A Microfluidics-Assisted Double-Barreled Nanobioconjugate Synthesis Introducing Aprotinin as a New Moonlight Nanocarrier Protein: Tested toward Physiologically Relevant 3D-Spheroid Models.

Proteins are promising substances for introducing new drug carriers with efficient blood circulation due to low possibilities of clearance by macrophages. However, such natural biopolymers have highly sophisticated molecular structures, preventing them from being assembled into nanoplatforms with manipulable payload release profiles. Here, we report a novel anticancer nanodrug carrier moonlighting protein, Aprotinin, to be used as a newly identified carrier for cytotoxic drugs. The Aprotinin-Doxorubicin (Apr-Dox) nanobioconjugate was prepared via a single-step microfluidics coflow mixing technique, a feasible and simple way to synthesize a carrier-based drug design with a double-barreled approach that can release and actuate two therapeutic agents simultaneously, i.e., Apr-Dox in 1:11 ratio (the antimetastatic carrier drug aprotinin and the chemotherapeutic drug DOX). With a significant stimuli-sensitive (i.e., pH) drug release ability, this nanobioconjugate achieves superior bioperformances, including high cellular uptake, efficient tumor penetration, and accumulation into the acidic tumor microenvironment, besides inhibiting further tumor growth by halting the urokinase plasminogen activator (uPA) involved in metastasis and tumor progression. Distinctly, in healthy human umbilical vein endothelial (HUVEC) cells, drastically lower cellular uptake of nanobioconjugates has been observed and validated compared to the anticancer agent Dox. Our findings demonstrate an enhanced cellular internalization of nanobioconjugates toward breast cancer, prostate cancer, and lung cancer both in vitro and in physiologically relevant biological 3D-spheroid models. Consequently, the designed nanobioconjugate shows a high potential for targeted drug delivery via a natural and biocompatible moonlighting protein, thus opening a new avenue for proving aprotinin in cancer therapy as both an antimetastatic and a drug-carrying agent.

One-Step Synthesis of Ultrasmall Nanoparticles in Glycerol as a Promising Green Solvent at Room Temperature Using Omega-Shaped Microfluidic Micromixers.

Despite innovations in the synthesis protocol of nanoparticles (NPs), the size distribution and uniformity of particles still remain as crucial attributes. Homogeneous and rapid nucleation is a critical phenomenon to obtain monodisperse nanoparticles. Herein, we have carried out the synthesis of metal nanoparticles in a customized microfluidic (MF) chip, with 18 omega-shaped micromixers, by using glycerol as a promising green solvent and reducing agent at various concentrations (10-80%), and simultaneous comparison of the results from batch synthesis. Initially, mixing characterization for 10-80% glycerol was obtained by adjusting the Peclet (Pe) number. Further, the effect of the Pe number, time, and concentrations of polyvinylpyrrolidone, metal source, and glycerol on the NP size was investigated. Interestingly, the experimental findings depicted that by varying different parameters, the spherical nanoparticles with an average ultrasmall particle diameter of <2 nm were obtained at all glycerol concentrations (10-80%), as compared to batch synthesis (giving a yield of ∼10-fold larger particles). The mixing efficiency in this MF chip design was analyzed by using a fluorescent dye in glycerol, while the particle morphology and size were characterized by using dynamic light scattering, transmission electron microscopy, and ultraviolet-visible spectroscopy. Hence, compared to the conventional benchtop-assisted NP synthesis, this study unveils the significant effect of the microfluidic technique on the synthesis of ultrasmall and homogeneous nanoparticles in a single step, using an environmentally friendly solvent.

Novel Size-Tunable and Straightforward Ultra-Small Nanoparticle Synthesis in a Varying Concentration Range of Glycerol as a Green Reducing Solvent.

Despite all the possibilities available so far for the synthesis of nanoparticles (NPs), synthesizing ultra-small (<10 nm) monodispersed particles is still demanding. Getting a particular size with a straightforward method is a trial-and-error approach. To explore this prospective, in the current study, we have introduced a protocol which offers a varying concentration range of glycerol to successfully generate the NPs of repeatable and consistent particle size in each synthesis, thus giving an alternative from lengthy tentative preparations and/or testing protocols. Since synthesizing controlled sized nanoparticles in aqueous medium is somewhat difficult as the balance of particle growth and nucleation is challenging to control, herein, we used a polyol method with glycerol both as a solvent medium as well as reducing species for silver nitrate, as an example model ion source, to execute the nanoparticle synthesis. In order to maintain the stability of the synthesized NPs, polyvinylpyrolidone (PVP) was added as a stabilizer. The synthesis, monodispersity, and stability were confirmed using techniques such as UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and X-ray powder diffraction (XRD), while morphological analysis and ultra-small size validation were conducted using TEM, SEM, and AFM. Interestingly, in the various concentrations of glycerol solution used (10-100%), we have observed a tunable linear size range to obtain ultra-small nanoparticles (<10 nm) up to 60% glycerol, while further increasing the glycerol component increased the size approximately to ∼160 nm, providing tunable properties in this synthesis procedure. Hence, this study provides a distinct possibility to obtain ultra-small nanoparticles with a tunable size feature for further applications in numerous fields.

Viruses as tools in gene therapy, vaccine development, and cancer treatment.

Using viruses to our advantage has been a huge leap for humanity. Their ability to mediate horizontal gene transfer has made them useful tools for gene therapy, vaccine development, and cancer treatment. Adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, alphaviruses, and herpesviruses are a few of the most common candidates for use as therapeutic agents or efficient gene delivery systems. Efforts are being made to improve and perfect viral-vector-based therapies to overcome potential or reported drawbacks. Some preclinical trials of viral vector vaccines have yielded positive results, indicating their potential as prophylactic or therapeutic vaccine candidates. Utilization of the oncolytic activity of viruses is the future of cancer therapy, as patients will then be free from the harmful effects of chemo- or radiotherapy. This review discusses in vitro and in vivo studies showing the brilliant therapeutic potential of viruses.

An Outline of Contributing Vaccine Technologies for SARS CoV2 Advancing in Clinical and Preclinical Phase-Trials.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) is an RNA virus involving 4 structural and 16 non-structural proteins, and exhibiting high transmission potential and fatality. The emergence of this newly encountered beta coronavirus-SARS CoV2 has brought over 2 million people to death, and more than 10 billion people got infected across the globe as yet. Consequently, the global scientific community has contributed to the synthesis and design of effective immunization technologies to combat this virus. OBJECTIVES: This literature review was intended to gather an update on published reports of the vaccines advancing in the clinical trial phases or preclinical trials, to summarize the foundations and implications of contributing vaccine candidates inferring their impact in the pandemic repression. In addition, this literature review distinctly facilitates an outline of the overall vaccine effectiveness at current doses. METHODS: The reported data in this review was extracted from research articles, review articles and patents published from January 2020 to July 2021, available on Google Scholar, Pubmed, Pubmed Central, Research Gate, Science direct, and Free Patent Online Database by using combination of keywords. Moreover, some information is retrieved from native web pages of vaccine manufacturing companies' due to progressing research and unavailability of published research papers. CONCLUSION: Contributing vaccine technologies include: RNA (Ribonucleic acid) vaccines, DNA (Deoxyribonucleic acid) vaccines, viral vector vaccines, protein-based vaccines, inactivated vaccines, viruses-like particles, protein superglue, and live-attenuated vaccines. Some vaccines are prepared by establishing bacterial and yeast cell lines and as self-assembling adenovirus- derived multimeric protein-based self-assembling nanoparticle (ADDOmer). On May 19, WHO has issued an emergency use sanction of Moderna, Pfizer, Sinopharm, AstraZeneca, and Covishield vaccine candidates on account of clinical credibility from experimental data.

Natural products embedded crown ethers as potent insulin secretory agents.

Natural products embedded crown ethers were prepared by utilizing bioactive natural products including chrysin, tetrahydroisoquinoline (THIQ), and biochanin-A. The prepared crown ether scaffolds were evaluated and compared with their natural product precursors for insulin secretory activity on isolated mice islets and for their fluorescent properties. All the crown adducts were found more active as compared to their natural product precursors. Bischrysin 32-crown-10 (6d), THIQ 15-Crown-5 (6a) and chrysin 16-crown-5 (6c) showed mild, moderate and strong insulin secretory activity, respectively when compared with the standard drug tolbutamide (TB). Particularly crown derivative 6c showed strong activity (31.10 ng/islet/h) that is almost two (02) fold higher than that of standard drug TB (16.82 ng/islet/h). To the best of our knowledge crown ethers based antidiabetic study is being reported for the first time in literature through this work. Furthermore, fluorescence study showed the significant increase in absorption and emission maximum (hypsochromic effect) in crown structures when compared with their natural product precursors. Present optimistic results obtained from this study may be a guided template for developing new effective insulin secretory agents.

Pharmacological evaluation of Vitis vinifera and Zingiber zerumbet on electrocardiographic, biochemical alterations and antioxidant status in isoproterenol-induced myocardial infarction in rats.

The Vitis vinifera (VV) and Zingiber zerumbet (ZZ) are popular functional foods which are used for the treatment of cardiovascular ailments. These possess antiproliferative, antiplatelet and antioxidant effects. The current study has been designed to ascertain their effectiveness against Isoproterenol (ISO)-induced myocardial infarction (MI). Chronic administration of VV and ZZ was accessed for its cardio-protective effect in ISO-induced MI rats. Male albino rats were treated with VV (250 mg/kg, p.o.), ZZ (200 mg/kg, p.o.) and its combination (Vitis vinifera + Zingiber zerumbet) VZ for 30 days prior to ISO administration (85 mg/kg, S/C). Electrocardiography (ECG) and Blood Pressure (BP) were measured using PowerLab data acquisition system. Biochemical serum markers, tissue histopathology and HPLC finger printing were performed. The VV, ZZ and its combination VZ showed significant protective effects on ST segment elevation, cardiac biomarkers; Troponin I (Trop I), creatine kinase-MB (CK-MB), alanine transaminase (ALT), aspartate amino transferase (AST), lactate dehydrogenase (LDH), enhanced the cardiac antioxidant defense system, restored the hematological (WBCs, RBCs, Platelets) & coagulation parameters and improved the lipid profile and histopathological alterations such as tissue necrosis, infiltration and edema which were observed only in ISO administered rats. These results indicate that V. vinifera and Z. zerumbet possess cardio protective effects possibly mediated through maintenance of endogenous antioxidant levels, cardiac biomarkers and lipid parameters.

Functional venomics of the Big-4 snakes of Pakistan.

In South Asia, the "Big-4" venomous snakes Naja naja, Bungarus caeruleus, Daboia russelii, and Echis carinatus are so-called because they are the most medically important snakes in the region. Antivenom is the only effective treatment option for snakebite envenoming but antivenom is not produced domestically in Pakistan making the country reliant on polyvalent products imported from India and Saudi Arabia. The present study investigated the toxin composition and activity of the venoms of Pakistani specimens by means of proteomic and physio/pharmacological experiments. To evaluate the composition of venoms, 1D/2D-PAGE of crude venoms and RP-HPLC followed by SDS-PAGE were performed. Enzymatic, hemolytic, coagulant and platelet aggregating activities of crude venoms were assayed and were concordant with expectations based on the abundance of protein species in each. Neutralization assays were performed using Bharat polyvalent antivenom (BPAV), a product raised against venoms from Big-4 specimens from southern India. BPAV exhibited cross-reactivity against the Pakistani venoms, however, neutralization of clinically relevant activities was variable and rarely complete. Cumulatively, the presented data not only highlight geographical variations present in the venoms of the Big-4 snakes of South Asia, but also demonstrate the neutralization potential of Indian polyvalent against the venom of Pakistani specimens. Given the partial neutralization observed, it is clear that whilst BPAV is a life-saving product in Pakistan, in future it is hoped that a region-specific product might be manufactured domestically, using venoms of local snakes in the immunising mixture.

Correction: Acridinedione as selective flouride ion chemosensor: a detailed spectroscopic and quantum mechanical investigation.

[This corrects the article DOI: 10.1039/C7RA11974G.].

Synthetic applications and methodology development of Chan-Lam coupling: a review.

Chan-Lam coupling is one of the most popular and easy methods to perform arylation of amines (N-arylations). This cross-coupling is generally performed by reacting aryl boronate derivatives with a variety of substrates involving nitrogen containing functional groups such as amines, amides, ureas, hydrazine, carbamates. This article summarizes the synthetic applications of this reaction and the efforts of scientists to develop novel and efficient methodologies for this reaction.

Novel acridine-based thiosemicarbazones as 'turn-on' chemosensors for selective recognition of fluoride anion: a spectroscopic and theoretical study.

New thiosemicarbazide-linked acridines 3a-c were prepared and investigated as chemosensors for the detection of biologically and environmentally important anions. The compounds 3a-c were found selective for fluoride (F-) with no affinity for other anions, i.e. -OAc, Br-, I-, HSO4-, SO42-, PO43-, ClO3-, ClO4-, CN- and SCN-. Further, upon the gradual addition of a fluoride anion (F-) source (tetrabutylammonium fluoride), a well-defined change in colour of the solution of probes 3a-c was observed. The anion-sensing process was studied in detail via UV-visible absorption, fluorescence and 1H-NMR experiments. Moreover, during the synthesis of acridine probes 3a-c nickel fluoride (NiF2), a rarely explored transition metal fluoride salt, was used as the catalyst. Theoretical studies via density functional theory were also carried out to further investigate the sensing and anion (F-) selectivity pattern of these probes.

Acridinedione as selective flouride ion chemosensor: a detailed spectroscopic and quantum mechanical investigation.

The use of small molecules as chemosensors for ion detection is rapidly gaining popularity by virtue of the advantages it offers over traditional ion sensing methods. Herein we have synthesized a series of acridine(1,8)diones (7a-7l) and explored them for their potential to act as chemosensors for the detection of various anions such as fluoride (F-), acetate (OAc-), bromide (Br-), iodide (I-), bisulfate (HSO4 -), chlorate (ClO3 -), perchlorate (ClO4 -), cyanide (CN-), and thiocyanate (SCN-). Acridinediones were found to be highly selective chemosensors for fluoride ions only. To investigate in detail the mechanism of selective fluoride ion sensing, detailed spectroscopic studies were carried out using UV-visible, fluorescence and 1H NMR spectroscopy. Fluoride mediated (NH) proton abstraction of acridinedione was found to be responsible for the observed selective fluoride ion sensing. Quantum mechanical computational studies, using time dependent density functional theory (TDDFT) were also carried out, whereupon comparison of acridinedione interaction with fluoride and acetate ions explained the acridinedione selectivity for the detection of fluoride anions. Our results provide ample evidence and rationale for further modulation and exploration of acridinediones as non-invasive chemosensors for fluoride ion detection in a variety of sample types.

Protein-drug nanoconjugates: Finding the alternative proteins as drug carrier.

Present study was conducted to establish the interaction of bovine fetuin-A to validate its binding modalities with doxorubicin (Dox). Fetuin-A was purified to highest purity and monodispersity. Green synthesis of fetuin-A conjugated gold nanoparticles (F-GNPs) has been performed giving typical UV-maxima with subtle variation in fourier transform infrared spectroscopy (FTIR). Atomic force microscopy (AFM) revealed spherical shaped, polydisperse F-GNPs of varying sizes, complementing the radius of hydration (19.5-62.4nm) by dynamic light scattering (DLS). Circular dichroism (CD) analysis of fetuin-A with respect to Dox interaction shows remarkable reduction in ellipticity with increasing concentrations of Dox (20-120µM). Fetuin-A:Dox and F-GNPs:Dox at variable concentrations revealed significantly enhanced absorption spectra, while a continuous decrease in florescence (560nm). This effect was more drastic when Dox interact with fetuin-A as compared to F-GNPs. Some known antimicrobial drugs were also investigated under similar conditions, giving strong quenching effect in a dose dependent manner suggesting the significant yet differential interactions. In cytotoxicity assay, fetuin-A:Dox conjugates revealed less toxicity as compared to F-GNPs:Dox and Dox alone. In-silico studies of the fetuin-A:Dox complex suggest that the drug binds in the major grove between beta-sheet and long loop region of D1 domain and stabilized by several hydrogen bonds.

Formulation and stabilization of norfloxacin in liposomal preparations.

A number of liposomal preparations of norfloxacin (NF) containing variable concentrations of phosphatidylcholine (PC) (10.8-16.2mM) have been formulated and an entrapment of NF to the extent of 41.7-56.2% was achieved. The values of apparent first-order rate constants (kobs) for the photodegradation of NF in liposomes (pH7.4) lie in the range of 1.05-2.40×10(-3)min(-1) compared to a value of 8.13×10(-3)min(-1) for the photodegradation of NF in aqueous solution (pH7.4). The values of kobs are a linear function of PC concentration indicating an interaction of PC and NF during the reaction. The second-order rate constant for the photochemical interaction of PC and NF has been determined as 8.92×10(-2)M(-1)min(-1). Fluorescence measurements on NF in liposomes indicate a decrease in fluorescence with an increase in PC concentration as a result of formation of NF(-) species which exhibits poor fluorescence. Dynamic light scattering has shown an increase in the size of NF encapsulated liposomes with an increase in PC concentration. The stabilization of NF in liposomes is achieved by the formation of a charge-transfer complex between NF and PC.

Formulation and stabilization of riboflavin in liposomal preparations.

A study of the formulation of liposomal preparations of riboflavin (RF) with compositional variations in the content of phosphatidylcholine (PC) and their entrapment efficiency (26-42%) have been conducted. Light transmission characteristics of the liposomal preparations have been determined to evaluate their effect on the amount of light passing through the system to initiate a photochemical reaction. Dynamic light scattering (DLS) and atomic force microscopy (AFM) have been used to study the physical characteristics of liposomes. The liposomal preparations of RF have been subjected to photolysis using visible light and the apparent first- order rate constant, kobs, for the degradation of RF have been determined. The values of kobs (1.73-2.29×10(-3)min(-1)) have been found to decrease linearly with an increase in PC concentration in the range of 12.15 to 14.85 mM. Thus, an increase in PC concentration of liposomes leads to an increase in the stability of RF. RF and its main photoproduct, lumichrome (LC), formed in liposomes have been assayed by a two-component spectrometric method at 356 and 445 nm using an irrelevant absorption correction to compensate for the interference of liposomal components. The fluorescence measurements of RF in liposomes indicate excited singlet state quenching and the formation of a charge-transfer complex between RF and PC. It results in electron transfer from PC to RF to cause photoreduction and stabilization of RF.

Pharmacological and biochemical studies on the venom of a clinically important viper snake (Echis carinatus) of Pakistan.

Echis carinatus (saw-scaled viper) has been the major culprit responsible for serious envenomation casualties throughout the subcontinent. The present study describes the electrophoretic and zymographic characterization of E. carinatus venom and its effect on mammalian smooth muscle. Crude venom showed the presence of disintegrin, PLA2, C-type lectin/lectin-like components, CRISP, Serine protease, l-amino acid oxidase and very high concentrations of SVMPs. E. carinatus venom (1, 10, 30, 50, 100 µg/ml) inhibited the active tension/force of muscle contraction in a time and concentration dependent manner. The observed effects abolished when the venom was heated at 100 °C for 5 min. However, a decrease in bath temperature from 37 °C to 26 °C or an increase in CaCl2 concentration to 5 mM did not prevent the inhibition of contractile activity. The contractile response elicited by exogenous application of 50 mM KCl and 1 µM acetylcholine (ACh) was also significantly inhibited by all venom concentrations. Prior administration of commercially available polyvalent anti-venom partially neutralized and prevented the effect of E. carinatus venom whereas addition of anti-venom at t50 failed to reverse the inhibitory effect. Studies on isolated intestinal muscle indicate involvement of myotoxic and apoptotic components in E. carinatus venom for irreversible damage to muscle tissue.