Laterally and Temporally Controlled Intracellular Staining by Light-Triggered Release of Encapsulated Fluorescent Markers.

Fluorescent molecular markers were encapsulated. The capsules were additionally modified with plasmonic nanoparticles. The encapsulated markers were endocytosed by cells. Upon light stimulation the plasmonic nanoparticles generated heat, which opened the encapsulation and transiently perforated the endosomal/lysosomal membrane surrounding the capsule, thus allowing for release of the marker into the cytosol. Fluorescence labeling of different intracellular compartments was demonstrated in this way. Most important, the cells do not need to be fixed and perforated, as the molecular markers are introduced into cells by endocytosis and subsequent light-induced release. Thus this technique allows for intracellular fluorescence labeling of living cells.

Comprehensive and Systematic Analysis of the Immunocompatibility of Polyelectrolyte Capsules.

The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.

The shedding-derived soluble receptor for advanced glycation endproducts sustains inflammation during acute Pseudomonas aeruginosa lung infection.

BACKGROUND: The membrane-bound isoform of the receptor for advanced glycation end products (FL-RAGE) is primarily expressed by alveolar epithelial cells and undergoes shedding by the protease ADAM10, giving rise to soluble cleaved RAGE (cRAGE). RAGE has been associated with the pathogenesis of several acute and chronic lung disorders. Whether the proteolysis of FL-RAGE is altered by a given inflammatory stimulus is unknown. Pseudomonas aeruginosa causes nosocomial infections in hospitalized patients and is the major pathogen associated with chronic lung diseases. METHODS: P. aeruginosa was injected in Rage-/- and wild-type mice and the impact on RAGE expression and shedding, levels of inflammation and bacterial growth was determined. RESULTS: Acute P. aeruginosa lung infection in mice induces a reduction of the active form of ADAM10, which determines an increase of FL-RAGE expression on alveolar cells and a concomitant decrease of pulmonary cRAGE levels. This was associated with massive recruitment of leukocytes and release of pro-inflammatory factors, tissue damage and relocation of cRAGE in the alveolar and bronchial cavities. The administration of sRAGE worsened bacterial burden and neutrophils infiltration. RAGE genetic deficiency reduced the susceptibility to P. aeruginosa infection, mitigating leukocyte recruitment, inflammatory molecules production, and bacterial growth. CONCLUSIONS: These data are the first to suggest that inhibition of FL-RAGE shedding, by affecting the FL-RAGE/cRAGE levels, is a novel mechanism for controlling inflammation to acute P. aeruginosa pneumonia. sRAGE in the alveolar space sustains inflammation in this setting. GENERAL SIGNIFICANCE: RAGE shedding may determine the progression of inflammatory lung diseases.

Some thoughts about the intracellular location of nanoparticles and the resulting consequences.

It is qualitatively demonstrated that the intracellular localization of particles depends on the way they are administered, their basic physicochemical properties, as well as on incubation time. For this purpose cells were exposed to fluorescently-labelled particles of different size under different exposure scenarios including incubation, microinjection, electroporation, and sonoporation. After co-exposure to cells the intracellular distribution of different particles was imaged with multicolor fluorescence microscopy. Qualitative co-localization analysis demonstrates, that different particles to which cells have been exposed in different ways did not automatically reside in the same compartment. As intracellular particle localization may influence potential toxic effects of particles on cells, studies attempting to unravel molecular mechanisms of toxicity should involve the determination of the intracellular particle distribution.

Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent.

The influence of the size and aspect ratio of anisotropic, porous CaCO3 particles on their uptake by cells.

BACKGROUND: Recent reports highlighting the role of particle geometry have suggested that anisotropy can affect the rate and the pathway of particle uptake by cells. Therefore, we investigate the internalization by cells of porous calcium carbonate particles with different shapes and anisotropies. RESULTS: We report here on a new method of the synthesis of polyelectrolyte coated calcium carbonate particles whose geometry was controlled by varying the mixing speed and time, pH value of the reaction solution, and ratio of the interacting salts used for particle formation. Uptake of spherical, cuboidal, ellipsoidal (with two different sizes) polyelectrolyte coated calcium carbonate particles was studied in cervical carcinoma cells. Quantitative data were obtained from the analysis of confocal laser scanning microscopy images. CONCLUSIONS: Our results indicate that the number of internalized calcium carbonate particles depends on the aspect ratio of the particle, whereby elongated particles (higher aspect ratio) are internalized with a higher frequency than more spherical particles (lower aspect ratio). The total volume of internalized particles scales with the volume of the individual particles, in case equal amount of particles were added per cell.

In vivo integrity of polymer-coated gold nanoparticles.

Inorganic nanoparticles are frequently engineered with an organic surface coating to improve their physicochemical properties, and it is well known that their colloidal properties may change upon internalization by cells. While the stability of such nanoparticles is typically assayed in simple in vitro tests, their stability in a mammalian organism remains unknown. Here, we show that firmly grafted polymer shells around gold nanoparticles may degrade when injected into rats. We synthesized monodisperse radioactively labelled gold nanoparticles ((198)Au) and engineered an (111)In-labelled polymer shell around them. Upon intravenous injection into rats, quantitative biodistribution analyses performed independently for (198)Au and (111)In showed partial removal of the polymer shell in vivo. While (198)Au accumulates mostly in the liver, part of the (111)In shows a non-particulate biodistribution similar to intravenous injection of chelated (111)In. Further in vitro studies suggest that degradation of the polymer shell is caused by proteolytic enzymes in the liver. Our results show that even nanoparticles with high colloidal stability can change their physicochemical properties in vivo.

Conjugation of Polymer-Coated Gold Nanoparticles with Antibodies-Synthesis and Characterization.

The synthesis of polymer-coated gold nanoparticles with high colloidal stability is described, together with appropriate characterization techniques concerning the colloidal properties of the nanoparticles. Antibodies against vascular endothelial growth factor (VEGF) are conjugated to the surface of the nanoparticles. Antibody attachment is probed by different techniques, giving a guideline about the characterization of such conjugates. The effect of the nanoparticles on human adenocarcinoma alveolar basal epithelial cells (A549) and human umbilical vein endothelial cells (HUVECs) is probed in terms of internalization and viability assays.

Comparison of the in Vitro Uptake and Toxicity of Collagen- and Synthetic Polymer-Coated Gold Nanoparticles.

We studied the physico-chemical properties (size, shape, zeta-potential), cellular internalization and toxicity of gold nanoparticles (NPs) stabilized with the most abundant mammalian protein, collagen. The properties of these gold NPs were compared to the same sized gold NPs coated with synthetic poly(isobutylene-alt-maleic anhydride) (PMA). Intracellular uptake and cytotoxicity were assessed in two cell lines (cervical carcinoma and lung adenocarcinoma cells) by employing inductively-coupled plasma-mass spectrometry (ICP-MS) analysis and a cell viability assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), respectively. We found that the collagen-coated gold NPs exhibit lower cytotoxicity, but higher uptake levels than PMA-coated gold NPs. These results demonstrate that the surface coating of Au NPs plays a decisive role in their biocompatibility.

Encapsulated enzymes with integrated fluorescence-control of enzymatic activity.

A fluorescence-based particle sensor for oxaloacetic acid is presented. In the presence of nicotinamide adenine dinucleotide as a cofactor, oxaloacetic acid is converted by malate dehydrogenase into l-malic acid. The progress of the reaction is monitored by sensing of proton consumption with an integrated pH sensor. The kinetics of this sensor are investigated on a single particle level. This work demonstrates the feasibility to detect analytes upon their enzymatic conversion into a product, which in turn can be sensed with a fluorophore responding to changes in the concentration of this product. Integration of enzymes and fluorophores into one carrier particle, as demonstrated here for the case of polyelectrolyte polymer capsules, allows the range of analytes that can be detected with fluorescence to be extended, as it enhances selectivity. This coupled system allows enzymatic activity, as well as the kinetics of malate dehydrogenase, to be monitored.

In vitro interaction of colloidal nanoparticles with mammalian cells: What have we learned thus far?

The interfacing of colloidal nanoparticles with mammalian cells is now well into its second decade. In this review our goal is to highlight the more generally accepted concepts that we have gleaned from nearly twenty years of research. While details of these complex interactions strongly depend, amongst others, upon the specific properties of the nanoparticles used, the cell type, and their environmental conditions, a number of fundamental principles exist, which are outlined in this review.

Magnetic layer-by-layer coated particles for efficient MRI of dendritic cells and mesenchymal stem cells.

AIM: Cell detection by MRI requires high doses of contrast agent for generating image contrast. Therefore, there is a constant need to develop improved systems that further increase sensitivity, and which could be used in clinical settings. In this study, we devised layer-by-layer particles and tested their potential for cell labeling. MATERIALS & METHODS: The advantages of layer-by-layer technology were exploited to obtain magnetic particles of controllable size, surface chemistry and magnetic payload. RESULTS: Flexibility in size and surface charge enabled efficient intracellular delivery of magnetic particles in mesenchymal stem cells and dendritic cells. Owing to the high magnetic payload of the particles, high MRI contrast was generated, even for very low cell numbers. Subcutaneous injection of the particles and subsequent uptake by dendritic cells enabled clear visualization of dendritic cells homing towards nearby lymph nodes in mice. CONCLUSION: The magnetic particles offer several possibilities as efficient cellular MRI contrast agents for direct in vitro or in vivo cell labeling.

Noncoding DNA in lipofection of HeLa cells-a few insights.

In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.

Transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy.

AIM: The aim of this study was to evaluate the effect of the surface functionalization of model nanoparticles on their mobility in bacterial biofilms and cystic fibrosis sputum. MATERIALS & METHODS: With single-particle tracking microscopy, the mobility of 0.1- and 0.2-µm fluorescent polyethylene glycol (PEG) modified, carboxylate- and N,N-dimethylethylenediamine-modified polystyrene nanospheres were evaluated in fresh cystic fibrosis sputum, as well as Burkholderia multivorans and Pseudomonas aeruginosa biofilms. RESULTS: PEGylation increased the mobility of the particles in sputum and biofilms, while the charged nanospheres were strongly immobilized. However, the transport of the PEGylated nanoparticles was lower in sputum compared with biofilms. Furthermore, the particle transport showed heterogeneity in samples originating from different patients. CONCLUSION: This study's data suggest that for future nanocarrier design it will be essential to combine PEGylation with a targeting moiety to ensure sufficient mobility in mucus and a better accumulation of the nanoparticles in the biofilm.

Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines.

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.

Lipoplexes carrying mRNA encoding Gag protein modulate dendritic cells to stimulate HIV-specific immune responses.

AIM: Cationic lipids (Lipofectamine™ [Invitrogen, Merelbeke, Belgium] and 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and polymers (jetPEI™ and in vivo-jetPEI™ [Polyplus-transfection, Illkirch, France]) were evaluated for their potential to deliver mRNA to monocyte-derived dendritic cells. MATERIALS & METHODS: Lipoplexes and polyplexes, containing mRNA encoding GFP or Gag protein, were incubated with human monocyte-derived dendritic cells and transfection efficiencies were assessed by flow cytometry. RESULTS: Lipofectamine was by far the most efficient in mRNA delivery, therefore it was used in further experiments. Incubation of monocyte-derived dendritic cells isolated from HIV-1-positive donors with mRNA encoding Gag protein complexed to Lipofectamine resulted in 50% transfection. Importantly, coculture of these Gag-transfected dendritic cells with autologous T cells induced an over tenfold expansion of IFN-γ- and IL-2-secreting CD4(+) and CD8(+) T cells. CONCLUSION: Cationic lipid-mediated mRNA delivery may be a useful tool for therapeutic vaccination against HIV-1. This approach can be applied to develop vaccination strategies for other infectious diseases and cancer.

Light-addressable capsules as caged compound matrix for controlled triggering of cytosolic reactions.

Release me: polyelectrolyte capsules with different cargo in their cavities and plasmonic and magnetic nanoparticles in their walls were synthesized. Enzymatic reactions were triggered inside cells by light-mediated opening of two individual capsules containing either an enzyme or its substrate, by using photothermal heating. Furthermore, this technique allows controlled release of mRNA from capsules, thereby resulting in synthesis of green fluorescent protein (GFP).

Internalization of mRNA lipoplexes by dendritic cells.

Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalized by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one-third of fluorescently labeled mRNA containing lipoplexes are colocalized with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, and clathrin- and caveolae-mediated endocytosis, on both the internalization of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasin D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalization (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfection-effective mRNA lipoplex internalization by DCs, i.e., uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis.

Efficient transfection of hepatocytes mediated by mRNA complexed to galactosylated cyclodextrins.

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.