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, C Z = 1 mg/mL, and varying concentrations of Laponite, C L (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 (C Z = 1 mg/mL, C L ≤ 0.00625% w/v) where soluble complexes was formed during interaction between oppositely charged zein and Laponite; Region II, the charge neutralization region (C Z = 1 mg/mL, 0.00625 < C L ≤ 0.05% w/v) where zein-Laponite complexes form neutral coacervates; and Region III, the interesting overcharged coacervates region (C Z = 1 mg/mL, C L > 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, CZ (1 mg/mL) and varying concentrations of Laponite, CL was investigated using viscosity measurement, the dynamic light-scattering (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 . However, the viscosity of the samples having fixed zein concentration, CZ (1 mg/mL), and varying Laponite concentrations, CL, 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 ) 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 (CZ = 1 mg/mL, CL = 0.00078–0.018% w/v), the viscosity remained almost constant indicating the formation of small-sized zein–Laponite complexes. Beyond CL = 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.

Figure 1

Variation in the viscosity of the zein–Laponite complex at different concentrations of Laponite, CL. Solid lines are the linear fitting to the data set. The arrow indicates the concentration where the slope of the fitted line changes.

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, g1(τ), in dynamic light scattering can be fitted with single exponential as given in eq .where D is the translational diffusion coefficient, τ is the delay time, and q is the magnitude of scattering vector as given in eq .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 aswhere Γ is the decay constant and G(Γ) is the decay rate distribution function obtained by performing inverse Laplace transformation on eq .

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 CL were plotted and tabulated in Figure and Table , respectively. The inset of Figure showed the variation of mean hydrodynamic diameter of the complexes with their standard deviation.

Figure 2

Variation of hydrodynamic diameter, d, of the zein–Laponite complex at different concentrations of Laponite, CL. The concentration of zein, CZ was fixed at 1 mg/mL. The Arrow indicates the concentration after which a drastic change in the hydrodynamic diameter was observed. Inset shows variation of d with their standard deviation.

Table 1

Mean Hydrodynamic Diameter (d), Standard Deviation, and Polydispersity Index (PDI) for Zein–Laponite Complexes for Fixed CZ (1 mg/mL) and Varying CL

CL (% w/v)	d/nm	standard deviation/nm	PDI
0	395.3	8.9	0.548
0.00078	439	16.69	0.694
0.00156	427.4	26.82	0.713
0.00312	559.8	78.9	0.905
0.00625	662.5	41.32	0.717

The variation of mean hydrodynamic diameter for zein–Laponite complexes at a fixed concentration of zein, CZ, and varying concentrations of Laponite, CL, was plotted in Figure . The figure suggested that the hydrodynamic diameter (d) of the complexes increased slowly up to CL = 0.00625% w/v, and then it rose drastically after CL > 0.00625% w/v. The drastic rise in complex size revealed the formation of larger complexes. Initially, up to five data points (Figure ), the solution was visibly clear; therefore, we could be able to measure size using DLS. However, at the sixth data point (CL = 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 CZ (1 mg/mL) and varying CL (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 shows the picture of the sample vials that was taken after 24 h.

Figure 3

Zein–Laponite complexes in 60% v/v aqueous ethanol solution at pH 4 after 24 h. Different regions based on visual inspection was classified as (a) Region I, CZ = 1 mg/mL, CL = 0.00078–0.00625% w/v, having smaller soluble complexes; (b) Region II, CZ = 1 mg/mL, CL = 0.0125–0.05% w/v, having a visually dense coacervate phase; and (c) Region III, CZ = 1 mg/mL, CL = 0.1–0.25% w/v, having a visually sparse coacervate phase.

Figure depicted three different regions based on visual inspection. Region I (CZ = 1 mg/mL, CL = 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 (CZ = 1 mg/mL, CL = 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 (CZ = 1 mg/mL, CL = 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 depicted the turbidity of the samples immediately (t = 0) after adding varying concentrations of Laponite, CL (7.8 × 10–4 – 0.15% w/v), to the fixed concentration of zein, CZ (1 mg/mL), in the 60% v/v ethanol solution. Stable soluble complexes with a slow rise in turbidity was observed for CL = 0.00078–0.00625% w/v, while a considerable increase in the turbidity was observed for CL = 0.025 and 0.05% w/v. At a further higher concentration of Laponite (CL = 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 (CZ = 1 mg/mL, CL ≤ 0.00625% w/v), (ii) Region II (CZ = 1 mg/mL, 0.00625 < CL ≤ 0.05% w/v), and (iii) Region III (CZ = 1 mg/mL, CL > 0.05% w/v).

Figure 4

Variation of turbidity of the solution at t = 0, i.e., immediately after mixing different concentrations of Laponite, CL, to a fixed concentration of zein (CZ = 1 mg/mL) in 60% v/v ethanol solution. Dotted lines are guides to the eye to demarcate different regions.

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 .

Figure 5

Turbidity of zein–Laponite complexes as a function of time for varying CL at pH = 4.

It was evident from Figure that time-dependent turbidity for zein–Laponite complexes remained almost constant for region I (CZ = 1 mg/mL, CL ≤ 0.00625% w/v). In region II (CZ = 1 mg/mL, 0.00625 < CL ≤ 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 . In region III (CZ = 1 mg/mL, CL > 0.05% w/v), the initial value of turbidity decreased as compared to region II. Nevertheless, the turbidity remained almost constant for each CL 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 ). 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 CL = 0.0125% w/v. For CL > 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 CL in deionized water at pH 7 and zein–Laponite complexes having fixed CZ (1 mg/mL) and varying CL in the 60% v/v ethanol solution at pH 4 have been plotted and tabulated in Figure and Table , respectively.

Figure 6

Variation of ξ potential for (a) various CL in deionized water at pH 7, (b) zein–Laponite complexes with varying CL and fixed CZ (1 mg/mL) in 60% v/v ethanol solution at pH 4. The shaded region indicates the range of ξ potential at which complexes will be considered to be neutral in charge. Dashed lines are guides to the eye for demarcation of region I, region II, and region III.

Table 2

Values of Zeta Potential and their Standard Deviations for (a) Various CL in Deionized Water at pH 7, (b) Zein–Laponite Complexes with Varying CL and Fixed CZ (1 mg/ml) in 60% v/v Ethanol Solution at pH 4

CZ = 0 mg/mL			CZ = 1 mg/mL
CL (% w/v)	ζ/mV	std. dev./mV	CL (% w/v)	ζ/mV	std. dev./mV
0.00078	–19.1	3.06	0	23.7	2.46
0.00312	–29.7	1.08	0.00078	11.4	0.25
0.00625	–19.8	0.98	0.00156	5.12	0.07
0.0125	–25.3	0.85	0.00312	5.06	0.3
0.025	–26.7	1.35	0.00625	3.88	0.38
0.05	–26.8	0.55	0.0125	2.45	0.08
0.1	–29.1	1.59	0.025	0.99	0.03
0.15	–32.63	5.27	0.05	–1.99	0.03
0.2	–29.4	3.16	0.1	–3.51	0.17
			0.15	–12.7	0.65

Figure 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, CL, the zeta potential tends to decrease, which is further followed by charge reversal (overcharging). The plot of the zeta potential as a function of CL suggested that Figure could be segregated in three regions based on the zeta potential of the complexes. Region I (CZ = 1 mg/mL, CL ≤ 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 formation of complexes. The region II (CZ = 1 mg/mL, 0.00625 < CL ≤ 0.05% w/v) consists of complexes that are charge-neutralized, and therefore they tend to aggregate. The aggregates are unstable in the solution phase and undergo liquid–liquid phase separation to give coacervates. The coacervates in region II were dense as seen from naked eyes. Beyond the charge neutralization point, region III (CZ = 1 mg/mL, CL > 0.05% w/v) was observed. This is the region where overcharging was observed. It was expected that beyond the charge neutralization point, if more complementary particles are added, further binding is not favored and the zeta potential would remain same. However, we saw charge reversal in zein–Laponite complexes when the excess of Laponite was added beyond the charge neutralization point. Many theoretical predictions and experimental data revealed that complementary particles can bind to the charge-neutralized complexes to give overcharged complexes, and this overcharging phenomenon is energetically favored.[52−54] Various other studies[55−59] revealed that non-DLVO (Derjaguin, Landau, Verwey, and Overbeek) 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 CL were shown in Figure . Figure a–c corresponds to CL = 0, 0.00156, and 0.0032% w/v, respectively, and belongs to region I. It could be seen that for CL = 0% w/v, i.e., pure zein (CZ = 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 concentration. Figure d–f corresponds to CL = 0.025, 0.05, and 0.2% w/v, respectively. Figure d,e belongs to region II where neutral coacervates were formed, while Figure f 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 f) looked sparse with voids in the aggregate phase as compared to densely packed aggregates in region II (Figure d,e). The possible reason for this may be attributed to electrostatic repulsion between the overcharged complexes in the coacervate phase of region III.

Figure 7

SEM images of samples at fixed CZ (1 mg/mL) and varying CL: (a) CL = 0% w/v, (b) CL = 0.00156% w/v, (c) CL = 0.00312% w/v, (d) CL = 0.025% w/v, (e) CL = 0.05% w/v, and (f) CL = 0.2% w/v.

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 showed the phase contrast image of coacervates in region II and region III.

Figure 8

Phase contrast image of coacervates and its size distribution for (a, d) CL = 0.025% w/v, (b, e) CL = 0.05% w/v, and (c, f) CL = 0.15% w/v, respectively.

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 a–c, respectively. The average size of coacervates along with its size distribution for region II and region III was shown in Figure d–f, respectively. It is pretty clear from Figure d that exactly at the charge-neutralized concentration (Region II, CZ = 1 mg/mL, CL = 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 charge-neutralized concentration but remained in region II (Region II, CZ = 1 mg/mL, CL = 0.05% w/v), we saw lesser aggregation with an average coacervate size of 699.2 nm (Figure e). Nevertheless, in the overcharged region (Region III, CZ = 1 mg/mL, CL = 0.15% w/v), aggregation was inhibited, and we saw smaller coacervates with an average particle size of 464.7 nm (Figure f).

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 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 .

Figure 9

Differential scanning calorimetry (DSC) thermogram of zein–Laponite complex coacervates obtained from region II (CZ = 1 mg/mL, 0.00625 < CL ≤ 0.05% w/v) and region III (CZ = 1 mg/mL, CL > 0.05% w/v).

In region II, the neutral complexes form tight and close-packed 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 . 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 FT represents the total interaction force, FE corresponds to the electrostatic force, and FV 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 should be modified. The modified interactions given by eq gives extended DLVO theory.[67,68]where FN 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 light-scattering (DLS) and static light-scattering (SLS) experiments was calculated using eq .where, t is the time, kDLSfast refers to the rate constant of actual measurement, kDLS refers to the fast rate constant, and Rh(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 .where A(t) = 100% – T(t), and the values of A(t) were obtained from Figure at different t, and refers to as the rate of change of turbidity of the actual measurement and the fast rate of change of turbidity, respectively, as t approaches zero. The values of for zein–Laponite complexes at different CL was obtained from the slope of the straight line by fitting few initial data points of Figure .

Figure depicted the plot of zeta potential and W for different zein–Laponite complexes, which was obtained from Figure and eq , respectively. The plot indicated that at CL = 0.025% w/v, the value of W = 1, which means that at this CL, the aggregation is diffusion limited and the aggregation process is fast. It should be noted that at this concentration, i.e., CL = 0.025% w/v, the zeta potential of zein–Laponite complexes goes down to almost zero. Figure also suggests that CL = 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 CL = 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 10

Variation of the zeta potential and stability ratio as a function of CL.

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 ). 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,[55−59,67] 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.

Table 3

Comparison of Binding Leading to Complex Coacervation in Diverse Systemsa

S. no.a	properties	GA + GB[72,73]	GA + L[74]	GA + DNA[54]	zein + Laponite (this work)
1	binding type	protein–protein	protein–colloid	protein–nucleic acid	protein–colloid
2	persistence length	10 nm (GA)	10 nm (GA)	10 nm (GA)	2 nm (Z)
		2 nm (GB)	30 nm (L)	50 nm (DNA)	30 nm (L)
4	zeta pot. ratio	2 (GB:GA)	5 (L:GA)	16 (DNA:GA)	1 (Z:L)
5	overcharging	absent	absent	present	present
6	pH	6.5	7.2	6.0	4.0

GA, GB, Z, and L represents Gelatin A, Gelatin B, Zein, and Laponite, respectively.

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.[81−83] Interestingly, we have seen that the size of our coacervates could vary over a large range (464–1223 nm) based on zein–Laponite interactions. Nevertheless, the size of zein–Laponite coacervates could further be tuned if we can vary the size of zein or the sample preparation parameter for Laponite. Different sizes of zein which depends upon the fabrication parameter, such as pH, solvent, and temperature,[84] and the sensitivity of Laponite towards sample preparation,[85] could affect the size of zein–Laponite complexes.

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, CL (CL ≤ 0.00625% w/v), soluble and stable zein–Laponite complexes were formed. However, for the concentration range of 0.00625 < CL ≤ 0.05% w/v and CL > 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 total nitrogen content of 14% and 0.2% drying loss. LAPONITE RD (Laponite) (Lot no. 0001603378, BYK-Additives and Instruments) was received from Aroma Chemical Agencies (India) Pvt. Ltd., New Delhi, as a gift. As indicated by the BYK datasheet, Laponite appeared as free flowing white powder having a bulk density of 1000 kg/m3 and a surface area of 370 m2/g. The technical datasheet from BYK additives and Instruments states that it is a synthetic clay having an empirical formula of Na+0.7[Si8Mg5.5Li0.3O20(OH)4]−0.7 and is in the form of disc-shaped crystals with a diameter of 25 nm and thickness of 1 nm. Absolute ethanol was purchased in Labogen Pvt. Ltd., India.

Preparation of Zein–Laponite Complexes

Stock solution of zein was prepared by dissolving a known amount of zein in 80% v/v ethanol solution. The stock solution of zein was maintained at pH = 4 using 0.1 M HCl. The obtained stock solution of zein, which appeared clear, was filtered with a 0.2 μm syringe filter. Laponite powder was dried in an oven for 4 h to remove moisture. The dried Laponite was then stirred in a known amount of deionized water (pH = 7) to get a clear stock solution. The stock solution of Laponite was also filtered with a 0.2 μm syringe filter. Finally, series of samples were prepared by mixing stock solution of zein and stock solution of Laponite in a known volume while maintaining the pH of the solution at 4 using 0.1 M HCl. The series of samples thus prepared should have a fixed concentration of zein, CZ = 1 mg/mL, and varying concentrations of Laponite, CL (7.8 × 10–4 – 0.25% w/v) in a 60% v/v ethanol solution at pH 4. All the samples were prepared at room temperature, 25 °C.

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.