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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. |
