Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands.

Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo.

miR-542-3p exerts tumor suppressive functions in neuroblastoma by downregulating Survivin.

MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma.

Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy.

Photodynamic therapy (PDT) of tumors causes skin photosensitivity as a result of unspecific accumulation behavior of the photosensitizers. PDT of tumors was improved by calcium phosphate nanoparticles conjugated with (i) Temoporfin as a photosensitizer, (ii) the RGDfK peptide for favored tumor targeting and (iii) the fluorescent dye molecule DY682-NHS for enabling near-infrared fluorescence (NIRF) optical imaging in vivo. The nanoparticles were characterized with regard to size, spectroscopic properties and uptake into CAL-27 cells. The nanoparticles had a hydrodynamic diameter of approximately 200 nm and a zeta potential of around +22mV. Their biodistribution at 24h after injection was investigated via NIRF optical imaging. After treating tumor-bearing CAL-27 mice with nanoparticle-PDT, the therapeutic efficacy was assessed by a fluorescent DY-734-annexin V probe at 2 days and 2 weeks after treatment to detect apoptosis. Additionally, the contrast agent IRDye® 800CW RGD was used to assess tumor vascularization (up to 4 weeks after PDT). After nanoparticle-PDT in mice, apoptosis in the tumor was detected after 2 days. Decreases in tumor vascularization and tumor volume were detected in the next few days. Calcium phosphate nanoparticles can be used as multifunctional tools for NIRF optical imaging, PDT and tumor targeting as they exhibited a high therapeutic efficacy, being capable of inducing apoptosis and destroying tumor vascularization.

A pH-sensitive poly(methyl methacrylate) copolymer for efficient drug and gene delivery across the cell membrane.

A versatile drug delivery system based on calcium phosphate/Eudragit®-E100 nanoparticles with a diameter below 200 nm was realized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. Hydrophilic drugs (siRNA for gene silencing and bovine serum albumin as model protein) and hydrophobic drugs (5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine, THPP, a photosensitizer for photodynamic therapy) were encapsulated as model drugs. Eudragit®-E100 is a poly(methyl methacrylate) copolymer with a low solubility at neutral pH and a high solubility at low pH. Thus, the particles are stable in cell culture media and rapidly dissolved in the lysosome after cellular uptake and delivery of their cargo into the cell. The particles had a positive charge (zeta potential +49 mV) and were taken up very well by epithelial cells (HeLa) as shown by fluorescence microscopy and confocal laser scanning microscopy. Gene-silencing experiments on HeLa-eGFP cells gave knockdown efficiencies of 39% with no toxic effects. The particles can be freeze-dried without cryoprotectant and easily redispersed in water, thus making their transport and storage convenient.

Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen.

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity.

An easy synthesis of autofluorescent alloyed silver-gold nanoparticles.

A one-pot synthesis of fluorescent bimetallic silver-gold nanoparticles in aqueous medium is presented. Carboxylic acid-functionalized nanoparticles were prepared with different metal compositions from 90 : 10 to 10 : 90 (n : n) for silver : gold with a diameter of 1.8 ± 0.4 nm. Pure silver and gold nanoparticles were prepared for comparison. Spectroscopic analyses showed that the ligand, i.e. 11-mercaptoundecanoic acid, binds to the particle surface by the thiol group, leaving the carboxylic acid accessible for further functionalization, e.g. by suitable coupling reactions. Nanoparticles with a silver content up to 60 : 40 showed autofluorescence with a large Stokes shift of about 250-300 nm (maximum wavelength of the emission between 608 nm and 645 nm). The intracellular localization of bimetallic silver-gold nanoparticles was studied in HeLa cells by confocal laser scanning microscopy (CLSM). The alloyed silver-gold nanoparticles showed no significant cytotoxicity at a metal concentration of 5 µg mL-1 for 24 h, but were cytotoxic to some degree at 50 µg mL-1 at higher silver content.

Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells.

Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥ 1 µM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms.

Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery.

Calcium phosphate/poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 µg siRNA, 5 µg DNA and 280 µg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin.

For subunit vaccines, adjuvants play a key role in shaping the magnitude, persistence and form of targeted antigen-specific immune response. Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application. Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA, RNA, peptides and proteins. Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system, i.e. the cytokine production, was studied. They induced the production of the proinflammatory cytokines IL-8 (Caco-2 cells) and IL-1ß (bone marrow-derived macrophages; BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice. The immunostimulation was more pronounced than with free flagellin.

Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells.

The uptake of calcium phosphate nanoparticles (diameter 120nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3h of incubation.