A novel high throughput plate-based method for 2-PE quantification in novel multidose vaccines (R21 malaria, Covishield and Covovax) and combination vaccines (Hexavalent).

Multidose presentation of vaccines is the most preferred choice, for mass immunization particularly during pandemics. WHO also recommends multidose containers of fill finished vaccines for programmatic suitability and global immunizations programmes. However, multidose vaccine presentations requires inclusion of preservatives to prevent contaminations. 2-Phenoxy ethanol (2-PE) is one such preservative which is being used in numerous cosmetics and many vaccines recently. Estimation of 2-PE content in multidose vials is a crucial quality control parameter to ensure in use stability of the vaccines. Presently available conventional methods, have their own limitation in terms of being time consuming, requiring sample extraction, large sample volume requirement etc. Therefore, a robust, simple, high-throughput method with a low turnaround time was required, which can quantitate 2-PE content in the conventional combination vaccines as well as new generation complex VLP based vaccines. In order to address this issue, a novel absorbance-based method has been developed. This novel method specifically detects 2-PE content in Matrix M1 adjuvanted R21 malaria vaccine, nano particle and viral vector based covid vaccines and combination vaccines like Hexavalent vaccine. The method has been validated for parameters such as linearity, accuracy and precision. Importantly, this method works even in presence of high amounts of proteins and residual DNA. Considering the advantages associated with method under study, this method can be used as an important in process or release quality parameter to estimate the 2-PE content in various vaccines containing 2-PE in multidose presentations.

Rapid, high throughput protein estimation method for saponin and alhydrogel adjuvanted R21 VLP Malaria vaccine based on intrinsic fluorescence.

Protein content estimation of recombinant vaccines at drug product (DP) stage is a crucial lot release and stability indicating assay in biopharmaceutical industries. Regulatory bodies such as US-FDA and WHO necessitates the quantitation of protein content to assess process parameters as well as formulation losses. Estimation of protein content at DP stage in presence of adjuvants (e.g AlOOH, AlPO4, saponin and squalene) is quite challenging, and the challenge intensifies when the target protein is in Virus like particles (VLP) form, owing to its size and structural complexity. Methods available for protein estimation of adjuvanted vaccines mostly suffer from inaccuracy at lower protein concentrations and in most cases require antigen desorption before analysis. Present research work is based on the development of a rapid plate-based method for protein estimation through intrinsic fluorescence by using Malaria vaccine R21 VLP as a model protein. Present method exhibited linearity for protein estimation of R21, in the range of 5-30 µg/mL in Alhydrogel and 4-20 µg/mL for Matrix M adjuvant. The method was validated as per ICH guidelines. The limit of quantification was found to be 0.94 µg/mL for both Alhydrogel and Matrix M adjuvanted R21. The method was found specific, precise and repeatable. This method is superior in terms of less sample quantity requirement, multiple sample analysis, short turnaround time and is non-invasive. This method was found to be stability indicating, works for other proteins containing tryptophan residues and operates well even in presence of host cell proteins. Based on the study, present method can be used in vaccine industries for routine in-process sample analysis (both inline and offline), lot release of VLP based drug products in presence of Alhydrogel and saponin based adjuvant systems.

Cytotoxicity of apo bovine α-lactalbumin complexed with La3+ on cancer cells supported by its high resolution crystal structure.

Cancer remains one of the biggest threats to human society. There are massive demands for compounds to selectively kill cancerous cells. Earlier studies have shown that bovine α -lactalbumin made lethal to tumor cells (BAMLET) becomes cytotoxic against cancer cells in complex with oleic acid {Hoque, M. et. al., PLoSOne 8, e68390 (2013)}. In our study, we obtained bovine α-lactalbumin complexed with lanthanum ion (La3+-B-α-LA) and determined its high resolution crystal structure. The natural calcium binding site of bovine α-lactalbumin is replaced by lanthanum. The La3+ complex formation by B-α-apo-LA was also supported by various biophysical methods. Interestingly, our complex, La3+-B-α-LA exhibits much greater anticancer activity against breast cancer cells as compared to the reported BAMLET-oleic acid complex. This study shows that La3+-B-α-LA complex is preferentially more toxic to MCF-7 cells as compared to KB (oral cancer) and HeLa (cervical) cells, while almost non-toxic to the healthy cells that we studied. Our data indicates that the cytotoxicity of La3+-B-α-LA against cancer cells is through apoptotic path way. The higher anticancer activity of La3+-B-α-LA is attributable to the requisite structural changes induced in the protein by La3+ binding as supported by the crystal structure of the complex.

Acetylcholinesterase and Aß Aggregation Inhibition by Heterometallic Ruthenium(II)-Platinum(II) Polypyridyl Complexes.

Two heteronuclear ruthenium(II)-platinum(II) complexes [Ru(bpy)2(BPIMBp)PtCl2]2+ (3) and [Ru(phen)2(BPIMBp)PtCl2]2+ (4), where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, and BPIMBp = 1,4'-bis[(2-pyridin-2-yl)-1H-imidazol-1-ylmethyl]-1,1'-biphenyl, have been designed and synthesized from their mononuclear precursors [Ru(bpy)2(BPIMBp)]2+ (1) and [Ru(phen)2(BPIMBp)]2+ (2) as multitarget molecules for Alzheimer's disease (AD). The inclusion of the cis-PtCl2 moiety facilitates the covalent interaction of Ru(II) polypyridyl complexes with amyloid ß (Aß) peptide. These multifunctional complexes act as inhibitors of acetylcholinesterase (AChE), Aß aggregation, and Cu-induced oxidative stress and protect neuronal cells against Aß-toxicity. The study highlights the design of metal based anti-Alzheimer's disease (AD) systems.

Fluorescent Copper Probe Inhibiting Aß1-16-Copper(II)-Catalyzed Intracellular Reactive Oxygen Species Production.

A variety of fluorescent probes are proposed to monitor the intracellular copper content. So far, none of the probes have been evaluated for their potential to inhibit copper-associated intracellular oxidative stress. Herein, we studied the ability of a fluorescent copper probe, OBEP-CS1, to inhibit intracellular oxidative stress associated with an amyloid ß (Aß) peptide-copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aß1-16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aß-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aß1-16 peptide can bind intracellular copper and thereby exert oxidative stress.

A Bimodal, Cationic, and Water-Soluble Calix[4]arene Conjugate: Design, Synthesis, Characterization, and Transfection of Red Fluorescent Protein Encoded Plasmid in Cancer Cells.

A new bimodal fluorescent cationic calix[4]arene (L1) conjugate has been synthesized in multiple steps and well characterized by NMR and electrospray ionization-mass spectrometry (ESI-MS) techniques. L1 has been investigated for its DNA binding ability by various spectroscopy techniques like absorption, fluorescence, and circular dichroism (CD). The formation of L1-DNA complex has been confirmed by the gel electrophoresis in the presence of incremental concentration of L1. To visualize the packing of the plasmid (pBR322), detailed tapping mode atomic force microscopy study has been performed, which revealed blob-like structure of plasmid upon addition of the incremental amount of L1. Concentration dependent transfection ability of L1 has been established in MCF-7 cells by confocal microscopy by carrying the red fluorescent protein (RFP) encoded plasmid pCMV-tdTomato-N1 to emit both intrinsic fluorescence of L1 as well as that from RFP. All this has been possible in the absence of any adjuvant phospholipids (DOPE) that are commonly used as helper. Further transfection efficiency of L1 has been compared with the commercially available lipofectamine (LTX) in two cancer cell lines, MCF 7 and SH-SY5Y, and found that the L1 is as efficient as that of LTX. Hence, L1 is an efficient and effective cargo to transport genetic material into the cells.

Oxidative Intramolecular 1,2-Amino-Oxygenation of Alkynes under AuI /AuIII Catalysis: Discovery of a Pyridinium-Oxazole Dyad as an Ionic Fluorophore.

Oxidative intramolecular 1,2-amino-oxygenation reactions, combining gold(I)/gold(III) catalysis, is reported. The reaction provides efficient access to a structurally unique ionic pyridinium-oxazole dyad with tunable emission wavelengths. The application of these fluorophores as potential biomarkers has been investigated.

Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aß peptide aggregation and protect against Aß-induced cytotoxicity.

Aggregation of amyloid ß peptide (Aß) is an important event in the progression of Alzheimer's disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aß aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer's disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aß peptide aggregation and the subsequent toxicity is little explored. Herein, we report synthesis and x-ray crystal structure of novel compound 3-acetyl coumarin thiosemicarbazone and its efficacy toward inhibition of Aß(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aß(1-42) peptide aggregation up to 80% compared to the parent 3-acetyl coumarin which inhibits 52%. Further, 3-acetyl coumarin thiosemicarbazone provides neuroprotection against Aß-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties.

Highly emissive organic solids with remarkably broad color tunability based on N,C-chelate, four-coordinate organoborons.

Molecular fluorophores based on N,C-chelate, four-coordinate organoborons exhibit tunable solid-state emission colors that cover the whole visible region from blue to red. The emission color can be tuned through the substituents on either quinolines or the boron center.