Development and characterization of immunoliposomes for Cidofovir and SiRNA delivery: a new strategy for the treatment of Primary Effusion Lymphoma

Primary Effusion Lymphoma (PEL) is an aggressive and consistently lethal non-Hodgkin's B-cell lymphoma growing as lymphomatous effusions in serous body cavities and invariably associated with HHV-8 [1,2].The majority of the patients affected with PEL, either elderly HIV-negative or immunocompromised AIDS patients, are typically characterized by several age-related co-morbidities or opportunistic infectious diseases. Clinical efficacy of conventional anti-neoplastic chemotherapy is commonly hampered by the excessive grade of systemic toxicity and low drug levels in the tumor area [3].New therapeutic challenges may arise from the use of SiRNA technology, which is based on cellular transfection of antisense small RNAs, specifically designed to recognize the target mRNA and able to turn off the changed cellular mechanism of tumor cells, or by using antineoplastic drug (Cidofovir). Considering the high instability and the poor cellular uptake of both these actives, tumor-specific delivery by means of targeted nanocarriers is strongly required. These strategies represent an attractive approach to enhance intra-tumoral cytotoxic effects together with the reduction of “off-target” side effects, possibly offering a radical improvement in the treatment of such fragile oncologic patients. Among the colloidal carrier systems for drug delivery, liposomes have received considerable attention. They allow to protect the drug from rapid degradation, being particularly suitable to form complexes with highly-degradable ribonucleic acids.In this study, we formulated and characterized immunoliposomal formulation direct to PEL cell line (BCBL-1 cell line) using the cationic lipid DOTAP and a pegylated cholesterol funzionalized with a maleidoimide moiety capable to interact with anti CD-138 antibody. The formulation was characterized (size, zeta potential and morphology) in comparison with untargeted DOTAP liposomes and pegylated systems.These liposomal systems were used to transfect a model FITC-ODN into a model PEL cell line (BCBL-1). The studies on cellular binding and on the internalization of oligo by flow cytometry and confocal analysis confirmed the higher transfection efficiency of the immunoliposomes when compared with DOTAP and pegylated liposomes. This targeted formulation could be reasonably considered as optimal candidates for therapeutic siRNA delivery and more generally for gene encapsulation and delivery against the poor curable PEL tumor. Concerning cidofovir, it has been demonstrated that this antiviral drug is able to induce cell apoptosis in different tumor included PEL. Indeed, the high pro-apoptotic concentrations of cidofovir are never achievable in situ after full-dose systemic administration (5mg/Kg i.v.), and however, this systemic treatment can frequently cause severe nephrotoxicity [4]. We proposed liposomes encapsulating cidofovir by a modified reversed phase evaporation method (mREV) followed by extrusion. The characterization of samples suggested that cationic liposomes are more suitable for cidofovir stabilization, taking advantage of the charge interaction between the anionic drug and the cationic lipid moieties. Using the in vitro model of PEL (BCBL-1 cell line), the carrier toxicity and the antineoplastic efficacy of liposomes were evaluated by flow cytometry, applying apoptosis and cell death analysis. This in vitro study showed the applicability of the liposomes within a restricted range of lipidic concentrations, mainly depending on the lipids used during the preparation. The cidofovir transfection mediated by liposome composed of PC:DOTAP and PC:DC-CHOL caused a moderate increase in the percentage of apoptotic/necrotic PEL cells with respect to the controls (free drug and empty liposomes) suggesting that liposomal delivery allows the release of cidofovir into BCBL-1 cells enabling an unexpected antineoplastic activity of this drug even at lower doses.