Deendayal Mandal

Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum

The development of reliable, eco-friendly processes for the synthesis of nanomaterials is an important aspect of nanotechnology today. One approach that shows immense potential is based on the biosynthesis of nanoparticles using biological micro-organisms such as bacteria. In this laboratory, we have concentrated on the use of fungi in the intracellular production of metal nanoparticles. As part of our investigation, we have observed that aqueous silver ions when exposed to the fungus Fusarium oxysporum are reduced in solution, thereby leading to the formation of an extremely stable silver hydrosol. The silver nanoparticles are in the range of 5-15 nm in dimensions and are stabilized in solution by proteins secreted by the fungus. It is believed that the reduction of the metal ions occurs by an enzymatic process, thus creating the possibility of developing a rational, fungal-based method for the synthesis of nanomaterials over a range of chemical compositions, which is currently not possible by other microbe-based methods.

The use of microorganisms for the formation of metal nanoparticles and their application

Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, and high monodispersity is one of the challenging issues in current nanotechnology. In the context of the current drive to develop green technologies in material synthesis, this aspect of nanotechnology is of considerable importance. Biological systems, masters of ambient condition chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macroscale. Recent studies on the use of microorganisms in the synthesis of nanoparticles are a relatively new and exciting area of research with considerable potential for development. This review describes a brief overview of the current research worldwide on the use of microorganisms in the biosynthesis of metal nanoparticles and their applications.

BIOREDUCTION OF AUCL4− IONS BY THE FUNGUS, VERTICILLIUM SP. AND SURFACE TRAPPING OF THE GOLD NANOPARTICLES FORMED

Fungi make piles of gold! A green-chemistry route, based on the bioreduction of AuCl 4 - ions by the fungus Verticillium sp., for the formation of gold nanoparticles is demonstrated. The TEM micrograph shows a single Verticillium cell after reaction with gold ions and entrapment of gold nanoparticles on the cell wall and cytoplasmic membrane.

Extracellular Synthesis of Gold Nanoparticles by the Fungus Fusarium oxysporum

A green chemistry approach to nanoparticle synthesis is the exciting possibility opened up by the fungus Fusarium oxysporum. The fungus, when exposed to aqueous AuCl 4 − ions, reduces the metal ions; this leads to the extracellular formation of gold nanoparticles.

Cell-Penetrating Homochiral Cyclic Peptides as Nuclear-Targeting Molecular Transporters†

The intracellular delivery of biologically active cargos by employing linear cell-penetrating peptides (CPPs) has been previously reported. Conjugation to linear cationic CPPs, such as TAT (trans-acting activator of transcription; a peptide derived from the HIV-1 transactivator protein), 3] penetratin, antennapedia, or oligoarginine, efficiently enhances the cellular uptake through different mechanisms. The cellular uptake and internalization of many CPPs along with the conjugated cargo occurs predominantly by an endocytic pathway that involves macropinocytosis, a caveolae pathway, clathrin-mediated endocytosis, or lipid-raft dependent endocytosis. Endosomal uptake represents a major challenge in targeted intracellular drug delivery since some compounds are trapped in endosomes and cannot reach the biological targets in the cytoplasm or nucleus. Thus, strategies that promote endosomal escape or avoid endosomal routes are required for improving bioavailability. Moreover, the nuclear delivery of cell-impermeable and water-insoluble molecules remains a major challenge. The nucleus is a desirable target because the genetic information of the cell and transcription machinery resides there. To date, most approaches for nuclear delivery of compounds have taken advantage of covalent conjugation, which requires release of the cargo from the conjugate and/or endosomal escape. There is therefore a need to develop alternative stable peptide carriers that avoid endosomal pathways and/or covalent conjugation. Compared to linear peptides that are susceptible to hydrolysis by endogenous peptidases, cyclic peptides are enzymatically more stable. The cell-penetrating properties and application of homochiral l-cyclic peptides in drug delivery remain unexplored. Previous studies on linear CPPs by our research group and others indicated that an optimal balance of positive charge and hydrophobicity is required for interactions with the cell membrane and deep penetration into the lipid bilayer. 4,8–10] Herein, we report the design and evaluation of amphipathic homochiral l-cyclic peptides for potential applications as CPPs and/or as molecular transporters of bioactive compounds. Eleven cyclic peptides, namely [WR]4, [FK]4, [AK]4, [EL]4, [RFEF]2, [EK]4, [ER]4, [FR]4, [RFE]3, [WR]3, and [WR]5 (Scheme 1), which contain l-amino acids, were synthesized by employing 9-fluorenylmethyloxycarbonyl (Fmoc) based peptide chemistry. The selection of the cyclic peptides was based on the presence of hydrophobic residues (e.g., W, F, L) and charged residues (e.g., K, R, E). We hypothesized that an optimal amphipathic cyclic peptide that contains appropriate residues, and that undergoes intermolecular and intramolecular interactions can act as a CPP and/or entrap and deliver a bioactive compound intracellularly. To examine the potential application of the cyclic peptides as molecular transporters, a model experiment was performed with lamivudine (( )-2’,3’-dideoxy-3’-thiacytidine, 3TC). 3TC is a nucleoside reverse transcriptase inhibitor that blocks HIV-1 and hepatitis B virus replication. The efficient cellular uptake of 3TC is critical for effective antiviral activity. To monitor the molecular transport ability of the cyclic peptides, a carboxyfluorescein derivative of 3TC (F-3TC) was synthesized. The cellular uptake of the fluorescently labeled 3TC (F3TC) was examined in the leukemia CCRF-CEM cell line in the presence or absence of cyclic peptides. After 1 h incubation at 37 8C, the cells were treated with trypsin to remove the cell-surface-bound drug. The cellular uptake of F3TC was monitored by fluorescence-activated cell sorting (FACS; Figure 1a) and fluorescence microscopy (Figure 1b). The cyclic peptides did not exhibit any cytotoxicity by using an MTT assay at an experimental concentration of 50 mm in four different cell lines, namely CCRF-CEM, HT-29, MDAMD-468, and SK-OV-3, thus showing consistent results (Figure S12). FACS and fluorescence microscopy showed significantly higher fluorescence signals in the cells treated with F-3TCloaded [WR]4 and [WR]5 compared to those treated with other F-3TC-loaded cyclic peptides and with F-3TC alone, thus suggesting that the uptake of F-3TC is facilitated by [WR]n (n = 4, 5) and is dependent on nature of amino acids. F3TC-loaded [WR]5 exhibited a cellular uptake that was approximately five times higher than that of F-3TC alone (Figure 1a). Phosphopeptides are valuable probes for studying phosphoprotein–protein interactions because these peptides mimic the interactions between the negatively charged phosphate group of phosphoproteins and positively charged amino acids in the binding pockets of a number of proteins. Studying negatively charged phosphopeptides in cellular systems is challenging because these peptides do not readily [*] Dr. D. Mandal, A. Nasrolahi Shirazi, Prof. K. Parang Department of Biomedical and Pharmaceutical Sciences University of Rhode Island 41 Lower College Road, Kingston, RI 02881 (USA) E-mail: kparang@uri.edu

Synthesis of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation of their Src kinase inhibitory and anticancer activities.

A series of two classes of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates were synthesized using click chemistry approach. All compounds were evaluated for inhibition of Src kinase and human ovarian adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-MB-361), and colon adenocarcinoma (HT-29). Hexyl triazolyl-substituted 3-phenylpyrazolopyrimidine exhibited inhibition of Src kinase with an IC(50) value of 5.6 μM. 4-Methoxyphenyl triazolyl-substituted 3-phenylpyrazolopyrimidine inhibited the cell proliferation of HT-29 and SK-Ov-3 by 73% and 58%, respectively, at a concentration of 50 μM.

Superparamagnetic nanoparticle-supported enzymatic resolution of racemic carboxylates.

Candida rugosa lipase immobilized on maghemite nanoparticles demonstrated high stereoselectivity in kinetic resolution of racemic carboxylates and improved long-term stability over its parent free enzyme, allowing the supported enzyme to be repeatedly used for a series of chiral resolution reactions.

Click chemistry inspired one-pot synthesis of 1,4-disubstituted 1,2,3-triazoles and their Src kinase inhibitory activity

Two classes of 1,4-disubstituted 1,2,3-triazoles were synthesized using one-pot reaction of α-tosyloxy ketones/α-halo ketones, sodium azide, and terminal alkynes in the presence of aq PEG (1:1, v/v) using the click chemistry approach and evaluated for Src kinase inhibitory activity. Structure-activity relationship analysis demonstrated that insertion of C(6)H(5)- and 4-CH(3)C(6)H(4)- at position 4 for both classes and less bulkier aromatic group at position 1 in class 1 contribute critically to the modest Src inhibition activity (IC(50) = 32-43 μM) of 1,4-disubstituted 1,2,3-triazoles.

Cellular uptake of gold nanoparticles directly cross-linked with carrier peptides by osteosarcoma cells

Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif’s that are known to deliver a ‘cargo’ into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such ‘carrier peptides’ can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles.

Synthesis, anticancer activities, and cellular uptake studies of lipophilic derivatives of doxorubicin succinate.

A number of lipophilic 14-substituted derivatives of doxorubicin were synthesized through conjugation of doxorubicin-14-hemisuccinate with different fatty amines or tetradecanol to enhance the lipophilicity, cellular uptake, and cellular retention for sustained anticancer activity. The conjugates inhibited the cell proliferation of human leukemia (CCRF-CEM, 69-76%), colon adenocarcinoma (HT-29, 60-77%), and breast adenocarcinoma (MDA-MB-361, 66-71%) cells at a concentration of 1 μM after 96-120 h of incubation. The N-tetradecylamido derivative of doxorubicin 14-succinate (10) exhibited consistently comparable antiproliferative activity to doxorubicin in a time-dependent manner (IC(50) = 77 nM in CCRF-CEM cells). Flow cytometry analysis showed a 3-fold more cellular uptake of 10 than doxorubicin in SK-OV-3 cells. Confocal microscopy revealed that the conjugate was distributed in cytoplasmic and perinuclear areas during the first 1 h of incubation and slowly relocalized in the nucleus after 24 h. The cellular hydrolysis study showed that 98% of compound 10 was hydrolyzed intracellularly within 48 h and released doxorubicin.