Matthieu Germain

Liposome-based nanocapsules

Here we present three different types of mechanically stable nanometer-sized hollow capsules. The common point of the currently developed systems in our laboratory is that they are liposome based. Biomolecules can be used to functionalize lipid vesicles to create a new type of intelligent material. For example, insertion of membrane channels into the capsule wall can modify the permeability. Covalent binding of antibodies allows targeting of the capsule to specific sites. Liposomes loaded with enzymes may provide an optimal environment for them with respect to the maximal turnover and may stabilize the enzyme. However, the main drawback of liposomes is their instability in biological media as well as their sensitivity to many external parameters such as temperature or osmotic pressure. To increase their stability we follow different strategies: 1) polymerize a two-dimensional network in the hydrophobic core of the membrane; 2) coat the liposome with a polyelectrolyte shell; or 3) add surface active polymers to form mixed vesicular structures.

Pp IX Silica Nanoparticles Demonstrate Differential Interactions with In Vitro Tumor Cell Lines and In Vivo Mouse Models of Human Cancers

Protoporphyrin IX (Pp IX) silica nanoparticles, developed for effective use in photodynamic therapy (PDT), were explored in in vitro and in vivo models with the ambition to improve knowledge on the role of biological factors in the photodamage. Pp IX silica nanoparticles are found efficient at temperature with extreme metabolic downregulation, which suggest a high proportion of passive internalization. For the first time, clearance of silica nanoparticles on tumor cells is established. Cell viability assessment in six tumor cell lines is reported. In all tumor types, Pp IX silica nanoparticles are more efficient than free Pp IX. A strong fluorescence signal of reactive oxygen species generation colocalized with Pp IX silica nanoparticles, correlates with 100% of cell death. In vivo studies performed in HCT 116, A549 and glioblastoma multiforme tumors‐bearing mice show tumor uptake of Pp IX silica nanoparticles with better tumor accumulation than the control alone, highlighting a high selectivity for tumor tissues. As observed in in vitro tests, tumor cell type is likely a major determinant but tumor microenvironment could more influence this differential time accumulation dynamic. The present results strongly suggest that Pp IX silica nanoparticles may be involved in new alternative local applications of PDT.

MRI contrast variation of thermosensitive magnetoliposomes triggered by focused ultrasound: a tool for image-guided local drug delivery

Improved drug delivery control during chemotherapy has the potential to increase the therapeutic index. MRI contrast agent such as iron oxide nanoparticles can be co-encapsulated with drugs in nanocarrier liposomes allowing their tracking and/or visualization by MRI. Furthermore, the combination of a thermosensitive liposomal formulation with an external source of heat such as high intensity focused ultrasound guided by MR temperature mapping allows the controlled local release of the content of the liposome. MRI-guided high-intensity focused ultrasound (HIFU), in combination represents a noninvasive technique to generate local hyperthermia for drug release. In this study we used ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) encapsulated in thermosensitive liposomes to obtain thermosensitive magnetoliposomes (TSM). The transverse and longitudinal relaxivities of this MRI contrast agent were measured upon TSM membrane phase transition in vitro using a water bath or HIFU. The results showed significant differences for MRI signal enhancement and relaxivities before and after heating, which were absent for nonthermosensitive liposomes and free nanoparticles used as controls. Thus, incorporation of USPIO as MRI contrast agents into thermosensitive liposomes should, besides TSM tumor accumulation monitoring, allow the visualization of TSM membrane phase transition upon temperature elevation. In conclusion, HIFU under MR image guidance in combination with USPIO-loaded thermosensitive liposomes as drug delivery system has the potential for a better control of drug delivery and to increase the drug therapeutic index.

Nano-sized cytochrome P450 3A4 inhibitors to block hepatic metabolism of docetaxel

Most drugs are metabolized by hepatic cytochrome P450 3A4 (CYP3A4), resulting in their reduced bioavailability. In this study, we present the design and evaluation of bio-compatible nanocarriers trapping a natural CYP3A4-inhibiting compound. Our aim in using nanocarriers was to target the natural CYP3A4-inhibiting agent to hepatic CYP3A4 and leave drug-metabolizing enzymes in other organs undisturbed. In the design of such nanocarriers, we took advantage of the nonspecific accumulation of small nanoparticles in the liver. Specific targeting functionalization was added to direct nanocarriers toward hepatocytes. Nanocarriers were evaluated in vitro for their CYP3A4 inhibition capacity and in vivo for their biodistribution, and finally injected 24 hours prior to the drug docetaxel, for their ability to improve the efficiency of the drug docetaxel. Nanoparticles of poly(lactic-co-glycolic) acid (PLGA) with a hydrodynamic diameter of 63 nm, functionalized with galactosamine, showed efficient in vitro CYP3A4 inhibition and the highest accumulation in hepatocytes. When compared to docetaxel alone, in nude mice bearing the human breast cancer, MDA-MB-231 model, they significantly improved the delay in tumor growth (treated group versus docetaxel alone, percent treated versus control ratio [%T/C] of 32%) and demonstrated a major improvement in overall survival (survival rate of 67% versus 0% at day 55).

Liposomes : support à la formation de capsules plus stables à base de polyéléctrolytes réticulés ou de silice☆

Resume L’utilisation d’enzyme en therapie ou en biodetection necessite de les isoler de leur environnement afin d’eviter leur degradation par des agents proteolytiques, par des variations de pH ou encore par effet de dilution, et d’eviter egalement le developpement de reaction immunitaire dans le cas d’injection. Cet isolement sous-entend de les encapsuler dans des conditions non denaturantes pour l’enzyme, dans une capsule suffisamment resistante et a l’echelle nanometrique pour diminuer la degradation par le systeme reticulo-endothelial. Les liposomes representent une methode de choix d’encapsulation de proteine dans des conditions non denaturantes mais ne sont pas suffisamment stables. En revanche, d’autres capsules offrent une tres bonne stabilite mais les conditions requises pour leur formation ne permettent pas de travailler en presence d’enzyme. Nos travaux utilisent les liposomes comme structure support a la formation de capsules plus stables a partir de deux types de materiaux. D’une part, nous avons forme en surface du liposome une capsule de polyelectrolytes qui est ensuite rigidifiee par pontage covalent entre les polyelectrolytes. D’autre part, nous avons utilise la silice afin de former une coque inorganique autour du liposome. Ces deux techniques ont permis d’obtenir une nanocapsule stable en presence de detergent et permettant de preserver de la degradation par des proteases de l’acetylcholinesterase, enzyme hautement sensible aux pesticides et utilisable pour le developpement de biodetecteurs.

Ultrasound-induced temperature elevation for in-vitro controlled release of temperature-sensitive liposomes

Drug loaded temperature-sensitive liposomes (TSLs) release their payload with mild hyperthermia near their phase transition temperature (Tm = 43–45 °C). Such a release may improve therapeutic efficacy and reduce toxic side effects in cancer treatment. In the present work, two different approaches are considered where focused ultrasound is used to induce the required temperature elevation for the release of doxorubicin from TSLs: (a) primary heating due to thermo-viscous absorption of ultrasound in absorptive media (oil, glycerol) and (b) secondary heating in non-absorptive media (blood, cell medium) due to heat transfer from the surroundings. Fine-wire thermocouple readings where in close agreement with theoretical predictions of temperature elevation with the Bioheat equation. Pulsing schemes to elevate and maintain the temperature at the desired value were designed with the Bioheat equation and validated with experiments. Fluorescence spectroscopy was used to assess the release of free doxorubicin that ...

Priming the body to receive the therapeutic agent to redefine treatment benefit/risk profile

Many therapeutic agents offer a low useful dose (dose responsible for efficacy)/useless dose (dose eliminated or responsible for toxicity) ratio, mainly due to the fact that therapeutic agents must ensure in one single object all the functions required to deliver the treatment, which leads to compromises in their physico-chemical design. Here we introduce the concept of priming the body to receive the treatment by uncorrelating these functions into two distinct objects sequentially administered: a nanoprimer occupying transiently the main pathway responsible for therapeutic agent limited benefit/risk ratio followed by the therapeutic agent. The concept was evaluated for different nature of therapeutic agents: For nanomedicines we designed a liposomal nanoprimer presenting preferential hepatic accumulation without sign of acute toxicity. This nanoprimer was able to increase the blood bioavailability of nanomedicine correlated with a lower hepatic accumulation. Finally this nanoprimer markedly enhanced anti-tumor efficacy of irinotecan loaded liposomes in the HT-29 tumor model when compared to the nanomedicine alone. Then, for small molecules we demonstrated the ability of a cytochrome inhibitor loaded nanoprimer to increase efficacy of docetaxel treatment. These results shown that specific nanoprimers could be designed for each family of therapeutic agents to answer to their specific needs.

High-intensity focused ultrasound-mediated doxorubicin delivery with thermosensitive liposomes

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin from thermosensitive liposomes (Dox-TSL) into human glioblastoma (U-87MG) cells. Using Dox-TSL, experiments were carried out in a water bath and showed that 15 min incubation of TSL at 43°C induced the release of 80% doxorubicin loaded TSL compared to the release at 37°C. The cytotoxicity of a range of concentrations of Dox-TSL was also evaluated on U-87MG cells. At 37°C, no cytotoxicity was observed, whereas at 43°C the results showed that the cytotoxicity is dose dependent. At maximal dose of doxorubicin (30 μg/mL), the cell viability was less than 20%. Application of 15 min of HIFU at 1 MHz, 1.5 MPa and 50% duty cycle induced the release of 100% of doxorubicin from Dox-TSL. In the same experimental condition, the cell viability decreased to 40% and 20% at 12h and 48h, respectively, in comparison to that obtained during the incubation of c...