extfieldsofstudy: Chemistry Medicine

text:
Hydrogen Bonding Regulates the Rigidity of Liposome‐Encapsulated Chlorin Photosensitizers

Abstract Liposomal formulations facilitate the administration of hydrophobic drugs, avoiding precipitation and aggregation phenomena when injected in polar solvents. The integration of the photosensitizer into the liposome may alter the fluidity of the system and, thus, modify the delivery process of the drug. Such a change has been observed for the liposomal formulation of Temoporfin, which is one of the most potent chlorin photosensitizers employed in photodynamic therapy. Here, all‐atom molecular dynamics simulations have been performed to identify the nature of the intermolecular interactions that might be responsible of the different lipids freedom of motion when the drug is introduced in the bilayer. It is found that Temoporfin participates as a hydrogen donor in strong hydrogen‐bonding interactions with the polar groups of the phospholipids. The theoretical analysis suggests that the rigidity of drug/liposome complexes can be modulated by considering the different hydrogen‐bond ability of the photosensitizer and the carrier material.

Introduction
Photodynamic therapy is at reatment that exploits the interaction of light with ap hotosensitive drug in the presence of molecular oxygen. [1][2][3] Such an interaction produces cytotoxic reactive oxygen speciest hat may induce the deatho ft he targeted tumor cells. [4,5] The advantage of photodynamic therapyl ies in its potential to achieve high selectivity towards the diseased tissues through the following therapeutic mechanism:t he drug is administered to the patient and, after as pecific interval, in which the drug accumulates in the tumor tissues,the interested region is irradiated with as ource of light with appropriate wavelength. [2,3,5] Chlorin derivatives are second-generation photosensitizers widely employedi na pproved clinical treatments and also in clinicalt rials. [1,6,7] These compounds are porphyrin-based species, [8] in which one of the double bonds has been reduced to enhancethe light absorption at lower energies, [9] in the so-called therapeuticw indow. [4] The best performance of second-generation photosensitizers over first-generation photosensitizers is well illustrated when comparing Te -moporfin (mTHPC) to Photofrin,w ith the former being 100 times more photoactive than the latter. [10,11] However,p orphyrin derivatives present severald isadvantages, especiallya tt he time of administration into patients. In polar media, likew ater or blood, porphyrins tend to form aggregates, [11,12] which are less photoactive [13] and considerably diminish the efficacy of the treatment. [14] This is the case for mTHPC( see Figure 1a), currently employed in photodynamic therapy to treat certain types of cancera nd precancerous conditions. [11,15,16] This drug is nowadays administrated as Foscan (biolitecp harma Ltd.) within an ethanol/propylene glycol mixture and it has shown aggregation and precipitation phenomena at the moment of injection, when the photosensitizer enters the blood stream. Liposomes have been identified as valuable nanocarriers of hydrophobic drugs, favoring the accumulationo ft he photosensitizer in the tumor tissues and solving the solubility issue in the administration phase of the treatment. [17] As shown in Figure 1b,liposomes consist of an assembly with ah ydrophilic inner cavity formed by the phosphate groups of the lipids, which contains water inside, ah ydrophobic inner part of the double layer formed by the alkyl chains, and an outer polar spherical surface also formed by the phosphate groups.
The efficacy of liposomal formulations of chlorin derivatives has been demonstrated for mTHPC, in which the drug has been embedded in the non-polar region of the liposome, as it is the case of the Foslip formulation. First, thesef ormulations solve the solubility problem, providing an easier administration of the drug to the patient,a st he final product can be dispersedi nw ater and administrated orally,i ntravenously,o r through skin permeation. [18,19] Second, Foslip and other liposome formulations show ah ighere fficiency with respectt o Foscan,p resentingalarger selectivity for tumor tissues with respect to healthy ones and provoking faster dissociation of Liposomal formulations facilitatet he administration of hydrophobic drugs, avoiding precipitation and aggregation phenomena when injected in polar solvents. The integration of the photosensitizer into the liposome may alter the fluidity of the system and, thus, modify the delivery process of the drug. Such ac hange has been observed for the liposomal formulation of Te moporfin, which is one of the most potent chlorin photosensitizers employedi np hotodynamic therapy.H ere, allatom molecular dynamics simulations have been performed to identifyt he nature of the intermoleculari nteractions that might be responsible of the different lipids freedom of motion when the drug is introduced in the bilayer.I ti sf ound that Temoporfin participates as ah ydrogen donor in strong hydrogen-bonding interactions with the polar groups of the phospholipids. The theoretical analysis suggestst hat the rigidity of drug/liposome complexes can be modulated by considering the different hydrogen-bond ability of the photosensitizer and the carrier material. the tumor cells, which resultsi nm ore effective growth inhibition. [17,[20][21][22][23] More importantly,l iposomal formulations of Temoporfin do not manifest any dark toxicity. [21,24] Finally,t he liposomal formulation ensures the presence of the drug in its monomeric form, which is the most photoactive species. [25,26] The factors that most influence the success of al iposomal formulation are the loading capacity of the carrier andt he release of the drug to the targeted cells, that is, the actual delivery process. Both factorsa re regulated by the interactions between the vehicle material and the drug. To provide an optimal formulation,s uch interactions should be of medium strength so that the drug is safely hosted in the carrier, but its releasei sn ot hindered. Intermolecular interactions, therefore, play av ery important role in the transport andd elivery of the drug, [27] and the elucidation of the nature of such interactions is relevant to designn anocarrierswith optimal properties. Different molecular compositions for possible mTHPCl iposomal formulationsh ave been investigated by differentials canning calorimetrya nd electron spin resonances pectroscopy. [28] The latter has shown that the photosensitizeri sl ocated in the hydrophobic part of the double layer and that its presence hinders the motiono ft he phospholipids, giving more rigid vesicles. [28] Thus, the drug-lipid interactionsl ower the molecular motion of the phospholipids. However,t he nature of these interactions, responsible for the high loading capacity of the carriera nd its different fluidity is unknown.
Coarse-grained methods are typically used to investigate the structuralb ehaviour of loaded and pure liposomes. [29,30] However,t hese methods are not able to provide atomistic details, such as interatomic interactions responsible for the final structural properties of the system. Therefore, in this work, we have performed all-atom classical molecular dynamics simulations to unveil the nature of the lipid-drug interactions that might be relevant in the liposomal formulation Foslip as am odel for chlorin-based photosensitizers. We show that hydrogen bonding between the photosensitizer and the phosphate heads of the lipids is the main interaction responsible for the large loading capacity of the carrier and the different mobility shownb y the phospholipids in calorimetric experimentsa nd electron spin resonance measurements. [28,31] 2. Results and Discussion

Convergencet oaSteady State
The first step in any molecular dynamics simulation of al arge system is to obtain an equilibrated structure. However,t his is very hard to achieve in liposomes of small size, whicha re metastable systems that converge very slowly to as teady state. [32] Liposomes with diameters smallert han 20 nm have a very high propensity to fuse with other liposomes, forming larger vesicless uch that the stress imposed by the highc urvature of small spherical shapes, which constrains the lipids to be very tightly packed, is released. [33] Therefore, the relatively small-sized liposomem odelled in this work (diametero f 12.6 nm;s ee Figure 2a)u ndergoes very strong curvature effects, which influence the packing of the phospholipids and make equilibration very difficult.
Commonly,c oarse-grained methodsa re employed to study lipid vesicles, allowing larger sizes (20-34 nm of diameter) and longer simulation times, [32,34] at the price of losing atomistic insight. Even in such cases,t he definition of at rue steady state is extremelyd ifficult and requires simulation times that can reach hundreds of microseconds. [34] Achieving atrue equilibrated structure, in fact, would requiret he simulationo fp henomena like the flip-flop of phospholipids between the inner and outer shell of the double layer as well as water exchange between the inner cavity of the liposomea nd the surrounding solvent. [32] In this work, we have monitored two parameters to analyze the structural quasi-equilibration of the system:t he area per lipid A L of the two layers of the membrane and the hydration number of the polar heads of the phospholipids. These two properties converge very slowly to as teady value in flat lipid membranes and are, therefore, employed as criteria to define the equilibrium of the system. [35] The area per lipid A L hasb een calculated considering the two spherical surfaces formed by the phosphorus atoms of the polar heads of the lipid chains, as shown in Figure 2a.T he radius of such spheres was calculated at intervals of 0.5 ns alongt he heatings imulation in the canonical ensemble (constant number of atoms, volume and temperature [NVT]) and at intervals of 10 ns along the simulation in the isothermal-isobarice nsemble( constant number of atoms, pressure and temperature [NPT]). The average radius (r) obtained for the inner and outer leaflets was employed to calculate the area of the two surfaces defined by the phosphate groups of the lipids (Figure 2a). Then, the area per lipid is given by the area of the surfaced ivided by the number of lipid moleculeso ft he leaflet (N L )[ Eq. (1)]: As seen in Figure 2b, A L presents strong oscillations along the NVT heating step, especially for the outer shell of lipid molecules. Instead, in the simulation at constant pressure (NPT), A L reaches ac onstant value almosti mmediately.T he area per lipid of the inner leaflet oscillates around 0.4 nm 2 with as mall deviation in the last 20 ns of the simulation, where it slightly increases up to 0.49 nm 2 .T his shows that af ull equilibrated structure is hard to achievef or relatively small liposomes,a s explained above. The A L value for the outers hell molecule is 1.02 nm 2 and remains stable throughout the whole NPTsimulation. The observed inner-leaflet structural fluctuationsi saconsequence of the curvature of the double layer,w hich influence the stabilityo ft he membrane inner leaflet to al arger extent. [32] The experimental A L for flat phospholipids double layers is found in ar ange of 0.55-0.72 nm 2 . [35] However,s uch values are only valid for very large vesiclesw here the surface locally approximate af lat bilayer.T he smalls ize of our theoretical model induces ah ighc urvature that constrains the inner shell lipids to be very tightly packed, whereas the ones in the outer layer are more loosely arranged. As there is no free lunch, such geometricala lterations introduce an error in the simulated A L .P revious molecular dynamics simulations, [29,30] employing a coarse-grained model tod escribe al iposome, reported A L valuesf or the inner and outer shells of 0.5 and 0.8 nm 2 ,respectively,t hat is, in better agreement with the experimental estimation, but still affected by the spherical shapeo ft he bilayer.
As intermoleculari nteractions are relevant for the structural properties of the bilayer, [36] we have selected the hydration number as the second parameter to monitor the equilibration process, whichd irectly correlatesw ith A L .T he hydration number represents the total number of water molecules per polar lipid head present in the first solvation shell of the liposome. This has been calculated by integrating the first peak of the radial distribution function of the water molecules and the negativelyc harged phosphate oxygen atom of the DPPC lipids. [35] As seen in Figure 2c,t he hydration number shows important oscillations during the heating simulation, but it convergest oavalue of 1.52 water molecules after 20 ns in the NPT simulation. We conclude, therefore, that the drug/liposome assembly is relativelyw ell equilibrated after 20 ns of dynamics in the NPT ensemble, attending to the time evolution of the area per lipid and the hydration number,w hich were recommended convergencec riteria. [35] Accordingly,t he following analyses are performed using framesf rom the dynamics between 20 and 100 ns.

Diffusion Coefficients
As shown by electron spin resonance spectroscopy experiments, [28] the integrationo fm THPC into the liposomei nduces al owering in the molecular freedom of motion of the lipids. This change in the mobility is ac onsequence of the different chemicale nvironmente xperienced by the lipid chains when they are in the presence of the drug versus when they are located in the pure liposome.T he appearance of new intermolecular interactions might induce an increaseo rd ecrease of the lipid motion, depending on the strength of the drug-lipid interactions. The lowering of the motion,e xperimentally observed, [28] indicates that the interactions in the loaded liposome are stronger than in the pure one.
To theoretically investigate the effect of the drug on the mobility of the lipids, we have calculated the root-mean-square displacement( RMSD) of the lipids along the 100 ns of the simulation in the NPTe nsemble by considering different regions of the liposome, definedb yt he separation between the lipids and ad rug molecule (see Figure 3a). We expect that the lipids in the immediate surroundings of the drug present less fluidity because of possible strong drug-lipid intermoleculari nteractions. First, the RMSD of the mTHPC molecules (without lipids) has been computed. Then, we have extended the analysist o the surrounding lipid molecules, gradually increasingt he distance from the drug molecules between2and 10 ,a ss ketched in Figure 3a.T he RMSD of the lipids located in the different regions,p lotted in Figure 3b,c learly evidences that the lipid molecules in the immediate surroundingso ft he drug molecules show less freedom of motion. In other words, the farther the residues are from the drug, the larger is their freedom of motion and, therefore, their displacementa long the simulation. This behaviori ndicatest hat the lipid molecules interact stronger with the drug than with other lipid molecules, and that their motion is reduced with respectt ot he situation where they are surrounded by only other lipid molecules. A more quantitative analysisc an be performed by calculating the diffusionc oefficient D from the mean-squared isplacement (MSD),a long the simulation time t,u sing the so-called Einstein relation[Eq. (2)]: [37,38] MSD ¼ 6Dt ð2Þ The MSD of the last 80 ns of the simulation is linearly fitted and the slope provides the diffusion constant D of the lipid residues located at different distances from the drug. Figure 3c shows that the variation of the diffusion coefficient with the separation r from the drug residues can be interpolated by the increasing exponentialf unction D(r) = Ae B(r) + D 0 ,w here A, B, and D 0 are fitting parameters with values of À0.008 2 ps À1 , À0.289 À1 ,a nd 0.01 2 ps À1 .T his indicates that the diffusion coefficient of the lipid chains increases with the separation between the lipids and the drug, and it reaches the value of 0.01 2 ps À1 at larger distances, where drug-lipid interactions are less significant or absenta nd are replaced by lipid-lipid interactions. In addition, the fast increase of D with the distance r from the drug residues (D reaches half of its maximum value already at ad rug-lipid separation of only 2.5 )c onfirmst hat the interactions controlling the system are of short-range character.
After confirming that the liposomep resents larger rigidity due to the intermolecular interactions between the drug and the phospholipids, the next step was to identify the nature of such interactions. To this aim, first, we investigated which fragments of the lipid chains,n amely heads or tails, play am ore significant role in the interactions with the drug molecules. Thus, the RMSD in the NPT ensemble is computed for the polar heads and the nonpolar tails of the phospholipids. In addition, this analysis ( Figure 4) is performed by considering two Figure 3. a) Schematic representation of the drug Temoporfin and the surrounding residues used in the RMSD analysis. The molecules represented with bolder lines are found in asphere of 2 radius centered at the drug. Thinnerl ines represent instead residues inside as phere of 10 radius. b) RMSD of the residues containedint he different size spheres surrounding the drug residuesd uring the NPT simulation.c)Diffusion coefficient reported against the separationfrom the drug molecules. The color scheme is the same as in (b).  Figure 3a). In the case of the residues in the immediate surroundings of the drug molecules (cutoff of 2 ,F igure 4a), the RMSD of the drug and the polar heads almosto verlap and oscillates aroundaRMSD of 13 . The nonpolar tails, instead, show al arger mobility with an average RMSD value of 20 throughout the simulation. The smaller mobility of the polar heads in comparison with the nonpolart ails clearly points to an existing interaction between the polar heads and mTHPC. Consideringn ow the RMSD of the residues at farther distances from the drug (cutoff of 10 , Figure 4b), the coupling between the polar heads and drug molecules is lost, and the tail and head residues show approximately the same freedomo fm otion.C learly,t he drug-head interactions, which are evident at short distances, vanish at larger separations, where the motion of the lipids is less perturbed by the presence of the drug.
The large number of hydrogen donora nd acceptor atoms present in both the drug and polar heads of the phospholipids (Figure 1a)a nd the short-range character of the drug/head interactions, as indicated by the rapid change of the diffusion coefficient of the lipids with the distance from the drug (Fig-ure 3c), suggest that hydrogen bonding is responsible for the rigidity in the proximity of the Te moporfin molecules.

Hydrogen-Bond Analysis
The presence of an etwork of hydrogen bonds between mTHPCm olecules and the polar heads of the lipids would explain the rigidity of the phospholipids in the direct proximity of the drug molecules. Hydrogen bonds are, in fact, interactions that are particularlyr elevant in ah ydrophobic environment, where water molecules cannot easily interfere. Hydrogen bonds are also somewhat directional interactions and can form rather ordered structures. Finally,t he presence of hydrogen bonds between the photosensitizer and the carrierm aterial would assure ag ood loading capacity of the vesicle. [27] The polar heads of the DPPC lipids are hydrogen acceptors, whereas the polar heads of the DPPG lipids and the drug moleculesc an act as both hydrogen donors and acceptors in the formation of hydrogen bonds. Therefore, we can classify the hydrogenb onds as drug-drug, head-drug, and head-head hydrogen bond pairs, see Scheme1.
According to the coupling of the polar head and the drug motion found in the analysis of the RMSD reported in The strength of ah ydrogen bond is intimately related with the geometrical parameters of the atoms involved in the interaction, specifically, with the hydrogen donor-hydrogen acceptor distance r D-A and with the angle q D-H-A formed by hydrogen donor,h ydrogen atom, andh ydrogen acceptor shown in Scheme 1d.T able 1s hows the total number of hydrogen bonds found along the full simulation, the number of hydrogen bondst hat last for more than half of the simulation time, the total number of fragment pairs that can potentially form Scheme1.Schematicrepresentation of the three categories considered for the hydrogen-bondanalysis:drug-drug pair forming ah ydrogen bond (a), drug-head pair involved in ah ydrogen bond (b), and two phospholipids connected through ah ydrogen bondbetween the polar head residues (c). The tails of the phospholipids are pictured in gray,whereas, for the residues involved in the bonds, the color scheme is red for oxygen atoms, blue for nitrogen,tan for phosphate,c yan for carbons, and white for hydrogens. d) A zoom on the atomsi nvolved in the hydrogenb ond(encircled in yellow in the previous panels), with the geometrical parametersshown.  Table 1, the number of long-lived head-drugh ydrogen bonds (27) is larger than the number of long-lived head-head hydrogen bonds (12), despite the number of head-drug pairs (113050) being smaller than the number of head-head pairs (116886). Althought he number of hydrogen bondsd epends on the number of speciesc onsidered in the box, the present numbers clearly indicatet hat the lipid chains are more prone to participate in hydrogen bondingw ith the drug than with other lipid chains. In addition, the head-drug hydrogen bondsa re not only more abundant buta lso stronger,a si ndicated by the smallest r D-A value (2.67 )a nd the largest q D-H-A value (1628).
Am ore quantitative picturei so btained by considering the distribution of the hydrogen-bond lifetimes and geometrical parameters obtained by the analysiso fa ll hydrogen bonds formed along the simulation. Figure 5a shows the number of drug-drug, head-drug, and head-head hydrogen bonds, grouped according to their lifetimes as ap ercentage of the total simulation time. Head-head hydrogen bonding dominates at short lifetimes (less than 10 %o fs imulation time), whereas they are surpassed by the drug-head hydrogen bonds at longer lifetimes, especially at lifetimes over 70 %o f the simulation time. The drug-head distribution in Figure 5a presents am inimum at al ife time of 50 %; such af eature is just ar esult of the way the distribution was built and has no physicalmeaning.
To comparet he strength of the interactions between the three different pairs of residues, the probability distribution of r D-A and q D-H-A is depicted in Figures 5b and 5c,r espectively. The r D-A distribution peaks at as horter distance for the drughead interaction (2.67 )t han those for the head-head (2.69 ) and the drug-drug (2.83 ), whereas the q D-H-A distribution peaks occur at larger angles for the drug-head interaction (1628 and 1648)t han for head-head (1618)a nd the drug-drug (1608)o nes.T herefore, both the hydrogen-bond lifetimes and the probability distributiono ft he geometrical parameters indicate that the hydrogen-bond strengths follow the order drughead > head-head > drug-drug. The strongerh ydrogen bonds found between Te moporfin and the polar headse xplain why the mobility of the phospholipids is smaller in close prox-imity to the drug. In addition, the hydrogen-bond behavior also explains the good loading capacity of the liposomes and the loss of mobility of severall iposomal formulations of mTHPC. [28] Finally,w ediscuss the reasonsb ehind the stronger interactions between the photosensitizer and the lipid heads than between different lipid heads. Ac loser inspection of the atoms involved in hydrogen bonding reveals that, in the drug-head interaction, mTHPCp lays the role of hydrogen donor from its phenolm oieties virtually all the time, whereas, in the headhead interaction, the hydrogen donor atoms are the Oa toms  . Owingt ot he electron-withdrawing effect of the aromatic ring of the phenolic substituent, mTHPCi sabetter hydrogen donor than the glycol moiety of the DPPG.I ng eneral, aromatica lcohols are better hydrogen donors than aliphatic ones. This is the reason why phenol acts as ah ydrogen donor and methanola sahydrogen acceptori nt he phenol-methanol dimer. [39] Based on our analysis, one can conclude that the rigidity of the liposomal formulation of mTHPC, closely related with the loading and release capacity of the liposome,c an be modulated considering the strength of the hydrogen bonds that the phenol moieties of the drug could form with phospholipids suitable for liposomal formulations. This consideration can be extended to different photosensitizers baring H-bonding groups in their substituents. In case of phenol moieties, furtherf unctionalization of the photosensitizer can be pursued. If the functionalization enhances the electron-withdrawing ability of the aromatic ring, the drug would be ab etter hydrogen donora nd the drug/liposome assembly would be more stable. Contrarily,i ft he aromatic ring loses its electron-withdrawinga bility upon functionalization, the drug is al ess effective hydrogend onor andt he drug/liposome assembly would be less stable. However,i ti si mportant to note that such af unctionalization should not modify other properties of the drug, for example, its hydrophobicity or the energy of the electronically excited states and, thus, the photosensitization efficiency and mechanism of the drug should not be altered.

Conclusions
In this work, we have performed all-atom molecular dynamics simulations to unravel the reasonsb ehind the smaller membrane fluidity experimentally found for several liposomeformulations of the photosensitizer Te moporfin, [28] ap rototypical chlorin photosensitizer.T he analysis of the motion of the different residues composing the liposome andt he calculation of diffusionc oefficients of the lipid chains in the immediate surroundings of the photosensitizer have shown that the interactions that confer rigidity to the drug/liposome assembly are of short-range character.T hese interactions are identified as relatively strong hydrogen bonds between the photosensitizer and the polar heads of the phospholipids. Owing to electron withdrawing effects, the drug is ap owerful hydrogen donor and, thus, the rigidity of the liposomei nduced by the drug into the phospholipids in its immediate surroundings derives from the tenacity of such interactions. Our theoretical analyses suggest that the stability of Temoporfin liposomal formulation could be also ascribed to the strengtho ft he hydrogen bond formed by the drug and the carrier material. Such considerations on the hydrogen-bonding capability of the drug and the phospholipids can be extended to other chlorinp hotosensitizers and, thus, can be relevant to design new drug formulations, as intermolecular interactions between the drug and the nanocarrier affect the loading capacity of the carrier, the drug releasep rocess, and the pharmacokinetic properties of the drug. [40] Computational Details The liposomei nitial structure was built with the Packmol software package, [41] with ac omposition of DPPC/DPPG phospholipids with ar atio of 9:1( w/w) and with am olar ratio of lipid/ mTHPCo f1 2:1. The inner leaflet of the liposomeb ilayer is formed by 118p hospholipid molecules confining4 72 molecules of water in the inner cavity.T he number of water molecules placed in the inner cavity was calculated so that the density of the solventi nt he inner cavity equals the one that surrounds the liposome. The second leaflet of the vesicle is assembled from 366 lipid molecules, resulting in al iposome with ad iameter of about 126 .T he liposome also contains 40 moleculeso fT emoporfin, placed in the hydrophobic region of the double layer,b ased on previous electron spin resonance spectroscopym easurements. [28] The resulting drug/liposome complex was solvated with 109 387 water molecules confined in a cubic box of 83 side. Na + counter ions were added to neutralize the DPPG negative charges and NaClw as added to reach as alt concentration of 0.15 M.
The energy of the system was first minimized in seven steps. In the first step, only 200 residues of the liposomew ere minimized,w hereas the other residues were spatially constrained by employing ah armonic force constant of 50 kcal mol À1 À2 . In each of the next fives teps, 200 more residues were allowed to move during minimization togetherw ith the ones already optimized in the previous steps.I nt he final minimization step, the whole liposomew as minimized withouta ny constrains. Each minimization calculation consists of 10 000 steps, where the first 5000 were performed by employing the steepestd escent algorithm whilet he last 5000 were performed by employing the conjugated gradient algorithm. The system was then slowly heated in the canonical ensemble (NVT ensemble) from 0t o3 00 Ki nf our different simulations of 1nse ach. In the first three heating simulations, the temperature was increasedb y1 00 Ki ntervals from 0t o3 00 K. Duringt he three heatings teps, the motion of the residues forming the liposome was constrained with decreasing harmonic force constants of 10, 5a nd 1kcal mol À1 À2 .T he last heating simulation was performed at af ixed temperature of 300 Ka nd with no constraints applied on the motion of the drug/liposome assembly.A fterwards, the system was run in the isothermal-isobaric ensemble( NPTe nsemble) to equilibrate the density of the system and the structure of the liposomef or 100 ns, and the snapshots were printed every 20 ps for analysis. As discusseda bove, after 20 ns, the system reached am etastable state and, therefore, the last 80 ns of the simulation were employedi nt he analyses.
For all of the simulations, periodic boundary conditions were applieda nd at ime step of 2f sw as employed.T he SHAKEa lgorithm [42] was used to freeze bonds involvingHa toms. The Langevint hermostat was used with ac ollision frequency gammao f1 .0 ps À1 while the pressure was kept fixed at 1atm with the Berendsen barostat and isotropic positions caling. For the non-bondedi nteractions calculation, ac utoff of 10.0 was employed together with the particle-meshE wald method to calculate the coulombic interactions. [43] The phospholipids were described by the Lipid17 [44] force field, available in the AmberTools17. [45] The intramoleculara nd Lennard-Jones parameterso ft he drug were taken from the generalA mberf orce field. [46] The restrained electrostatic potential( RESP) charges were computed by density functional theory, employing the B3LYP [47] functional andt he 6-31G* basis set [48] using the Gaus-sian09 [49] softwarep ackage. We employed the TIP3P model [50] for the water molecules and appropriate Amberp arameters to describe the NaCl ions. [51] All the simulations were performed with the GPU-based module PMEMD implemented in AMBER16. [45] The visualization of the simulationt rajectories and the RMSD calculations were performed with the visual molecular dynamics (VMD) program, [52] the hydrogen-bond analysis was performed with the CPPTRJ software [53] by employing ac utoffo f 3 for the D-A distance and 1358 for the D-H-A angle. By using the Einstein relation, Equation (2), the RMSD curves were linearly fitted to calculate the diffusion coefficient of the drug and lipid molecules.


s2fieldsofstudy:
Chemistry
Materials Science

text:
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.


EFFICIENTONE-POT SYNTHESIS OF IMIDAZOLES CATALYZED BY SILICA- SUPPORTED La0.5Pb0.5MnO3 NANO PARTICLES AS ANOVEL AND REUSABLE PEROVSKITE OXIDE

Silica-supported La0.5Pb0.5MnO3 nanoparticles was prepared and used as a new perovskite-type catalyst for rapid and efficient synthesis of substituted imidazoles by an one-pot three-component condensation of [9,10]-phenanthraquinone, aryl aldehydes and ammonium acetate in excellent yield under reflux, and also solventfree conditions.

INTRODUCTION
Imidazole derivatives are an important group of heterocyclic compounds that have biological and chemical properties [1]. Nowadays these compounds are used in pharmacology. They have different capacity such as anti-bacterial, anti-viral, and anti-cancer, anti-inflammatory [2][3][4]. Also, the imidazole ring system is one of the main substructures found in most of natural products and pharmacologically active compounds, such as the hypnotic agent [5]. Therefore, a great number of synthetic methods have been reported for the synthesis of multi substituted imidazoles [6].
Perovskite-type oxides with the chemical formula ABO 3 can be crystallized with cubic structure. The level of oxygen and defects is affected by the composition because of a wide constancy range of the structure. The greater A-site cation mostly represents a metal cation belonging to one of groups of rare earth metals (La, Sm, Pr), alkaline earth metals (Sr, Ba, Ca), or alkali metals (Na, K) coordinated to 12 oxygen anions. The B-site cation represents typical normally a smaller transition metal cation, which occupying occupies octahedral spaces in the oxygen framework. Some hybrids of A-and B-site cations can create a steady perovskite-like structure [18]. The catalytic activity of perovskite-type catalysts is mainly because of the existence of the multiplicity of oxidation states [19]. Perovskite-type oxides possess adsorption, acid-base, as well as redox properties, which leads to attractive catalytic activities. They can fulfil the requirements (excellent activity, constancy, and potential to be processed into structured catalysts) [20].

General
All materials and reagents were purchased from Merck and Aldrich and utilized with no more purification. Determination of melting points were carried out using an Electro thermal type 9100 melting point apparatus and are uncorrected. The IR spectra were recorded on a Thermo Nicolet AVATAR-370 FT-IR spectrophotometer. A Bruker DRX250 spectrometer was used to obtain 1H NMR spectra.

Preparation of La 0.5 Pb 0.5 MnO 3 nano particles
In current study, the perovskite precursor was synthesized using the citrate-based sol-gel improved Pechini technique [21]. Chemicals of La(NO 3 ) 3 .6H 2 O, C 6 H 9 MnO 6 .2H 2 O, Pb(NO 3 ) 2 and citric acid (99.5%) were utilized as raw materials. Next, a mixture of metal nitrates solutions with nominal La:Pb:Mn ratios of 0.5:0.5:1 (LPMO) was prepared in deionized water. The addition of citric acid to the metal solution was performed proportionally to have the similar equivalents. The solution was concentrated through evaporation at nearly 50 o C with agitating for 1 h for converting them to steady (La, Pb)/CA complexes. The solution was agitated on a hot plate at about 75 o C for removing excess water and accelerating polyesterification reaction. Next, the dry gel was achieved thought heating gradually to 120 o C in an oven. The gel pieces were milled to create a fine powder. Finally, La 0.5 Pb 0.5 MnO 3 NPs were synthesized through thermal treating the precursor at 650 o C for 9 h in air. Most of the residual carbon was removed trough annealing of the amorphous precursor and the hexagonal perovskite phase was achieved.

Preparation of silica-supported La 0.5 Pb 0.5 MnO 3 nanoparticles (30% w/w)
Initially silica was put into oven for 2 hours with 90 o C, then catalyst (0.3 g) with silica (0.7 g) were worn out into glass mortar about 1 hour when mixture were bright and unify.

Typical procedure synthesizing imidazole derivatives in reflux condition
A mixture of aldehyde (1 mmol), 9,10-phenanthraquinone (1 mmol), ammonium acetate (2.5 mmol) and S-LPMO nanoperovskite (0.04 g) in 10 mL ethanol was stirred. The resulted mixture was refluxed for described time. At the end of the reaction (the reaction progress was monitored by TLC using n-hexan:ethylacetateas eluent solution) the catalyst was separation with filtering, and solid perovskite catalyst was isolated and could be reused. The solid product resulted after evaporation of organic phase, was washed with cold water (3 × 20 mL), and recrystallized in ethanol to provide pure product.

Typical process for synthesizing imidazole derivatives in solvent-free condition
A mixture of aldehyde (1 mmol), 9,10-phenanthraquinone (1 mmol), ammonium acetate (2.5 mmol) and S-LPMO nano perovskite (0.04 g) was stirred. Then, the mixture was heated at a temperature of 100 o C for described time (the reaction progress was monitored by TLC using nhexan:ethylacetate as eluent). After cooling, the filtration of mixture was carried out and the filtrated mixture was washed using ethanol to separated catalyst. The filtrate was concentrated through evaporation under reduced pressure using a rotary evaporator. To obtain pure products, the recrystallization of remaining solid from ethanol was carried out.
Analytical data for selected compounds

RESULTS AND DISCUSSION
After synthesis of La 0.5 Pb 0.5 MnO 3 nanoparticles [21], for characterization of this catalyst, the morphology of the sample was determined using SEM analysis. Figure 1 demonstrates the micrograph related to the samples prepared through the sol-gel modified Pechini technique and calcined 650 o C. According to the SEM images, the surface is obviously porous and it appears that the size of grown particles is uniform. The size of pores is in the range of 30-188 nm. Moreover, SEM 160 image shows that besides the presence of bigger particles, there were relatively smaller particles on the surface. However, the main particles on the surface were the bigger ones. The aggregation of smaller particles (in the nm scale) may cause the creation of larger LPMO NPs on the surface. FT-IR spectra were obtained for single LPMO and S cm -1 . Figure 2 shows the FT-IR spectrum of LPMO perovskite nanoparticle, and Fig the FT-IR spectrum of S-LPMO stretching and bending vibration of H 1113 cm -1 is usually assigned to the Si cm -1 corresponding to LPMO perovskite coated on SiO [18,21].
For the purpose of optimization of the amount of catalyst and the reaction time, were executed various amount catalyst in presence ethanol solvent. A out on the synthesis of 2 condensing [9,10] phenanthraquinone acetate (2.5 mmol) in ethanol. For establish effectiveness o catalyst at ambient temperature. We were performed trace catalyst (entry 1). We increased temperature up to reflux condition, but final product was appreciable (entry 2). After th catalyst. Results summarized in Table 1 indicated the best result was with 0.04 g amount of catalyst but increasing the amount of catalyst when we supported LPMO on silica (S IR spectra were obtained for single LPMO and S-LPMO samples in range of 400 IR spectrum of LPMO perovskite nanoparticle, and Figure 3 shows LPMO sample. The IR bands at 3430 and 1638 cm -1 is due to the stretching and bending vibration of H 2 O molecules, respectively. The very strong and IR band at is usually assigned to the Si-O-Si asymmetric vibrations. The picks at 668 and 592 sponding to LPMO perovskite coated on SiO 2 are clearly observed in this spectrum For the purpose of optimization of the amount of catalyst and the reaction time, initially we were executed various amount catalyst in presence ethanol solvent. A model study was carried out on the synthesis of 2-(4-chlorophenyl)-1H-phenanthro[9,10-d]imidazole (3 g) via [9,10] phenanthraquinone (1 mmol), 4-choloro benzaldehyde (1 mmol), ammonium acetate (2.5 mmol) in ethanol. For establish effectiveness of LPMO, a test reaction done without catalyst at ambient temperature. We were performed trace amount of product in the absence of catalyst (entry 1). We increased temperature up to reflux condition, but final product was appreciable (entry 2). After that, reactions were reacted with different amounts of LPMO Results summarized in Table 1 indicated the best result was with 0.04 g amount of increasing the amount of catalyst did not improve the yield (entry 6 and 7). Also, supported LPMO on silica (S-LPMO), the amount of product was increased (entry 8 LPMO samples in range of 400-4000 3 shows is due to the O molecules, respectively. The very strong and IR band at Si asymmetric vibrations. The picks at 668 and 592 are clearly observed in this spectrum initially we model study was carried g) via ammonium f LPMO, a test reaction done without amount of product in the absence of catalyst (entry 1). We increased temperature up to reflux condition, but final product was not of LPMO Results summarized in Table 1 indicated the best result was with 0.04 g amount of ). Also, entry 8). Considering solvent plays a key role in reactions, reaction was reacted in presence different solvents. Results are presented in Table 2. We observed protic solvent including ethanol and methanol have higher yield in this method. The best result was achieved using ethanol(entry 3). This reaction had proper result in free solvent condition. Thus this reaction was considered in free solvent reaction similarly. At the end of the model reaction, the nano catalyst was separated from the reaction mixture, washed with acetone and dried at 100°C under vacuum for 2 h and reapplied four times for the same reaction. As presented in Table 3, the catalyst can be reused at least four times with no change in its activity.  Finally, after optimizing the reaction conditions, we prepared a range of replaced imidazoles (Table 4). In all cases, aldehydes reacted effectively with replaced carrying either electrondonating (entries 7-10) or electron-withdrawing (entry 2-4) groups and provided the estimated products in good to yields.
The mechanism of the reaction was proposed in Scheme 2. Ammonia molecules are obtained from ammonium acetate. We think that the aldehyde and 1,2-dicarbonyl compounds including 9,10-phenanthraquinone at first activated by S-LPMO as Lewis acid, in the rate determining step.

CONCLUSION
In conclusion, we benefit from one pot synthesis for the preparation of substituted imidazoles through three-component condensation of aldehyde, 9,10-phenanthroquinone, and ammonium acetate in presence of silica-supported La 0.5 Pb 0.5 MnO 3 nanoparticles as an efficient, re-usable and green solid acid catalyst, in reflux and solvent-free conditions. This catalyst has enhanced specific surface area, therefore increasing the contact between the catalyst and reactant. Excellent yields, short reaction times, simplicity of operation and easy separation and reusability of catalyst are several advantages of this technique. Recovery of both products and inorganic support/catalyst is generally possible, leading to an effective and low-waste route to a range of products.