biopolymer photoluminescent nanoprobes for bioimaging brain cancer cells

Materials Chemistry and Physics 244 (2020) 122716

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Green synthesis of ZnS quantum dot/biopolymer photoluminescent nanoprobes for bioimaging brain cancer cells Anderson J. Caires a, Alexandra A.P. Mansur a, Isadora C. Carvalho a, Sandhra M. Carvalho a, b, Herman S. Mansur a, * a b

Center of Nanoscience, Nanotechnology and Innovation - CeNano2I, Federal University of Minas Gerais - UFMG, Av. Ant^ onio Carlos, 6627, Belo Horizonte, MG, Brazil Department of Preventive Veterinary Medicine, Veterinary School, Federal University of Minas Gerais - UFMG, Brazil

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

� Facile green synthesis of ZnS quantum dot-carboxymethylcellulose nano­ conjugates. (79). � CMC pH-sensitive ligand tailored nucleation/growth processes of ZnS nanocrystals. (80). � Colloidal process parameters regulated the optical properties of ZnS@CMC nanohybrids. (84). � ZnS@CMC colloids behaved as active photoluminescent biological nanop­ robes. (73). � Fluorescent nanoconjugates were effec­ tive for bioimaging brain cancer cells in vitro. (84).

A R T I C L E I N F O

A B S T R A C T

Keywords: Fluorescent nanoprobes Luminescent nanoconjugates Green nanotechnology ZnS quantum dots Luminescent nanomaterials Cancer bioimaging

Semiconductor quantum dots (QDs) are one of the most interesting photoluminescent nanomaterials with very promising applications in cancer nanomedicine. In this work, ZnS fluorescent quantum dots (ZnS-QDs) were synthesized and stabilized by carboxymethylcellulose (CMC) as a pH-sensitive biopolymer using a facile one-step green aqueous colloidal process at distinct pH conditions (acidic, neutral and alkaline) and chemical proportions of precursors (Zn2þ, S2 ). The optical properties of these nanoconjugates (ZnS@CMC) were characterized by UV–visible and photoluminescence spectroscopy. The morphological features and physicochemical properties were evaluated by TEM, FTIR spectroscopy, zeta potential, and dynamic light scattering (DLS) analyses. The cytocompatibility in vitro of ZnS@CMC was assessed by MTT assay using normal and malignant glioma cells. The UV–Vis results indicated that ZnS-QDs were effectively produced with different bandgap energies (from 4.5 to 3.8 eV) blue-shifted from bulk (Ebulk ¼ 3.61 eV), and sizes (typically from 3.3 to 4.5 nm), dependent on the pH and concentration ratio of precursors during the synthesis. Analogously, the changes of synthesis parameters significantly altered the photoluminescence emission energies and intensities within the visible range of spectra (PL maxima from λ ¼ 400–430 nm, at pH ¼ 3.5, [Zn:S] ratio ¼ 1:2). The cell viability results in vitro (>90%) demonstrated no cytotoxicity of ZnS@CMC nanohybrids towards both cell types. Importantly, these ZnS@CMC

* Corresponding author. Federal University of Minas Gerais, Av. Ant^ onio Carlos, 6627, Escola de Engenharia, Bloco 2 – Sala 2233, 31.270-901, Belo Horizonte, MG, Brazil. E-mail address: [email protected] (H.S. Mansur). https://doi.org/10.1016/j.matchemphys.2020.122716 Received 7 July 2019; Received in revised form 6 December 2019; Accepted 22 January 2020 Available online 23 January 2020 0254-0584/© 2020 Elsevier B.V. All rights reserved.

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nanoconjugates behaved as active fluorescent nanoprobes for bioimaging malignant glioma cells proving the high potential for applications in cancer nanomedicine.

1. Introduction

Advances in the early diagnosis of cancer and prompt treatment are essential to a better prognosis and to improve the quality of patients’ life. To this end, bioimaging of cancer cells is among the most efficient methods for the detection and confirmation of the cancer diagnosis. Nowadays, the most prominent applications of quantum dots are asso­ ciated with bioimaging of cells and their complex signaling mechanisms for the development of more accurate and reliable techniques for detecting primary tumor cells at the very early stages of the disease [6,7, 24]. To this end, in this work, we report the synthesis and comprehensive characterization of novel ZnS quantum dots stabilized and functional­ ized by carboxymethylcellulose produced via an environmentallyfriendly aqueous colloidal process at room temperature. The depen­ dence of optical properties on the synthesis parameters, i.e., pH of the solution and concentration of precursors, were systematically investi­ gated and interpreted, bearing in mind the colloidal chemistry approach. The results evidenced a close relation between the conditions used in the synthesis on the nucleation and growth processes of ZnS QDs produced, where their sizes directly correlated with their absorption and emission properties. In addition, the cytotoxicity of the novel ZnS@CMC nanoconjugates was investigated towards brain cancer (U-87 MG) and healthy cells (HEK 293T) using MTT in vitro assays, to validate their suitability for biomedical applications. Importantly, as a proof of concept, these ZnS@CMC nanoconjugates were applied as fluorescent biological nanoprobes for in vitro bioimaging of malignant glioma brain cells.

Nanotechnology has emerged in recent years as a field of integrated disruptive knowledge with several prospective revolutionary applica­ tions [1]. These new technologies rely on the ability of nanomaterials to have their properties adjusted by the size, morphology, composition, surface capping agents, atomic and molecular structures, and the in­ teractions with other molecules [2,3]. Optical properties such as pho­ toluminescence have attracted much interest from the scientific community, due to the broad range of biomedical applications, including for diagnosis of diseases, biosensing, and bioimaging [4–7]. Semiconductor quantum dots (QDs) are the most interesting lumines­ cent nanomaterials because of their unique optoelectronic and physi­ cochemical properties [8–10], with intense research in different materials and designs to improve light emission efficiency. Nowadays, the processes of synthesis are commonly based on well-established colloidal chemistry [11,12], where the most popular are cadmium-based quantum dots such as CdS, CdSe and CdTe [13,14], and their core/shell structure derivatives (CdSe/ZnS and CdTe/ZnS) [15, 16]. However, traditionally, the QD research has been primarily based on standard protocols using organic solvents at high temperatures with high toxicity associated with heavy metal semiconductors, which are not environmentally sustainable and biocompatible. Thus, more recently, innovative processes for the synthesis of non-toxic and eco-friendly QDs have been intensified. Nanoalloys of binary and ternary systems combining different semiconductor materials have been reported as al­ ternatives to produce greener QDs associated with improved emission efficiency, such as zinc-based quantum dots [17–24]. ZnS quantum dots (ZnS-QDs) are promising due to their visible light emission, biocompatibility, and the ability to be produced using several types of surface capping agents [20,21,23]. Pure or doped ZnS nano­ materials are being used in a vast realm of applications such as lumi­ nescent nanoprobes for biomedicine, optical nanosensors, and nano-photocatalysts, as well as in FRET processes [22–27]. Recently, our group reported the synthesis of ZnS QDs with different surface capping ligands, using green and facile single-pot methods in aqueous media [20,21,23]. The surface capping agents played a key role due to their influence on the nucleation and growth processes of the nano­ crystals and, therefore, controlling their optical and physicochemical properties [20,21,23,27–29]. In that sense, the development of new applications of nanomaterials is dependent on “green” synthesis routes and the understanding of how the optical and physicochemical prop­ erties can be tuned by using distinct parameters and conditions of synthesis. Currently, biopolymers such as polysaccharides have been inten­ sively researched for the production of novel nanomaterials. Among several alternatives, cellulose derivatives, such as carboxymethylcellu­ lose (CMC), are promising candidates for the synthesis of semiconductor nanocrystals. CMC has been used as a simple and intrinsically biocom­ patible surface capping macromolecule for the synthesis of nano­ materials [4,18,24,30]. Essentially, CMC is an anionic water-soluble polysaccharide derived from cellulose, with a variable number of carboxymethyl groups (known as the degree of substitution, DS) in the polymer chain, which is pHsensitive, non-toxic, biocompatible and environmentally friendly. Thus, these characteristics of CMC offer numerous possibilities for biomedical applications such as functional capping ligands for devel­ oping luminescent nanoprobes directed to cancer research [4,18,24]. Considering serious chronic and devastating illnesses, cancer re­ mains one of the deadliest diseases worldwide, and glioblastoma is regarded as the utmost common and aggressive malignant brain tumor.

2. Materials and methods 2.1. Materials Sodium carboxymethylcellulose (CMC, Sigma-Aldrich, USA) with degree of substitution (DS ¼ 0.77, average molar mass MM ¼ 250 kDa, and viscosity of 735 cps, 2% in H2O at 25 � C), zinc chloride (ZnCl2, Sigma-Aldrich, USA, � 98%), sodium sulfide nonahydrate (Na2S⋅9H2O, Synth, Brazil, > 98%), sodium hydroxide (NaOH, Merck, USA, � 99%), and hydrochloric acid (HCl, Sigma-Aldrich, USA, 36.5–38.0%) were used as received without any further preparation. Deionized water with a resistivity of 18 MΩ cm (DI water, Millipore Simplicity, USA) was used to prepare all solutions, and the procedures were performed at room temperature (RT, 23 � 2 � C) unless specified otherwise. 2.2. Green synthesis of colloidal ZnS@CMC quantum dots ZnS@CMC quantum dots were synthesized via an aqueous route at room temperature with different Zn:S molar ratios: 1:2 ([Zn2þ]/[S2 ] ¼ 0.5, excess of sulfur); 1:1 ([Zn2þ]/[S2] ¼ 1.0, stoichiometric); and 2:1 ([Zn2þ]/[S2 ] ¼ 2.0, excess of zinc). Additionally, the synthesis was performed at different pH values (�0.1): 3.5 and 5.5 (acidic), 7.5 (neutral), and 8.5 and 10.5 (alkaline). These parameters were chosen based on the pH-sensitive behavior of CMC associated with the effect of the relative concentrations of precursors on the nucleation and growth of ZnS nanoparticles in solution. First, CMC solution (0.3% w/v) was prepared by adding sodium carboxymethylcellulose powder (0.6 g) to a 200 mL of deionized water and stirring at room temperature until complete solubilization (pH ¼ 7.5 � 0.1). For the chemical synthesis at alkaline conditions (pH ¼ 8.5 and 10.5), the pH of the CMC solution was adjusted with NaOH (1.0 mol L 1) added dropwise. For the acidic synthesis (pH ¼ 3.5 and 5.5), the pH was adjusted with HCl (0.5 mol L 1) added dropwise. After pH adjust­ ment, the required amount of DI water was added to obtain a CMC 2

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concentration of 0.2 wt %. Considering the relative molar ratio of salt precursors, the syntheses of the ZnS@CMC nanoparticles were carried out as follows: 10 mL of Zn2þ precursor solution (ZnCl2, 6.0 � 10 3 mol L 1 for ratios 1:2 and 1:1, and 12.0 � 10 3 mol L 1 for 2:1) was added under moderate magnetic stirring to 15 mL of CMC solution (0.2% wt %, after pH adjustment) and incubated at 6 � 2 � C for 24 h. Then, under moderate stirring, 2 mL of S2 precursor solution (Na2S.9H2O, 3.0 � 10 2 mol L 1 for ratios 1:1 and 2:1, and 6.0 � 10 2 mol L 1 for 1:2) was dropped into the flask and stirred for 30 min resulting in the final concentrations of the [Zn2þ] and [S2 ] precursors in the synthesis shown in Table 1. The obtained ZnS@CMC quantum dots suspensions were clear and colorless, and the samples were stored at 6 � 2 � C. Fig. 1 summarizes the ZnS@CMC synthesis procedure. 2.3. Characterization of the CMC and ZnS@CMC quantum dots Ultraviolet–visible (UV–vis) spectroscopy analysis was performed using Lambda EZ-210 equipment (PerkinElmer, USA) in transmission mode. The samples were positioned in a quartz cuvette over a wave­ length ranging from 600 to 190 nm. Photoluminescence (PL) characterization of the ZnS–CMC was con­ ducted based on steady-state spectra and 3D excitation-emission contour mapping acquired using a FluoroMax-Plus-CP equipment (Horiba Sci­ entific, Japan) measured at room temperature in a quartz cuvette (excitation wavelength, λexc ¼ 200–400 nm; emission wavelength, λem ¼ 300–700 nm; and slit ¼ 2.5 nm). The quantum yield (QY) of the sample with the highest PL intensity was estimated by the comparative standard procedure using quinine bisulfate (Sigma-Aldrich, USA) in sulfuric acid (H2SO4 0.1 mol L 1, Sigma-Aldrich, USA, 95.0–98.0%) as the standard reference at λexc ¼ 310 nm [31,32]. For the other samples, QY was estimated using the single-point method. Morphological and element chemical characterizations were per­ formed using transmission electron microscopy (TEM, Tecnai G2-20FEI, FEI Company, USA) at 200 kV coupled with energy-dispersive xray (EDX) spectroscopy. In all the TEM analyses, the samples were prepared by droplets of the colloidal dispersions placed onto porous carbon copper grids. The QD size and size distribution data were assessed based on the TEM images with at least 100 random measure­ ments of nanoparticles using an image processing program (Digital­ Micrograph® software, version 3.4, Gatan Microscopy Suite Software). Atomic force microscopy (AFM) analysis was performed with an XE-70 (Parker) microscope operating in non-contact tapping mode (frequency ¼ 325 Hz; scanning rate ¼ 1.0 Hz; pixel resolution ¼ 256 � 256). The samples were prepared by dropping the ZnS colloidal suspension onto a mica plate. Electrokinetic potential (or zeta potential, ZP) and dynamic light scattering (DLS) analyses were carried out for characterizing the colloidal dispersions using Brookhaven Instruments’ ZetaPlus equip­ ment (Brookhaven Instruments Corporation, USA) with laser light at λ ¼ 660 nm (35-mW red diode laser), angle 90� , temperature of 26 � C, and with a minimum of ten replicates (n � 10). Fourier transformed infrared spectroscopy (FTIR) spectra were ob­ tained using attenuated total reflectance method (ATR, from 4000 to 650 cm 1, with 32 scans, and 4 cm 1 resolution, Nicolet 6700, ThermoFischer) with background subtraction. ZnS@CMC samples were washed with DI water and centrifuged using Amicon® Ultra Centrifuge Filter

Fig. 1. Schematic representation of the environmentally-friendly aqueous colloidal process of ZnS@CMC quantum dots synthesis (not to scale).

(30 kDa cut-off cellulose membrane, Sigma-Aldrich, 4 cycles � 5 min, at 12,000 rpm). The retained materials were poured into plastic molds and dried in a hot-air oven at 40 � 1 � C for 6 h. All FTIR experiments were conducted in duplicates (n ¼ 2) unless expressly noted. X-ray photoelectron spectroscopy (XPS) analytical technique was used for surface characterization (Mg-Kα, 1253.6 eV, Amicus spec­ trometer, Shimadzu, Japan). The positions of peaks were adjusted using the C 1s binding energy (at 284.6 eV). For sample preparation, washed and concentrated QD colloidal solution was dropped onto a plastic mold and dried in an oven (see FTIR section for sample preparation). In the sequence, the samples were dehydrated in absolute ethanol (3 immer­ sions of 30 s) and dried at room temperature for 2 h using a vacuum desiccator. Quantitative elemental composition was estimated from the integrated area of Zn 2p and S 2p signals corrected by atomic sensitivity factors using the Vision Processing software (Kratos).

Table 1 The final concentration of the [Zn2þ] and [S2 ] precursors in reaction flask during synthesis after adding all of the reagents and CMC ligand (final volume ¼ 27 mL). Sample

[Zn2þ] (mol L

Zn:S 1:2 Zn:S 1:1 Zn:S 2:1

2.2 � 10 2.2 � 10 4.4 � 10

3 3 3

1

)

[S2 ] (mol L 1) 4.4 � 10 2.2 � 10 2.2 � 10

3 3 3

[Zn2þ]/[S2 ] 0.5 1.0 2.0

3

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2.4. Biological characterization

solution of paraformaldehyde (4.0% in PBS) for 30 min, gently washed three times with PBS, and coverslips were mounted with hydromount®. In the sequence, digital images were captured with an Eclipse Ti confocal microscope (Nikon Instruments, USA) using the oil immersion objective (63 � Plan-Apo/1.4 NA). Excitation was performed by UV at λexc ¼ 405 nm, and emission was collected at λem ¼ 417–477 nm (DAPI filter cube).

All of the biological assays were performed according to the inter­ national standard ISO 10993–5:2009/(R)2014 (Part 5: Tests for in vitro cytotoxicity; Biological evaluation of medical devices -). Human em­ bryonic kidney cells (HEK 293T, American Type Culture Collection ATCC CRL-1573) were kindly provided by Federal University of Minas Gerais/UFMG. Human brain glioblastoma cells (U-87 MG, ATCC HTB14) were purchased from Brazilian cell repository (Banco de C�elulas do Rio de Janeiro: BCRJ, Brazil; cell line authentication by molecular technique, Short tandem repeat DNA (STR); quality assurance and cer­ tification based on international standard NBR ISO/IEC 17025:2005). HEK 293T (on passage 25) and U-87 MG (on passage 47) cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL, USA) with 10% FBS (fetal bovine serum, Gibco BRL, USA), streptomycin sulfate (10 mg mL 1, Gibco BRL, USA), amphotericin-b (0.025 mg mL 1, Gibco BRL, USA), and penicillin G sodium (10 units mL 1, Gibco BRL, USA) in a humidified atmosphere of 5% CO2 at 37 � C.

3. Results and discussion 3.1. Design of ZnS@CMC nanoconjugates Carboxymethylcellulose (CMC) is a cellulose derivative that is a pHsensitive multifunctional polysaccharide, mostly due to the deprotona­ tion of the carboxylic acid group (-R-COOH) to anionic carboxylate (RCOO-) with increasing pH according to Eq. (2), resulting in negatively charged polyelectrolyte chains. CMC-O-CH2COOH(aq) þ OH

2.4.1. Cell viability in vitro – mitochondrial activity (MTT) assay MTT (3-(4,5-dimethylthiazol-2yl-) 2,5-diphenyl tetrazolium bro­ mide) experiments were performed to evaluate the cytotoxicity of ZnS@CMC conjugates after incubation with HEK 239T and U87 cells for 24 h. Cells were plated (1 � 104 cells/well) in 96-well plates, and the cell populations were synchronized in serum-free media for 24 h. Then, the media volume was suctioned and replaced with DMEM media containing 10% FBS for 24 h. In the sequence, ZnS@CMC was added to individual wells at final concentrations of 2.5 nmol L 1 of QD nanoparticles. Control samples were designed and produced as follows: control group (cell culture with DMEM and 10% FBS); positive control (cell culture with DMEM, 10% FBS and 1.0% v/v Triton™ X-100 (SigmaAldrich, USA); and negative control (cell culture with DMEM, 10% FBS and chips of sterile polypropylene Eppendorf®, 1 mg mL 1, Eppendorf, Germany). After 24 h of incubation, all media volume were aspirated from each well and replaced with 60 μL of culture solution containing serum. MTT (5 mg mL 1, >98%) was added to each well and maintained under incubation for 4 h at 37 � C and 5% CO2 in an oven. Next, 40 μL SDS (sodium dodecyl sulfate, � 99.0%, Sigma-Aldrich, USA) solution/ 4% HCl (37%, Sigma-Aldrich, USA) was placed in each well and incu­ bated for 16 h in an oven at 37 � C and 5% CO2. Then, 100 μL were removed from each well and transferred to a 96-well plate, and the absorbance was measured on iMark™ Microplate Absorbance Reader (Bio-Rad, USA) with a wavelength filter at λ ¼ 595 nm. The percentage of cell viability was calculated according to Eq. (1), where the values of the controls (i.e., wells with cells but without nanoconjugate sample) were fixed to 100% cell viability.

(aq)

↔ CMC-O-CH2COO

(aq)

þ H2O(l)

(2)

As CMC is usually produced as a sodium salt (CMC-O-CH2COO / Naþ), Naþ is readily dissociated in aqueous solution (Eq. (3)) and, above the pKa (~4.3) [33], CMC is fully deprotonated (-COO-). CMC-O-CH2COONa(aq) þ H2O(l) ↔ CMC-O-CH2COO

(aq)

þ Naþ(aq)

(3)

As the pH is reduced below the pKa (i.e., < 4.3 by adding H ), the carboxylate groups (-COO-) are mostly protonated, forming carboxylic acid groups (-COOH, Eq. (4)). þ

CMC-O-CH2COO

(aq)

þ Hþ(aq) ↔ CMC-O-CH2COOH(aq)

(4)

CMC polymer is expected to act as stabilizing ligand for ZnS nano­ crystals bearing in mind two major aspects: (a) the interactions of ZnS inorganic core through surface metallic cations (Zn2þ) with negative charges of CMC (R-COO-), forming COO /Zn2þ chelates according to Eq. (5); (b) due to colloidal stabilization by steric hindrance and/or elec­ trostatic repulsion between negatively charged solvated polymeric chains in the water medium. In addition, according to the literature [4], primary and secondary alcohols (hydroxyl groups, –OH, from cellulose) may be also reactive sites for electron donation for passivation of ZnS nanoparticle dangling bonds. CMC-O-CH2COO

(aq)

þ Zn2þ(surface) ↔ CMC-O-CH2COO /Zn2þ (chelates)(5)

Moreover, the CMC with a low degree of substitution (DS ¼ 0.7) was selected to favor the biodegradability combined with the molecular mass (MM ¼ 250 kDa) above the renal threshold. These features are crucial to prevent the QD-polymer fluorescent nanoconjugates from rapid elimination of the body, considering the perspective of future biomedical applications. Finally, by considering the physicochemical background, the stabil­ ity of ZnS-CMC nanoconjugates can be assigned to the highly favorable thermodynamically and kinetically formation of ZnS solid-phase based on the extremely low product of solubility (Ksp ¼ 1.0 � 10 23) [34], combined with the resistance of CMC and ZnS to degradation under the acidic-alkaline range of pH selected for the experimental procedures (above 3.5 and below 10.5).

Cell viability ¼ (Absorbance of sample and cells)/(Absorbance of control) � 100% (1) Statistical significance was evaluated using One-way ANOVA fol­ lowed by Bonferroni’s method. At α confidence level value α < 0.05 was considered statistically significant (n � 6). 2.4.2. Cell uptake of fluorescent ZnS@CMC The evaluation of the ZnS@CMC nanoconjugates as fluorescent biological probes for in vitro bioimaging was performed using confocal laser scanning microscopy (CLSM) after exposing U-87 MG and HEK 293T cell lines to ZnS@CMC nanoconjugates. Cells were plated (5 � 105 cells per well) in a 6-well plate, incubated in 5% CO2 at 37 � C for 24 h, and synchronized for 24 h. Then, ZnS@CMC colloidal suspension (1:1, v/v) with the medium solution (DMEM with 10% FBS) was added to the cells and incubated in 5% CO2 at 37 � C for 120 min, followed by washing with PBS solution. In the sequence, the cells (U-87 MG and HEK 293T) were fixed with a

3.2. Optical characterization of ZnS@CMC nanoconjugates Essentially, this research was designed and developed for investi­ gating the effect of processing parameters during the synthesis for tun­ ing the optical properties of ZnS colloidal nanoparticles via nucleation and growth processes stabilized by the CMC biopolymer. Therefore, the results of UV–visible spectroscopy (UV–vis) and photoluminescence (PL) spectroscopy of the ZnS@CMC nanoconjugates under acidic, neutral, and alkaline aqueous media and with three concentrations of precursors are shown in Fig. 2. As a general trend, all of the UV–vis spectra (Fig. 2 4

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Fig. 2. UV–vis (a–c) and PL (d–f) spectra of the ZnS@CMC colloidal solution at different pH ((A) 3.5; (B) 7.5; and (C) 10.5) and Zn:S molar ratios ((a,d) 1:2; (b, e) 1:1; and (c,f) 2:1).

(a-c)) exhibited well-defined onset absorption bands between 270 and 330 nm (4.49–3.76 eV), blue-shifted (i.e., hypsochromic shift) compared with the bulk bandgap (absorption edge at λ ¼ 343 nm, 3.61 eV), which is associated with the first excitonic transition of semiconductors under the quantum confinement regime [35,36]. The bandgap energy values (Eg) of the ZnS semiconductor nanoparticles were estimated using the “Tauc relation” [13,37] presented in Fig. 3, which confirmed they were formed with Eg higher than the pristine bulk value (Ebulk ¼ 3.61 eV). Additionally, according to the literature [35,36], the absorption bandgap energy of the semiconductor nanoparticle is drastically affected by the size of the nanocrystals, which imparts them a unique set of size-dependent optoelectronic properties. Thus, based on the

Fig. 3. Optical bandgap using ‘‘Tauc’’ relation of the ZnS@CMC colloidal so­ lutions at different pH ((A) 3.5; (B) 7.5; and (C) 10.5) and Zn:S molar ratios ((a) 1:2; (b) 1:1; and (c) 2:1).

semi-empirical correlation equation (Eq. (6)) [38], the diameters (2r) of the ZnS nanoparticles produced at distinct pH and concentrations were calculated based on Eg values and typically ranged from 3.3 to 4.5 nm, which are lower than ZnS Bohr radius (2rB ~ 5.5 nm) [39], commonly referred to as quantum dots (QDs). 5

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Table 2 Summary of properties of ZnS@CMC nanoconjugates. pH

Zn:S

Band Gap (Eg) (eV)

2r (nm)

Stokes shift (nm)

PL Max (nm)

PL FWHMa (nm)

3.5

1:2 1:1 2:1 1:2 1:1 2:1 1:2 1:1 2:1

3.80 � 0.05 3.90 � 0.05 3.93 � 0.05 3.79 � 0.05 3.97 � 0.05 4.04 � 0.05 3.76 � 0.05 3.98 � 0.05 4.05 � 0.05

4.31 � 3.84 � 3.72 � 4.37 � 3.59 � 3.39 � 4.56 � 3.56 � 3.36 �

106 116 112 106 121 121 116 131 132

411 � 411 � 407 � 417 � 413 � 410 � 428 � 423 � 421 �

89 � 3 96 � 3 89 � 3 97 � 3 98 � 3 97 � 3 105 � 3 106 � 3 106 � 3

7.5 10.5

2r ¼ 2*{[0.32–2.9*(Eg – 3.49)1/2] / [2*(3.50 – Eg)]}

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

� � � � � � � � �

2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2 2

nanoparticles to reaching bigger sizes. As expected, at the stoichiometric ratio of precursors (Zn:S ¼ 1:1), the intermediate size of ZnS nano­ crystals was detected in the colloidal solution. Analogously, the effect of pH on the nucleation and growth processes for producing ZnS nanoparticles in aqueous solution was clearly observed, as depicted in Fig. 4, associated with the bandgap energy (Tauc, Eg) and estimated average size, respectively. As CMC is a pHsensitive biopolymer (RCOO þ Hþ ↔ COOH), the macromolecule

(6)

Analogously, as a common tendency, all of the photoluminescence (PL) spectra (Fig. 2(d–f)) of the ZnS@CMC quantum dots typically exhibited maximum emission peaks predominantly within the wave­ length range from 400 to 430 nm (violet). It was also observed large Stokes shift (>100 nm) and the “full width at half maximum” (FWHM) ranging from 89 to 106 nm. These results are in good agreement with the typical behavior of ZnS, where emissions are predominantly based on defect-activated sites, and the band-to-band optical transition is usually absent [4,20–24,30]. That means lattice point defects (sulfur (VS) and metal (VZn) vacancies, and interstitials atoms, IS and IZn) act as efficient traps for electrons, holes, and exciton charge carriers, leading to radia­ tive recombination at energies lower (higher wavelengths) than the excitonic emission. According to the energy levels diagrams reported in the literature for ZnS mid-gap states [40–44], the VS and IZn trap states are localized closer to the conduction band while sulfur at interstitial sites (IS) and vacancies of zinc (VZn) are closer to the valence band. In this sense, it was expected to create different populations of the types of point defects (VZn, VS, IZn and IS) affected by the changes of processing parameters in the synthesis and interactions with carboxylate species from CMC that resulted in the broad emission curves. Therefore, these UV–vis and PL results demonstrated that ZnS quantum dots were effectively produced and stabilized by CMC biopolymer ligand using a facile green aqueous colloidal process at room temperature and also proved photoluminescent activity under excitation. More specifically, these optical properties were analyzed considering the effects of the pH (acidic, neutral, and alkaline) and the concentration of precursors (excess of cations, stoichiometric, and excess of anions) on the mechanism of nanocrystal formation and stabilization associated with the chemistry involved in the colloidal process. Therefore, the re­ sults were extracted from Figs. 2 and 3 and summarized in Table 2, and the plots were depicted in Fig. 4. Additionally, the results for syntheses at smaller pH intervals (i.e., pH ¼ 5.5 � 0.1 and pH ¼ 8.5 � 0.1) were also included (Fig. 4), where the major trend was retained. Initially, the influence of the concentration ratio of precursors Zn:S during the synthesis on the optical properties of ZnS nanoparticles was analyzed considering the size-dependence with the UV–vis absorption. Thus, at the same pH, in the case of excess of zinc ions (Zn:S ¼ 2:1) in solution, the interactions between the cations (Zn2þ) and the carbox­ ylate groups from CMC favored the formation of more stable metalligand complexes (Zn2þ/RCOO ). Consequently, the injection of relatively lower concentration of sulfides (S2 ) favored the burst nucleation of ZnS nanocrystals, imme­ diately depleting the concentration of S2 in solution and restricting the growth, leading to the formation of smaller QDs. Conversely, at higher concentrations of sulfides during the synthesis (Zn:S ¼ 1:2) the opposite tendency was observed due to the competition of the anionic species (RCOO and S2 ) for interactions with Zn2þ species, leading to less stable complexes (Zn2þ/RCOO ) and thus favoring the growth of ZnS

Fig. 4. Evolution of (A) optical bandgap (Eg) and (B) diameter (2r) of ZnS@CMC as a function of pH at different Zn:S molar ratios: (a) 1:2; (b) 1:1; and (c) 2:1 (error bar: standard deviation (SD), replicates (n) � 3). 6

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played an important role in the stabilization of the ZnS@CMC nano­ crystals. Under acidic (pH ¼ 3.5) and neutral (pH ¼ 7.5) conditions the Eg values were smaller (Fig. 4(A)) than under alkaline conditions (pH ¼ 10.5) for the syntheses under excess of Zn2þ (2:1) and stoichiometric (1:1). These results were explained based on the majority of protonated carboxylic groups (Zn2þ/RCOO ↔ RCOOH) at lower pH values (pH < or ~ pKa) reducing the anionic attraction with the metallic cations in solution (excess ratio of Zn2þ) or with the surface of ZnS nanoparticles (stoichiometric ratio of Zn2þ) formed. As stated before, lower stabilization of Zn2þ by the CMC capping ligand produced larger ZnS nanoparticles. On the contrary, under alkaline conditions (pH ¼ 10.5 ≫ pKa ~4.3), the carboxylic groups of CMC are predominately deprotonated (RCOOH → RCOO ) forming negatively charged carboxylates in the medium. Therefore, they acted as multidentate capping ligands by improving the electrostatic attraction with Zn2þ ions in solution and producing smaller ZnS nanoparticles (i.e., higher Eg). However, for the synthesis at excess of S2 (Zn:S ¼ 1:2), this effect was not clearly evidenced, where practically no relevant changes of Eg values (Fig. 4(A)) were observed from acidic to alkaline conditions, despite minor variations of the estimated sizes (Fig. 4(B)), which are dependent of the non-linear semi-empirical equation. These results were interpreted as the predominant effect of the excess of sulfides surpassing the dependence of pH for the nucleation and growth of ZnS nano­ particles. In fact, the synthesis under the excess of S2 in solution, regardless of the pH, always produced larger ZnS nanoparticles (i.e., smaller Eg) compared to the other conditions. This trend was attributed to the different nucleation/growth kinetics and thermodynamics caused by a more complex balance of electrostatic charges in solution. A similar approach was used for the analysis of the effects of pH and concentration of precursors during the synthesis on the PL results of ZnS@CMC nanoconjugates, but considering that the emission properties are related to very different mechanisms compared to the absorption phenomena. Hence, all the photoluminescence spectra of the ZnS@CMC nanoconjugates (Fig. 2) typically exhibited maxima emission peaks in the wavelength range from 410 to 430 nm with the Stokes shift related to the corresponding absorption spectra (details in Table 2). It was clearly observed that, under the same pH, the higher concentration of sulfides (Zn:S ¼ 1:2) produced higher PL intensities. Conversely, the higher concentration of Zn2þ (Zn:S ¼ 2:1) caused lower PL intensities. Regarding the pH of the medium, for specific concentrations of pre­ cursors, the synthesis at acidic conditions (i.e., pH ¼ 3.5) provoked higher PL intensities compared to neutral and alkaline solutions. Thus, the ZnS@CMC quantum dots synthesized with a non-stoichiometric molar ratio of excess of sulfides (Zn:S ¼ 1:2) and under acidic condi­ tions (pH ¼ 3.5 < pKa) demonstrated the highest PL emission, which is consistent with the literature of ZnS quantum dots [20,21]. This trend was explained by considering the processes of nucleation and growth of the ZnS nanocrystals, which were affected by the synthesis parameters producing quantum dots with variable sizes. As previously discussed, the combination of an excess of Zn2þ with neutral or alkaline conditions during the syntheses, generated relatively smaller ZnS quantum dots (Fig. 4(B)). Therefore, these smaller ZnS nanocrystals were produced with higher surface disorders and dangling bonds, which are the dominant physical process in the luminescence properties, creating non-radiative pathways that can dissipate light emission, reducing the PL emission intensity and QY [21]. On the other hand, the synthesis of ZnS QDs using the excess of S2 at a molar ratio of Zn:S ¼ 1:2 produced larger nanocrystals and, therefore, reduced the relative number of atoms at the surfaces, which may have significantly favored the PL emission. Moreover, the higher PL emission can also be attributed to the fact that CMC acted as a capping anionic polymer, repelling the excess of sulfides at the surfaces of ZnS QDs reducing the charge-transfer (via non-radiative pathways) of the core with the shell and solution, which may have contributed with the PL

emission. However, it is important to highlight these are insights of a preliminary interpretation of the very complex phenomena associated with the light-matter interactions in these semiconductor-based nano­ hybrids (i.e., ZnS@CMC) generating excitonic pairs (e /hþ), which have numerous recombination pathways. There are intricate mechanisms, which can be significantly affected by several factors, including the conditions during the synthesis, the concentration and nature of ligand (i.e., anionic, or cationic), relative ratio of precursors (i.e., metallic ions and chalcogenides), presence of scavenger agents (for holes or elec­ trons), among others. Therefore, the PL is highly dependent on the overall contributions of these factors, which must be taken into account for every particular nanoconjugate system under investigation. In order to further investigate the optical properties of the ZnS quantum dots synthesized, bearing in mind their designed applications as fluorescent nanoprobes for bioimaging, 3D excitation and emission contour maps were obtained, and the results are presented in Fig. 5. These 3D results are complementary to the PL spectra (Fig. 2) as they showed the dependence of the emission by varying the energy of the irradiation source. As a general trend, it was observed significant PL emissions typically within the visible region of the light spectrum on the 3D color plot (λem range from ¼ 350 nm to 500 nm (violet-blue); maxima ~410–430 nm) upon excitation with wavelength (λexc) from ~250 nm to ~320 nm. In these curves, it was confirmed the results of Fig. 2, where the highest photoluminescence PL emission was obtained for the ZnS@CMC nanoconjugates synthesized with an excess of sulfides (Zn:S ¼ 1:2) and under acidic conditions (pH ¼ 3.5 < pKa). In addition, minor shifts towards lower wavelengths of the excitation region for the ZnS@CMC samples with Zn:S ¼ 1:2 compared to the other concentra­ tions, which was attributed to the sum of contributions of density of surface defects and trap states, scavenging anions in solution, and size and crystalline structure of the QDs, affecting the mechanisms of charge generation and recombination. Also, a relatively more diffuse excitation/emission map was observed under alkaline conditions (pH ¼ 10.5) assigned to the excess of hydroxides species in the medium affecting the overall balance of charges at the solution-quantum dot nanointerfaces. As previously stated, the excess of [S2 ] relative to [Zn2þ] produced larger ZnS nanoparticles and also provoked the availability of sulfide species in solution, which can act as hole scavengers influencing the charge-transfer process at the nanocrystal-liquid interfaces and there­ fore, the optical properties measured. Importantly, regarding optical properties, these results proved that these ZnS@CMC nanoconjugates are appropriate for using as fluorescent nanoprobes for several biomedical applications, offering a broad range of possibilities for excitation energy combined with a reasonably wide range of visible emission for allowing luminescent detection. Addition­ ally, the quantum yield (QY) of ZnS@CMC quantum dots was measured using the quinine-based protocol as a reference [32]. The QY values are presented in Fig. 5 (insert) for the ZnS nanocrystals, clearly showing their dependence with the synthesis parameters supported by the pre­ vious discussion. It was evident the relation between ZnS QD size and QY results (Fig. 6) associated with the large surface to volume ratio of smaller nanocrystals that favors nonradiative decay of charge carriers (i. e., lower QY). The ZnS@CMC nanoconjugates with the highest PL emission (Zn:S 1:2 at pH 3.5) showed an average QY ¼ 0.3%, which has already been proved as suitable fluorophores for bioimaging applica­ tions [18] and consistent with the literature [35,45]. It is broadly accepted that, despite several advantages of producing semiconductor quantum dots using aqueous colloidal process, including environmental and biological compatibilities associated with facile routes, commonly, it has lower quantum yield (QY) compared to organometallic synthesis [35,45]. To surpass this effect, the QY can be significantly improved by several methods, including growing a shell layer of another semiconductor and a thermal process for passivating surface defects and annealing crystalline defects, respectively. However, the improvement and optimization of QY is not the goal of this study, 7

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Fig. 5. 3D excitation-emission curves measured at the different ratios of precursors and pH and QY values.

Fig. 6. Relation of QY with ZnS quantum dot size (2r) as a function of pH: (a) 3.5 (acidic); (b) 7.5 (neutral); and (c) 10.5 (alkaline).

which focused on tuning the optical properties of ZnS@CMC nano­ conjugates by varying the synthesis parameters producing photo­ luminescent nanoprobes for testing in bioimaging applications. 3.3. Morphological and compositional analysis of ZnS@CMC quantum dots Morphological features, sizes, composition, and crystalline structure of the ZnS stabilized by CMC biopolymer were investigated by TEM coupled to EDX analysis, AFM images, and XPS spectroscopy. First, TEM analysis was performed directly onto the ZnS quantum dot samples with

Fig. 7. TEM images and histogram of nanoparticle size distribution of ZnS@CMC quantum dots synthesized at pH 3.5 at different Zn:S molar ratios: (A) 1:2; (B) 1:1; and (C) 2:1. 8

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crystalline nature of the synthesized QD due to the presence of the lattice fringes. The interplanar distance (d) of approximately 2.9 � 0.2 Å is compatible with the (111) plane of zinc blende lattice structure of ZnS (JCPDS 05–0566). Despite wurtzite (hexagonal) being the stable form of zinc sulfide bulk at RT, the cubic polymorph is mainly observed for ZnS nanocrystals [20]. Similar images and d values were obtained for all of the evaluated pH and Zn2þ/S2 molar ratios indicating that synthesis parameters did not significantly affect the structural ordering of ZnS atoms. As TEM images are predominantly related to the inorganic ZnS core due to the lower contrast of CMC organic shell, AFM technique was selected in order to further assessing the size of the ZnS QD surrounded by the stabilizing coating of polymer. The AFM images (Fig. 10) clearly showed the ZnS nanoparticle embedded in a polymer matrix with an estimated size (~9 nm). As expected, this dimension is relatively larger than the values calculated for the inorganic core from UV–vis and TEM analyses, as it summed the contributions of both inorganic (ZnS, core) and organic (CMC, layer shell) components in “unswollen” conditions (no water). The elemental composition of the ZnS nanoparticles based on the typical EDX (Fig. 8(D)) spectra indicated the presence of Zn and S from inorganic core and C and O from CMC polymer ligand. In the spectra, peaks of copper (Cu) and silicon (Si) were also detected associated with the grid used as solid support for sample deposition and the from the microscope detector, respectively. Furthermore, the chemical state of Zn and S in the ZnS QD was evaluated by XPS spectroscopy after removing the organic shell and surface layer using ion bombardment with argon ions (Arþ, 2 cycles, 3 s/ cycle, the emission current 55 mA, and beam voltage 0.5 kV). In the spectra obtained from Zn 2p region (Fig. 11(A)) the peaks at 1021.7 � 0.2 eV and 1044.7 � 0.2 eV match to the Zn 2p3/2 and Zn 2p1/2 levels, respectively, which were associated with the zinc in ZnS [46–49]. Additionally, Zn 2p3/2 and Zn 2p1/2 peaks are separated by a binding energy interval of approximately 23.0 eV, in agreement with the liter­ ature [46]. The S 2p region (Fig. 11(B)) comprises of two peaks with binding energy (BE) 161.6 � 0.2 eV (S 2p3/2) and 162.8 � 0.2 eV (S 2p1/2) identified via deconvolution with a Δ ¼ 1.2 eV, which can be assigned to sulfur in metal sulfides (M S) [46–49]. The atomic con­ centrations of Zn and S were estimated of 55 � 5% and 45 � 5%, respectively, in agreement with the theoretical ZnS stoichiometry. This slight tendency to higher metal content may be assigned to the presence of an outmost layer surface comprising complexes between the Zn2þ cations and anionic CMC polymer passivating dangling bonds. These Zn

Fig. 8. TEM and HRTEM images of ZnS@CMC conjugates synthesized at molar ratio 1:1 and distinct pH: (A) 3.5; (B) 7.5; and (C) 10.5. (D) Typical EDX spectra of ZnS@CMC conjugates (Zn:S 2:1, pH 3.5).

the highest photoluminescence emission, which were synthesized under acidic conditions (pH ¼ 3.5) and three concentrations of precursors. The TEM images and histograms of the size distribution of ZnS@CMC are shown in Fig. 7. In the sequence, the effects of pH during the synthesis on morphology and composition/structure of ZnS nanocrystals were eval­ uated considering the stoichiometric molar ratio Zn:S ¼ 1:1 under acidic, neutral, and alkaline conditions (Fig. 8(A-C)). As a general trend, it was observed fairly well-dispersed and uniform nanoparticles with predominant spherical shape for all samples. More­ over, at the same pH (Fig. 7), it was also detected a trend of increasing the average size of ZnS QDs by increasing the concentration ratio of sulfides relative to Zn2þ in the synthesis. This tendency endorsed the results estimated by the “Tauc relation” in section 3.2, and the effects of [S2 ] on the average size of ZnS nanocrystals are depicted in Fig. 9. As stated before, this effect was interpreted by considering the nucleation and growth processes of the ZnS semiconductor nanocrystals affected by the protonated (RCOOH) or deprotonated (RCOO ) functional groups of CMC polymer (i.e., pH-sensitive polymer) associated with ions in solu­ tion (Zn2þ or S2 ) forming complexes. High-resolution TEM images (HRTEM, inset in Fig. 8) revealed the

Fig. 9. Analysis of influence of concentration of precursors Zn:S on the average size (2r) of ZnS@CMC measured by TEM (error bar ¼ SD; n � 100) and estimated using “Tauc relation” (error bar ¼ SD; n ¼ 3) at pH ¼ 3.5. Inset: Schematic representation of size dependence of ZnS@CMC nanoconjugates with [Zn2þ]/[S2 ] in the synthesis. 9

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Fig. 10. AFM topographic image of ZnS quantum dot stabilized by CMC ligand (Zn:S 1:1, pH 7.5) showing the contribution of inorganic core and polymer shell to the size of nanoconjugate in “dry state”.

Fig. 11. Typical XPS spectra of (A) Zn 2p and (B) S 2p regions after etching (Zn:S 2:1, pH 7.5).

2p and S 2p spectral regions were similar for all of the synthesized ZnS independent of pH and Zn:S molar ratio, which indicated the formation of ZnS nanoparticles compatible with stoichiometric relative proportion (1:1).

consumption of all precursors forming the ZnS@CMC nanocolloids and therefore, with no residual ions of precursors (Zn2þ or S2 ) remained in solution. Thus, these results proved that ZnS@CMC conjugates were effectively stabilized as colloidal nanostructures in water medium pre­ dominantly by electrostatic forces and polymer macromolecule ligands. In addition to UV–Vis, TEM, and AFM analyses, DLS experiments were also performed with aqueous dispersions of ZnS nanoconjugates to determine the nanoparticle hydrodynamic diameter (dh) and the size distribution of the nanocolloids. For colloidal QDs, the dh measurement is a valuable tool for researching the chemical stability and possible interactions with other molecules involved in the formation of nano­ structures. The results of hydrodynamic dh using DLS analysis are pre­ sented in Fig. 12(B). The samples with an excess of sulfides (Zn:S ¼ 1:2) showed higher average dh values under the three conditions of pH tested. These findings were associated with the conformation of CMC polymer macromolecule, forming a “shell” layer onto the quantum dot core surface, producing ZnS@CMC hybrid nanostructures. As discussed before, the interactions between the cations (Zn2þ) and the carboxylate groups from CMC forming M2þ/RCOO complexes favored more sig­ nificant interactions between the polymer and the ZnS@CMC surface and consequently, reducing the dh for Zn:S ¼ 2:1 and 1:1. Conversely, under the condition of excess of sulfides (Zn:S ¼ 1:2), the formation of complexes is disadvantageous and, therefore, decreasing the interaction between the ZnS nanocrystals and carboxylates of CMC. This effect favors the repulsion between the CMC chains provoking the relative increase of the hydrodynamic diameter. It is essential to high­ light that the dh values are different from the primary semiconductor nanoparticle size calculated in previous sections from UV–Vis absor­ bance curves and TEM, because the hydrodynamic dimensions of these

3.4. Physicochemical properties analysis of ZnS@CMC quantum dots Surface charge measurements using ξ-potential (ZP) technique are shown in Fig. 12(A). The ZP values indicated that all samples presented negatively charged surfaces typically ranging from 15 to 45 mV associated with the presence of carboxylic groups in the CMC polymer assigning anionic characteristics. CMC, as a pH-sensitive polymer, under acidic conditions (pH ¼ 3.5 < pKa) the carboxylic functional groups are mostly protonated (RCOO þ Hþ ↔ RCOOH) reducing the negative surface charges of the nanoconjugates assessed by ZP measurements at approximately 20 mV for the three ratios of Zn:S, (1:2, 1:1, and 2:1). Conversely, at nearly neutral pH (pH ¼ 7.5 > pKa), the carboxylic groups are partially deprotonated and converted to carboxylates (RCOOH ↔ RCOO þ Hþ) increasing the negative charges of the surface of the ZnS@CMC nanoconjugates. Under alkaline conditions (pH ¼ 10.5 > pKa), the ZP values demonstrated highly negative surfaces (~ 30 to 45 mV) due to the deprotonation forming predominantly carboxylates (RCOO ) associated with the presence of hydroxide ions (OH ). Similar trends on ZP values were observed for the three relative concentrations of precursors by changing the pH, but with more or less pronounced effects attributed to the overall balance of electrostatic interactions including the contributions of charged species in solution, cations (i.e., Zn2þ) and anions (i.e., S2 ). The lowest variation of ZP with pH was observed for the stoichiometric ratio of Zn:S ¼ 1:1 due to the 10

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Fig. 13. Effect of pH on FTIR spectra of Zn:S 1:2: (a) pH 10.5; (b) pH 7.5; and (c) pH 3.5.

Fig. 12. (A) Zeta potential (error bar: SD, n � 10) and (B) dh (error bar: SD, n � 3) measurements at different molar ratio of precursors ((a) Zn:S 1:2; (b) Zn:S 1:1; and (c) Zn:S 2:1) and pH.

colloidal nanostructures comprise the sum of several contributions, including the inorganic core made of ZnS, the CMC organic polymer shell layer, excluded volume interactions, electrolyte effects, as well as the solvation layers of water molecules and constraints in bond and rotation angles [36,50]. These results are very relevant, bearing in mind the potential biomedical applications of these nanoconjugates, as the hydrodynamic size is one of the critical aspects of the interactions of nanomaterials with living cells and biological systems [4,50]. FTIR is a powerful technique for evaluation of the interactions occurring between the functional groups of the CMC ligand and the ZnS quantum dots affected by the surrounding microenvironment in the colloidal media. In this sense, the influence of pH on the FTIR spectra of the ZnS@CMC at Zn:S molar ratio of 1:2 is presented in Fig. 13. The main difference among the spectra was associated with the pH-sensitive behavior of CMC, mostly due to the protonation-deprotonation of car­ boxylic groups (R–COOH/R-COO-). At pH < pKa (i.e., acidic, pH 3.5, Fig. 13(c)), the characteristic band of COOH groups was observed at approximately 1730 cm 1 assigned to the antisymmetric stretching vi­ – O. At pH > pKa (pH 7.5 and pH 10.5, Fig. 13(a,b)), as the bration of C– carboxylic acids are fully deprotonated (COO ), the band at 1730 cm 1 disappeared, and the vibrations related to carboxylates were clearly detected at 1650 and 1592 cm 1 (asymmetric stretching) and 1416 and

Fig. 14. Effect of Zn:S molar ratio ((a) Zn:S 2:1; (b) Zn:S 1:1; and (c) Zn:S 1:2) compared to CMC (d) at (A) pH 3.5 (pH < pKa) and (B) pH 10.5 (pH > pKa).

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1324 cm 1 (symmetric stretching) [51]. Also, the bands of primary (C6–OH at 1028 cm 1) and secondary (C2–OH at 1112 cm 1 and C3–OH at 1060 cm 1) alcohols and the vibration of glycoside bonds (β 1–4 at 895-900 cm 1) were observed for all of the samples [51]. In addition, the effect on the FTIR spectra of Zn:S molar ratio for QD produced below (pH 3.5, Fig. 14(A)) and above pKa (pH 10.5, Fig. 14 (B)) compared with CMC at the same pHs were evaluated. Based on the spectra, for both pH, upon conjugation with ZnS, no significant change was detected in the energy of the vibrations of COOH/COO of CMC capping ligand, independent of the stoichiometry. However, changes in the intensity of carboxylic/carboxylate bands were observed, mostly at pH 3.5 (
[53,54], monodentate coordination. The differences between the stretch vibrations at approximately 1592 and 1416 cm 1 are related (Δν2 ¼ 176 cm 1) and revealed a bidentate bridging mode of interaction of COO with bivalent metal (Zn2þ) at the nanoparticle surface. Based on this analysis, a combination of monodentate and bidentate bridging modes was observed in the formation of the complexes Zn2þ/COO at quantum dots surfaces in agreement with previous analysis regarding enrichment of metal cations at QD outmost layer, passivation of dangling bonds, and formation of chelates for stabilizing the ZnS nanoparticles. 3.5. Stability of ZnS@CMC quantum dots The long-term stability of ZnS@CMC nanoconjugates commercially referred to as “shelf-life”, is vital for the future clinical use in biomedical applications and nanomedicine. Thus, all of the systems investigated showed suitable colloidal stability for over 12 months after the synthesis stored at 6 � 2 � C in the dark. Visually, the aqueous suspensions were homogenous, free of agglomerations or aggregation. The optical prop­ erties (UV–vis absorption/PL emission) remained relatively stable after 12 months with relative signal changes smaller than 10%. These prop­ erties were evaluated and presented in Fig. 15 (for Zn:S 1:1): (a) bandgap energy (obtained from UV–vis data using “TAUC” equation); (b) size 2r (estimated from Eg using Eq. (6)); (c) PL maxima; and (d) PL parameter “FWHM”. Moreover, hydrodynamic diameter (dh) and ZP values presented changes of ca. 10% after 12 months of storage in comparison to the measurements within 24 h after synthesis. This noteworthy behavior was assigned to the high stability promoted by the CMC biopolymer as macromolecule ligand and predominantly nega­ tively charged moiety, which caused stabilization by electrostatic repulsion and steric hindrance. These physicochemical characteristics stabilized the core-shell nanocolloids in aqueous media and avoided the agglomeration/coalescence of the nanoparticles. 3.6. Biological analysis of ZnS@CMC quantum dots 3.6.1. Cell viability in vitro – mitochondrial activity (MTT) assay The cytocompatibility of ZnS@CMC nanoconjugates was assessed using in vitro MTT assay using HEK 293T and U-87 MG cell lines,

Fig. 15. ZnS@CMC nanoconjugates properties and parameters (a) “as-synthesized” and (b) after 12 months of storage for Zn:S 1:1 as a function of pH: (A) Eg; (B) 2r; (C) PL maxima; and (D) “FWHM” (error bar: SD, n � 3). 12

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Fig. 16. Cell viability response of (A) HEK 293T and (B) U-87 MG cell lines based on the MTT assay after 24 h of incubation with ZnS@CMC (Zn:S 1:2, pH 3.5) (error bar: SD, n ¼ 6).

corresponding to normal and cancer cells, respectively. All the biological assays were conducted according to ISO 10993–5:2009/(R)2014 (Bio­ logical evaluation of materials and medical devices: Tests for in vitro cytotoxicity) globally accepted as a preliminary evaluation of toxicity of (nano)materials and devices. The cell viability responses of the ZnS@CMC are presented in Fig. 16 ((A) HEK 293T and (B) U-87 MG) based on the MTT assays after 24 h of incubation. The results clearly demonstrated that ZnS@CMC nanoconjugates have cell viability higher than 90% for both types of cells with no significant differences (within the statistical variation). The results evidenced that CMC stabilized ZnS quantum dots were produced using a facile green aqueous process rendering cytocompatible fluorescent nanoprobes suitable for in vitro biomedical applications such as bioimaging and bio-labeling living cells.

Fig. 17. Confocal microscopy imaging of the cellular uptake of the ZnS@CMC blue emission by (A) HEK 293T cells and (B) U-87 MG cells with incubation time 120 min: (a) bright-field image; (b) PL image; and (c) merged PL þ brightfield image (scale bar ¼ 10 μm).

4. Conclusions Novel ZnS@CMC hybrid nanoconjugates composed of ZnS quantum dot core and carboxymethylcellulose (CMC) polymer capping ligand were successfully synthesized via a facile green aqueous colloidal pro­ cess at room temperature. These ZnS@CMC nanoconjugates were synthesized under three pH conditions (acidic, neutral, and alkaline) and concentration of pre­ cursors for tuning their optical properties. They were extensively char­ acterized for their structural, morphological, and physicochemical properties and associated with their optical absorption behavior and emission activity. It was verified the pH-dependent effect on the nucleation and growth of the ZnS nanocrystals in colloidal dispersions assigned to the extension of protonation/deprotonation of carboxylic functional groups of CMC producing relatively larger nanoparticles at low pH (3.5) and, on the contrary, smaller at high pH (10.5). Analo­ gously, the excess of sulfides in the synthesis provoked a similar response with bigger ZnS QDs compared to the excess of metallic zinc ions, which was interpreted as the formation of metal-polymer com­ plexes affecting the kinetics of nucleation and growth mechanisms. As expected, based on the size-dependent behavior of optoelectronic properties of QDs, these changes were detected by alterations on the absorption and emission spectra of ZnS@CMC nanoconjugates, where the excess of sulfides and acidic medium showed the highest PL emis­ sion. The estimated QY (~0.3%) was less affected by the changes in the synthesis of these nanoconjugates because the relatively high density of surface defects usually present in QDs produced via aqueous colloidal route at room temperature. New 3D emission and excitation mapping plots evidenced the dependence of the optical properties with the con­ ditions used in the synthesis rendering nanoconjugates with tunable PL emission in a relatively broad range of the visible spectrum (~400–650 nm). Consequently, these features assure appropriate optical properties associated with cytocompatibility for using these ZnS@CMC nano­ colloids as fluorescent nanoprobes for bioimaging cells in biomedical applications. As a proof-of-concept, these ZnS@CMC nanoconjugates proved significant PL activity when used as a cytocompatible fluorescent biological nanoprobe for bioimaging malignant glioma cells in vitro showing efficient cytoplasm internalization for potential applications in cancer research.

3.6.2. Cell uptake of fluorescent ZnS@CMC In this work, as a proof of concept, the ZnS@CMC quantum dots were applied as biocompatible fluorescent biological nanoprobes for in vitro bioimaging malignant glioma cells (U-87 MG) through cell internaliza­ tion assessed by using confocal laser scanning microscopy (CLSM) and HEK293T as reference normal cell line [23,24]. HEK 293T was used as a cell model due to higher transfection efficiency. On the other hand, the U-87 MG cell line is a human primary glioblastoma cell line widely used for brain cancer research. Nowadays, the research for novel nano­ materials for glioblastoma targeting is very important due to the high lethality of brain tumors [18]. The ZnS@CMC cellular uptake by HEK 293T and U-87 MG is shown in Fig. 17. As can be observed, the ZnS@CMC nanoconjugates presented the characteristic blue emission as expected by photoluminescence emission results presented in section 3.1. These results demonstrated high internalization efficiency of ZnS@CMC quantum dots by malignant glioma cells U-87 MG cell lines. In addition, the fluorescent emission of the quantum dots was detected mostly scattered in the cytoplasm of the cells, and it is clearly observed that U-87 MG malignant glioma cancer cells exhibit higher lumines­ cence intensity when compared to HEK 237T cells. This may be explained by the accelerated metabolism associated with cancer cells when compared to normal cells [55]. It should be noted that, although beyond the scope of this study, specific targeting to tumor cells could be achieved by the bio­ functionalization of the ZnS@CMC nanoconjugates. CMC biopolymer possesses functional reactive sites (hydroxyl and carboxylate groups) that could be covalent or electrostatically bonded to affinity bio­ molecules (e.g., proteins, receptors, antibodies, etc.) that can endow specific target properties to the nanoprobe. Thus, these findings are very important because not only they prove the feasibility of using these ZnS@CMC nanoconjugates as fluorescent biological probes for bio­ imaging live cells but also offer great potential to be expanded for the early and reliable diagnosis of cancers, using simple, green and cyto­ compatible nanoplatforms, which will undoubtedly be addressed in future studies for active targeting of cancer cells.

Funding sources This work was supported by the following Brazilian research agencies: CAPES (PROEX- 433/2010; PNPD; PROINFRA2010–2014); 13

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FAPEMIG (PPM-00760-16; UNIVERSAL-APQ-00291-18; PROBIC-2018); CNPq (PQ1B-306306/2014-0; PQ1A-303893/2018-4; UNIVERSAL457537/2014-0; 421312/2018-1; PIBIC-2017-18; GM/GD 140775/ 2016-1; 140810/2015-3); and FINEP (CTINFRA-PROINFRA 2008/ 2010/2011/2018).

[18] A.A.P. Mansur, H.S. Mansur, S.M. Carvalho, A.J. Caires, One-pot aqueous synthesis of fluorescent Ag-in-Zn-S quantum dot/polymer bioconjugates for multiplex optical bioimaging of glioblastoma cells, Contrast Media Mol. Imaging (2017) 3896107, https://doi.org/10.1155/2017/3896107, 2017. [19] Y. Su, I. Cockerill, Y. Wang, Y.-X. Qin, L. Chang, Y. Zheng, D. Zhu, Zinc-based biomaterials for regeneration and therapy, Trends Biotechnol. 37 (2019) 428–441, https://doi.org/10.1016/j.tibtech.2018.10.009. [20] F.P. Ramanery, A.A.P. Mansur, H.S. Mansur, One-step colloidal synthesis of biocompatible water-soluble ZnS quantum dot/chitosan nanoconjugates, Nanoscale Res. Lett. 8 (2013) 512, https://doi.org/10.1186/1556-276X-8-512. [21] F.P. Ramanery, A.A.P. Mansur, F.G.L.M. Borsagli, H.S. Mansur, Green and facile synthesis of water-soluble ZnS quantum dots nanohybrids using chitosan derivative ligands, J. Nano Res. 16 (2014) 16, https://doi.org/10.1007/s11051-014-2504-1. [22] A.A.P. Mansur, H.S. Mansur, F.P. Ramanery, L.C. Oliveira, P.P. Souza, “Green” colloidal ZnS quantum dots/chitosan nano-photocatalysts for advanced oxidation processes: study of the photodegradation of organic dye pollutants, Appl. Catal., B 158–159 (2014) 269–279, https://doi.org/10.1016/j.apcatb.2014.04.026. [23] A.A.P. Mansur, H.S. Mansur, S.M. Carvalho, Z.I.P. Lobato, M.F. Leite, L.L. Mansur, Fluorescent ZnS quantum dots-phosphoethanolamine nanoconjugates for bioimaging live cells in cancer research, ACS Omega 3 (2018) 15679–15691, https://doi.org/10.1021/acsomega.8b02098. [24] A.A.P. Mansur, F.G. de Carvalho, R.L. Mansur, S.M. Carvalho, L. Carlos de Oliveira, H.S. Mansur, Carboxymethylcellulose/ZnCdS fluorescent quantum dot nanoconjugates for cancer cell bioimaging, Int. J. Biol. Macromol. 96 (2017) 675–686, https://doi.org/10.1016/j.ijbiomac.2016.12.078. [25] M. Shamsipur, H.R. Rajabi, Pure zinc sulfide quantum dot as highly selective luminescent probe for determination of hazardous cyanide ion, Mater. Sci. Eng. C 36 (2014) 139–145, https://doi.org/10.1016/j.msec.2013.12.001. [26] H.R. Rajabi, M. Shamsipur, A.A. Khosravi, O. Khani, M.H. Yousefi, Selective spectrofluorimetric determination of sulfide ion using manganese doped ZnS quantum dots as luminescent probe, Spectrochim. Acta, Part A 107 (2013) 256–262, https://doi.org/10.1016/j.saa.2013.01.045. [27] H.R. Rajabi, M. Farsi, Study of capping agent effect on the structural, optical and photocatalytic properties of zinc sulfide quantum dots, Mater. Sci. Semicond. Process. 48 (2016) 14–22, https://doi.org/10.1016/j.mssp.2016.02.021. [28] H.R. Rajabi, F. Karimi, H. Kazemdehdashti, L. Kavoshi, Fast sonochemicallyassisted synthesis of pure and doped zinc sulfide quantum dots and their applicability in organic dye removal from aqueous media, J. Photochem. Photobiol. B Biol. 181 (2018) 98–105, https://doi.org/10.1016/j. jphotobiol.2018.02.016. [29] N.T.K. Thanh, N. Maclean, S. Mahiddine, Mechanisms of nucleation and growth of nanoparticles in solution, Chem. Rev. 114 (2014) 7610–7630, https://doi.org/ 10.1021/cr400544s. [30] A.A.P. Mansur, H.S. Mansur, A.J. Caires, R.L. Mansur, L.C. Oliveira, Compositiontunable optical properties of ZnxCd(1 x)S quantum dot–carboxymethylcellulose conjugates: towards one-pot green synthesis of multifunctional nanoplatforms for biomedical and environmental applications, Nanoscale Res. Lett. 12 (2017) 443, https://doi.org/10.1186/s11671-017-2212-8. [31] A.N. Fletcher, Quinine sulfate as a fluorescence quantum yield standard, Photochem. Photobiol. 9 (1969) 439–444, https://doi.org/10.1111/j.17511097.1969.tb07311.x. [32] A.M. Brouwer, Standards for photoluminescence quantum yield measurements in solution, Pure Appl. Chem. 83 (2011) 2213–2228, https://doi.org/10.1351/PACREP-10-09-31. [33] Y. Gong, D. Zhao, In situ immobilization of mercury in water, soil, and sediment using carboxymethyl cellulose stabilized iron sulfide nanoparticles, in: S. Ahuja, K. Hristovski (Eds.), Novel Solutions to Water Pollution, ACS Symposium Series vol. 1123, 2013, pp. 61–77. Washington. [34] L.G. Sillen, A.E. Martell, Stability constants of metal-ion complexes, in: Lange’s Handbook, The Chemical Society, London, 1964, 8.6–8.11. [35] H.S. Mansur, Quantum Dots and Nanocomposites, vol. 2, Wiley Interdiscip, 2010, pp. 113–129, https://doi.org/10.1002/wnan.78. Rev.: Nanomed. Nanobiotechnol. [36] H.S. Mansur, A.A.P. Mansur, A. Soriano-Araújo, Z.I.P. Lobato, Beyond biocompatibility: an approach for the synthesis of ZnS quantum dot-chitosan nanoimmunoconjugates for cancer diagnosis, Green Chem. 17 (2015) 1820–1830, https://doi.org/10.1039/C4GC02072C. [37] J. Tauc, A. Menth, States in the gap, J. Non-Cryst. Solids 8–10 (1972) 569–585, https://doi.org/10.1016/0022-3093(72)90194-9. [38] M. Mall, L. Kumar, Optical studies of Cd2þ and Mn2þ Co-doped ZnS nanocrystals, J. Lumin. 130 (2010) 660–665, https://doi.org/10.1016/j.jlumin.2009.11.012. [39] R.K. Rana, L. Zhang, J.C. Yu, Y. Mastai, A. Gedanken, Mesoporous structures from supramolecular assembly of in situ generated ZnS nanoparticles, Langmuir 19 (2003) 5904–5911, https://doi.org/10.1021/la0343627. [40] M.A. Osman, A.A. Othman, W.A. El-Said, A.G. Abd-Elrahim, A.A. Abu-sehly, Thermal annealing and UV irradiation effects on structure, morphology, photoluminescence and optical absorption spectra of EDTA-capped ZnS nanoparticles, J. Phys. D Appl. Phys. 49 (2016), https://doi.org/10.1088/00223727/49/5/055304, 055304. [41] R. Chen, D. Li, B. Liu, Z. Peng, G.G. Gurzadyan, O. Xiong, H. Sun, Optical and excitonic properties of crystalline ZnS nanowires: toward efficient ultraviolet emission at room temperature, Nano Lett. 10 (2010) 4956–4961, https://doi.org/ 10.1021/nl102987z. [42] S. Wageh, Z.S. Ling, X. Xu-Rong, Growth and optical properties of colloidal ZnS nanoparticles, J. Cryst. Growth 255 (2003) 332–337, https://doi.org/10.1016/ S0022-0248(03)01258-2.

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors acknowledge the financial support from the Brazilian research agencies (CNPq, CAPES, FAPEMIG, FINEP). The authors ex­ press their gratitude to the staff at the Center of Nanoscience, Nano­ technology and Innovation-CeNano2I/CEMUCASI/UFMG for spectroscopy analyses and to the staff at the Microscopy Center at UFMG for their assistance with TEM analysis. References [1] V.P. Sharma, U. Sharma, Ma Chattopadhyay, V.N. Shukla, Advance applications of nanomaterials: a review, Mater. Today: SAVE Proc. 5 (2018) 6376–6380, https:// doi.org/10.1016/j.matpr.2017.12.248. [2] W.R. Algar, K. Susumu, J.B. Delehanty, I.L. Medintz, Semiconductor quantum dots in bioanalysis: crossing the valley of death, Anal. Chem. 83 (2011) 8826–8837, https://doi.org/10.1021/ac201331r. [3] M.A. Boles, D. Ling, T. Hyeon, D.V. Talapin, The surface science of nanocrystals, Nat. Mater. 15 (2016) 141–153, https://doi.org/10.1038/nmat4526. [4] A.A.P. Mansur, H.S. Mansur, R.L. Mansur, F.G. de Carvalho, S.M. Carvalho, Bioengineered II–VI semiconductor quantum dot–carboxymethylcellulose nanoconjugates as multifunctional fluorescent nanoprobes for bioimaging live cells, Spectrochim. Acta, Part A 189 (2018) 393–404, https://doi.org/10.1016/j. saa.2017.08.049. [5] Z. Jin, N. Hildebrandt, Semiconductor quantum dots for in vitro diagnostics and cellular imaging, Trends Biotechnol. 30 (2012) 394–403, https://doi.org/10.1016/ j.tibtech.2012.04.005. [6] K.J. McHugh, L.H. Jing, A.M. Behrens, S. Jayawardena, W. Tang, M.Y. Gao, R. Langer, A. Jaklenec, Biocompatible semiconductor quantum dots as cancer imaging agents, Adv. Mater. 30 (2018) 1706356, https://doi.org/10.1002/ adma.201706356. [7] B. Liu, B. Jiang, Z. Zheng, T. Liu, Semiconductor quantum dots in tumor research, J. Lumin. 209 (2019) 61–68, https://doi.org/10.1016/j.jlumin.2019.01.011. [8] J. Owen, L. Brus, Chemical synthesis and luminescence applications of colloidal semiconductor quantum dots, J. Am. Chem. Soc. 139 (2017) 10939–10943, https://doi.org/10.1021/jacs.7b05267. [9] X. Qiu, N. Hildebrandt, Rapid and multiplexed MicroRNA diagnostic assay using quantum dot-based f€ orster resonance energy transfer, ACS Nano 9 (2015) 8449–8457, https://doi.org/10.1021/acsnano.5b03364. [10] J. Yao, P. Li, L. Li, M. Yang, Biochemistry and biomedicine of quantum dots: from biodetection to bioimaging, drug discovery, diagnostics, therapy, Acta Biomater. 74 (2018) 36–55, https://doi.org/10.1016/j.actbio.2018.05.004. [11] G. Whitesides, Nanoscience, Nanotechnology, and Chemistry, Small 1 (2005) 172–179, https://doi.org/10.1002/smll.200400130. https://search.crossref.org/? q¼Nanoscience%2CþNanotechnology%2CþandþChemistry%2CþSmall. [12] Y. Pu, F. Cai, D. Wang, J.-X. Wang, J.-F. Chen, Colloidal synthesis of semiconductor quantum dots toward large-scale production: a review, Ind. Eng. Chem. Res. 57 (2018) 1790–1802, https://doi.org/10.1021/acs.iecr.7b04836. [13] H.S. Mansur, A.A.P. Mansur, J.C. Gonz� alez, Synthesis and characterization of CdS quantum dots with carboxylic-functionalized poly (vinyl alcohol) for bioconjugation, Polymer 52 (2011) 1045–1054, https://doi.org/10.1016/j. polymer.2011.01.004. [14] H.S. Mansur, A.A.P. Mansur, CdSe quantum dots stabilized by carboxylicfunctionalized PVA: synthesis and UV–vis spectroscopy characterization, Mater. Chem. Phys. 125 (2011) 709–717, https://doi.org/10.1016/j. matchemphys.2010.09.068. � Jurgelen _ e, _ M. Stankevi�cius, M. Stankevi�ci� _ N. Kazlauskiene, _ [15] R. Rotomskis, Z. ute, _ V. Kulvietis, V. Karabanovas, Interaction of K. Jok�sas, D. Montvydiene, carboxylated CdSe/ZnS quantum dots with fish embryos: towards understanding of nanoparticles toxicity, Sci. Total Environ. 635 (2018) 1280–1291, https://doi.org/ 10.1016/j.scitotenv.2018.04.206. [16] D. Saikia, S. Chakravarty, N.S. Sarma, S. Bhattacharjee, P. Datta, N.N. Adhikary, Aqueous synthesis of highly stable CdTe/ZnS Core/Shell quantum dots for bioimaging, Luminescence 32 (2017) 401–408, https://doi.org/10.1002/bio.3193. [17] R.E. Bailey, S. Nie, Alloyed semiconductor quantum Dots: tuning the optical properties without changing the particle size, J. Am. Chem. Soc. 125 (2003) 7100–7106, https://doi.org/10.1021/ja035000o.

14

A.J. Caires et al.

Materials Chemistry and Physics 244 (2020) 122716

[43] W.G. Becker, A.J. Bard, Photoluminescence and photoinduced oxygen adsorption of colloidal zinc sulfide dispersions, J. Phys. Chem. 87 (1983) 4888–4893, https:// doi.org/10.1021/j150642a026. [44] D. Denzler, M. Olschewski, K. Sattler, Luminescence studies of localized gap states in colloidal ZnS nanocrystals, J. Appl. Phys. 84 (1998) 2841–2845, https://doi. org/10.1063/1.368425. [45] G. Xu, S. Zeng, B. Zhang, M.T. Swihart, K.-T. Yong, P.N. Prasad, New generation cadmium-free quantum dots for biophotonics and nanomedicine, Chem. Rev. 116 (2016) 12234–12327, https://doi.org/10.1021/acs.chemrev.6b00290. [46] J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-Ray Photoelectron Spectroscopy, Perkin-Elmer Corporation, Eden Prairie, Minnesota, USA, 1992. [47] G. Zorn, S.R. Dave, T. Weidner, X. Gao, D.G. Castner, Direct characterization of polymer encapsulated CdSe/CdS/ZnS quantum dots, Surf. Sci. 648 (2016) 339–344, https://doi.org/10.1016/j.susc.2015.10.013. [48] D. Barreca, A. Gasparotto, C. Maragno, E. Tondello, T.R. Spalding, Analysis of nanocrystalline ZnS thin films by XPS, Surf. Sci. Spectra 9 (2002) 51–64, https:// doi.org/10.1116/11.20030117. [49] M.C. L� opez, J.P. Espinos, F. Martín, D. Leinen, J.R. Ramos-Barrado, Growth of ZnS thin films obtained by chemical spray pyrolysis: the influence of precursors, J. Cryst. Growth 285 (2008) 66–75, https://doi.org/10.1016/j. jcrysgro.2005.07.050. [50] A.A.P. Mansur, H.S. Mansur, A. Soriano, Z.I.P. Lobato, Fluorescent nanohybrids based on quantum dot–chitosan–antibody as potential cancer biomarkers, ACS

[51]

[52] [53] [54]

[55]

15

Appl. Mater. Interfaces 6 (2014) 11403–11412, https://doi.org/10.1021/ am5019989. N.S.V. Capanema, A.A.P. Mansur, A.C. de Jesus, S.M. Carvalho, L.C. de Oliveira, H. S. Mansur, Superabsorbent crosslinked carboxymethyl cellulose-PEG hydrogels for potential wound dressing applications, Int. J. Biol. Macromol. 106 (2017) 1218–1234, https://doi.org/10.1016/j.ijbiomac.2017.08.124. M.F. Shukur, R. Ithnin, M.F.Z. Kadir, Electrical properties of proton conducting solid biopolymer electrolytes based on starch–chitosan blend, Ionics 20 (2014) 977–999, https://doi.org/10.1007/s11581-013-1033-8. �k, Z. Vargov� V. Zele� na a, K. Gy€ oryov� a, Correlation of infrared spectra of zinc (II) carboxylates with their structures, Spectrochim. Acta, Part A 66 (2007) 262–272, https://doi.org/10.1016/j.saa.2006.02.050. C.C.R. Sutton, G. da Silva, G.V. Franks, Modeling the IR spectra of aqueous metal carboxylate complexes: correlation between bonding geometry and stretching mode wavenumber shifts, Chem. Eur J. 21 (2015) 6801–6805, https://doi.org/ 10.1002/chem.201406516. A.A.P. Mansur, S.M. Carvalho, Z.I.P. Lobato, M.F. Leite, A.S. Cunha Jr., H. S. Mansur, Design and development of polysaccharide-doxorubicin-peptide bioconjugates for dual synergistic effects of integrin-targeted and cell-penetrating peptides for cancer chemotherapy, Bioconjugate Chem. 29 (2018) 1973–2000, https://doi.org/10.1021/acs.bioconjchem.8b00208. https://search.crossref.org/? q¼Designþandþdevelopmentþofþpolysaccharide-doxorubicin-peptideþbioconju gatesþforþdualþsynergisticþeffectsþofþintegrin-targetedþandþcell-penetrating þpeptidesþforþcancerþchemotherap.