Diana Kozlova

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.

Cell targeting by antibody-functionalized calcium phosphate nanoparticles

Calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization, either with thiol or with amino groups. This permits the covalent attachment of molecules like dyes or antibodies. Between the calcium phosphate surface and the outer silica shell, biomolecules like nucleic acids (DNA or siRNA) can be incorporated as cargo. This leads to cell-specific carriers of biomolecules into cells, e.g. for transfection, gene silencing or cell activation. The cellular uptake of antibody-coated calcium phosphate nanoparticles was demonstrated on two cell lines: HeLa (epithelial cell line) and MG-63 (osteoblast-like cell line). Furthermore, the functionalization of calcium phosphate nanoparticles with a dendritic cell-specific antibody (CD11c) led to a cell-specific targeting as shown with primary murine splenocytes. Thus, the successful coating of calcium phosphate nanoparticles with cell-specific antibodies makes them suitable for many clinical applications.

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.

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.

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.

Chlorhexidine-loaded calcium phosphate nanoparticles for dental maintenance treatment: combination of mineralising and antibacterial effects

One of the main problems in dental medicine is the growth of bacterial biofilms on tooth surfaces which cause caries and periodontitis. We have developed a new system for oral hygiene and dental treatment that consists of either a paste or a rinsing solution containing calcium phosphate nanoparticles, functionalized with the antibacterial agent chlorhexidine. As calcium phosphate is the natural component of tooth mineral, it can lead to the remineralization of damaged enamel, while chlorhexidine prevents the colonization of the tooth surface by bacteria. In the form of a paste, a bifunctional system with both mineralizing and antibacterial properties is obtained. The nanoparticles may also stick to open dentin tubules at the root surface due to their coating with carboxymethyl cellulose. In vitro studies on teeth show that the paste sticks well to the root surface and closes dentin tubules.

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-1β (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.

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.

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.

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.