Water-mediated green synthesis of PbS quantum dot and its glutathione and biotin conjugates for non-invasive live cell imaging

This study addresses the cellular uptake of nanomaterials in the field of bio-applications. In the present study, we have synthesized water-soluble lead sulfide quantum dot (PbS QD) with glutathione and 3-MPA (mercaptopropionic acid) as the stabilizing ligand using a green approach. 3-MPA-capped QDs were further modified with streptavidin and then bound to biotin because of its high conjugation efficiency. Labelling and bio-imaging of cells with these bio-conjugated QDs were evaluated. The bright red fluorescence from these types of QDs in HeLa cells makes these materials suitable for deep tissue imaging.


Introduction
Although green technology is advancing at a rapid rate, it does not meet the current demand in medicine and health field. The most challenging task is to diagnose diseases, including human immunodeficiency virus, Zika virus and cancer, using simple and cost-effective materials. With the advances in the field of nanoscience and nanotechnology, the above problem could be solved to some extent. Design and synthesis of bioconjugated quantum dots (QDs) is a hot field in current medical research because of their vast advantages over organic dyes [1].
Semiconductor QD has immeasurable applications in the medical field due to its various advantages such as broad excitation spectra, narrow emission bands, tunable emission peaks with respect to QD size, long fluorescence lifetimes, negligible photobleaching [2][3][4]. QD has high quantum yield, high molar extinction coefficients [3][4][5], large effective Stokes shift [6], the ability to conjugate to proteins and single-particle tracking regulating photochromic fluorescence resonance energy transfer [7]. Colloidal semiconductor PbS QD, a p-type semiconductor with a direct band gap of 0.37 eV at room temperature and vast exciton Bohr radius of 18 nm [8], offers tunable luminescence over visible and near-infrared (NIR) regions (400-2500 nm) by controlling the dot size [9][10][11]. PbS QD is one of the hot topics in the field of nanoscience and technology because of its wide application in biology [12] and solar energy field [13,14]. PbS QD for anti-HER2 bioconjugates shows a promising field for NIR-targeted molecular imaging with SK-BR-3 breast cancer cells [15]. The fluorescence peak in NIR IIa region is applied for deep tissue imaging because of negligible scattering of light [12]. Potential applications in bio-imaging using PbS and Ag 2 S QD in the second NIR window are reported in various aspects such as reduced toxicity [16] and KPL-4 cells with in vivo dual fluorescence [17]. RNase-A-capped PbS QD is applied for deep tissue imaging with ultra-low concentration with excellent fluorescence and reduced cytotoxicity [18]. A green strategy has been developed for the synthesis of whey-protein-capped PbS QD reusing unreacted precursors for bio-imaging application in the second NIR window [19]. Enhanced fluorescence of PbS QD by employing self-assembled molecular J-aggregates was also highlighted [20]. This type of QD has been applied for bioimaging and photoacoustic imaging [21,22].
Xu et al. compared the capping effect of PbS with those of benzodithiol, 1,2-ethanedithiol and mercaptopropionic acid (MPA) ligands, where PbS is passivated with MPA ligand, shortens the interdot separation, enhances the QD passivation and emission [23]. PbS QD capped with β-lactoglobulin was synthesized in an aqueous medium using microwave and examined on 293T cells in the second NIR optical window [24]. Kim et al. reported the cytotoxicity of bare PbS-MPA QD on human kidney cells (HEK 293) [25]. Hence, further surface modification of PbS-MPA QD is necessary for the diagnosis and selective delivery of a drug in cancer cells.
Sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene) is an essential transmembrane protein responsible for translocation of vitamins and other essential cofactors such as biotin, pantothenic acid and lipoic acid [26]. As cancer cells have higher SMVT expression or higher biotin uptake capability than normal cells [26], the diagnosis of cancer and uptake of a drug in the cancer cells will be effective using biotin-conjugated QDs. While the affinity of streptavidin (SA) and biotin is high, the biotin conjugation to QD occurs via SA modification on QD [27]. Biotin conjugation to SA for biological imaging, sensing and target delivery has already been reported [28,29].
However, PbS QDs coated with thiol compounds, such as 1-thioglycerol and dithioglycerol, are nonbiocompatible and cytotoxic. Intracellular glutathione is the most abundant non-protein mono-thiol (1-10 mM) in cells that maintains intramolecular redox homeostasis through an equilibrium between its reduced thiol form (GSH) and its oxidized disulfide form [26,34,35]. To maintain the intramolecular redox homeostasis and to enhance the bio-imaging property of PbS QD, the antioxidant GSH modification on the surface of QD is highly warranted. We, therefore, considered GSH as a coating agent for the preparation of biocompatible second NIR-emitting PbS QDs. In this paper, we have synthesized PbS QD, capped with 3-MPA and the good biocompatible ligand GSH. In addition, surface modifications of MPA-coated QD were also performed with SA followed by conjugation with biotin. GSH-coated QDs can also be conjugated to biomolecules such as an antibody as they have two carboxyls and one primary amino group in their surface [36].

Results and discussion
In continuation of our previous work [37], herein, we have used a sonication technique for the preparation of PbS-MPA and PbS-GSH QDs for further bioconjugation. Firstly, the sulfur and lead precursors were prepared by dissolving sodium sulfide and lead acetate trihydrate in deionized water. The synthesis was carried out separately for 3-MPA-capped and GSH-capped PbS QDs. Lead precursor was added to 3-MPA and GSH solutions by maintaining the required pH; after which sulfur precursor was added, turning both solutions darkish brown indicating the formation of PbS QD. The reaction was continued under sonication for appropriate time for the growth of PbS QD under nitrogen protection.   After that, the reaction mixture was cooled down to room temperature, centrifuged and dried for further use. Figure 1a demonstrates the absorption spectra of 3-MPA-capped PbS QD with an absorption peak centred at 550 nm. No other visible absorption peaks can be seen which implies that the transitions, which can be attributed to 1se-1sh transitions individually, are occurring from other quantized states of the QD. More excitonic peaks in the absorption spectrum of PbS-GSH are attributed to the good crystalline quality of the nanoparticles. Broad absorption spectrum in the visible region indicates the fluorescence efficiency of PbS-GSH in the second NIR optical window which is very useful for deep tissue in vivo imaging (figure 1b). All these exciton transitions also demonstrate great quantum confinement impact as contrasted with bulk PbS. The absorption spectra started from the UV-visible region and extended up to 1800 nm (electronic supplementary material, figure S1), indicating the blue shift and significant quantum size. Nanocrystals exhibited threshold energy in the optical absorption measurement, which could be affected by blue shifting of the absorption edge with decreasing particle size. SA modification in 3-MPA-capped PbS QD occurred through EDC coupling reaction. SA-modified PbS QD was further conjugated with biotin. In the absorption spectra (figure 1a), it was observed that 3-MPA-capped PbS QD has a higher absorbance value at 550 nm than SA-modified PbS QD. Next orange graph is for biotin linked to PbS-SA QD shows slightly less intensity than PbS-SA QD, but both have the same wavelength of 550 nm [38].
In the fluorescence (PL) spectra in figure 2a, the PbS-MPA QD emitted at 925 nm when excited at 550 nm, showing emission in the first NIR optical window which is suitable for bio-imaging. Chu et al. discovered that PL intensity increases, when the QD is synthesized in alkaline medium [39]. Herein, the PbS-MPA QD was synthesized in basic pH medium and then conjugated with SA using EDC coupling and further linked with biotin for efficient cellular uptake of the QD, having the emission at 925 nm ( figure 2a). We also observed that PL spectra for PbS-SA and PbS-SA-biotin show fluorescence quenching. In figure 2b, emission of GSH-modified PbS was observed at 1010 nm when excited at 725 nm. The   emission is in the second NIR-a optical window and the same can be extended up to the third NIR (1600-1870 nm) window with different excitation values, provided we have a source for excitation. Figure 3 shows the powder X-ray diffraction (XRD) pattern for PbS QD, which confirms the crystalline structure of the PbS-MPA and PbS-GSH QDs. The broad peak at 2θ = 30.342°indicates the 1-10 nm nano size range of the QD. As revealed by the diffraction patterns, the peaks are at 25°,   For cellular delivery of the nanomaterials, size is one of the main criteria which can be determined by transmission electron microscopy (TEM). High-resolution transmission electron microscopy (HRTEM) confirms the shape and size of the QD. Figure 5a shows the TEM image of PbS-MPA; the image indicates the particle is spherical, and has good homogeneity in the particle size distribution. Figure 5b shows the HRTEM image of the sample giving a clear indication of the size of 6 ± 0.5% nm and lattice plane in each QD. Figure 5c gives the shape and size of PbS-GSH QD of about 5 ± 0.5% nm which can be correlated with XRD data. Stability of the PbS QD was also well correlated with the zeta potential value (electronic supplementary material, figure S2).
Electronic supplementary material, figure S3, shows Fourier transform infrared (FTIR) spectra of surface-modified PbS QD in the range from 4000 cm −1 to 500 cm −1 . In the electronic supplementary material, figure  Undoubtedly, cellular imaging is one of the essential practices in biology and medicine, and it is a vital method for cellular analysis, especially analysis of biological processes in cells. However, two major factors could control the usage of QDs in cellular imaging: one is cytotoxicity and the other is fluorescence stability. Fluorescence stability was evaluated by time-dependent fluorescence spectroscopy. It is shown in electronic supplementary material, figure S4, that with the extension of storage time, the fluorescence intensities of GSH-modified PbS QD and MPA-capped SA-biotin-modified PbS QD are increasing which sustain in their imaging efficiency. This is because of surface capping or surface modification [40]. The fluorescence stability of GSH-capped CdTe QD was highlighted by Yuan et al. [41]. Likewise, good photostability of CdSe QD was reported in a review article [42]. Long-term fluorescence retention of colloidal PbS QD was also described by Pichaandi et al. [43].
The cytotoxicity of as-prepared PbS QDs (PbS-SA-biotin and PbS-GSH), which was used to evaluate their biocompatibility, was studied using cervical cancer cell HeLa and human embryonic kidney cell

Conclusion
In  without affecting the normal cells. The bright red fluorescence from these types of surface-modified QDs in HeLa cells makes these materials suitable for deep tissue imaging.

Experimental section
4.1. Synthesis of glutathione-modified PbS quantum dot GSH modified PbS was synthesized as per the following protocol. Firstly, 4 ml of 0.1 M L-glutathione was dissolved in 50 ml of Milli-Q water in a two-necked flask, stirred for 10 min under N 2 protection by adjusting pH of the solution to 10 using 1N NaOH solution and then the solution was further stirred for another 10 min. One millilitre of 0.1 M lead precursor was added to the above mixture and stirred for another 10 min, after which 1 ml of 0.1 M sulfur precursor was added and immediately the pH was adjusted to 11 using NaOH solution and then stirred for 10 min with a Pb : S ratio of 1 : 1. The solution turned dark brown. To enhance the PL of the QD, another 1 ml of lead precursor was added to the above solution again by adjusting the pH to 11, the final ratio of GSH : Pb : S being 4 : 2 : 1. The final solution turned dark brown which confirmed the formation of GSH-modified PbS nanocrystals. For further growth of QDs, the solution was bath sonicated for 30 min at 50°C. After sonication, QDs were precipitated with an equivalent amount of 2-propanol, followed by resuspension in a minimal amount of ultrapure water. Excess salts were removed by repeating this procedure three times, and the purified QDs were dried overnight at room temperature in vacuum.