B

B. that the antiproliferative effects of each drug treatment were mainly due to the induction of apoptosis. In conclusion, liposomes armed with gH625 are able to overcome doxorubicin resistance in lung adenocarcinoma cell lines. degradation [13]. Cationic cell-penetrating peptide-mediated endocytosis is one of the mechanisms by which drug carriers cross the membrane bilayer [14]; subsequently, the cargo is trapped in endosomes, eventually landing in lysosomes where its intracellular bioavailability is decreased. In order to avoid the endocytic pathway, it is of GW 4869 great importance to discover new molecules exploiting different GW 4869 mechanisms of uptake. Hydrophobic peptides that efficiently cross biological membranes, promoting lipid membrane-reorganizing processes represent a powerful alternative [15C17]. Viral-derived peptides can be useful as Trojan horses due to their intrinsic properties of inducing membrane perturbations [16C18]. The twenty residue peptide gH625, previously identified as a membrane-perturbing domain in the glycoprotein H (gH) of Herpes simplex virus 1 (HSV-1), is able to cross the membrane bilayer [19] and has been extensively used for vector-mediated strategies anti-cancer activity of Doxo-encapsulating liposomes, constituted by soy phospholipids, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), in order to improve biocompatibility and lead to a prolonged presence in the systemic circulation. The anti-proliferative effects of liposomal formulations functionalized or not with gH625 were investigated on non-small cell lung cancer (NSCLC) A549 cells either sensitive or resistant to Doxo. The differential accumulation and the oxidative stress caused by the two different formulations in resistant and parental A549 cells were also evaluated. RESULTS Peptide synthesis and conjugation of gH625 to liposomes surface The peptide gH625-Pra and the liposome component (C18)2L-N3 were synthesized according to standard solid phase peptide synthesis (SPPS) protocols with Fmoc/tBu (tBu = tert-butyl) chemistry. The alkyne moiety of gH625-Pra was introduced in the peptide sequence at the C-terminal position as L-propargylglycine. (C18)2L-N3 was synthesized on solid phase following a modified protocol of the classical Fmoc/tBu strategy [22]. Both gH625-Pra and (C18)2L-N3 were collected in good yields ( 40% and 85%, respectively) after HPLC-RP purification, and analyzed by mass spectrometry, 1H and 13C NMR spectroscopy (for (C18)2L-N3), and HPLC to confirm the compound identity and the purity. The coupling of gH625 on the surface of preformed liposomes was performed by click chemistry (Figure ?(Figure1).1). This procedure involves a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes yielding 1,4-disubstituted 1,2,3-triazole-linked conjugates [25]. The click reaction was performed in an aqueous solution and was catalyzed by CuI generated, < 0.01; LipoDoxo-gH625 vs Doxo < 0.01. Effects of liposomes encapsulating doxorubicin conjugated or not with TNFSF10 gH625 viral peptide on A549 and A549 Dx cell proliferation The effects of Doxo, empty liposomes (Lipo) and liposomes encapsulating Doxo conjugated or not with gH625 on the proliferation of either parental A549 GW 4869 or Doxo-resistant cells (A549 Dx) were evaluated by MTT assay as reported in Materials and Methods. Doxo, LipoDoxo and LipoDoxo-gH625 induced a dose-dependent growth inhibition in both cell lines after 72 h (Figure ?(Figure3),3), while treatment with Lipo produced no significant cytotoxic effects in both cell lines (Figure ?(Figure3).3). In Table ?Table2,2, results are reported as concentrations inhibiting 50% of cell growth (IC50) after 72 h of treatment. The IC50 was reached with 0.8 M and 5 M of Doxo (Figure ?(Figure3A3A and ?and3B,3B, Table ?Table2),2), with 1.6 M and about 5 M of LipoDoxo (Figure ?(Figure3A3A and ?and3B,3B, Table ?Table2),2), with 1 M and 0.3 M of LipoDoxo-gH625 (Figure ?(Figure3A3A and ?and3B,3B, Table ?Table2)2) in A549 and A549 Dx cells, respectively. These data suggested that A549 cells were more sensitive to the treatment with Doxo compared to A549 Dx, confirming the drug-resistant phenotype of this cell line. Both cell lines were more responsive to LipoDoxo-gH625 compared to LipoDoxo. In particular, LipoDoxo-gH625 was able to overcome resistance in A549 Dx compared to free Doxo. These data suggested that the conjugation of liposomes with gH625 probably facilitated the entry and retention of doxorubicin in both sensitive and drug-resistant tumor cell lines allowing an increase of cell growth inhibition. Open in a separate window Figure 3 Evaluation of cell growth in lung adenocarcinoma cell line sensitive (A549) and resistant (A549 Dx) to doxorubicin after 72 h of treatment with Lipo, LipoDoxo, LipoDoxo-gH625 and doxorubicin (DOXO) (ACB)The figure shows representative experiments performed in triplicate with SDs. Statistical analysis: LipoDoxo vs LipoDoxo-gH625 < 0.01; LipoDoxo vs Doxo < 0.01; LipoDoxo-gH625 vs Doxo.