Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters.

BACKGROUND: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis. RESULT: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu2+) ions. The off state of LA@AuAg_Cu2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu2+. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu2+ ions, and again quenched off state was retrieved due to interaction of free Cu2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples. SIGNIFICANCE: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

Chitosan functionalized doxorubicin loaded poly(methacrylamide) based copolymeric nanoparticles for enhanced cellular internalization and in vitro anticancer evaluation.

Doxorubicin (Dox), a chemotherapeutic agent, encounters challenges such as a short half-life, dose-dependent toxicity, and low solubility. In this context, the present study involved the fabrication of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA) bearing P(HPMA-s-APMA) copolymeric nanoparticles (P(HPMA-s-APMA) NPs) and their investigation for efficient delivery of Dox. Furthermore, the synthesized nanoparticles (NPs) were coated with chitosan (Cht) to generate positively charged nanoformulations. The prepared formulations were evaluated for particle size, morphology, surface charge analysis, percentage encapsulation efficiency (EE%), and drug release studies. The anticancer activity of Cht-P(HPMA-s-APMA)-Dox NPs was assessed in the HeLa cancer cell line. The prepared P(HPMA-s-APMA)-Dox NPs exhibited an average particle size of 240-250 nm. Chitosan decorated P(HPMA-s-APMA)-Dox NPs displayed a significant increase in particle size, and the zeta potential shifted from negative to positive. The EE% for Cht-P(HPMA-s-APMA)-Dox NPs was calculated to be 68.06 %. The drug release studies revealed a rapid release of drug from Cht-P(HPMA-s-APMA)-Dox NPs at pH 4.8 than pH 7.4, demonstrating the pH-responsiveness of nanoformulation. Furthermore, the cell viability assay and internalization studies revealed that Cht-P(HPMA-s-APMA)-Dox NPs had a high cytotoxic response and significant cellular uptake. Hence, the Cht-P(HPMA-s-APMA)-Dox NPs appeared to be a suitable nanocarrier for effective, and safe chemotherapy.

Tuning the Luminescence of Microwave-Assisted N-Doped Fluorescent Carbon Dots: Bioimaging Applications and Label-Free Anti-Cancer Drug Delivery.

Nanosized fluorescent carbon dots (Cdots) have gained a lot of attention in the recent years because of their superior properties, such as good biocompatibility, low toxicity, excellent chemical stability, resistance to photobleaching, and ease of chemical modification. Cdots are promising candidates for considerable applications in various fields: sensors, bioimaging, and drug delivery. Specifically, nitrogen-doped Cdots have attracted a huge interest because of their applicability in bioimaging and drug delivery. Conventional methods for the synthesis of Cdots have drawbacks, such as the use of organic solvents, the presence of side products, and the time required for synthesis. Keeping all these points in mind, herein, we report green methodology for the synthesis of water-soluble, blue-emitting, nitrogen-doped multifunctional Cdots under microwave irradiation within 3 min. The Cdots were prepared using citric acid and arginine as source materials and were characterized using various physicochemical techniques. A pH-responsive drug delivery system was then designed using anticancer drug doxorubicin and the synthesized Cdots. The biocompatibility of synthesized Cdots was analyzed against L929 normal cell line. The Cdots-DOX conjugates exhibited efficient anticancer activity against HeLa cells and also acted as excellent bioimaging agents.

Self-Assembled Peptide Hydrogel for Accelerated Wound Healing: Impact of N-Terminal and C-Terminal Modifications.

Wound dressings are required to provide a moist environment for wounds, protect against invading infections, expedite tissue regeneration, and improve wound healing efficiency. Developing biomaterials with all aforesaid properties is still a big challenge. However, peptide-based hydrogels have the potential to overcome these challenges as they are biocompatible, biodegradable as well as have the ability to mimic the extracellular matrix and provide an appropriate moist environment which is important for wound healing. With this in mind, we report the preparation and comparison of three hexapeptide-based hydrogels, LIVAGD, with the aim to understand the importance of the N-terminal protecting group as well as the C-terminal amino acid substitution on its various biological efficacies. Fmoc and acetyl groups were used for N-terminal peptide protection, while aspartic acid was substituted with lysine at the C-terminus. The resulting peptide-based hydrogels were compared. Fmoc peptide-based hydrogels exhibited efficient anti-inflammatory action along with improved biocompatibility while lysine provided enhanced antibacterial effect to the hydrogel. Additionally, in vivo efficacy was examined using a mouse model, and Fmoc hydrogels demonstrated an improved wound healing ability with ∼40 % faster healing rate in comparison to the reported acetylated peptide hydrogels.

Optical pico-biosensing of lead using plasmonic gold nanoparticles and a cationic peptide-based aptasensor.

A novel biosensor for the rapid detection of lead ions employing the optical properties of AuNPs, a lead-specific aptamer and a cationic peptide has been demonstrated. The limit of detection of the biosensor was 98.7 pM, the lowest so far obtained using colorimetry.

Insights into cell penetrating peptide conjugated gold nanoparticles for internalization into bacterial cells.

Gold nanoparticles (AuNPs) functionalized with different biomolecules find extensive application in therapy, clinical diagnosis and biomedical imaging. Herein, two derivatives of TAT peptide with sequences YGRKKRRQRRR and YGRKKRRQRRR-(ß-ala)3-Cys-amide were conjugated with tannic acid capped gold nanoparticles which acted as a carrier for cell penetrating peptides (CPPs) into the bacterial cells. The interaction of YGRKKRRQRRR peptide with AuNPs was non-covalent in nature whereas YGRKKRRQRRR-(ß-ala)3-Cys-amide interacted covalently with the AuNPs due to presence of thiol group in cysteine which bind strongly to gold nanoparticles surface. Further, tannic acid functionalised AuNPs conjugated CPPs constructs were duly characterized using critical flocculation essay test, UV-visible and TEM. FITC was tagged over AuNPs-CPPs in order to study the intracellular distribution using confocal microscopy. The confocal results revealed that nanoconjugates (AuNP-CPPs) of 5 nm diameter exhibited strong fluorescent signal in Gram positive and Gram negative bacterial strains. The present method can also be used for the killing of bacterial cells using photo-thermal therapy and therefore can be highly useful for targeting multi-drug resistant bacteria.

A Facile Approach for Synthesis and Intracellular Delivery of Size Tunable Cationic Peptide Functionalized Gold Nanohybrids in Cancer Cells.

Peptide-based drug delivery systems have become a mainstay in the contemporary medicinal field, resulting in the design and development of better pharmaceutical formulations. However, most of the available reports employ tedious multiple reaction steps for the conjugation of bioactive cationic peptides with drug delivery vehicles. To overcome these limitations, the present work describes a one-step approach for facile and time efficient synthesis of highly cationic cell penetrating peptide functionalized gold nanoparticles and their intracellular delivery. The nanoconstruct was synthesized by the reduction of gold metal ions utilizing cell penetrating peptide (CPP), which facilitated the simultaneous synthesis of metal nanoparticles and the capping of the peptide over the nanoparticle surface. The developed nanoconstruct was thoroughly characterized and tested for intracellular delivery into HeLa cells. Intriguingly, a high payload of cationic peptide over gold particles was achieved, in comparison to conventional conjugation methods. Moreover, this method also provides the ability to control the size and peptide payload of nanoparticles. The nanoconstructs produced showed enhanced cancer cell penetration (µM) and significant cytotoxic effect compared to unlabeled gold nanoparticles. Therefore, this novel approach may also have significant future potential to kill intracellular hidden dreaded pathogens like the human immunodeficiency virus, Mycobacterium tuberculosis, and so forth.

Synthesis, stability and mechanistic studies of potent anticryptococcal hexapeptides.

The growing incidents of cryptococcosis in immuno-compromised patients have created a need for novel drug therapies capable of eradicating the disease. The peptide-based drug therapy offers many advantages over the traditional therapeutic agents, which has been exploited in the present study by synthesizing a series of hexapeptides that exhibits promising activity against a panel of Gram-negative and Gram-positive bacteria and various pathogenic fungal strains; the most exemplary activity was observed against Cryptococcus neoformans. The peptides 3, 24, 32 and 36 displayed potent anticryptococcal activity (IC50 = 0.4-0.46 µg/mL, MIC = 0.63-1.25 µg/mL, MFC = 0.63-1.25 µg/mL), and stability under proteolytic conditions. Besides this, several other peptides displayed promising inhibition of pathogenic bacteria. The prominent ones include peptides 18-20, and 26 that exhibited IC50 values ranged between 2.1 and 3.6 µg/mL, MICs of 5-20 µg/mL and MBCs of 10-20 µg/mL against Staphylococcus aureus and methicillin-resistant S. aureus. The detailed mechanistic study on selected peptides demonstrated absolute selectivity towards the bacterial membranes and fungal cells by causing perturbations in the cell membranes, confirmed by the scanning electron microscopy and transmission electron microscopy studies.

Discovery of Short Peptides Exhibiting High Potency against Cryptococcus neoformans.

Rapid increase in the emergence of resistance against existing antifungal drugs created a need to discover new structural classes of antifungal agents. In this study we describe the synthesis of a new structural class of short antifungal peptidomimetcis, their activity, and plausible mechanism of action. The results of the study show that peptides 11e and 11f are more potent than the control drug amphotericin B, with no cytotoxicity to human cancer cells and noncancerous mammalian kidney cells. The selectivity of peptides to fungus is depicted by transmission electron microscopy studies, and it revealed that 11e possibly disrupts the model membrane of the fungal pathogen.

Design of single peptides for self-assembled conduction channels.

Self-assembly of peptides provides the possibility of achieving relatively long range order on surfaces. These ordered peptides can also form channels that can be used as conduction channels. In the past, studies were focused on electron conduction through the secondary structure and amine bond of peptides and these restrict conduction of electrons over a short range (a few nanometers). In this work, we demonstrate the realization of electron conduction over a longer range of a few hundred nanometers via π-π stacking of the phenyl groups in the tyrosine residue of a single peptide. The peptide used in this work was designed with a phenyl ring for π-π stacking at one end and a carboxylic group at the other end for binding to aminopropyltriethoxysilane (APTES) treated silicon wafer. The distance between the peptides is controlled by a disulfide bond formed between neighboring cysteine residue and also by the amine groups of aminopropyltriethoxysilane. We demonstrate that the self-assembled peptide is conducting in the dry state over hundreds of nanometers, realizing the possibility of using peptide as a molecular wire.

New antimicrobial hexapeptides: synthesis, antimicrobial activities, cytotoxicity, and mechanistic studies.

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug-resistant pathogens. We identified a novel hexapeptide, Orn-D-Trp-D-Phe-Ile-D-Phe-His(1-Bzl)-NH(2), which exhibits broad-spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non-proteinogenic amino acids depending on the hydrophobic or positive-charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis, as well as the Gram-negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1-5 microg mL(-1) (IC(50) values in the range of 0.02-2 microg mL(-1)). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 microg mL(-1). A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.