Design and synthesis of novel palladium cyclometallate-based fluorescent probe: Studies on interaction with cell membrane by confocal and fluorescence lifetime imaging.

Coordination complexes offer great potential as cellular imaging probes, which allow to examine specific cell organelle structures in their physiological conditions to better understand the biological system. Understanding the heterogeneous nature of the cell membrane could unveil details of their functionality. Here, we have developed a new anthracene conjugated fluorescent palladium(II) cyclometallate [PdL1Cl] where L1H = [2-(2- (anthracen-9-ylmethylene)-1-phenylhydrazineyl)pyridine] (H stands for dissociable proton), which not only specifically stains the cell membrane, but could be utilized to visualise the membrane by the confocal and fluorescence lifetime imaging microscopy (FLIM). This probe is unable to enter inside the cell as it did not pass through the cell membrane via diffusion or various organic and metal transporters. However, the great lipophilicity of fluorescein improves the interaction of the probe with the peptidoglycan layer of the cell membrane. Probable dissociation of chloride ion and formation of positively charged palladium complex resulted in staining the negatively charged cell membrane. The 3D confocal imaging clearly expressed sole membrane staining by the probe. The probe efficiently stains both cancer cells (HeLa and MCF-7 cell lines) and normal cell (HEK 293 T), confirming the universality of the probe in membrane staining.

Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy.

Doxorubicin is an anthracycline drug most commonly used in cancer therapy. It intercalates with the nuclear DNA and induces toxicity by causing DNA breaks and histone eviction. However, the kinetics of its action on the nucleus has not been mapped effectively. This study shows successful PEGylation and DOX loading through π-π interaction onto carbogenic fluorescent nanodots (FNDs), which have an affinity for the nucleolus. Then the drug release from the nanoparticle and its action on the nuclear environment were aptly mapped using both fluorescence lifetime imaging and superresolution radial fluctuation (SRRF) techniques. Here for the first time, the nuclear degradation kinetics caused by the released DOX from the FNDs as a result of DNA double-strand breaks and histone eviction was visualized. This led to the observation of decreasing length, breadth, and complex structure of the nuclear clusters from 6 h to 24 h, resulting in isolated cluster visualization. However, the superresolution images for free DOX and untreated cells reveal no such drastic effects at the same concentration and time points, unlike DOX loaded particles.

Structural and spectroscopic characterization of pyrene derived carbon nano dots: a single-particle level analysis.

The bottom-up approach has been widely used for large-scale synthesis of carbon nanodots (CNDs). However, the structure and origin of photoluminescence in CNDs synthesized by the bottom-up approach is still a subject of debate. Here, using a series of separation techniques like solvent extraction, column chromatography, gel electrophoresis and dialysis, we present three distinct fluorescent components in CNDs synthesized from pyrene, a well-known precursor molecule. The separated components have qualitative and quantitatively different absorption and emission spectral features including quantum yield (QY). Optical and vibrational spectroscopy techniques combined with electron microscopy indicate that a subtle balance between the extent of graphitization and the presence of molecular fluorophores determines the nature of fluorescence emission. A substantial difference in photons/cycle, single-particle fluorescence blinking, ON-OFF photoswitching strongly supports the distinct nature of the components.

Absorption and emission of light in red emissive carbon nanodots.

The structure-function relationship, especially the origin of absorption and emission of light in carbon nanodots (CNDs), has baffled scientists. The multilevel complexity arises due to the large number of by-products synthesized during the bottom-up approach. By performing systematic purification and characterization, we reveal the presence of a molecular fluorophore, quinoxalino[2,3-b]phenazine-2,3-diamine (QXPDA), in a large amount (∼80% of the total mass) in red emissive CNDs synthesized from o-phenylenediamine (OPDA), which is one of the well-known precursor molecules used for CND synthesis. The recorded NMR and mass spectra tentatively confirm the structure of QXPDA. The close resemblance of the experimental vibronic progression and the mirror symmetry of the absorption and emission spectra with the theoretically simulated spectra confirm an extended conjugated structure of QXPDA. Interestingly, QXPDA dictates the complete emission characteristics of the CNDs; in particular, it showed a striking similarity of its excitation independent emission spectra with that of the original synthesized red emissive CND solution. On the other hand, the CND like structure with a typical size of ∼4 nm was observed under a transmission electron microscope for a blue emissive species, which showed both excitation dependent and independent emission spectra. Interestingly, Raman spectroscopic data showed the similarity between QXPDA and the dot structure thus suggesting the formation of the QXPDA aggregated core structure in CNDs. We further demonstrated the parallelism in trends of absorption and emission of light from a few other red emissive CNDs, which were synthesized using different experimental conditions.

Effect of Protein Corona on the Drug Delivery of Carbogenic Nanodots and Their Mapping by Fluorescence Lifetime Imaging Microscopy.

It is practically impossible to avoid the nonspecific binding of protein to a nanocarrier when it enters a biological fluid. This hinders the chemotherapeutic efficacy of the nanocarrier to a large extent. Surface functionalization, in the recent past, helped in reducing such nonspecific interactions. However, there is a lack of understanding as to how they help in the case of nanocarriers with size <6 nm. Here, we show that the glutathione and folic acid functionalization to a small carbogenic nanocarrier leads to substantial improvement in cell internalization and chemotherapeutic efficacy. The functionalization on smaller size of the nanocarrier helped in manipulating the binding affinity of the protein, which in turn helped in easy dynamic exchange with the surrounding environment. Using fluorescence lifetime imaging, we directly visualized and mapped the released drug at a very high resolution and provide a comprehensive mechanism of the drug distribution inside a cancer cell, as a consequence of the different affinity of protein corona on the carbon nanoparticle.

Fluorescent Probes for Super-Resolution Microscopy of Lysosomes.

Lysosomes are membrane-enclosed small spherical cytoplasmic organelles. Malfunctioning and abnormalities in lysosomes can cause a plethora of neurodegenerative diseases. Consequently, understanding the structural information on lysosomes down to a subnanometer level is essential. Recently, super-resolution imaging techniques enable us to visualize dynamical processes occurring in suborganelle structures inside living cells down to subnanometer accuracy by breaking the diffraction limit. A brighter and highly photostable fluorescent probe is essential for super-resolution microscopy. In this regard, this mini-review deals with the various types of super-resolution techniques and the probes that are used to specifically stain and resolve the structure of the lysosomes.

Direct visualization of the protein corona using carbon nanodots as a specific contrasting agent.

The cellular uptake of the nanoparticles is greatly affected by the formation of protein corona. As a result, an in-depth knowledge of direct visualization of the corona and quantification thereof is extremely important. Although transmission electron microscopy is one of the best techniques for visualization, the heavy metals that are used to increase the contrast of protein are non-specific and may lead to artifacts and erroneous conclusions. Here, we present a new strategy using carbogenic nanodots that showed excellent contrast, under a transmission electron microscope for the direct visualization and quantification of the single particle protein corona.

Magnetofluorescent Nanoprobe for Multimodal and Multicolor Bioimaging.

Although, superparamagnetic iron oxide nanoparticles (SPIONs) have extensively been used as a contrasting agent for magnetic resonance imaging (MRI), the lack of intrinsic fluorescence restricted their application as a multimodal probe, especially in combination with light microscopy. In Addition, the bigger size of the particle renders them incompetent for bioimaging of small organelles. Herein, we report, not only the synthesis of ultrasmall carbon containing magneto-fluorescent SPIONs with size ∼5 nm, but also demonstrate its capability as a multicolor imaging probe. Using MCF-7 and HeLa cell lines, we show that the SPIONs can provide high contrast mulicolor images of the cytoplasm from blue to red region. Further, single particle level photon count data revealed that the SPIONs could efficaciously be utilized in localization based super resolution microscopy in future.

Intrinsically disordered proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis.

Intrinsically disordered proteins (IDPs) possess the property of inherent flexibility and can be distinguished from other proteins in terms of lack of any fixed structure. Such dynamic behavior of IDPs earned the name "Dancing Proteins." The exploration of these dancing proteins in viruses has just started and crucial details such as correlation of rapid evolution, high rate of mutation and accumulation of disordered contents in viral proteome at least understood partially. In order to gain a complete understanding of this correlation, there is a need to decipher the complexity of viral mediated cell hijacking and pathogenesis in the host organism. Further there is necessity to identify the specific patterns within viral and host IDPs such as aggregation; Molecular recognition features (MoRFs) and their association to virulence, host range and rate of evolution of viruses in order to tackle the viral-mediated diseases. The current book chapter summarizes the aforementioned details and suggests the novel opportunities for further research of IDPs senses in viruses.

Bovine Serum Albumin-Conjugated Red Emissive Gold Nanocluster as a Fluorescent Nanoprobe for Super-resolution Microscopy.

The gold nanocluster (GNC), because of its interesting photoluminescence properties and easy renal clearance from the body, has tremendous biomedical applications. Unfortunately, it has never been explored for super-resolution microscopy (SRM). Here, we present a protein-conjugated red emissive GNC for super-resolution radial fluctuation (SRRF) of the lysosome in HeLa cells. The diameter of the lysosome obtained in SRRF is ∼59 nm, which is very close to the original diameter of the smallest lysosome in HeLa cells. Conjugation of protein to GNC aided in the specific labeling of the lysosome. We hope that GNC not only will replace some of the common dyes used in SRM but due to its electron beam contrast could also be used as a multimodal probe for several other correlative bioimaging techniques.

Dual responsive specifically labelled carbogenic fluorescent nanodots for super resolution and electron microscopy.

Due to their high biocompatibility and nontoxic nature, carbogenic fluorescent nanodots (FNDs) have already shown their application in bioimaging. However, their non-specific labeling has restricted their application in live cell super resolution microscopy (SRM). Here we introduce, for the first time, an orange emissive FND, specifically conjugated to the HeLa cell actin filament, for successful single molecule stochastic optical reconstruction microscopy (STORM) and super resolution radial fluctuation (SRRF) microscopy. The resolution obtained in SRRF (∼35 nm) was almost an order of magnitude less than the diffraction limited spot. Interestingly, in addition, the FND also showed electron microscope (EM) contrast inside the cell. We hope that this FND will not only replace some of the common dyes used for SRM, but will also be used as a dual responsive marker in correlative super resolution microscopy (CLEM).

Labelling Proteins with Carbon Nanodots.

We present efficient labelling of several proteins with orange-emissive carbon dots. N-Hydroxysuccinimide was used to activate the carboxyl groups of carbon dots, which subsequently reacted with the lysine groups present on the protein. Labelling was confirmed by UV absorption spectroscopy, PAGE and fluorescence correlation spectroscopy. Protein-conjugated carbon dots showed an enhancement in fluorescence lifetime and intensity owing to reduced intramolecular dynamic fluctuations. Single-molecule fluorescence measurements showed reduced fluorescence fluctuations and higher photon budget after protein tagging. Our study opens up opportunities to use carbon dots as highly precise biolabelling probes.