Particle-based optical sensing of intracellular ions at the example of calcium - what are the experimental pitfalls?

Colloidal particles with fluorescence read-out are commonly used as sensors for the quantitative determination of ions. Calcium, for example, is a biologically highly relevant ion in signaling, and thus knowledge of its spatio-temporal distribution inside cells would offer important experimental data. However, the use of particle-based intracellular sensors for ion detection is not straightforward. Important associated problems involve delivery and intracellular location of particle-based fluorophores, crosstalk of the fluorescence read-out with pH, and spectral overlap of the emission spectra of different fluorophores. These potential problems are outlined and discussed here with selected experimental examples. Potential solutions are discussed and form a guideline for particle-based intracellular imaging of ions.

pH-sensitive capsules as intracellular optical reporters for monitoring lysosomal pH changes upon stimulation.

The concept of a long-term sensor for ion changes in the lysosome is presented. The sensor is made by layer-by-layer assembly of oppositely charged polyelectrolytes around ion-sensitive fluorophores, in this case for protons. The sensor is spontaneously incorporated by cells and resides over days in the lysosome. Intracellular changes of the concentration of protons upon cellular stimulation with pH-active agents are monitored by read-out of the sensor fluorescence at real time. With help of this sensor concept it is demonstrated that the different agents used (Monensin, Chloroquine, Bafilomycin A1, Amiloride) possessed different kinetics and mechanisms of action in affecting the intracellular pH values.

Common Aspects Influencing the Translocation of SERS to Biomedicine.

This review overviews the impact in biomedicine of surface enhanced. Raman scattering motivated by the great potential we believe this technique has. We present the advantages and limitations of this technique relevant to bioanalysis in vitro and in vivo and how this technique goes beyond the state of the art of traditional analytical, labelling and healthcare diagnostic technologies.

Effects of Graphene Quantum Dots on the Self-Renewal and Differentiation of Mesenchymal Stem Cells.

The influence of graphene quantum dots (GQDs) on key characteristics of bone marrow derived mesenchymal stem cells (MSCs) phenotype (i.e., self-renewal, differentiation potential, and pluripotency) is systematically investigated in this work. First, the viability and impact of GQDs on the self-renewal potential of MSCs is evaluated in order to determine a threshold for the exposing dose. Second, GQDs uptake by MSCs is confirmed due to the excellent fluorescent properties of the particles. They exhibit a homogenous cytoplasmatic distribution that increases with the time and concentration. Third, the impact of GQDs on the osteogenic differentiation of MSCs is deeply characterized. An enhanced activity of alkaline phosphatase promoted by GQDs indicates early activation of osteogenesis. This is also confirmed upon GQD-induced up-regulation of phenotypically related osteogenic genes (Runx2, osteopontin, and osteocalcin) and specific biomarkers expression (osteopontin and osteocalcin). GQDs also effectively enhance the formation of calcium-rich deposits characteristics of osteoblasts. Furthermore, genes microarray results indicate that the enhanced osteogenic differentiation of MSCs by GQDs is in progress through a bone morphogenetic protein and transforming growth factor-ß relative signaling pathways. Finally, intracytoplasmatic lipid detection shows that GQDs can also promote the adipogenic differentiation of MSCs, thus confirming the prevalence of their pluripotency potential.

α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

Interaction of stable colloidal nanoparticles with cellular membranes.

Due to their ultra-small size, inorganic nanoparticles (NPs) have distinct properties compared to the bulk form. The unique characteristics of NPs are broadly exploited in biomedical sciences in order to develop various methods of targeted drug delivery, novel biosensors and new therapeutic pathways. However, relatively little is known in the negotiation of NPs with complex biological environments. Cell membranes (CMs) in eukaryotes have dynamic structures, which is a key property for cellular responses to NPs. In this review, we discuss the current knowledge of various interactions between advanced types of NPs and CMs.

Protein-mediated synthesis, pH-induced reversible agglomeration, toxicity and cellular interaction of silver nanoparticles.

Casein, a milk protein, is used to produce biotolerable and highly stable silver nanoparticles with a fair control over their size without using any additional reducing agent. These silver nanoparticles undergo reversible agglomeration to form protein-silver nanoparticle composite agglomerates as pH approaches to the isoelectric point of casein protein (pI=4.6). These agglomerates can then easily be re-dispersed in alkaline aqueous media with no obvious change in their optical properties. The nanoparticles can withstand high salt concentration (~0.5M), and can also be freeze-dried, stored as dry powder and then dispersed in aqueous media whenever required. More interestingly, by controlling the concentration of casein protein and pH, it was also possible to control the self-assembly of silver nanoparticles to produce fairly uniform spherical agglomerates. The nanoparticles and their agglomerates were thoroughly characterized using UV-visible and FTIR spectroscopy, TEM, SEM and DLS, etc. Cytotoxicity of the hybrid materials was examined using a Resazurin based cytotoxicity assay. After determining the LD(50) using NIH/3T3 fibroblast cells, the cellular interaction of these hybrid nanoparticles was studied to examine the behavior of casein-coated nanoparticles for their potential bio-applications.

Antimicrobial hydantoin-containing polyesters.

A new N-hydantoin-containing biocompatible and enzymatically degradable polyester with antibacterial properties is presented. Different polyesters of dimethyl succinate, 1,4-butanediol, and 3-[N,N-di(ß-hydroxyethyl)aminoethyl]-5,5-dimethylhydantoin in varying molar ratios are prepared via two-step melt polycondensation. The antibacterially active N-halamine form is obtained by subsequent chlorination of the polyesters with sodium hypochlorite. Chemical structures, thermal properties, and spherulitic morphologies of the copolymers are studied adopting FT-IR, NMR, TGA, DSC, WAXD, and POM. The polyesters exhibit antibacterial activity against Escherichia coli. The adopted synthetic approach can be transferred to other polyesters in a straightforward manner.

Sphingosine 1-phosphate mediates chemotaxis of human primary fibroblasts via the S1P-receptor subtypes S1P1 and S1P3 and Smad-signalling.

The sphingolipid sphingosine 1-phosphate (S1P) induces chemotaxis of primary fibroblasts. Thus, S1P exhibited a chemotactic effect in a concentration-dependent manner from 10⁻6 to 10⁻9 M; higher concentrations resulted in a loss of migration, and lower amounts were ineffective to evoke movement toward a concentration gradient of S1P. In congruence with the migratory response, S1P caused an extension of lamellipodia at the cell periphery of human fibroblasts and a rearrangement of the cytoskeleton. These effects were visible by phalloidin staining of actin filaments as well as focal adhesion turnover. As the molecular mechanism of S1P-mediated migration of fibroblasts has not been well characterized, we investigated whether S1P-receptors are involved in the chemotactic response. Indeed, inhibition of G(i) signalling markedly reduced motility towards S1P, suggesting an involvement of S1P-receptor subtypes. Moreover, downregulation of S1P1 and S1P3 indicated that these S1P-receptor subtypes are responsible for the chemotactic action of the bioactive sphingolipid. After having identified a crosstalk between Smad-proteins and S1P-signalling, we investigated whether Smad-activation is involved in the chemotactic response induced by S1P. Indeed S1P caused a Smad-activation via the S1P receptor subtypes S1P1 and S1P3. Moreover, downregulation of Smad3 diminished the ability of S1P to mediate a chemotactic response in fibroblasts, indicating a crosstalk between TGF-ß- and S1P-signalling.

The effect of PEG-coated gold nanoparticles on the anti-proliferative potential of Specific Nutrient Synergy.

The role of PEG-coated gold nanoparticles (Au NPs) on the anti-proliferative effect of Specific Nutrient Synergy (SNS) on HTLV-1 infected (C91-PL and HuT-102) and non-infected (CEM and Jurkat) malignant T-lymphocytes cells, was investigated. When PEG-coated Au NPs (of different molecular weights) were added alone, there was no effect on either viability or proliferation of the leukemic cell lines studied. Treatment of cells with SNS and PEG (5 or 10 kDa) coated Au NP reduced significantly the proliferation in all cell lines tested; this reached more than 50% reduction as compared to the control for cells treated for 96 h. Data showed that the best anti-proliferative effect was obtained using SNS and Au NP coated with PEG of molecular weights of 5 and 10 kDa with almost no effect of PEG of lower molecular weights (0.75 and 2 kDa) or higher ones (20 kDa). This was true as well for HTLV-1 infected as for non-infected malignant T-lymphocytes. Electron microscopy results showed uptake of the gold particles to Jurkat cells. All described effects are specific to leukemia cell lines, and no effects were observed with freshly activated human mononuclear lymphocytes as control.

One example on how colloidal nano- and microparticles could contribute to medicine.

Nanomedicine, nowadays, is a popular keyword in the media, although everyone seems to associate it with different visions, hopes and even fears. This article gives a perspective from two sides. From the point of view of a materials scientist, it will be pointed out what new materials will be possible, how they will be designed and which properties they could offer for diagnosis and treatment. From the point of view of a medical doctor, it will be pointed out which properties are actually desired and what materials are hoped for practical applications. The two different points of view indicate that, although sophisticated materials with advanced novel properties will be available in the future, they do not automatically match the requirements and demands of clinicians. The discussion is centerd around one example, multifunctional polyelectrolyte capsules, which might act as a 'nanosubmarine' for in vivo sensing and delivery, which is used to highlight promising interfaces between both disciplines.

Composite nanoparticles take aim at cancer.

Nanoparticles have properties that are useful for the diagnosis and treatment of cancer, including their size-dependent properties, stability in solvent, ideal size for delivery within the body, and tunable surface chemistry for targeted delivery. Several different nanoparticle building blocks possessing varied functionality can be assembled into one multifunctional composite nanoparticle, further expanding their potential use in cancer diagnostics and therapeutics. Here, we present several examples of the types of functional composite nanoparticles that have been studied, in addition to highlighted applications of their uses.