Synthesis of a benzyl-grafted alginate derivative and its effect on the colloidal stability of nanosized titanium dioxide aqueous suspensions for Pickering emulsions

TiO2 nanoparticles (nano-TiO2) as one of the most extensively used nanoscale materials easily undergo spontaneous aggregation and gravity sedimentation ascribed to their high adsorption energy, which significantly restricts their actual applications. For this reason, a benzyl-grafted alginate derivative (BAD) with good colloidal interface activity, prepared by a bimolecular nucleophilic substitution (SN2) reaction, was used as the dispersant to stabilize nano-TiO2. The structure and colloidal properties of BAD was evaluated by FT-IR spectroscopy, 1H NMR spectroscopy, thermal gravimetric analysis (TGA) and dynamic light scattering (DLS). The effects of pH and ionic strength on the dispersion stability of BAD/nano-TiO2 suspensions were also examined by DLS. To further probe its feasibility as a drug delivery system, the BAD/nano-TiO2 complex was applied as particulate emulsifiers to fabricate drug-loaded Pickering emulsions. Meanwhile, the morphology properties and the sustained release performance of the drug-loaded Pickering emulsions were also investigated. Experimental results showed that the adsorption of BAD on nano-TiO2 was achieved by an intermolecular hydrogen bond between the carboxylic functional groups of BAD and the Ti–OH of TiO2. The adsorption of BAD enhanced the electrostatic repulsion and steric hindrance between nano-TiO2 improving the dispersion stability of nano-TiO2 at different pH and ionic strength. Additionally, the obtained Pickering emulsions displayed good drug-loading capacity and sustained release performance with the release mechanism of non-Fickian transport, which exhibited great potential in the pharmaceutical field.


Introduction
TiO 2 nanoparticles (nano-TiO 2 ) are considered to be one of the most extensively used nanoscale materials for their unique advantages, such as good stability, high photocatalytic activity, strong UV resistance, etc. 1,2 They have been commonly utilized as additives to commercial products such as cosmetics, pharmaceuticals, and foodstuffs. [3][4][5] However, nano-TiO 2 in suspension readily show a strong tendency to spontaneously agglomerate and then sediment due to their strong hydrophilicity, large specic surface area, and high adsorption energy, which signicantly restrict their actual application in the manufacture of commercial products. 6,7 The aggregation of aqueous nano-TiO 2 suspensions is owing to the London-van der Waals attractive forces between the nano-TiO 2 of the dispersed phase, which can be overcome by means of electrostatic repulsions between similarly charged particles arising from solvation of adsorbed layers, or most frequently by an electrostatic mechanism with the use of dispersants. 6,8 Currently, most of reports have focused on the ways to stabilize TiO 2 particles against aggregation by using surfactants or polymers as dispersants. [9][10][11][12][13] The rationale underlying this approach is that the adsorption of dispersants on the nanoparticles surfaces not only affects the electrostatic interactions, but also introduces additional steric interactions, which can prevent the nanoparticles surfaces from coming into direct contact. 14 For examples, Tkachenko et al. 15 studied the inuence of concentration of nonionic TRITON X-100 and anionic ATLAS G-3300 surfactants on the size and zeta potential of TiO 2 particles in the water suspensions. The results showed that anionic ATLAS G-3300 surfactant was more effective stabilizator of aqueous suspensions of titanium dioxide, than nonionic surfactants of TRITON X-100. And it was also found that hydrophobic interaction had important role in the processes of stabilization of suspensions. Yang et al. 13 investigated the effect of anionic sodium dodecyl sulfate monomer and micelles on the stability of aqueous dispersions of nano-TiO 2 using dynamic light scattering (DLS) techniques. They found the strong steric repulsion generated by sodium dodecyl sulfate micelles can effectively prevent the aggregation and sedimentation of nano-TiO 2 . Liufu et al. 16 applied the poly (acrylic acid) (PAA) to stabilize the suspension of nano-TiO 2 , and the results showed that the stabilization of PAA/nano-TiO 2 suspension was achieved through electrostatic repulsion, and the adsorption of PAA on the TiO 2 surface may take place by hydrogen bonding and chemical interaction between the nanoparticles and the carboxyl groups of the polymer. From the above examples, we can nd that the dispersant may either possess colloidal interface activity or be exible polyelectrolytes. The employed polymer dispersants should be capable of hydrogen bonding with the metal oxide surface. 17,18 And the carboxylic functional groups are oen used to anchor polymeric chains to oxide nanoparticles surfaces. 19,20 Although the addition of different surfactants and synthetic polymers can effectively improve the dispersion stability of nano-TiO 2 , the extensive use of them is bound to cause some pollution to the environment. 21 Therefore, it is necessary to seek a green alternative to reduce environmental damages and environmental consequences. To note, alginate, as a natural anionic polyelectrolyte, has abundant carboxylic functional groups and other merits, such as source availability, non-toxicity, biocompatibility and biodegradability. [22][23][24] It is these characteristics that possibly endow it with the potential to become a green dispersant for the stabilization of nano-TiO 2 aqueous suspension.
Alginate, derived from marine algae and various bacteria, as a natural anionic unbranched biopolymer is a heteropolysaccharide comprised of (1-4)-linked b-D-mannuronic acid and a-L-guluronic acid blocks distributed in an irregular blockwise pattern. 25,26 As alginate molecular chain contains large amounts of free hydroxyl and carboxyl groups, intramolecular hydrogen bonds are easily formed within the molecules. 27,28 These intramolecular hydrogen bonds lead to highly stretched rigid structure of the alginate chain in aqueous solution, which is not conducive to the adsorption of alginate as a dispersant on the surface of nano-TiO 2 . In this case, the chemical modication of alginate by hydrophobic groups may be an effective method to enhance its molecular exibility or colloidal interface activity, 29,30 thus improving the ability to disperse nano-TiO 2 . As a result of available hydroxyl and carboxyl groups, chemical modication of alginate could be achieved by the acetylation, phosphorylation, sulfation, oxidation, esterication and amidation. 27 Most of the modications need the catalyst to activate the hydroxyl or carboxyl groups attributed to the rigidity of alginate chain, 29,30 but they oen exhibited low chemical activity. Recently, Pawar et al. reported a strategy to dissolve tetrabutylammonium (TBA) salts of alginic acid in polar aprotic solvents containing tetrabutylammonium uoride (TBAF) for synthesis of carboxyl-modied alginate esters. This strategy makes the esterication of TBA-alginate be performed under homogeneous conditions via bimolecular substitution nucleophilic (S N 2) reactions without the aid of the catalyst, resulting in the high chemical activity with the total degree of substitution (DS) close to 1.0. 31,32 In spite of high chemical activity for S N 2 reaction, the chemical characterization and colloidal properties of the corresponding alginate derivative synthesized by S N 2 reaction is still scarce, especially when the prepared alginate derivative was used as a dispersant for the dispersion of nano-TiO 2 in aqueous media.
In the present study, the benzyl-graed alginate derivative (BAD) was prepared by S N 2 reaction to improve the colloidal interface activity of alginate. To retain partial carboxylic functional groups of alginate that could be used to self-assemble into core-shell architectures and anchor polymeric chains to nano-TiO 2 's surfaces, the molar ratio of benzyl bromide and uronic acid of TBA-alginate was xed at 50%. The structure and colloidal property of BAD was evaluated by FT-IR spectrometer, 1 H NMR spectrometer, thermal gravimetric analysis (TGA) and dynamic light scattering (DLS). And then we attempted to use BAD as the dispersant to stabilize commercial nano-TiO 2 with the crystalline phase of anatase in aqueous solution. The effects of pH and ionic strength on the dispersion stability of BAD/ nano-TiO 2 suspensions were also examined by DLS. Finally, the drug-loaded Pickering emulsions were prepared by high-speed shearing method using BAD/nano-TiO 2 complex as the particulate emulsiers and l-cyhalothrin as the model pesticide to probe its feasibility as a drug delivery system. The morphology properties and the release performance of the drug-loaded Pickering emulsions were also investigated. This work combined renewable alginate with inorganic nanomaterials, aiming at paving a new path for the extensive application of nano-TiO 2 .

Synthesis and characterization of benzyl-graed alginate derivative
Benzyl-graed alginate derivative (BAD) was synthesized according to previous method with some modications. 31,32 The synthesis route, as displayed in Scheme 1, involved the neutralization of the alginic acid with TBAOH and its bimolecular substitution nucleophilic (S N 2) reaction, which were detailedly described as follows.
Firstly, 4.0 g of alginic acid was dispersed in 100 mL distilled water under stirring, and the aqueous TBAOH was added dropwise until the pH was adjusted to 8-9. Then, the mixture was ltered under vacuum using coarse lter paper to remove any unreacted particulates. Aerward, the obtained solution was dialyzed in distilled water for 3 days and lyophilized to get the pure TBA-alginate polymer.
Subsequently, 1.5 g of TBA-alginate was dissolved in 100 mL DMF containing 1.0 g of TBAF and stirred vigorously to allow its complete dissolution. Then 0.19 mL of benzyl bromide was directly added to the TBA-alginate solution and the mixture was le to react at ambient temperature under stirring. Aer 24 h of reaction, 50 mL of 2.5 mol L À1 NaCl aqueous solution was added to replace the existing TBA + ions with Na + ions under another 2 h of stirring. Finally, the resultant polymer was completely precipitated by adding ethyl acetate, then separated by centrifuge at 7000 rpm and washed thoroughly with ethanol to remove excess reactants repeatedly. The product was dialyzed in distilled water for 3 days and lyophilized to get the pure BAD. For comparison, sodium alginate (SA) was prepared by neutralizing raw alginic acid with NaOH solution.
The degree of substitution (DS) for BAD during the S N 2 reaction was determined by titration according to previous reports, 32 which were performed by reacting alginate esters with excess NaOH to hydrolyze the ester groups. The chemical structure of BAD was characterized by FT-IR, 1 H NMR and thermogravimetric analysis. FT-IR spectra of samples were recorded on a Thermo Scientic Nicolet 6700 Fourier transform infrared spectrometer. The samples were mixed with KBr and compressed to semitransparent disks for spectroscopic analysis. 1 H NMR was performed on a Bruker AV 400 nuclear magnetic resonance spectrometer. The samples were dissolved in D 2 O to the concentration of 10 mg mL À1 . Thermogravimetric analysis was carried out on a TA Q600 Thermal Analyzer in N 2 at a heating rate of 20 C min À1 to examine the thermal properties of the samples. Fluorescence measurement was performed on a Hitachi F7000 uorescence spectrophotometer, which is an efficient way to detect the colloidal interface activity of BAD. Pyrene as a uorescence probe was excited at 335 nm and the emission spectrum was collected in the range of 335-600 nm at an integration time of 1 s with the slit width of 2.5 nm. The size and zeta potential of BAD micelle-like self-aggregates were measured by DLS with a Malvern Nano-ZS90 Zetasizer (UK) at a scattering angle of 90 at 25 C, employing an (He-Ne) argon laser (l ¼ 633 nm). The polymeric micelle-like self-aggregates solution was prepared at 60 C and diluted to the concentration of 1.0 mg mL À1 to avoid multiple scattering.

Preparation and evaluation of BAD/nano-TiO 2 suspensions
The BAD/nano-TiO 2 suspensions were prepared by the mechanical mixing method. Briey, 5 mg of BAD and 10 mg of nano-TiO 2 were ultrasonically dispersed in 10 mL deionized water. Then the dispersions were shaken on a shaker overnight to promote the adsorption of BAD on nano-TiO 2 , thus preparing the BAD/nano-TiO 2 suspensions.
The effects of pH and ionic strength on the dispersion stability of BAD/nano-TiO 2 suspensions were examined by DLS, which were expressed by the change of their size and zeta potential. The pH effect test was performed by adjusting pH of the suspensions from 3 to 9 using 0.1 mol L À1 HCl solution and 0.1 mol L À1 NaOH solution. Similarly, the ionic strength effect test was performed by adjusting ionic strength of the suspensions to 50, 100, 150, 200 and 300 mmol L À1 by the addition of NaCl.
To further conrm the morphology and structure of BAD/ nano-TiO 2 suspensions and the interactions between BAD and nano-TiO 2 , the scanning electron microscopy (SEM) observation, transmission electron microscope (TEM) observation and FT-IR analysis were conducted for the freeze-dried BAD/nano-TiO 2 suspensions. The SEM measurement was performed on a Hitachi S-3000N scanning electron microscope (Japan) aer gold coating, while the TEM measurement was carried out using a JEM 2100 TEM (JEOL Co., Japan) at an acceleration voltage of 200 kV.

Fabrication of Pickering emulsions by BAD/nano-TiO 2 suspensions
The Pickering emulsions with an oil/water ratio of 1 : 9 (v/v) were prepared by high-speed shearing method with a high shear homogenizer (T10 basic ULTRA-TURRAX, IKA, Germany), using BAD/nano-TiO 2 complex as the particulate emulsiers and toluene as the oil phase, as shown in Scheme 2. Detailedly, 1.0 mL of toluene was added to 9.0 mL of BAD/nano-TiO 2 suspensions with the concentrations of 5.0 mg mL À1 for nano-TiO 2 and 2.5 mg mL À1 for BAD to form the emulsions with the aid of 20 000 rpm high-speed shearing.
The stability to creaming of the resultant emulsions was checked by storing them at ambient temperature for 24 h. Since emulsion droplets had a lower density than the surrounding liquid, they moved upwards rapidly and a cream-depleted aqueous phase was emerged at the bottom of the emulsions. To observe the microstructures of emulsion droplets, the oil phase was stained with Nile red. The emulsion droplets were placed directly onto a glass microscope slide and covered the coverslip. Then the microstructure of Pickering emulsion droplets was observed by using a Nikon Ti-S uorescent microscope (Japan).

Loading and release studies of l-cyhalothrin
l-Cyhalothrin is a kind of broad-spectrum, highly-efficient, and long-lasting biomimetic pesticide, which can not only control various pests on crops, such as cotton, fruit trees, vegetables, and soybeans, but also control parasites on animals. For this reason, l-cyhalothrin was chosen as the model pesticide for loading and release studies to verify the application of Pickering emulsions stabilized by BAD/nano-TiO 2 complex as an efficient pesticide carrier. In our work, about 20 mg of l-cyhalothrin was fully dissolved in oil phase under stirring prior to the fabrication of Pickering emulsions. The loading of l-cyhalothrin into Pickering emulsions was achieved based on the emulsication of BAD/nano-TiO 2 complex.
The release of l-cyhalothrin from the drug-loaded Pickering emulsions was performed in 50% methanol solution at ambient temperature in a stoppered conical ask. 2 mL of drug-loaded Pickering emulsions were enclosed into a dialysis bag (cut-off 3500 MW), and then the bag was immersed in 50 mL of 50% methanol solution. 50 mL of the solution was replaced with the same volume of fresh 50% methanol solution at different time intervals, which could avoid the inuences of the saturated solutions. 1 mL of the solution was removed at different time intervals and ltered with 0.22 mm membrane for the determination of l-cyhalothrin by GC analysis using a HP6890N Gas Chromatography (Agilent, USA). The drug release procedure was performed in triplicate to calculate the standard deviation.

Preparation and characterization of BAD
Alginic acid can be easily neutralized by TBAOH to form TBAalginate that is only partially soluble in organic solvents such as DMF and DMSO. The solubility of TBA-alginate in DMF could be signicantly improved in the presence of a small amount of TBAF, which achieves the S N 2 reaction for the synthesis of BAD in a homogeneous solution. 31,32 To keep partial carboxylic functional groups of alginate that would be applied to adsorb nano-TiO 2 , benzyl bromide as the hydrophobic modier was added at 50% stoichiometry to react with TBA-alginate. Due to the presence of p-p conjugate effect, the benzyl group has high stability and strong activity. The degree of substitution (DS) of the resultant BAD reached about 46.3%, revealing high reactivity and reaction rate for S N 2 reaction, which consisted with previous report by Pawar et al. 32 FT-IR and 1 H NMR analyses are useful methods for elucidating the specic functional groups and structure of synthetic BAD. The FT-IR spectra of SA and BAD were displayed in Fig. 1. From the FT-IR spectrum of SA (Fig. 1a), the characteristic peaks at 2928.88, 1092.95 and 1031.74 cm À1 were respectively assigned to C-H and C-O-C stretching vibrations of the saccharide structure. 30 The peaks at 1620.13, 622.22 and 1417.40 cm À1 were attributed to asymmetric and symmetric stretching vibrations of -COO-. 33 In comparison with SA, BAD exhibited the similar FT-IR spectrum as shown in Fig. 1b. However, BAD showed the additional new peak at 1742.79 cm À1 for the presence of ester bonds, indicating the successful esterication of alginate by benzyl groups. Additionally, the peak of O-H at 3000-4000 cm À1 in the spectrum of BAD became sharp. The results indicated that the graing of hydrophobic benzyl groups could destroy the original intramolecular hydrogen bonding of alginate, which could enhance its molecular exibility. [33][34][35] The successful synthesis of BAD was further proved by 1 H NMR. As shown in Fig. 2, the proton peaks of SA and BAD were marked in 1 H NMR spectra. The proton peaks ranging from 5.0  to 3.5 ppm were ascribed to the H of native alginate chains. 30 By contrast, the spectrum of BAD showed additional new proton peaks at 7.35 and 5.45 ppm, which were respectively assigned to benzyl aromatic and benzyl -CH 2signals. 32 The protons assignment of the new functional groups were further conrm the successful graing of hydrophobic benzyl groups onto alginate main chains.
Thermogravimetric analysis is the most effective method for characterizing the thermal stability of polymer materials. 36 The thermal gravity analysis (TGA) and derivative thermograms (DTG) for SA and BAD were shown in Fig. 3. The weight retentions of SA and BAD at 800 C were respectively 30% and 24% ( Fig. 3a and b), and their DTG curves displayed two main mass loss stages, which was ascribed to the losses of physically adsorbed water at low temperature (80-120 C) 37 and the decomposition of polymer moieties at high temperature (200-250 C). 38 The thermal decomposition of polymer moieties resulted in the formation of CO, CO 2 and H 2 O, resulting in the rapid decrease in weight. 39 To note, the initiating decomposition temperature of 227 C for BAD was obviously smaller than that of 237 C for SA, indicating that BAD had lower thermal stability. As the benzyl groups was covalently bonded to the carboxyl groups of alginate during the S N 2 reaction, the carboxyl groups of BAD was reduced. And the decrease of carboxyl moieties may easily resulted in the destruction of the polymeric intramolecular hydrogen molecule, thus causing the reduction of its initiating decomposition temperature. These results further implied the esterication of alginate by S N 2 reaction was feasible.
Owing to the partial graing of hydrophobic benzyl groups onto the main chains of alginate molecule, which could destroy the original intramolecular hydrogen bonding to enhance its molecular exibility, the BAD may exhibit certain colloidal interface activity. The colloidal interface activity could be indirectly examined with the uorescence measurement using hydrophobic pyrene as the uorescent probe. 37 There are ve electronic vibration peaks in the pyrene uorescence spectroscopy, and the ratio value of the rst peak at 372 nm (I 1 ) to the third peak at 383 nm (I 3 ) is related to the micro-environmental polarity surrounding pyrene molecules, which decreases with the reduction of the micro-environmental polarity. 36,37 When the BAD molecules aggregated to form micelle-like selfaggregates due to the hydrophobic association of benzyl groups, the hydrophobic pyrene can be encapsulated in the hydrophobic microdomains formed by benzyl groups, thus resulting the signicant decrease of the ratio value of I 1 /I 3 . As shown in Fig. 4, the I 1 /I 3 value decreased with the increase of BAD concentration. The corresponding onset concentration is the critical aggregation concentration (CAC) of BAD, which is the lowest concentration of micelle-like self-aggregates by hydrophobic association. The BAD exhibited relative low CAC value of 0.50 g L À1 in comparison with that of alginate (1.90 g L À1 ), indicating good amphipathy for BAD. Therefore, it is the amphiphilic property of AAD that could achieve the adsorption of BAD at the oil/water interface. 30 The colloidal property of BAD was further investigated by DLS with the results showed in Fig. 5. As can be seen from Fig. 5a, the BAD exhibited a narrow size distribution involving   Paper two continuous ranges, which may be ascribed to the unequal graing of hydrophobic benzyl groups onto the alginate main chains or the degradation of molecular chains during the S N 2 reaction. Their average hydrodynamic diameter was 423.4 nm with the PDI of 0.68. The results indicated that due to the hydrophobic association of benzyl groups, the BAD molecules aggregate to form micelle-like self-aggregates. 37 This results further validated that the molecular exibility of BAD was enhanced by hydrophobic association. 34 In addition, the BAD micelle-like self-aggregates showed a relatively high negative zeta potential at about À36.4 mV as a result of the presence of the residual free-curling carboxyl groups (Fig. 5b). This result indicated the BAD molecules, containing both the hydrophilic main chains and hydrophobic side groups, could self-assemble into core-shell architectures, where the hydrophilic domains were exposed. It was generally considered to be stable in aqueous solution when the absolute value of zeta potential was higher than 30 mV, as the strong repulsion forces prevented the aggregation among the micelle-like self-aggregates. 40 On the basis of the above results, the BAD displayed good colloidal interface activity.

Effect of BAD on the colloidal stability of nano-TiO 2
As the BAD possessed good colloidal interface activity, it can be utilized to regulate the colloidal properties of nano-TiO 2 through electro-sterical repulsion. 15 The method to achieve the dispersion stability of nano-TiO 2 is that the adsorption of BAD on the nano-TiO 2 's surfaces via carboxylic functional groups to prevent the nanoparticles' surfaces from coming into direct contact. 14 Since the change of pH and ionic strength could inuence the charge density and size of BAD, the adsorption of polymer onto the surfaces of nanoparticles depends notably on the pH and ionic strength of the solution. 16 3.2.1 Effect of pH on the zeta potential and size of BAD/ nano-TiO 2 suspensions. Commercial nano-TiO 2 with the crystalline phase of anatase contain a large amount of Ti-OH on their surfaces, and they will show different chargeabilities ascribed to the inuence of the pH of the aqueous phase. As shown in Fig. 6a, with the increase of pH, the zeta potential of the nano-TiO 2 gradually decreased from a positive value to a negative value. Meanwhile, it could be also observed that the point of zero charge (PZC) of the nano-TiO 2 suspension was at pH value of 5.6, which was close to the previous reports of pH at the PZC for anatase nano-TiO 2 . 41 When the pH value of the aqueous phase was less than 5.6, the surface of nano-TiO 2 was positively charged attributed to the protonation of Ti-OH. But, when the pH of the aqueous phase was higher than 5.6, the surface of nano-TiO 2 was negatively charged owing to the deprotonation of Ti-OH. 42 In the vicinity of the PZC, due to the least charge on the surface of the nano-TiO 2 , the electrostatic repulsion between the nanoparticles was the weakest. So the agglomeration phenomenon between the particles was the most signicant, making nano-TiO 2 suspension exhibit a higher average hydrodynamic diameter (d H ) as shown in Fig. 6b. However, the zeta potential and d H of BAD was less inuenced by pH value. When BAD, as a dispersant, was adsorbed on nano-TiO 2 , BAD/nano-TiO 2 complex exhibited strong negative charge with good colloidal properties, and their zeta potentials were lower than À30 mV, which was favorable for reducing the aggregation of nano-TiO 2 , thus improving their dispersion stability. As we can see from the inset, the pictures of BAD/nano-TiO 2 suspensions at various pH exhibited good dispersion stability. It was the higher electrostatic and steric effects of BAD that made the average d H of BAD/nano-TiO 2 complex signicantly smaller than that of the single TiO 2 nanoparticle. 14 3.2.2 Effect of ionic strength on the zeta potential and size of BAD/nano-TiO 2 suspensions. The effect of ionic strength on the dispersion stability of BAD/nano-TiO 2 suspensions was evaluated in the NaCl solution. There was an ionotropic effect existed in the aqueous NaCl solution, which could induce the electrostatic shielding effects to reduce the chargeability of the charged nanoparticles. 43 As shown in Fig. 7a, with the increase of ionic strength, the zeta potential of nano-TiO 2 and BAD/ nano-TiO 2 complex gradually increased because of the counter ion Na + that aggregated around the negatively charged Ti-O À and carboxyl groups. When BAD was adsorbed on nano-TiO 2 , their electrostatic shielding effect in NaCl solution was signicantly weakened. Moreover, as shown in the inset (Fig. 7b), the adsorption of BAD greatly reduces the agglomeration behavior of nano-TiO 2 in different ionic strength aqueous phases, resulting in a signicant decrease in hydrodynamic diameter. As reported, salt can not only reduce the surface potential of the colloidal particles, but also cause their coagulation/occulation, which is attributed to the electrostatic contribution to the interparticle interactions. 44 With the gradual increase of ionic strength, their d H showed a trend of rst increasing and then decreasing, resulted from the existence of certain coagulation/occulation effects.
Although the higher electrostatic and steric effects of BAD could signicantly decrease the average d H of BAD/nano-TiO 2 complex in comparison to the nano-TiO 2 , the d H of BAD/nano-TiO 2 complex was much higher than the expected individual nano-TiO 2 (10-25 nm). The results perhaps could be explained by the fact that nano-TiO 2 were too small to achieve the dispersion stability of individual nano-TiO 2 due to the high adsorption energy between nanoparticles. Additionally, a hydrodynamic diameter was the actual object's diameter plus a strongly bound water shell generated by the continuous phase and the BAD on the surface of nano-TiO 2 . 37 So the BAD/nano-TiO 2 complex revealed higher d H than the actual size of the individual nano-TiO 2 .
3.2.3 Morphology and FT-IR analysis of BAD/nano-TiO 2 complex. The morphology of the freeze-dried BAD/nano-TiO 2 suspensions was evaluated by SEM observation. As shown in Fig. 8a, the single nano-TiO 2 were easily agglomerated because of the lack of interfacial activity and gravity sedimentation of nano-TiO 2 , which may severely restrict its actual applications. However, the adsorption of BAD on the surface of nano-TiO 2 signicantly improved the dispersibility of the nano-TiO 2 , and the BAD lm could be observed between the dried nano-TiO 2 , as shown in Fig. 8b. Therefore, the adsorption of BAD enhanced the steric hindrance between nano-TiO 2 to make them stably dispersive.  The structure of BAD/nano-TiO 2 complex was also examined by TEM measurement. As we can see from Fig. 9, most of nano-TiO 2 aggregated to form a cluster of nanoparticles with the size of 140 nm, which were in the middle of a micelle-like structure, whereas the BAD was in the corona areas. As the zeta potential of BAD/nano-TiO 2 complex was almost constant with the change of the pH values, the hydrophilic domain of BAD could be still exposed outside, 45 i.e., the cluster of nano-TiO 2 was in the hydrophobic core of micelle-like self-aggregates. These results showed that nano-TiO 2 can interact with benzyl group to achieve the adsorption of BAD on the surface of nano-TiO 2 .
To further investigate the interaction between BAD and nano-TiO 2 in BAD/nano-TiO 2 suspensions, the FT-IR measurement was performed on the freeze-dried BAD/nano-TiO 2 suspensions. In the FT-IR spectrum of nano-TiO 2 (Fig. 10c), the broad peak at 3442.08 cm À1 was assigned to the stretching vibration of Ti-OH. The characteristic peaks at 1632.65 and 650.58 cm À1 were respectively attributed to the bending and stretching vibration of Ti-O. 16 In Fig. 10b, the BAD/nano-TiO 2 complex contained both the main characteristic diffraction peaks of nano-TiO 2 and that of the BAD, in spite of the decrease of diffraction intensity, indicating that BAD was adsorbed onto the nanoparticles' surfaces by ultrasound and shaking. It was worth noting that the peaks of hydroxyl stretching in the spectrum of the BAD/nano-TiO 2 complex became much broader aer absorbing BAD in comparison with that of nano-TiO 2 . Meanwhile, the peaks of hydroxyl stretching at 3442.08 cm À1 for nano-TiO 2 red shied to 3431.04 cm À1 , indicating the formation of the intermolecular hydrogen bond between the carboxylic functional groups of BAD and the Ti-OH of nano-TiO 2 in BAD/nano-TiO 2 suspensions. 46,47

Preparation and release studies of the drug-loaded pickering emulsions
To further illustrate the colloidal stability of BAD/nano-TiO 2 complex and expand their applications in pharmacology eld, the drug-loaded Pickering emulsions were fabricated using BAD/nano-TiO 2 complex as the particulate emulsiers. As shown in Fig. 11, both of the freshly prepared Pickering emulsions stabilized by nano-TiO 2 and BAD/nano-TiO 2 complex were homogeneous and milky. Aer storing them at ambient temperature for 24 h, both of the Pickering emulsions revealed emulsion phase and cream-depleted aqueous phase, owing to the upward movement of the emulsion droplets. By contrast, gravity sedimentation was emerged at the bottom of the Pickering emulsions stabilized by nano-TiO 2 , attributed to the poor dispersibility of single nano-TiO 2 . 48 And the Pickering emulsions stabilized by BAD/nano-TiO 2 complex revealed good emulsion stability.
As the oil/water ratio was xed at 1 : 9 (v/v) during the preparation of the Pickering emulsion, the emulsion phase would emerge at the surface of the cream-depleted aqueous phase because of its lower density. The emulsion droplets were taken from the upper emulsion phase, and their    microstructures were observed by uorescent microscope aer uorescence staining. The uorescent images of the dyed Pickering emulsions stabilized by nano-TiO 2 and BAD/nano-TiO 2 complex were shown in Fig. 12. The emulsion droplets appeared red for the Nile red excitation. As oil-soluble Nile red only existed in oil phase and appeared red for its excitation, the emulsion type was conrmed to be oil-in-water type. 49 The emulsion droplets stabilized by BAD/nano-TiO 2 complex exhibited good spherical shape, while the emulsion stabilized by nano-TiO 2 exhibited the coalescence phenomenon owing to the demulsication, indicating that BAD/nano-TiO 2 complex displayed better emulsifying performance than single nano-TiO 2 .
The release of l-cyhalothrin was achieved through its diffusion and the slow destruction of the drug-loaded Pickering emulsion, which was applied to indirectly analyze the emulsion stability. The release curves of l-cyhalothrin from the drugloaded Pickering emulsions respectively stabilized by nano-TiO 2 and BAD/nano-TiO 2 complex were presented in Fig. 13. Both of the release curves showed a slow release in the whole stage. The irreversible adsorption of the particulate emulsiers at the oil-water interface, which probably formed a mechanically robust layer may account for the slow release of l-cyhalothrin. 49 Within the initial 150 min, both of Pickering emulsions respectively stabilized by nano-TiO 2 and BAD/nano-TiO 2 complex had the same release speed owing to the release of dissociative drug. But, aer 150 min, the drug-loaded Pickering emulsions stabilized by BAD/nano-TiO 2 complex exhibited a slower release than the drug-loaded Pickering emulsions stabilized by nano-TiO 2 , attributed to the electrostatic forces and intermolecular hydrogen bonds formed between BAD that was absorbed on the surfaces of nano-TiO 2 , which could retard the release of l-cyhalothrin. 50 To further investigate the release mechanisms, Ritger & Peppas model was used to analyze the release data. The model is expressed by the following equation. 51 where M t /M N is the fractional release of drug in time t, K is a constant incorporating structural and geometrical characteristics of the delivery system and n is the diffusion exponent characteristic of the release mechanism. For normal Fickian diffusion the value of n ¼ 0.5, for case II diffusion n ¼ 1.0 and values of n intermediate between the above limits indicate non-Fickian transport.
This model was applied for analysis of the data in Fig. 13 where M t /M N # 0.6. The high correlation coefficient (r > 0.99) indicated that the release data could be tted well with the model equation. The n values of the drug-loaded Pickering emulsions stabilized by nano-TiO 2 and the drug-loaded Pickering emulsions stabilized by BAD/nano-TiO 2 complex were respectively 0.8234 and 0.8989, which ranged between 0.5 and 1.0, indicating that the release mechanism of l-cyhalothrin from the drug-loaded Pickering emulsions was non-Fickian diffusion transport, 52 which implied that water migration into the Pickering emulsions and diffusion of the drug through the Pickering emulsions as well as the slow destruction of the Pickering emulsion were carried out simultaneously. Consequently, it is the drug-loading and sustained release properties that make the Pickering emulsions stabilized by BAD/nano-TiO 2 complex possess great potentials to become the good candidates for their use in pharmaceutical applications.

Conclusions
In summary, the benzyl-graed alginate derivative (BAD) was successfully prepared by S N 2 reaction to improve its colloidal interface activity. Aerwards, the BAD, as the dispersant, was adsorbed on the surfaces of commercial nano-TiO 2 by the mechanical mixing method. Due to the good colloidal interface activity, the BAD not only made BAD/nano-TiO 2 complex possess strong negative charge to enhance the electrostatic repulsions among the nano-TiO 2 , but also weakened the electrostatic shielding effect in NaCl solution to reduce the agglomeration of nano-TiO 2 . It was the adsorption of BAD that enhanced the electrostatic repulsion and steric hindrance between nano-TiO 2 to improve the dispersion stability of nano-TiO 2 at different pH and ionic strength. Moreover, the adsorption of BAD on nano-TiO 2 was achieved by the intermolecular  hydrogen bond between the carboxylic functional groups of BAD and the Ti-OH of nano-TiO 2 . The resultant BAD/nano-TiO 2 complex was used as the particulate emulsiers to stabilize the drug-loaded Pickering emulsions. The obtained Pickering emulsions exhibited good drug-loading capacity and sustained release performance with the release mechanism of non-Fickian transport, which may have great potentials as the carrier for drug delivery.

Conflicts of interest
There are no conicts to declare.