Cyclic peptide-capped gold nanoparticles for enhanced siRNA delivery.
ABSTRACT: Previously, we have reported the synthesis of a homochiral l-cyclic peptide [WR]5 and its use for delivery of anti-HIV drugs and biomolecules. A physical mixture of HAuCl4 and the peptide generated peptide-capped gold nanoparticles. Here, [WR]5 and [WR]5-AuNPs were tested for their efficiency to deliver a small interfering RNA molecule (siRNA) in human cervix adenocarcinoma (HeLa) cells. Flow cytometry investigation revealed that the intracellular uptake of a fluorescence-labeled non-targeting siRNA (200 nM) was enhanced in the presence of [WR]5 and [WR]5-AuNPs by 2- and 3.8-fold when compared with that of siRNA alone after 24 h incubation. Comparative toxicity results showed that [WR]5 and [WR]5-AuNPs were less toxic in cells compared to other available carrier systems, such as Lipofectamine.
Project description:Gold nanoparticles (AuNPs) were synthesized in situ in a green and rapid method from the reaction of reducing linear and cyclic peptides containing tryptophan and lysine residues, (KW)5 and cyclic [KW]5, with an aqueous solution of HAuCl4 and were evaluated as cellular nanodrug delivery systems. The cyclic or linear nature of the peptide was found to determine the morphology and size of the formed peptide-AuNPs and their in vitro molecular transporting efficiency. While cyclic [KW]5-AuNPs formed sponge-like agglomerates, linear (KW)5-AuNPs demonstrated ball-shaped structures. A comparative flow cytometry study showed that the cellular uptake of fluorescence-labeled anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC)) in human leukemia (CCRF-CEM) cells, and a negatively charged cell-impermeable phosphopeptide (GpYEEI) in human ovarian adecarcinoma (SK-OV-3) cells was significantly higher in the presence of cyclic [KW]5-AuNPs than that of linear (KW)5-AuNPs, parent cyclic [KW]5, and linear (KW)5 peptides. For example, the cellular uptake of F'-GpYEEI was enhanced 12.8-fold by c[KW]5-AuNPs. Confocal microscopy revealed the localization of fluorescence-labeled-3TC in the presence of c[KW]5-AuNPs mostly in nucleus in SK-OV-3 cells after 1 h. On the other hand, l(KW)5-AuNPs delivered fluorescence-labeled-3TC in cytoplasm. These data suggest that noncell penetrating peptides can be converted to efficient molecular transporters through peptide-capped AuNPs formation.
Project description:In this study, we explored the biocompatibility of Au nanoparticles (NPs) capped with peptide-biphenyl hybrid (PBH) ligands containing glycine (Gly), cysteine (Cys), tyrosine (Tyr), tryptophan (Trp) and methionine (Met) amino acids in the human hepatocellular carcinoma cell line Hep G2. Five AuNPs, Au[(Gly-Tyr-Met)2B], Au[(Gly-Trp-Met)2B], Au[(Met)2B], Au[(Gly-Tyr-TrCys)2B] and Au[(TrCys)2B], were synthesised. Physico-chemical and cytotoxic properties were thoroughly studied. Transmission electron micrographs showed isolated near-spherical nanoparticles with diameters of 1.5, 1.6, 2.3, 1.8 and 2.3 nm, respectively. Dynamic light scattering evidenced the high stability of suspensions in Milli-Q water and culture medium, particularly when supplemented with serum, showing in all cases a tendency to form agglomerates with diameters approximately 200 nm. In the cytotoxicity studies, interference caused by AuNPs with some typical cytotoxicity assays was demonstrated; thus, only data obtained from the resazurin based assay were used. After 48-h incubation, only concentrations ?50 ?g/ml exhibited cytotoxicity. Such doses were also responsible for an increase in reactive oxygen species (ROS). Some differences were observed among the studied NPs. Of particular importance is the AuNPs capped with the PBH ligand (Gly-Tyr-TrCys)2B showing remarkable stability in culture medium, even in the absence of serum. Moreover, these AuNPs have unique biological effects on Hep G2 cells while showing low toxicity. The production of ROS along with supporting optical microscopy images suggests cellular interaction/uptake of these particular AuNPs. Future research efforts should further test this hypothesis, as such interaction/uptake is highly relevant in drug delivery systems.
Project description:Nanocellulose-assisted gold nanoparticles are considered promising materials for developing eco-friendly diagnostic tools for biosensing applications. In this study, we synthesized 2,2,6,6-tetramethylpiperidin-1-piperidinyloxy (TEMPO)-oxidized cellulose nanocrystal (TEMPO-CNC)-capped gold nanoparticles (AuNPs) for the colorimetric detection of unamplified pathogenic DNA oligomers of methicillin-resistant <i>Staphylococcus aureus</i>. The fabricated TEMPO-CNC-AuNPs (TC-AuNPs) were characterized using UV-visible spectroscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering. The average diameter of the synthesized AuNPs was approximately 30 nm. The aqueous solution of TC-AuNPs was stable and exhibited an absorption peak at 520 nm. The chemical interaction between TC-AuNPs and the surface charge of the target and non-target DNA determined the colorimetric differences under ionic conditions. A dramatic color change (red → blue) was observed in the TC-AuNP solution with the target DNA under ionic conditions due to the aggregation of AuNPs. However, no observable color change occurred in the TC-AuNP solution with the non-target DNA under similar conditions owing to the better shielding effects of the charged moieties. The colorimetric detection limit of the TC-AuNPs was demonstrated to be as low as 20 fM pathogenic DNA. Therefore, the use of TEMPO-oxidized CNC-capped AuNPs is efficient and straightforward as a biosensor for the colorimetric detection of pathogenic DNA.
Project description:A colorimetric assay was developed for the detection of biothiols, based on the peroxidase-like activity of iodine-capped gold nanoparticles (AuNPs). These AuNPs show a synergetic effect in the form of peroxidase-mimicking activity at the interface of AuNPs, while free AuNPs and iodine alone have weak catalytic properties. Thus, iodine-capped AuNPs possess good intrinsic enzymatic activity and trigger the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), leading to a change in color from colorless to yellow. When added to solution, biothiols, such as cysteine, strongly bind to the interface of AuNPs via gold-thiol bonds, inhibiting the catalytic activity of AuNPs, resulting in a decrease in oxidized TMB. Using this strategy, cysteine could be linearly determined, at a wide range of concentrations (0.5 to 20 ?M), with a detection limit of 0.5 ?M using UV-Vis spectroscopy. This method was applied for the detection of cysteine in diluted human urine.
Project description:Glyco-gold nanoparticles (AuNPs) in aqueous dispersions were prepared by two approaches, namely direct reduction and ligand substitution methods. In the direct method, potassium salts of glyco thiols, with the general formula (C<sub>6</sub>H<sub>11</sub>O<sub>6</sub>)NH(CH<sub>2</sub>) <sub><i>n</i></sub> CH<sub>2</sub>SK (where <b>L1</b>, <i>n</i> = 1; <b>L2</b>, <i>n</i> = 2; <b>L3</b>, <i>n</i> = 3, <b>L4</b>, <i>n</i> = 4; <b>L5</b>, <i>n</i> = 5), were used as reducing and capping agents to give the glyco thiolate capped gold nanoparticles (AuNPs <b>G1</b>-<b>G5</b>); meanwhile in the ligand exchange experiments, <b>L1</b>-<b>L5</b> and their acetylated forms (<b>L6</b>-<b>L8</b>) replaced citrate ions in citrate-capped gold nanoparticles to give additional AuNPs <b>G6</b>-<b>G11</b>. UV-visible spectroscopy, surface charge (<i>?</i>-potential,) measurements and transmission electron microscopy (TEM) were used for physical and chemical characterization of all the resultant AuNPs. The <i>?</i>-potential studies of AuNPs prepared through the direct method revealed that the surface charge is dependent on the length of the alkyl unit of (C<sub>6</sub>H<sub>11</sub>O<sub>6</sub>)NH(CH<sub>2</sub>) <sub><i>n</i></sub> CH<sub>2</sub>S<sup>-</sup> ligands. TEM images of the acetylated and non-acetylated glyco thiolate capped gold nanoparticles (AuNPs <b>G6</b>-<b>G11</b>) prepared <i>via</i> the ligand exchange method indicate that the size and shape of the gold nanoparticles remained the same as those of the citrate-capped gold nanoparticles used to prepare them. Selected AuNPs were tested on peripheral blood mononuclear cells (PBMCs) and the A549 cancer cell line to investigate their respective toxicity and cytotoxicity profiles. All AuNPs showed indiscriminate activity against both PBMCs and A4549 cells, although the gold nanoparticles having an acetylated glyco moiety with an amino propyl thiol linker as the ligand (<b>G10</b>) prepared <i>via</i> the citrate exchange method had better selectivity (PBMCs >59 mg mL<sup>-1</sup> and for A549 ?7 ?g mL<sup>-1</sup>).
Project description:In this work, we explored the formation processes of suspended hybrid thin films of thiol-capped Au nanoparticles (AuNPs) inside metal oxide tubular structures. We found that a balance between in-film interactions of the AuNPs and boundary interactions with metal oxides is a key in making these special organic-inorganic thin films. The hybrid films process many processing advantages and flexibilities, such as controllable film thickness, interfacial shape and inter-AuNPs distance, tuning of particle sizes, thiol population, chain lengths, and other new properties by introducing functional groups to thiol chains. Among their many unique features, the assembly-disassembly property may be useful for future on-off or store-release applications.
Project description:Delivery of small interfering RNA (siRNA) provides one of the most powerful strategies for downregulation of therapeutic targets. Despite the widely explored capabilities of this strategy, intracellular delivery is hindered by a lack of carriers that have high stability, low toxicity and high transfection efficiency. Here we propose a layer by layer (LBL) self-assembly method to fabricate chitosan-coated gold nanoparticles (CS-AuNPs) as a more stable and efficient siRNA delivery system. Direct reduction of HAuCl<sub>4</sub> in the presence of chitosan led to the formation of positively charged CS-AuNPs, which were subsequently modified with a layer of siRNA cargo molecules and a final chitosan layer to protect the siRNA and to have a net positive charge for good interaction with cells. Cytotoxicity, uptake, and downregulation of enhanced Green Fluorescent Protein (eGFP) in H1299-eGFP lung epithelial cells indicated that LBL-CS-AuNPs provided excellent protection of siRNA against enzymatic degradation, ensured good uptake in cells by endocytosis, facilitated endosomal escape of siRNA, and improved the overall silencing effect in comparison with commercial transfection reagents Lipofectamine and jetPEI<sup>®</sup>. Therefore, this work shows that LBL assembled CS-AuNPs are promising nanocarriers for enhanced intracellular siRNA delivery and silencing.
Project description:The preparation of gold nanoparticles (AuNPs) of high purity and stability remains a major challenge for biological applications. This paper reports a simple synthetic strategy to prepare water-soluble peptide-stabilized AuNPs. Reduced glutathione, a natural tripeptide, was used as a synthon for the growth of two peptide chains directly on the AuNP surface. Both nonpolar (tryptophan and methionine) and polar basic (histidine and dansylated arginine) amino acids were conjugated to the GSH-capped AuNPs. Ultracentrifugation concentrators with polyethersulfone (PES) membranes were used to purify precursor materials in each stage of the multi-step synthesis to minimize side reactions. Thin layer chromatography, transmission electron microscopy, UV-Visible, 1H-NMR, and fluorescence spectroscopies demonstrated that ultracentrifugation produces high purity AuNPs, with narrow polydispersity, and minimal aggregation. More importantly, it allows for more control over the composition of the final ligand structure. Studies under conditions of varying pH and ionic strength revealed that peptide length, charge, and hydrophobicity influence the stability as well as solubility of the peptide-capped AuNPs. The synthetic and purification strategies used provide a facile route for developing a library of tailored biocompatible peptide-stabilized AuNPs for biomedical applications.
Project description:Bacteriogenic synthesis of metal nanoparticles is ecofriendly and greatly influenced by physico-chemical reaction parameters with respect to shape and size. Thus, present work aimed to synthesize and optimization of bacteriogenic gold nanoparticles (AuNPs) and study their antioxidant activity. Acinetobacter sp. cells were able to synthesize AuNPs, when challenged with tetra-chloroauric acid (HAuCl4). By physicochemical optimization, maximum synthesis was obtained with 72 h old culture using 2.1 × 109 CFU/ml cell density. Whereas, pH-7 is suitable for AuNPs synthesis. HAuCl4 concentration (0.5 mM) enhanced the formation of monodispersed and spherical nanoparticles (15 ± 10 nm). At 37°C temperature, Acinetobacter sp. released nanoparticles in supernatant. From characterization, AuNPs were found to be crystalline in nature with negative surface charge. AuNPs showed up to 86% different radical scavenging ability, exhibiting antioxidant activity. In conclusion, spherical AuNPs can be synthesized using Acinetobacter sp. through physicochemical optimization. This is the first report of antioxidant activity exhibited by monodispersed bacteriogenic AuNPs synthesized using Acinetobacter sp.
Project description:Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer.