África González-Fernández

Minireview: Nanoparticles and the Immune System

Today nanotechnology is finding growing applications in industry, biology, and medicine. The clear benefits of using nanosized products in various biological and medical applications are often challenged by concerns about the lack of adequate data regarding their toxicity. One area of interest involves the interactions between nanoparticles and the components of the immune system. Nanoparticles can be engineered to either avoid immune system recognition or specifically inhibit or enhance the immune responses. We review herein reported observations on nanoparticle-mediated immunostimulation and immunosuppression, focusing on possible theories regarding how manipulation of particle physicochemical properties can influence their interaction with immune cells to attain desirable immunomodulation and avoid undesirable immunotoxicity.

Antibody-conjugated nanoparticles for biomedical applications

Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc.), they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc.) and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

Assessing Methods for Blood Cell Cytotoxic Responses to Inorganic Nanoparticles and Nanoparticle Aggregates

Inorganic nanoparticles (NPs) show great potential for medicinal therapy. However, biocompatibility studies are essential to determine if they are safe. Here, five different NPs are compared for their cytotoxicity, internalization, aggregation in medium, and reactive oxygen species (ROS) production, using tumoral and normal human blood cells. Differences depending on the cell type are analyzed, and no direct correlation between ROS production and cell toxicity is found. Results are discussed with the aim of standardizing the procedures for the evaluation of the toxicity.

Chitosan-based nanoparticles for improving immunization against hepatitis B infection

The design of effective vaccine delivery vehicles is opening up new possibilities for making immunization more equitable, safe and efficient. In this work, we purpose polysaccharidic-based nanoparticles as delivery structures for virus-like particle antigens, using recombinant hepatitis B surface antigen (rHBsAg) as a model. Polysaccharidic-based nanoparticles were prepared using a very mild ionic gelation technique, by cross-linking the polysaccharide chitosan (CS) with a counter ion. The resulting nanoparticles could be easily isolated with a size in the nanometric range (160-200 nm) and positive surface charge (+6 to +10 mV). More importantly, CS-based nanoparticles allowed the efficient association of the antigen (>60%) while maintaining the antigenic epitope intact, as determined by ELISA and Western blot. The entrapped antigen was further released in vitro from the nanoparticles in a sustained manner without compromising its antigenicity. In addition, loaded CS-based nanoparticles were stable, and protected the associated antigen during storage, either as an aqueous suspension under different temperature conditions (+4 degrees C and -20 degrees C), or as a dried form after freeze-drying the nanoparticles. Finally, immunization studies showed the induction of important seroprotection rates after intramuscular administration of the nanoparticles, indicating their adjuvant capacity. In fact, CS-based nanoparticles were able to induce anti-HBsAg IgG levels up to 5500 mIU/ml, values 9-fold the conventional alum-adsorbed vaccine. In conclusion, we report here a polysaccharidic nanocarrier which exhibits a number of in vitro and in vivo features that make it a promising adjuvant for vaccine delivery of subunit antigens.

Monosaccharides versus PEG-Functionalized NPs: Influence in the Cellular Uptake

Magnetic nanoparticles (NPs) hold great promise for biomedical applications. The core composition and small size of these particles produce superparamagnetic behavior, thus facilitating their use in magnetic resonance imaging and magnetically induced therapeutic hyperthermia. However, the development and control of safe in vivo applications for NPs call for the study of cell-NP interactions and cell viability. Furthermore, as for most biotechnological applications, it is desirable to prevent unspecific cell internalization of these particles. It is also crucial to understand how the surface composition of the NPs affects their internalization capacity. Here, through accurate control over unspecific protein adsorption, size distribution, grafting density, and an extensive physicochemical characterization, we correlated the cytotoxicity and cellular uptake mechanism of 6 nm magnetic NPs coated with several types and various densities of biomolecules, such as glucose, galactose, and poly(ethylene glycol). We found that the density of the grafted molecule was crucial to prevent unspecific uptake of NPs by Vero cells. Surprisingly, the glucose-coated NPs described here showed cellular uptake as a result of lipid raft instead of clathrin-mediated cellular internalization. Moreover, these glucose-functionalized NPs could be one of the first examples of NPs being endocytosed by caveolae that finally end up in the lysosomes. These results reinforce the use of simple carbohydrates as an alternative to PEG molecules for NPs functionalization when cellular uptake is required.

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

Macrophage scavenger receptor A mediates the uptake of gold colloids by macrophages in vitro.

AIMS While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. MATERIALS & METHODS The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. RESULTS Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. CONCLUSION Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.

Pathogen-mimetic stealth nanocarriers for drug delivery: a future possibility

UNLABELLED The Mononuclear Phagocyte System (MPS) is a major constraint to nanocarrier-based drug-delivery systems (DDS) by exerting a negative impact on blood circulation times and biodistribution. Current approaches rely on the protein- and cell-repelling properties of inert hydrophilic polymers, to enable escape from the MPS. Poly(ethylene glycol) (PEG) has been particularly useful in this regard, and it also exerts positive effects in other blood compatibility parameters, being correlated with decreased hemolysis, thrombogenicity, complement activation and protein adsorption, due to its uncharged and hydrophilic nature. However, PEGylated nanocarriers are commonly found in the liver and spleen, the major MPS organs. In fact, a hydrophilic and cell-repelling delivery system is not always beneficial, as it might decrease the interaction with the target cell and hinder drug release. Here, a full scope of the immunological and biochemical barriers is presented along with some selected examples of alternatives to PEGylation. We present a novel conceptual approach that includes virulence factors for the engineering of bioactive, immune system-evasive stealth nanocarriers. FROM THE CLINICAL EDITOR The efficacy of nanocarrier-based drug-delivery systems is often dampened by the Mononuclear Phagocyte System (MPS). Current approaches to circumvent MPS rely on protein- and cell-repelling properties of inert hydrophilic polymers, including PEG. This paper discusses the full scope of the immunological and biochemical barriers along with selected examples of alternatives to PEGylation.

Gold nanoparticle-based electrochemical magnetoimmunosensor for rapid detection of anti-hepatitis B virus antibodies in human serum

A sandwich immunoassay using magnetic beads as bioreaction platforms and AuNPs as electroactive labels for the electrochemical detection of human IgG antibodies anti-Hepatitis B surface antigen (HBsAg), is here presented as an alternative to the standard methods used in hospitals for the detection of human antibodies directed against HBsAg (such as ELISA or MEIA). The electrochemical detection of AuNPs is carried out approaching their catalytic properties towards the hydrogen evolution in an acidic medium, without previous nanoparticle dissolution. The obtained results are a good promise toward the development of a fully integrated biosensing set-up. The developed technology based on this detection mode would be simple to use, low cost and integrated into a portable instrumentation that may allow its application even at doctor-office. The sample volumes required can be lower than those used in the traditional methods. This may lead to several other applications with interest for clinical control.

Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: palytoxin.

Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R(2)=0.9945), showing a working range of 0.0005-500ngmL(-1). Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.