Streptolysin O and NAD-Glycohydrolase Prevent Phagolysosome Acidification and Promote Group A Streptococcus Survival in Macrophages

ABSTRACT Group A Streptococcus (GAS, Streptococcus pyogenes) is an ongoing threat to human health as the agent of streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. In animal models of infection, macrophages have been shown to contribute to host defense against GAS infection. However, as GAS can resist killing by macrophages in vitro and induce macrophage cell death, it has been suggested that GAS intracellular survival in macrophages may enable persistent infection. Using isogenic mutants, we now show that the GAS pore-forming toxin streptolysin O (SLO) and its cotoxin NAD-glycohydrolase (NADase) mediate GAS intracellular survival and cytotoxicity for macrophages. Unexpectedly, the two toxins did not inhibit fusion of GAS-containing phagosomes with lysosomes but rather prevented phagolysosome acidification. SLO served two essential functions, poration of the phagolysosomal membrane and translocation of NADase into the macrophage cytosol, both of which were necessary for maximal GAS intracellular survival. Whereas NADase delivery to epithelial cells is mediated by SLO secreted from GAS bound to the cell surface, in macrophages, the source of SLO and NADase is GAS contained within phagolysosomes. We found that transfer of NADase from the phagolysosome to the macrophage cytosol occurs not by simple diffusion through SLO pores but rather by a specific translocation mechanism that requires the N-terminal translocation domain of NADase. These results illuminate the mechanisms through which SLO and NADase enable GAS to defeat macrophage-mediated killing and provide new insight into the virulence of a major human pathogen

Lauryl-gemcitabine loaded nanomedicine hydrogel for the local treatment of glioblastoma

Glioblastoma (GBM) is one of the greatest challenges in oncology. The standard of care therapy of this highly malignant brain tumor includes surgical resection followed, one month after, by radiotherapy and chemotherapy with Temozolomide. However, GBM still remains incurable mainly because of its anatomical location, high intra - and inter-tumor heterogeneity and intrinsic characteristics that inevitably lead to the formation of recurrences [1]. Considering that 80-90% of GBM recurrences are localized in proximity of resection cavity borders we hypothesized to deliver an injectable nanomedicine hydrogel directly in the tumor resection cavity after surgery in order to obtain a sustained release of the drug. This could avoid the formation of recurrences before starting the conventional treatment. The hydrogel that we have developed and selected is formed of lipid nanocapsules (LNC) loaded with the prodrug Lauroyl -gemcitabine (GemC12), which shows excellent radio-sensitizing properties, could potentiate cancer immunotherapy and has a MGMT -independent mechanism of action [2,3]. This nanomedicine hydrogel is injectable, adapted for brain implantation and able to release the drug over one month in vitro [2]. In vivo, the anti-tumor efficacy studies in a subcutaneous and ortothopic GBM rodent models have shown, respectively, to decrease the tumor growth and increase the survival of the mice after intratumoral injection of the hydrogel compared to the controls. Also, to better mimic the clinical conditions, we have developed and validated a resection model of the GBM orthotopic tumor and on -going anti -tumor efficacy studies after administration of the treatment in the resection cavity are showing promising results. Moreover, short -, mid- and long- term tolerability studies (1 week, 2 months and 6 months) indicated that this system is well tolerated in the brain. In conclusion, we have demonstrated the fe asibility, safety and efficiency of the GemC12 -LNC hydrogel for the local treatment of GBM. This system, which has a very simple formulation and combines the properties and advantages of nanomedicines and hydrogels, could be considered as a promising platform for the delivery of GemC12 for the local treatment of GBM

OPA quantification of amino groups at the surface of Lipidic NanoCapsules (LNCs) for ligand coupling improvement

Lipidic NanoCapsules (LNCs) were prepared via an emulsion phase inversion method. Nanoparticles with hydrodynamic diameter of 25, 50 and 100nm were easily obtained. Their surfaces are covered with short PEG chains (PEG 660) which are not bearing any chemical reactivities. Thus, in order to overcome this handicap towards post-functionalization possibilities, post-insertion of DSPE-PEG2000 amino (DSPA) can be employed. In order to characterize the insertion step, we have developed a chemical assay for the quantification of amino group inside the PEG shell of LNCs. Subsequently, the post-insertion yield was found to be comprised between 60 and 90% whatever the hydrodynamic diameter of the LNCs is. By means of simple calculations, the density of amino group is estimated to be closed to 0.2 and 1.2molecules/nm(2). The formulation of LNCs and their controlled functionalization represent an interesting system for the development of bionanoconjugates in a short and effective process

Glioblastoma-targeting peptide adsorbed on lipid nanocapsule surface: optimization of the process and targeting efficacy

The current standard of care of glioblastoma (GBM): the highest grade brain tumor is not a curative treatment and does not prevent high patient mortality (median survival of 14 months, and 5-year survival rate lower than 10% (Stupp R et al., N. Engl. J. Med, 2005; Stupp R et al., Lancet Oncol., 2009)). The scientific community must address solutions to these unmet medical and patient needs and opportunities can be found to develop innovative and complementary treatment to the standard therapeutic scheme. The GLIOGEL project (ERA-NET Cofund EuroNanoMed III) focuses on a hydrogel of GBM-targeting, drug-loaded lipid nanocapsules (LNCs). This new drug delivey system will be implantable directly after GBM resection, and will close the treatment gap until Stupp protocol (radiotherapy and/or adjuvant chemotherapy, 4 to 6 weeks after resection). The sustained LNCs release will specifically target the residual infiltrating GBM cells at resection border, main cause of tumor recurrences. The proof of concept of adsorption of a targeting peptide: the NFL-TBS.40-63 (NFL) at LNC surface was already done, with the specific targeting property for GBM cells (Berges R et al., Mol. Ther., 2012 ; Balzeau J. et al., Biomaterials, 2013). So the first part of the project was to confirm and optimize the association NFL-LNCs, and to show correlations regarding NFL and LNC physicochemical properties.   LNCs in suspension with different sizes (from 30 to 100nm), surface charges (positive, neutral and negative) and Span® 80 composition (from 0 to 15% w/wLNCs) have been performed according to a phase inversion process (Heurtault et al., WO2001 / 064328, 2001). Different LNC concentrations (0.001 to 275mg/mL) were incubated at room temperature for 12h with fixed NFL concentrations (50 to 200μg/mL). Free NFL proportions were quantified in the free NFL and NFL-adsorbed LNC mixtures without prior separation, i.e. directly after incubation, to avoid all the bias that can be observed using physical separations such as filtration by centrifugation and dialysis. A Steric Exclusion Chromatography method was developed for this purpose. Due to NFL properties (slightly positively charged and capacity to form H-bond), we showed that the NFL adsorption at LNC surface was enhanced with negatively charged LNCs, and when Span® 80 proportion at LNC surface increase. These results were confirmed using two other methods without prior separation: NMR diffusometry using the diffusion coefficients for both free and adsorbed NFLs, and Fluorescence Correlation Spectroscopy using FITC-labeled NFL. Other protein or peptide-adsorbed nanoparticles could be characterized using these three methods, and the absence of physical separation before the quantification is a real benefit for the accuracy and veracity of data. Finally, the best LNC candidates with total NFL adsorption were tested on a large library of GBM cell lines, with different incubation times in order to verify the targeting efficacy. The LNCs internalization is faster when the NFL is present at their surface. Nevertheless, we observed that the efficiency depends on different parameters: the amount of NFLs at LNC surface and also the cellular models

Formulation and characterization of a 0.1% rapamycin cream for the treatment of Tuberous Sclerosis Complex-related angiofibromas

Medicines for the treatment of rare diseases frequently do not attract the interest of the pharmaceutical industry, and hospital pharmacists are thus often requested by physicians to prepare personalized medicines. Tuberous Sclerosis Complex (TSC) is a rare disease that causes disfiguring lesions named facial angiofibromas. Various topical formulations of rapamycin (= sirolimus) have been proved effective in treating these changes in small case series. The present study provides for the first time characterization of a 0.1% rapamycin cream formulation presenting good rapamycin solubilisation. The first step of the formulation is solubilisation of rapamycin in Transcutol®, and the second step is the incorporation of the mixture in an oil-in-water cream. A HPLC stability-indicating method was developed. Rapamycin concentration in the cream was tested by HPLC and confirmed that it remained above 95% of the initial concentration for at least 85 days, without characteristic degradation peaks. The preparation met European Pharmacopoeia microbial specifications throughout storage in aluminum tubes, including when patient use was simulated. Odour, appearance and colour of the preparation were assessed and no change was evidenced during storage. The rheological properties of the cream also remained stable throughout storage. To conclude, we report preparation of a novel cream formulation presenting satisfactory rapamycin solubilisation for the treatment of TSC cutaneous manifestations, with stability data. The cream is currently being used by our patients. Efficacy and tolerance will be reported later

Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering

International audienceThe challenge of tissue engineering of the infarcted heart is how to improve stem cell engraftment, survival, homing, and differentiation for myocardial repair. We here propose to integrate human adipose-derived stem cells (ADSCs) and pharmacologically active microcarriers (PAMs), a three-dimensional (3D) carrier of cells and growth factors, into an injectable hydrogel (HG), to obtain a system that stimulates the survival and/or differentiation of the grafted cells toward a cardiac phenotype. PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) (PLGA) microspheres conveying cells on their 3D surface that deliver continuously and in a controlled manner a growth factor (GF) acting on the transported cells and on the microenvironment to improve engraftment. The choice of the appropriate GF and its protection during the formulation process and delivery are essential. In this study two GFs, hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1), have been encapsulated under a solid state in order to limit their interaction with the polymer and conserve their integrity. GF precipitation conditions and release profile from PAMs have been first investigated before combining them to ADSCs. The released IGF-1 and HGF induced the protein synthesis of cardiac differentiation markers GATA4, Nkx2.5, cTnI and CX43 after 1 week in vitro. Moreover, the GFs accelerated cell cycle progression, as suggested by the increased expression of Cyclin D1 mRNA and the widespread distribution of Ki67 protein. Integrating PAMs within the thermosensitive P407 hydrogel increased their elastic properties but decreased the transcription of most cardiac markers. In contrast, CX43 expression increased in ADSC–PAM–GF complexes embedded within the hydrogel compared to the ADSCs cultured alone in the absence of P407. These results suggest that particulate scaffolds releasing HGF and IGF-1 may be beneficial for applications in tissue-engineering strategies for myocardial repair and the association with a P407 hydrogel can increase substrate elasticity and junction connections in ADSCs