The effect of magnetic targeting on the uptake of magnetic-fluid-loaded liposomes by human prostatic adenocarcinoma cells.

Interactions of magnetic-fluid-loaded liposomes (MFL) with human adenocarcinoma prostatic cell line PC3 were investigated in vitro. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 180 nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5 mol.% of distearylphosphatidylcholine poly(ethylene glycol)(2000) (DSPE-PEG(2000)) in the vesicle bilayer. The association processes with living cells, including binding and effective internalization, were followed versus time at two levels. On one hand, the lipid vesicles labeled by 1 mol.% of rhodamine-marked phosphatidylethanolamine were imaged by confocal fluorescence microscopy. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. This allowed modeling of MFL uptake kinetics as a two-step process involving first binding adsorption onto the outer cell membrane followed by subsequent internalization. Capture efficiency was significantly improved by guiding MFL in the near vicinity of the cells by means of a 0.29-T external magnet developing a magnetic field gradient close to 30 mT/mm. Double detection of lipids by fluorescence tracking and of iron oxide by magnetophoresis showed excellent correlation. This demonstrated that MFL associate with tumor cells as intact vesicle structures which conserve their internal content.

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

Magnetic targeting of nanometric magnetic fluid loaded liposomes to specific brain intravascular areas: a dynamic imaging study in mice.

PURPOSE: To prospectively determine, by using dynamic imaging, whether a magnet placed over a specific area of the mouse brain could target systemically administered rhodamine-labeled magnetic fluid-loaded liposomes (MFLs) to that brain region. MATERIALS AND METHODS: Experiments were performed with a French Ministry of Agriculture permit and regional ethics committee authorization. In seven anesthetized C57BL/6 mice, a closed cranial window was implanted above the left parieto-occipital cortex. A laser-scanning confocal fluorescence microscope (LSCFM) was used to track the intravenously injected rhodamine-labeled MFLs within this cortical area, through the cranial window. The MFLs were video monitored for 2 minutes every 15 minutes for 1 hour after injection. A magnet was placed on the cranial window implanted in four mice, while no magnet was placed in three (control) mice. After dynamic in vivo imaging, static in vivo imaging was performed with a different LSCFM. Ex vivo fluorescence histologic analysis was then performed. Paired Student t testing was used to compare the cerebral blood flow and two-dimensional flow values before and 1 hour after MFL injection. For image analysis, intergroup comparisons were performed by using an independent t test. RESULTS: In vivo video monitoring through the window revealed that the rhodamine-labeled MFLs accumulated in the mouse brain microvasculature exposed to the magnet-first within superficial brain venules and then within intracerebral venules-with no significant change in blood flow (P > .05). MFLs accumulated neither in the arterioles of the mice with a magnet nor in the arterioles of the control mice. Static in vivo imaging findings confirmed the microvascular localization of the rhodamine-labeled MFLs, and histologic findings specified their accumulation on the side of the magnet only. CONCLUSION: Real-time in vivo imaging of rhodamine-labeled MFLs in the mouse brain cortex revealed that these nanosystems can be magnetically targeted, through microvessels, to selected brain areas.

Magnetic targeting of rhodamine-labeled superparamagnetic liposomes to solid tumors: in vivo tracking by fibered confocal fluorescence microscopy.

Polyethylene glycol (PEG)ylated and rhodamine-labeled liposomes loaded with maghemite nanocrystals provide a novel nanoscaled hybrid system for magnetic targeting to solid tumors in possible combination with double in vivo imaging by fluorescence microscopy and magnetic resonance imaging (MRI). Human prostate adenocarcinoma tumors implanted in mice were used as a system model. A magnetic field gradient was produced at the tumor level by external apposition of a magnet. Noninvasive fibered confocal fluorescence microscopy was successfully used to track the liposomes in vivo within organs and tumor blood vessels. Active targeting to the magnet-exposed tumors was clearly shown, in agreement with previous MRI studies. The liposomes were driven and accumulated within the microvasculature through a process that preserved vesicle structure and content.

Magnetic targeting of magnetoliposomes to solid tumors with MR imaging monitoring in mice: feasibility.

PURPOSE: To establish the feasibility of magnetoliposome tumor targeting with an extracorporeal magnet. MATERIALS AND METHODS: Animal experiments were performed in compliance with Institut National de la Santé Et de la Recherche Médicale animal protection guidelines and were approved by local government authorities. Magnetophoresis was used to measure the velocity of magnetoliposomes constituted of polyethylene glycol-lipids and anionic maghemite nanocrystals in a calibrated magnetic field in vitro. For in vivo studies, 38 male Swiss nude mice bearing a PC3 human prostate carcinoma tumor in each flank received an intravenous injection of magnetoliposomes (n = 27), saline (n = 9), or nonencapsulated superparamagnetic particles (n = 2) after a small magnet with a magnetic field of 0.3 T and a field gradient of 11 T/m was fixed to the skin above one tumor. The animals were examined at magnetic resonance (MR) imaging with eight different sequences, iron doses (13 mice), and magnet-application durations (12 mice). Their excised tumors were then stained with Perls Prussian blue and hematoxylin-eosin and were examined histologically. With use of the paired Student t test, signal intensity, tumor surface enhancement, and number of stained cells were compared between the control and magnet-exposed tumors to determine significant differences (P

Generation of superparamagnetic liposomes revealed as highly efficient MRI contrast agents for in vivo imaging.

Maghemite (gamma-Fe2O3) nanocrystals stable at neutral pH and in isotonic aqueous media were synthesized and encapsulated within large unilamellar vesicles of egg phosphatidylcholine (EPC) and distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG(2000), 5 mol %), formed by film hydration coupled with sequential extrusion. The nonentrapped particles were removed by flash gel exclusion chromatography. The magnetic-fluid-loaded liposomes (MFLs) were homogeneous in size (195 +/- 33 hydrodynamic diameters from quasi-elastic light scattering). Iron loading was varied from 35 up to 167 Fe(III)/lipid mol %. Physical and superparamagnetic characteristics of the iron oxide particles were preserved after liposome encapsulation as shown by cryogenic transmission electron microscopy and magnetization curve recording. In biological media, MFLs were highly stable and avoided ferrofluid flocculation while being nontoxic toward the J774 macrophage cell line. Moreover, steric stabilization ensured by PEG-surface-grafting significantly reduced liposome association with the macrophages. The ratios of the transversal (r2) and longitudinal (r1) magnetic resonance (MR) relaxivities of water protons in MFL dispersions (6 < r2/r1 < 18) ranked them among the best T2 contrast agents, the higher iron loading the better the T2 contrast enhancement. Magnetophoresis demonstrated the possible guidance of MFLs by applying a magnetic field gradient. Mouse MR imaging assessed MFLs efficiency as contrast agents in vivo: MR angiography performed 24 h after intravenous injection of the contrast agent provided the first direct evidence of the stealthiness of PEG-ylated magnetic-fluid-loaded liposomes.