Locating Functionalized Gold Nanoparticles Using Electrical Impedance Tomography.

OBJECTIVE: An imaging device to locate functionalised nanoparticles, whereby therapeutic agents are transported from the site of administration specifically to diseased tissues, remains a challenge for pharmaceutical research. Here, we show a new method based on electrical impedance tomography (EIT) to provide images of the location of gold nanoparticles (GNPs) and the excitation of GNPs with radio frequencies (RF) to change impedance permitting an estimation of their location in cell models Methods: We have created an imaging system using quantum cluster GNPs as contrast agent, activated with RF fields to heat the functionalized GNPs, which causes a change in impedance in the surrounding region. This change is then identified with EIT. RESULTS: Images of impedance changes of around 80 ± 4% are obtained for a sample of citrate stabilized GNPs in a solution of phosphate-buffered saline. A second quantification was carried out using colorectal cancer cells incubated with culture media, and the internalization of GNPs into the colorectal cancer cells was undertaken to compare them with the EIT images. When the cells were incubated with functionalised GNPs, the change was more apparent, approximately 40 ± 2%. This change was reflected in the EIT image as the cell area was more clearly identifiable from the rest of the area. SIGNIFICANCE: EIT can be used as a new method to locate functionalized GNPs in human cells and help in the development of GNP-based drugs in humans to improve their efficacy in the future.

Exploiting the efficacy of Tyro3 and folate receptors to enhance the delivery of gold nanoparticles into colorectal cancer cells in vitro.

Colorectal cancer (CRC) is the fourth most common cancer in the world. Due to its asymptomatic nature, CRC is diagnosed at an advanced stage where the survival rate is <5%. Besides, CRC treatment using chemotherapy, radiotherapy and surgery often causes undesirable side-effects. As such, gold nanoparticles (GNPs) are envisaged in the field for the diagnosis and treatment of CRC. GNPs have unique physical, chemical and electrical properties at the nanoscale which make them suitable for application in biomedicine. However, for GNPs to become clinically effective, their internalisation efficiency in cancer cells must be enhanced. Folate receptor-α (FR) is overexpressed in CRC cells wherein FR helps in the uptake of folic acid within the cells. Tyro3, a novel tyrosine kinase receptor, drives cell proliferation and its overexpression is correlated with poor prognosis in CRC. Their upregulated expression in CRC cells relative to normal cells makes them an ideal target for GNPs using active targeting. Therefore, in this study receptors FR and Tyro3 were simultaneously targeted using specific antibody-coated GNPs in order to enhance the uptake and internalisation of GNPs in CRC cells in vitro. Four different types of coated-GNPs were synthesised GNPs-PEG, GNPs-anti-FR, GNPs-anti-Tyro3 and GNPs-anti-(FR + Tyro3) and incubated (0-50 ng) with three CRC cell lines namely CRL1790, CRL2159 and HCT116. Simultaneous targeting of these receptors by GNPs-anti-(FR + Tyro3) was found to be the most effective in internalisation in CRC cells compared with GNPs targeted singly to FR or Tyro3 (p <0.05). Besides this, results show that Tyro3 mediated similar internalisation efficacy to FR (p <0.05) in CRC cells using ICP-OES.

Emerging applications of nanotechnology for diagnosis and therapy of disease: a review.

Nanotechnology is of increasing interest in the fields of medicine and physiology over recent years. Its application could considerably improve disease detection and therapy, and although the potential is considerable, there are still many challenges that need to be addressed before it is accepted in routine clinical use. This review focuses on emerging applications that nanotechnology could enhance or provide new approaches in diagnoses and therapy. The main focus of recent research centres on targeted therapies and enhancing imaging; however, the introduction of nanomaterial into the human body must be controlled, as there are many issues with possible toxicity and long-term effects. Despite these issues, the potential for nanotechnology to provide new methods of combating cancer and other disease conditions is considerable. There are still key challenges for researchers in this field, including the means of delivery and targeting in the body to provide effective treatment for specific disease conditions. Nanoparticles are difficult to measure due to their size and physical properties; hence there is still a great need to improve physiological measurement methods in the field to ascertain how effective their use is in the human subject. This review is a brief snapshot into the fast changing research field of measurement and physiological links to nanoparticle use and its potential in the future.

Detection of the tau protein in human serum by a sensitive four-electrode electrochemical biosensor.

This study presents a novel approach based on a four-electrode electrochemical biosensor for the detection of tau protein - one of the possible markers for the prediction of Alzheimer's disease (AD). The biosensor is based on the formation of stable antibody-antigen complexes on gold microband electrodes covered with a layer of a self-assembled monolayer and protein G. Antibodies were immobilized on the gold electrode surface in an optimal orientation by protein G interaction. Electrochemical impedance spectroscopy was used to analyze impedance change, which revealed a linear response with increasing tau concentrations. The assay is fast (<1h for incubation and measurement) and very sensitive. The limit of quantification for the full-length 2N4R tau protein is 0.03pM, a value unaltered when the assay was processed in bovine serum albumin or human serum. This technology could be adapted for the detection of other biomarkers to provide a multiple assay to identify AD progression in a point of care setting.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine.

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro.

The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier.

The influence of ligand organization on the rate of uptake of gold nanoparticles by colorectal cancer cells.

We have explored the uptake of different hydrophilic mono- and dual-ligand gold nanoparticles in colorectal cancer cells in vitro and find that the rate of uptake is dependent on the structural organization of the ligands on the surface of the particles rather than their charge or chemical properties. Gold nanoparticles with 50%PEG-NH(2)/50% glucose are taken up eighteen fold faster than nanoparticles carrying only PEG-NH(2) or glucose. Glutathione-coated gold particles are by far the most efficiently internalized; however, glucose-glutathione dual-ligand nanoparticles are taken up at a thirty fold reduced rate. We found furthermore that the rates are influenced by the cell density and concentration of glucose in the growth medium. Rather than being internalized through a conventional receptor-mediated mechanism the particles appear to be taken up by the cells via an energy-independent diffusion across the cell membrane through pre-existing pores or openings in the lipid bi-layer created by ligands on the gold nanoparticles.

A human domain antibody and Lewis b glycoconjugate that inhibit binding of Helicobacter pylori to Lewis b receptor and adhesion to human gastric epithelium.

Increasing antibiotic resistance has prompted development of alternative approaches to antimicrobial therapy, including blocking microbial adhesion to host receptors. The BabA adhesin of Helicobacter pylori binds to fucosylated blood group antigens, such as the Lewis(b) antigens in human primate gastric mucosa. We have isolated a human domain antibody specific for BabA that inhibits binding of BabA to Lewis(b) and prevents adhesion of H. pylori to human gastric epithelium. In addition, Lewis(b) oligosaccharides covalently linked to poly-D-lysine inhibited BabA binding to Le(b). The poly-D-lysine-Le(b) hexasaccharide and an Le(b) human serum albumin conjugate not only inhibited adherence of H. pylori to gastric epithelium but also displaced adherent bacteria when added to human stomach sections. Combinations of Le(b) and sialyl Le(x) or domain antibody 25 and sialyl Le(x) acted synergistically. Domain antibody 25 inhibitor may have potential for prophylactic use and, in combination with Le(b) glycoconjugates, therapeutic use in treatment of drug-resistant H. pylori infection.

Screening autoantibody profiles in systemic rheumatic disease with a diagnostic protein microarray that uses a filtration-assisted nanodot array luminometric immunoassay (NALIA).

BACKGROUND: We developed a cost-efficient modular system for multiplex analysis of the multiple autoantibodies that characterize systemic rheumatoid diseases. METHODS: The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls. The test fluid, control sera, and subsequent reagents are drawn through the membrane. The captured analytes are quantified by monitoring chemiluminescence with a charge-coupled device (CCD) and analyzed with commercial array software. RESULTS: The assay can detect <20 x 10(3) IU/L of anti-dsDNA. The interwell CV was 10%-14%. There was an 83% concordance (kappa = 0.56) between the NALIA results obtained for anti-dsDNA assayed by beta-testing in a routine immunology diagnostic laboratory and the results obtained with a conventional ELISA reagent set. The concordance values for Ro, La, Sm, and RNP were 98% (kappa, 0.92), 93% (kappa, 0.41), 97% (kappa, 0.62), and 97% (kappa, 0.73), respectively. CONCLUSION: The NALIA approach promises to provide a highly economical platform for a wide range of applications that require assays of multiple analytes. The degree of concordance of our results with a conventional reagent set was no less than that occurring between different commercial products. A sample of serum from a finger stick provides a volume sufficient to perform the array assay.

Comparison of the Calibration Standards of Three Commercially Available Multiplex Kits for Human Cytokine Measurement to WHO Standards Reveals Striking Differences.

Serum parameters as indicators for the efficacy of therapeutic drugs are currently in the focus of intensive research. The induction of certain cytokines (or cytokine patterns) is known to be related to the status of the immune response e.g. in regulating the T(H)1/T(H)2 balance. Regarding their potential value as surrogate parameters in clinical trials and subsequently for the assignment of treatment efficacy, the accurate and reliable determination of cytokines in patient serum is mandatory. Because serum samples are precious and limited, test methods-like the xMAP multiplex technology-that allow for the simultaneous determination of a variety of cytokines from only a small sample aliquot, can offer great advantages.We here have compared multiplex kits from three different manufactures and found striking differences upon standardizing using WHO standards for selected cytokines. We therefore extended our xMAP multiplex measurements investigations to an ex-vivo situation by testing serum samples and found that the cytokine amounts measured was critically influenced by the actual kit used. The presented data indicate that statements regarding the quantitive determination of cytokines-and therefore their use as biomarkers-in serum samples have to be interpreted with caution.

Human domain antibodies against virulence traits of Candida albicans inhibit fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis.

Antibody variable domains (domain antibodies [DAbs]) are genetically engineered antibody fragments that include individual heavy-chain (VH) or kappa-chain (Vkappa) variable domains and lack the Fc region. Human DAbs against the 65-kDa mannoprotein (MP65) or the secretory aspartyl proteinase (SAP)-2 of Candida albicans (monospecific DAbs) or against both fungal antigens (heterodimeric, bispecific DAbs) were generated from phage expression libraries. Both monospecific and bispecific DAbs inhibited fungus adherence to the epithelial cells of rat vagina and accelerated the clearance of vaginal infection with the fungus. When heterodimeric DAbs were used, the clearance of infection was at least equivalent to treatment with fluconazole. The in vivo protective effects of DAbs were demonstrated by both pre- and postchallenge schedules of DAb administration and with both fluconazole-susceptible and fluconazole-resistant strains of C. albicans. This is the first demonstration that human DAbs lacking the Fc constituent can efficiently control an infection and can act largely by inhibiting adherence.

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.

Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy.

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1x10(6)-1x10(9) cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our 'Region of Interest' method, with the software's 'Standard Area Method' of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 mug/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells.

Tumour burden and interleukin-2 dose affect the interaction between low-dose total body irradiation and interleukin 2.

Low-dose total body irradiation (LTBI) has a synergistic immune-mediated antitumour effect when used in combination with interleukin 2 (IL-2) in a murine metastatic malignant melanoma model. To optimise the use of this combination treatment this study was performed to test the effect of tumour burden and dose of both LTBI and IL-2 on the therapeutic potential of this treatment strategy. Ten-week-old female C57BL/6 mice were inoculated intravenously (day 0) with 1 million B16F1 malignant melanoma cells. Groups of mice received no treatment, a single fraction of LTBI alone, IL-2 treatment alone, or a combination of LTBI and IL-2. Two doses of LTBI and IL-2 were tested. LTBI was given on day +10 and IL-2 treatment started on day +11. On day +18 the mice were killed. The lungs were removed and analysed for tumour burden. Lung sections were also tested for infiltrating leucocytes using immunohistochemical staining. In one experiment, mice were treated at day +7 with low-dose IL-2 with and without LTBI. LTBI (in the two tested doses) showed no independent therapeutic effects. An IL-2 dose of 300,000 Cetus units (CU) that was effective and showed synergism with LTBI when mice were treated on day +7 failed to show a therapeutic effect when mice were treated on day +10, at which time the initial tumour burden had doubled. High-dose IL-2 (600,000 CU), in contrast, led to a significant reduction in metastatic burden compared to the control group. Combining high-dose IL-2 with LTBI led to a further significant reduction in tumour burden. Moreover, this combination was associated with a less severe vascular leakage syndrome compared to IL-2 alone. IL-2 and combination treatment was associated with an increase in the number of tumour-infiltrating immune cells, but only the number of tumour-infiltrating natural killer cells reflected therapeutic efficacy. It was concluded that tumour burden at the time of treatment and IL-2 dose are two crucial factors affecting the synergism between LTBI and IL-2. The combination may not only be more effective than IL-2 alone but also less toxic.

Low-dose total body irradiation augments the therapeutic effect of interleukin-2 in a mouse model for metastatic malignant melanoma.

BACKGROUND: Low-dose total body irradiation (LTBI) is known for its antitumor immune modulatory effects. Moreover, there is theoretical ground suggesting that combining LTBI with interleukin-2 (IL-2) will have a synergistic immune-mediated antitumor effect. However, the use of LTBI in combination with IL-2 or other forms of immunotherapy has not been tested before. AIM OF THE WORK: To test the efficacy of combining LTBI and IL-2 in controlling lung metastases in a murine model for malignant melanoma compared to IL-2 alone. MATERIAL AND METHODS: Ten-week-old female C57BL/6 mice were inoculated intravenously (on day 0) with 1 million B16F1 malignant melanoma cells. The mice received either no treatment (control group), LTBI alone (single fraction of 0.75 Gy), IL-2 treatment alone (30,000 CU x 2 daily for 5 consecutive days), or a combination of LTBI and IL-2. LTBI was given on day +7 and IL-2 treatment started day +8. On day +14, the mice were sacrificed and the lungs were removed and analyzed for tumor burden. Lung sections were also tested for tumor-infiltrating cells using immunohistochemical staining. Peripheral blood and splenic cells were collected and tested for the percentage of the various lymphocytic subsets using immunostaining and flow cytometry. RESULTS: Tumor burden expressed as the percentage of lung area occupied with metastases (+/- 1 SD), was the same in the control group (8.1 +/- 4.9%), and in the group receiving LTBI alone (8.3 +/- 4.5%). Tumor burden was reduced to 6.4% (+/- 3.4%) in the IL-2 alone group (P = 0.3) and further reduced to 3% (+/- 1%) in the combined treatment group (P = 0.004). The difference in tumor burden between the IL-2 alone group and the combined treatment group was statistically significant (P = 0.006). The combined treatment caused a significant increase in the number of natural killer (NK) cells and macrophages infiltrating the metastatic sites. This was associated with a significant increase in the percentage of CD122+ (IL-2R beta) cells and NK cells in both peripheral blood and spleens. CONCLUSION: We conclude that combining LTBI and IL-2 treatment is synergistic and therapeutically more effective than IL-2 alone. The data points to NK cells and macrophages as likely major effectors of the synergistic outcome of the combined treatment. This observation may have important clinical implications in the treatment of patients with metastatic malignant melanoma.

Innate immunity and pathogen-host interaction.

The skin and contiguous mucosal surfaces define the primary locus of interaction between host and micro-organisms. In this review, we focus on the innate immune system in the mucosa, which manages to deal with invading pathogens, the mechanisms that organisms have evolved in order to circumvent this primary defensive barrier and, finally, potential therapeutic manipulation of the innate immune system that was the focus of meeting at a Euroconference/Workshop on "Novel Strategies of Mucosal Immunisation through Exploitation of Mechanisms of Innate Immunity in Pathogen-Host Interaction", which was held in Siena, Italy, November 2002.