Green synthesis of ZnS quantum dot/biopolymer photoluminescent nanoprobes for bioimaging brain cancer cells

Highlights

• Facile green synthesis of ZnS quantum dot-carboxymethylcellulose nanoconjugates. (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 nanoprobes. (73).

• Fluorescent nanoconjugates were effective for bioimaging brain cancer cells in vitro. (84).

Abstract

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 (Zn²⁺, 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 (E_bulk = 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 = 400–430 nm, at pH = 3.5, [Zn:S] ratio = 1:2). The cell viability results in vitro (>90%) demonstrated no cytotoxicity of ZnS@CMC nanohybrids towards both cell types. Importantly, these ZnS@CMC nanoconjugates behaved as active fluorescent nanoprobes for bioimaging malignant glioma cells proving the high potential for applications in cancer nanomedicine.

Introduction

Nanotechnology has emerged in recent years as a field of integrated disruptive knowledge with several prospective revolutionary applications [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 interactions with other molecules [2,3]. Optical properties such as photoluminescence 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], [5], [6], [7]]. Semiconductor quantum dots (QDs) are the most interesting luminescent nanomaterials because of their unique optoelectronic and physicochemical properties [[8], [9], [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 alternatives to produce greener QDs associated with improved emission efficiency, such as zinc-based quantum dots [[17], [18], [19], [20], [21], [22], [23], [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 nanomaterials are being used in a vast realm of applications such as luminescent nanoprobes for biomedicine, optical nanosensors, and nano-photocatalysts, as well as in FRET processes [[22], [23], [24], [25], [26], [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 nanocrystals and, therefore, controlling their optical and physicochemical properties [20,21,23,[27], [28], [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 properties can be tuned by using distinct parameters and conditions of synthesis.

Currently, biopolymers such as polysaccharides have been intensively researched for the production of novel nanomaterials. Among several alternatives, cellulose derivatives, such as carboxymethylcellulose (CMC), are promising candidates for the synthesis of semiconductor nanocrystals. CMC has been used as a simple and intrinsically biocompatible surface capping macromolecule for the synthesis of nanomaterials [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 pH-sensitive, non-toxic, biocompatible and environmentally friendly. Thus, these characteristics of CMC offer numerous possibilities for biomedical applications such as functional capping ligands for developing luminescent nanoprobes directed to cancer research [4,18,24].

Considering serious chronic and devastating illnesses, cancer remains one of the deadliest diseases worldwide, and glioblastoma is regarded as the utmost common and aggressive malignant brain tumor. 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 associated 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 functionalized by carboxymethylcellulose produced via an environmentally-friendly aqueous colloidal process at room temperature. The dependence of optical properties on the synthesis parameters, i.e., pH of the solution and concentration of precursors, were systematically investigated 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.