The detection and localization of polymer-based nanoparticles in human bone marrow-derived stromal cells (hBMSC) by time-of-flight secondary ion mass spectrometry (ToF-SIMS) is reported as an example for the mass spectrometry imaging of organic nanoparticles in cell environments. Polyelectrolyte complex (PEC) nanoparticles (NP) made of polyethylenimine (PEI) and cellulose sulfate (CS), which were developed as potential drug carrier and coatings for implant materials, were chosen for the imaging experiments. To investigate whether the PEI/CS-NP were taken up by the hBMSC ToF-SIMS measurements on cross sections of the cells and depth profiling of whole, single cells were carried out. Since the mass spectra of the PEI/CS nanoparticles are close to the mass spectra of the cells principal component analysis (PCA) was performed to get specific masses of the PEI/CS-NP. Mass fragments originating from the NP compounds especially from cellulose sulfate could be used to unequivocally detect and image the PEI/CS-NP inside the hBMSC. The findings were confirmed by light and transmission electron microscopy. Graphical Abstract During ToF-SIMS analysis Bi3 (+) primary ions hit the sample surface and so called secondary ions (SI) are emitted and detected in the mass analyser. Exemplary mass images of cross sections of human mesenchymal stromal cells (red; m/z = 86.1 u) cultured with organic nanoparticles (green; m/z = 143.0 u) were obtained.

Herein, the authors report and review polyelectrolyte complex (PEC) nanoparticles (NPs) loaded with zoledronate (ZOL) and simvastatin and their effects on bone cells. PEC NPs are intended for modification of bone substitute materials. For characterization, they can be solution casted on germanium (Ge) substrates serving as analytically accessible model substrate. PEC NPs were generated by mixing poly(ethyleneimine) (PEI) either with linear cellulose sulfate (CS) or with branched dextransulfate (DS). Four important requirements for drug loaded PEC NPs and their films are addressed herein, which are the colloidal stability of PEC dispersions (1), interfacial stability (2), cytocompatibility (3), and retarded drug release (4). Dynamic light scattering measurements (DLS) showed that both PEI/CS and PEI/DS PEC NP were obtained with hydrodynamic radii in the range of 35-170 nm and were colloidally stable up to several months. Transmission FTIR spectroscopy evidenced that films of both systems were stable in contact to the release medium up to several days. ZOL-loaded PEI/CS nanoparticles, which were immobilized on an osteoblast-derived extracellular matrix, reduced significantly the resorption and the metabolic activity of human monocyte-derived osteoclasts. FTIR spectroscopy at cast PEC/drug films at Ge substrates revealed retarded drug releases in comparison to the pure drug films.

Drug loaded PEC NP consist of oppositely charged biorelated Polyelectrolytes (PEL) like ionic polysaccharides or ionic polypeptides and also synthetic PEL, which are mixed and processed in aqueous media. At first, freshly prepared drug/PEC NP exhibit time dependent colloidal stability in the range of weeks and months, which enables and simplifies storage, transport and application in the medical field. Secondly, after deposition and drying of drug/PEC NP a local wet adhesive PEC matrix at the BSM remains in contact to relevant aqueous media (e.g. buffer, cell culture medium), which minimizes asepsis, systemic toxicity, immune or inflammatory reaction. Thirdly, cell compatible PEC NP coatings were identified, which showed only minimal effects on various relevant bone related cells due to biorelateness, complexation, local confinement and low surface area. Fourthly, PEC NP elute drugs for bone healing like bisphosphonates, antibiotics and growth factors (e.g. bone morphogenetic proteins) in delayed and sustained manner. Moreover, the onset of elution could be triggered by thermoresponsive PEL via temperature increase giving clinicians a tool into hand allowing spatiotemporal drug release on demand. Finally, drug/PEC NP could be integrated into commercial or still developed allotropic stabilizing or defect filling BSM systems.

Short TitlePolyelectrolyte nanoparticles and vascular endothelial cells.AbstractAngiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.