In vitro photothermal therapy of pancreatic cancer mediated by immunoglobulin G-functionalized silver nanoparticles.

Pancreatic cancer is one of the most aggressive forms of cancer, and treatment options are limited. One therapeutic approach is to use nanoparticles to deliver the active agent directly to pancreatic cancer cells. Nanoparticles can be designed to specifically target cancer cells, minimizing damage to healthy tissues. Silver nanoparticles have the unique ability to absorb light, especially in the near-infrared (NIR) region. In this study, silver nanoparticles functionalized with IgG molecules were synthesized and administered to pancreatic cancer cell lines. Subsequently, the cells were photo-excited using a 2 W 808 nm laser and further examined in PANC-1 pancreatic cancer cell lines. Flow cytometry and confocal microscopy combined with immunochemical staining were used to examine the interaction between photo-excited silver nanoparticles and pancreatic cancer cells. The photothermal therapy based on IgG-functionalized silver nanoparticles in pancreatic cancer induces dysfunction in the Golgi apparatus, leading to the activation of the caspase-3 apoptotic pathway and ultimately resulting in cellular apoptosis. These findings suggest that our proposed IgG nanoparticle laser treatment could emerge as a novel approach for the therapy of pancreatic cancer.

Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules.

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking. Here we describe the systematic and quantitative characterisation of passive EV loading with small molecules based on hydrophobic interactions - either through direct adsorption of hydrophobic compounds, or by membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by single vesicle imaging, both ligand types bind to CD63 positive EVs (exosomes), however also non-specifically to other vesicles, particles, and serum proteins. The hydrophobic compounds Curcumin and Terbinafine aggregate on EVs with no apparent saturation up to 106-107 molecules per vesicle as quantified by liquid chromatography - high resolution mass spectrometry (LC-HRMS). For both compounds, high density EV loading resulted in the formation of a population of large, electron-dense vesicles as detected by quantitative cryo-transmission electron microscopy (TEM), a reduced EV cell uptake and a toxic gain of function for Curcumin-EVs. In contrast, cholesterol tagging of a hydrophilic mdm2-targeted cyclic peptide saturated at densities of ca 104-105 molecules per vesicle, with lipidomics showing addition to, rather than replacement of endogenous cholesterol. Cholesterol anchored ligands did not change the EVs' size or morphology, and such EVs retained their cell uptake activity without inducing cell toxicity. However, the cholesterol-anchored ligands were rapidly shed from the vesicles in presence of serum. Based on these data, we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding or redistribution to other components if present in the sample, (2) cholesterol anchoring needs substantial optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with hydrophobic compounds to mitigate changes in physicochemical properties, loss of EV function and potential cell toxicity.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Silver Nanoparticles for the Therapy of Tuberculosis.

Rapid emergence of aggressive, multidrug-resistant Mycobacteria strain represents the main cause of the current antimycobacterial-drug crisis and status of tuberculosis (TB) as a major global health problem. The relatively low-output of newly approved antibiotics contributes to the current orientation of research towards alternative antibacterial molecules such as advanced materials. Nanotechnology and nanoparticle research offers several exciting new-concepts and strategies which may prove to be valuable tools in improving the TB therapy. A new paradigm in antituberculous therapy using silver nanoparticles has the potential to overcome the medical limitations imposed in TB treatment by the drug resistance which is commonly reported for most of the current organic antibiotics. There is no doubt that AgNPs are promising future therapeutics for the medication of mycobacterial-induced diseases but the viability of this complementary strategy depends on overcoming several critical therapeutic issues as, poor delivery, variable intramacrophagic antimycobacterial efficiency, and residual toxicity. In this paper, we provide an overview of the pathology of mycobacterial-induced diseases, andhighlight the advantages and limitations of silver nanoparticles (AgNPs) in TB treatment.

Nanotechnology in metastatic cancer treatment: Current Achievements and Future Research Trends.

The systemic spread of malignant cells from a primary site, a process termed metastasis represents a global challenge in cancer treatment. There is a real need to develop novel therapy strategies and nanomedicine may have remarkable and valuable contribution through specific and selective delivery of chemotherapeutic agents, through its intrinsic cytotoxic activity or through imaging applications, appealing also in the context of cancer personalized therapy. This review is focused on the applications of nanoparticles in the treatment of metastatic cancer, particularly on the possible effect on cell stabilization, growth inhibition, eventual interaction with adhesion molecules and antiangiogenic effect.

Quantum dots in imaging, drug delivery and sensor applications.

Quantum dots (QDs), also known as nanoscale semiconductor crystals, are nanoparticles with unique optical and electronic properties such as bright and intensive fluorescence. Since most conventional organic label dyes do not offer the near-infrared (>650 nm) emission possibility, QDs, with their tunable optical properties, have gained a lot of interest. They possess characteristics such as good chemical and photo-stability, high quantum yield and size-tunable light emission. Different types of QDs can be excited with the same light wavelength, and their narrow emission bands can be detected simultaneously for multiple assays. There is an increasing interest in the development of nano-theranostics platforms for simultaneous sensing, imaging and therapy. QDs have great potential for such applications, with notable results already published in the fields of sensors, drug delivery and biomedical imaging. This review summarizes the latest developments available in literature regarding the use of QDs for medical applications.

Carbon nanotubes as anti-bacterial agents.

Multidrug-resistant bacterial infections that have evolved via natural selection have increased alarmingly at a global level. Thus, there is a strong need for the development of novel antibiotics for the treatment of these infections. Functionalized carbon nanotubes through their unique properties hold great promise in the fight against multidrug-resistant bacterial infections. This new family of nanovectors for therapeutic delivery proved to be innovative and efficient for the transport and cellular translocation of therapeutic molecules. The current review examines the latest progress in the antibacterial activity of carbon nanotubes and their composites.

Development of nanoparticle-based optical sensors for pathogenic bacterial detection.

BACKGROUND: Pathogenic bacteria contribute to various globally important diseases, killing millions of people each year. Various fields of medicine currently benefit from or may potentially benefit from the use of nanotechnology applications, in which there is growing interest. Disease-related biomarkers can be rapidly and directly detected by nanostructures, such as nanowires, nanotubes, nanoparticles, cantilevers, microarrays, and nanoarrays, as part of an accurate process characterized by lower sample consumption and considerably higher sensitivity. There is a need for accurate techniques for pathogenic bacteria identification and detection to allow the prevention and management of pathogenic diseases and to assure food safety. CONCLUSION: The focus of this review is on the current nanoparticle-based techniques for pathogenic bacterial identification and detection using these applications.

Laser thermal ablation of multidrug-resistant bacteria using functionalized gold nanoparticles.

The issue of multidrug resistance (MDR) has become an increasing threat to public health. One alternative strategy against MDR bacteria would be to construct therapeutic vectors capable of physically damaging these microorganisms. Gold nanoparticles hold great promise for the development of such therapeutic agents, since the nanoparticles exhibit impressive properties, of which the most important is the ability to convert light into heat. This property has scientific significance since is exploited to develop nano-photothermal vectors to destroy bacteria at a molecular level. The present paper summarizes the latest advancements in the field of nanotargeted laser hyperthermia of MDR bacteria mediated by gold nanoparticles.

Selective ex vivo photothermal nano-therapy of solid liver tumors mediated by albumin conjugated gold nanoparticles.

We have used albumin (BSA) bound to gold nanoparticles (GNPs) as active vectors to target liver cells. Our incentive to develop an original model of living liver cancer sprang from the ethical drawbacks that hindered the assessment of the selective character and the therapeutic capacity of these nano-biosystems in cancer patients. Ex vivo-perfused liver specimens were obtained from hepatocellular carcinoma patients similarly to the surgical technique of transplantation. Albumin bound to GNPs was inoculated intra-arterially onto the resulting specimen and determined the specific delivery of the nano-bioconjugate into the malignant tissue by means of the capillary bed. The extent of necrosis was considerable following laser therapy and at the same time surrounding parenchyma was not seriously affected. The selective photothermal ablation of the malignant liver tissue was obtained after the selective accumulation of BSA bound to GNPs into tumor cells following ex-vivo intra-vascular perfusion.

Selective in vitro photothermal nano-therapy of MRSA infections mediated by IgG conjugated gold nanoparticles.

There are serious systemic infections associated with methicillin-resistant Staphylococcus aureus (MRSA) and several other types of bacteria leading to the deaths of millions of people globally. This type of mortality is generally caused by the increasing number of antibiotic-resistant organisms, a consequence of evolution via natural selection. After the synthesis of gold nanoparticles (GNPs) by wet chemistry, bio-functionalization with IgG molecules was performed. Following administration of IgG-GNPs to MRSA cultures at various concentrations and various incubation time laser irradiation was performed. To assess the selectivity and specificity of the proposed treatment the following methods were used: flow cytometry, contrast phase microscopy, and by fluorescence microscopy. The results in our study indicate that following administration of IgG-GNPs biomolecule an extended and selective bacterial death occurs following laser irradiation in a dose dependent manner. Therefore, the new findings might impel studies on these antibacterial nanomaterials and their biological and medical applications.

Selective Laser Ablation of Methicillin-Resistant Staphylococcus Aureus with IgG Functionalized Multi-Walled Carbon Nanotubes.

Severe infections caused by Methicillin-resistant Staphylococcus aureus (MRSA) and other bacteria are responsible for millions of deaths each year. One of the main objectives of future antibiotic strategies is to develop new anti-infective agents, which would be highly effective and drug-resistant (antimicrobial resistance being currently exhibited by MRSA), using specific antibodies conjugated to thermally active nanomaterials such as NIR-responsive photothermal contrast agents. Multi-walled carbon nanotubes (MWCNTs) covalently functionalized with immunoglobulin G (IgG, an antagonist of Staphylococcal protein A-SpA, which is a MRSA membrane associated protein) were selectively delivered (at various concentrations and incubation times) into MRSA bacteria. Following treatment, cultures were irradiated using an 808 nm 2 w laser diode. The post irradiation death rate ranged from 39.6% (for 1 mg/L) to 79.2% (for 50 mg/L) at 60 seconds (p < 0.001), while at 30 minutes, the death rate increased from 45.2% (1 mg/L) to 85.72% (50 mg/L), p < 0.001. Irradiated MRSAs treated with MWCNTs alone (control) for 60 seconds and 30 minutes, at concentrations ranging from 1 mg/L to 50 mg/L, resulted in significantly lower death rates (7.1-34.1% for 60 seconds, 11.7-48.8% for 30 minutes). Using IgG molecules bound to MWCNTs, followed by laser irradiation, we obtained a very efficacious nanoshell-mediated laser therapy of individual MRSA agents providing highly localized killing effects for IgG-MWCNTs targeted bacteria.

Advances in cancer research using gold nanoparticles mediated photothermal ablation.

Recent research suggests that nanotechnologies may lead to the development of novel cancer treatment. Gold nanoparticles with their unique physical and chemical properties hold great hopes for the development of thermal-based therapies against human malignancies. This review will focus on various strategies that have been developed to use gold nanoparticles as photothermal agents against human cancers.

Hypersensitivity and nanoparticles: update and research trends.

Nanotechnology holds a great promise for a wide range of medical-intent applications (diagnostic, treatment and prophylaxis of various diseases). Their advantages are due to their size, versatility and potential for multiple simultaneous applications. However, concerns have been formulated by scientific world due to insufficient data on toxicity of nanomaterials. One area of interest is represented by the interactions between nanoparticles and the components of the immune system. We review herein reported data on hypersensitivity reactions. The role exerted by nanoparticles in both immunostimulation and immunosuppression in allergen-driven mechanisms was studied, as well as future trends in worldwide research.

A novel immunoglobulin G monolayer silver bio-nanocomposite.

BACKGROUND: Nanoparticles have a large number of surface atoms, which translates into a significant increase in the surface energy. Once introduced in a biological environment they tend to interact with proteins and form a protein corona shell. The aim of this study was to develop a novel, silver based, bio-nanocomposite for biological applications. Immunoglobulin G (IgG) molecule was chosen for the passivation of the silver nanoparticles (AgNPs) in order to avoid macrophage recognition of the synthesized structures. RESULTS: Monodisperse IgG-folinate functionalized silver nanoparticles were obtained, with sizes around 39 nm. UV-Vis and UATR-FT-IR spectroscopies were employed to confirm the successful functionalization of the silver nanoparticles. Atomic force microscopy and dynamic light scattering measurements gave information about the size and shape of the nanoparticles prior and after the passivation with IgG. CONCLUSIONS: Immunoglobulin G formed a monolayer around the nanoparticles with the binding site seemingly in the Fc domain, leaving the two Fab regions available for antigen binding. To our knowledge, this is the first report of an IgG-folinate functionalized AgNP bionanostructure developed for biological applications. Graphical abstract:Graphical illustration for IgG-folinate silver nanoparticles functionalization steps.

Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor.

We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 µg/mL) to 48.2% (for 50 µg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 µg/mL) to 52.3% (50 µg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus-endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating.

Pharmacokinetics Evaluation of Carbon Nanotubes Using FTIR Analysis and Histological Analysis.

Carbon nanotubes (CNTs) are biologically non-toxic and long-circulating nanostructures that have special physical properties. This study was focused on developing alternative methods that track carbon nanotubes, like FR-IR to classical tissue histological procedure. FT-IR absorption spectra were used to confirm the carboxylation of carbon nanotubes and to evaluate the presence of carbon nanotubes from bulk spleen samples and histologically prepared samples (control spleen and spleen with SWCNT-COOH). FT-IR spectrum of spleen sample from animals injected with CNTs shows major spectral differences consisting in infrared bands located at ~1173 cm(-1), ~ 1410 cm(-1); ~1658 cm(-1), ~1737 cm(-1) and around 1720 cm(-1) respectively. In terms of localization of carbon nanotubes, selective accumulation of marginal zone macrophages and splenic red pulp is observed for all treated groups, indicating the presence of carbon nanotubes even at 3, 4 and 7 days after treatment. In summary, we believe that histological evaluation and FT-IR can provide more characteristic information about the pharmacokinetcis and the clearance of carbon nanotubes.

Rational design of gold nanocarrier for the delivery of JAG-1 peptide.

BACKGROUND: Unique properties exhibited by nanoparticles makes them great candidates for applications in physics, chemistry, biology, material science and medicine. The biological applications of water-soluble gold nanoparticles range from contrast agents, delivery vehicles to therapeutics. Notch signaling is a complex network that orchestrates cell fate decisions, which involves proliferation, migration, differentiation and cell death in organisms ranging from insects to humans. Studies have showed that a correct orientation of the Jag-1 signalling protein on the substrates proves to be of great importance when promoting Jagged-1 Notch interactions, also the availability of the ligands, super cedes the importance of their concentration. RESULTS: The aim of the present study was to synthetize a Jag-1 functionalized nanocarrier, which would promote an efficient interaction between the Jag-1 peptide and the Notch receptor. To this end, two routes for gold nanoparticle-peptide assembly were investigated, and the synthetized bio-nanostructures were characterized and compared by means of UV-Vis, FT-IR, DLS and AFM techniques. CONCLUSIONS: We have obtained a stable, monodisperse, hetero-functionalized GNP-PEG-JAG-1 bio-nanostructure for Notch pathway activation applications.

In Vitro Administration of Gold Nanoparticles Functionalized with MUC-1 Protein Fragment Generates Anticancer Vaccine Response via Macrophage Activation and Polarization Mechanism.

Therapeutic cancer vaccines (or active immunotherapy) aim to guide the patient's personal immune system to eradicate cancer cells. An exciting approach to cancer vaccines has been offered by nanoscale drug delivery systems containing tumor associated antigens (TAAs). Their capacity to stimulate the immune system has been suggested during late years. However, the role of the macrophages as key-elements in antigen-presentation process following TAAs-containing nanosystems is not completely understood. We aimed to evaluate the effect of gold nanoparticles functionalized with mucin-1 peptide (MUC-1) on murine peritoneal macrophages. Gold nanoparticles, obtained using a modified Turkevich method, were functionalized with MUC-1 protein using Clealand's reagent. The obtained GNP-MUC-1 solution was used to treat at various concentrations monolayers of peritoneum-derived macrophages that were further analyzed using confocal and hyperspectral microscopy, ELISA assays and spectroscopic techniques. The GNP-MUC-1 nano-construct had proven to function as a powerful macrophage activator with consequent release of cytokines such as: TNF-ɑ, IL-6, IL-10 and IL-12 on peritoneal macrophages we have isolated from mice. Our results demonstrate optimization of antigen-presenting process and predominant M1 polarization following exposure GNP-MUC-1. To our best knowledge this is the first study to evaluate the anticancer effects of a newly designed nano-biocompound on the complex antigen- processing apparatus of peritoneal macrophages.