Granulocyte Colony-Stimulating Factor Nanocarriers for Stimulation of the Immune System (Part I): Synthesis and Biodistribution Studies.

In the field of cancer immunotherapy, an original approach consists of using granulocyte colony-stimulating factor (G-CSF) to target and activate neutrophils, cells of the innate immune system. G-CSF is a leukocyte stimulating molecule which is commonly used in cancer patients to prevent or reduce neutropenia. We focused herein on developing a G-CSF nanocarrier which could increase the in vivo circulation time of this cytokine, keeping it active for targeting the spleen, an important reservoir of neutrophils. G-CSF-functionalized silica and gold nanoparticles were developed. Silica nanoparticles of 50 nm diameter were functionalized by a solid phase synthesis approach. The technology enabled us to incorporate multiple functionalities on the surface such as a PEG as hydrophilic polymer, DTPA as 111In chelating agent and G-CSF. The gold nanocarrier consisted of nanoparticles of 2-3 nm diameter elaborated with DTPA groups on the surface and functionalized with G-CSF. We studied the particle biodistribution in mice with special attention to organs involved in the immune system. The two nanocarriers with similar functionalization of surface showed different pathways in mice, probably due to their difference in size. Considering the biodistribution after G-CSF functionalization, we confirmed that the protein was capable of modifying the pharmacokinetics by increasing the nanocarrier concentration in the spleen, a reservoir of G-CSF receptor expressing cells.

Granulocyte-Colony Stimulating Factor Nanocarriers for Stimulation of the Immune System (Part II): Dose-Dependent Biodistribution and In Vivo Antitumor Efficacy in Combination with Rituximab.

The purpose of immuno-modulation is to increase or restore the action of immunocompetent cells against tumors with or without the use of monoclonal antibodies. The innate immune system is a key player in various pathological situations, but cells of this system appear to be inhibited or insufficiently active in malignancy or severe infectious diseases. The present study was designed to investigate therapeutic value of nanoparticles (NPs) coupled with bioactive hematopoietic growth factors acting on the innate immune system. The use of nanoparticles (NPs) allowing multimodal detection and multifunctional grafting are currently of great interest for theranostic purposes. In the present work, we have evaluated the impact of the number of granulocyte-colony stimulating factor (G-CSF) grafted on the surface on the NPs on the biodistribution in mice thanks to indium 111 radiolabeling. Furthermore, we have investigated whether grafted G-CSF NPs could stimulate the immune innate system and enhance the therapeutic efficacy of the monoclonal antibody rituximab in mice bearing human lymphoma xenografts. Following intravenous (i.v.) administration of NP-DTPA and NP-DTPA/G-CSF-X high levels of radioactivity were observed in the liver. Furthermore, spleen uptake was correlated with the number of G-CSF molecules grafted on the surface of the NPs. Combining NP-DTPA/G-CSF-34 with rituximab strongly reduced RL tumor growth compared to rituximab alone or in combination with conventional G-CSF + rituximab. The use of highly loaded G-CSF NPs as immune adjuvants could enhance the antitumor activity of therapeutic monoclonal antibodies by amplifying tumor cell destruction by innate immune cells.

Methylene blue phosphoramidite for DNA labelling.

We report the first synthesis of a methylene blue (MB) phosphoramidite derivative suitable for DNA solid-phase synthesis. The electrochemical and optical properties of the resulting MB modified oligonucleotides were confirmed. This new molecule is an important breakthrough in the design of new probes labelled with MB.

Label-free electrochemical monitoring of protein addressing through electroactivated "click" chemistry on gold electrodes.

In this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at -300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/mL.

Impact of immobilization support on colorimetric microarrays performances.

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.