pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein

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pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein Induced Morphology Control, Bioapplicability, and Bioextraction of Nanomaterials Aabroo Mahal, Lavanya Tandon, Poonam Khullar, Gurinder K. Ahluwalia, and Mandeep Singh Bakshi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00991 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 7, 2016

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ACS Sustainable Chemistry & Engineering

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pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein Induced Morphology Control, Bioapplicability, and Bioextraction of Nanomaterials

Aabroo Mahal3,4, Lavanya Tandon3, Poonam Khullar3*, Gurinder Kaur Ahluwalia2, Mandeep Singh Bakshi1* 1

Department of Natural and Applied Sciences, University of Wisconsin - Green Bay, 2420

Nicolet Drive, Green Bay, WI 54311-7001, USA. 2Materials & Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7 Canada. 3Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India.

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

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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 grey (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. Key words: pH responsive lead sulfide nanoparticles, bioactive, hemolysis, bioextraction.

Introduction 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 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 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


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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 cell wall. It 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 environmental friendly, clear, odorless, tasteless, and edible nature. Its highly robust structure made up of nine homologous repeat units arranged in an anti-parallel distorted cylindrical form and stabilized by the hydrogen bonds13-15 makes it highly stable protein. Because of its non-toxic nature, zein coated PbS NPs can find their applications in biological systems. Similarly, Lys is an important 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 the steric hindrances while travelling across the cells and also have a limited role in eliminating the bacteria from the blood stream a most favourite place for the bacterial growth. However, Lys functionalized16-18 PbS NPs can be employed to transport drugs through the blood stream 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, protein and nucleic acid purification as well as separation.19-22 Its non-toxic nature allows it to use in oral formulations especially designed to decrease serum cholesterol and triglycerides. Thus, DEAE coated NPs are also expected to have several exciting applications in the biological systems. Herein, we are using these industrial important biomolecules in shape control synthesis of one of the most important semiconducting material PbS NPs. This is to highlight a relatively much unexplored role of biomolecules in the shape controlled crystal growth of nanomaterials.



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While focussing 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 the in situ synthesis where proteins find their way to participate in the crystal growth by selectively adsorbing at certain crystal planes of the nucleating centres 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. Experimental 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 dodecylsulfate (SDS) were purchased from Aldrich. Double distilled water was used for all preparations. Zein was aqueous solubilized by taking 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 bottom 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 oC control for 24 hours under static conditions. The color of the solution changed from colorless to light orange, then to a pinkish black, and finally it attained a grey-black color within 16 hours which indicated the formation of PbS NPs. NPs were collected by centrifuging at 10,000 rpm for 5 minutes and washed with distilled water at least 2-3 times. These reactions were also simultaneously monitored under the


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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 to measure the spectrum at a constant temperature within ± 1 oC. PbS NPs were characterized by 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 the air. The x-ray diffraction (XRD) patterns were characterized by using a Bruker-AXS D8-GADDS with Tsec = 480. Samples were prepared on glass slides by spotting a concentrated drop of aqueous suspension and dried in a vacuum desiccator. Hemolytic assay: Hemolytic assay was performed to evaluate the response of biomoleculeconjugated 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. 1 ml 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 pellet was resuspended in 4 ml of water. The negative control was PBS. All the readings were taken at 540 nm i.e. absorption maxima of hemoglobin. Results and discussion Morphology control by zein In this section, we discuss that how appropriately selected biomolecules can have inherent ability to control the shape and size of the growing PbS NPs. Fig 1 shows the morphology control of PbS NPs prepared in the presence of zein. 0.1 % (Fig 1a, b) and 0.2 % zein (Fig 1c, d) produce fine monodisperse spheres while 0.4 % zein converts the spheres into monodisperse cubes (Fig 1e, f). All NPs are of ~ 47 ± 7 nm in size and fully coated with zein (indicated by block arrows in Fig 1b,d,f). XRD patterns (Fig 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



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experimental), 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 controls the crystal growth of PbS nucleating centres. Zein is predominantly hydrophobic and highly robust protein while its unfolded state is highly surface active and show significant surface adsorption that is instrumental in the morphology control.23 It happens through the wellknown protein seeding process.3 Free surface electrostatically attracts the protein and allows it to surface adsorb that simultaneously induces the unfolding. Unfolded surface adsorb protein attracts the aqueous solubilized protein through protein – protein interactions and hence, triggers the seeding. This process continues till 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, it also promotes the seeding due to its fusogenic behavior24,25 with the result 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 non-polar 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

pH Responsive Bioactive Lead Sulfide Nanomaterials: Protein

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