Some of the landmark findings have been highlighted to convey the importance of multifunctional nanomaterials in different arenas. Fast and easy synthetic procedures for multifunctional nanomaterials using biopolymers as well as small molecules have been developed.
Fabrication of Nanomaterials
The fabrication of nanomaterials is generally classified into two main classes of top-down and bottom-up approaches (10). Green synthesis of nanoparticles using biomolecules as precursors has attracted attention due to their easy synthesis, environmental friendliness, and nontoxic nature. (12) Proteins, biopolymers, phytogenic extracts, bacterial cells, and even mammalian cells have been widely studied for the synthesis of nanoparticles.
Characterization of Nanomaterials
The crystal properties and information regarding lattice spacing of nanoparticles can be verified by XRD. This technique yields information about the important optical properties such as absorption, luminescence, reflectance and phosphorescence of nanoparticles.
Types of Nanomaterials
Natural polymer-based nanoparticles provide a significant improvement over traditional drug delivery systems in terms of biocompatibility, biodegradability, and efficacy for potential use in drug and gene delivery, tissue engineering, and immunotherapy.(17) The various natural polymers such as chitosan, gelatin, albumin, and alginate. used to produce the nanoparticles. Due to their ultra-small size, these nanoclusters have been reported to have ROS-generating ability, which can be exploited in cancer therapy.
Multifunctional Nanomaterials and their Applications
In addition, the blue emission of most carbon dots makes them susceptible to interference from cellular autofluorescence. Suicide gene therapy is one of the prominent forms of gene therapy being conducted in clinical trials.
Scope and Challenges
A bimetallic module composed of Au NCs (gold nanoclusters) and Ag NPs (silver nanoparticles) was synthesized and characterized as effective in theranostic application. The synthesized Au NCs were also applied for targeted bioimaging and therapy of cancer cells and were found to be more effective against HeLa cells.
Polycation-functionalized water-soluble gold nanoclusters: A potential platform for simultaneous gene delivery and cell imaging. Peptide-patterned gold nanoclusters as a novel label-free biosensor for the detection of protease activity.
Bimetallic Silver Nanoparticle–Gold Nanocluster Embedded Composite
These can be broadly classified into co-reduction methods involving addition of both metal ion salt precursors, followed by reduction and post-treatment methods such as core etching, galvanic replacement, etc.(21,22) The synthesis of bimetallic hollow NPs has also been reported by the galvanic substitution method.(23) Silver and gold are one of the most used bimetallic systems due to the ease in synthesis.(24) The bimetallic nanocluster systems studied so far have mainly focused on improved luminescence intensity and the catalytic properties of bimetallic systems. on monatomic.(25–27) However, fewer reports are available on the evaluation of the therapeutic potential of bimetallic nanocluster systems. Thus, these theranostic composite nanoparticles killed cancer cells and provided an option for concomitant cellular imaging.
Synthesis and Characterisation of AgNP–AuNCs
Probably, along with Ag NPs, the presence of thiol (in MPA) as a stabilizing ligand and the appropriate amount of template (chitosan) provided protection due to their bulk nature, which facilitated the formation of AgNP-AuNCs. The quantum yield of AgNP–AuNCs was measured to be 2.3% using quinine sulfate as a standard (Appendix A, Fig. A2.7a and b) and was considered suitable for imaging applications. (51) The FTIR spectra of AgNPs compared to the chitosan control showed a marked shift of the –NH bending peak from 1609 cm-1 to 1570 cm-1, indicating the interaction of the formed AgNPs with the –NH group of chitosan.
Synthesis and Characterisation of Composite NPs (AgNP–AuNC–CSNPs)
The luminescence intensity decay rate (F/F0) of the NC was 0.0025% per second, while the rate was found to be 0.095% per second for a commonly used fluorescent dye such as rhodamine 6G. The presence of unreacted ions was eliminated by repeatedly washing the sample after centrifugation.
Uptake and Bio-imaging Application of Composite NPs
Incorporation of the compound (Ag NPs with Au NCs) inside the cell was clearly observed and plasma membrane localization suggests possible endocytosis-mediated uptake of the composite NPs (56) (Figure 2.4K and L). In addition to conventional methods, this approach provides a new insight to monitor the uptake of composite NPs by cancer cells, where cell membrane alteration was also seen in TEM images.
Cell Viability Assay and Mechanism of Cell Death
The results (Figure 2.5B) indicated that the IC50 value of the composite NPs had an Ag concentration of 3.6 μg ml−1. It was observed that a slightly higher amount of ROS was generated in the case of composite NPs compared to AgNPs alone, possibly as a result.
Rapid Microwave-Assisted Synthesis of Ultrabright Fluorescent Carbon Dots for Live Cell Staining, Cell Targeting, and In Vivo Imaging. Total green synthesis of gold nanoparticles using laser ablation in deionized water containing chitosan and starch.
The cells were treated with compound NPs and imaged under a Delta Vision deconvolution microscope (GE Healthcare). Initially, HeLa cells (1 × 10 cells, 6-well plates) were cultured and then treated with compound NPs for 24 h.
Particle size distribution of the AgNP-AuNCs as obtained from the TEM image in Figure 2.2(C). Plasma membrane extensions at 3 h suggest endocytosis-mediated uptake of assembled NPs; membrane disruption after 24 hours is visible. f, g) FACS analysis of the incorporation of compound NPs into FL1-H and FL2-H, respectively. a-b) FESEM images of control and compound NPs treated HeLa cells. a) EtBr/AO double staining of control HeLa cells, (b) composite NPs treated (for 24 hours at IC5) HeLa cells, (c) AgNPs treated only HeLa cells for 24 hours at IC50. a-c) Cell cycle analysis of control HeLa cells, AgNPs-treated HeLa cells, composite NPs-treated HeLa cells for 24 hours at IC50, respectively. a-b) Overexpression of Caspase-3 was observed from gene expression analysis of compound NPs-treated cells and control cells, using actin as endogenous control.
Cationic BSA Templated Au–Ag Bimetallic Nanoclusters as a Theranostic Gene
Herein, the synthesis of a cationic BSA-embedded Au-Ag bimetallic nanocluster system cast into a composite nanoparticle as a carrier for the pDNA encoding the CD-UPRT enzyme is reported. Synthesis of cationic BSA Au-Ag NCs composite NPs to achieve bioimaging alongside combinatorial therapy as a result of suicide gene delivery and Au-Ag NC-induced ROS generation in cancer cells.
Synthesis and Characterisation of Au–Ag NCs, Au–Ag NC-Embedded Cationic BSA
The presence of amine groups was also analyzed using a standard TNBS test for primary amines. (42) It was revealed that the cationic BSA (embedded with Au–Ag NCs) had increased amine group content, evidenced by increased absorbance at 420 nm for cationic BSA (embedded with Au–.Ag NCs) at all concentrations μg/mL ) compared to natural BSA (embedded with Au–Ag NCs; Appendix B, Figure B3.1d). Synthesis and characterization of Au-Ag NCs embedded cationic BSA composite nanoparticles and pDNA loading.
Synthesis and Characterisation of Au–Ag NCs Embedded Cationic BSA Composite
The quantum yield of cationic BSA Au–Ag NCs is found to be 11.7% and that of pDNA-loaded composite NPs is found to be 8.8%, which is suitable for bioimaging purposes (Appendix B, Figure B3 .5a-c). . The concentration of Au and Ag was found to be 13.25 μg/mL and 3.11 μg/mL, respectively, in the pDNA-loaded composite NPs (Appendix B, Figure B3.6c and d).
Uptake and Bio-imaging Application of pDNA Loaded Composite NPs
Time-dependent confocal microscopy studies provided better insight into the uptake of assembled pDNA-loaded NPs. In cells treated with sodium azide (Appendix B, Fig. B3.10a-f), a difference in the uptake of assembled pDNA-loaded NPs was visible after 4 h of treatment.
Cell Viability and Mechanism of Cell Death
A significant increase in the sub G1 population was observed in the case of pDNA-loaded composite NPs compared to control cells and pDNA-loaded negatively charged composite. Additional results include the simultaneous cell cycle analysis of pDNA-loaded positively charged composite NPs without Au-Ag NCs, where a smaller percentage of cells in sub G1 was observed.
He found that cells treated with pDNA-loaded composite NPs showed a significant increase in the apoptotic population compared to control cells and pDNA-loaded negatively charged composite NPs. Also, pDNA-loaded positively charged composite NPs without Au-Ag NCs were simultaneously evaluated for their caspase-3 activity, which again showed a nominal increase in the apoptotic population (Figure 3.6A-F).
Polycation-functionalized water-soluble gold nanoclusters: a potential platform for simultaneous enhanced gene delivery and cell imaging at the nanoscale. Bovine serum albumin nanoparticles as a controlled-release carrier for local drug delivery to the inner ear.
Then, the cells were treated with pDNA-loaded composite NPs for the required time intervals. For ROS generation studies, HeLa cells (1 × 105 cells/well, seeded in 6-well plate) were grown for 24 h and then treated for 3 h with pDNA-loaded composite NPs, composite NPs negatively charged pDNA and positively charged pDNA. loaded composite NPs without Au–Ag NCs.
Figures and Tables
Time-dependent fluorescence spectra of pDNA-loaded composite NPs, (b) quantum yield, and (c) photostability of cationic BSA Au-Ag NCs and pDNA-loaded composite NPs. FESEM images of (a) control and (b) treated HeLa cells with composite NPs + DNA in the presence of 5-FC.
Gold Nanoclusters Embedded Mucin Nanoparticles for Photodynamic Therapy
Reduced opsonization effect with promising hemo- and cyto-compatibility are prominent qualities of mucillated nanocarriers (eg, polylactic-co-glycolic acid). polyethylene terephthalate, which effectively helps reduce the host's immune response resulting from biomaterials. (15) Drug-encapsulated mucin-coated micro- or nanotubes can be used for longer targeted delivery. attitude. It should be noted that one of the main obstacles in photodynamic therapy arises from the inability to track photosensitizers according to conventional tracking techniques. (22) The presumed behavior of PS in the accumulation at the cancer site and the lack of consecutive therapy follow-up lead to the limited use of such therapeutic practices.
Synthesis and Characterisation
Photostability studies of the emission intensity of the Au NC-mucin nanoparticles showed that the NCs were stable compared to the standard rhodamine 6G. The release profile of MB from Au NC-mucin NPs was studied at pH 4.5 and 7.5.
Detection of Singlet Oxygen Generation
The decay rate of luminescence intensity (F/F0) of the NCs was 0.27% per minute, while in the case of the commonly used fluorescent dye rhodamine 6G, the rate was found to be 0.80%. The sustained release profile followed by the initial burst release helps maintain the photosensitizer at its optimal activity (Appendix C, Figure C4.5).
Uptake and Delivery of MB Loaded Au NC-mucin NPs
Photodynamic Therapy and Mechanism of Cell Death
ROS generation profile of (A) HeLa cells treated with Au NC-mucin NP, (B) MB, (C) MB loaded Au NC-mucin NP under different conditions. Caspase-3 assay of HeLa cells showing percentage of apoptosis in control (A), treated with (B,C) MB, Au NC-mucin NP-loaded MB in the dark, (D) MB under irradiation and (E ) MB Au loaded NC-mucin NPs under irradiation.
Therefore, from all the above findings, it can be concluded that the MB-loaded Au NC-mucin NPs effectively delivered the photosensitizer and killed the cancer cells due to the generation of singlet oxygen. Also, the luminescence property of Au NC-mucin NPs loaded with MB provided bi-labeling properties, thus helping to track the delivery process of MB.
It revealed that MB, MB-loaded Au NC-mucin NPs-treated cells irradiated with 640 nm light showed a significant increase in the active caspase-3 positive cell population (apoptotic cells) compared to other controls (Figure 4.8A-E) . Alternatives to outdoor daylighting for photodynamic therapy - use of greenhouses and artificial light sources.
Then, the cells were treated with MB, Au NC-mucin NPs, and MB-loaded Au NC-mucin NPs for 3 h. Cells were then harvested by trypsinization and all samples were centrifuged (650 rcf, 6 min).
Fluorescence data for 15000 cells were recorded with the Cell Quest program (BD) in each sample for further analysis. a-c) Zeta potential of mucin, Au NC-mucin NPs, MB loaded Au NC-mucin NPs.
Phenylboronic Acid Templated Gold Nanoclusters for Mucin Detection Using a
The overall work is illustrated in Figure 5.1, which shows multiple applications of the as-synthesized PB-Au NC probe for mucin detection using a smartphone-based platform, targeted bioimaging, therapeutic activity towards cancer cells and multicellular spheroids. Schematic representation of the rapid synthesis procedure of PB-Au NCs and their application in targeted imaging and therapy of cancer cells, as well as smartphone-based mucin detection.
Synthesis and Characterisation of PB-Au NCs
Uptake and Targeting of Cancer Cells by PB-Au NCs
Thereby, the inhibition assay indicated that the uptake of PB-Au NCs was mediated through the interaction of PB and sialic acid residues on the cancer cell surface (Appendix D, Figure D5.6a-c). The semiquantitative real-time polymerase chain reaction of sialyltransferase genes also revealed the overexpression of ST3GALIII (in HeLa and Hep G2) and ST6GALI (in HeLa) compared to the normal cell line HEK, which in turn affects the sialic acid expression on the cancer cell surface (Appendix D, Figure D5.7).
Cell Viability and Mechanism of Cell Death
The results revealed a higher amount of ROS generation in the case of PB-Au NC-treated cells. The depth projection of the confocal image showed uptake of PB-Au NCs inside the spheroids (Appendix D,.
Confocal images of control HeLa spheroids without PB-Au NC treatment showed no fluorescence (Appendix D, Figure D5.12a-c). These results indicated the successful application of a PB-Au NC probe for labeling and therapeutic response to multicellular spheroids.
Detection of Mucin using Smartphone Based Platform
Upon addition of increasing amounts of mucin to the PB-Au NC probe, the luminescence increased proportionally (Appendix D, Figure D5.14a). The luminescence intensity of PB-Au NCs was found to increase specifically with respect to mucin (Appendix D, Figure D5.14b.).
It was found that the obtained intensities and concentrations of mucin (in the range 0–1000 μg/ml) would best fit a second-order polynomial. The fluorescence intensity of the standard spectrofluorometer and the concentration of mucin were also found to best fit a second-order polynomial similar to that obtained by the device.
Graphene Oxide-Based Fluorescent Aptasensor for On-Line Detection of Epithelial Tumor Marker Mucin 1 Nanoscale. Aptamer-based detection of the epithelial tumor marker mucin 1 by quantum dot-based fluorescence readout anal.
The emitted light passed through the emission filter (cutoff ~400 nm) and an additional lens (placed between the cuvette and the camera) and was imaged with the camera on the mobile phone. With the handheld POC platform, 24-bit images (ARGB) of the emitted luminescence from different samples were acquired using the mobile phone camera.
-c) Uptake of PB-Au NCs by HEK, HeLa, Hep G2 cells studied using FACS by tracking the fluorescence of PB-Au NCs. a) (i-iii) Bright field image, fluorescent image and merged image of control HEK cells. Semi-quantitative RT-PCR of genes ST3GAL-III, ST6GAL-I, β-actin expressed in HeLa, HEK, Hep G2 cell lines. a) Cell viability assay of HeLa cells treated with free PB.
Protein Expression Analyses Using Luminescent Gold Nanoclusters
Described here is the synthesis of luminescent Au nanoclusters (a signal-generating probe) by a rapid one-step method for array-based multiprotein analysis as shown in Scheme 1. This synthesis of Au nanoclusters can be performed on proteins in solution or purified proteins, bound to an antibody attached to a polyvinylidene difluoride (PVDF) membrane.
Rapid Synthesis of Au Nanoclusters with Proteins
Au nanoclusters for a range of protein concentrations (BSA) and HAuCl4 are shown in Appendix E, Figure E6.1a,b. Also, circular dichroism (CD) spectroscopy revealed that the formation of Au nanoclusters did not significantly change the 3D structure of BSA.
Application of Au Nanoclusters for Protein Expression Studies
Then the membrane was imaged using the visualization unit of the bench top device and the luminescence intensity of the Au nanoclusters was found to be the highest in the case of GST – anti-GST antibody conjugate followed by GST-hGMCSF – anti-GST antibody. conjugate , compared to only GST, GST-hGMCSF, anti-GST antibody as shown in Figure 6.4. As a control experiment, specific GST protein and a non-specific BSA protein were interacted with anti-GST antibodies and it was found that the luminescence is not enhanced in the case of BSA as with increasing concentrations of GST not, possibly due to washout of the non-specific BSA antigen (Appendix E, Figure E6.7).
Enhancement of Sensitivity with Zinc Ions
Polyethyleneimine-protected silver nanoclusters luminescence probe for sensitive detection of cobalt (II) in living cells. Blue-emitting gold nanoclusters formed by polycytosine DNA at low pH and polyadenine DNA at neutral pH.
The flow-through fractions were collected, followed by washing the column eight times with PBS. The membrane was blocked for 30 min using a blocking solution (as mentioned above) to prevent non-specific binding. Then the membrane was incubated with the respective GST antigens for 30 min and washed with PBST buffer (phosphate buffered saline with Tween 20) to reduce non-specificity. . f) Synthesis of Au nanoclusters on PVDF membrane: After antigen-antibody interactions, Au nanoclusters were synthesized on the spots by adding 1.5 μl 0.7 mM HAuCl4 and 0.5 μl 0.01 M MPA, followed by heating the membrane using a thermocycler at 95 ºC for 2 minutes and then cooling at 15 ºC for 3 minutes. g) Image acquisition and analysis: The membrane with synthesized Au nanoclusters was imaged and analyzed using the visualization unit using custom-developed software under UV illumination (254 nm).
The suicide gene in the presence of the prodrug 5 FU induced apoptosis in HeLa cancer cells. The activity of the gold nanoclusters was also studied in a more realistic tumor-like environment using in vitro 3D multicellular spheroids.