The menage a trois of autophagy, lipid droplets and liver disease

Autophagic pathways cross with lipid homeostasis and thus provide energy and essential building blocks that are indispensable for liver functions. Energy deficiencies are compensated by breaking down lipid droplets (LDs), intracellular organelles that store neutral lipids, in part by a selective type of autophagy, referred to as lipophagy. The process of lipophagy does not appear to be properly regulated in fatty liver diseases (FLDs), an important risk factor for the development of hepatocellular carcinomas (HCC). Here we provide an overview on our current knowledge of the biogenesis and functions of LDs, and the mechanisms underlying their lysosomal turnover by autophagic processes. This review also focuses on nonalcoholic steatohepatitis (NASH), a specific type of FLD characterized by steatosis, chronic inflammation and cell death. Particular attention is paid to the role of macroautophagy and macrolipophagy in relation to the parenchymal and non-parenchymal cells of the liver in NASH, as this disease has been associated with inappropriate lipophagy in various cell types of the liver. Abbreviations: ACAT: acetyl-CoA acetyltransferase; ACAC/ACC: acetyl-CoA carboxylase; AKT: AKT serine/threonine kinase; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BECN1/Vps30/Atg6: beclin 1; BSCL2/seipin: BSCL2 lipid droplet biogenesis associated, seipin; CMA: chaperone-mediated autophagy; CREB1/CREB: cAMP responsive element binding protein 1; CXCR3: C-X-C motif chemokine receptor 3; DAGs: diacylglycerols; DAMPs: danger/damage-associated molecular patterns; DEN: diethylnitrosamine; DGAT: diacylglycerol O-acyltransferase; DNL: de novo lipogenesis; EHBP1/NACSIN (EH domain binding protein 1); EHD2/PAST2: EH domain containing 2; CoA: coenzyme A; CCL/chemokines: chemokine ligands; CCl(4:) carbon tetrachloride; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FA: fatty acid; FFAs: free fatty acids; FFC: high saturated fats, fructose and cholesterol; FGF21: fibroblast growth factor 21; FITM/FIT: fat storage inducing transmembrane protein; FLD: fatty liver diseases; FOXO: forkhead box O; GABARAP: GABA type A receptor-associated protein; GPAT: glycerol-3-phosphate acyltransferase; HCC: hepatocellular carcinoma; HDAC6: histone deacetylase 6; HECT: homologous to E6-AP C-terminus; HFCD: high fat, choline deficient; HFD: high-fat diet; HSCs: hepatic stellate cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; ITCH/AIP4: itchy E3 ubiquitin protein ligase; KCs: Kupffer cells; LAMP2A: lysosomal associated membrane protein 2A; LDs: lipid droplets; LDL: low density lipoprotein; LEP/OB: leptin; LEPR/OBR: leptin receptor; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIR: LC3-interacting region; LPS: lipopolysaccharide; LSECs: liver sinusoidal endothelial cells; MAGs: monoacylglycerols; MAPK: mitogen-activated protein kinase; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCD: methionine-choline deficient; MGLL/MGL: monoglyceride lipase; MLXIPL/ChREBP: MLX interacting protein like; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver disease; NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; NPC: NPC intracellular cholesterol transporter; NR1H3/LXRα: nuclear receptor subfamily 1 group H member 3; NR1H4/FXR: nuclear receptor subfamily 1 group H member 4; PDGF: platelet derived growth factor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA: patatin like phospholipase domain containing; PNPLA2/ATGL: patatin like phospholipase domain containing 2; PNPLA3/adiponutrin: patatin like phospholipase domain containing 3; PPAR: peroxisome proliferator activated receptor; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARD/PPARδ: peroxisome proliferator activated receptor delta; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SE: sterol esters; SIRT1: sirtuin 1; SPART/SPG20: spartin; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; TAGs: triacylglycerols; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TGFB1/TGFβ: transforming growth factor beta 1; Ub: ubiquitin; UBE2G2/UBC7: ubiquitin conjugating enzyme E2 G2; ULK1/Atg1: unc-51 like autophagy activating kinase 1; USF1: upstream transcription factor 1; VLDL: very-low density lipoprotein; VPS: vacuolar protein sorting; WIPI: WD-repeat domain, phosphoinositide interacting; WDR: WD repeat domai

Design and Evaluation of Autophagy-Inducing Particles for the Treatment of Abnormal Lipid Accumulation

Autophagy is a fundamental housekeeping process by which cells degrade their components to maintain homeostasis. Defects in autophagy have been associated with aging, neurodegeneration and metabolic diseases. Non-alcoholic fatty liver diseases (NAFLDs) are characterized by hepatic fat accumulation with or without inflammation. No treatment for NAFLDs is currently available, but autophagy induction has been proposed as a promising therapeutic strategy. Here, we aimed to design autophagy-inducing particles, using the autophagy-inducing peptide (Tat-Beclin), and achieve liver targeting in vivo, taking NAFLD as a model disease. Polylactic acid (PLA) particles were prepared by nanoprecipitation without any surfactant, followed by surface peptide adsorption. The ability of Tat-Beclin nanoparticles (NP T-B) to modulate autophagy and to decrease intracellular lipid was evaluated in vitro by LC3 immunoblot and using a cellular model of steatosis, respectively. The intracellular localization of particles was evaluated by transmission electron microscopy (TEM). Finally, biodistribution of fluorescent NP T-B was evaluated in vivo using tomography in normal and obese mice. The results showed that NP T-B induce autophagy with a long-lasting and enhanced effect compared to the soluble peptide, and at a ten times lower dose. Intracellular lipid also decreased in a cellular model of NAFLD after treatment with T-B and NP T-B under the same dose conditions. Ultrastructural studies revealed that NP T-B are internalized and located in endosomal, endolysosomal and autolysosomal compartments, while in healthy and obese mice, NP T-B could accumulate for several days in the liver. Given the beneficial effects of autophagy-inducing particles in vitro, and their capacity to target the liver of normal and obese mice, NP T-B could be a promising therapeutic tool for NAFLDs, warranting further in vivo investigation

Design and Evaluation of Autophagy-Inducing Particles for the Treatment of Abnormal Lipid Accumulation

Autophagy is a fundamental housekeeping process by which cells degrade their components to maintain homeostasis. Defects in autophagy have been associated with aging, neurodegeneration and metabolic diseases. Non-alcoholic fatty liver diseases (NAFLDs) are characterized by hepatic fat accumulation with or without inflammation. No treatment for NAFLDs is currently available, but autophagy induction has been proposed as a promising therapeutic strategy. Here, we aimed to design autophagy-inducing particles, using the autophagy-inducing peptide (Tat-Beclin), and achieve liver targeting in vivo, taking NAFLD as a model disease. Polylactic acid (PLA) particles were prepared by nanoprecipitation without any surfactant, followed by surface peptide adsorption. The ability of Tat-Beclin nanoparticles (NP T-B) to modulate autophagy and to decrease intracellular lipid was evaluated in vitro by LC3 immunoblot and using a cellular model of steatosis, respectively. The intracellular localization of particles was evaluated by transmission electron microscopy (TEM). Finally, biodistribution of fluorescent NP T-B was evaluated in vivo using tomography in normal and obese mice. The results showed that NP T-B induce autophagy with a long-lasting and enhanced effect compared to the soluble peptide, and at a ten times lower dose. Intracellular lipid also decreased in a cellular model of NAFLD after treatment with T-B and NP T-B under the same dose conditions. Ultrastructural studies revealed that NP T-B are internalized and located in endosomal, endolysosomal and autolysosomal compartments, while in healthy and obese mice, NP T-B could accumulate for several days in the liver. Given the beneficial effects of autophagy-inducing particles in vitro, and their capacity to target the liver of normal and obese mice, NP T-B could be a promising therapeutic tool for NAFLDs, warranting further in vivo investigation

Conception et évaluation de particules thérapeutiques modulant l'autophagie

Autophagy is a fundamental house-cleaning process, which is widely conserved among all cells and across all species. Dysregulation of autophagy has been associated with various human diseases, most notably aging, neurodegeneration, cancer and metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is the most common metabolic liver disease worldwide. In recent years, many FDA-approved medicines which induce autophagy have shown to be beneficial in NAFLD mouse models. Nevertheless, direct evidence linking the autophagy-modulatory properties of these agents to the observed therapeutic effects is lacking. Furthermore, targeted autophagy modulation in the liver, while sparing other tissues, is a formidable challenge. Here, we wish to investigate the therapeutic potential of specific and targeted autophagy in the liver in the context of NAFLD. In Review (Annex 1) we unravel the complex relationship between autophagy, the selective degradation of lipid (lipophagy) and fatty liver disease. We propose that targeted autophagy/lipophagy induction in the liver together with interventions targeting early biosynthetic pathways of lipids, could be beneficial in the context of NAFLD and NASH. In Article 1, we describe the development and characterisation of autophagy inducing particles. We prove the superiority of this system to induce autophagy in vitro, compared to the non-formulated autophagy inducer. We also show that autophagy-inducing particles can induce lipid droplet degradation (lipophagy) in an in vitro model of NAFLD. Finally, to prove the translational potential of using such system in vivo, we evaluate the biodistribution of these particles in healthy and obese mice. We confirm liver targeting and accumulation of autophagy-inducing particles in the liver of both models. These data clearly demonstrate the potential of autophagy inducing particles for the treatment of NAFLD. Further studies will examine whether autophagy inducing particles can improve the metabolic profile and the fatty liver phenotype in animal models of NAFLD.L'autophagie est un processus de recyclage cellulaire qui est largement conservé dans toutes les cellules et chez toutes les espèces. La dérégulation de l'autophagie a été associée à diverses maladies humaines, notamment le vieillissement, la neurodégénération, le cancer et les maladies métaboliques. La stéatose hépatique non alcoolique (NAFLD) est la maladie métabolique du foie la plus répandue dans le monde. Ces dernières années, de nombreux médicaments approuvés par la FDA qui induisent l'autophagie se sont révélés bénéfiques dans des modèles de souris NAFLD. Néanmoins, les preuves directes reliant les propriétés modulatrices de l'autophagie de ces agents aux effets thérapeutiques observés font défaut. En outre, la modulation ciblée de l'autophagie dans le foie, tout en épargnant d'autres tissus, est un formidable défi. Nous souhaitons ici étudier le potentiel thérapeutique d'une autophagie spécifique et ciblée dans le foie dans le contexte de la NAFLD. Dans la revue, nous démêlons la relation complexe entre l'autophagie, la dégradation sélective des lipides (lipophagie) et la maladie du foie gras. Nous proposons qu'une induction ciblée de l'autophagie/lipophagie dans le foie, associée à des interventions ciblant les premières voies de biosynthèse des lipides, pourrait être bénéfique dans le contexte de la NAFLD et de la NASH. Dans l'article, nous décrivons le développement et la caractérisation de particules induisant l'autophagie. Nous prouvons la supériorité de ce système pour induire l'autophagie in vitro, par rapport à l'inducteur d'autophagie non formulé. Nous montrons également que les particules induisant l'autophagie peuvent induire la dégradation des gouttelettes lipidiques dans un modèle in vitro de NAFLD. Enfin, pour prouver le potentiel translationnel de l'utilisation de ce système in vivo, nous évaluons la biodistribution de ces particules chez des souris saines et obèses. Nous confirmons le ciblage du foie et l'accumulation des particules induisant l'autophagie dans le foie des deux modèles. Ces données démontrent clairement le potentiel des particules induisant l'autophagie pour le traitement de la NAFLD. D'autres études examineront si les particules induisant l'autophagie peuvent améliorer le profil métabolique et le phénotype du foie gras dans les modèles animaux de la NAFLD

Conception et évaluation de particules thérapeutiques modulant l'autophagie

L'autophagie est un processus de recyclage cellulaire qui est largement conservé dans toutes les cellules et chez toutes les espèces. La dérégulation de l'autophagie a été associée à diverses maladies humaines, notamment le vieillissement, la neurodégénération, le cancer et les maladies métaboliques. La stéatose hépatique non alcoolique (NAFLD) est la maladie métabolique du foie la plus répandue dans le monde. Ces dernières années, de nombreux médicaments approuvés par la FDA qui induisent l'autophagie se sont révélés bénéfiques dans des modèles de souris NAFLD. Néanmoins, les preuves directes reliant les propriétés modulatrices de l'autophagie de ces agents aux effets thérapeutiques observés font défaut. En outre, la modulation ciblée de l'autophagie dans le foie, tout en épargnant d'autres tissus, est un formidable défi. Nous souhaitons ici étudier le potentiel thérapeutique d'une autophagie spécifique et ciblée dans le foie dans le contexte de la NAFLD. Dans la revue, nous démêlons la relation complexe entre l'autophagie, la dégradation sélective des lipides (lipophagie) et la maladie du foie gras. Nous proposons qu'une induction ciblée de l'autophagie/lipophagie dans le foie, associée à des interventions ciblant les premières voies de biosynthèse des lipides, pourrait être bénéfique dans le contexte de la NAFLD et de la NASH. Dans l'article, nous décrivons le développement et la caractérisation de particules induisant l'autophagie. Nous prouvons la supériorité de ce système pour induire l'autophagie in vitro, par rapport à l'inducteur d'autophagie non formulé. Nous montrons également que les particules induisant l'autophagie peuvent induire la dégradation des gouttelettes lipidiques dans un modèle in vitro de NAFLD. Enfin, pour prouver le potentiel translationnel de l'utilisation de ce système in vivo, nous évaluons la biodistribution de ces particules chez des souris saines et obèses. Nous confirmons le ciblage du foie et l'accumulation des particules induisant l'autophagie dans le foie des deux modèles. Ces données démontrent clairement le potentiel des particules induisant l'autophagie pour le traitement de la NAFLD. D'autres études examineront si les particules induisant l'autophagie peuvent améliorer le profil métabolique et le phénotype du foie gras dans les modèles animaux de la NAFLD.Autophagy is a fundamental house-cleaning process, which is widely conserved among all cells and across all species. Dysregulation of autophagy has been associated with various human diseases, most notably aging, neurodegeneration, cancer and metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is the most common metabolic liver disease worldwide. In recent years, many FDA-approved medicines which induce autophagy have shown to be beneficial in NAFLD mouse models. Nevertheless, direct evidence linking the autophagy-modulatory properties of these agents to the observed therapeutic effects is lacking. Furthermore, targeted autophagy modulation in the liver, while sparing other tissues, is a formidable challenge. Here, we wish to investigate the therapeutic potential of specific and targeted autophagy in the liver in the context of NAFLD. In Review (Annex 1) we unravel the complex relationship between autophagy, the selective degradation of lipid (lipophagy) and fatty liver disease. We propose that targeted autophagy/lipophagy induction in the liver together with interventions targeting early biosynthetic pathways of lipids, could be beneficial in the context of NAFLD and NASH. In Article 1, we describe the development and characterisation of autophagy inducing particles. We prove the superiority of this system to induce autophagy in vitro, compared to the non-formulated autophagy inducer. We also show that autophagy-inducing particles can induce lipid droplet degradation (lipophagy) in an in vitro model of NAFLD. Finally, to prove the translational potential of using such system in vivo, we evaluate the biodistribution of these particles in healthy and obese mice. We confirm liver targeting and accumulation of autophagy-inducing particles in the liver of both models. These data clearly demonstrate the potential of autophagy inducing particles for the treatment of NAFLD. Further studies will examine whether autophagy inducing particles can improve the metabolic profile and the fatty liver phenotype in animal models of NAFLD

The menage a trois of autophagy, lipid droplets and liver disease

Autophagic pathways cross with lipid homeostasis and thus provide energy and essential building blocks that are indispensable for liver functions. Energy deficiencies are compensated by breaking down lipid droplets (LDs), intracellular organelles that store neutral lipids, in part by a selective type of autophagy, referred to as lipophagy. The process of lipophagy does not appear to be properly regulated in fatty liver diseases (FLDs), an important risk factor for the development of hepatocellular carcinomas (HCC). Here we provide an overview on our current knowledge of the biogenesis and functions of LDs, and the mechanisms underlying their lysosomal turnover by autophagic processes. This review also focuses on nonalcoholic steatohepatitis (NASH), a specific type of FLD characterized by steatosis, chronic inflammation and cell death. Particular attention is paid to the role of macroautophagy and macrolipophagy in relation to the parenchymal and non-parenchymal cells of the liver in NASH, as this disease has been associated with inappropriate lipophagy in various cell types of the liver.Abbreviations: ACAT: acetyl-CoA acetyltransferase; ACAC/ACC: acetyl-CoA carboxylase; AKT: AKT serine/threonine kinase; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BECN1/Vps30/Atg6: beclin 1; BSCL2/seipin: BSCL2 lipid droplet biogenesis associated, seipin; CMA: chaperone-mediated autophagy; CREB1/CREB: cAMP responsive element binding protein 1; CXCR3: C-X-C motif chemokine receptor 3; DAGs: diacylglycerols; DAMPs: danger/damage-associated molecular patterns; DEN: diethylnitrosamine; DGAT: diacylglycerol O-acyltransferase; DNL: de novo lipogenesis; EHBP1/NACSIN (EH domain binding protein 1); EHD2/PAST2: EH domain containing 2; CoA: coenzyme A; CCL/chemokines: chemokine ligands; CCl4: carbon tetrachloride; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FA: fatty acid; FFAs: free fatty acids; FFC: high saturated fats, fructose and cholesterol; FGF21: fibroblast growth factor 21; FITM/FIT: fat storage inducing transmembrane protein; FLD: fatty liver diseases; FOXO: forkhead box O; GABARAP: GABA type A receptor-associated protein; GPAT: glycerol-3-phosphate acyltransferase; HCC: hepatocellular carcinoma; HDAC6: histone deacetylase 6; HECT: homologous to E6-AP C-terminus; HFCD: high fat, choline deficient; HFD: high-fat diet; HSCs: hepatic stellate cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; ITCH/AIP4: itchy E3 ubiquitin protein ligase; KCs: Kupffer cells; LAMP2A: lysosomal associated membrane protein 2A; LDs: lipid droplets; LDL: low density lipoprotein; LEP/OB: leptin; LEPR/OBR: leptin receptor; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIR: LC3-interacting region; LPS: lipopolysaccharide; LSECs: liver sinusoidal endothelial cells; MAGs: monoacylglycerols; MAPK: mitogen-activated protein kinase; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCD: methionine-choline deficient; MGLL/MGL: monoglyceride lipase; MLXIPL/ChREBP: MLX interacting protein like; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver disease; NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; NPC: NPC intracellular cholesterol transporter; NR1H3/LXRα: nuclear receptor subfamily 1 group H member 3; NR1H4/FXR: nuclear receptor subfamily 1 group H member 4; PDGF: platelet derived growth factor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA: patatin like phospholipase domain containing; PNPLA2/ATGL: patatin like phospholipase domain containing 2; PNPLA3/adiponutrin: patatin like phospholipase domain containing 3; PPAR: peroxisome proliferator activated receptor; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARD/PPARδ: peroxisome proliferator activated receptor delta; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SE: sterol esters; SIRT1: sirtuin 1; SPART/SPG20: spartin; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; TAGs: triacylglycerols; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TGFB1/TGFβ: transforming growth factor beta 1; Ub: ubiquitin; UBE2G2/UBC7: ubiquitin conjugating enzyme E2 G2; ULK1/Atg1: unc-51 like autophagy activating kinase 1; USF1: upstream transcription factor 1; VLDL: very-low density lipoprotein; VPS: vacuolar protein sorting; WIPI: WD-repeat domain, phosphoinositide interacting; WDR: WD repeat domain.</p

Development and characterization of liposomal formulation of bortezomib

Bortezomib is a proteasome inhibitor used for the treatment of multiple myeloma. The poor pharmacokinetic profile and off-target adverse effects provide a strong incentive to develop drug delivery systems for bortezomib. In the past, liposomal encapsulation has been proven to improve the therapeutic index of a variety of anti-neoplastic therapeutics. Here, we developed and characterized liposomal bortezomib formulations in order to find the most optimal loading conditions. Polyols were used to entrap bortezomib inside the liposomes as boronate ester via a remote loading strategy. Effect of various polyols, incubation duration, temperature, and total lipid concentration on loading efficiency was examined. Moreover, the effect of drug/lipid ratio on the release kinetics was studied. Loading efficiency was maximal when using meglumine plus mannitol as entrapping agents. Loading at room temperature was better than at 60 °C and loading efficiency was increased with increasing total lipid concentrations. There was a positive correlation between drug/lipid ratio and released amount of bortezomib. In vitro release kinetics in HBS and human plasma showed time dependent release. In HBS, at 4 °C, only 20% of the drug was released in three weeks, whereas at 37 °C 85% of the drug was released in 24 h. In human plasma, 5% of the drug retained after 24 h indicating faster release. Taken together, the most favorable liposomal formulation of bortezomib should be further exploited to study in vitro and in vivo efficacy performance

Development and characterization of liposomal formulation of bortezomib

Bortezomib is a proteasome inhibitor used for the treatment of multiple myeloma. The poor pharmacokinetic profile and off-target adverse effects provide a strong incentive to develop drug delivery systems for bortezomib. In the past, liposomal encapsulation has been proven to improve the therapeutic index of a variety of anti-neoplastic therapeutics. Here, we developed and characterized liposomal bortezomib formulations in order to find the most optimal loading conditions. Polyols were used to entrap bortezomib inside the liposomes as boronate ester via a remote loading strategy. Effect of various polyols, incubation duration, temperature, and total lipid concentration on loading efficiency was examined. Moreover, the effect of drug/lipid ratio on the release kinetics was studied. Loading efficiency was maximal when using meglumine plus mannitol as entrapping agents. Loading at room temperature was better than at 60 °C and loading efficiency was increased with increasing total lipid concentrations. There was a positive correlation between drug/lipid ratio and released amount of bortezomib. In vitro release kinetics in HBS and human plasma showed time dependent release. In HBS, at 4 °C, only 20% of the drug was released in three weeks, whereas at 37 °C 85% of the drug was released in 24 h. In human plasma, 5% of the drug retained after 24 h indicating faster release. Taken together, the most favorable liposomal formulation of bortezomib should be further exploited to study in vitro and in vivo efficacy performance

Development and characterization of liposomal formulation of bortezomib

Bortezomib is a proteasome inhibitor used for the treatment of multiple myeloma. The poor pharmacokinetic profile and off-target adverse effects provide a strong incentive to develop drug delivery systems for bortezomib. In the past, liposomal encapsulation has been proven to improve the therapeutic index of a variety of anti-neoplastic therapeutics. Here, we developed and characterized liposomal bortezomib formulations in order to find the most optimal loading conditions. Polyols were used to entrap bortezomib inside the liposomes as boronate ester via a remote loading strategy. Effect of various polyols, incubation duration, temperature, and total lipid concentration on loading efficiency was examined. Moreover, the effect of drug/lipid ratio on the release kinetics was studied. Loading efficiency was maximal when using meglumine plus mannitol as entrapping agents. Loading at room temperature was better than at 60 °C and loading efficiency was increased with increasing total lipid concentrations. There was a positive correlation between drug/lipid ratio and released amount of bortezomib. In vitro release kinetics in HBS and human plasma showed time dependent release. In HBS, at 4 °C, only 20% of the drug was released in three weeks, whereas at 37 °C 85% of the drug was released in 24 h. In human plasma, 5% of the drug retained after 24 h indicating faster release. Taken together, the most favorable liposomal formulation of bortezomib should be further exploited to study in vitro and in vivo efficacy performance