Luminescent gold and copper nanoclusters for theranostic applications

Introduction and Literature Review 1

Introduction 3

Due to its small size, the nanoscale particles, including metals, metal oxides and semiconductors, exhibit high surface-to-volume ratios with distinct physical, chemical, optical, electrical and biological properties1–6 with widespread applications in the fields of opto- electronics,7,8 catalysis,9,10 sensors,11 white light emission,12,13 bioimaging,14–16 biosensing,15,16 medicine,17,18 nanobiotechnology.19 Nanobiotechnology has applied the nanoscale principles and techniques to create new biomaterials that have extensive applications in the field. of biology and biomedical healthcare, such as diagnostics (imaging), detection of pathogens, biosensing, nanomedicine, drug delivery, gene delivery and tissue engineering.20.

Nanoparticles 3

Bright fluorescence was observed under trans UV illuminator (Excitation 305 nm). The luminescence peak was obtained at 582 nm. Similarly, the emission spectrum of the Dox control, i.e., the initial concentration used during NP synthesis was recorded (λex = 480 nm).

Figure 1.3.Illustration of essential steps for the synthesis of Cu NCs taking protein as a template

Metal Nanoclusters 4

Applications of Metal Nanoclusters 10

Key Areas and Scopes 20

Exploration of the application of new metal nanoclusters in the field of energy (white light emission). The design of biocompatible biopolymer and drug-based theranostic nanocarriers for improved in vitro and in vivo combination therapy for cancer.

Significance and the salient features of the present study 20

The role of ART and NaB in DNA damage and HDAC inhibition resulting in cell death was observed both in vitro and in vivo. Multifunctional targeted nanoparticle system or nanodrug using stable blue-emitting transferrin-stabilized copper nanoclusters in combination with the drug doxorubicin as precursors (Tf-CuNC-Dox-NPs) has been formulated for diagnostic imaging, targeted drug delivery assisted via FRET, and therapy for in vitro as well as in vivo .

In Chapter 2, a novel, facile synthesis of luminescent gold nanoclusters (Au NCs) using bacteria as a template is described. Furthermore, it was used to detect bacterial contaminants from water sources and kanamycin-resistant strains.

Introduction 31

Interestingly, the luminescence intensity of the Au NCs changes with the number of bacteria, providing a rapid method to enumerate the number of bacteria present in the samples. Therefore, the current study emphasizes the development of a simple, rapid detection method for point of care diagnosis and prognosis based on the luminescence property of the Au NCs on the bacterial surface.

Outline of the Present Work 32

The method is very versatile, where gram positive and gram negative bacteria have been used to synthesize Au NCs and at the same time this method can be used to detect bacterial contamination in various water sources (samples) based on its luminescence . This rapid synthesis of Au NCs on the bacterial template attributes a facile and rapid method for the enumeration, detection of bacterial contaminants and kanamycin-resistant strains.

Experimental Section 33

The current method could also rapidly detect bacterial contaminants from water sources and kanamycin-resistant strains.

Characterization 34

For the Delta Vision Deconvolution Microscope (GE Healthcare), imaging was performed in agar pads prepared with 0.6% agar in LB medium, where 10 µL of Au NCs synthesized bacteria were drop-cast for imaging. To study the cell viability of Au NCs synthesized bacteria, MTT assay was performed after 24 h treatment on HEK-293 cells.

Results and Discussions 37

The formation of the Au NCs was confirmed by fluorescence spectroscopy by observing the luminescence peak at 580 nm after the formation of Au NCs (Figure 2.2a); However, the plasmon peak around 520 nm, which is a typical signature of the gold nanoparticles (Au NPs), was not seen in UV-Vis spectroscopy (Figure 2.2b). The control bacteria, i.e. without Au NCs synthesis has no peak in red region, whereas after synthesis of the Au NCs, strong luminescence was observed at 580 nm when excited at 320 nm (Figure 2.2a). a) Luminescence spectra of control bacteria and Au NCs synthesized on bacteria together with the inset image (b) UV-Vis spectra of Au NCs synthesized on bacteria. In this respect, white light emitting (WLE) materials that provide energy efficiency are a research area where new ideas could make a potential difference to the technology and devices.5-7 The importance of white light emitting materials lies in their potential as the main component of displays and light-emitting devices (LEDs).8 A general strategy that has been widely used to achieve white light is. If, for example, inorganic nanocrystals (such as ZnS) could be surface functionalized with inorganic complexes – in addition to being doped with cations such as Mn2+ – the total emission from the single nanoparticle could be white light.10 Furthermore, the color and other indexes could be tuned not only based on the doping substances, but also on the concentration and nature of complex(es) present on the surface.

It was found that there was a rapid increase in liver function marker enzymes (SGOT, SGPT & .ALP) in the case of DLA-induced mice compared to that of normal mice (Table A 4.4, Appendix). Treatment with Au NCs also revealed improved liver function marker enzymes. Moreover, the histogram plot of the as-synthesized Tf-Cu NC-Dox NPs is shown in Figure 5.4d. This is mainly due to the selective targeting of the Tf-Cu NC-Dox NPs.

Figure 2.2. (a) Luminescence spectra of control bacterium and Au NCs synthesized on bacterium along with the inset image (b) UV-Vis spectra of Au NCs synthesized on bacterium

Conclusion 52

Combination index (CI) revealed synergistic activity of Tf-Cu NCs and Dox in Tf-Cu NC-Dox NPs. The emission intensities of the supernatant of Tf-Cu NC−Dox NPs and Dox only at λem= 590 nm were then measured. The overgrown HeLa cells during treatment with transferrin inhibitor blocked the uptake of Tf-Cu NC−Dox NPs.

Cells treated with Tf-Cu NC-Dox NPs showed a prominent shift compared to the control cells. The uptake of Tf-Cu NC-Dox NPs on DLA cells was confirmed by confocal microscopy (Figure 5.15b). FESEM images of (a) control DLA cells without treatment and DLA cells after 16 days of treatment with (b) Tf-Cu NCs, (c) Dox, and (d) Tf-Cu NC-Dox NPs.

Table S1. Surface roughness and indentation of statistics of control bacteria and bacteria after Au NCs synthesis quantified using Gwiddion software analysis

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Introduction 59

As global consumption increases, conventional energy-intensive incandescent lights are being phased out in favor of devices using more efficient advanced light-emitting materials.1–4 For example, current stress on the development of luminescent quantum dots, carbon dots, and atoms metal-based nanoclusters (NCs) as powerful nanoscale light-emitting materials are driven by achievable high emission/quantum efficiency and control over chromaticity, color rendering indices, and correlated color temperature. A way out of this limitation could be based on the development of composite materials with many components (mainly inorganic), which would emit white light as a combination of the basic colors from the components. On the other hand, nanomaterial-based white light-emitting organic sources, such as carbon quantum dots and graphene, are also in focus for development.11 Moreover, single organic molecules have been reported recently.

On the other hand, for stability and ease of adaptability of color indices, inorganic components can be essential. The luminescent properties of the clusters can be modified by using appropriate stabilizing ligands during synthesis or by forming an assembly.17 Importantly, while there have been a growing number of reports on their utility as probes for sensing and analysis, the focus is on the use their as powerful light-emitting materials have not yet begun. It could be remarkable if light-emitting materials could be obtained using biochemical pathways involving prokaryotic cells, which are easy enough to grow in the laboratory.

Outline of the Present Work 61

Experimental Section 61

To synthesize Au NCs on bacterial template, 18 µl of HAuCl4 (10 mM) and 6 µl of 0.11 M MPA were added to a dispersion containing bacteria. The pellet was then redispersed in water after repeated washing and used for further experiments. The quantum yield of white luminescent bacteria was calculated based on quinine sulfate in 0.1 M H2SO4.

Characterization 63

The as-synthesized Au NCs on bacteria were cast onto a glass slide and covered with a coverslip that was further sealed on both sides using nail paint.

Results and Discussions 64

It is interesting to observe that the peak due to GFP (in the bacteria) at 510 nm (Figure 3.1e) was no longer prominent in the spectrum of the reaction product. a) UV-Vis spectrum of Au NCs synthesized on GFP-expressing E.Coli. FESEM image of (a) control GFP-expressing E.Coli (b) High magnification image of (a) FESEM image of (c) GFP-expressing E.Coli after synthesis of Au NCs and (d) High magnification image of (c ). CLSM image of Au NCs synthesized on GFP-expressing E.Coli after 405 nm laser excitation in the (a) green (b) blue and (c) red channels.

In a related vein, the mixture of Au NCs synthesized on GFP-expressing bacteria and control GFP bacteria was studied under CLSM. CLSM images of GFP-expressing bacteria after the synthesis of Au nanoclusters obtained under different thermal conditions. CLSM images of Escherichia coli expressing GFP after the synthesis of Au nanoclusters obtained at different time intervals.

Figure 3.1. (a) UV-Vis spectrum of Au NCs synthesized on GFP-expressing E.Coli. (b) UV- UV-Vis absorbance spectrum of GFP-expressing E.Coli

Conclusion 74

The as-synthesized Tf-Cu NC−Dox NPs were then collected by centrifugation at 10,000 rpm for 30 min. The Tf-Cu NC−Dox NPs were then collected after centrifugation at 10,000 rpm for 30 min. The FESEM image (Figure 5.6b, Appendix) of Tf-Cu NC− Dox NPs revealed an average particle size of 47.79.

Bright field (a and c) and confocal microscopy (b and d) images of 3T3-L1 and HeLa cells treated with Tf-Cu NC-Dox-NPs taken after 4 h of treatment. The HeLa cells were incubated at different concentrations of Tf-CuNCs, free Dox and Tf-Cu NC-Dox NPs for 48 h at 37 °C. Interestingly, the same concentration of Dox on cells treated with Tf-Cu NC−Dox NPs viability of only 22%.

For this, HeLa cells were treated with an IC50 dose of Tf-Cu NC-Dox NPs with respective Tf-Cu NC and Dox concentrations as mentioned above. In contrast, more blistering and deformation was observed in the case of cells treated with Tf-Cu NC-Dox NPs (Figure 5.11c). In contrast, Tf-Cu NCs and Dox induced less oxidative stress than Tf-Cu NC-Dox NPs.

More importantly, the hepatic physiology of Tf-Cu NC-Dox NP-treated DLA mice was comparable to normal mice.