N A N O P R O B E S     E - N E W S

Vol. 7, No. 10          October 26, 2006

The penton base protein is multifunctional: in addition to its structural role and binding the fiber, it is involved in viral cell entry through binding to cellular alphav integrins, and in viral release from endosomes on the pathway towards the cell nucleus. Although at 80 Å (8 nm) in diameter, its internal cavity is too small to accommodate a gene, it is a highly promising vector for targeted drug delivery to specific cell types.

To better evaluate this potential, the authors explored various conditions for dodecahedral assembly, using negative-stain electron microscopy using sodium silicotungstate as the contrasting agent. In order to test the ability of the particles to incorporate payloads, they were assembled in the presence of Monomaleimido Nanogold and examined by electron microscopy to determine the extent of Nanogold encapsulation. In general, low pH values and high salt concentrations were found to promote dodecahedron assembly.

A second goal was the demonstration of 'passive encapsulation' of a payload within the dodecahedron by shifting buffer conditions from those favoring disassembly to conditions favoring assembly, after incubation with a marker molecule which could be imaged by cryoelectron microscopy. Nanogold is ideal for this purpose because it is small enough to fit inside the dodecahedral cavity, yet large enough to be visualized under the conditions used. Furthermore, its organic ligand shell protects it from non-specific interactions with proteins and tissues. Unlike larger colloidal gold, Nanogold does not adhere spontaneously to protein; instead, it is usually derivatized with a selectively reactive functional group, such as maleimide, and used to specifically label unique target groups, such as the thiol group of the cysteine side chain. Although Monomaleimido Nanogold was used for this experiment, Non-functional Nanogold would also have been a good choice. Dodecahedra were disassembled at basic pH (8-9) and mixed with Nanogold particles. Subsequently, the sample was dialyzed at 4°C into 'assembly' buffer. Several approaches were used to remove free gold particles, including filtration by 100K membrane centrifuge filters, dialysis (100,000 MW cut-off membrane) and sucrose density-gradient ultracentrifugation.

For cryo-EM, samples were applied to holey carbon on copper grids, blotted and plunge-frozen into liquid ethane. Frozen grids were transferred onto a cryoholder, and imaged in at 200 kV and a nominal magnification of 638,000. Low-dose techniques were used to focus and expose suitable areas onto film with an estimated dose of 612 eÅ^-2. Although only a limited number of successful encapsulations were expected, a number of promising particles with electron density in the core were identified for analysis. These were compared with control particles prepared in the absence of Nanogold. To enhance signal from encapsulated moieties, the overlaid density due to the dodecahedron was removed by projecting equivalently oriented images from the reconstruction of the empty dodecahedra, and subtracting these from the EM images. Small cores of significantly higher density than the noisy background remained after subtraction, indicating that gold clusters were successfully encapsulated in these particles. Control images subjected to the same procedure resulted only in background.

Successful manipulation the assembly status of the dodecahedron was achieved and demonstrated by changing buffer conditions, and additionally, successful passive encapsulation of a Nanogold marker was achieved. This represents an important stage towards development of this dodecahedral particle for use as a delivery vehicle capable of targeting therapeutic molecules to specific cell types.

Reference:

• Fuschiotti, P.; Fender, P.; Schoehn, G., and Conway, J. F.: Development of the dodecahedral penton particle from adenovirus 3 for therapeutic application. J. Gen. Virol.,, 87, 2901-2905 (2006).
  • Abstract (courtesy of the Journal of General Virology).

More information:

• Nanogold labeling reagents - catalog information
• Non-functional Nanogold - product info and instructions
• Technical help - Nanogold labeling reagents
• Nanogold for cryo-electron microscopy - other references
• Nanogold references, sorted by application

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How Well did my Reaction Work: Calculating Labeling

The most useful extinction coefficients if you are labeling proteins are those at 280 nm, where most proteins absorb strongly and which is used for protein quantitation, and at 420 nm where Nanogold and undecagold absorb strongly and which are used for quantitation of the two gold labels. If you are labeling oligonucleotides, you will need the extinction coefficients at 260 nm, which is the value usually used for oligonucleotide quantitation. These values are given below, along with the extinction coefficients for IgG antibody molecules and Fab' fragments:

It should be noted that the absorption of your conjugate biomolecule may be small compared with that of Nanogold; for example, if you are labeling a small peptide or oligonucleotide with a relatively small extinction coefficient. In this situation, small errors in the overall measurements can produce large errors in the final calculation. It is therefore important to ensure that measurements are made with the highest possible accuracy and precision. You can ensure that your measurements are as accurate as possible by averaging multiple scans, running a blank cell with buffer as a baseline, and using a slow scan speed on your instrument.

The procedure for calculating labeling, for a biomolecule with negligible absorption at 420 nm and a characteristic extinction coefficient or optical density at 280nm, is as follows:

1. Since the absorption at 420 nm arises solely from the Nanogold, first calculate the concentration of Nanogold. Use the extinction coefficient of Nanogold at 420 nm (110,000) to calculate the concentration of Nanogold directly from the conjugate spectrum:

   [Nanogold concentration] = A_420nm / 110,000 M

2. You will calculate the protein concentration from the absorption at 280 nm. First, calculate the absorption at 280 nm due to Nanogold. Multiply the absorption at 420 nm by the ratio of the extinction coefficients of Nanogold at 280 nm and 420 nm: this will give you the absorption at 280 nm that is due to the Nanogold label:

   A_{280nm (Nanogold)} = A_420nm x 300,000 / 110,000

3. Subtract the absorption due to Nanogold from that measured for the conjugate at 280 nm. The difference is the absorption due to protein:

   A_{280nm (protein)} = A_{280nm (conjugate)} - A_{280nm (Nanogold)}

4. Use the absorption arising from protein to calculate the protein concentration in the same manner as for the Nanogold in step (1): divide the absorption by the extinction coefficient of the protein:

   [Protein concentration] = A_{280nm (protein)} / E_{280nm (protein)}

   where E_{280nm (protein)} is the extinction coefficient of the protein at 280 nm. If you have only the optical density to work from, you can convert optical density to extinction coefficient using the molecular weight, as follows:

   E = OD (1%) X MW(protein) / 10

5. The efficiency of labeling, expressed as the number of Nanogold particles per protein, is simply the concentration of Nanogold divided by the concentration of protein:

   Labeling = [Nanogold concentration] / [protein concentration]

For systems where the protein or biomolecule has significant absorbance in the visible range, the labeling calculation is more complex, and is based on the solution of a pair of simultaneous equations which describe the absorbance at two wavelengths in terms of the contribution from each species. This method is given in full on a separate page.

Reference:

• Hainfeld, J.F. and Furuya, F.R. A 1.4nm Gold cluster covalently attached to antibodies improves immunolabeling, J. Histochem. Cytochem., 40, 177-184 (1992).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

More information:

• Guide to Gold Cluster Labeling - labeling calculation
• Guide to Gold Cluster Labeling - labeling calculation
• Guide to Gold Cluster Labeling - advanced labeling calculation
• Nanogold labeling reagents: catalog information
• Nanogold labeling reagents: technical help
• Nanogold labeling references, sorted by application

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GoldEnhance Visualizes Gold Nanoparticle Cancer Targeting

• Gold enhancement may safely be used before osmium tetroxide - it is not etched.
• May be used in physiological buffers (chlorides precipitate silver, but not gold).
• The metallographic reaction is less pH sensitive than that of silver.
• Gold gives a much stronger backscatter signal than silver.
• GoldEnhance is near neutral pH for best ultrastructural preservation.
• Low viscosity, so the components may be dispensed and mixed easily and accurately.

Dixit, Andres and co-workers used GoldEnhance to develop and evaluate water-soluble gold nanoparticles for selective uptake by folate receptor-positive (FR+) cancer cells as a potential method for therapeutic delivery, and describe their work in the current Bioconjugate Chemistry. They used a novel synthetic poly(ethylene glycol) (PEG) construct bearing thioacetic acid and folic acid coupled to opposite ends of the polymer chain to target 10 nm gold nanoparticles to FR+ tumor cells via receptor-mediated endocytosis.

In order to effectively target tumor cells, a therapeutic delivery agent should be small enough to be transported through the circulatory system, yet large enough not to be rapidly cleared, in order to remain in the bloodstream long enough to accumulate in cancer cells. These requirements are met by 10 nm gold nanoparticles. For in vivo use, gold nanoparticles must also be stable to a wide range of pH and electrolyte concentrations in order to effectively target tumor cells. Polyethylene glycol is a highly effective stabilizing agent that also increases bioavailability and biocompatibility, as has been demonstrated in a number of systems, including stealth liposomes and implant surfaces. For this study, a polyethylene glycol construct, Folate-NH-PEG-NH-thioctamide (F-PEG-1500-T) was synthesized using 1,500 MW polyethylene glycol. Citrate-stabilized 10 nm gold colloid solution (3 mL, used as supplied at about 5.7 x 10¹² particles/mL) was adjusted to pH 9-11 with 0.5 M sodium hydroxide, then combined with 3 mL of 0.25 mM mPEG2000-T (control) or F-PEG1500-T; the solution was stirred at 25°C for 16-18 hours, then split into two equal volumes and centrifuged twice at 13,000 rpm for 60 minutes at 8°C, the supernatent was decanted off, and the functionalized nanoparticles recombined in 1 mL of DI water. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range from 2 to 12, in electrolyte concentrations of up to 0.5 M NaCl, at particle concentrations as high as 1.5 x 10¹³ particles/mL.

KB cells, a human cancer cell line of nasopharyngeal origin known to overexpress folate receptors, were cultured in folate-deficient Dulbeccos modified Eagle medium (FD-DMEM) with 10% heat inactivated fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin for 3 weeks to establish a folate deficiency. The folate-deficient KB cells were then subcultured in 75 cm2 flasks at 37°C in a 5% CO₂ atmosphere, used during the log phase of growth and discarded after the sixth passage. WI-38 cells, a normal embryonic human diploid cell line, were used as a control and cultured and used similarly. Both were plated at ~250,000 cells/well in six-well plates and incubated for 48 hours. 100 µL of gold nanoparticle solution (~1.5 x 10¹³ particles/mL, filtered through a 0.22 µm filter) was added to three of the wells. After 1 hour incubation, 100 µL of nanoparticle solution was added to the remaining three wells. All were incubated for a further 1 hour before rinsing with fresh media to remove free gold particles before fixation.

Transmission electron microscopy was used to evaluate nanoparticle performance in cell culture. Gold enhancement was used to enhance the observability of the 10 nm particles, followed by osmium fixation to delineate the cell membranes. Gold enhancement, unlike silver intensification, is not degraded by exposure to osmium tetroxide. Samples were processed in a microwave equipped with variable wattage, a Coldspot temperature regulator, and vacuum chamber. Cells were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 2 mM MgCl₂, 1 mM CaCl₂ and 40 mM NaCl. Samples were then washed with buffer three times followed by a water wash. Cells were scraped from the wells, pelleted by centrifugation, then incubated for 2 minutes at 25°C in GoldEnhance EM, washed with water and post-fixed in reduced osmium (1% OsO₄ + 1.5% K₄Fe(CN)₆) (2 x 40 seconds). The samples were enrobed with 1.5% agarose and processed as tissue blocks. Sample blocks were dehydrated through a graded ethanol series (P1: 30, 50, 70, 90 and 100%, two cycles each, 40 seconds per change), followed by propylene oxide (40 seconds) and infiltrated in propylene oxide (LX-112 resin mix, 3:1, 1:1 for 30 minutes each followed by overnight in 1:3 mix plus accelerator on a rotator at 25°C). Blocks were embedded in LX-112 resin mixture and polymerized at 60°C for 48 hours. Ultrathin sections (~100 nm) were picked up on Formvar+C coated 100 mesh copper grids. The samples were viewed in the transmission electron microscope with a 80 kV accelerating voltage.

Selective uptake of folate-PEG grafted gold nanoparticles by the KB cells was observed: gold nanoparticle uptake was minimal in cells that do not overexpress the folate receptor, in cells exposed to gold particles lacking the folate-PEG conjugate, and in cells co-incubated with free folic acid in large excess relative to the folate-PEG grafted gold particles. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.

Reference:

• Dixit, V.; Van den Bossche, J.; Sherman, D. M.; Thompson, D. H.; and Andres, R. P.: Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells. Bioconjug. Chem., 17, 603-609 (2006).
  • Abstract (courtesy of Bioconjugate Chemistry

More information:

• Gold Enhancement: Catalog and general information
• GoldEnhance EM: product and technical information
• Technical help - Gold enhancement
• Other references: gold enhancement
• Gold based autometallography: our paper from Microscopy and Microanalysis 1999

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Nanogold® Penetrates the Nucleus to Find HuD

• High penetration: Because of their small size, Nanogold-Fab' conjugates penetrate readily up to 40 microns into cells and tissues.
• May be used to label interior targets such as nuclear antigens, and hindered antigens inaccessible to larger gold probes.
• High-resolution, more quantitative labeling.

HuD is an ELAV/Hu RNA-binding protein that stabilizes the GAP-43 mRNA in response to nerve growth factor (NGF) stimulation in PC12 cells: the authors were interested in determining the nuclear distribution of HuD, and whether neurotrophic stimulation induced changes in its distribution. In preparation for study, PC12 cells were transfected with c-mycHuD using Lipofectamine 2000 (Invitrogen) and examined 3 days after transfection. HuR and HuD were localized using three different microscopic methods. With confocal microscopy, optically sectioned cells were reconstructed to show that HuD granules were clearly within the nucleus. Wide-field fluorescence microscopy with cryo-ultramicrotome sections demonstrated that HuD granules were found in nuclei of cells sectioned at 400 nm. Pre-embedding electron microscopic immunocytochemistry was then used to localize the HuD more specifically.

Cultures were prepared for pre-embedding electron microscopic immunocytochemistry with Nanogold conjugates and N-propyl gallate (NPG)-based silver enhancement. Following incubation in 1:500 monoclonal anti-HuD followed by 1:50 Nanogold-conjugated goat anti-mouse antibody, cells were silver enhanced with a modified silver-enhancement solution. Silver enhancement was conducted using a N-propyl gallate (NPG)-based silver enhancement method described previously, with some modifications. NPG concentration was reduced from 0.36 mg/ml to 0.09 mg/ml, which gave more reproducible silver-enhancement times. Sections were rinsed three times in 50 mM MES buffer with 200 mM sucrose (pH 5.8), then incubated under a sodium vapor safelight with N-propyl gallate (NPG) enhancement solution and agitated. The NPG-silver-enhancement solution had a final concentration of 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.15), 0.25 g/mL gum arabic, 0.09 mg/mL NPG NPG, and 1.1 mg/mL silver lactate, and may be prepared stock solutions of MES (0.5 M MES adjusted to pH 6.15 with sodium hydroxide), gum arabic (50 g / 100 mL gum arabic dissolved over 2 days), NPG (made on the day of use by dissolving 10 mg of NPG in 0.25 ml ethyl alcohol and adding 4.75 ml of water), and silver lactate stock (36 mg / 5 mL silver lactate in water in a light-tight film box) in a darkroom equipped with a sodium vapor safelight. The reaction was stopped with three changes over 5 minutes of neutral fixer solution (250 mM sodium thiosulfate and 20 mM HEPES, pH 7.4) and sections were rinsed three times in PBS. After enhancement, sections were rinsed and incubated in 0.1% Os0₄ for 30 minutes, acetone dehydrated and embedded in Spurrs resin. Pale gold thin sections were cut on an ultramicrotome with a diamond knife, stained with uranyl acetate and lead citrate, and examined in the transmission electron microscope. Control samples with no primary antibody showed no particles.

In PC12 cells, it was found that in response to heat shock, HuR translocates from the nucleus to the cytoplasm, as expected, while HuD forms large cytoplasmic stress granules, consistent with a role for HuD in the cessation of translation. In unstimulated cells, HuD was found to be distributed in small granules in the cytoplasm. Using Nanogold labeling with electron microscopic immunocytochemistry, silver-enhanced gold particles were consistently found within nuclei, indicating a small but consistent level of HuD in the nucleus. Stimulation of PC12 cells with NGF induces neuronal differentiation, including outgrowth of neurites and increased levels of GAP-43 protein, whereas HuD remains localized in small cytoplasm granules and is still present in the nucleus. These results suggest that after neurotrophic stimulation, the lack of changes in HuD distribution are due to continued steady state of HuD nuclear shuttling in PC12 cells, or because HuD is not normally shuttled from the nucleus in response to NGF.

Reference:

• Burry, R. W., and Smith, C. L.: HuD distribution changes in response to heat shock but not neurotrophic stimulation. J. Histochem. Cytochem., 54, 1129-1138 (2006).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Silver enhancement details and formulation:

• Burry, R. W., and Smith, C. L.: HuD distribution changes in response to heat shock but not neurotrophic stimulation. J. Histochem. Cytochem., 54, 1129-1138 (2006).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

More information:

• Nanogold Conjugates: catalog information
• Nanogold-antibody conjugates: product information and instructions
• Technical help - Nanogold conjugates
• Nanogold labeling references, sorted by application

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Ordering and Shipping Deadlines

If you have any questions about shipping, or about an order that you have placed, remember to contact our main sales office nano¤nanoprobes.com, not our technical support address - our technical support personnel do not have direct access to inventory, shipping or package tracking, and will have to forward your question to our main office for help. If you are ordering from outside the US, you may wish to order from one of our international distributors instead: contact your distributor directly for ordering information.

More information:

• Ordering information
• International Distributors
• Online order form
• About us: Company information

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Other Recent Publications

Reference:

• Si-Tayeb, K.; Monvoisin, A.; Mazzocco, C.; Lepreux, S.; Decossas, M.; Cubel, G.; Taras, D.; Blanc, J. F.; Robinson, D. R., and Rosenbaum, J.: Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis. Am. J. Pathol., 169, 1390-1401 (2006).
  • Abstract (courtesy of the American Journal of Pathology).

Reference:

• Pineau, I.; Barrette, B.; Vallieres, N., and Lacroix, S.: A Novel Method for Multiple Labeling Combining In Situ Hybridization with Immunofluorescence. J. Histochem. Cytochem., 54, 1303-1313 (2006).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Meanwhile, Morales and group added immunogold flavor to their autoradiographic labeling: they used autoradiography with immunohistochemistry and immunogold labeling to collect evidence for the involvement of Megalin (gp330/LRP-2), an endocytic receptor belonging to the low-density lipoprotein receptor family, in the in vivo epithelial endocytosis and trafficking of non-lipid-modified Sonic hedgehog (ShhN). Distribution of ShhN was studied using a radiolabeled glutathione-S-transferase ShhN fusion protein, [³²P]-GST-ShhN. Labeled [³²P]-GST-ShhN in PIPES buffer was infused into the efferent ducts of anesthetized 4-month-old Sprague Dawley rats via microinjection; to block megalin-mediated interaction, ShhN was coinjected with either anti-megalin IgG (5 mg/ml) or the megalin antagonist, RAP (2 mg/ml). Efferent ducts were removed by dissection, fixed, and embedded either in paraffin (for light microscopy) or in Lowicryl K11M (for electron microscopy). Autoradiography was performed on 5-µm-thick deparaffinized tissue sections. After passage in alcohol, slides were air-dried, dipped in LM-1 emulsion, exposed in the dark at 4°C for 8 days, then developed with Kodak D-170 developer for 7 minutes at 15°C, fixed for 4 minutes in Kodak Rapid Fixer, and counterstained with hematoxylin and eosin. Shh and mouse patched-1 (Ptc-1) were localized immunohistochemically by staining with primary antibodies, biotinylated secondaries and streptavidin-peroxidase developed with diaminobenzidine (DAB). Post-embedding double immunogold labeling was then used to localize Shh and megalin, using secondary antibodies conjugated to 10 and 15 nm gold particles. Initially, exogenous ShhN is detected in endocytic vesicles and early endosomes located near the apical plasma membrane of non-ciliated cells. Within 3060 min following infusion, ShhN can be detected in lysosomes and at basolateral regions of non-ciliated cells. Basolaterally, ShhN was observed along the extracellular surfaces of interdigitated plasma membranes of adjacent cells and in the extracellular compartment underlying the efferent duct epithelium. Uptake and subcellular trafficking of infused ShhN by non-ciliated cells could be blocked by either anti-megalin IgG or the megalin antagonist, RAP. Ciliated cells, which do not express megalin, displayed little if any apical internalization of ShhN even though they were found to express Ptc-1. However, ShhN was found in coated pits of lateral plasma membranes of ciliated cells as well as in underlying endocytic vesicles. It was concluded that megalin-mediated endocytosis of ShhN can occur in megalin-expressing epithelia in vivo, and that the internalized ShhN can be targeted to the lysosome or transcytosed in the plane of the epithelium or across the epithelium. There are therefore multiple mechanisms by which megalin may influence Shh morphogen gradients in vivo.

Reference:

• Morales, C. R.; Zeng, J.; El Alfy, M.; Barth, J. L.; Chintalapudi, M. R.; McCarthy, R. A.; Incardona, J. P.; Argraves, W. S.: Epithelial trafficking of Sonic hedgehog by megalin. J. Histochem. Cytochem., 54, 1115-1127 (2006).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

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