From Pipeline to Plant Protection Products: Using New Approach Methodologies (NAMs) in Agrochemical Safety Assessment.

The human population will be approximately 9.7 billion by 2050, and food security has been identified as one of the key issues facing the global population. Agrochemicals are an important tool available to farmers that enable high crop yields and continued access to healthy foods, but the average new agrochemical active ingredient takes more than ten years, 350 million dollars, and 20,000 animals to develop and register. The time, monetary, and animal costs incentivize the use of New Approach Methodologies (NAMs) in early-stage screening to prioritize chemical candidates. This review outlines NAMs that are currently available or can be adapted for use in early-stage screening agrochemical programs. It covers new in vitro screens that are on the horizon in key areas of regulatory concern. Overall, early-stage screening with NAMs enables the prioritization of development for agrochemicals without human and environmental health concerns through a more directed, agile, and iterative development program before animal-based regulatory testing is even considered.

Gd3+:DOTA-Modified 2-Hydroxypropyl-ß-Cyclodextrin/4-Sulfobutyl Ether-ß-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd3+:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-ß-cyclodextrin and 4-sulfobutylether-ß-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd3+ chelation. The PR products were characterized by 1H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T1 relaxation and spin-lattice T2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T1-weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different ß-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.

Development of dihydrochalcone-functionalized gold nanoparticles for augmented antineoplastic activity.

BACKGROUND: Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodium-glucose linked transport 2 (SGLT2) inhibitor. While the aglycone metabolite of phloridzin, phloretin, displays a reduced capacity of SGLT2 inhibition, this nutraceutical displays enhanced antineoplastic activity in comparison to phloridzin. PURPOSE: The objective of this study was to develop gold nanoparticle (AuNP) mediated delivery of phloridzin and phloretin and explore their anticancer mechanism through conjugation of the dihydrochalcones and the AuNP cores. METHODS: Phloridzin and phloretin conjugated AuNPs (Phl-AuNP and Pht-AuNP) were synthesized in single-step, rapid, biofriendly processes. The synthesized AuNPs morphology was characterized via transmission electron microscopy and ultraviolet-visible spectroscopy. The presence of phloridzin or phloretin was confirmed using scanning electron microscopy-energy dispersive x-ray spectroscopy. The percentage of organic component (phloridzin/phloretin) onto AuNPs surface was characterized using thermogravimetric analysis. Assessment of the antineoplastic potency of the dihydrochalcones conjugated AuNPs against cancerous cell lines (HeLa) was accomplished through monitoring via flow cytometry. RESULTS: The functionalized AuNPs were synthesized via a single-step method that relied only upon the redox potential of the conjugate itself and required no toxic chemicals. The synthesized Phl-AuNPs were found to be in the size range of 15±5 nm, whereas the Pht-AuNP were found to be 8±3 nm, placing both conjugated AuNPs well within the size range necessary for successful pharmaceutical applications. These assays demonstrate a significant increase in the cancerous cell toxicities as a result of the conjugation of the drugs to AuNPs, as indicated by the 17.45-fold increase in the efficacy of Pht-AuNPs over pure phloretin, and the 4.49-fold increase in efficacy of Phl-AuNP over pure phloridzin. CONCLUSION: We report a simple, biofriendly process using the reducing and capping potential of the dihydrochalcones, phloridzin and phloretin, to synthesize stable AuNPs that have promising futures as potential antineoplastic agents.

Novel Synthesis of Kanamycin Conjugated Gold Nanoparticles with Potent Antibacterial Activity.

With a sharp increase in the cases of multi-drug resistant (MDR) bacteria all over the world, there is a huge demand to develop a new generation of antibiotic agents to fight them. As an alternative to the traditional drug discovery route, we have designed an effective antibacterial agent by modifying an existing commercial antibiotic, kanamycin, conjugated on the surface of gold nanoparticles (AuNPs). In this study, we report a single-step synthesis of kanamycin-capped AuNPs (Kan-AuNPs) utilizing the combined reducing and capping properties of kanamycin. While Kan-AuNPs have increased toxicity to a primate cell line (Vero 76), antibacterial assays showed dose-dependent broad spectrum activity of Kan-AuNPs against both Gram-positive and Gram-negative bacteria, including Kanamycin resistant bacteria. Further, a significant reduction in the minimum inhibitory concentration (MIC) of Kan-AuNPs was observed when compared to free kanamycin against all the bacterial strains tested. Mechanistic studies using transmission electron microscopy and fluorescence microscopy indicated that at least part of Kan-AuNPs increased efficacy may be through disrupting the bacterial envelope, resulting in the leakage of cytoplasmic content and the death of bacterial cells. Results of this study provide critical information about a novel method for the development of antibiotic capped AuNPs as potent next-generation antibacterial agents.

Mechanistic Insight into Receptor-Mediated Delivery of Cationic-ß-Cyclodextrin:Hyaluronic Acid-Adamantamethamidyl Host:Guest pDNA Nanoparticles to CD44(+) Cells.

Targeted delivery is a key element for improving the efficiency and safety of nonviral vectors for gene therapy. We have recently developed a CD44 receptor targeted, hyaluronic acid-adamantamethamidyl based pendant polymer system (HA-Ad), capable of forming complexes with cationic ß-cyclodextrins (CD-PEI(+)) and pDNA. Complexes formed using these compounds (HA-Ad:CD-PEI(+):pDNA) display high water solubility, good transfection efficiency, and low cytotoxicity. Spatial and dynamic tracking of the transfection complexes by confocal microscopy and multicolor flow cytometry techniques was used to evaluate the target specificity, subcellular localization, and endosomal escape process. Our data shows that cells expressing the CD44 receptor undergo enhanced cellular uptake and transfection efficiency with HA-Ad:CD-PEI(+):pDNA complexes. This transfection system, comprised noncovalent assembly of cyclodextrin:adamantamethamidyl-modified hyaluronic acid via host:guest interactions to condense pDNA, is a potentially useful tool for targeted delivery of nucleic acid therapeutics.

Structure-property relationship for in vitro siRNA delivery performance of cationic 2-hydroxypropyl-ß-cyclodextrin: PEG-PPG-PEG polyrotaxane vectors.

Nanoparticle-mediated siRNA delivery is a promising therapeutic approach, however, the processes required for transport of these materials across the numerous extracellular and intracellular barriers are poorly understood. Efficient delivery of siRNA-containing nanoparticles would ultimately benefit from an improved understanding of how parameters associated with these barriers relate to the physicochemical properties of the nanoparticle vectors. We report the synthesis of three Pluronic(®)-based, cholesterol end-capped cationic polyrotaxanes (PR(+)) threaded with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) for siRNA delivery. The biological data showed that PR(+):siRNA complexes were well tolerated (∼90% cell viability) and produced efficient silencing (>80%) in HeLa-GFP and NIH 3T3-GFP cell lines. We further used a multi-parametric approach to identify relationships between the PR(+) structure, PR(+):siRNA complex physical properties, and biological activity. Small angle X-ray scattering and cryoelectron microscopy studies reveal periodicity and lamellar architectures for PR(+):siRNA complexes, whereas the biological assays, ζ potential measurements, and imaging studies suggest that silencing efficiency is influenced by the effective charge ratio (ρeff), polypropylene oxide (PO) block length, and central PO block coverage (i.e., rigidity) of the PR(+) core. We infer from our findings that more compact PR(+):siRNA nanostructures arising from lower molecular weight, rigid rod-like PR(+) polymer cores produce improved silencing efficiency relative to higher molecular weight, more flexible PR(+) vectors of similar effective charge. This study demonstrates that PR(+):siRNA complex formulations can be produced having higher performance than Lipofectamine(®) 2000, while maintaining good cell viability and siRNA sequence protection in cell culture.

Efficient pDNA Delivery Using Cationic 2-Hydroxypropyl-ß-Cyclodextrin Pluronic-Based Polyrotaxanes.

A family of cationic Pluronic-based polyrotaxanes (PR(+)), threaded with 2-hydroxypropyl-ß-cyclodextrin (HPCD), was synthesized for pDNA delivery into multiple cell lines. All PR(+) formed highly stable, positively charged pDNA complexes that were < 250 nm in diameter. The cellular uptake and pDNA transfection efficiencies of the PR(+):pDNA complexes was enhanced relative to the commercial transfection standards L2K and bPEI, while displaying similar or lower toxicity profiles. Charge density and threading efficiency of the PR(+) agent significantly influenced the colloidal stability and physical properties of the complexes, which impacted their intracellular transfection efficiencies. Taken together, our results suggest that HPCD: Pluronic PR(+) can be used as potent vectors for pDNA-based therapeutics.

Nanotechnology's Impact on Medicinal Chemistry.

Nanotechnology has intrigued a large number of researchers the world over owing to its unique properties as compared to bulk materials, and the novelty of applications made possible across many fields of science. Researchers, taking advantage of the unique properties of particles in nano (1-100 nm) form, have been developing nanoformulations of various medicinal compounds to enhance drug solubility, dissolution, and bioavailability. There are various methods by which drug compounds are conjugated to nanoparticles, and some bioactive compounds are attached by intermediary agents which are themselves usually part of the formation reaction of nanoparticles. Nanoformulations have been developed involving a range of medicinal compounds of biological and syntheticorigin intended to enhance the compound's pharmacokinetic and pharmacological profiles, or to capitalize on unique properties of nanoparticles for therapeutic or diagnostic purposes. A number of nanodrugs exist on the market today, and many more are in the clinical or pre-clinical pipeline. There are a number of challenges commonly encountered when designing nanodrug formulations as well as challenges to the long term viability of nanodrug formulation strategies, especially in regards to environmental and safety concerns. Some researchers have harnessed the structural and functional relationship of various medicinal compounds to enhance the design of nanoformulations. Other researchers have used structure-activity relationships as a means of enhancing safety and efficacy testing through in silico modeling. This article will touch on each of the above issues within the context of the impact each facet of nanotechnology has on medicinal chemistry.

Gold nanoparticles: various methods of synthesis and antibacterial applications.

Colloidal gold is very attractive for several applications in biotechnology because of its unique physical and chemical properties. Many different synthesis methods have been developed to generate gold nanoparticles (AuNPs). Here, we will introduce these methods and discuss the differences between fabrication techniques. We will also discuss ecofriendly synthesis methods being developed to efficiently generate AuNPs without the use of toxic substrates. Finally, we will discuss the medical applications for AuNPs by highlighting the potential use of intact or functionalized AuNPs in combating bacterial infections.

Biological applications of gold nanoparticles.

This article reviews some of the recent biological applications of gold nanoparticles (GNPs) which have been discovered lately by individual studies all around the world. GNPs have emerged as a promising candidates for various biological applications due to their unique physical properties (size and shape dependent), excellent biocompatibility, facile synthesis, ease of bioconjugation, etc. This review starts with a brief introduction about nanotechnology followed by an insight into the history, emergence, and enhanced properties of various gold nanostructures, which form the basis for their numerous biomedical applications. In addition, a brief overview on some of the commonly used fabrication techniques for synthesizing GNPs is also discussed. Finally, a miscellany of the latest biological applications of GNPs, such as cancer diagnostics and therapy, biological probes, drug delivery, gene delivery, vaccine preparation, brain implants, artificial skin, sterilization system, and improving electrical signaling in the heart, published in different articles in reputed journals are highlighted.

Effect of pendant group on pDNA delivery by cationic-ß-cyclodextrin:alkyl-PVA-PEG pendant polymer complexes.

We have previously shown that cationic-ß-cyclodextrin:R-poly(vinyl alcohol)-poly(ethylene glycol) (CD+:R-PVA-PEG) pendant polymer host:guest complexes are safe and efficient vehicles for nucleic acid delivery, where R = benzylidene-linked adamantyl or cholesteryl esters. Herein, we report the synthesis and biological performance of a family of PVA-PEG pendant polymers whose pendant groups have a wide range of different affinities for the ß-CD cavity. Cytotoxicity studies revealed that all of the cationic-ß-CD:pendant polymer host:guest complexes have 100-1000-fold lower toxicity than branched polyethylenimine (bPEI), with pDNA transfection efficiencies that are comparable to bPEI and Lipofectamine 2000. Complexes formed with pDNA at N/P ratios greater than 5 produced particles with diameters in the 100-170 nm range and ζ-potentials of 15-35 mV. Gel shift and heparin challenge experiments showed that the complexes are most stable at N/P ≥ 10, with adamantyl- and noradamantyl-modified complexes displaying the best resistance toward heparin-induced decomplexation. Disassembly rates of fluoresceinated-pDNA:CD(+):R-PVA-PEG-rhodamine complexes within HeLa cells showed a modest dependence on host:guest binding constant, with adamantyl-, noradamantyl-, and dodecyl-based complexes showing the highest loss in FRET efficiency 9 h after cellular exposure. These findings suggest that the host:guest binding constant has a significant impact on the colloidal stability in the presence of serum and cellular uptake efficiency, whereas endosomal disassembly and transfection performance of cationic-ß-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer complexes appears to be controlled by the hydrolysis rates of the acetal grafts onto the PVA main chain.

Antibacterial gold nanoparticles-biomass assisted synthesis and characterization.

Xylose is a natural monosaccharide found in biomass such as straw, pecan shells, cottonseed hulls, and corncobs. Using this monosaccharide, we report the facile, green synthesis and characterization of stable xylose encapsulated gold nanoparticles (Xyl-GNPs) with potent antibacterial activity. Xyl-GNPs were synthesized using the reduction property of xylose in an aqueous solution containing choloraurate anions carried out at room temperature and atmospheric pressure. These nanoparticles were stable and near spherical in shape with an average diameter of 15 +/- 5 nm. Microbiological assay results showed the concentration dependent antibacterial activity of these particles against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus epidermidis) bacteria. Thus the facile, environmentally friendly Xyl-GNPs have many potential applications in chemical and biomedical industries, particularly in the development of antibacterial agents in the field of biomedicine.

Bactericidal activity of starch-encapsulated gold nanoparticles.

We report the bactericidal applications of eco-friendly starch encapsulated gold nanoparticles (St-AuNPs). The mechanism of interaction of the properly characterized St-AuNPs with both Gram negative and Gram positive bacteria were investigated using spread plate assay, transmission electron microscopy (TEM), and fluorescent propidium iodide (PI) exclusion assay. The St-AuNPs were found to possess significant dose dependent antibacterial activity against both types of bacteria. St-AuNPs at 1.2 mg/mL caused 98 % eradication of Gram positive bacteria that was exposed over a period of 12 h. Similarly, 4.8 mg/mL St-AuNPs caused 98 % eradication of Gram negative bacteria over a period of 12h. The St-AuNPs are biocompatible and present a useful solid porous carbohydrate-based polymer vehicle with excellent antimicrobial activity against [Frontiers in Bioscience 18, 982-992, June 1, 2013].

Size-dependent antimicrobial properties of sugar-encapsulated gold nanoparticles synthesized by a green method.

The antimicrobial properties of dextrose-encapsulated gold nanoparticles (dGNPs) with average diameters of 25, 60, and 120 nm (± 5) and synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of dGNPs on the growth, morphology, and ultrastructural properties of bacteria. dGNPs were found to have significant dose-dependent antibacterial activity which was also proportional to their size. Experiments revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action by disrupting the bacterial cell membrane which leads to the leakage of cytoplasmic content. The overall outcome of this study suggests that green-synthesized dGNPs hold promise as a potent antibacterial agent against a wide range of disease-causing bacteria by preventing and controlling possible infections or diseases.

Efficient and inexpensive method for purification of heparin binding proteins.

Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

Single-step biofriendly synthesis of surface modifiable, near-spherical gold nanoparticles for applications in biological detection and catalysis.

There is an increased interest in understanding the toxicity and rational design of gold nanoparticles (GNPs) for biomedical applications in recent years. Such efforts warrant reliable, viable, and biofriendly synthetic methodology for GNPs with homogeneous sizes and shapes, particularly sizes above 30 nm, which is currently challenging. In the present study, an environmentally benign, biofriendly, single-step/single-phase synthetic method using dextrose as a reducing and capping agent in a buffered aqueous solution at moderate temperature is introduced. The resulting GNPs are near-spherical, stable, catalytically active, place exchangeable, and water-soluble within the size range of 10-120 nm. The added advantage of the biologically friendly reaction medium employed in this new synthetic approach provides a method for the direct embedment/integration of GNPs into biological systems such as the E. coli bacterium without additional capping ligand or surface modification processes.