Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.

Mesoporous silica nanoparticles (MSNs) are considered as one of the most promising nanovectors for controlled drug delivery. For the design of ideal drug nanocarriers, several factors have to be taken into account, such as size and surface chemistry. Here, we report how MSNs surface functionalization and particle size critically affect the drug release performances and therapeutic capabilities. We illustrate the size effect of these functionalized MSNs on in vitro, intracellular, and in vivo drug release efficiency, as well as on nanoparticle and drug diffusion into the targeted tissues (tumor). For this, dispersible MSNs with different particle sizes (from 500 down to 45 nm), similar physicochemical properties (e.g., structural and textural properties), and high colloidal stability (even in saline conditions), were synthesized. Their surface was specifically functionalized with a phosphonate-silane according to a novel postgrafting strategy, for better control over loading and release of positively charged drugs. An efficient particle-size-dependent and pH-dependent release of the loaded drug (i.e., doxorubicin) was achieved in physiological conditions with phosphonated-MSNs compared to pure-MSNs. The cellular uptake efficiency is much higher with the smallest phosphonated-nanoparticles (45 nm). Furthermore, doxorubicin is efficiently released from the nanoparticles into the intracellular compartments, and the drug reaches the nucleus in a time- and particle size-dependent manner. Intratumoral diffusion of the developed nanoparticles, as well as the drug release and its diffusion into the tumor matrix, is clearly enhanced with the smallest phosphonated-nanoparticles (45 nm), leading ultimately to a superior cell and tumor growth inhibition.

The majority of contrast agents used in magnetic resonance imaging (MRI) is based on the rare-earth element gadolinium. Gadolinium-based nanoparticles could find promising applications in pre-clinical diagnostic procedures of certain types of cancer, such as glioblastoma multiforme. This is one of the most malignant, lethal and poorly accessible forms of cancer. Recent advances in colloidal nanocrystal synthesis have led to the development of ultra-small crystals of gadolinium oxide (US-Gd(2)O(3), 2-3 nm diameter). As of today, this is the smallest and the densest of all Gd-containing nanoparticles. Cancer cells labeled with a sufficient quantity of this compound appear bright in T(1)-weighted MRI images. Here we demonstrate that US-Gd(2)O(3) can be used to label GL-261 glioblastoma multiforme cells, followed by localization and visualization in vivo using MRI. Very high amounts of Gd are efficiently internalized and retained in cells, as confirmed with TEM and ICP-MS. Labeled cells were visualized in vivo at 1.5 T using the chicken embryo model. This is one more step toward the development of "positively contrasted" cell tracking procedures with MRI.

Stress granules (SG) are cytoplasmic multimeric RNA bodies that form under stress conditions known to inhibit cap-dependent translation. SG contain translation initiation factors, RNA binding proteins, and signaling molecules. SG are known to inhibit apoptotic pathways, thus contributing to chemo-and radioresistance in tumor cells. However, whether stress granule formation involves oncogenic signaling pathways is currently unknown. Here, we report a novel role of the mTORC1-eukaryotic translation initiation factor 4E (eIF4E) pathway, a key regulator of cap-dependent translation initiation of oncogenic factors, in SG formation. mTORC1 specifically drives the eIF4E-mediated formation of SG through the phosphorylation of 4E-BP1, a key factor known to inhibit formation of the mTORC1-dependent eIF4E-eIF4GI interactions. Disrupting formation of SG by inactivation of mTOR with its specific inhibitor pp242 or by depletion of eIF4E or eIF4GI blocks the SG-associated antiapoptotic p21 pathway. Finally, pp242 sensitizes cancer cells to death in vitro and inhibits the growth of chemoresistant tumors in vivo. This work therefore highlights a novel role of the oncogenic mTORC1-eIF4E pathway, namely, the promotion of formation of antiapoptotic SG.

Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.