Comparison of classical, stealth and super-stealth liposomes for intravenous delivery of lumefantrine: Formulation, characterization and pharmacodynamic study

Purpose: To develop and compare classical liposomes (CL), stealth liposomes (SL) and super-stealth liposomes (SSL) encapsulating lumefantrine for intravenous administration. Method: CL, SL or SSL were prepared by thin-layer evaporation method and evaluated for particle size, polydispersity index (PdI), encapsulation efficiency and short-term stability. Pharmacodynamic study using mice infected with Plasmodium berghei was also carried out. Results: The particle sizes (nm) and PDI of the liposomes were: CL (248 ± 44.89; 0.78 ± 0.02), SL (235.8 ± 45.18; 0.39 ± 0.06) and SSL (238.2 ± 23.0; 0.24 ± 0.04). Encapsulation efficiency was highest in SSL (66 %), followed by SL (44.4 %) and then by CL (42.5 %). SSL was the most stable after 72 h of storage. In vivo, lumefantrine produced significant reduction in parasitaemia after 7 days (p < 0.05) by SSL (68.3 ± 8.9 %) followed by CL (55.8 ± 15.2 %) and then SL (53.4 ± 14.9 %). Conclusion: SSL formulation of lumefantrine exhibits good physicochemical and pharmacodynamic potentials and should be further investigated in future studies for the treatment of malaria

Anti-HER2 Super Stealth Immunoliposomes for Targeted-Chemotherapy

: Liposomes play an important role in the field of drug delivery by virtue of their biocompatibility and versatility as carriers. Stealth liposomes, obtained by surface decoration with hydrophilic polyethylene glycol (PEG) molecules, represented an important turning point in liposome technology, leading to significant improvements in the pharmacokinetic profile compared to naked liposomes. Nevertheless, the generation of effective targeted liposomes - a central issue for cancer therapy - has faced several difficulties and clinical phase failures. Active targeting remains a challenge for liposomes. In this direction, we designed a new Super Stealth Immunoliposomes (SSIL2) composed of a PEG-bi-phospholipids derivative that stabilizes the polymer shielding over the liposomes. Furthermore, its counterpart, conjugated to the fragment antigen-binding of trastuzumab (Fab'TRZ -PEG-bi-phospholipids), is firmly anchored on the liposomes surface and correctly orients outward the targeting moiety. Throughout this study, the performances of SSIL2 are evaluated and compared to classic stealth liposomes and stealth immunoliposomes in vitro in a panel of cell lines and in vivo studies in zebrafish larvae and rodent models. Overall, SSIL2 shows superior in vitro and in vivo outcomes, both in terms of safety and anticancer efficacy, thus representing a step forward in targeted cancer therapy, and valuable for future development. This article is protected by copyright. All rights reserved

Development of novel super stealth immunoliposomes for anticancer drug delivery

Nowadays, liposomes play an important role in the field of drug delivery since they are biocompatible and versatile carriers. Their surface modification with hydrophilic polymers, usually polyethylene glycol (PEG), confers “stealth” properties, thus avoiding the fast clearance by the reticuloendothelial system and thereby increasing their circulation half-life in vivo. In such way, passive accumulation in the tumor site, exploiting the enhanced vascular permeability and lack of lymphatic drainage (EPR effect [1]) typically found in tumor tissues, is favoured. The present work aimed at formulating new Super Stealth Immunoliposomes (SSILs), which should be both stable in the bloodstream and able to reach selectively the tumor site. The enhanced stability was achieved by using PEG dendron molecules conjugated to 2 or 4 molecules of distearoylphosphatidylethanolamine (DSPE) [2]. This allowed to increase the hydrophobic interactions with the phospholipid bilayer with respect to the classical PEG-single phospholipid derivatives, thus avoiding the polymer detachment. Active targeting, instead, was obtained by conjugation of these PEG dendron-lipids derivatives to a targeting moiety. In this case, the Fab’ (fragment, antigen-binding) derived from Trastuzumab is used to target with high affinity HER-2 (human epidermal growth factor receptor 2), which is overexpressed on the surface of certain tumor cells. Doxorubicin, an antineoplastic drug commonly used in the treatment of a wide range of cancers (leukaemia, lymphoma, many types of carcinoma and soft tissue sarcomas), was chosen to be loaded into these nanocarriers. Since Doxyl®/Caelyx®, doxorubicin stealth liposomal formulation on the market, contains mPEG2kDa-DSPE, it was initially decided to formulate SSILs using as well PEG2kDa dendron-lipids derivatives. Preliminary in vitro and in vivo experiments on super stealth liposomes (SSLn, n=2 or 4 DSPE), including either 5% or 10% mol of mPEG2kda-(DSPE)n and formulated without the targeting ligand, evidenced a negative trend of stability with the increase of the hydrophobic anchor (PEG-DSPE > PEG-DSPE2 > PEG-DSPE4). This behavior was confirmed by in vivo pharmacokinetics since stealth liposomes (SL) presented a prolonged half-life (t½ ~22h) compared to super stealth liposomes (SSL2 t½ ~8h and SSL4 t½ ~7h), which were eliminated even faster from the bloodstream than naked liposomes (t½ ~10h). For this reason, it was decided to formulate super stealth immunoliposomes using a higher MW polymer, namely PEG 5kDa. mPEG5kDa-(DSPE)n and Boc-NH-PEG5kDa-(DSPE)n derivatives were successfully synthesized, purified and characterized by 1H NMR spectroscopy. The Fab’ fragment of Trastuzumab, chosen as targeting agent. Functionalization of NH2-PEG-phopsholipids derivatives with N-(ß-maleimidopropyloxy)succinimide ester (BMPS) provided the best yields of monoPEGylated Fab’- PEG5kDa-DSPE, Fab’-PEG5kDa-(DSPE)2 and PEG5kDa-(DSPE)4, as evidenced by SDS-PAGE analysis. Post-insertion of mPEG-lipid(s) derivatives or/and ligand-coupled PEG-lipid(s) derivatives into drug-loaded pre-formed naked liposomes either failed or caused aggregation of the liposomal vesicles. TEM analysis evidenced jagged and discontinuous surfaces, justifying the physical instability of the formulated vesicles. Post-insertion of Fab’-PEG dendron-lipids derivatives on doxorubicin-loaded pre-formed stealth (SL) and super stealth liposomes (SSLn) to obtain the corresponding super stealth immunoliposomes (SSILn) provided stable and homogeneous SSIL2 (102.11 ± 3.68 nm) and SIL (128.23 ± 1.02 nm) with low polydispersity index (PdI 0.1). According to drug release experiments doxorubicin was efficiently entrapped inside the nanocarrier and drug leakage was not observed within the 16 h of incubation in all tested formulations. Preliminary in vitro cytotoxicity studies were performed on human breast ductal carcinoma cell line (BT-474) overexpressing HER-2, evidenced that SL-DXR could not reduce the cell viability below 50% after a 24 h-treatment with 10 µM DXR, whereas both SIL-DXR and SSIL2-DXR reduced cell viability to about 40% in the same experimental conditions. Preliminary IC50 calculation evidenced that, at the tested conditions, all the doxorubicin-loaded liposomal formulations possessed the same potency in inducing cell death, whereas small differences were observed taking into consideration the efficacy of each formulation (SL-DXR < SIL-DXR < SSIL2-DXR). Interestingly, SSIL2-DXR possessed the same efficacy of free doxorubicin. In vivo pharmacokinetic studies in rats evidenced the prolonged half-life of SSIL2 (t½ = 37.80 h) compare to SL (t½ = 10.77 h), confirming the stabilizing effect of PEG dendron-lipids derivatives over mPEG-DSPE. Accordingly, a reduction in the clearance rate of SSIL2 (~0.2 ml/h) was observed with respect to SL (~0.5 ml/h), resulting in increased bioavailability (AUC) and distribution volume (Vd), also compared to all the other tested formulations. In vivo organ toxicity evaluation after single dose administration of 2.5 mg/kg in DXR evidenced that the gene expression of the three pro-inflammatory cytokines interleukin β1 (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNFα) was enhanced in rats treated with SL-DXR and SIL-DXR, especially in liver, spleen and heart tissues. Histological analyses performed on liver and spleen sections of rats treated with SL-DXR and SIL-DXR showed remarkable alterations (granulomatous lesions, apoptotic bodies, etc.), whereas those treated with free DXR and SSIL2-DXR resulted generally healthy. Heart, lungs and brain didn’t show any pathological alteration in all the groups of rats examined. Overall, SSIL2 proved to be the best and safest formulation both in terms of pharmacokinetic profile, cytokines expression and histological analysis of RES organs, thus representing a promising system to improve conventional cancer therapy by enhancing drug delivery and antitumor efficacy.Attualmente, i liposomi rivestono un ruolo importante nel campo del drug delivery per la loro biocompatibilità e versatilità. La modifica della superficie liposomiale con polimeri idrofilici, generalmente polietilenglicole (PEG), conferisce proprietà “stealth” consentendo di evitare la rapida eliminazione da parte del sistema reticoloendoteliale, aumentando così il tempo di emivita in vivo. In questo modo, è favorito l’accumulo passivo nel sito tumorale, sfruttando l’aumentata permeabilità vascolare e il ridotto drenaggio linfatico (effetto EPR [1]) tipici dei tessuti tumorali. Il presente lavoro è mirato alla formulazione di nuovi immunoliposomi super stealth (SSILs) dotati sia di maggiore stabilità nel circolo sanguigno sia di un targeting selettivo al sito tumorale. L’aumentata stabilità è stata ottenuta mediante l’uso di molecole di PEG dendrone coniugate a 2 o 4 molecole di distearoilfosfoetanolamina (DSPE) [2]. Questo ha consentito di aumentare le interazioni idrofobiche con il doppio strato fosfolipidico rispetto al classico derivato PEG-fosfolipide ed evitare il distacco del polimero durante il circolo ematico. Il targeting attivo, invece, è stato ottenuto mediante coniugazione di questi derivati PEG dendrone-lipidi ad un agente di targeting. In questo caso, il Fab’ (fragment, antigen-binding) del Trastuzumab che ha una elevata affinità verso il recettore HER-2 (human epidermal growth factor receptor 2), sovraespresso sulla superficie di alcune cellule tumorali. La doxorubicina, agente antineoplastico comunemente utilizzato nel trattamento di una grande varietà di tumori (lucemie, linfomi, vari tipi di carcinoma e sarcomi), è stata scelta per essere incapsulata all’interno di questi nanocarriers. Dal momento che Doxyl®/Caelyx®, formulazione liposomiale stealth contenente doxorubicina presente in commercio, contiene mPEG2kDa-DSPE, è stato inizialmente deciso di formulare gli SSILs usando dei derivati PEG2kDa-dendrone-lipidi. Studi preliminari in vitro e in vivo effettuati su liposomi super stealth (SSLn, n=2 o 4 DSPE) contenenti il 5% o 10% mol di mPEG2kda-(DSPE)n e privi di agente di targeting, hanno evidenziato un trend negativo di stabilità all’aumentare dell’ancoraggio idrofobico (PEG-DSPE > PEG-DSPE2 > PEG-DSPE4). Questo comportamento è stato confermato dagli studi di farmacocinetica in vivo poiché i liposomi stealth (SL) presentavano un’emivita prolungata (t½ ~22h) rispetto ai liposomi super stealth (SSL2 t½ ~8h e SSL4 t½ ~7h), i quali venivano eliminati dal circolo sanguigno anche più rapidamente dei liposomi convenzionali (t½ ~10h). Per questo motivo è stato deciso di formulare gli immunoliposomi super stealth usando un polimero a maggior peso molecolare (PEG 5kDa). I derivati mPEG5kDa-(DSPE)n e Boc-NH-PEG5kDa-(DSPE)n sono stati sintetizzati, purificati e caratterizzati con successo mediante spettroscopia 1H NMR. La funzionalizzazione dei derivati H2N-PEG-lipidi con il cross-linker BMPS (estere N-(ß-maleimidopropilossi)succinimide) ha fornito rese migliori ottenendo i monoPEGhilati Fab’-PEG5kDa-DSPE, Fab’-PEG5kDa-(DSPE)2 and PEG5kDa-(DSPE)4, come dimostrato dalla caratterizzazione mediante SDS-PAGE. La post-insertion dei derivati mPEG-(DSPE)n e/o Fab’-PEG-(DSPE)n in liposomi convenzionali preformati contenenti il farmaco non è avvenuta con successo o ha portato all’aggregazione delle vescicole liposomiali. Le immagini acquisite mediante TEM hanno evidenziato superfici discontinue, motivando l’instabilità delle vescicole formulate. La post-insertion dei derivati Fab’-PEG dendrone-lipidi su liposomi stealth (SL) e super stealth (SSLn) preformati contenenti doxorubicina, per ottenere i corrispondenti immunoliposomi super stealth (SSILn), ha fornito formulazioni stabili ed omogenee di SSIL2 (102.11 ± 3.68 nm) e SIL (128.23 ± 1.02 nm) con basso indice di polidispersività (PdI 0.1). Studi di rilascio in vitro hanno confermato che tutte le formulazioni testate sono in grado di trattenere efficacemente la doxorubicina incapsulata, la quale non viene rilasciata durante le 16 ore di incubazione. Studi preliminari in vitro di citotossicità, eseguiti su cellule umane di carcinoma mammario (BT-474) che sovraesprimono HER-2, hanno dimostrato che SL-DXR non sono in grado di ridurre la vitalità cellulare al di sotto del 50% dopo un trattamento di 24 ore con DXR 10 µM, mentre risulta ridotta al 40% dopo trattamento con SIL-DXR e SSIL2-DXR nelle stesse condizioni sperimentali. Un calcolo preliminare dell’IC50 ha dimostrato che tutte le formulazioni liposomiali possiedono la stessa potenza nell’indurre la morte cellulare, mentre minime differenze sono state osservate prendendo in considerazione l’efficacia di ciascuna formulazione (SL-DXR < SIL-DXR < SSIL2-DXR). È interessante notare che SSIL2-DXR dimostrano la stessa efficacia della DXR libera. Studi di farmacocinetica in vivo hanno evidenziato la prolungata emivita di SSIL2 (t½ = 37.80 h), confermando l’effetto stabilizzante dei derivati PEG dendrone-lipidi rispetto a mPEG-DSPE. Di conseguenza, una riduzione della clearance plasmatica di SSIL2 (~0.2 ml/h) è stata osservata rispetto a SL (~0.5 ml/h), con aumento della biodisponibilità (AUC) e del volume di biodistribuzione (Vd) rispetto alle altre formulazioni testate. La valutazione della tossicità d’organo in vivo dopo una singola somministrazione di 2.5 mg/kg in DXR ha messo in evidenza che l’espressione genica delle tre citochine proinfiammatorie interleukina β1 (IL-1β), interleukina 6 (IL-6) e tumor necrosis factor α (TNFα) è aumentata nei ratti trattati con SL-DXR e SIL-DXR, specialmente nel fegato, nella milza e nel cuore. L’analisi istologica effettuata su sezioni di tessuto di fegato e milza di ratti trattati con SL-DXR e SIL-DXR ha dimostrato la presenza di notevoli alterazioni patologiche (lesioni granulomatose, corpi apoptotici, ecc), mentre gli organi dei ratti trattati con DXR e SSIL2-DXR sono risultati complessivamente sani. Nel cuore, nei polmoni e nel cervello non è stata evidenziata alcuna alterazione patologica in tutti i gruppi di animali esaminati. In generale, SSIL2 sono emersi come la formulazione migliore e più sicura in termini di profilo farmacocinetico, espressione di citochine infiammatorie e analisi istologica degli organi del sistema reticolo endoteliale (RES), rappresentando perciò un promettente sistema per migliorare il veicolamento di farmaci antitumorali convenzionali

A maladaptive ER stress response triggers dysfunction in highly active muscles of mice with SELENON loss

open12noThis study was supported by a Telethon (Italy) career award (TDEZ00112T), an ERC Cariplo grant (2014-1856), a Cariplo Biomedical Science (Italy) for young scientist grant (2014-1075) to EZ and a STARS consolidator grant, AFM Telethon (France) grant (21865) to BB.Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca 2+ uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca 2+ uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca 2+ uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.openPozzer, Diego; Varone, Ersilia; Chernorudskiy, Alexander; Schiarea, Silvia; Missiroli, Sonia; Giorgi, Carlotta; Pinton, Paolo; Canato, Marta; Germinario, Elena; Nogara, Leonardo; Blaauw, Bert; Zito, EsterPozzer, Diego; Varone, Ersilia; Chernorudskiy, Alexander; Schiarea, Silvia; Missiroli, Sonia; Giorgi, Carlotta; Pinton, Paolo; Canato, Marta; Germinario, Elena; Nogara, Leonardo; Blaauw, Bert; Zito, Este

Super Stealth immunoliposomes as a strategy to overcome liposome-induced liver toxicity

Background and Aims: Immunoliposomes (ILs) are nano-delivery systems functionalized with monoclonal antibodies or antibody fragments, with the double aim of ameliorating the pharmacokinetics and tolerability of incapsulated drugs and permitting targeted therapy. Binding poly-ethylene-glycol (PEG) chains to their surface, thus obtaining stealth ILs, delays their elimination, which is mainly due to the clearance operated by the reticuloendothelial system (RES). Recently, to further improve their biopharmaceutical and pharmacokinetic features, the so called \u201csuper stealth liposomes\u201d have been proposed, by adding mPEG-dendron-phospholipids. However, these nano-sized materials can accumulate in the liver and cause hepatic toxicity. On the basis of these considerations, the aim of this study was to evaluate, at the histological and molecular level, the in vivo liver toxicity of two formulations, one of stealth (SIL) and one of new super-stealth IL (SSIL2), both loaded with doxorubicin, in Sprague-Dawley rats. Method: A dose of 2.5 mg/kg of doxorubicin-loaded immunoliposomes (SIL and SSIL2) was administered via caudal vein to Sprague-Dawley female rats (n=3 per group) and vehicle-administered rats were used as controls. Rats were sacrificed 48 hours after the treatment. Hepatic toxicity of the formulations was assessed by: 1) standard histological analysis performed on 5-\ub5m sections of liver tissues stained with H&E; 2) mRNA hepatic expression of IL-1\uf062, IL-6 and TNF-\uf061; 3) reactive oxygen species (ROS) concentration in liver tissues. The results were compared by one-way ANOVA followed by Dunnett\u2019s post-hoc test. p<0.05 was considered statistically significant. Results: Rats treated with SIL showed hepatic histological alterations, i.e. numerous granulomatous lesions, sometimes associated with apoptotic bodies, whereas in rats treated with SSIL2 only few isolated granulomas could be observed in the otherwise healthy livers. The expression of both IL-1\u3b2 and TNF\u3b1 was significantly increased only in in SIL-treated rats (p <0.001 vs controls) and did not change in SSIL2 \u2013 treated rats with respect to controls. Accordingly, the concentration of hepatic ROS increased significantly in SIL-treated rats (p<0.001 vs controls) and was comparable to that of controls in SSIL2 \u2013 treated rats. Conclusion: SILs are able to induce dramatic alterations of the hepatic parenchyma, probably due to their preferential deposition in hepatic tissue, which is particularly rich in RES cells. Conversely, SSIL2 caused only limited histological liver alterations. Therefore, SSIL2s, besides their pharmacokinetic advantages, permit to overcome the hepatic toxicity caused by SIL administration, thus representing a smart strategy to improve the tolerability of cancer therapy

Novel Super Stealth immunoliposomes for cancer targeted delivery of doxorubicin: an innovative strategy to reduce liver toxicity

Introduction: Immunoliposomes (ILs) are nano-delivery systems functionalized with monoclonal antibodies, with the aim of ameliorating the pharmacokinetics and tolerability of incapsulated drugs and permitting targeted therapy. Binding poly-ethylene-glycol (PEG) chains to their surface, thus obtaining stealth ILs (SILs), delays their elimination, which is mainly due to the clearance operated by the reticuloendothelial system (RES). Recently, to further improve their pharmacokinetic features, the super stealth immunoliposomes (SSIL) have been proposed, by adding mPEG-dendron-phospholipids. However, these nano-sized materials can accumulate in the liver and cause hepatic toxicity. Aims: To evaluate the in vivo liver toxicity of two formulations, one of stealth (SIL) and one of new super-stealth IL (SSIL2), both loaded with doxorubicin, in rats. Methods: A dose of 2.5 mg/kg of doxorubicin-loaded immunoliposomes (SIL and SSIL2) was administered via caudal vein to Sprague-Dawley female rats (n=3 per group) and vehicle-administered rats were used as controls. Rats were sacrificed 48 hours after the treatment. Hepatic toxicity of the formulations was assessed by: 1) standard histological analysis performed on 5-\ub5m sections of liver tissues stained with H&E; 2) mRNA hepatic expression of IL-1b, IL-6 and TNF-a; 3) reactive oxygen species (ROS) in liver tissues. Results were compared by one-way ANOVA followed by Dunnett\u2019s post-hoctest. p<0.05 was considered statistically significant. Results: SIL-treated rats showed histological alterations, i.e. numerous granulomatous lesions, sometimes associated with apoptotic bodies, whereas in SSIL2-treated animals only few isolated granulomas could be observed in the otherwise healthy livers. The hepatic expression of IL-1\u3b2 and TNF\u3b1, and ROS concentration in the liver increased only in in SIL-treated rats (p <0.001 vs controls) and they were comparable to that of controls in SSIL2\u2013treated rats. Conclusions:SSIL2s, besides their pharmacokinetic advantages, permit to overcome the hepatic toxicity caused by SIL, thus representing a smart strategy to improve the tolerability of cancer therapy