Aqueous and solid magnetoliposomes containing MnFe2O4 nanoparticles were prepared and evaluated as nanocarriers for a new antitumor drug.

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.

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Nickel ferrite nanoparticles with superparamagnetic behavior at room temperature were synthesized using a coprecipitation method. These magnetic nanoparticles were either covered with a lipid bilayer, forming dry magnetic liposomes (DMLs), or entrapped in liposomes, originating aqueous magnetoliposomes (AMLs). A new and promising method for the synthesis of DMLs is described. The presence of the lipid bilayer in DMLs was confirmed by FRET (Förster Resonance Energy Transfer) measurements between the fluorescent-labeled lipids NBD-C12-HPC (NBD acting as a donor) included in the second lipid layer and rhodamine B-DOPE (acceptor) in the first lipid layer. An average donor-acceptor distance of 3 nm was estimated. Assays of the non-specific interactions of magnetoliposomes with biological membranes (modeled using giant unilamellar vesicles, GUVs) were performed. Membrane fusion between both aqueous and dry magnetoliposomes and GUVs was confirmed by FRET, which is an important result regarding applications of these systems both as hyperthermia agents and antitumor drug nanocarriers.

The lipid/surfactant mixed interactions between the lipid dipalmitoylphosphatydilcholine (DPPC) and the nonionic surfactant C12E7 (C12H25(OCH2CH2)7OH) were studied by the use of fluorescence anisotropy of nile red and DCM laser dye. Three different regions consisting of mixed micelles, mixed vesicles, and both aggregates were identified. Nile red fluorescence anisotropy is wavelength-dependent, the spectra being decomposed into two log-normal functions. These results are consistent with a solvent relaxation process, with distinct anisotropies for the relaxed and unrelaxed states. The spectral properties (anisotropy and maximum emission wavelength) of the relaxed state show higher sensitivity to the environment than those of the unrelaxed state. DCM fluorescence anisotropy shows similar trends. This was interpreted as a result of a trans−cis photoisomerization process. The observed anisotropy spectrum was decomposed in two Gaussian functions reflecting a distinct anisotropy for each isomer.

Magnetoliposomes containing superparamagnetic manganese ferrite nanoparticles were tested as nanocarriers for two new promising antitumor drugs, a N-(3-methoxyphenyl)thieno[3,2-b]pyridin-7-amine (1) and a N-(2-methoxy-phenyl)thieno[3,2-b]pyridin-7-amine (2). The fluorescence emission of both compounds was studied in different polar and non-polar media, evidencing a strong intramolecular charge transfer character of the excited state of both compounds. These in vitro potent antitumor thienopyridine derivatives were successfully incorporated in both aqueous and solid magnetoliposomes, with encapsulation efficiencies higher than 75%. The magnetic properties of magnetoliposomes containing manganese ferrite nanoparticles were measured for the first time, proving a superparamagnetic behaviour. Growth inhibition assays on several human tumor cell lines showed very low GI 50 values for drug-loaded aqueous magnetoliposomes, comparing in most cell lines with the ones previously obtained using the neat compounds. These results are important for future drug delivery applications using magnetoliposomes in oncology, through a dual therapeutic approach (simultaneous chemotherapy and magnetic hyperthermia).

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