Caliper, contrast enhanced-ultrasound or laser speckle contrast imaging: Techniques to follow mice melanoma growth.

Due to morphological characteristics, metastatic melanoma is a cancer for which vascularization is not a diagnostic criterion. Laser speckle contrast imaging (LSCI) and contrast enhanced ultrasound (CEUS) are two imaging techniques that will be explored in this study, which aims to confirm these two techniques for monitoring tumor vascularization. B16F10 cells were xenografted to C57BL/6 mice treated with anti-PD1 or 0.9% NaCl. Tumor volume was measured daily while CEUS and LSCI were performed weekly. LSCI and CEUS analyses showed a decrease in tumor perfusion in both groups of mice. Although both CEUS and LSCI are useful for measuring tumor volume, LSCI appears to be more robust and effective for monitoring tumor microcirculation. Non-invasive investigations are needed to better predict tumor vascularization: CEUS and LSCI have a good applicability in a mice model.

The use of liposomes functionalized with the NFL-TBS.40-63 peptide as a targeting agent to cross the in vitro blood-brain barrier and target glioblastoma cells.

Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood-brain barrier (BBB), which limits the delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40-63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood-brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.

Glioblastoma-targeted, local and sustained drug delivery system based on an unconventional lipid nanocapsule hydrogel.

The objective of this work was to develop an implantable therapeutic hydrogel that will ensure continuity in treatment between surgery and radiochemotherapy for patients with glioblastoma (GBM). A hydrogel of self-associated gemcitabine-loaded lipid nanocapsules (LNC) has shown therapeutic efficacy in vivo in murine GBM resection models. To improve the targeting of GBM cells, the NFL-TBS.40-63 peptide (NFL), was associated with LNC. The LNC-based hydrogels were formulated with the NFL. The peptide was totally and instantaneously adsorbed at the LNC surface, without modifying the hydrogel mechanical properties, and remained adsorbed to the LNC surface after the hydrogel dissolution. In vitro studies on GBM cell lines showed a faster internalization of the LNC and enhanced cytotoxicity, in the presence of NFL. Finally, in vivo studies in the murine GBM resection model proved that the gemcitabine-loaded LNC with adsorbed NFL could target the non-resected GBM cells and significantly delay or even inhibit the apparition of recurrences.

Pharmacokinetics of intact lipid nanocapsules using new quantitative FRET technique.

The present study investigated the pharmacokinetics of intact lipid nanocapsules (LNCs) after intravenous administration in rats. Six different Förster resonance energy transfer LNCs (FRET-LNCs) have been studied with 2 sizes (50 and 85 nm) and 3 coating types (none, DSPE-mPEG 2000 or stearylamine). A FRET-LNCs blood extraction method was developed to retain an accurate FRET signal. Intact FRET-LNCs were specifically quantified through combination of FRET signal and Nano Tracker Analysis. Pharmacokinetic data were first described by non-compartmental analysis, then used to develop a population pharmacokinetic model. The pharmacokinetic elimination of FRET-LNCs was non-linear and dependent on size and surface modification, while the distribution was dependent on size. The LNCs 85 nm volume of distribution was lower than LNCs 50 nm. As expected, LNCs 85 nm with PEG coating displayed a lower clearance than other formulations. Surprisingly, this study highlighted a faster elimination of LNCs 50 nm with PEG compared to other formulations which could be explained by instability in blood. This first pharmacokinetic model of intact LNCs allowed a thorough understanding of the influence of size and coating on pharmacokinetic properties and paves the way for future mechanistic modeling approaches to predict the fate of LNCs in vivo.

pH-Responsive Lipid Nanocapsules: A Promising Strategy for Improved Resistant Melanoma Cell Internalization.

Despite significant advances in melanoma therapy, low response rates and multidrug resistance (MDR) have been described, reducing the anticancer efficacy of the administered molecules. Among the causes to explain these resistances, the decreased intratumoral pH is known to potentiate MDR and to reduce the sensitivity to anticancer molecules. Nanomedicines have been widely exploited as the carriers of MDR reversing molecules. Lipid nanocapsules (LNC) are nanoparticles that have already demonstrated their ability to improve cancer treatment. Here, LNC were modified with novel copolymers that combine N-vinylpyrrolidone (NVP) to impart stealth properties and vinyl imidazole (Vim), providing pH-responsive ability to address classical chemoresistance by improving tumor cell entry. These copolymers could be post-inserted at the LNC surface, leading to the property of going from neutral charge under physiological pH to positive charge under acidic conditions. LNC modified with polymer P5 (C18H37-P(NVP21-co-Vim15)) showed in vitro pH-responsive properties characterized by an enhanced cellular uptake under acidic conditions. Moreover, P5 surface modification led to an increased biological effect by protecting the nanocarrier from opsonization by complement activation. These data suggest that pH-sensitive LNC responds to what is expected from a promising nanocarrier to target metastatic melanoma.

p722 ferrocifen loaded lipid nanocapsules improve survival of murine xenografted-melanoma via a potentiation of apoptosis and an activation of CD8+ T lymphocytes.

Metastatic melanoma is a malignant tumor with a poor prognosis. Recent new therapeutics improved the survival of patients at a metastatic stage. However, the low response rate to immunotherapy, explained in part by resistance to apoptosis, needs to develop new strategies. The ferrocifen family represents promising bioorganometallic molecules for melanoma treatment since they show potent anticancer properties. The aim of this study is (i) to evaluate the benefits of a strategy involving encapsulated p722 in lipid nanocapsules (LNC) in B16F10 melanoma mice models and (ii) to compare the beneficial effects with an existing therapy such as anti-CTLA4 mAb. Interestingly, LNC-p722 induces a significant decrease of melanoma cell viability. In vivo data shows a significant improvement in the survival rate and a slower tumor growth with p722-loaded LNC in comparison with anti-CTLA4 mAb. Western blots confirm that LNC-p722 potentiates intrinsic apoptotic pathway. Treatment with LNC-p722 significantly activates CD8+ T lymphocytes compared to treatment with anti-CTLA4 mAb. This study uncovers a new therapeutic strategy with encapsulated p722 to prevent B16F10 melanoma growth and to improve survival of treated mice.

Melanoma tumour vasculature heterogeneity: from mice models to human.

Tumour angiogenesis is defined by an anarchic vasculature and irregularities in alignment of endothelial cells. These structural abnormalities could explain the variability in distribution of nanomedicines in various tumour models. Then, the main goal of this study was to compare and to characterize the tumour vascular structure in different mouse models of melanoma tumours (B16F10 and SK-Mel-28) and in human melanomas from different patients. Tumours were obtained by subcutaneous injection of 106 B16F10 and 3.106 SK-Mel-28 melanoma cells in C57BL/6 and nude mice, respectively. Tumour growth was evaluated weekly, while vasculature was analysed through fluorescent labelling via CD31 and desmin. Significant differences in tumour growth and mice survival were evidenced between the two melanoma models. A fast evolution of tumours was observed for B16F10 melanoma, reaching a tumour size of 100 mm3 in 7 days compared to SK-Mel-28 which needed 21 days to reach the same volumes. Important differences in vascularization were exposed between the melanoma models, characterized by a significant enhancement of vascular density and a significant lumen size for mice melanoma models compared to human. Immunostaining revealed irregularities in endothelium structure for both melanoma models, but structural differences of vasculature were observed, characterized by a stronger expression of desmin in SK-Mel-28 tumours. While human melanoma mainly develops capillaries, structural irregularities are also observed on the samples of this tumour model. Our study revealed an impact of cell type and tumour progression on the structural vasculature of melanoma, which could impact the distribution of drugs in the tumour environment.