Franchissement de barrières biologiques, mécanisme d'action et devenir subcellulaire de nanovecteurs d'agents anticancéreux pour la thérapie des gliomes

By focusing on drug administration within and to the central nervous system and notably on glioblastoma, the aim of the present PhD thesis work was to develop tools and evaluate the behavior of particulate nanocarriers as regards to biological barrier crossing. Three types of nanocarriers with sizes varying from 20 to 100 nm were evaluated: polysaccharide nanoparticles, PLGA nanoparticles, and lipid nanocapsules. The behavior of those objects concerning blood constituents allowed to define that coating with transferrine of PLGA nanoparticles or insertion of phospholipids or BSA within polysaccharide nanoparticles reduce their recognition by the reticulo-endothelial system and improve their plasma-resident time. Those surface modifications are also associated with a possibility of internalization in F98 glioma target cells essentially influenced by the lipid or polymeric nature or the carrier. Clear-cut evaluation of the cellular and subcellular behavior of LNC within F98 cells allowed to demonstrate that if the nature of the carrier is involved, notably considering the recruitment of cholesterol-dependant endocytic pathways, the size, correlated to amounts of surfactant provided, had also its significance. 20nm LNC are consequently the more apt to allow lysosome escape of transported drug and demonstrated higher pharmacological activities in inducing cell death through paclitaxel loading. Those results provide, therefore, original links between the subcellular behavior of nanocarrier and the bioavailability anticancer drugs. New potentialities of biological barrier crossing and size-dependent abilities were demonstrated. Those observations emphasize the interest of developing comparative studies in order to rationalize the use of a define nanocarrier for a given drug on a given target. Regarding new nanomedicines development, it demonstrates the importance to establish justification between biological behavior of nanocarriers and their therapeutic relevance.En se focalisant sur l'administration de médicaments dans et vers le système nerveux central et notamment pour le traitement du glioblastome, ce travail de thèse a eu pour but la mise en place d'outils expérimentaux et l'évaluation du comportement de nanovecteurs au cours du franchissement de barrières biologiques. Trois types de nanovecteurs de taille variant entre 20 et 100nm ont été appréhendés : des nanoparticules de polysaccharide, de PLGA et des nanocapsules lipidiques (LNC). Le comportement de ces objets vis-à-vis des éléments du sang a permis de définir que le revêtement par la transferrine de nanoparticules de PLGA et l'insertion de phospholipides ou de BSA dans des nanoparticules polysaccharidiques diminuait leur reconnaissance par le système réticulo-endothélial et améliorait leur temps de résidence plasmatique. Ces modifications de surface sont également associées à une possibilité d'internalisation dans les cellules cibles F98 de gliomes influencée essentiellement par la nature lipidique ou polymérique du vecteur. L'évaluation précise du comportement cellulaire et subcellulaire des LNC dans les cellules F98 a permis de démontrer que si la nature du vecteur est impliquée notamment en ce qui concerne le recrutement de voies d'endocytoses cholestéroldépendantes, la taille, corrélée au taux de surfactant véhiculé, est également impliquée. Les LNC de 20nm sont ainsi les plus aptes à permettre l'échappement lysosomal des principes actifs véhiculés et démontrent des activités pharmacologiques renforcées notamment pour ce qui concerne la mort cellulaire induite par le paclitaxel. Ces résultats établissent donc un lien original entre le comportement subcellulaire des vecteurs et la biodisponibilité des agents anticancéreux. De nouvelles potentialités de franchissement de barrières ligand- ou taille-dépendants ont été soulignées. Ces observations renforcent donc l'intérêt d'études comparatives permettant de rationaliser l'utilisation d'un vecteur donné pour un médicament et une cible donnés. Elles démontrent également tout l'intérêt d'établir des justifications entre le comportement biologique et la pertinence thérapeutique des nanovecteurs

Franchissement de barrières biologiques, mécanisme d'action et devenir subcellulaire de nanovecteurs d'agents anticancéreux pour la thérapie des gliomes

En se focalisant sur l'administration de médicaments dans et vers le système nerveux central et notamment pour le traitement du glioblastome, ce travail de thèse a eu pour but la mise en place d'outils expérimentaux et l'évaluation du comportement de nanovecteurs au cours du franchissement de barrières biologiques. Trois types de nanovecteurs de taille variant entre 20 et 100nm ont été appréhendés : des nanoparticules de polysaccharide, de PLGA et des nanocapsules lipidiques (LNC). Le comportement de ces objets vis-à-vis des éléments du sang a permis de définir que le revêtement par la transferrine de nanoparticules de PLGA et l'insertion de phospholipides ou de BSA dans des nanoparticules polysaccharidiques diminuait leur reconnaissance par le système réticulo-endothélial et améliorait leur temps de résidence plasmatique. Ces modifications de surface sont également associées à une possibilité d'internalisation dans les cellules cibles F98 de gliomes influencée essentiellement par la nature lipidique ou polymérique du vecteur. L'évaluation précise du comportement cellulaire et subcellulaire des LNC dans les cellules F98 a permis de démontrer que si la nature du vecteur est impliquée notamment en ce qui concerne le recrutement de voies d'endocytoses cholestéroldépendantes, la taille, corrélée au taux de surfactant véhiculé, est également impliquée. Les LNC de 20nm sont ainsi les plus aptes à permettre l'échappement lysosomal des principes actifs véhiculés et démontrent des activités pharmacologiques renforcées notamment pour ce qui concerne la mort cellulaire induite par le paclitaxel. Ces résultats établissent donc un lien original entre le comportement subcellulaire des vecteurs et la biodisponibilité des agents anticancéreux. De nouvelles potentialités de franchissement de barrières ligand- ou taille-dépendants ont été soulignées. Ces observations renforcent donc l'intérêt d'études comparatives permettant de rationaliser l'utilisation d'un vecteur donné pour un médicament et une cible donnés. Elles démontrent également tout l'intérêt d'établir des justifications entre le comportement biologique et la pertinence thérapeutique des nanovecteurs.By focusing on drug administration within and to the central nervous system and notably on glioblastoma, the aim of the present PhD thesis work was to develop tools and evaluate the behavior of particulate nanocarriers as regards to biological barrier crossing. Three types of nanocarriers with sizes varying from 20 to 100 nm were evaluated: polysaccharide nanoparticles, PLGA nanoparticles, and lipid nanocapsules. The behavior of those objects concerning blood constituents allowed to define that coating with transferrine of PLGA nanoparticles or insertion of phospholipids or BSA within polysaccharide nanoparticles reduce their recognition by the reticulo-endothelial system and improve their plasma-resident time. Those surface modifications are also associated with a possibility of internalization in F98 glioma target cells essentially influenced by the lipid or polymeric nature or the carrier. Clear-cut evaluation of the cellular and subcellular behavior of LNC within F98 cells allowed to demonstrate that if the nature of the carrier is involved, notably considering the recruitment of cholesterol-dependant endocytic pathways, the size, correlated to amounts of surfactant provided, had also its significance. 20nm LNC are consequently the more apt to allow lysosome escape of transported drug and demonstrated higher pharmacological activities in inducing cell death through paclitaxel loading. Those results provide, therefore, original links between the subcellular behavior of nanocarrier and the bioavailability anticancer drugs. New potentialities of biological barrier crossing and size-dependent abilities were demonstrated. Those observations emphasize the interest of developing comparative studies in order to rationalize the use of a define nanocarrier for a given drug on a given target. Regarding new nanomedicines development, it demonstrates the importance to establish justification between biological behavior of nanocarriers and their therapeutic relevance.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

SERS spectroscopic approach to study doxorubicin complexes with Fe(2+) ions and drug release from SPION-based nanocarriers.

International audienceThe aim of this work is to present a surface-enhanced Raman scattering (SERS) spectroscopic approach to study complexes of a frequently used antineoplastic agent, doxorubicin (DOX), with ferrous ions, at sub-micromolar concentrations in aqueous solution. The SERS bands of DOX were assigned according to critical analysis of literature. Prior to the complexation study, the spectral changes related to the drug orientation on the silver surface and to its protonation state were highlighted. The SERS spectra of DOX-Fe(2+) complexes showed several features distinguishing them from the free drug, protonated or not on the phenolic part of its chromophore. The lowest detectable content of the DOX-iron complex in the presence of free DOX was estimated to be 5-10%. This property is particularly interesting from the analytical point of view, since it allows for study of drug-iron interactions upon the drug loading on and release from magnetic drug carriers based on superparamagnetic iron oxide nanoparticles (SPIONs), stabilized with citrate ions or coated with polyethylene glycol (PEG) polymer. Our SERS data indicate that the drug loaded on magnetic nanocarriers as DOX-iron chelate was mainly released in the free DOX form. These results demonstrate the strength of the SERS approach for the study of DOX-iron interactions in relation to delivery issues and drug action mechanisms

Positively-charged, porous, polysaccharide nanoparticles loaded with anionic molecules behave as 'stealth' cationic nanocarriers.

International audiencePURPOSE: Stealth nanoparticles are generally obtained after modifying their surface with hydrophilic polymers, such as PEG. In this study, we analysed the effect of a phospholipid (DG) or protein (BSA) inclusion in porous cationic polysaccharide (NP(+)) on their physico-chemical structure and the effect on complement activation. METHODS: NP(+)s were characterised in terms of size, zeta potential (zeta) and static light scattering (SLS). Complement consumption was assessed in normal human serum (NHS) by measuring the residual haemolytic capacity of the complement system. RESULTS: DG loading did not change their size or zeta, whereas progressive BSA loading lightly decreased their zeta. An electrophoretic mobility analysis study showed the presence of two differently-charged sublayers at the NP(+) surface which are not affected by DG loading. Complement system activation, studied via a CH50 test, was suppressed by DG or BSA loading. We also demonstrated that NP(+)s could be loaded by a polyanionic molecule, such as BSA, after their preliminary filling by a hydrophobic molecule, such as DG. CONCLUSION: These nanoparticles are able to absorb large amounts of phospholipids or proteins without change in their size or zeta potential. Complement studies showed that stealth behaviour is observed when they are loaded and saturated either with anionic phospholipid or proteins

Bilateral Nonpenetrating Deep Sclerectomy: Difference in Outcomes Between First- and Second-Operated Eyes at 24 Months

BACKGROUND/AIM: The aim of this study was to assess the difference in outcome between the first-operated and the second-operated eyes after nonpenetrating deep sclerectomy (DS), and to identify potential success predictors for the second eye. METHODS: This single-surgeon, retrospective study analyzed the outcomes of all bilateral nonsimultaneous DS with at least 24 months of follow-up. Its main outcome measure was surgical success, defined as unmedicated intraocular pressure (IOP) ≤15 mm Hg associated with a relative reduction ≥20%. RESULTS: In all, 104 eyes of 52 patients who underwent bilateral (standalone or combined) DS, within a mean of 344.3±526.3 days of each other, were analyzed. Postoperatively, the mean medicated IOP decreased from 20.7±7.9 (first-operated eyes) and 19.3±6.6 mm Hg (second-operated eyes) at baseline (P=0.107) to 13.8±4.8 [(-33.3%; P<0.001) first-operated eyes) and 12.7±3.8 mm Hg [(-34.2%; P<0.001) second-operated eyes] after 2 years (P=0.619). Postoperative IOP and treatment reduction, respectively, showed fair (r=0.53) and good (r=0.71) levels of correlation between fellow eyes. The rates of complete success were comparable between first-operated and second-operated eyes (32.7% and 40.4%, respectively; P=0.364). At 2 years, among patients whose first-operated eyes were considered a success, 82.4% of surgeries in second eyes were successful (P=0.001). The odds ratio of a second-operated eye experiencing complete success were 6.32 (P=0.011) if the first-operated eye experienced complete success. CONCLUSIONS: The present study demonstrated a strong association between first-operated and second-operated eyes after DS, in terms of surgical outcomes and IOP reduction. In effect, surgical success in the first-operated eye increases the odds of success in the second eye by 6-fold

XEN 45 Gel Stent Implantation in Open Angle Glaucoma : 5-Year Results of a Prospective Study

Précis: XEN 45 Gel Stent is safe and effective for 3 years. The study results provide useful insight into the outcome of XEN 45 Gel Stent surgery over 5 years in daily clinical practice. Purpose: To evaluate 5-year outcomes of XEN 45 gel stent implantation (XEN) in patients with open angle glaucoma. Methods: This is a prospective, single-center, interventional study. XEN implantation either alone (XEN) or combined with phacoemulsification (Phaco + XEN) was performed on 170 consecutive eyes (126 patients) with uncontrolled intraocular pressure (IOP) or disease progression despite medical treatment. “Complete” surgical success at 60 months was defined as unmedicated IOP ≤15 mm Hg and a relative IOP reduction ≥20% from medicated baseline, while “qualified” success allowed fewer ocular hypotensive medications than at baseline. Other definitions of success with various IOP targets were also analyzed. Secondary outcomes included mean IOP and IOP-lowering medication changes and rates of reoperations. Results: Mean age was 78.1±9.2 years, and 70.3% were female. Mean medicated IOP decreased from 19.8±7.7 mm Hg [19.6±7.1 (XEN) vs. 19.8±7.0 mm Hg (Phaco+XEN)] at baseline to 12.6±3.1 mm Hg [12.5± 3.1 (XEN) vs. 12.6±3.1 (Phaco+XEN)] at 5 years (−37.0%; P &lt; 0.001). Medications decreased from 2.0±1.3 [2.0±1.3 (XEN) vs. 2.0±1.3 (Phaco+XEN)] to 0.8±1.1 [0.8±1.1 (XEN) vs. 0.8±1.1 (Phaco + XEN)] (−60%; P &lt;0.001). Needling was performed in 84 eyes (49%), and 19.4% underwent a secondary surgical intervention. Complete success at 3 years was a strong predictor of success at 5 years (odds ratio: 3.06, P &lt;0.01), while needling was associated with higher rates of failure (odds ratio: 3.6, P &lt;0.01). Conclusions: At 5 years, XEN gel stent implantation was a safe procedure and achieved clinically meaningful IOP and medication reduction. Success at 3 years is a predictor of success at 5 years. Needling correlates with higher failure rates.</p

Bilateral Nonpenetrating Deep Sclerectomy:Difference in Outcomes Between First- and Second-Operated Eyes at 24 Months

BACKGROUND/AIM: The aim of this study was to assess the difference in outcome between the first-operated and the second-operated eyes after nonpenetrating deep sclerectomy (DS), and to identify potential success predictors for the second eye. METHODS: This single-surgeon, retrospective study analyzed the outcomes of all bilateral nonsimultaneous DS with at least 24 months of follow-up. Its main outcome measure was surgical success, defined as unmedicated intraocular pressure (IOP) ≤15 mm Hg associated with a relative reduction ≥20%. RESULTS: In all, 104 eyes of 52 patients who underwent bilateral (standalone or combined) DS, within a mean of 344.3±526.3 days of each other, were analyzed. Postoperatively, the mean medicated IOP decreased from 20.7±7.9 (first-operated eyes) and 19.3±6.6 mm Hg (second-operated eyes) at baseline (P=0.107) to 13.8±4.8 [(-33.3%; P<0.001) first-operated eyes) and 12.7±3.8 mm Hg [(-34.2%; P<0.001) second-operated eyes] after 2 years (P=0.619). Postoperative IOP and treatment reduction, respectively, showed fair (r=0.53) and good (r=0.71) levels of correlation between fellow eyes. The rates of complete success were comparable between first-operated and second-operated eyes (32.7% and 40.4%, respectively; P=0.364). At 2 years, among patients whose first-operated eyes were considered a success, 82.4% of surgeries in second eyes were successful (P=0.001). The odds ratio of a second-operated eye experiencing complete success were 6.32 (P=0.011) if the first-operated eye experienced complete success. CONCLUSIONS: The present study demonstrated a strong association between first-operated and second-operated eyes after DS, in terms of surgical outcomes and IOP reduction. In effect, surgical success in the first-operated eye increases the odds of success in the second eye by 6-fold

Bilateral Non-penetrating Deep Sclerectomy

BACKGROUND/AIM: The aim of this study was to assess the difference in outcome between the first-operated and the second-operated eyes after nonpenetrating deep sclerectomy (DS), and to identify potential success predictors for the second eye.METHODS: This single-surgeon, retrospective study analyzed the outcomes of all bilateral nonsimultaneous DS with at least 24 months of follow-up. Its main outcome measure was surgical success, defined as unmedicated intraocular pressure (IOP) ≤15 mm Hg associated with a relative reduction ≥20%.RESULTS: In all, 104 eyes of 52 patients who underwent bilateral (standalone or combined) DS, within a mean of 344.3±526.3 days of each other, were analyzed. Postoperatively, the mean medicated IOP decreased from 20.7±7.9 (first-operated eyes) and 19.3±6.6 mm Hg (second-operated eyes) at baseline (P=0.107) to 13.8±4.8 [(-33.3%; P&lt;0.001) first-operated eyes) and 12.7±3.8 mm Hg [(-34.2%; P&lt;0.001) second-operated eyes] after 2 years (P=0.619). Postoperative IOP and treatment reduction, respectively, showed fair (r=0.53) and good (r=0.71) levels of correlation between fellow eyes. The rates of complete success were comparable between first-operated and second-operated eyes (32.7% and 40.4%, respectively; P=0.364). At 2 years, among patients whose first-operated eyes were considered a success, 82.4% of surgeries in second eyes were successful (P=0.001). The odds ratio of a second-operated eye experiencing complete success were 6.32 (P=0.011) if the first-operated eye experienced complete success.CONCLUSIONS: The present study demonstrated a strong association between first-operated and second-operated eyes after DS, in terms of surgical outcomes and IOP reduction. In effect, surgical success in the first-operated eye increases the odds of success in the second eye by 6-fold.</p

A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drug targeting.

International audienceOne of the new strategies to improve cancer chemotherapy is based on new drug delivery systems, like the polyethylene glycol-coated superparamagnetic iron oxide nanoparticles (PEG-SPION, thereafter called PS). In this study, PS are loaded with doxorubicin (DOX) anticancer drug, using a pre-formed DOX-Fe(2+) complex reversible at lower pH of tumour tissues and cancer cells. The DOX loaded PS (DLPS, 3% w/w DOX/iron oxide) present a hydrodynamic size around 60nm and a zeta potential near zero at physiological pH, both parameters being favourable for increased colloidal stability in biological media and decreased elimination by the immune system. At physiological pH of 7.4, 60% of the loaded drug is gradually released from the DLPS in ∼2h. The intracellular release and distribution of DOX is followed by means of confocal spectral imaging (CSI) of the drug fluorescence. The in vitro cytotoxicity of the DLPS on MCF-7 breast cancer cells is equivalent to that of a DOX solution. The reversible association of DOX to the SPION surface and the role of polymer coating on the drug loading/release are discussed, both being critical for the design of novel stealth magnetic nanovectors for chemotherapy