Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

A biomolecular recognition approach for the functionalization of cellulose with gold nanoparticles

Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles (AuNPs) via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre‐synthesized AuNPs. Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin‐AuNPs that relies on a complex of 2 recognition elements: a ZZ‐CBM3 fusion that combines a carbohydrate‐binding module (CBM) with the ZZ fragment of the staphylococcal protein A and an anti‐biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ‐CBM3:anti‐biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials. Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ‐CBM3:anti‐biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM‐immobilized antibody and its specific, AuNP‐conjugated antigen is signaled by red color. This opens up the way for the development of simple and straightforward paper/cellulose‐based tests where detection of a target analyte can be made by direct use of color signaling.     

High-resolution mapping of satellite DNA using biotin-labeled DNA probes

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity- purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Green synthesis and stabilization of gold nanoparticles in chemically modified chitosan matrices

Chitosan-N-2-methylhydroxypyridine-6-methylcorboxylate (Ch-PDC) and chitosan-N-2-methylhydroxypyridine-6-methylhydroxy thiocarbohydrazide (Ch-PDC-Th) were synthesized for the first time using chitosan as precursor. Chitosan, Ch-PDC, Ch-PDC-Th were used in the synthesis of gold nanoparticles (AuNP) in aqueous medium. Chitosan and Ch-PDC-Th possess reducing properties which enabled the 'green' synthesis of AuNPs. The stabilization of the AuNPs was as a result of the thiocarbide (SC) and amine (NH(2)) groups in the chitosan matrix. The modified chitosan, its derivatives and the resulting AuNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Ultraviolet-visible (UV-vis) spectroscopy, Raman scattering measurements, powder X-ray diffraction (PXRD) and thermo gravimetric analysis (TGA). Particle size, morphology, segregation and individuality of the AuNPs were examined by transmission electron microscope (TEM) and energy dispersion spectroscopy (EDS). An average AuNPs size of 20 nm was observed for chitosan and Ch-PDC-Th while Ch-PDC was 50 nm. In comparison, AuNPs resulting from Ch-PDC-Th precursor has the most enhanced Raman and fluorescent intensities and was stable for over 2 months.     

Competitive cellular uptake of nanoparticles made from polystyrene, poly(methyl methacrylate), and polylactide

The uptake behavior of negatively charged fluorescent nanoparticles made from different polymers (PS, PMMA, and PLLA) is studied on HeLa cells. All particles are obtained by the miniemulsion process using sodium dodecylsulfate as anionic surfactant. The size of the particles is in the range 105-125 nm. Cell uptake is analyzed by flow cytometry and reveals a higher uptake of PLLA particles compared to PMMA and PS particles. In competitive uptake studies two different types of particles are co-incubated with the HeLa cells; the results indicate a mutual influence of the particles on their uptake behavior. A reduced internalization of PLLA particles in the presence of PS particles is observed, although neither the co-incubation of PMMA and PLLA nor of PMMA and PS shows similar effect.     

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.     

An Improved Method for Standardization of Microspectrofluorometers

The use of meshed cadmium-zinc sulfide fluorescent particles in combination with an electron microscope locator grid greatly facilitates the standardization of miam spectrofluorometers. The fluorescent particles emit maximally at 570 nm, and the intensity of fluorescence is directly proportional to cross-sectional area when epi-illumination is used Details concerning technique and fluorescent particle characteristics are reported.     

Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects

Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO2 layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO2 thicknesses that are accurate with respect to the known SiO2 thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.     

Ultrabright fluorescein-labeled antibodies near silver metallic surfaces

Fluorescein-labeled antibodies are widely used in clinical assays and fluorescence microscopy. The fluorescent signal per labeled antibody is limited by fluorescein self-quenching, which occurs when the antibody is heavily labeled with multiple fluoresceins. We examined immunoglobulin G (IgG) when labeled with 0.7 to about 30 fluoresceins per antibody molecule. The extent of self-quenching was decreased, and the signal increased, when the labeled antibody was in close proximity to metallic silver particles. Time-resolved measurements showed that the intensity increase was due in part to a silver-induced increase in the radiative decay rate. These results suggest the use of labeled antibodies conjugated to silver particles as ultrabright probes for imaging or analytical applications.     

Immunogold localization of photosynthetic fructose-1,6-bisphosphatase in pea leaf tissue

An enriched IgG serum fraction obtained from rabbits immunized against pea chloroplast fructose-1,6-bisphosphatase (FBPase) was used, coupled to colloidal gold (15 nanometer particles) goat anti-rabbit IgG, to analyze by electron microscopy the location of photosynthetic FBPase in pea (Pisum sativum L.) leaf ultrathin sections. In accordance with earlier biochemical studies on distribution of FBPase activity, the enzyme was visualized both in the stromal space and bound to the chloroplast membranes. Some gold particles also appear in the cytoplasm, which can be related to the presence in the cytosol of a high molecular weight precursor of this nuclear coded enzyme.     

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II)

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.     

Quantitative detection of single molecules using enhancement of Dye/DNA conjugate-labeled nanoparticles

An ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules is developed on the basis of counting single magnetic nanobeads (MNBs) with combined amplification of DNA and dye/DNA conjugate. Highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidin-coated magnetic nanobeads (SA-MNBs) and magnetic separation. In this method, human IgG (Ag) is captured on the silanized glass substrate surface, followed by immunoreaction with biotinylated mouse antihuman antibody (BT-Ab). Then, SA-MNBs are attached to the BT-Ab through the biotin/streptavidin interaction at a ratio of 1:1. Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The resultant Ag-BT-Ab-SA-MNBs/BT-dsDNA/SYBR Green I is achieved after a fluorescent DNA probe, SYBR Green I, is added to the substrate and bound to the oligonucleotide at high ratios. Finally, epifluorescence microscopy coupled with a high-sensitivity electron multiplying charge-coupled device is employed for human IgG fluorescence imaging and detection. The number of fluorescent spots corresponding to single protein molecules on the images is counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/SYBR Green I immuotargeted on the glass slides is correlated with the concentration of human IgG target antigen in the range 3.0-50 fM.     

Gold nanoparticles–conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt–mediated pathway in breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles–conjugated quercetin (AuNPs‐Qu‐5) in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs‐Qu‐5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V‐FITC staining were performed. AuNPs and AuNPs‐Qu‐5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs‐Qu‐5 exhibited lower IC₅₀ value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs‐Qu‐5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)–mediated PI3K/Akt/GSK‐3β signalling by immunoblotting and immunocytochemistry. The pro‐apoptotic proteins (Bax, Caspase‐3) were found to be up regulated and anti‐apoptotic protein (Bcl‐2) was down regulated on treatment with AuNPs‐Qu‐5. Additionally, AuNPs‐Qu‐5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK‐3β. In conclusion, administration of AuNPs‐Qu‐5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs‐Qu‐5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy.     

Controlling the diameter,monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry

Nanolipoprotein particles (NLPs) are nanometer‐scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein “scaffold,” which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell‐free protein synthesis followed by self‐assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo‐lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine “branches” are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell‐free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo‐EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6–30 nm) and polydispersity. The telodendrimer NLPs (TD‐NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.     

Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells

This paper reports the creation of Au nanoparticles (AuNP) that are soluble in aqueous solution over a broad range of pH and ionic strength values and that are capable of selective uptake by folate receptor positive (FR+) cancer cells. A novel poly(ethylene glycol) (PEG) construct with thioctic acid and folic acid coupled on opposite ends of the polymer chain was synthesized for targeting the AuNP to FR+ tumor cells via receptor-mediated endocytosis. These folic acid-PEG-thioctic acid conjugates were grafted onto 10-nm-diameter Au particles in aqueous solution. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range of from 2 to 12 and at electrolyte concentrations of up to 0.5 M NaCl with particle concentrations as high as 1.5 x 10(13) particles/mL. Transmission electron microscopy was used to document the performance of these coated nanoparticles in cell culture. Selective uptake of folate-PEG grafted AuNPs by KB cells, a FR+ cell line that overexpress the folate receptor, was observed. AuNP uptake was minimal in cells that (1) do not overexpress the folate receptor, (2) were exposed to AuNP lacking the folate-PEG conjugate, or (3) were co-incubated with free folic acid in large excess relative to the folate-PEG grafted AuNP. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.     

Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV–visible–NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I—control cells, Group II—cells treated with laser light alone, Group III—cells treated with unconjugated AuNP and further laser irradiation and Group IV—anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.     

The use of markers for correlative light electron microscopy

Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a “warning” that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Electron density imaging of protein films on gold-particle surfaces with transmission electron microscopy

BACKGROUND: Surface bound proteins on colloid particles are widely used in biotechnological applications such as diagnostics or separation. Analysis of colloid surfaces by imaging methods provides information on the structure of these protein films, and an understanding of the functional relationships of biomolecules immobilised on solid surfaces. METHODS: In order to visualise protein molecules organised in films on surfaces of nano-sized gold-particles, an electron-microscopic approach based on the scattering absorption contrast of the specimen was applied. RESULTS: Analysing protein conjugated gold particles with a transmission electron microscope, protein films on gold particle surfaces cause a significant scattering absorption contrast based on the materials' electron density. Thus, the thickness of such films becomes directly measurable in planar projection and the shape of these films are visualised without negative staining methods. The insertion of Ruthenium-labelled antibodies instead of non-labelled antibodies as a marker with increased electron-density in these films yields a contrast enhancement of the whole film. Additional labelling with anti-Mouse IgG Gold conjugates localises the position of the surface bound antibodies in such protein films. CONCLUSIONS: The power of transmission electron microscopy to resolve protein-films on colloid surfaces without staining or labelling as a sample preparation procedure has been demonstrated. Thus, this direct method provides an analytical tool for studying protein films and their structural features on particle surfaces.