In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti‐Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16‐OVA tumor model. AuNP delivery of OVA (AuNP‐OVA) and of CpG (AuNP‐CpG) enhanced the efficacy of both agents and induced strong antigen‐specific responses. In addition, it is found that AuNP‐OVA delivery alone, without CpG, is sufficient to promote significant antigen‐specific responses, leading to subsequent anti‐tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP‐mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.     

Self‐Assembly of Poly‐Adenine‐Tailed CpG Oligonucleotide‐Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP‐CpG nanoconjugates for cost‐effective drug delivery. It is found that non‐thiolated, diblock ODNs containing a CpG motif and a poly‐adenine (polyA) tail can readily self‐assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll‐like receptor 9 (TLR9)‐dependent manner. Under optimal conditions, polyA‐CpG‐AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG‐AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA‐tailed CpG‐AuNP nanoconjugates.     

Gold nanoparticles: sonocatalytic synthesis using ethanolic extract of Andrographis paniculata and functionalization with polycaprolactone–gelatin composites

Gold nanoparticles (AuNPs) were synthesized by sonication using ethanolic leaf extract of Andrographis paniculata. We investigated the optimum parameters for AuNP synthesis and functionalization with polycaprolactone–gelatin (PCL–GL) composites. The AuNPs were characterized with various biophysical techniques such as TEM, XRD, FT-IR and EDX spectroscopy. TEM images showed that nanoparticles were spherical in shape with a size range from 5 to 75 nm. EDX analysis revealed the presence of molecular oxygen and carbon on the surface of AuNPs. The synthesized AuNPs were tested for their effect on HeLa (human cervical cancer) and MCF-7 (human breast cancer) cell lines and found to be nontoxic and biocompatible, which are potential carriers for hydrophobic drugs.     

Combined Positron Emission Tomography and Cerenkov Luminescence Imaging of Sentinel Lymph Nodes Using PEGylated Radionuclide‐Embedded Gold Nanoparticles

New imaging probes with high sensitivity and stability are urgently needed to accurately detect sentinel lymph nodes (SLNs) for successful cancer diagnosis. Herein, the use of highly sensitive and stable PEGylated radionuclide‐embedded gold nanoparticles (PEG‐RIe‐AuNPs) is reported for the detection of SLNs by combined positron emission tomography and Cerenkov luminescence imaging (PET/CLI). PEG‐RIe‐AuNPs show high sensitivity and stability both in vitro and in vivo, and are not toxic to normal ovarian and immune cells. In vivo PET/CLI imaging clearly reveals SLNs as early as 1 h post PEG‐RIe‐AuNP‐injection, with peak signals achieved at 6 h postinjection, which is consistent with the biodistribution results. Taken together, the data provide strong evidence that PEG‐RIe‐AuNPs are promising as potential lymphatic tracers in biomedical imaging for pre and intraoperative surgical guidance.     

TGF‐β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor‐beta 1 (TGF‐β1), a member of the cysteine‐knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF‐β1 and the surface of AuNPs when incubated with serum‐containing medium, and demonstrate a time‐ and dose‐dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP–TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial–mesenchymal transition (EMT) upon treatment with TGF‐β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF‐β1 is found to bind to AuNPs through S–Au bonds by X‐ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF‐β1 on the surface of AuNPs. The results indicate that TGF‐β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF‐β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF‐β1‐secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD‐SCID mice, and this is associated with an increase in the number of tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF‐β1 signaling.     

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers

Understanding the molecular mechanisms governing nanoparticle–membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse‐grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di‐stearoyl‐phosphatidylcholine (DSPC) and anionic di‐stearoyl‐phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC–DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP–membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse‐grained MD simulations as a lipid‐crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.     

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.     

Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.     

Tumor‐Responsive Fluorescent Light‐up Probe Based on a Gold Nanoparticle/Conjugated Polyelectrolyte Hybrid

A tumor‐responsive nanoprobe based on a conjugated polyelectrolyte and gold nanoparticle (AuNP) hybrid was designed to response to the low pH extracellular microenvironment in tumor with light‐up fluorescence. AuNPs with positive surface charges were prepared by direct reducing Au salt with sodium borohydride and stabilized by cystamine. A pH triggered charge‐reversible polymer and a water‐soluble cationic conjugated polyelectrolyte (CPE) were sequentially deposited onto the AuNP surface through electrostatic interaction. The obtained hybrid probe is monodispersed with an average diameter of 68.3 nm by dynamic light scattering measurement. In physiological conditions (pH ≈ 7.4), the hybrid probe is almost non‐fluorescent due to the super‐quenching of CPE by AuNPs via energy/charge transfer and efficient exciton migration along the polymer backbone. When exposed to acidic extracellular microenvironments in tumor (pH_e ≈ 6.5), the acid‐labile amides hydrolyze into primary amines. The generated amine groups result in strong electrostatic repulsion between CPE and AuNPs, leading to recovered probe fluorescence. The fluorescence turn‐on is further utilized for tumor extracellular acidic microenvironment imaging. In addition, under in vivo conditions, the nanosized hybrid probe exhibits specific accumulation in tumor tissue with light‐up fluorescence, which provides new opportunities for easy tumor imaging and identification.     

Facile synthesis of anisotropic gold nanoparticles and its synergistic effect on breast cancer cell lines

Gold nanoparticles (AuNPs) possess colourful light‐scattering properties due to different composition, size and shape. Their unique physical, optical and chemical properties coupled with advantages, have increased the scope of anisotropic AuNPs in various fields. This study reports a green methodology developed for the synthesis of anisotropic AuNPs. The aqueous extracts of Alternanthera sessilis (PGK), Portulaca oleracea (PAK) and Sterculia foetida (SF) with gold ions produced violet, purple and pink coloured AuNPs, respectively, under sonication and room temperature methods revealing the formation of different shapes of AuNPs. The results of TEM analysis of AuNPs confirmed the formation of triangular plate AuNPs of the size 35 nm for PAK extract. Spherical‐shaped AuNPs (10–20 nm) were obtained using an extract of PGK. SF extract produced rod, hexagon, pentagon‐shaped AuNPs and nanorice gold particles. The cell viability studies of the PGK, PAK and SF‐mediated AuNPs on MCF‐7 cell lines by MTT assay revealed the cytotoxic activity of AuNPs to depend on the size, shape and the nature of capping agents. The synthesised AuNPs significantly inhibited the growth of cancer cells (MCF‐7) in a concentration‐dependent manner. The size and shape of these anisotropic AuNPs also reveal its potency to be used as sensors, catalysis, photothermal and therapeutic agents.Inspec keywords: toxicology, gold, transmission electron microscopy, catalysis, nanofabrication, biomedical materials, nanomedicine, particle size, cellular biophysics, nanoparticles, cancer, biological organsOther keywords: Au, size 10.0 nm to 20.0 nm, temperature 293.0 K to 298.0 K, size 35.0 nm, TEM analysis, Sterculia foetida, Portulaca oleracea, Alternanthera sessilis, chemical properties, colourful light‐scattering properties, anisotropic AuNP, triangular plate AuNP, spherical‐shaped AuNP, SF‐mediated AuNP, cancer cells, MCF‐7 cell lines, cell viability, nanorice gold particles, gold ions, optical properties, breast cancer cell lines, anisotropic gold nanoparticles     

Gold Nanoparticles of Diameter 1.4 nm Trigger Necrosis by Oxidative Stress and Mitochondrial Damage

Gold nanoparticles (AuNPs) are generally considered nontoxic, similar to bulk gold, which is inert and biocompatible. AuNPs of diameter 1.4 nm capped with triphenylphosphine monosulfonate (TPPMS), Au1.4MS, are much more cytotoxic than 15‐nm nanoparticles (Au15MS) of similar chemical composition. Here, major cell‐death pathways are studied and it is determined that the cytotoxicity is caused by oxidative stress. Indicators of oxidative stress, reactive oxygen species (ROS), mitochondrial potential and integrity, and mitochondrial substrate reduction are all compromised. Genome‐wide expression profiling using DNA gene arrays indicates robust upregulation of stress‐related genes after 6 and 12 h of incubation with a 2 × IC50 concentration of Au1.4MS but not with Au15MS nanoparticles. The caspase inhibitor Z‐VAD‐fmk does not rescue the cells, which suggests that necrosis, not apoptosis, is the predominant pathway at this concentration. Pretreatment of the nanoparticles with reducing agents/antioxidants N‐acetylcysteine, glutathione, and TPPMS reduces the toxicity of Au1.4MS. AuNPs of similar size but capped with glutathione (Au1.1GSH) likewise do not induce oxidative stress. Besides the size dependency of AuNP toxicity, ligand chemistry is a critical parameter determining the degree of cytotoxicity. AuNP exposure most likely causes oxidative stress that is amplified by mitochondrial damage. Au1.4MS nanoparticle cytotoxicity is associated with oxidative stress, endogenous ROS production, and depletion of the intracellular antioxidant pool.     

Anionic Lipid,pH‐Sensitive Liposome‐Gold Nanoparticle Hybrids for Gene Delivery – Quantitative Research of the Mechanism

Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE‐AuNPs), which possess 3‐ and 1.5‐fold higher transfection efficiency than those of DODAB‐AuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.     

Chirality‐Selective Photoluminescence Enhancement of ssDNA‐Wrapped Single‐Walled Carbon Nanotubes Modified with Gold Nanoparticles

In this work, a convenient method to enhance the photoluminescence (PL) of single‐walled carbon nanotubes (SWNTs) in aqueous solutions is provided. Dispersing by single‐stranded DNA (ssDNA) and modifying with gold nanoparticles (AuNPs), about tenfold PL enhancement of the SWNTs is observed. More importantly, the selective PL enhancement is achieved for some particular chiralities of interest over all other chiralities, by using certain specific ssDNA sequences that are reported to recognize these particular chiralities. By forming AuNP–DNA–SWNT nanohybrids, ssDNA serves as superior molecular spacers that on one hand protect SWNT from direct contacting with AuNP and causing PL quench, and on the other hand attract the AuNP in close proximity to the SWNT to enhance its PL. This PL enhancement method can be utilized for the PL analysis of SWNTs in aqueous solutions, for biomedical imaging, and may serve as a prescreening method for the recognition and separation of single chirality SWNTs by ssDNA.     

Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been self-assembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of approximately 915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted "V" shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 +/- 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADH-dependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors.     

Electrophoretic Build‐Up of Alternately Multilayered Films and Micropatterns Based on Graphene Sheets and Nanoparticles and their Applications in Flexible Supercapacitors

Graphene nanosheets and metal nanoparticles (NPs) have been used as nano‐building‐blocks for assembly into macroscale hybrid structures with promising performance in electrical devices. However, in most graphene and metal NP hybrid structures, the graphene sheets and metal NPs (e.g., AuNPs) do not enable control of the reaction process, orientation of building blocks, and organization at the nanoscale. Here, an electrophoretic layer‐by‐layer assembly for constructing multilayered reduced graphene oxide (RGO)/AuNP films and lateral micropatterns is presented. This assembly method allows easy control of the nano‐architecture of building blocks along the normal direction of the film, including the number and thickness of RGO and AuNP layers, in addition to control of the lateral orientation of the resultant multilayered structures. Conductivity of multilayered RGO/AuNP hybrid nano‐architecture shows great improvement caused by a bridging effect of the AuNPs along the out‐of‐plane direction between the upper and lower RGO layers. The results clearly show the potential of electrophoretic build‐up in the fabrication of graphene‐based alternately multilayered films and patterns. Finally, flexible supercapacitors based on multilayered RGO/AuNP hybrid films are fabricated, and excellent performance, such as high energy and power densities, are achieved.     

PEI-conjugated AuNPs as a sensing platform for arsenic (AS-III)

We have developed a simple method for synthesis of spherical gold nanoparticles (AuNPs) with enhanced surface properties. The polyethyleneimine (PEI) has good potential to minimise the size of the precursor. The UV–vis spectra of synthesised AuNPs with reducing agents (PEI) have been characterised with a peak at 530?nm. The size and shape measurement of AuNPs was confirmed by transmission electron microscopy (TEM) which shows that the mean diameter is 3.9?nm. The optimal concentration of reducing agents was found to be 1% for synthesis of AuNPs. PEI-conjugated AuNP shows binding with arsenic III (0.1?ppm) as confirmed by scanning electron microscope (SEM)/energy dispersive X-rays mapping. TEM revealed the particle shape and size. Zeta potential, zeta deviation, effective particle size, Z-average diameter, polydispersity index and electrophoretic mobility have been observed in order to understand the stability of AuNPs. The image of SEM confirmed that As (III) particles were eventually distributed in PEI-conjugated AuNPs matrix. Further, this study demonstrated that PEI-conjugated AuNPs is a sensing platform of As (III).     

High‐Sensitivity Real‐Time Analysis of Nanoparticle Toxicity in Green Fluorescent Protein‐Expressing Zebrafish

Gold nanoparticles (AuNP) show great potential for diagnostic and therapeutic application in humans. A great number of studies have tested the cytotoxicity of AuNP using cell culture. There is, however, an urgent need to test AuNP in vertebrate animal models that interrogate biodistribution and complex biological traits like organ development, whole body metabolism, and cognitive function. The sheer number of different compounds precludes the use of small rodent model for initial screening. The extended fish embryo test (FET) is used here to bridge the gap between cell culture and small animal models. A study on the toxicity of ultrasmall AuNP in wild type and transgenic zebrafish is presented. FET faithfully reproduce all important findings of a previous study in HeLa cells and add new important information on teratogenicity and hepatotoxicity that could not be gained from studying cultured cells.     

Ultrasmall dopamine-coated nanogolds: preparation,characteristics, and CT imaging

ABSTRACT

Water-dispersible ultrasmall nanogolds (WDU AuNPs) and their dopamine-coated nanogolds (WDU AuNPs@DPAs) were prepared by a reduction method with sodium borohydride as a reducing agent and a stabilised agent of 2-mercaptosuccinic acid in aqueous solution. The effects of these nanoparticles on computed tomography (CT) imaging were evaluated. The size distributions and Zeta potential of the nanoparticles were measured with a Malvern size analyser, and nanoparticle morphology was observed by transmission electron microscopy. These characteristics were confirmed by Fourier transform spectroscopy and ultraviolet/visible spectra. It was found that WDU AuNPs@DPAs were 5.4 nm in size with clear core–shell structure. The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide assay results showed that the WDU AuNPs and WDU AuNPs@DPAs were hypotoxic to different cells. The WDU AuNPs@DPAs showed a much longer circulation time and a larger CT attenuation coefficient than iohexol and could be excreted by the kidney and bladder. These nanoparticles showed considerable potential for future application in CT imaging.