Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells.

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.

Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells.

INTRODUCTION: Since most biologically active macromolecules are natural nanostructures, operating in the same scale of biomolecules gives the great advantage to enhance the interaction with cellular components. Noteworthy efforts in nanotechnology, particularly in biomedical and pharmaceutical fields, have propelled a high number of studies on the biological effects of nanomaterials. Moreover, the determination of specific physicochemical properties of nanomaterials is crucial for the evaluation and design of novel safe and efficient therapeutics and diagnostic tools. In this in vitro study, we report a physicochemical characterisation of fluorescent silica nanoparticles (NPs), interacting with biological models (U937 and PBMC cells), describing the specific triggered biologic response. METHODS: Flow Cytometric and Confocal analyses are the main method platforms. However TEM, NTA, DLS, and chemical procedures to synthesize NPs were employed. RESULTS: NTB700 NPs, employed in this study, are fluorescent core-shell silica nanoparticles, synthesized through a micelle-assisted method, where the fluorescence energy transfer process, known as FRET, occurs at a high efficiency rate. Using flow cytometry and confocal microscopy, we observed that NTB700 NP uptake seemed to be a rapid, concentration-, energy- and cell type-dependent process, which did not induce significant cytotoxic effects. We did not observe a preferred route of internalization, although their size and the possible aggregated state could influence their extrusion. At this level of analysis, our investigation focuses on lysosome and mitochondria pathways, highlighting that both are involved in NP co-localization. Despite the main mitochondria localization, NPs did not induce a significant increase of intracellular ROS, known inductors of apoptosis, during the time course of analyses. Finally, both lymphoid and myeloid cells are able to release NPs, essential to their biosafety. DISCUSSION: These data allow to consider NTB700 NPs a promising platform for future development of a multifunctional system, by combining imaging and localized therapeutic applications in a unique tool.

Trehalose preserves the integrity of lyophilized phycoerythrin-antihuman CD8 antibody conjugates and enhances their thermal stability in flow cytometric assays.

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption-emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8-RPE (CD8-RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose-CD8-RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8(+) lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the "cold chain" distribution, particularly suited for use in less-developed countries of the tropical areas.

Testing oligothiophene fluorophores under physiological conditions. Preparation and optical characterization of the conjugates of bovine serum albumin with oligothiophene N-hydroxysuccinimidyl esters.

Bovine serum albumin (BSA) was reacted with linear and newly synthesized branched oligothiophene N-hydroxysuccinimidyl ester fluorophores (TSEs) in moderately basic carbonate buffer solution. Optically stable BSA-TSE conjugates were obtained with a degree of labeling depending on experimental conditions. Conjugates with high fluorophore to BSA ratios (F/BSA = 8) displayed fluorescence quantum yields in the range of 10-30% in water at pH = 7.2, comparable to the quantum yield (25%) of the BSA-FITC conjugate prepared under the same conditions and with the same degree of labeling.

Bright oligothiophene N-succinimidyl esters for efficient fluorescent labeling of proteins and oligonucleotides.

The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported.

Rigid-core fluorescent oligothiophene-S,S-dioxide isothiocyanates. Synthesis, optical characterization, and conjugation to monoclonal antibodies.

In this paper we report the synthesis of a new class of fluorescent thiophene-based isothiocyanates containing a 3,5-disubstituted dithieno[3,2-b:2',3'-d]thiophene-4,4-dioxide moiety as the rigid core, using the palladium-catalyzed cross-coupling reaction of aryl stannanes with aryl bromides (Stille coupling). By changing the molecular structure through the progressive addition of thienylene or phenylene units, light emission from blue to orange was obtained. Photoluminescence quantum yields ranged from 0.65 to 0.90 for blue and green light emitters to 0.10-0.35 for yellow and orange ones. Optically and chemically stable fluorescent bioconjugates were prepared by spontaneous reaction of the isothiocyanates with monoclonal antibodies anti-CD3 and anti-CD8 in slightly basic solutions.