Hybrid inorganic-organic fluorescent silica nanoparticles—influence of dye binding modes on dye leaching

Silica nanoparticles with embedded fluorescent dyes represent an important class of markers for example in biological imaging. We systematically studied the various incorporation mechanisms of fluorescent xanthene dyes in 30–40 nm silica nanoparticles. An important parameter was the interaction of the dye with the matrix material, either by weak electrostatic or strong covalent interactions, which also has implications on the stability of fluorescence and brightness of the dyes. Factors that can influence leaching of dyes such as the position of the dyes in particles and the intensity of the particle-dye interaction were investigated by using the solvatochromic effect of xanthene dyes and by stationary fluorescence anisotropy measurements. We compared uranine and rhodamine B, which were physically embedded, with modified fluorescein isothiocyanate and rhodamine B isothiocyanate, which were covalently bound to the silica matrix within a usual Stöber synthesis. Systematic leaching studies of time spans up to 4 days revealed that covalent bonding of dyes like fluorescein isothiocyanate or rhodamine B isothiocyanate is necessary for fluorescence stability, since dyes bound by physical interaction tend to leach out of porous silica networks. Coverage of silica particles with hydrophobic protection layers of alkyltrialkoxysilanes or hydrophilic polyethylene glycol (PEG) groups resulted in a better retention of physisorbed dyes and provides the possibility to adapt the particles to the polarity of the medium. Best results were archived with PEG groups, but even small trimethylsilyl (TMS) groups already reduce leaching.

Fluorescent Polyelectrolyte System to Track Anthocyanins Delivery inside Melanoma Cells

Over the past decades, there has been a growing interest in using natural molecules with therapeutic potential for biomedical applications. In this context, our aim is focused on anthocyanins (AN) as molecules with anticancer properties that could be used in melanoma local therapies. Due to their susceptibility to environmental changes, current study is based on the design and development of a fluorescent system for carrying and trafficking AN inside melanoma cells. The architectural structure of the proposed system CaCO3(PAH)@RBITC@AN reflects a spherical shape, 1080 nm diameter and a solid groundwork CaCO3(PAH), on which rhodamine B isothiocyanate (RBITC) fluorophore was firstly added; then, poly(acrylic acid) (PAA) polyelectrolytes and poly(allylamine hydrochloride) (PAH) were successfully deposited. Purified AN from chokeberries were entrapped between PAA layers (rate of 94.6%). In vitro tests confirmed that CaCO3(PAH)@RBITC@AN does not affect the proliferation of melanoma B16-F10 cells and proved that their internalization and trafficking can be followed after 24 h of treatment. Data presented here could contribute not only to the existing knowledge about the encapsulation technology of AN but also might bring relevant information for a novel formula to deliver therapeutic molecules or other bio-imaging agents directly into melanoma cells, a strategy that could positively improve tumor therapies.

Silver Nanoparticles Functionalized by Fluorescein Isothiocyanate or Rhodamine B Isothiocyanate: Fluorescent and Plasmonic Materials

This paper presents the synthesis of silver nanoparticles (AgNPs) functionalized with fluorescent molecules, in particular with xanthene-based dyes, i.e., fluorescein isothiocyanate (FITC, λmax = 485 nm) and rhodamine B isothiocyanate (RITC, λmax = 555 nm). An in-depth characterization of the particle–dye systems, i.e., AgNPs–RITC and AgNPs–FITC, is presented to evaluate their chemical structure and optical properties due to the interaction between their plasmonic and absorption properties. UV–Vis spectroscopy and the dynamic light scattering (DLS) measurements confirmed the nanosize of the AgNPs–RITC and AgNPs–FITC. Synchrotron radiation X-ray photoelectron spectroscopy (SR-XPS) was used to study the chemical surface functionalization by structural characterization, confirming/examining the isothiocyanate–metal interaction. For AgNPs–RITC, in which the plasmonic and fluorescence peak are not superimposed, the transient dynamics of the dye fluorescence were also studied. Transient absorption measurements showed that by exciting the AgNPs–RITC sample at a wavelength corresponding to the AgNP plasmon resonance, it was possible to preferentially excite the RITC dye molecules attached to the surface of the NPs with respect to the free dye molecules in the solution. These results demonstrate how, by combining plasmonics and fluorescence, these AgNPs can be used as promising systems in biosensing and imaging applications.

Effect of silica-coated magnetic nanoparticles on rigidity sensing of human embryonic kidney cells

Background Nanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell’s rigidity sensing, but how these NPs affect rigidity sensing is unknown. Results Here, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC). Conclusions Our results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Effect of silica-coated magnetic nanoparticles on rigidity sensing of human embryonic kidney cells

BackgroundNanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell’s rigidity sensing, but how these NPs affect rigidity sensing is unknown.ResultsHere, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC).ConclusionsOur results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Effect of Silica-Coated Magnetic Nanoparticles on Rigidity Sensing of Human Embryonic Kidney Cells

Background: Nanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell’s rigidity sensing, but how these NPs affect rigidity sensing is unknown.Results: Here, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffness k) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC).Conclusions: Our results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Real-time detection of single-molecule reaction by plasmon-enhanced spectroscopy

Determining structural transformations of single molecules (SMs) is an important fundamental scientific endeavor. Optical spectroscopies are the dominant tools used to unravel the physical and chemical features of individual molecules and have substantially contributed to surface science and biotechnology. In particular, Raman spectroscopy can identify reaction intermediates and reveal underlying reaction mechanisms; however, SM Raman experiments are subject to intrinsically weak signal intensities and considerable signal attenuation within the spectral dispersion systems of the spectrometer. Here, to monitor the structural transformation of an SM on the millisecond time scale, a plasmonic nanocavity substrate has been used to enable Raman vibrational and fluorescence spectral signals to be simultaneously collected and correlated, which thus allows a detection of photo-induced bond cleavage between the xanthene and phenyl group of a single rhodamine B isothiocyanate molecule in real time. This technique provides a novel method for investigating light-matter interactions and chemical reactions at the SM level.

Effect of magnesium ions/Type I collagen promote the biological behavior of osteoblasts and its mechanism

Type I collagen (Col I) is a main component of extracellular matrix (ECM). Its safety, biocompatibility, hydrophilicity and pyrogen immunogenicity make it suitable for tissues engineering applications. Mg2+ also control a myriad of cellular processes, including the bone development by enhancing the attachment and differentiation of osteoblasts and accelerating mineralization to enhance bone healing. In our studies, Mg2+ bind collagen to promote the proliferation and differentiation of osteoblasts through the expression of integrins and downstream signaling pathways. In order to clarify the biological behavior effect of 10 mM Mg2+/Col I coating, we performed 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP), 4′6-diamidino-2-phenylindole (DAPI), Alizarin red staining and Rhodamine B-isothiocyanate (RITC)-labeled phalloidin experiments and found that 10 mM Mg2+ group, Col I-coating group, 10 mM Mg2+/Col I-coating group, respectively, promoted the proliferation and differentiation of osteoblasts, especially 10 mM Mg2+/Col I-coating group. We detected the mRNA expression of osteogenic-related genes (Runx2, ALP and OCN, OPN and BMP-2) and the protein expression of signaling pathway (integrin α2, integrin β1, FAK and ERK1/2), these results indicated that 10 mM Mg2+/Col I coating play an critical role in up-regulating the MC3T3-E1 cells activity. The potential mechanisms of this specific performance may be through activating via integrin α2β1-FAK-ERK1/2 protein-coupled receptor pathway.

Insights on the relationship between structure vs. toxicological activity of antibacterial rhodamine-labelled 3-hydroxy-4-pyridinone iron(III) chelators in HepG2 cells

In the present study we investigated the in vitro hepatotoxicity of a set of rhodamine-labelled 3-hydroxy-4-pyridinones (3,4-HPO) that had previously demonstrated significant inhibitory effect in the intramacrophagic growth of Mycobacterium avium. Our aim was to establish a correspondence between the molecular structure and the in vitro toxicological activity of these compounds. The impact of a set of bidentate (MRB2, MRB7, MRB8, and MRB9) and hexadentate (MRH7, MRH8, and MRH10) chelators on cellular metabolic competence and membrane integrity was investigated in HepG2 cells. Our findings indicate that: a) hexadentate chelators are more cytotoxic than parent bidentate ligands; b) disruption of cell membrane and metabolic competence only occurred after 5 days, at the highest concentrations tested; c) strict correlation between bacteriostatic activity and in vitro toxicity was observed, which seems to be directly dependent on the size of the molecule and on the hydrophilic/lipophilic balance; d) among the set of bidentate ligands, carboxyrhodamine derivatives (amide linker) presented lower detrimental effects, when compared with rhodamine B isothiocyanate chelators (thiourea linker); e) contrarily, for the hexadentate series, rhodamine B isothiocyanate derivatives are less cytotoxic to HepG2 cells than carboxyrhodamine molecules; and f) for all compounds tested, when the substituents of the nitrogen atom were switched from ethyl to methyl, an increment of toxicity was observed. Overall, all chelators seem to display suitable in vitro toxicological potential to combat fast grow bacteria. According to their in vitro pharmacological: toxicological potential ratio, MRH7 and MRH8 may be considered as the most suitable compounds to undergo further pre-clinical development studies.