Complex Coacervation and Overcharging during Interaction between Hydrophobic Zein and Hydrophilic Laponite in Aqueous Ethanol Solution

In this paper, for the first time, we have reported the formation of complex coacervate during interaction between hydrophobic protein, zein, and hydrophilic nanoclay, Laponite, in a 60% v/v ethanol solution at pH 4. Dynamic light scattering and viscosity measurements revealed the formation of zein–Laponite complexes during the interaction between zein at fixed concentration, CZ = 1 mg/mL, and varying concentrations of Laponite, CL (7.8 × 10–4 – 0.25% w/v). Further investigation of the zein–Laponite complexes using turbidity and zeta potential data showed that these complexes could be demarcated in three different regions: Region I, below the charge neutralization region (CZ = 1 mg/mL, CL ≤ 0.00625% w/v) where soluble complexes was formed during interaction between oppositely charged zein and Laponite; Region II, the charge neutralization region (CZ = 1 mg/mL, 0.00625 < CL ≤ 0.05% w/v) where zein–Laponite complexes form neutral coacervates; and Region III, the interesting overcharged coacervates region (CZ = 1 mg/mL, CL > 0.05% w/v). Investigation of coacervates using a fluorescence imaging technique showed that the size of neutral coacervates in region II was large (mean size = 1223.7 nm) owing to aggregation as compared to the small size of coacervates (mean size = 464.7 nm) in region III owing to repulsion between overcharged coacervates. Differential scanning calorimeter, DSC, revealed the presence of an ample amount of bound water in region III. The presence of bound water was evident from the presence of an additional peak at 107 °C in region III apart from normal enthalpy of evaporation of water from coacervates.


■ INTRODUCTION
Compartmentalization has long been postulated as an important means of concentrating the substances crucial for the origin of life. 1 This is driven by a spontaneous and self-assembly process by concentrating the substances like RNA, DNA and proteins. Spatial localization and concentration of a substance were very crucial during origin of life to start any biochemical reaction. 2 One of the possible known route to achieve this was described as the coacervation phenomenon, which is a complex process of liquid−liquid phase separation. In the process, generally, two oppositely charged polymers like nucleotides, polypeptides, or lipids interact to give a polymer-rich phase called coacervates. 3−6 Moreover, being stable over a wide range of physiochemical conditions, coacervates provide a suitable compartment to accumulate and up-concentrate different molecules. 7,8 The role of coacervation was found to take place in the extracellular matrix (ECM) during the process of elastogenesis. 9 During elastogenesis, tropoelastin undergo a coacervation process where they self-aggregate to give a concentrated and ordered structure. 10−13 The coacervate phase was also found to occur during the interaction between polynucleotides and polypeptide. 3,14 Coacervation also imparts its importance in many organisms for their survival. In the case of sandcastle worms and mussels, the coacervation phenomenon was found to play an important role in the formation of adhesive in a wet environment. 15−18 The study of the beak of the humboldt squid (Dosidicus gigas) revealed that the gradient of a soft base to an exceptionally hard tip (rostrum) of the beak comes from the self-coacervation of the beak proteins. 19 Apart from the role of coacervates in natural phenomena of a biological system, coacervates showed a promising role in various applications, such as biomedicine, 20 small molecule uptake, 21 nanobioreactors, 22 pharmaceutical and food industries, 23 encapsulation of molecules, 24 gene delivery, 25 cartilage mimics, 26 tissue culture scaffold, 27 and drug delivery vehicle. 28 Researchers have focused a lot to explore the application of coacervates in the field of drug delivery. Li et al. 29 showed the use of zein−chitosan complex coacervate particles in the slow release of curcumin. Zein−chitosan complex coacervation was studied by Ren et al. 30 to investigate the effect of ultrasound frequency in the encapsulation of resveratrol. Thermodynamics and wetting kinetics of zein coacervate was studied by Li et al. 31 Their study also revealed the formation of zein coacervate in a water/propylene glycol solvent and its ability to encapsulate limonene. Injectable hydrogel coacervate was used by Lee et al. 32 for the delivery of anticancer drug bortezomib. Huei et al. 33 have reported iron cross-linked carboxymethyl cellulose complex coacervate beads for the sustained release of ibuprofen drug. Chenglong et al. 34 reported a dextran-based coacervate nanodroplet as potential gene carriers for efficient cancer therapy. A water-soluble starch derivative anionic and cationic polymer that undergoes nanoparticle formation via coacervation was reported by Barthold et al. 35 The group discussed the potential use of the nanoparticles in pulmonary delivery of protein/peptides. While exploring the efficiency of coacervates in drug delivery, a very interesting work was carried by Lim et al. 36 They showed that a Humboldt squid beak-derived biomimetic peptide coacervate can be used for encapsulating insulin with high efficiency along with its controlled release. Chitosan-based coacervates for propolis encapsulation and its release and cytotoxic effect was reported by Sato et al. 37 The above literature 29−37 suggests that coacervates can be potentially used as drug delivery systems. As far as zein is concerned, it has been used in adhesive, 38 food industry, 39 biodegradable plastic, 40 delivery of small molecules, 41,42 encapsulation of molecules, 43 etc. The complex coacervation of zein with polymer-like chitosan, 37 ds-DNA, 44 etc. has been studied; however, complex coacervation of zein with a clay nanomaterial has not been studied and explored for its potential application as a drug delivery system. Future prospect of the zein−Laponite coacervates laid in the fact that both cationic and hydrophobic drugs could be loaded to these coacervates. In the coacervates, the cationic drug can be attached to the negatively charged surface of Laponite, while the hydrophobic drug can be loaded to zein via electrostatic and hydrophobic interactions, respectively. The coacervates thus will have the potential to carry cationic and hydrophobic drugs simultaneously and thus possibly can be used as a dual drug delivery system.
In this paper, we have studied the interaction between a nanoclay disc, Laponite, and a hydrophobic protein, zein. The interaction was studied at pH 4 where Laponite and zein exhibits negative and positive charges, respectively. The paper reports the formation of neutral coacervates and an interesting overcharged coacervates phase during the interaction between zein and Laponite.

■ RESULTS AND DISCUSSION
Zein is a positively charged hydrophobic protein soluble in aqueous ethanol at pH 4, whereas Laponite is a hydrophilic particle soluble in water having a negatively charged surface at all pH. This property of zein and Laponite led to the complex formation between zein and Laponite in a 60% v/v aqueous ethanol solution at pH 4 via electrostatic interaction. The complex formation between the fixed concentration of zein, C Z (1 mg/mL) and varying concentrations of Laponite, C L was investigated using viscosity measurement, the dynamic lightscattering (DLS) technique, zeta potential, turbidity, imaging, and differential scanning calorimetric data.
Viscosity Measurements. Viscosity of zein (1 mg/mL) in a 60% v/v ethanol solution at pH 4 was observed to be 2.93 mPa·s. The viscosity of Laponite in deionized water at various concentrations remained almost constant as shown in Figure   1. However, the viscosity of the samples having fixed zein concentration, C Z (1 mg/mL), and varying Laponite concentrations, C L , showed a rise in viscosity. As the concentration of the Laponite increases in samples, we saw a rise in the relative viscosity values, which indicated the formation of large zein− Laponite complexes. The relative viscosity profile ( Figure 1) for zein−Laponite complexes was fitted linearly, which gave two distinct regions. The two regions were demarcated by the change in the slope of the viscosity profile. In the first region (C Z = 1 mg/mL, C L = 0.00078−0.018% w/v), the viscosity remained almost constant indicating the formation of small-sized zein− Laponite complexes. Beyond C L = 0.018% w/v, we saw a second region where linear fitting of viscosity data points had a positive slope. The positive slope indicated the formation of larger zein− Laponite complexes in samples having higher Laponite concentrations.
Dynamic Light Scattering. Dynamic light scattering, DLS, is used to calculate the size of the particle undergoing Brownian motion. 45 For a dilute system of spherical and monodisperse particles undergoing Brownian motion, a field autocorrelation function, g 1 (τ), in dynamic light scattering can be fitted with single exponential as given in eq 1.
where D is the translational diffusion coefficient, τ is the delay time, and q is the magnitude of scattering vector as given in eq 2.
where, n is the refractive index of the solution, λ is the wavelength of laser, and θ is the scattering angle. However, for polydisperse samples, the CONTIN method 45 was used where the field autocorrelation function is given as where Γ is the decay constant and G(Γ) is the decay rate distribution function obtained by performing inverse Laplace transformation on eq 3. The mean size and polydispersity index (PDI) of the complexes formed during the interaction between zein and Laponite were calculated using the CONTIN method by the software in the dynamic light-scattering experiment. The mean hydrodynamic diameter and PDI values of zein−Laponite complexes at various C L were plotted and tabulated in Figure 2 and Table 1, respectively. The inset of Figure 2 showed the variation of mean hydrodynamic diameter of the complexes with their standard deviation.
The variation of mean hydrodynamic diameter for zein− Laponite complexes at a fixed concentration of zein, C Z , and varying concentrations of Laponite, C L , was plotted in Figure 2. The figure suggested that the hydrodynamic diameter (d) of the complexes increased slowly up to C L = 0.00625% w/v, and then it rose drastically after C L > 0.00625% w/v. The drastic rise in complex size revealed the formation of larger complexes. Initially, up to five data points (Figure 2), the solution was visibly clear; therefore, we could be able to measure size using DLS. However, at the sixth data point (C L = 0.00625% w/v) and after that, the solution becomes turbid. The turbid sample suffers multiple scattering and does not give the correct size and so measuring the size using a turbid solution does not make any sense. However, we have taken the sixth data point just to demarcate region I (clear solution region) and region II (turbid solution region).
Visual Inspection of the Zein−Laponite Complexes. Zein−Laponite complexes for fixed C Z (1 mg/mL) and varying C L (7.8 × 10 −4 − 0.25% w/v) formed in the 60% v/v aqueous ethanol solution at pH 4 was stored in sample vials at 25°C. Figure 3 shows the picture of the sample vials that was taken after 24 h. Figure 3 depicted three different regions based on visual inspection. Region I (C Z = 1 mg/mL, C L = 0.00078−0.00625% w/v) was classified based on the stable suspension of the complexes in the solution. The solution in this region remained stable, and no aggregation or flakes were found except that we could see a small amount of turbidity. The small amount of turbidity occurred due to the formation of zein−Laponite complexes in the solution. As the concentration of Laponite was further increased, we observed region II (C Z = 1 mg/mL, C L = 0.0125−0.05% w/v). Region II showed a liquid−liquid phase separation state. The upper part of the phase-separated state in sample vials that are transparent and contains small traces of the complexes are called supernatant, whereas the lower part of the phase-separated state, which was opaque and rich in complexes are called coacervates. The phase-separated state was also observed in region III at further higher concentrations of Laponite (C Z = 1 mg/mL, C L = 0.1−0.25% w/v). However, the reason to segregate region II and region III was based on visual inspection. Visual inspection suggests that the coacervate phase in region II looked more dense and opaque as compared to region III where the coacervate phase looked sparse. The reason for visually dense coacervate in region II and sparse coacervate in region III has been discussed in detail in the Zeta Potential section. It has been argued in the Zeta Potential section that neutral zein−Laponite complexes aggregate and tend to give a dense phase in region II, while overcharged coacervates in region III try to repel each other due to electrostatic repulsion and thus inhibit aggregation and form a sparse coacervate phase.
Turbidity Measurements. Figure 4 depicted the turbidity of the samples immediately (t = 0) after adding varying concentrations of Laponite, C L (7.8 × 10 −4 − 0.15% w/v), to the fixed concentration of zein, C Z (1 mg/mL), in the 60% v/v ethanol solution. Stable soluble complexes with a slow rise in turbidity was observed for C L = 0.00078−0.00625% w/v, while a considerable increase in the turbidity was observed for C L = 0.025 and 0.05% w/v. At a further higher concentration of Laponite (C L = 0.1 and 0.15% w/v), the turbidity decreased drastically. Therefore, on the basis of variation of turbidity, different regions were classified as (i) Region I (C Z = 1 mg/mL, The study of turbidity as a function of time was done to further get insights on the complex formation. The change of turbidity with time gave information about the liquid−liquid phase separation phenomenon as depicted in Figure 5. It was evident from Figure 5 that time-dependent turbidity for zein−Laponite complexes remained almost constant for region I (C Z = 1 mg/mL, C L ≤ 0.00625% w/v). In region II (C Z = 1 mg/   ACS Omega http://pubs.acs.org/journal/acsodf Article mL, 0.00625 < C L ≤ 0.05% w/v), the turbidity grew much larger at the initial time followed by a decrease in turbidity with the passage of time. The reason for this behavior 5,46−49 owes its explanation from the fact that at the initial time, the high values of turbidity indicated the formation of large-sized zein− Laponite complexes. However, with the passage of time, these complexes due to large size became unstable in the solution phase and undergo liquid−liquid phase separation (coacervation) indicated by the drop in the turbidity values. The phase-separated state in region II could be seen in Figure 3. In region III (C Z = 1 mg/mL, C L > 0.05% w/v), the initial value of turbidity decreased as compared to region II. Nevertheless, the turbidity remained almost constant for each C L in this region for almost an hour, and we could not see a drop in turbidity till that time to indicate liquid−liquid phase separation phenomena. Instead of the above fact, liquid−liquid phase separation was observed in region III at a much longer waiting time with visually sparse density of coacervates ( Figure 3). The reason for the large waiting time for the liquid−liquid phase separation (coacervation) and visually sparse density of coacervates in region III as compared to region II has been discussed in the Zeta Potential section. Zeta, ξ, Potential. Zeta potential experiment was done to ascertain that zein−Laponite complexes were formed owing to electrostatic interaction between positively charged zein and a negatively charged Laponite surface in the 60% v/v aqueous ethanol solution at pH 4. Moreover, zeta potential data was also used to understand the reason for the fast coacervation process and the visually dense coacervate phase in region II as compared to the long waiting time for coacervation and visually sparse density of coacervates in region III.
The size of pure zein calculated from DLS measurement was nearly 400 nm, and the size of the zein−Laponite complex grew large, i.e., more than 7000 nm for C L = 0.0125% w/v. For C L > 0.0125% w/v, the samples grew turbid indicating the further large size of the complexes. Therefore, the Smoluchowski equation was used to convert electrophoretic mobility to zeta potential because this equation is used when κa ≫ 1, where a is the radius of the particle and κ −1 is the Debye length. 50,51 Zeta potential and the standard deviation for varying C L in deionized water at pH 7 and zein−Laponite complexes having fixed C Z (1 mg/mL) and varying C L in the 60% v/v ethanol solution at pH 4 have been plotted and tabulated in Figure 6 and Table 2, respectively. Figure 6 depicts that the ξ potential of zein was +23 mV at pH = 4 in the 60% v/v ethanol solution. With the increase in the concentration of Laponite, C L , the zeta potential tends to decrease, which is further followed by charge reversal (overcharging). The plot of the zeta potential as a function of C L suggested that Figure 6 could be segregated in three regions based on the zeta potential of the complexes. Region I (C Z = 1 mg/mL, C L ≤ 0.00625% w/v) consists of complexes that are not fully charge-neutralized and forms soluble and stable complexes. The solution phase in this region looked a little turbid due to    interaction, such as charge patch interaction, was responsible for the overcharging phenomenon. The overcharged complexes in solution try to repel each other and therefore inhibit fast coacervation. Accordingly, zein−Laponite complexes before and after the charge-neutralized region was stabilized by electrostatic and charge patch repulsion, respectively. On the other hand, in the charge-neutralized region, electrostatic repulsion between complexes vanishes and van der Waals interactions dominate, which causes unstable dispersion and therefore rapid aggregation and coacervation. Nevertheless, overcharging that inhibited fast coacervation resulted in the visually sparse coacervate phase due to charge patch repulsion between complexes in region III as compared to the visually dense coacervate phase in region II due to aggregation of neutral zein−Laponite complexes.
Imaging. SEM images of complexes having different C L were shown in Figure 7. Figure 7a−c corresponds to C L = 0, 0.00156, and 0.0032% w/v, respectively, and belongs to region I. It could be seen that for C L = 0% w/v, i.e., pure zein (C Z = 1 mg/mL), the sample is polydisperse, and the size of zein varies from 94 to 360 nm. At higher Laponite concentrations (0.00156 and 0.0032% w/v), samples remain polydisperse and the size of zein− Laponite complexes increased with the increase in Laponite   Figure 7d,e belongs to region II where neutral coacervates were formed, while Figure 7f belongs to region III where overcharged coacervates were formed. The coacervate phase is a liquid phase with densely packed zein− Laponite complexes in a mobile state, and therefore, the dehydrated SEM images of the coacervates will appear as aggregates. The SEM image of aggregates in region III ( Figure  7f) looked sparse with voids in the aggregate phase as compared to densely packed aggregates in region II (Figure 7d,e). The possible reason for this may be attributed to electrostatic repulsion between the overcharged complexes in the coacervate phase of region III.
It should be noted that SEM images were taken after dehydrating the samples, and therefore the coacervate phase looked like aggregates. The image of coacervates in the hydrated state was thus obtained using phase contrast imaging. Figure 8 showed the phase contrast image of coacervates in region II and region III.
The image of bigger size coacervates in region II due to aggregation and smaller size coacervates in region III due to repulsion between overcharged complexes can be seen in Figure  8a−c, respectively. The average size of coacervates along with its size distribution for region II and region III was shown in Figure  8d−f, respectively. It is pretty clear from Figure 8d that exactly at the charge-neutralized concentration (Region II, C Z = 1 mg/mL, C L = 0.025% w/v), we saw large coacervate particles with an average size of 1223.7 nm due to aggregation of neutral coacervates. However, if we slightly deviate from the chargeneutralized concentration but remained in region II (Region II, C Z = 1 mg/mL, C L = 0.05% w/v), we saw lesser aggregation with an average coacervate size of 699.2 nm (Figure 8e). Nevertheless, in the overcharged region (Region III, C Z = 1 mg/mL, C L = 0.15% w/v), aggregation was inhibited, and we saw smaller coacervates with an average particle size of 464.7 nm (Figure 8f).
Differential Scanning Calorimeter. Hydration of polymers was driven by interaction between polymer−water and water−water interactions. 60−64 Water molecules that are not in the vicinity of the polymers interact with each other to give

ACS Omega
http://pubs.acs.org/journal/acsodf Article water−water interaction. The water−water interaction between water molecules gives rise to bulk water. However, water molecules that are in the close vicinity to polymers render polymer−water interaction, and we call these water molecules as bound water. It was therefore felt important to understand the hydration behavior of coacervates in terms of bulk and bound water. The hydration behavior of coacervates in the two regions (region II and region III) was therefore studied using differential scanning calorimeter, DSC, as shown in Figure 9.
In region II, the neutral complexes form tight and closepacked aggregates of coacervates, and therefore a small area would be available for the water molecule to hydrate the densely packed aggregates of coacervates. We believe that because of this reason, the water−water interaction will be favored to give bulk water. The enthalpy for evaporation of bulk water in this region was observed between 90−100°C as depicted in Figure 9. However, in the region III, the overcharged complexes in the coacervates repel each other. This repulsion will create voids and facilitate a large amount of water molecules to interact with the coacervate phase. The interaction will enrich polymer−water interaction to give a sufficient amount of bound water in region III. We believe that enthalpy of evaporation of these bound water gave an extra peak at 107°C in region III.
DLVO Theory. Stability of charged particles and colloids in the solution phase has been well described by DLVO theory. 65−70 According to DLVO theory, the stability of charged colloids or particles was governed by the sum of two forces, i.e., the electrostatic force and van der Waals force.
where F T represents the total interaction force, F E corresponds to the electrostatic force, and F V is the van der Waals force. For highly charged colloids or particles, the electrostatic repulsive force is more than van der Waals attractive force, and so the colloids/particles will remain stable in the solution phase. However, if some ions were added to screen the charged particles, then the van der Waals attractive force will dominate and particles will aggregate.
Nevertheless, some non-DLVO terms, such as hydrophobic and charge patch interactions, may exist in some colloidal systems, and so the total interactions in eq 4 should be modified. The modified interactions given by eq 5 gives extended DLVO theory. 67,68 = 5) where F N represents forces arising due to non-DLVO terms.
It is to be noted that zein is a hydrophobic protein, and at a high laponite concentration, the zein−Laponite complexes acquire the charge reversal phenomenon (overcharged phenomenon) probably because of charge patch interactions. Thus, as far as zein−Laponite complexes are concerned, we believe that these complexes should follow extended DLVO theory to give liquid−liquid phase separation.
Nevertheless, the stability ratio (W) is often calculated to understand the aggregation process predicted using DLVO or extended DLVO theory. 55,66,71 For W = 1, the aggregation is diffusion limited; therefore, fast aggregation occurs, while values of W between 1 and 100 corresponds to the slow aggregation process. The stability ratio 55,66 (W) using dynamic lightscattering (DLS) and static light-scattering (SLS) experiments was calculated using eq 6.
()where, t is the time, k DLS fast refers to the rate constant of actual measurement, k DLS refers to the fast rate constant, and R h (t) is the hydrodynamic radius of the particle at time t.
Literature 5,46−49 suggests that the rise in turbidity can be hypothesized as the aggregation of inter-and intrapolymeric complexes in a cooperative manner. Thus, size can be directly related to turbidity, and therefore we can redefine our stability factor using turbidity data as eq 7. for zein−Laponite complexes at different C L was obtained from the slope of the straight line by fitting few initial data points of Figure  5. Figure 10 depicted the plot of zeta potential and W for different zein−Laponite complexes, which was obtained from Figure 6 and eq 7, respectively. The plot indicated that at C L = 0.025% w/v, the value of W = 1, which means that at this C L , the aggregation is diffusion limited and the aggregation process is fast. It should be noted that at this concentration, i.e., C L = 0.025% w/v, the zeta potential of zein−Laponite complexes goes down to almost zero. Figure 10 also suggests that C L = 0.025% w/v is the critical coagulation concentration, because at this concentration, we saw a transition between the slow (W between 1 and 100) and fast (W = 1) aggregation regime. Before and beyond C L = 0.025% w/v, the aggregation rate is slow because of the positively charged and negatively charged (overcharged) zein−Laponite complexes, respectively. The restabilization or slow aggregation process of particles in the presence of excess ionic liquids, polymers, surfactants, etc. has been associated with charge reversal or the overcharging phenomenon as reported in various studies. 56−59 Figure 9. Differential scanning calorimetry (DSC) thermogram of zein−Laponite complex coacervates obtained from region II (C Z = 1 mg/mL, 0.00625 < C L ≤ 0.05% w/v) and region III (C Z = 1 mg/mL, C L > 0.05% w/v).

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http://pubs.acs.org/journal/acsodf Article Overcharging Phenomena in Coacervates. Coacervates formed due to zein and Laponite interactions can be broadly divided into two regions. These regions can be identified as at and after the charge neutralization point of zein−Laponite complexes, i.e., region II and region III, respectively. At the charge neutralization region, neutral complexes aggregate to form larger complexes. These large complexes become unstable in the solution to give a neutral coacervate phase via the liquid− liquid phase separation mechanism. Beyond the neutralization point, interesting overcharging behavior of the complexes was noticed. The overcharged complexes played an important role in suppressing the dynamics of coacervation due to electrostatic repulsion; however, at a sufficiently long time, we get overcharged coacervates. Thus, it felt important to compare different systems 54,72−75 where complex coacervation and overcharging were observed due to intermolecular binding (Table 3). Various other studies are available in which layered double hydroxide 55 (diameter, 334 nm), latex 56 (diameter, 220 nm), Laponite 57 (diameter, 30 nm), halloysite 58 (length, 200− 500 nm), and hematite 59 (diameter, 140 nm) particles have shown an overcharging effect in the presence of polyelectrolyte, ionic liquid, polymer, protamine, and surfactant, respectively. As mentioned in studies, [55][56][57][58][59]67 various reasons, such as hydrophobicity, charge patch interaction, chain length, etc., were responsible for the overcharging phenomenon. Interestingly, we may notice that in all the above cases, overcharging was observed when one molecule is stiffer than the counter molecule. Therefore, we believe that apart from various reasons cited above, relatively high stiffness of one molecule as compared to its partner molecule could be a possible reason for getting the overcharging phenomenon.
Future Prospect of Coacervates as Dual Drug Delivery System. The idea of encapsulating drugs in coacervates, 43,76,77 making films of coacervates 20,78,79 and loading cationic drugs in laponite−polymer hydrogels 80 for the release of drug has been studied in many cases. The same idea could be used in our zein− Laponite coacervates for using it as a drug delivery system. Moreover, a combinatorial drug delivery system provides a therapeutic effect to overcome drug resistance along with lower toxicity and improved efficacy. Therefore, designing new vehicles to carry more than one drug at a time seems quite reasonable and promising. Zein−Laponite coacervates could be a possible solution for such a dual drug delivery carrier. Future prospect of zein−Laponite coacervates as a dual drug delivery system laid in the fact that both cationic and hydrophobic drugs could be loaded to these coacervates. In the coacervates, the cationic drug could be attached to the negatively charged surface of Laponite, while the hydrophobic drug could be attached to zein via electrostatic and hydrophobic interactions, respectively.
An important factor that determines the role of particles to be used as a drug carrier is related to its size. 81

■ CONCLUSION
For the first time, the paper reports complexation between the hydrophobic corn protein, zein, and a negatively charged nanodisc, Laponite in a 60% v/v ethanol solution at pH 4. It was observed that electrostatic interaction between zein and Laponite at pH 4 was responsible for the complexation. The complexation between the fixed concentration of zein and varying concentrations of Laponite led to various phase states. It was observed that at a low concentration of Laponite, C L (C L ≤ 0.00625% w/v), soluble and stable zein−Laponite complexes were formed. However, for the concentration range of 0.00625 < C L ≤ 0.05% w/v and C L > 0.05% w/v, neutral charged and overcharged complex coacervates were formed, respectively. The neutral coacervates tend to aggregate to give large-sized coacervates, whereas overcharged coacervates have relatively smaller sizes due to repulsion between the coacervates. It was also revealed that in overcharged coacervates, bound water was responsible for giving an extra peak for the enthalpy of evaporation at 107°C.

■ EXPERIMENTAL SECTION
Materials. Zein was purchased from TCI chemicals (CAS no. 9010-66-6), India and used as received. The specification sheet for zein reports that it has been obtained from corn with a  Instrumentation and Characterization. Samples were analyzed with a Zetasizer Nano-ZS instrument (Malvern instrument Ltd., India) for the mean particle size and for the zeta potential. Dynamic light scattering measurements were collected at the 173°detector angle at 25°C. Viscosity of samples was measured by sine-wave vibro viscometer (model SV: 10−100, A&D co. Ltd., Japan). This instrument was equipped with a matched pair of gold plated electrodes. In this technique, the mechanical vibration given to one electrode propagates through the sample and is picked by the other electrode to give viscosity reading. Phase separation was studied by continuously measuring transmittance (% T) using a colorimeter (Brinkmann-910, Brinkmann Instruments, U.S.) operating at 450 nm. Thermal analysis of coacervates were carried out using differential scanning calorimetry, DSC (Setaram instrumentation, model no. DSC-131). The instrument gave information about the enthalpy of evaporation of water for the coacervates. For DSC experiment, samples having a coacervate phase were centrifuged at 10,000 rpm for 30 mins. Coacervates was then separated from the supernatant and used for the DSC experiment. Structural morphology of the zein− Laponite coacervate was examined by using an Axio-observer 7.0 fluorescence microscope (ZEISS). For imaging, coacervates (dense phase) was separated from the supernatant using a syringe. The coacervates were then placed into the depression slides and covered with a cover slip for imaging. Bright-field snapshots (Phase-contrast images) of coacervates were taken using Plan-Apochromat 63x oil (NA = 1.40) objective lens and with a CMOS sensor 2.3 mega pixel camera (702 monoD, Zeiss). Distribution of coacervate size in each of the concentration was done using ImageJ online free software. SEM images were captured by drop casting the samples on the cover slip. The cover slips were then coated with gold and then imaged using a Nova Nano SEM 450, FEI.