This thesis was submitted by me to the Department of Chemistry, Indian Institute of Technology Guwahati for the award of the degree of Doctor of Philosophy. She was, is and will continue to be the best thing in my life.
Catalysis
Therapeutics and theranostics
Moreover, NCs that are a functional component in the nanocomposite can be used as an optical and flow cytometric (FACS) probe.34 As per another report, multifunctional nanocomposites consisting of Au NCs can be loaded with doxorubicin and used for theranostic purposes. In addition, the fabricated nanocomposite can be used to investigate cancer cells using one and two photon excitations.35 On the other hand, it has been demonstrated that copper nanoclusters generate reactive oxygen species (ROS).
Assembly of Nanoclusters
Ligand mediated assembly of NCs
Atomically precise NCs have been crystallized in various geometries based on the interactions between the ligands that stabilize the NCs. In an allied manner, Liu and co-workers have reported the observation of Au38S2 (S-Adm) 20 body-centered structured NCs (BCCs). hcp) structure.45.
Self-assembly of NCs
Working for “A step ahead” in assembly of NCs
Despite the advantages of these methods in easily forming “assembly of NCs”, a crucial concern that continues to demand resolution is whether a method could be introduced for the formation of “assembly of NCs” that makes the assembled NCs superior could make in terms of chemical properties. and optical properties relative to the constituent NCs.
Context of the current thesis work
Early and easy disease detection, using inexpensive and point-of-care equipment, not only provides opportunities for early treatment, but also reduces the risk of spread. Additionally, the sensitivity of bilirubin detection was tuned to the amount of Cu2+ added, thus facilitating the detection of the desired bilirubin concentration range.
Experimental Section
- Materials
- Synthesis of luminescent Au nanoclusters
- Preparation of glycine buffer
- Preparation of Au nanocluster dispersion for luminescence experiment
- Preparation of bilirubin solution
- Preparation of Au nanocluster containing film
- Treatment of the Au nanocluster containing film with copper sulphate
- Interaction of bilirubin solution with copper sulphate added Au nanocluster containing film
- Interaction of copper salt with luminescent Au nanoclusters in liquid phase
- Interaction of bilirubin with copper salt added luminescent Au nanoclusters in liquid phase
- Control experiments
- Collection of serum and thumb imprint of patients affected with hyperbilirubinemia
The luminescence intensity of the film was significantly reduced (when exposed to 254 nm UV light) after addition of the copper salt. Furthermore, it was observed that addition of bilirubin to a system consisting of Cu2+ added Au nanoclusters (whose luminescence was quenched due to the presence of Cu2+ ions) led to the restoration of luminescence.
Experimental Section .1 Materials
Instruments
A Perkin Elmer 4000 was used to record the TGA spectrum of the crystal complex of gold nanoclusters. Sorption of hydrogen gas inside the crystal complex of gold nanoclusters was performed with a high-pressure gas sorption analyzer Quantachrome instruments isorb HP1.
Synthetic Methods
Results and discussions
3.6 (A) Transmission electron microscopy (TEM) image of crystalline precipitate obtained after addition of Zn2+ salt to Au14 nanoclusters in liquid medium. The crystal structure of the complex, obtained by electron diffraction and other analyses, was further confirmed by computational modeling using Avogadro.
Conclusion
3.14 (A) (a) Adsorption and (b) desorption isotherms of hydrogen on zinc-doped Au14 nanoclusters. B) Photoluminescence spectra of (a) zinc added Au14. Also, it is important to note that our findings led to a new concept of nanocluster crystallization via assembly after the complexation reaction, using metal ions and ligands on the nanoclusters, which can give rise to the rich chemistry involving the nanoclusters. noble metals. The three-dimensional organization of gold nanoclusters stabilized with tryptophan D or L and with mercaptopropionic acid has been achieved by the complexation reaction involving the ligands that stabilize the gold nanoclusters and zinc ions.
The hierarchical arrangement of nanoclusters exhibited superior optical properties (namely, enhanced photoluminescence and excited state lifetime) compared to unassembled nanoclusters. Further, the photoluminescence of the crystalline assembly of the nanoclusters served as a visual marker for chiral recognition of the D and L enantiomers of tryptophan with simultaneous separation of the corresponding enantiomer. The mechanistic aspect of cleavage complemented by chiral recognition has been proposed to occur through binding of tryptophan D and L to coordinatively unsaturated zinc ions, thereby forming supercomplexes, the degree of stabilization of the supercomplex being dictated by "three points versus two - point interaction” between enantiomers and chiral selector.
Experimental Section
Results and discusssions
The photoluminescence intensity due to Au nanoclusters was significantly enhanced after the addition of the zinc ions (Fig. 4.4 A). Moreover, the photoluminescence lifetime of the clusters showed significant improvement after the addition of zinc ions. Thus, the results of the photoluminescence studies clearly indicated significant change of the emission properties of the Au nanoclusters in the presence of Zn2+ ions.
The structure of the crystalline product was further confirmed from high resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) analyses. Computational modeling, based on 'Avogadro', further confirmed the experimentally obtained structure of the crystalline assembly. Based on the symmetry of the composition obtained from XRD analysis, the mentioned structure of Au14 clusters was adopted for further analysis.
Tryptophan and MPA are symmetrically positioned on the Au atoms of the cage-like Au14 clusters (scheme 1). A representative structure of the crystalline assembly based on experimental and computational evidence is depicted in Figure 4.5 G.
Schematic representation of (A) the structure of D tryptophan and MPA stabilized Au nanoclusters prior to and following complexation with zinc ions. (B) The three
Conclusions
In summary, we have designed a new strategy for chiral recognition as well as separation based on a novel crystalline assembly of chiral ligand stabilized Au14 nanoclusters. It is based on complexation reaction-assisted three-dimensional assembly of tryptophan (of a specific enantiomer) and MPA-stabilized Au nanoclusters. Herein, we report the formation of crystalline assembly of gold (Au) nanoclusters for cancer therapy via active targeting of mitochondria.
To the best of our knowledge, this is the first report of target-specific activity of “assembly of gold nanoclusters”. Limited solubility of L-tyrosine in methanolic solution led to the formation of polycrystalline assembly of Au nanoclusters via interactions between ligands (tyrosine) that stabilize the clusters. Furthermore, complexation reaction between zinc ions and ligands stabilizing Au nanoclusters led to the formation of single crystalline assembly of gold nanoclusters.
The crystalline assembly of Au nanoclusters, formed through ligand binding and complexation reaction, showed mitochondrial-directed anticancer activity - as confirmed by Mito tracer staining experiments. However, MTT assay, FACS analysis, and JC-1 staining experiments indicated that zinc-mediated assembly of Au nanoclusters exhibited superior therapeutic activity compared to methanol-driven cluster assembly.
Experimental .1 Materials
Confocal microscopy: To verify the internalization of Au nanocrystals and ZnAu NCs, confocal microscopy studies were performed using a Zeiss LSM 880 microscope, where 5 × 103 cells were first grown on a coverslip in a 35 mm culture plate and maintained at 5% CO2. JC-1: For JC-1 staining, live cell confocal imaging was performed where cells were grown on a special 35 mm live cell culture plate overnight. Once sufficiently confluent, the cells were treated with the IC50 dose of Au nanocrystals and Zn Au NCs for 24 h.
Then the cells were thoroughly washed twice with PBS and incubated for 10 minutes with the dye JC-1 (2.7 µM) and live cells were imaged in red and green with a diode laser λex= 525 nm and λex= 480, respectively. Mitotracker Green: For imaging with Mitotracker green, cells were incubated with Au nanocrystals and Zn Au NCs for 3 h and washed twice with PBS, followed by incubation with 300 nM mitotracker green for 30 min and fixed with 70% chilled ethanol. For this, cells were seeded at 5 × 103 cells/well in a 6-well plate and incubated overnight, followed by treatment with 77.5 µg/mL Au nanocrystals and Zn Au NCs, respectively, for 3 h.
Then, the cells were collected after trypsinization and redispersed in DMEM before being analyzed in a fluorescence-activated cell sorter (FacsCalibur, BD Biosciences, NJ) in FL1-H channel (530/30 nm), corresponding to green emission. ICP atomic emission spectrometric analysis: For ICP atomic emission spectrometric analysis, 5 x 10 HeLa cells were seeded into 6-well plates and left for 24 hours. The overgrown cells were treated with Au nanocrystals (77.5 μg/mL) and Zn Au NCs (77.5 μg/mL) for 6 h.
Results and discussions
Time-resolved photoluminescence spectra of (B) Au nanoclusters and (C) the reaction product between Au nanoclusters and zinc ions. Interestingly, TEM analysis of the centrifuged product of the reaction between Au nanoclusters and zinc ions revealed the formation of crystalline nanoparticles with distortion. The crystalline nanoparticles formed as a result of the reaction between zinc ions and Au nanoclusters are further referred to as Zn Au NCs.
Fourier transform infrared (FTIR) analysis of Au nanoclusters and the product of its reaction with zinc ions revealed the possible binding mode between the ligands that stabilize Au nanoclusters and zinc ions through carboxylate groups of MPA and Tyr. 5.9 (A) (a) Excitation and emission spectrum of mercaptonedecanoic acid stabilized Au nanoclusters (B) TEM image of mercaptonedecanoic acid stabilized Au nanoclusters. C) MTT assay-based cell viability of HeLa cells after treatment with mercaptodecanoic acid-stabilized Au nanoclusters. The results show that ultrasmall Au nanoclusters have negligible cytotoxic effect on HeLa cells.
CLSM images of Hela cells treated with JC-1 dye after treatment with zinc added Au nanoclusters obtained in (G) red channel (H) green channel and (I) merged image of G and H. A plausible explanation for anticancer activity of assembled Au nanoclusters and higher cytotoxic activity of Au nanocrystals compared to Zn Au NCs are given below.
Au-nanocrystals Se -
- Conclusion
- Experimental Section
- Results and discussions
- Conclusions
- Summary
- Future Prospects
This could possibly explain the difference between the cytotoxic activity of Au nanocrystals and Zn Au NCs. 5,16, the red luminescence obtained from Au nanocrystals and Zn Au NCs (λex-405 and λem-590) overlaps with the green fluorescence from the Mito tracer green (λex-488 and λem-530). Confocal laser scanning microscopy (CLSM) analysis revealed the localization of Au nanocrystals and Zn Au NCs within the mitochondria.
Also, as mentioned above, the generation of reactive oxygen species (ROS) was observed in the presence of Au nanocrystals and Zn Au NCs. Interestingly, the luminescence of Au nanocrystals and Zn Au NCs was significantly quenched by the addition of H2O2. The structural deformation of Au nanocrystals and Zn Au NCs was further confirmed by transmission electron microscopic (TEM) analysis.
Further, we have also measured the stability of Au nanocrystals and ZnAu NCs in human blood serum. The origin of the aforementioned emission (at 590 and 425 nm) in the luminescence spectra of Au NCs is described as follows (Fig. 6.2). 6.3 (A) Transmission electron microscopy (TEM) image and (B) selected area electron diffraction (SAED) pattern of the as-synthesized Cys-Au14 NCs.
6.20 (A) Transmission electron microscopy (TEM) images and (B) selected area electron diffraction (SAED) patterns of the evaporated dispersion containing Cys, MPA and Zn2+ ions and obtained at two different positions (1 and 2) of the sample.
List of Publications in Peer reviewed Journals
List of Patents
