Vaccination with trifunctional nanoparticles that address CD8+ dendritic cells inhibits growth of established melanoma.

AIM: We wanted to assess the potency of a trifunctional nanoparticle (NP) that targeted and activated CD8+ dendritic cells (DC) and delivered an antigen to induce antitumor responses. MATERIALS & METHODS: The DC targeting and activating properties of ferrous NPs conjugated with immunostimulatory CpG-oligonucleotides, anti-DEC205 antibody and ovalbumin (OVA) as a model antigen to induce antigen-specific T-cell responses and antitumor responses were analyzed. RESULTS: OVA-loaded NP conjugated with immunostimulatory CpG-oligonucleotides and anti-DEC205 antibody efficiently targeted and activated CD8+ DC in vivo, and induced strong OVA-specific T-cell activation. Vaccination of B16/OVA tumor-burdened mice with this NP formulation resulted in tumor growth arrest. CONCLUSION: CD8+ DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.

Multifunctional two-photon active silica-coated Au@MnO Janus particles for selective dual functionalization and imaging.

Monodisperse multifunctional and nontoxic Au@MnO Janus particles with different sizes and morphologies were prepared by a seed-mediated nucleation and growth technique with precise control over domain sizes, surface functionalization, and dye labeling. The metal oxide domain could be coated selectively with a thin silica layer, leaving the metal domain untouched. In particular, size and morphology of the individual (metal and metal oxide) domains could be controlled by adjustment of the synthetic parameters. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g., antibodies, proteins) in a single step for converting the photoluminescent and superparamagnetic Janus nanoparticles into multifunctional efficient vehicles for theranostics. The Au@MnO@SiO2 Janus particles were characterized using high-resolution transmission electron microscopy (HR-)TEM, powder X-ray diffraction (PXRD), optical (UV-vis) spectroscopy, confocal laser fluorescence scanning microscopy (CLSM), and dynamic light scattering (DLS). The functionalized nanoparticles were stable in buffer solution or serum, showing no indication of aggregation. Biocompatibility and potential biomedical applications of the Au@MnO@SiO2 Janus particles were assayed by a cell viability analysis by coincubating the Au@MnO@SiO2 Janus particles with Caki 1 and HeLa cells. Time-resolved fluorescence spectroscopy in combination with CLSM revealed the silica-coated Au@MnO@SiO2 Janus particles to be highly two-photon active; no indication for an electronic interaction between the dye molecules incorporated in the silica shell surrounding the MnO domains and the attached Au domains was found; fluorescence quenching was observed when dye molecules were bound directly to the Au domains.

Damage-associated molecular pattern activated Toll-like receptor 4 signalling modulates blood pressure in L-NAME-induced hypertension.

AIMS: Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in an experimental model of hypertension thus contributing to blood pressure regulation. METHODS AND RESULTS: NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension was blunted in TLR4(-/-) when compared with wild-type mice. Treatment with l-NAME was associated with a release of DAMPs, leading to reactive oxygen species production of smooth muscle cells in a TLR4-dependent manner. As oxidative stress leads to an impaired function of the NO-sGC-cyclic GMP (cGMP) pathway, we were able to demonstrate that TLR4(-/-) was protected from sGC inactivation. Consequently, arterial contractility was reduced in TLR4(-/-). CONCLUSIONS: Cell damage-associated TLR4 signalling might act as a direct mediator of vascular contractility providing a molecular link between inflammation and hypertension.

Inorganic Janus particles for biomedical applications.

Based on recent developments regarding the synthesis and design of Janus nanoparticles, they have attracted increased scientific interest due to their outstanding properties. There are several combinations of multicomponent hetero-nanostructures including either purely organic or inorganic, as well as composite organic-inorganic compounds. Janus particles are interconnected by solid state interfaces and, therefore, are distinguished by two physically or chemically distinct surfaces. They may be, for instance, hydrophilic on one side and hydrophobic on the other, thus, creating giant amphiphiles revealing the endeavor of self-assembly. Novel optical, electronic, magnetic, and superficial properties emerge in inorganic Janus particles from their dimensions and unique morphology at the nanoscale. As a result, inorganic Janus nanoparticles are highly versatile nanomaterials with great potential in different scientific and technological fields. In this paper, we highlight some advances in the synthesis of inorganic Janus nanoparticles, focusing on the heterogeneous nucleation technique and characteristics of the resulting high quality nanoparticles. The properties emphasized in this review range from the monodispersity and size-tunability and, therefore, precise control over size-dependent features, to the biomedical application as theranostic agents. Hence, we show their optical properties based on plasmonic resonance, the two-photon activity, the magnetic properties, as well as their biocompatibility and interaction with human blood serum.

Covalent grafting of the RGD-peptide onto polyetheretherketone surfaces via Schiff base formation.

In recent years, the synthetic polymer polyetheretherketone (PEEK) has increasingly been used in a number of orthopedic implementations, due to its excellent mechanical properties, bioinertness, and chemical resistance. For in vivo applications, the surface of PEEK, which does not naturally support cell adhesion, has to be modified to improve tissue integration. In the present work we demonstrate a novel wet-chemical modification of PEEK to modify the surface, enabling the covalent grafting of the cell-adhesive RGD-peptide. Modification of the polymer surface was achieved via Schiff base formation using an aliphatic diamine and subsequent crosslinker-mediated immobilization of the peptide. In cell culture experiments with primary osteoblasts it was shown that the RGD-modified PEEK not only significantly promoted cellular adhesion but also strongly enhanced the proliferation of osteoblasts on the modified polymer surface.

Multi-photon imaging of amine-functionalized silica nanoparticles.

A convenient and simple strategy for preparing water soluble, photoluminescent functionalized silica nanoparticles (M-dots) in the absence of fluorophores or metal doping is demonstrated. These M-dots can be used for bioimaging using one and two-photon microscopy. Because of their high photostability, low toxicity and high biocompatibility compared with Lumidot™ CdSe/ZnS quantum dots, functionalized silica particles are superior alternatives for current bioimaging platforms. Moreover, the presence of a free amine group at the surface of the M-dots allows biomolecule conjugation (e.g. with antibodies, proteins) in a single step for converting these photoluminescent SiO(2) nanoparticles into multifunctional efficient vehicles for theragnostics.

Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells.

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

Staining of mitochondria with Cy5-labeled oligonucleotides for long-term microscopy studies.

Labeling of organelles for microscopy is achieved generally by specific dyes that either accumulate in a cellular compartment such as cyanine dyes in mitochondria or are only fluorescent under specific conditions such as the low pH in the lysosome. Here we demonstrate that Cy5--a fluorescent molecule that does not enter cells by itself--can be loaded into cells by attaching a short oligonucleotide. This very inexpensive labeling procedure can be done in the presence of serum. Therefore, very sensitive cell types should also be amenable to this procedure, and longer observations can be achieved compared to other commercially available dyes as the labeling reagent does not need to be washed out. This also points to the pitfall of using fluorescently labeled oligonucleotides for live cell imaging where the oligonucleotide is supposed to detect a specific target sequence in its subcellular distribution.

DNA amplification via polymerase chain reaction inside miniemulsion droplets with subsequent poly(n-butylcyanoacrylate) shell formation and delivery of polymeric capsules into mammalian cells.

There is a growing interest in the development of stable nanocapsules that could deliver the bioactive compounds within the living organism, and to release them without causing any toxic effects. Here the miniemulsion droplets were first used as "nanoreactors" for the amplification of single-molecule dsDNA template (476 and 790 base pairs) through PCR. Afterwards, each droplet was surrounded with a biodegradable PBCA shell by interfacial anionic polymerization, enabling therefore to deliver the PCR products into the cells. The size of the initial miniemulsion droplets and the final polymeric capsules was in the range of 250 and 320 nm, mainly depending on the type of the continuous phase and presence of dsDNA template molecules. The formation of PCR products was resolved with gel electrophoresis and detected with fluorescence spectroscopy in the presence of DNA specific dye (SYBRGreen). TEM studies were performed to prove the formation of the polymeric shell. The shell thickness was measured to be within 5-15 nm and the average molecular weight of the formed PBCA polymer was around 75000 g · mol(-1) . For the cell uptake experiments, the obtained nanocapsules were transferred from the organic phase into aqueous medium containing a water-soluble surfactant. The effect of the surfactant type (anionic, cationic or non-ionic) on the HeLa cell viability and nanocapsule uptake behavior was studied by CLSM and FACS. Confocal analysis demonstrated that nanocapsules stabilized with cationic (CTMA-Cl) and non-ionic (Lutensol AT50) surfactants show almost the same uptake, whereas capsules redispersed in anionic (SDS) surfactant possess a 30% higher uptake. The release of the encapsulated material within the cell was studied on the example of Cy5-labeled oligonucleotides showing the colocalization with mitochondria of MSCs cells.

Annihilation upconversion in cells by embedding the dye system in polymeric nanocapsules.

The first energetically conjoined TTA-assisted photon energy upconversion operating in cell tissue is described. The synthesized nanocapsules with the encapsulated UC dye system consisting of an emitter and a sensitizer show very efficient UC emission in aqueous dispersion under extremely low excitation intensity down to 0.05 W · cm(-2) so that tissue and cells are not affected by the excitation light. The demonstrated sub-linear intensity dependence of the UC emission is of crucial importance for life-science applications as the UC photon could serve as a local or in situ optical excitation source for subsequent light-triggered processes.

Direct and indirect effects of functionalised fluorescence-labelled nanoparticles on human osteoclast formation and activity.

Recently, it was demonstrated that phosphonate-functionalised nanoparticles were successfully taken up by mesenchymal stem cells without influencing their viability and differentiation capacity, suggesting that they may provide a promising basis for the development of nanoparticles for drug delivery or cell labelling. The present study aimed to investigate the effects of these nanoparticles on osteoclast formation and activity as well as on the inflammatory response of osteoclasts and osteoblasts. The intracellular uptake of the particles by human osteoclasts and osteoblasts was demonstrated by confocal laser scanning microscopy, transmission electron microscopy and fluorescence microscopy. The expression of tartrate-resistant acid phosphatase, carboanhydrase II, cathepsin K, calcitonin receptor and osteoclast-specific vacuolar proton pump subunit TCIRG1 as well as actin ring formation were not significantly altered in osteoclasts by particle treatment, as demonstrated by cytochemical staining and immunostaining. Active calcium phosphate resorption by osteoclasts was also not significantly influenced by the particles. The expression and secretion of pro-inflammatory cytokines (IL-6, IL-1ß) by osteoclasts and osteoblasts and the expression of osteoclast-regulating genes (M-CSF, OPG, RANKL) in osteoblasts were similarly not significantly affected. In conclusion, phosphonate-functionalised nanoparticles did not affect osteoclast formation and activity either directly or indirectly, suggesting that they could provide a promising tool for the development of particle-based treatments for anti-resorptive therapies.

The softer and more hydrophobic the better: influence of the side chain of polymethacrylate nanoparticles for cellular uptake.

Intracellular uptake of nanoparticles is highly interesting for labeling of cells, drug delivery, or non-viral gene delivery. In this study we have synthesized a wide variety of poly(alkyl methacrylate) nanoparticles with the same size and investigated their uptake into cells. The nanoparticles were prepared from alkylmethacrylates with different linear and branched ester chains as well as from benzylmethacrylate using the miniemulsion polymerizaiton technique. By adding a fluorescent dye as a marker, the internalization of the nanoparticles could be investigated quantitatively with flow cytometry and qualitatively with confocal laser scanning microscopy. With increasing side chain of the ester and therefore increasing hydrophobicity and at glass transition temperature (T(g)), below the incubation temperature of 37 degrees C the uptake of the nanoparticles into cells is favored.

Effect of functionalised fluorescence-labelled nanoparticles on mesenchymal stem cell differentiation.

The combined use of nanoparticles and mesenchymal stem cells (MSC) in regenerative medicine requires the incorporation of the particles and, at the same time, undisturbed cell viability and maintenance of the multi-lineage potential of MSC. The aim of this study was to investigate the uptake of novel phosphonate-functionalised polystyrene nanoparticles prepared by miniemulsion polymerisation. After exposition of human MSC to the particles, their uptake and localisation were analysed by flow cytometry, confocal laser scanning microscopy (CLSM), and transmission electron microscopy (TEM). The osteogenic, adipogenic and chondrogenic differentiation potential was examined by analysing representative marker genes by RT-PCR. Flow cytometry revealed that after 5 and 16 days more than 98% of the MSC and of the cells, which underwent osteogenic and adipogenic differentiation were positive for particle association. CLSM and TEM demonstrated the successful intracellular incorporation of the particles without using any transfection agents and their presence over the cultivation period. The cell viability was found to be unaffected. Particle treated MSC maintained their potential for osteogenic, adipogenic and chondrogenic differentiation. It was concluded that the surface functionalisation with phosphonate groups provides a promising basis for the development of nanoparticles with high intracellular uptake rates for drug delivery or cell labelling.

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors.

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.