Impact of RAFT chain transfer agents on the polymeric shell density of magneto-fluorescent nanoparticles and their cellular uptake.

The impact of nanoparticle surface chemistry on cell interactions and especially cell uptake has become evident over the last few years in nanomedicine. Since PEG polymers have proved to be ideal tools for attaining stealthiness and favor escape from the in vivo mononuclear phagocytotic system, the accurate control of their geometry is of primary importance and can be achieved through reversible addition-fragmentation transfer (RAFT) polymerization. In this study, we demonstrate that the residual groups of the chain transfer agents (CTAs) introduced in the main chain exert a significant impact on the cellular internalization of functionalized nanoparticles. High-resolution magic angle spinning 1H NMR spectroscopy and fluorescence spectroscopy permitted by the magneto-fluorescence properties of nanoassemblies (NAs) revealed the compaction of the PEG comb-like shell incorporating CTAs with a long alkyl chain, without changing the overall surface potential. As a consequence of the capability of alkyl units to self-assemble at the NA surface while hardly contributing more than 0.5% to the total polyelectrolyte weight, denser PEGylated NAs showed notably less internalization in all cells of the tumor microenvironment (tumor cells, macrophages and healthy cells). Interestingly, such differentiated uptake is also observed between pro-inflammatory M1-like and immunosuppressive M2-like macrophages, with the latter more efficiently phagocytizing NAs coated with a less compact PEGylated shell. In contrast, the NA diffusion inside multicellular spheroids, used to mimic solid tumors, appeared to be independent of the NA coating. These results provide a novel effort-saving approach where the sole variation of the chemical nature of CTAs in RAFT PEGylated polymers strikingly modulate the cell uptake of nanoparticles upon the organization of their surface coating and open the pathway toward selectively addressing macrophage populations for cancer immunotherapy.

A partially demineralized allogeneic bone graft: in vitro osteogenic potential and preclinical evaluation in two different intramembranous bone healing models.

In skeletal surgical procedures, bone regeneration in irregular and hard-to-reach areas may present clinical challenges. In order to overcome the limitations of traditional autologous bone grafts and bone substitutes, an extrudable and easy-to-handle innovative partially demineralized allogenic bone graft in the form of a paste has been developed. In this study, the regenerative potential of this paste was assessed and compared to its clinically used precursor form allogenic bone particles. Compared to the particular bone graft, the bone paste allowed better attachment of human mesenchymal stromal cells and their commitment towards the osteoblastic lineage, and it induced a pro-regenerative phenotype of human monocytes/macrophages. The bone paste also supported bone healing in vivo in a guide bone regeneration model and, more interestingly, exhibited a substantial bone-forming ability when implanted in a critical-size defect model in rat calvaria. Thus, these findings indicate that this novel partially demineralized allogeneic bone paste that combines substantial bone healing properties and rapid and ease-of-use may be a promising alternative to allogeneic bone grafts for bone regeneration in several clinical contexts of oral and maxillofacial bone grafting.

High Oncolytic Activity of a Double-Deleted Vaccinia Virus Copenhagen Strain against Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a cancer of the pleura that lacks efficient treatment. Oncolytic immunotherapy using oncolytic vaccinia virus (VV) may represent an alternative therapeutic approach for the treatment of this malignancy. Here, we studied the oncolytic activity of VV thymidine kinase (TK)-ribonucleotide reductase (RR)-/green fluorescent protein (GFP) against MPM. This virus is a VV from the Copenhagen strain that is deleted of two genes encoding the TK (J2R) and the RR (I4L) and that express the GFP. First, we show in vitro that VVTK-RR-/GFP efficiently infects and kills the twenty-two human MPM cell lines used in this study. We also show that the virus replicates in all eight tested MPM cell lines, however, with approximately a 10-fold difference in the amplification level from one cell line to another. Then, we studied the therapeutic efficiency of VVTK-RR-/GFP in non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice that bear peritoneal human MPM tumors. One intraperitoneal infection of VVTK-RR-/GFP reduces the tumor burden and significantly increases mice survival compared to untreated animals. Thus, VVTK-RR - may be a promising oncolytic virus (OV) for the oncolytic immunotherapy of MPM.

Involvement of the M-CSF/IL-34/CSF-1R pathway in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The tumor microenvironment content, particularly the presence of macrophages, was described as crucial for the development of the disease. This work aimed at studying the involvement of the M-CSF (CSF-1)/IL-34/CSF-1R pathway in the formation of macrophages in MPM, using samples from patients. METHODS: Pleural effusions (PEs), frozen tumors, primary MPM cells and MPM cell lines used in this study belong to biocollections associated with clinical databases. Cytokine expressions were studied using real-time PCR and ELISA. The Cancer Genome Atlas database was used to confirm our results on an independent cohort. An original three-dimensional (3D) coculture model including MPM cells, monocytes from healthy donors and a tumor antigen-specific cytotoxic CD8 T cell clone was used. RESULTS: We observed that high interleukin (IL)-34 levels in PE were significantly associated with a shorter survival of patients. In tumors, expression of CSF1 was correlated with 'M2-like macrophages' markers, whereas this was not the case with IL34 expression, suggesting two distinct modes of action of these cytokines. Expression of IL34 was higher in MPM cells compared with primary mesothelial cells. Particularly, high expression of IL34 was observed in MPM cells with an alteration of CDKN2A. Finally, using 3D coculture model, we demonstrated the direct involvement of MPM cells in the formation of immunosuppressive macrophages, through activation of the colony stimulating factor-1 receptor (CSF1-R) pathway, causing the inhibition of cytotoxicity of tumor antigen-specific CD8+ T cells. CONCLUSIONS: The M-CSF/IL-34/CSF-1R pathway seems strongly implicated in MPM and could constitute a therapeutic target to act on immunosuppression and to support immunotherapeutic strategies.

Hybrid Azo-fluorophore Organic Nanoparticles as Emissive Turn-on Probes for Cellular Endocytosis.

The development of fluorescent organic nanoparticles, serving as bioimaging agents or drug cargos, represents a buoyant field of investigations. Nevertheless, their ulterior fate and structural integrity after cell uptake remain elusive. Toward this aim, we have elaborated original photoactive organic nanoparticles (dTEM ∼ 35-50 nm wide) with an off-on signal upon cellular internalization. Such nanoparticles are based on the noncovalent association of red-emitting benzothiadiazole (BDZ) derivatives and azo dyes, acting as fluorescence quenchers. Upon varying the azo/BDZ ratio, we found that quantitative emission quenching could be obtained with only a 0.2:1 azo/BDZ ratio and originated from exergonic oxidative and reductive photoinduced electron transfer from the azo units (ΔelG0 = -0.21 and -0.29 eV, respectively). Such results revisited the origin of emission quenching, often confusedly ascribed to Förster resonance energy transfer. A nonlinear and sharp drop of the emission intensity with the increase in the azo unit density n was observed and presents comparable evolution to a n-1/3 mathematical law. Thorough biological examinations involving cancer cells prove a receptor-independent endocytosis pathway, leading to progressive cell lighting upon nanoparticle accumulation in the late endosomal/lysosomal compartments. Complete emission recovery of the initially quenched azo/BDZ nanosystems could be achieved by using mefloquine, which caused endosomal/lysosomal disruption, and release of their content in the cytoplasm. Such results demonstrate that the dotlike emission from endosomes actually stems from fully dissociated individual dyes and not integer nanoparticles. They conclude on the high spatial confinement promoted by organelles and finally question its severe impact on functional compounds or nanoparticles whose properties are strongly distance dependent.

Small Molecule-Based Fluorescent Organic Nanoassemblies with Strong Hydrogen Bonding Networks for Fine Tuning and Monitoring Drug Delivery in Cancer Cells.

Bright supramolecular fluorescent organic nanoassemblies (FONs), based on strongly polar red-emissive benzothiadiazole fluorophores containing acidic units, are fabricated to serve as theranostic tools with large colloidal stability in the absence of a polymer or surfactant. High architectural cohesion is ensured by the multiple hydrogen-bonding networks, reinforced by the dipolar and hydrophobic interactions developed between the dyes. Such interactions are harnessed to ensure high payload encapsulation and efficient trapping of hydrophobic and hydrogen-bonding drugs like doxorubicin, as shown by steady state and time-resolved measurements. Fine tuning of the drug release in cancer cells is achieved by adjusting the structure and combination of the fluorophore acidic units. Notably delayed drug delivery is observed by confocal microscopy compared to the entrance of hydrosoluble doxorubicin, demonstrating the absence of undesirable burst release outside the cells by using FONs. Since FON-constituting fluorophores exhibit a large emission shift from red to green when dissociating in contact with the lipid cellular content, drug delivery could advantageously be followed by dual-color spectral detection, independently of the drug staining potentiality.

Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine. Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PD-L1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi. Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response.

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis.

BACKGROUND: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. RESULTS: The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. CONCLUSIONS: The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. GENERAL SIGNIFICANCE: Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.

Pleural Effusions from Patients with Mesothelioma Induce Recruitment of Monocytes and Their Differentiation into M2 Macrophages.

INTRODUCTION: Mesothelioma is a rare and aggressive cancer related to asbestos exposure. We recently showed that pleural effusions (PEs) from patients with mesothelioma contain high levels of the C-C motif chemokine ligand 2 (CCL2) inflammatory chemokine. In the present work, we studied the effect of CCL2 contained in mesothelioma samples, particularly on monocyte recruitment. Then, we studied the fate of these monocytes in malignant pleural mesothelioma (MPM) PEs and their impact on tumor cells' properties. METHODS: The implication of CCL2 in monocyte recruitment was evaluated using transmigration assays and a CCL2 blocking antibody. The phenotype of macrophages was determined by flow cytometry and enzyme-linked immunosorbent assay. Immunohistochemical analysis was used to support the results. Cocultures of macrophages with mesothelioma cells were performed to study cancer cell proliferation and resistance to treatment. RESULTS: We showed that CCL2 is a major factor of monocyte recruitment induced by MPM samples. Macrophages obtained in MPM samples were M2 macrophages (high CD14, high CD163, and interleukin-10 secretion after activation). The colony-stimulating factor 1 receptor/macrophage colony-stimulating factor (M-CSF) pathway is implicated in M2 polarization, and high levels of M-CSF were measured in MPM samples compared with benign PE (4.17 ± 2.75 ng/mL and 1.94 ± 1.47 ng/mL, respectively). Immunohistochemical analysis confirmed the presence of M2 macrophages in pleural and peritoneal mesothelioma. Finally, we showed that M2 macrophages increased mesothelioma cell proliferation and resistance to treatment. CONCLUSIONS: These results demonstrate the implication of CCL2 in MPM pathogenesis and designate M-CSF as a new potential biomarker of MPM. This study also identifies CCL2 and colony-stimulating factor 1 receptor/M-CSF as interesting new targets to modulate pro-tumorigenic properties of the tumor microenvironment.