pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein

Research Article pubs.acs.org/journal/ascecg

pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein Induced Morphology Control, Bioapplicability, and Bioextraction of Nanomaterials Aabroo Mahal,§ Lavanya Tandon,§ Poonam Khullar,*,§ Gurinder Kaur Ahluwalia,‡ and Mandeep Singh Bakshi*,†

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Department of Natural and Applied Sciences, University of WisconsinGreen Bay, 2420 Nicolet Drive, Green Bay, Wisconsin 54311-7001, United States ‡ Materials & Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2 V 2K7 Canada § Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India S Supporting Information *

ABSTRACT: Precise morphologies of pH responsive bioactive lead sulfide nanoparticles (PbS NPs) were synthesized by using industrially and environmentally important proteins like zein and lysozyme (Lys), and a bioactive polymer diethylaminoethyl dextran chloride (DEAE). Though, proteins are not known morphology control agents, zein demonstrated a fine crystal growth control of PbS NPs better than Lys as well as DEAE, and even better than conventional surfactants known for their shape control behavior. Proteins and DEAE coated NPs thus obtained were highly pH responsive in terms of a color change from light gray (at low pH) to dark brown (at high pH). Bioapplicability of coated NPs was done by subjecting them to hemolysis. Both Lys and DEAE coated NPs did not induce any significant hemolysis and demonstrated their good compatibility and usability in systemic circulation. For their industrial scale uses, different extraction methods were proposed by using other industrially important biomolecules and ionic liquids. Alginic acid and xanthan gum were excellent complexing agents for an instant extraction of Lys and DEAE coated NPs from aqueous phase. Ionic liquid exhibited excellent extraction ability in both organic as well as aqueous phases. KEYWORDS: pH responsive lead sulfide nanoparticles, Bioactive, Hemolysis, Bioextraction

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INTRODUCTION

induces significant cytotoxic effects in the biological system and hence, surfactant coated nanomaterials are usually not suitable for possible drug release vehicles in the systemic circulation.12 An appropriate choice of a surface active biomolecule with amphiphathic behavior is a better candidate than the conventional surfactant for the shape controlled synthesis as well as for the formation of bioactive nanomaterials.3 Three important biomolecules viz. zein, Lys, and DEAE with diverse properties and several industrial applications have been selected for this study. Zein is an industrial corn storage protein and is extensively used in food and pharmaceutical coating applications due to its environmentally friendly, clear, odorless, tasteless, and edible nature. Its highly robust structure made up of nine homologous repeat units arranged in an antiparallel distorted cylindrical form and stabilized by the hydrogen bonds13−15 makes it a highly stable protein. Because of its nontoxic nature, zein coated PbS NPs can find their applications in biological systems. Similarly, Lys is an important

pH responsive biosustainable semiconducting nanomaterials is a unique class of materials with remarkable applications in biological systems.1,2 They perform specific functions for drug release under different conditions and act as biological markers due to the quantum confinement effects. A semiconducting NP can be made pH responsive if it is appropriately coated with pH sensitive biomolecules suitable for the biological environment. In this study, we are presenting the synthesis and pH responsive properties of bioactive PbS NPs.3,4 Selection of PbS is mainly due to its narrow band gap of 0.41 eV in the bulk, a large excitation Bohr radius of 18 nm, and diverse morphologies.5−7 Morphology control4,8,9 is an important parameter to design suitable sustainable semiconducting nanomaterials for different biological applications because the drug carrying ability of biomolecule coated NPs as well as their uniform photophysical properties are entirely related to the shape, size, and monodisperse nature. Several reports on the morphology control of PbS NPs are related to the use of conventional surfactants.10,11 They have little biological applicability because their surfactant coated nature has high affinity to interact with the lipid bilayer of the cell wall. It © 2016 American Chemical Society

Received: May 8, 2016 Revised: October 25, 2016 Published: October 31, 2016 119

DOI: 10.1021/acssuschemeng.6b00991 ACS Sustainable Chem. Eng. 2017, 5, 119−132

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ACS Sustainable Chemistry & Engineering

Figure 1. TEM micrographs of zein coated PbS monodisperse spherical NPs prepared in the presence of 0.1% zein (a) low resolution and (b) high resolution images. Block arrow indicates the zein coating. Similar images c and d for the NPs prepared in the presence of 0.2% zein. Images e and f represent the cubic NPs of PbS prepared in the presence of 0.4% zein. (g) XRD patterns of fcc cubic geometry of PbS NPs. See text for details.

place for bacterial growth. However, Lys functionalized16−18 PbS NPs can be employed to transport drugs through the bloodstream to target the bacterial infections or other site specific therapeutic applications where photophysical properties of PbS allow them to act as markers. DEAE, on the other hand, is a bioactive polymer. Its cellulose counterpart is used in ion exchange chromatography and protein and nucleic acid

model protein which is abundantly available in human tears, saliva, breast milk, and mucus, where it demonstrates its ability to kill bacteria by attacking peptidoglycans through the hydrolysis of glycosidic bonds. Lys cannot be used as a drug because of its large size which causes steric hindrances while traveling across the cells and also have a limited role in eliminating the bacteria from the bloodstream, a most favorite 120


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ACS Sustainable Chemistry & Engineering purification as well as separation.19−22 Its nontoxic nature allows it to be used in oral formulations especially designed to decrease serum cholesterol and triglycerides. Thus, DEAE coated NPs are also expected to have several exciting applications in biological systems. Herein, we are using these industrially important biomolecules in shape control synthesis of one of the most important semiconducting material PbS NPs. This is to highlight a relatively unexplored role of biomolecules in the shape controlled crystal growth of nanomaterials. While focusing on the shape controlled behavior of biomolecules, we also simultaneously demonstrate that the best way to coat the nanomaterials with proteins or other biomolecules is to use them in in situ synthesis where proteins find their way to participate in crystal growth by selectively adsorbing at certain crystal planes of the nucleating centers and hence producing fine coated nanomaterials suitable for their use in the biological systems. Although, there are several studies of the shape control synthesis of semiconducting NPs, the use of proteins and DEAE in achieving the precise shape control morphologies is a step forward in designing new biomaterials. We show some of the remarkable results of bioactive PbS NPs in terms of their unique pH-responsive behavior, shape control synthesis, bioapplicability in systematic circulation, and most importantly their extraction from the aqueous phase by using other industrially important biologically important molecules. The role of environmental friendly ionic liquids in extracting these NPs both in aqueous as well as organic phases simply by choosing an appropriate water insoluble ionic liquid has also been highlighted.

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spotting a concentrated drop of aqueous suspension and dried in a vacuum desiccator. Hemolytic Assay. A hemolytic assay was performed to evaluate the response of biomolecule-conjugated NPs on blood group B of red blood cells (RBCs) from a healthy human donor. Briefly, 5% suspension of RBCs was used for this purpose after giving three washings along with three concentrations (i.e., 25, 50, and 100 μg/ mL) of each NPs sample. One milliliter packed cell volume (i.e., hematocrit) was suspended in 20 mL of 0.01 M phosphate buffered saline (PBS). The positive control was RBCs in water, and it was prepared by spinning 4 mL of 5% RBCs suspension in PBS. PBS as supernatant was discarded, and the pellet was resuspended in 4 mL of water. The negative control was PBS. All of the readings were taken at 540 nm, i.e., absorption maxima of hemoglobin.

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RESULTS AND DISCUSSION

Morphology Control by Zein. In this section, we discuss how appropriately selected biomolecules can have inherent ability to control the shape and size of the growing PbS NPs. Figure 1 shows the morphology control of PbS NPs prepared in the presence of zein. 0.1% (Figure 1a,b) and 0.2% zein (Figure 1c,d) produce fine monodisperse spheres, while 0.4% zein converts the spheres into monodisperse cubes (Figure 1e,f). All NPs are of ∼47 ± 7 nm in size and fully coated with zein (indicated by block arrows in Figure 1b,d,f). XRD patterns (Figure 1g) demonstrate the rock salt crystal structure of NPs with prominent growth at {100} crystal planes. Since all reactions contain equal amount of precursors with identical reaction conditions except the amount of zein (see Experimental Section), the shape transformation from spheres to nanocubes is mainly induced by an increase in the amount of zein from 0.1 to 0.4%. Thus, zein provides colloidal stability as well as control of the crystal growth of PbS nucleating centers. Zein is a predominantly hydrophobic and highly robust protein, while its unfolded state is highly surface active and shows significant surface adsorption that is instrumental in morphology control.23 It happens through the well-known protein seeding process.3 The free surface electrostatically attracts the protein and allows it to surface adsorb, which simultaneously induces unfolding. Unfolded surface adsorbed protein attracts the aqueous solubilized protein through protein−protein interactions and thus triggers the seeding. This process continues until a particular crystal plane which promotes its surface adsorption is passivated. A transformation of fine sphere bound with all {111}, {110}, and {100} crystal planes to a nanocube morphology mainly bound with {100} promotes the zein adsorption on {100} crystal planes, thus passivating them from further participating in the nucleation process. This happens with the increase in the amount of zein from 0.1 to 0.4%. Greater amount of zein not only helps in a better colloidal stabilization but also promotes the seeding due to its fusogenic behavior24,25 with the result that it completely passivates the {100} crystal planes to produce cubic morphology. Preference for the {100} crystal planes arises from the zero dipole moment of PbS which facilitates the nonpolar adsorption of predominantly hydrophobic zein.11 The shape control ability of zein is compared with that of highly hydrophobic twin tail surfactants which are known for their role as morphology control agents11 under identical reaction conditions. The hydrophobicity of twin tail surfactants increases in the order of didodecyldimethylammonium bromide < ditetradecyldimethylammonium bromide < dihexadecyldimethylammonium bromide as the length of the hydrocarbon tail increases from C12 to C16. These surfactants along with

EXPERIMENTAL SECTION

Materials. Chloroauric acid (HAuCl4), zein protein (molar mass 21 kDa), lysozyme (Lys) chicken egg white, diethylaminoethyl dextran chloride (DEAE) (white powder, average molar mass = 500,000, hygroscopic, lot # 39 H1323), lead acetate (99.9%), thioacetamide, (TAA, 98%), acetic acid (99.5%), and sodium dodecyl sulfate (SDS) were purchased from Aldrich. Double distilled water was used for all preparations. Zein was aqueous solubilized by taking a 24 mM SDS solution. Preparation of Bioactive PbS Nanoparticles. In a typical procedure, 10 mL of aqueous biomolecules (zein, Lys, and DEAE 0.1− 0.4%) solution was taken in a round-bottomed glass flask. Under constant stirring, 1 mL of 0.5 M aqueous acetic acid was added. This was followed by the addition of 0.5 mL of 100 mM aqueous lead acetate and 0.5 mL of aqueous 100 mM thioacetamide. After mixing all the components at room temperature, the reaction mixture was kept in a water thermostated bath (Julabo F 25) at precise 70 °C control for 24 h under static conditions. The color of the solution changed from colorless to light orange, then to a pinkish black, and finally it attained a gray-black color within 16 h which indicated the formation of PbS NPs. NPs were collected by centrifuging at 10,000 rpm for 5 min and washed with distilled water at least 2−3 times. These reactions were also simultaneously monitored under the effect of reaction time with the help of UV−visible (Shimadzu-Model No. 2450, double beam) and steady state fluorescence spectroscopy (PTI QuantaMaster) measurements. Both instruments were equipped with a TCC 240A thermoelectrically temperature controlled cell holder that allowed one to measure the spectrum at a constant temperature within ±1 °C. PbS NPs were characterized by a transmission electron microscopic (TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of a solution on a carbon coated Cu grid and allowed to dry in air. The X-ray diffraction (XRD) patterns were characterized by using a Bruker-AXS D8GADDS with Tsec = 480. Samples were prepared on glass slides by 121


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Figure 2. TEM micrographs of Lys coated PbS cubic NPs prepared in the presence of 0.4% (a), 0.6% (b), 0.8% (c), and 1% (d) Lys. High resolution images of crystal planes is shown in e with its corresponding selected area diffraction image f. See text for details.

produces fine monodisperse spherical PbS NPs of ∼150 nm in size (Figure S1). Their shape distorts in the presence of ditetradecyldimethylammonium bromide (C14) + CTAB mixed micelles (Figure S2) and transforms into roughly cubic morphologies for didodecyldimethylammonium bromide + CTAB mixed micelles (Figure S3). This sequence though is similar to that of Figure 1 where increasing amount of zein is used as a shape directing agent instead of surfactants; zein

cetyltrimethylammonium bromide (CTAB) are used for the synthesis of PbS NPs under identical reaction conditions. A combination of twin tail surfactants with CTAB provides a strong mixed micellar hydrophobic environment for the morphology control of growing PbS nucleating centers. TEM images of PbS NPs thus synthesized are shown in the Supporting Information (Figures S1−S3). A combination of dihexadecyldimethylammonium bromide (C16) with CTAB 122


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Figure 3. TEM micrographs a, b, and c of self-crystallized Lys in roughly cubic geometries. They exist in a chain like arrangement (d) or in a highly aggregated state (e). The TEM micrograph (f) is taken when all self-crystallized Lys roughly cubic morphologies are dismantled under the intense electron beam leaving behind only PbS NPs. (g) UV−visible scan of a reaction of the synthesis of PbS NPs in the presence of Lys and (h) plots of intensity and wavelength shift versus reaction time. See text for details.

zein, but it is highly water-soluble with stronger amphiphilicity. Because of the predominantly amphiphilic nature, it promotes the smaller Lys coated NPs to self-aggregate in small groups when the amount of Lys is increased.28 High resolution (Figure 2e) and selected area diffraction images (SAED) (Figure 2f) show that small NPs are bound with {100} crystal planes, which means that the surface preference of Lys is also {100} crystal planes just like that of zein. However, the lack of preserving monodisperse nanocube morphologies with greater amount of Lys indicates its lack of fusogenic behavior which is prominently demonstrated by zein because of its hydrophobic nature. Interestingly, all concentrations of Lys also crystallize29−32 it in monodisperse protein nanocubes of identical shape and size to that of PbS nanocubes (Figure 3a−c). This happens when the sample is placed for aging over a period of a month. Lys is known to form protein crystals of different shapes and

demonstrates much better shape directing ability. That is, NPs are much smaller and nearly monodisperse over 0.1−0.4% zein, while they are only so for the C16 + CTAB combination (Figure S1). The better shape controlling ability of zein in comparison to highly hydrophobic surfactants arises from its macromolecular nature demonstrates better surface coverage and passivation of crystal planes during the growth process. Morphology Control by Lysozyme, and Its SelfCrystallization in Nanocubes. Lys, on the other hand, also produces fine PbS nanocubes of ∼50 nm (Figure 2a). However, when the amount of Lys is increased from 0.4 to 0.6% (Figure 2b), dendritic cubic morphologies start to emerge. They become quite prominent when 0.8% of Lys is used (Figure 2c) and eventually convert into star shaped morphologies at 1% of Lys. In fact, latter morphologies emerge from the selfaggregation and fusion among the smaller Lys coated NPs. Lys is also a highly surface active protein26,27 just like that of 123


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Figure 4. TEM micrographs of DEAE coated PbS NPs prepared in the presence of 0.4% (a and b), 0.8% (c), and 1.6% (d) DEAE. Block arrow in d indicates the DEAE coating. (e) UV−visible scan of a reaction of the synthesis of PbS NPs in the presence of DEAE and (f) plots of intensity and wavelength shift versus reaction time. See text for details.

sizes,29−32 but the formation of nearly monodisperse nanocubes of Lys arises from the catalytic and templating effect of PbS nanocubes. It seems that the predominantly amphiphilic nature of Lys is instrumental in its self-aggregation in the form of nanocubes and PbS nanocubes provide the templating effect for this process. Figure 3d shows both Lys and PbS nanocubes in a chain like arrangement where one can easily differentiate the transparent Lys nanocubes (indicated by block arrows) from dark metallic PbS nanocubes. Figure 3e shows the large presence of both particles. However, under the effect of an intense electron beam during the TEM measurement, Lys

nanocubes melt away and get separated (as shown in Figure 3c) leaving behind the chain like arrangement of only PbS nanocubes (Figure 3f) with interesting UV−visible behavior. A time dependent UV−visible scan of a reaction (Figure 3g) demonstrates an emergence of a broad band around 580 nm (indicated by an arrow) which red shifts and becomes prominent with the passage of time. Its intensity and wavelength variation with time is depicted in Figure 3h. Both quantities increase with almost equal contribution during the reaction due to the time dependent self-aggregation in a chain like arrangement. 124


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Figure 5. UV−visible plots of zein coated PbS NPs prepared in the presence of 0.4% zein with respect to change in the pH. Sample photos show a dramatic change in the color of the sample with the change in pH. Inset, intensity and wavelength shift versus pH plots. (b) Fluorescence intensity versus pH plots for the PbS NPs samples prepared with different amounts of zein. Inset, fluorescence spectra of a zein coated PbS NP sample with change in pH of the medium. See text for details.

Morphology Control by DEAE. Apart from the abovementioned proteins, DEAE, a cationic polysaccharide has already been used as a shape directing agent in the synthesis of fine plate like gold NPs.33 It stabilizes and controls the crystal growth of PbS NPs to some extent but does not produce fine nanocubes as observed for zein and Lys. Its 0.4% mainly produces roughly cubic morphologies (Figure 4a) which are present in the form of a self-assembled state of small groups (Figure 4b). Further increase in its amount to 0.8 (Figure 4c) and 1.6% (Figure 4d) does not impart appreciable change in the morphology except a large amount of polymer coating (see

block arrow) which engulfs several NPs in large groups. A UV− visible scan (Figure 4e) of such a reaction depicts an emerging broad absorbance around 420 nm which red-shifts and becomes prominent with the passage of time just like that of Figure 3g. This absorbance represents the small aggregated state of PbS NPs in a similar manner as happened in the presence of Lys. Figure 4f shows the variation of the intensity and wavelength almost in a similar manner as observed in Figure 3h. A large red shift of about 300 nm in Figure 3h in comparison to around 70 nm in Figure 4f is due to the greater extent of self-aggregation in the former rather than in the latter case. Thus, DEAE also 125


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Figure 6. (a) UV−visible plots of Lys coated PbS NPs prepared in the presence of 0.6% zein with respect to change in the pH. (b) Intensity and wavelength shift versus pH plots. (c) Fluorescence spectra of a Lys coated PbS NPs sample with change in pH of the medium. (d) Fluorescence intensity versus pH plots for the PbS NP samples prepared with different amounts of Lys. See text for details.

induces the time dependent self-aggregation though it is simply in the random groups rather than in a chain like arrangement as observed for Lys. pH Effect. Self-assembled arrangement of PbS NPs in the presence of zein, Lys, and DEAE demonstrates a remarkable pH effect,34,35 which is clearly shown by the UV−visible and fluorescence measurements. Figure 5a depicts the dependence of UV−visible absorbance of purified zein coated NPs on pH. At pH 2.55, PbS NPs produce weak absorbance close to 310 nm which red shifts, and its intensity increases (indicated by the

black arrows) with the further increase in pH. Simultaneously, the color of the PbS NPs suspension also changes from light gray to light yellow-brown, and eventually dark brown at pH 11.5 (see sample photos). The variation in the intensity and wavelength with pH is illustrated in the inset where a large increase in the intensity and wavelength occurs around pH 10. Likewise, the fluorescence emission spectra of the same reaction (inset) and the variation of its intensity is depicted in Figure 5b. In all cases, intensity starts to rise from pH 4, remains prominent between pH 5−9, and then diminishes at 126


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Figure 7. (a) Molecular structure of DEAE. (b) UV−visible plots of DEAE coated PbS NPs prepared in the presence of 0.8% DEAE with respect to change in the pH. Inset, intensity and wavelength shift versus pH plots. (c) Fluorescence intensity versus pH plots for the PbS NPs samples prepared with different amounts of DEAE. Inset, fluorescence spectra of a DEAE coated PbS NP sample with change in pH of the medium. See text for details.

fusogenic behavior36 of zein due to the screening of surface negative charge (zein is solubilized in aqueous SDS). Increase in the pH reduces the charge screening and increases the Columbic repulsions with the result that NPs start to disperse and show increasing absorbance. As more and more NPs get

pH 10. This is the region (pH 5−9) where color transformation from light gray to light yellow-brown occurs, which eventually converts into dark brown. At pH 2.55 (acetic acid reaction medium, see Experimental Section), zein coated NPs exist in self-aggregated state (Figure 1), which is prompted by the 127


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Figure 8. (a, b, and c) Typical UV−visible profiles of heme absorption of different doses of zein coated PbS NPs prepared in the presence of 0.4% zein, 0.8% DEAE, and 0.6% Lys, respectively, along with positive and negative controls. Photos represent the sample tubes with positive control, negative control, and 25, 50, and 100 μg/mL of zein coated PbS NPs. (d, e, and f) Corresponding plots of hemolysis % versus the dose of zein, DEAE, and Lys coated PbS NPs, respectively. See details in the text.

quenched at pH 2.55. The rise in pH brings deaggregation of

free, their absorbance increases, and a highly basic pH 10 reduces the self-aggregation to a minimum and produces maximum absorbance with dark brown color (Figure 5a). Fluorescence emission (Figure 5b) supplements this information. The emission of PbS NPs of ∼47 nm in aggregated state is

NPs with the result that their emission increases. However, at pH 10 where maximum dispersal and minimum aggregation of NPs occurs, the radiative decay converts into nonradiative 128


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Figure 9. (a) UV−visible plot of Lys coated NPs suspension (100 μg/mL) with alginic acid. Sample photo shows the degree of aggregation among the Lys coated PbS NPs upon complexation with alginic acid. (b) Plots of intensity versus the amount of alginic acid, DNA, carboxymethyl cellulose, and xanthan gum. Table 1, complexation concentration required for the extraction of PbS NPs (100 μg/mL) in each case. NA refers to the reaction in which no complexation occurred. (c) Demonstrates the extraction of PbS NPs in organic phase by using 100 mM IL. Concentrated suspension of Lys and zein coated PbS NPs were taken to show the color contrast between the aqueous and organic layers, and hence, 100 mM IL in the organic layer was insufficient to transfer all PbS NPs in a single extraction cycle.

decay due to increasing interparticle collisions that reduces the emission to a minimum. Similar pH dependent behavior of Lys coated PbS NPs is observed. Figure 6a shows the variation in the absorbance of Lys coated PbS NPs. It demonstrates a dramatic dependence on the pH.37 Lys coated PbS NPs in aggregated state show weak absorbance around 310 nm that becomes prominent and much broader and shifts in the visible region with the rise in pH. It is also much more prominent and significant in comparison to that of Figure 5a. A rise in pH brings a rapid change in the color from light gray to dark brown from pH 4 to 8 (Figure 6a, inset) as observed previously for zein coated NPs

(Figure 5) which is due to a much greater extent of unfolding of Lys in the acidic environment38 in comparison to predominantly hydrophobic zein. A fully unfolded state of Lys is responsible for the chain like arrangement of NPs as shown in Figure 3f. This arrangement dismantles with the rise in pH (Figure 6a) and is demonstrated by the variation in intensity and wavelength plots (Figure 6b). Florescence emission spectra of the same experiment are shown in Figure 6c, and their variation in the intensity and wavelength with pH are presented in Figure 6d. A dramatic rise in the emission intensity along with a red shift of ∼17 nm occurs in a narrow pH range of 5.5− 129


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NPs through different modes of interactions. Lys coated NPs show strong complexing ability with all bioactive molecules, while DEAE and zein coated NPs are quite specific. Figure 9a shows a simple titration of purified Lys coated NPs with aqueous solution of alginic acid which leads to a coagulation of the NPs at a certain concentration (see the sample photos before and after the titration with alginic acid). The onset of the fall in the intensity of PbS NPs indicates the loading ability of Lys coated NPs. Similar behavior is observed when Lys coated NPs are titrated with carboxy methyl cellulose, xanthum gum, and DNA. A plot of intensity (around 620 nm) versus concentration (Figure 9b) depicts quantitative amount of bioactive molecules required for the complexation. In each case except xanthan gum, the intensity of NPs initially increases upon complexation with bioactive molecules. It is due to an enhancement in the colloidal stability of the NPs.18 However, after a certain concentration, NPs start coagulating (indicated by an arrow in Figure 9b) due to the charge neutralization between predominantly positive charged Lys coated NPs (Lys isoelectric point ∼11) and anionic alginic acid, carboxymethyl cellulose, or DNA. In the case of xanthan gum, no initial increase in the intensity of NPs is observed; rather, it shows a slight decrease and eventually drops at a certain concentration. Xanthan gum47 undergoes extensive hydrogen bonding in aqueous phase that leads to a helical structure which may entrap Lys coated NPs and leads to the aggregation of the NPs. Although, its scaffolding structure makes xanthan gum an excellent suspending agent, in the present case, it induces strong coagulation mainly due to its complexation with Lys coated NPs. Extraction concentration of the additive molecules is listed in Table 1 (Figure 9) for DEAE, Lys, and zein coated PbS NPs. Xanthan gum induces an instant aggregation of DEAE coated NPs which demonstrates a strong affinity between DEAE and xanthan gum because of the strong electrostatic interactions between cationic DEAE and anionic xanthan gum. The same is true for alginic acid, while little interactions of DEAE coated NPs are observed with CMC and DNA. Zein coated NPs demonstrate the loading ability of DNA only, which can be attributed to the electrostatic interactions between the surface amino acids with DNA. Extraction of NPs can also be achieved by using an ionic liquid48,49 such as 1-butyl-3-methyl imidazolium hexafluorophosphate (IL1), a well studied and water insoluble IL. By using the IL, the extraction can be done in both organic as well as aqueous phases, i.e., some amount of the NPs move from the aqueous to organic layer, while IL moves in the opposite direction and complexes with DEAE, Lys, or zein. In both ways, we can extract the NPs in the organic as well as in the aqueous phases. Figure 9c shows the photographs of one sample of each DEAE, Lys, and zein coated NPs in aqueous phase along with the 100 mM IL in the toluene phase in the respective first tube before mixing. The photographs of the respective second tubes are taken after shaking and mixing both phases for 30 min and leaving them standing overnight. DEAE coated NPs show a complete transfer from the aqueous to organic phase, while it is partial for both Lys and zein coated NPs samples. Interestingly, if we perform another extraction with the leftover samples especially in the case of Lys and zein coated NPs or take double the amount of IL (200 mM), a complete separation of the NPs takes place in the aqueous phase where all NPs settle at the bottom of the tube due to the loss of colloidal stability. It happens because of the transfer of IL from the organic to aqueous phase leading to a much stronger complexation

7.7 and extends to a broader pH range when a larger amount of Lys is used. Low pH drastically dehydrates sugars,39,40 and hence, DEAE is also expected to be highly pronated at low pH (Figure 7a). Thus, a strongly cationic DEAE with a relatively nonpolar backbone provides a strong amphiphilic nature that drives DEAE coated PbS NPs to self-assemble.41,42 As pH rises, DEAE slowly gets deprotonated and hence induces the deaggregation. This is well depicted in Figure 7b where a significant increase in the intensity and a red shift are observed with pH. A sharp variation in intensity and wavelength (Figure 7b, inset) occurs around pH 6 that brings a color change from light gray to dark brown. A complementary fluorescence scan produces significant emission dependence on pH which is also related to the amount of DEAE (Figure 7c). A greater amount of DEAE, i.e., 1.6% requires a wider pH range for deaggregation and hence produces emission over a wider pH range. Hemolysis and Bioapplicability. Coated NPs in comparison to the naked ones are always considered to be the best suited vehicles for drug delivery in systemic circulation.43,44 This is demonstrated by performing the hemolytic analyses of the present PbS NPs. Figure 8 shows some of the representative UV−visible plots of heme absorption within 500 to 600 nm of wavelength range with positive and negative controls. Zein coated PbS NPs induce hemolysis (Figure 8a) that increases with the increase in the dose amount from 25 to 100 μg/mL, while Lys (Figure 8b) and DEAE (Figure 8c) coated NPs show almost insignificant hemolysis. The percentage hemolysis = [(sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100] evaluated from the UV−visible spectra for different samples is compared in the corresponding bar graphs (Figure 8d−f). Hemolysis occurs when the free NP surface (uncoated) interacts with the various components of the blood cell membrane through various forms of interactions viz. electrostatic, nonelectrostatic, and hydrogen bonding.43,44 Coated NPs usually do not induce hemolysis because complete passivation of the NPs surface is achieved by coated molecules.12,18 However, if coated molecules have the ability to disrupt the blood cell membrane, hemolysis cannot be avoided. That is why conventional surfactant coated NPs have high potential to induce significant hemolysis.18 However, predominantly hydrophobic zein also interacts with the blood cell wall through its unfolded hydrophobic domains. The blood cell membrane consists of three layers with glycocalyx on the exterior, protein network on the anterior, and lipid bilayer in between the two. Glycoprotein45 and lipid bilayer46 are highly susceptible to complexation due to the predominant hydrophobic interactions of hydrophobic domains that cause the rupturing of blood cells. Such interactions are expected to be minimized for the Lys and DEAE coated NPs because of their predominantly amphiphilic nature. Several coated layers of Lys and DEAE help in the reduction of surface charge, thereby diminishing the probability to interact with the blood cell membrane through electrostatic interactions. Thus, Lys and DEAE coated PbS NPs can act as promising drug delivery vehicles in the systemic circulation in comparison to zein coated NPs. Bioextraction of Nanoparticles. In order to demonstrate the drug loading and complexing ability of the present PbS NPs, a series of other industrially important bioactive molecules (such as alginic acid, carboxymethyl cellulose, xanthan gum, and DNA) have been selected which can interact with coated PbS 130


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Janshoff, A. Cytotoxicity of Metal and Semiconductor Nanoparticles Indicated by Cellular Micromotility. ACS Nano 2009, 3, 213−222. (3) Bakshi, M. S. Nanoshape Control Tendency of Phospholipids and Proteins: Protein−Nanoparticle Composites, Seeding, SelfAggregation, and Their Applications in Bionanotechnology and Nanotoxicology. J. Phys. Chem. C 2011, 115, 13947−13960. (4) Bakshi, M. S. How Surfactants Control Crystal Growth of Nanomaterials. Cryst. Growth Des. 2016, 16, 1104−1133. (5) Khan, A. H.; Thupakula, U.; Dalui, A.; Maji, S.; Debangshi, A.; Acharya, S. Evolution of Long Range Bandgap Tunable Lead Sulfide Nanocrystals With Photovoltaic Properties. J. Phys. Chem. C 2013, 117, 7934−7939. (6) Fan, D.; Thomas, P. J.; O’Brien, P. Pyramidal Lead Sulfide Crystallites With High Energy {113} Facets. J. Am. Chem. Soc. 2008, 130, 10892−10894. (7) Zhang, F.; Wong, S. S. Controlled Synthesis of Semiconducting Metal Sulfide Nanowires. Chem. Mater. 2009, 21, 4541−4554. (8) Peng, Z.; Jiang, Y.; Song, Y.; Wang, C.; Zhang, H. Morphology Control of Nanoscale PbS Particles in a Polyol Process. Chem. Mater. 2008, 20, 3153−3162. (9) Li, W.; Zamani, R.; Ibanez, M.; Cadavid, D.; Shavel, A.; Morante, J. R.; Arbiol, J.; Cabot, A. Metal Ions To Control the Morphology of Semiconductor Nanoparticles: Copper Selenide Nanocubes. J. Am. Chem. Soc. 2013, 135, 4664−4667. (10) Bakshi, M. S.; Kaur, G.; Possmayer, F.; Petersen, N. O. ShapeControlled Synthesis of Poly(Styrene Sulfonate) and Poly(Vinyl Pyrolidone) Capped Lead Sulfide Nanocubes, Bars, and Threads. J. Phys. Chem. C 2008, 112, 4948−4953. (11) Bakshi, M. S.; Thakur, P.; Sachar, S.; Kaur, G.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. Aqueous Phase Surfactant Selective Shape Controlled Synthesis of Lead Sulfide Nanocrystals. J. Phys. Chem. C 2007, 111, 18087−18098. (12) Khullar, P.; Singh, V.; Mahal, A.; Dave, P. N.; Thakur, S.; Kaur, G.; Singh, J.; Singh Kamboj, S.; Singh Bakshi, M. Bovine Serum Albumin Bioconjugated Gold Nanoparticles: Synthesis, Hemolysis, and Cytotoxicity Toward Cancer Cell Lines. J. Phys. Chem. C 2012, 116, 8834−8843. (13) Argos, P.; Pedersen, K.; Marks, M. D.; Larkins, B. A. A structural model for maize zein proteins. J. Biol. Chem. 1982, 257, 9984−9990. (14) Shukla, R.; Cheryan, M. Zein: The industrial protein from corn. Ind. Crops Prod. 2001, 13, 171−192. (15) Momany, F. A.; Sessa, D. J.; Lawton, J. W.; Selling, G. W.; Hamaker, S. A. H.; Willett, J. Structural Characterization of α-Zein. J. Agric. Food Chem. 2006, 54, 543−547. (16) Bamrungsap, S.; Shukoor, M. I.; Chen, T.; Sefah, K.; Tan, W. Detection of Lysozyme Magnetic Relaxation Switches Based on Aptamer-Functionalized Superparamagnetic Nanoparticles. Anal. Chem. 2011, 83, 7795−7799. (17) Kao, K. C.; Lin, T. S.; Mou, C. Y. Enhanced Activity and Stability of Lysozyme by Immobilization in the Matching Nanochannels of Mesoporous Silica Nanoparticles. J. Phys. Chem. C 2014, 118, 6734−6743. (18) Goshisht, M. K.; Moudgil, L.; Rani, M.; Khullar, P.; Singh, G.; Kumar, H.; Singh, N.; Kaur, G.; Bakshi, M. S. Lysozyme Complexes for the Synthesis of Functionalized Biomaterials To Understand Protein−Protein Interactions and Their Biological Applications. J. Phys. Chem. C 2014, 118, 28207−28219. (19) Raksakulthai, R.; Haard, N. F. Purification and characterization of a Carboxypeptidase from Squid Hepatopancreas (Illex illecebrosus). J. Agric. Food Chem. 2001, 49, 5019−5030. (20) Dorsey, J. G.; Cooper, W. T.; Siles, B. A.; Foley, J. P.; Barth, H. G. Liquid chromatography:theory and methodology. Anal. Chem. 1998, 70, 591−644. (21) Larive, C. K.; Lunte, S. M.; Zhong, M.; Perkins, M. D.; Wilson, G. S.; Gokulrangan, G.; Williams, T.; Afroz, F.; Schoneich, C.; Derrick, T. S.; Middaugh, C. R.; Bogdanowich-Knipp, S. Separation and analysis of peptides and proteins. Anal. Chem. 1999, 71, 389−423.

between the protein and IL than the NPs surface adsorption of protein. In this way, protein leaves the NPs coating and complexes with IL with the result NPs lose their colloidal stability. Thus, IL provides a dual advantage of extracting NPs in both the aqueous as well as the organic phase.

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CONCLUDING REMARKS From the present results, we have demonstrated that appropriately selected proteins like zein and Lys can act as excellent shape directing agents of PbS NPs. The morphology control of zein is even better in terms of the monodisperse nature and small size of NPs in comparison to highly hydrophobic double tail conventional surfactants which are known for their precise shape control ability of nanomaterials. The biomolecule coated PbS NPs thus obtained are highly pH responsive and demonstrate strong color change from light gray to dark brown when the pH is increased from low to high value. pH responsive color change is accomplished by pH induced change in the aggregation behavior of NPs and has been demonstrated by the UV−visible and fluorescence spectroscopy. Insignificant hemolytic response of especially Lys and DEAE coated PbS NPs allows for their bioapplicability as fluorescence marker in the systemic circulation. Industrial scale accessibility can be achieved through an efficient extraction process. Lys and DEAE coated PbS NPs are easily extracted by using complexing agents like alginic acid and xanthum gum in aqueous phase, while zein coated NPs are extracted by using DNA. However, water insoluble ionic liquids are more versatile extracting agents because they can even extract the NPs in the organic phase due to their complexing ability with zein, Lys, and DEAE. Thus, IL helps to extract the NPs both in the organic as well as aqueous phases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b00991. TEM images (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(P.K.) E mail: [email protected]. *(M.S.B) E mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS These studies were partially supported by the financial assistance from DST under nanomission research project [ref no: SR/NM/NS-1057/2015(G)], New Delhi. Dr. Gurinder Kaur thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA.

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REFERENCES

(1) Erathodiyil, N.; Ying, J. Y. Functionalization of Inorganic Nanoparticles for Bioimaging Applications. Acc. Chem. Res. 2011, 44, 925−935. (2) Tarantola, M.; Schneider, D.; Sunnick, E.; Adam, H.; Pierrat, S.; Rosman, C.; Breus, V.; Sonnichsen, C.; Basche, T.; Wegener, J.; 131


Research Article

ACS Sustainable Chemistry & Engineering (22) Ghimici, L.; Constantin, M.; Fundueanu, G. Novel biodegradable flocculanting agents based on pullulan. J. Hazard. Mater. 2010, 181, 351−358. (23) Mahal, A.; Khullar, P.; Kumar, H.; Kaur, G.; Singh, N.; Jelokhani-Niaraki, M.; Bakshi, M. S. Green Chemistry of Zein Protein Toward the Synthesis of Bioconjugated Nanoparticles: Understanding Unfolding, Fusogenic Behavior, and Hemolysis. ACS Sustainable Chem. Eng. 2013, 1, 627−639. (24) Ryan, M. A.; Qi, X.; Serrano, A. G.; Ikegami, M.; Perez-Gil, J.; Johansson, J.; Weaver, T. E. Mapping and Analysis of the Lytic and Fusogenic Domains of Surfactant Protein B. Biochemistry 2005, 44, 861−872. (25) Carrica, M. d. C.; Craig, P. O.; Alonso, S. d. V.; Goldbaum, F. A.; Cravero, S. L. Brucella Abortus MFP: A Trimeric Coiled-Coil Protein With Membrane Fusogenic Activity. Biochemistry 2008, 47, 8165−8175. (26) Kubiak-Ossowska, K.; Mulheran, P. A. Mechanism of Hen Egg White Lysozyme Adsorption on a Charged Solid Surface. Langmuir 2010, 26, 15954−15965. (27) Yu, G.; Liu, J.; Zhou, J. Mesoscopic Coarse-Grained Simulations of Lysozyme Adsorption. J. Phys. Chem. B 2014, 118, 4451−4460. (28) Bakshi, M. S.; Kaur, H.; Banipal, T. S.; Singh, N.; Kaur, G. Biomineralization of Gold Nanoparticles by Lysozyme and Cytochrome c and Their Applications in Protein Film Formation. Langmuir 2010, 26, 13535−13544. (29) Jones, W. F.; Arnold, M. A.; Wiencek, J. M. PrecipitantControlled Growth of Lysozyme Crystals in Sodium Thiocyanate. Cryst. Growth Des. 2004, 4, 1387−1393. (30) Heijna, M. C. R.; Enckevort, W. J. P.; Vlieg, E. Growth Inhibition of Protein Crystals: A Study of Lysozyme Polymorphs. Cryst. Growth Des. 2008, 8, 270−274. (31) Aldabaibeh, N.; Jones, M. J.; Myerson, A. S.; Ulrich, J. The Solubility of Orthorhombic Lysozyme Crystals Obtained at High PH. Cryst. Growth Des. 2009, 9, 3313−3317. (32) Tsekova, D. S. Formation and Growth of Tetragonal Lysozyme Crystals at Some Boundary Conditions. Cryst. Growth Des. 2009, 9, 1312−1317. (33) Singh, V.; Khullar, P.; Dave, P. N.; Kaur, G.; Bakshi, M. S. Ecofriendly Route To Synthesize Nanomaterials for Biomedical Applications: Bioactive Polymers on Shape-Controlled Effects of Nanomaterials Under Different Reaction Conditions. ACS Sustainable Chem. Eng. 2013, 1, 1417−1431. (34) Kim, J. H.; Lee, T. R. Thermo- and PH-Responsive HydrogelCoated Gold Nanoparticles. Chem. Mater. 2004, 16, 3647−3651. (35) Isojima, T.; Lattuada, M.; Vander Sande, J. B.; Hatton, T. A. Reversible Clustering of PH- and Temperature-Responsive Janus Magnetic Nanoparticles. ACS Nano 2008, 2, 1799−1806. (36) Navdeep; Banipal, T. S.; Kaur, G.; Bakshi, M. S. Nanoparticle Surface Specific Adsorption of Zein and Its Self-Assembled Behavior of Nanocubes Formation in Relation to On−Off SERS: Understanding Morphology Control of Protein Aggregates. J. Agric. Food Chem. 2016, 64, 596−607. (37) Ghimire, A.; Kasi, R. M.; Kumar, C. V. Proton-Coupled Protein Binding: Controlling Lysozyme/Poly(Acrylic Acid) Interactions With PH. J. Phys. Chem. B 2014, 118, 5026−5033. (38) Raccosta, S.; Martorana, V.; Manno, M. Thermodynamic Versus Conformational Metastability in Fibril-Forming Lysozyme Solutions. J. Phys. Chem. B 2012, 116, 12078−12087. (39) Roudsari, M.; Nakamura, A.; Smith, A.; Corredig, M. Stabilizing Behavior of Soy Soluble Polysaccharide or High Methoxyl Pectin in Soy Protein Isolate Emulsions at Low PH. J. Agric. Food Chem. 2006, 54, 1434−1441. (40) Kim, J. W.; Mazza, G. Extraction and Separation of Carbohydrates and Phenolic Compounds in Flax Shives With PHControlled Pressurized Low Polarity Water. J. Agric. Food Chem. 2009, 57, 1805−1813. (41) Lee, S.; Perez-Luna, V. c. H. Dextran−Gold Nanoparticle Hybrid Material for Biomolecule Immobilization and Detection. Anal. Chem. 2005, 77, 7204−7211.

(42) Dai, Q.; Zhu, X.; Abbas, S.; Karangwa, E.; Zhang, X.; Xia, S.; Feng, B.; Jia, C. Stable Nanoparticles Prepared by Heating Electrostatic Complexes of Whey Protein Isolate−Dextran Conjugate and Chondroitin Sulfate. J. Agric. Food Chem. 2015, 63, 4179−4189. (43) Lin, Y. S.; Haynes, C. L. Impacts of mesoporous silica nanoparticle size, pore ordering, and pore integrity on hemolytic activity. J. Am. Chem. Soc. 2010, 132, 4834−4842. (44) Dobrovolskaia, M. A.; Clogston, J. D.; Neun, B. W.; Hall, J. B.; Patri, A. K.; McNeil, S. E. Method for analysis of nanoparticle hemolytic properties in vitro. Nano Lett. 2008, 8, 2180−2187. (45) Derjaguin, B.; Landau, L. Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes. Prog. Surf. Sci. 1993, 43, 30−59. (46) Dedinaite, A.; Lundin, M.; Macakova, L.; Auletta, T. Mucinchitosan complexes at the solid-liquid interface: multilayer formation and stability in surfactant solutions. Langmuir 2005, 21, 9502−9509. (47) Kocherbitov, V.; Ulvenlund, S.; Briggner, L. E.; Kober, M.; Arnebrant, T. Hydration of a Natural Polyelectrolyte Xanthan Gum: Comparison With Non-Ionic Carbohydrates. Carbohydr. Polym. 2010, 82, 284−290. (48) Cao, Y.; Xing, H.; Yang, Q.; Bao, Z.; Su, B.; Yang, Y.; Ren, Q. Separation of Soybean Isoflavone Aglycone Homologues by Ionic Liquid-Based Extraction. J. Agric. Food Chem. 2012, 60, 3432−3440. (49) Seitkalieva, M. M.; Kachala, V. V.; Egorova, K. S.; Ananikov, V. P. Molecular Extraction of Peptides in Ionic Liquid Systems. ACS Sustainable Chem. Eng. 2015, 3, 357−364.

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pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein

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