Magnetoliposomes Research Articles

Magnetoliposomes: Liposomal Contrast Agents

Magnetoliposomes (MLs) are phospholipid vesicles encapsulating magnetic nanoparticles utilised as contrast agents for targeted molecular Magnetic Resonance Imaging (MRI). [...]

The role of nanoparticle concentration-dependent induction of cellular stress in the internalization of non-toxic cationic magnetoliposomes

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide–lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys) 4]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 ± 5.83 pg Fe and 87.46 ± 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys) 4-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell–nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.

Addressing the problem of cationic lipid-mediated toxicity: The magnetoliposome model

The high biocompatibility and versatile nature of liposomes made these particles keystone components in many hot-topic research areas. For transfection and cell labelling purposes, synthetic cationic lipids are often added, but in most studies, little attention has been paid to their cytotoxic effects. In the present work, cationic magnetoliposomes (MLs), i.e. iron oxide cores enwrapped by a phospholipid bilayer (dimyristoylphosphatidylcholine or sphingomyelin) doped with cationic lipids (1,2-distearoyl-3-trimethylammonium propane), serve as a model to examine cationic lipid toxicity. Mechanisms of cytotoxic effects were found to be either dependent or independent of actual particle internalisation according to data obtained in the absence or presence of several endocytosis inhibitors. The former seem to be caused by the generation of reactive oxygen species (ROS) leading to a Ca 2+ influx at high ROS levels. The latter are due to a destabilisation of the cell plasma membrane upon transfer of the cationic lipid from the ML bilayer into the plasma membrane. However, these adverse effects can be diminished by the use of a ROS scavenger, a Ca 2+-channel blocker or by modulating the liposome size, lipid bilayer constitution or by stabilising the membrane by anchoring it on a solid core. Careful attention must be paid in terms of assessing cell viability as the effects are highly time dependent and the data suggest the incompatibility of using the well-known MTT assay when high levels of ROS species are generated.

Absorption of 10-hydroxycamptothecin into the coat of magnetoliposomes

The work deals with the binding of 10-hydroxycamptothecin (HCPT) in the coating of so-called magnetoliposomes (MLs). The latter consist of nanometer-sized crystalline magnetite (Fe 3O 4) cores surrounded by a phospholipid bilayer, which for the present study is built up of either dipalmitoylphosphatidylcholine (DPPC) or mixtures of DPPC with dipalmitoylphosphatidylglycerol (DPPG) (DPPC/DPPG molar ratios of 4/1, 2/1, 1/1 and 1/3). Partitioning of HCPT in the ML coat significantly improves as the DPPG content in the ML coat increases, suggesting the importance of H-bridges between DPPG and HCPT in the absorption process. In contrast to the behaviour of ‘classical’ phospholipid membranes which expand upon drug binding, it is calculated that with the phospholipid bilayer immobilized on the Fe 3O 4 surface 2.6 ± 0.5 phospholipid molecules have to leave the membrane for insertion of 1 HCPT molecule. The potential of the ML-drug structures as promising theranostics (i.e. nanocolloids with both therapeutic and diag nostic features) is briefly discussed.

Design and Development of Magnetoliposome-Based Theranostics

Magnetoliposomes (MLs) consist of nanometre-sized magnetite cores, enwrapped by a bilayer of phospholipid molecules. In the past we showed that these nanocolloids can be exploited as powerful biocompatible magnetic resonance imaging (MRI) contrast agents. In the present work, we report on the partitioning of the amphiphilic drug, (R/S)-propranolol, within the lipidic envelope of MLs, built up of the zwitterionic dimyristoylphosphatidylcholine. Furthermore, it is also shown that MLs are easily internalized by 3T3 fibroblasts, used in this study as a representative cell model, without damaging cell viability. Overall, the results deliver the proof-of-concept that drug-loaded MLs have great potential as unique intracellular theranostics, i.e., as a nanoscale delivery system with combinatory therapeutic-diagnostic imaging modalities.

Partitioning of propranolol in the phospholipid bilayer coat of anionic magnetoliposomes

This work deals with the partitioning of the cationic amphiphilic drug, propranolol, in thecoating of so-called magnetoliposomes (MLs), which consist of nanometre-sized,magnetizable iron oxide cores covered with a phospholipid bilayer. MLs of two types wereused: either the ML coat consisted entirely of anionic dimyristoylphosphatidylglycerol,or it was mixed with zwitterionic dimyristoylphosphatidylcholine in a5/95 molar ratio. To separate sorbed from non-sorbed propranolol, high-gradient magnetophoresiswas used. The sorption profiles clearly show that electrostatic interactions play a key rolein the sorption process as drug incorporation in the ML coat was favoured byincreasing the anionic character of the ML envelope and by reducing the saltconcentration of the medium. Also, upon drug binding some phospholipid moleculeswere expelled from the ML coat. The observations may be of relevance in thebiomedical field, i.e. in the development of ML-based, intracellular theranostics.

Optimal Conditions for Labelling of 3T3 Fibroblasts with Magnetoliposomes without Affecting Cellular Viability

A comparative study that deals with the internalisation of different types of magnetoliposomes (MLs) by 3T3 fibroblasts revealed that cationic MLs proved to be superior to neutral and anionic ones. Internalisation was visualised both by optical light and transmission electron microscopy. The latter showed that the cationic MLs ultimately ended up in lysosomal structures. The effect of increasing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) concentrations in the cationic ML coat has been elucidated. High uptake efficiency was only achieved with MLs that carry a high DOTAP payload. However, these structures also demonstrated toxic effects. The use of the saturated distearoyl analogue (DSTAP) at identical concentrations led to improved uptake efficiency and lower toxicity. By using iron-oxide-free vesicles, it was shown that the toxicity was due to lipid bilayer constituents and not the iron oxide. In conclusion, the use of DMPC-DSTAP (96.67:3.33; molar ratio) MLs results in an extremely high labelling of 3T3 fibroblasts with iron oxides (47.66 pg Fe per cell) without evoking any influence on cell viability.

A Successful Strategy for the Production of Cationic Magnetoliposomes

Summary The present work describes a strategy and the mechanistic background for synthesizing magnetoliposomes (MLs) in which the bi-layered coating is partly composed of positively charged lipids. In a first step, neutral MLs are prepared from zwitterionic phosphatidylcholine vesicles and magnetite nanocores stabilized with an anionic surfactant, and then incubated with cationic vesicles formed from phosphatidylcholine and dioleoyltrimethylammoniumpropane. During this latter step, spontaneous intermembrane lipid transfer occurs. After reaching an equilibrium state, the desired cationic MLs are captured from the mixture in a high-gradient magnetophoresis setup.

Selective hyperthermia using magnetoliposomes to target cervical lymph node metastasis in a rabbit tongue tumor model.

The effect of hyperthermia on cervical lymph node metastasis of VX7 tongue cancer in female Japanese white rabbits was investigated. Magnetoliposomes (MLs) with a neutral surface charge and a size of 94.1 nm were used as heating mediators. MLs were injected into the tongue 20 days after tumor transplantation, and we examined whether they reached the metastatic deep cervical lymph node. The highest magnetite concentration 24 h after ML injection was detected in the lymph node, followed by tongue, spleen, blood, and liver. Rabbits were separated into three groups: group I as the control; group II with ML injection alone; and group III with ML injection and hyperthermia 24 h after ML injection, generated by applying an alternating magnetic field (118 kHz, 384 Oe) to the neck region. The hyperthermic effect was evaluated in terms of the percentage of necrosis in proportion to the metastatic tumor and the apoptotic index (AI), defined as the ratio of TUNEL-positive cells. The temperature of lymph nodes in group III reached over 44 degrees C. The mean area of necrosis in group III was 58.0%, which was significantly higher than that in group I (19.6%) or group II (20.4%). The AI in group III was 22.9%, significantly higher than in group I (1.67%) or II (1.42%). The difference between group I and II was not statistically significant. Group III tumor sites around MLs showed necrosis or apoptosis-positive cells induced by hyperthermia. These results indicate that MLs injected into the tongue can target cervical lymph node metastases and accumulate there at concentrations sufficient to generate therapeutically effective temperatures.

Birefringence and transmission electron microscopy of monolayer and bilayer magnetoliposomes

In this study, static magnetic birefringence (SMB) and transmission electron microscopy (TEM) were used to investigate magnetite-based monolayer and bilayer magnetoliposomes (MLs). The SMB data were analyzed using the recent model proposed by Skeff Neto et al. (J. Appl. Phys. 89 (2001) 3362). The SMB data indicate that monolayer-based MLs internalize magnetic nanoparticles as dimers while bilayer-based MLs internalize both isolated nanoparticles and dimers. The higher content of dimers inside monolayer MLs has been confirmed by TEM data.

AC-magnetic field controlled drug release from magnetoliposomes: design of a method for site-specific chemotherapy

Large unilamellar magnetoliposomes (MLs) with encapsulated doxorubicin (DOX) (anticancer drug) were prepared by reverse-phase evaporation. They were exposed to an alternating magnetic field with a frequency of 3.5 MHz and an induction of 1.5 mT produced in three-turn pancake coil. The results showed that magnetoliposomes could be specifically heated to 42 °C (phase transition temperature of a used lipid) in a few minutes and during this, the encapsulated doxorubicin is massively released.

Targeting Hyperthermia for Renal Cell Carcinoma Using Human MN Antigenspecific Magnetoliposomes

Magnetoliposomes (MLs) conjugated with an antibody fragment to give specificity to a tumor were applied to hyperthermia for cancer. The Fab' fragment of the G250 antibody, which binds to MN antigen on many types of human renal cell carcinoma, was cross–linked to Af–(6–maleimidocaproyl–oxy)–dipalmitoyl phosphatidylethanolamine (EMC–DPPE) in liposomal membrane. The targetabil–ity of the G250–Fab' fragment–conjugating MLs (G250–FMLs) was investigated using the mouse renal cell carcinoma (mRCC) and MN antigen–presenting cell, MN–mRCC. The amount of G250–FMLs uptake reached 67 pg/cell against MN–mRCC cells in an in vitro experiment using plastic dishes and this value was about 6 tunes higher than that in the case of MLs. In an in vivo experiment using MN–mRCC–harboring mice, 1.5 mg of the FMLs per carcinoma tissue accumulated (tumor weight was 0.19 g), which corresponded to approximately 50% of the total injection. This value was 27 tunes higher than that of the MLs. After injection of the FMLs, mice were exposed to intracellular hyperthermia using alternating magnetic field irradiation. The temperature of tumor tissue increased to 43°C and the growth of the carcinoma was strongly arrested for at least 2 weeks. These results indicate the G250–FMLs could target renal cell carcinoma cells in vitro and in vivo, and are efficiently applicable to the hyper thermic treatment of carcinoma.

Preparation of Tumor-Specific Magnetoliposomes and Their Application for Hyperthermia.

Magnetoliposomes (MLs) were conjugated with an antibody fragment to give specificity to a tumor. The antibody fragment was cross-linked to N-(6-maleimidocaproyloxy)-dipalmitoyl phosphatidylethanolamine (EMC-DPPE) in liposomal membrane. The immobilization of the antibody fragment was optimal when the content of EMC-DPPE was 10-wt% and the reaction time for immobilization was 18 h. The Fab' fragment-conjugating MLs (FMLs) were 2.4 times higher molar immobilization density compared with the method using the whole antibody. The targetability of the FMLs to the glioma cells, U251-SP, was then investigated. The amount of FMLs uptake reached 85 pg/cell in an in vitro experiment using plastic dishes. In an in vivo experiment using glioma-harboring mice, 260 μg of the FMLs per 1 g of tumor tissue accumulated (tumor sizes was 0.1 cm3), which corresponded to approximately 60% of the total injection. This value was 7 times higher than that of the MLs. After injection of the FMLs, mice were exposed to intracellular hyperthermia using the alternating magnetic field irradiation. The temperature of tumor tissue increased to 43°C and the growth of the tumor was found to be arrested over 2 weeks. These results indicate the FMLs could target the glioma cells in vitro and in vivo, and are efficiently applicable to the hyperthermia of tumor.