan/TiO2 biomaterial Figure 3 FTIR-ATR spectrum of a) base chitosan, b) base TiO2 c) TiO2/chitosan biomaterial Figure 4 Relationship of zeta potential for base TiO2 and TiO2/chitosan suspension as a function of pH Figure 5 Transmission of light for TiO2/chitosan (0.8x10-8mol/dm3) particles as a function of time and height of cuvette Figure 6 The effective diameter and zeta potential of titanium dioxide as a function of time for different chitosan solution concentration (up). Model transmission of light for TiO2/chitosan (0.2 x 10-7mol/dm3) particles as a function of time and height of cuvette (down) Figure 7 The effective diameter and zeta potential of titanium dioxide and titanium dioxide/chitosan as a function of time at acidic pH Figure 8 Transmission of light for TiO2 particles (up) and TiO2/chatoyant particles (down) as a function of time and height of curette at pH 3 and pH 4.8, respectively


9

Physicochemical Characteristics of ChitosanâTiO2 Biomaterial. 1

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