US20140227343A1 - Method of monitoring the release from liposomes of a product of interest using superparamagnetic nanoparticles - Google Patents

Method of monitoring the release from liposomes of a product of interest using superparamagnetic nanoparticles Download PDF

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US20140227343A1
US20140227343A1 US13/981,776 US201213981776A US2014227343A1 US 20140227343 A1 US20140227343 A1 US 20140227343A1 US 201213981776 A US201213981776 A US 201213981776A US 2014227343 A1 US2014227343 A1 US 2014227343A1
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liposome
nanoparticles
thermosensitive
membrane
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Agnès Pottier
Laurent Levy
Marie-Edith Meyre
Matthieu Germain
Cyril Lorenzato
Chrit Moonen
Pierre Smirnov
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Centre National de la Recherche Scientifique CNRS
Universite Victor Segalen Bordeaux 2
Nanobiotix SA
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Nanobiotix SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5094—Microcapsules containing magnetic carrier material, e.g. ferrite for drug targeting
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
    • G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
    • G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent

Definitions

  • the present invention is to be used in the health sector, in particular in human health.
  • the methods and compositions herein disclosed generally relate to employing magnetic resonance techniques to monitor the delivery of a compound of interest in vivo. More particularly, the disclosure relates to the use of superparamagnetic nanoparticles the electrostatic surface charge of which is below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH to monitor the membrane permeabilization of thermosensitive liposomes and the incidental release, to a desired site in vivo, of a compound of interest.
  • MRI nuclear magnetic resonance imaging
  • Proton MRI is based on the principle that the concentration and relaxation characteristics of protons in tissues and organs, when submitted to a static magnetic field, can influence the intensity of a magnetic resonance image.
  • this technique brings a high contrast resolution, which allows for morphological imaging thanks to contrast tissue differentiation due to differences in protons density of the different tissues (depending on fat or water content).
  • a functional imaging of certain organs can also be performed, i.e.
  • Contrast enhancement agents that are useful for proton MRI are responsible for a change in the relaxation characteristics of protons, which can result in image enhancement and improved soft-tissue differentiation.
  • thermosensitive liposomes losing their structural integrity within a given temperature range is a promising approach to target a tumor or other tissue, but so far no efficient liposomal MRI contrast agents has been proposed.
  • US 2004/0101969 describes a method of monitoring the localisation and distribution of a compound of interest released from an envirosensitive liposome, using a molecular compound, MnSO 4 , as a contrast agent.
  • WO 2008/035985 describes a trackable particulate material for drug delivery comprising a matrix or membrane material, a drug, an internal T1 magnetic resonance metal chelate contrast agent and an external T1 magnetic resonance metal chelate contrast agent, wherein the internal T1 agent is shielded from bulk water and the external T1 agent is exposed to bulk water.
  • thermosensitive liposomes for drug delivery which comprise a paramagnetic metal compound.
  • the paramagnetic metal compound may be a metallic nanoparticle.
  • the paramagnetic metal compound comprises a chelating structure allowing the metal to interact with water or with another suitable source of protons as chemical exchange-dependent saturation transfer (CEST) contrast agent.
  • CEST chemical exchange-dependent saturation transfer
  • contrast agents which are T1 magnetic resonance paramagnetic molecular compounds and offer only a limited sensitivity when measured in vivo. As a result, relatively large, and possibly toxic, doses of such contrast agents are to be administered to a given subject. This and other problems are addressed by the compositions and methods herein disclosed.
  • WO2008/033031 describes a trackable MRI drug delivery particle comprising two distinct chemical contrast agents, one of them (T1 agent) exhibiting a T1 signal used to monitor drug release, and the other one (T2* agent) exhibiting a T2* signal. This document does not suggest using the T2* agent to monitor drug release.
  • US2009/004258 describes thermosensitive liposomes encapsulating iron oxide nanoparticles and carbofluorosceine (CF), CF being used as a drug model. Nanoparticles are capable of generating heat when activated by an alternative magnetic field, thereby allowing permeabilization of the membrane towards CF. In US2009/004258, monitoring of the CF's release is performed by fluorescence detection.
  • This method in particular uses low doses of non toxic charged superparamagnetic nanoparticles and allows the monitoring of liposomes delivery on a target site and also, surprisingly, the efficient monitoring of the product of interest release from said liposomes.
  • Inventors herein provide a method of monitoring the liposome membrane permeabilization and the incidental release of a product of interest, the liposome comprising a thermosensitive lipidic membrane encapsulating superparamagnetic nanoparticles, the electrostatic surface charge of which is below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH, the method comprising, in a first embodiment, the following steps of:
  • the method further comprises measuring T2*, or T2 and T2*, during the heating step b).
  • the method comprises the following steps of:
  • the method further comprises measuring T2* and T1, and optionally T2, during the heating step b).
  • thermosensitive liposome membrane permeabilization upon heating, and the incidental release of a product of interest, results in a variation of the magnetic resonance imaging (MRI) signal, allowed by the superparamagnetic nanoparticles encapsulated in the liposome, which is of high value and easily measurable.
  • MRI magnetic resonance imaging
  • thermosensitive liposome comprising a thermosensitive lipidic membrane encapsulating superparamagnetic nanoparticles, the electrostatic surface charge of nanoparticles being below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH, and the nanoparticles being preferably usable as a diagnostic or monitoring agent.
  • a liposome is for use in a method, according to the present invention and as herein described, of monitoring the liposome membrane permeabilization and the incidental release of a product of interest.
  • thermosensitive liposome is a liposome comprising a thermosensitive lipidic membrane encapsulating nanoparticles, the nanoparticles being usable as a therapeutic or as a diagnostic agent, each nanoparticle i) comprising an inorganic core the largest dimension of which is less than about 100 nm, and ii) being fully coated with an agent responsible for the presence of an electrostatic charge below ⁇ 20 mV or above +20 mV at the surface of the nanoparticle, the electrostatic charge being determined by zeta potential measurements in an aqueous medium between pH 6 and 8, for a concentration of nanoparticles in suspension varying between 0.2 and 8 g/L.
  • kits comprising any one or more of the herein-described products, i.e., thermosensitive liposomes and compositions, together with a labeling notice providing instructions for using the product(s) in the context of the herein described methods.
  • FIG. 2 Typical elution profile obtained for iron oxide-containing liposomes
  • FIG. 2 shows the elution profile for iron oxide-containing liposomes determined by quantification of magnetic nanoparticles by UV-visible spectroscopy (Cary 100 Varian spectrometer) via a colorimetric reaction between ferrous ions and phenanthroline.
  • concentration of iron oxide in liposomes ranges from 1 to 2.5 g/L.
  • FIG. 3 Release profile of doxorubicin
  • FIG. 3 shows the release kinetic profile of doxorubicin (DOX)-loaded iron oxide-containing thermosensitive liposomes (TSL), determined by fluorescence measurements.
  • Samples were aged in a medium comprising Phosphate Buffer Saline 1 ⁇ (PBS 1 ⁇ ) and Fetal Bovine Serum (FBS) (volume ratio 1:1), at 37° C., 41° C., 43° C., and 45° C. Heating is performed via a water bath.
  • PBS 1 ⁇ Phosphate Buffer Saline 1 ⁇
  • FBS Fetal Bovine Serum
  • FIG. 4 Evolution upon heating of Magnetic Resonance (MR) relaxivities at 1.5 Tesla of iron oxide nanoparticles encapsulated in thermosensitive (TSL) and non-thermosensitive liposomes (NTSL), and free nanoparticles (free NP)
  • MR Magnetic Resonance
  • FIG. 4A shows longitudinal (r 1 ) relaxivities of thermosensitive liposomes (TSL), non-thermosensitive liposomes (NTSL), both loaded with 5 nm-sized iron oxide nanoparticles, and free nanoparticles (free NP) at 1.5 Tesla, before (black) and after (grey) heating at 45° C. Heating is performed via a water bath.
  • FIG. 4B shows transverse (r 2* ) relaxivities of thermosensitive liposomes (TSL), non-thermosensitive liposomes (NTSL), both loaded with 5 nm-sized iron oxide nanoparticles, and free nanoparticles (free NP) at 1.5 Tesla, before (black) and after (grey) heating at 45° C. Heating is performed via a water bath.
  • FIG. 5 Evolution upon heating of Magnetic Resonance (MR) signal at 1.5 Tesla of iron oxide encapsulated in thermosensitive (TSL) and non-thermosensitive liposomes (NTSL), and free nanoparticles (free NP).
  • MR Magnetic Resonance
  • FIG. 5 shows the MR images at 1.5 Tesla, of solutions of iron oxide nanoparticles encapsulated within thermosensitive (TSL) and non-thermosensitive liposomes (NTSL), and free nanoparticles (free NP), at 37° C. and 45° C. for the following dilutions (40, 20, 10 and 5 ⁇ g/mL in iron oxide).
  • TSL thermosensitive
  • NTSL non-thermosensitive liposomes
  • free NP free nanoparticles
  • FIG. 6 MR signal and T2*-mapping of iron oxide nanoparticles-encapsulated in thermosensitive liposomes (TSL) before and after High Intensity Focused Ultrasound (HIFU)-induced heating.
  • TSL thermosensitive liposomes
  • HIFU High Intensity Focused Ultrasound
  • FIG. 6 shows MR images and T2*-mapping of iron oxide nanoparticles encapsulated in thermosensitive liposomes (TSL), before and after heating with a HIFU device.
  • Red arrows show changes in T2* at the HIFU focal point corresponding to permeabilization of the thermosensitive liposome upon heating at or above Tm.
  • spin-lattice relaxation also longitudinal or T1 relaxation
  • the excited nucleus gives off its energy to the surrounding environment (lattice) at a particular rate characterized by a time-constant T1
  • spin-spin relaxation also transverse or T2 relaxation
  • a nucleus in the high energy state exchanges energy with a nucleus in the low energy state at a particular rate characterized by a time-constant T2.
  • T1 and T2 depend upon the chemical and magnetic environment in which a particular nucleus is situated and different structures and tissues within the body have different T1 and T2 values (Tilcock et al. Adv Drug Delivery Reviews, 1999; 37:33-51).
  • MR contrast agents Magnetic resonance (MR) contrast agents, thus shortening the longitudinal (T1) and transverse (T2) relaxation times and, therefore, increasing the intensity of the MR signal in the regions where they are distributed (S. Aime, et al., Biopolymers 2002; 66:419-428).
  • the efficacy of a MR contrast agent is commonly evaluated in terms of its impact on the proton relaxation rates, or relaxivities, r 1 and r 2 . They are determined from the linear relationship:
  • the first class includes mainly chelates of ions presenting a high number of unpaired spins, i.e. Mn(II), Mn(III) and Gd(III) ions, conferring them a paramagnetic behavior.
  • Gadolinium-based agents are the most commonly used.
  • Paramagnetic agents increase r 1 and r 2 by similar amounts, but are best visualized using T1-weighted images since the percentage change in r 1 in the tissue is much greater than that in r 2 (Koslowska et al., Adv. Drug Deliv. Rev. 2009; 61:1402-1411).
  • the second class of contrast agents is constituted of superparamagnetic iron oxide nanoparticles, iron oxide being maghemite ( ⁇ -Fe 2 O 3 ) or magnetite (Fe 3 O 4 ) crystalline structure (De Cuyper et al. Methods in Enzymology, 2003; 373:175-198).
  • Superparamagnetism occurs when the size of the crystals is smaller than ferromagnetic domains (approximately 30 nm) and, consequently, they do not show any magnetic remanence (i.e. restoration of the induced magnetization to zero upon removal of the external magnetic field), unlike ferromagnetic materials.
  • Each crystal is then considered to be a fully magnetized single magnetic monodomain, and can be considered to be a monomagnet which is a direct consequence of the spinel structure of the crystal, allowing strong magnetic coupling and consequently perfect alignment of the individual magnetic spins (Corot et al. Adv. Drug Deliv. Rev. 2006; 58:1471-1504).
  • T2* Another type of relaxation time, T2*, which is commonly due to a combination of magnetic field inhomogeneities and spin-spin transverse relaxation, with the result of rapid loss in transverse magnetization.
  • Superparamagnetic contrast agents are then highly sensitive to T2*-weighted scans.
  • Paramagnetic agents directly affect water protons in their close vicinity and are highly dependent on local water flux. Hence, the influence of these agents is very local and they should ideally be in contact with water with adequate exchange rates. In contrast, superparamagnetic agents affect the magnetic field independent of their environment and thus their influence in terms of contrast extends well beyond their immediate surroundings (G. M. Lanza, et al., J. Nucl. Cardiol. 2004; 11:733-743). On conventional spin-echo MR images, the presence of T2 (superparamagnetic) agents leads to a dark (hypointense) appearance of tissue, whereas T1 agents cause the opposite (hyperintensity).
  • thermosensitive liposomes used in the context of the present invention can be administered in a subject by different routes such as local (intra-tumoral (IT) for example), subcutaneous, intra venous (IV), intra-dermic, intra-arterial, airways (inhalation), intra peritoneal, intra muscular and oral route (per os).
  • IT intra-tumoral
  • IV intravenous
  • IV intra-dermic
  • intra-arterial airways
  • inhalation intra peritoneal
  • intra muscular and oral route per os
  • the term “subject” means any organism.
  • the term need not refer exclusively to human being, which is one example of a subject, but can also refer to animals, in particular warm-blooded vertebrates, typically mammals, and even tissue cultures.
  • Inventors herein provide a method of monitoring, in particular in vivo, the liposome membrane permeabilization and the incidental release of a product of interest, the liposome comprising a thermosensitive lipidic membrane encapsulating superparamagnetic nanoparticles the “electrostatic surface charge” (also herein identified as “charge” or “surface charge”) of which is below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH (between 6 and 8).
  • electrostatic surface charge also herein identified as “charge” or “surface charge”
  • the method comprises the following steps of:
  • the r 2* ratio varies in response to the liposome membrane permeabilization, i.e., in response to the product of interest release from the liposome. More specifically, the r 2* ratio varies from 1.2 up to 10, preferably the r 2* ratio is above 1.5.
  • the method further comprises measuring T2*, or T2 and T2*, during the heating step b).
  • the method comprises the following steps of:
  • the r 2* /r 1 ratio varies in response to the liposome membrane permeabilization, i.e., in response to the product of interest release from the liposome. More specifically, the r 2* /r 1 ratio varies from 1.2 up to 20, preferably the r 2* /r 1 is above 2, more preferably above 5.
  • the method further comprises measuring T2* and T1, and optionally T2, during the heating step b).
  • measuring refers to a complete acquisition, or to a real-time acquisition of T2, T2* or T1 in the context of real-time imaging techniques.
  • the heating step b) at Tm or above Tm of herein described methods is performed locally in order to monitor the liposome membrane permeabilization and the incidental release of a product of interest in a specific area (corresponding to the heat-treated area) of a subject.
  • Such a locally performed method allows the assessment of spatial distribution of the released product.
  • thermosensitive liposome with a Tm (gel-to-liquid crystalline phase transition temperature).
  • This liposome comprises a thermosensitive lipidic membrane encapsulating nanoparticles the electrostatic surface charge of which is below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH (typically between pH 6 and pH 8).
  • These nanoparticles are usable as a diagnostic agent to monitor the delivery of thermosensitive liposomes on a target site and the efficient monitoring of the products of interest release from said thermosensitive liposomes when said liposomes are activated by heat.
  • inventors thus herein provide a liposome comprising a thermosensitive lipidic membrane encapsulating nanoparticles the electrostatic surface charge of which is below ⁇ 20 mV or above +20 mV when measured in an aqueous medium between pH 6 and 8, for use in a method of monitoring the liposome membrane permeabilization and the incidental release of a product of interest (the product of interest being originally present in addition to nanoparticles in said liposome).
  • the electrostatic charge is typically determined by zeta potential measurements in an aqueous medium between pH 6 and 8, for a concentration of nanoparticles in suspension varying between 0.2 and 8 g/L.
  • liposome refers to a spherical vesicle composed of at least one bilayer of amphipathic molecules which forms a membrane separating an intravesicular medium from an external medium.
  • the intravesicular medium constitutes the internal aqueous core of the liposome.
  • Hydrophilic molecules or components can be encapsulated inside the internal aqueous core of the liposome via active methods of encapsulation known by the skilled person and further herein below described. Hydrophobic molecules or components can be entrapped inside the membrane.
  • the amphipathic molecules constituting the bilayer are lipids, more particularly phospholipids.
  • the amphipathic characteristic of phospholipid molecules lies in the presence of a hydrophilic head, constituted of a phosphate group and of a glycerol group, and a hydrophobic tail, constituted of one or two fatty acids.
  • phospholipids tend to self-assemble to minimize contact of the fatty acyl chains with water, and they tend to adopt different types of assembly (micelles, lamellar phase, etc) according to their chemical structure. More particularly, phosphatidylcholines are known to form a lamellar phase consisting in stacked bilayers undergoing a “spontaneous” curvature and finally forming vesicles (Lasic et al. Adv. Colloid. Interf. Sci. 2001; 89-90:337-349). The phospholipidic lamellar phase constitutes a thermotropic liquid crystal. This means that the ordering degree of the amphipathic molecules depends on the temperature.
  • a phospholipidic bilayer demonstrates a main phase transition temperature T m (for temperature of “melting”), corresponding to a transition between the “gel-like” lamellar phase L ⁇ to the “fluid-like” lamellar phase L ⁇ .
  • T m for temperature of “melting”
  • the bilayer is only permeable to small ions.
  • the hydrophobic tails are moving due to the thermal motion which causes a loss of ordering of the phospholipid molecules and leads to a “liquid crystalline” phase: the bilayer becomes permeable to molecules such as a drug.
  • the “gel-to-liquid crystalline” phase transition temperature T m depends on the chemical structure of the phospholipid molecule: hydrocarbon chain length, unsaturation, asymmetry and branching of fatty acids, type of chain-glycerol linkage (ester, ether, amide), position of chain attachment to the glycerol backbone (1,2- vs 1,3-) and head group modification.
  • type of chain-glycerol linkage ester, ether, amide
  • position of chain attachment to the glycerol backbone (1,2- vs 1,3-)
  • head group modification In the case of phosphatidylcholines, the structure and conformation of fatty acyl chains is of particular relevance (Koynova et al., Biochim. Biophys. Acta 1998; 1376:91-145).
  • the effect of unsaturation on the main gel-to-liquid crystalline phase transition temperature depends on the conformation (cis or trans type), the position in the fatty acyl chains and the number of double bonds.
  • cis or trans type conformation
  • introducing a single site of unsaturation of the cis type on the sn-2 chain only and in both chains of a phosphatidylcholine comprising 18-carbons can have the effect of lowering the chain melting transition temperature by 50° C. (from 54.5° C. to 3.8° C.) and 75° C. (from 54.5° C. to ⁇ 21° C.), respectively.
  • T m depends critically on the position of the cis-double bond.
  • T m is minimized when the double bond is located near the geometric center of the hydrocarbon chain, and progressively increases as the double bond migrates toward either end of the chain.
  • the chain melting temperature decreases monotically when the chain length inequivalence parameter ⁇ C/CL is increased to about 0.4.
  • ⁇ C/CL goes above ca. 0.4, packing perturbation, caused by the methyl ends of the acyl chains, becomes so overwhelming that the asymmetric phosphatidylcholine molecules adopt a new packing arrangement referred to as mixed interdigitation.
  • T m increases with chain length asymmetry.
  • a sterol component may be included to confer the liposome suitable physicochemical and biological behavior.
  • a sterol component may be selected from cholesterol or its derivative e.g., ergosterol or cholesterolhemisuccinate, but it is preferably cholesterol.
  • Cholesterol is often used in lipidic formulation of liposomes because it is generally recognized that the presence of cholesterol decreases their permeability and protects them from the destabilizing effect of plasma or serum proteins.
  • the cholesterol molecule contains three well-distinguished regions: a small polar hydroxyl group, a rigid plate-like steroid ring, and an alkyl chain tail.
  • a small polar hydroxyl group When cholesterol intercalates into the membrane, its polar hydroxyl groups positioned near the middle of the glycerol backbone region of the phosphatidylcholine molecule (Kepczynski M. et al., Chemistry and Physics of Lipids, 2008; 155:7-15).
  • Incorporation of modifiers as cholesterol into the lipid bilayers changes greatly the structural or physical properties of the liposomal membrane such as its organization, free volume, thickness, fluidity (viscosity) and polarity (hydrophobicity).
  • the bilayer's viscosity depends on the position of cholesterol within the bilayer which influences the free volume of the membrane and on the temperature.
  • the effect of cholesterol on the bilayer's microviscosity is rather complex. It is well-known that cholesterol increases the apparent microviscosities (reduces fluidity) of membranes being in liquid phase (Cournia et al. J. Phys. Chem. B, 2007; 111:1786-1801).
  • Papahadjopoulos et al. showed that the protective effect of cholesterol for liposomes depends on the physical state, i.e. “gel” or “fluid”, of the lipidic membrane when in contact with serum or plasma.
  • gel state the presence of cholesterol affects the ordering parameter of the phospholipid acyl chains within the bilayer and enhances the release of entrapped molecule.
  • fluid state cholesterol stabilizes the liposomes and prevents leakage of encapsulated material (Papahadjopoulos et al. Pharm. Research, 1995; 12(10): 1407-1416).
  • the liquid-ordered phase is formed when cholesterol associates with saturated, high-melting lipids, such as dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin, to create dynamic complexes in model membranes, so-called “lipid-rafts”.
  • DPPC dipalmitoylphosphatidylcholine
  • sphingomyelin lipid-rafts.
  • Cholesterol promotes a phase separation in model membranes where cholesterol-rich and cholesterol-poor microdomains are formed (Radhakrishnan et al. Proc. Natl. Acad. Sci., 2000; 97:12422-12427; Mc Connell et al. Biochim. Biophys. Acta, 2003; 1610:159-173).
  • Gaber et al. Pharm.
  • Typical “thermosensitive” liposomes usable in the context of the present invention comprises at least a phosphatidylcholine.
  • the phosphatidylcholine may be selected from dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), monopalmitoylphosphatidylcholine (MPPC), monostearylphosphatidylcholine (MSPC) and any mixture thereof.
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearylphosphatidylcholine
  • HSPC hydrogenated soybean phosphatidylcholine
  • MPPC monopalmitoylphosphatidylcholine
  • MSPC monostearylphosphatidylcholine
  • thermosensitive liposome further comprises distearylphosphatidylethanolamine (DSPE), distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG) (DSPE-PEG).
  • DSPE distearylphosphatidylethanolamine
  • DSPE distearylphosphatidylethanolamine
  • PEG polyethylene glycol
  • cholesterol is added in a molar ratio inferior to 25 mol %.
  • thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG), for example PEG2000 (DSPE-PEG2000).
  • DPPC dipalmitoylphosphatidylcholine
  • HSPC hydrogenated soybean phosphatidylcholine
  • DSPE distearylphosphatidylethanolamine
  • PEG distearylphosphatidylethanolamine-methoxypolyethylene glycol
  • PEG2000 DSPE-PEG2000
  • the molar ratio of the previously identified compounds is preferably 100:50:30:6 or 100:33:27:7
  • thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), monopalmitoylphosphatidylcholin (MPPC) and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG), for example methoxypolyethylene glycol-2000 (DSPE-PEG2000).
  • DPPC dipalmitoylphosphatidylcholine
  • MPPC monopalmitoylphosphatidylcholin
  • DSPE distearylphosphatidylethanolamine
  • PEG methoxypolyethylene glycol-2000
  • the molar ratio of the previously identified compounds is preferably 100:12:5.
  • thermosensitive lipidic membrane comprises dipalmitoylphosphatidylcholine (DPPC), monostearylphosphatidylcholine (MSPC), and distearylphosphatidylethanolamine (DSPE)-methoxypolyethylene glycol (PEG), for example methoxypolyethylene glycol-2000 (DSPE-PEG2000).
  • DPPC dipalmitoylphosphatidylcholine
  • MSPC monostearylphosphatidylcholine
  • DSPE distearylphosphatidylethanolamine
  • PEG methoxypolyethylene glycol-2000
  • the molar ratio of the previously identified compounds is preferably 100:12:5.
  • lipids are solubilized in an organic solvent such as chloroform. After homogenization of the solution, the organic solvent is evaporated under a nitrogen stream. The as-obtained dried lipid film is then hydrated by an aqueous medium at a temperature above the main phase transition temperature T m , leading to the formation of multilamellar vesicles with sizes ranging from 100 to 800 nm (Mills J. K. et al. Methods in Enzymology 2004; 387:82-113).
  • Cycles of dehydration and rehydration by respectively freezing (in liquid nitrogen) and thawing the solution (at a temperature above T m ), allow increasing the aqueous internal volume by forming unilamellar vesicles.
  • a process allowing vesicles size calibration is then applied to obtain a homogeneous size distribution.
  • Sonication produces Small Unilamellar Vesicles (SUV) with size ranging from 20 to 50 nm
  • extrusion process through a filter membrane produces Large Unilamellar Vesicles (LUV) with size ranging from 50 to 500 nm depending on the size of the filter pores. Both processes, sonication and extrusion, have to be performed at a temperature above T m .
  • thermosensitive liposomes typically comprised between 50 and 500 nm, preferably between 50 and 250 nm, for example between about 50 nm and about 150 nm.
  • Thermosensitive liposomes used in the present invention preferably comprise a biocompatible coating to ensure or improve their biocompatibility and specific biodistribution.
  • the biocompatible coating allows or favours the liposomes stability in a biocompatible suspension, such as a physiological fluid (blood, plasma, serum, etc.), any isotonic media or physiologic medium, for example media comprising glucose (5%) and/or NaCl (0.9%), which is required for a pharmaceutical administration.
  • a biocompatible suspension such as a physiological fluid (blood, plasma, serum, etc.), any isotonic media or physiologic medium, for example media comprising glucose (5%) and/or NaCl (0.9%), which is required for a pharmaceutical administration.
  • Such a biocompatible coating is obtained by treating the liposome with a surface treating agent.
  • Stability may be confirmed by dynamic light scattering of the liposomes in biocompatible suspension.
  • Said coating advantageously preserves the integrity of the liposome in vivo, ensures or improves its biocompatibility, and facilitates its optional functionalization (for example with spacer molecules, biocompatible polymers, targeting agents, proteins, etc.).
  • the coating can be non-biodegradable or biodegradable. Both options can be used in the context of the present invention.
  • non-biodegradable coatings are one or more materials or surface treating agents selected in the group consisting of sugar (agarose for example), saturated carbon polymers (polyethylene oxide for example), reticulated or not, modified or not (polymethacrylate or polystyrene for example), as well as combinations thereof.
  • biodegradable coatings are for example one or more materials or surface treating agents selected from the group consisting of a biological molecule, modified or not, natural or not and a biological polymer; modified or not, of natural shape or not.
  • the biological polymer may be a saccharide, an oligosaccharide or a polysaccharide, polysulfated or not, for example dextran.
  • the aforementioned materials, compounds or surface treating agents can be used alone or in combinations, mixtures or assemblies, composite or not, covalent or not, optionally in combination with other compounds.
  • Thermosensitive liposomes according to the present invention can further comprise a surface component enabling specific targeting of biological tissues or cells.
  • a surface component is preferably a targeting agent allowing the liposome interaction with a recognition element present on the target biological structure.
  • Such targeting agents can act only once the liposomes are accumulated in the tumor.
  • the density of said targeting agent is to be controlled carefully according to methods known by the skilled person.
  • a high density thereof can indeed perturb the targeting agent conformation and in consequence its recognition by the target cell (see for example J. A. Reddy et al. Gene therapy 2002; 9:1542; Ketan B. Ghaghada et al. Journal of Controlled Release 2005; 104:113).
  • a high target agent density may favour liposomes clearance by the Reticulo Endothelial System (RES) during circulation in the vasculature.
  • RES Reticulo Endothelial System
  • the targeting agent may be selected from an antigen, a spacer molecule, a biocompatible polymer.
  • the targeting agent can be any biological or chemical structure displaying affinity for molecules present in the human or animal body. For instance it can be a peptide, oligopeptide or polypeptide, a protein, a nucleic acid, a hormone, a vitamin, an enzyme and the like and in general any ligand of molecules (for example receptors, markers, antigens, etc.).
  • Ligands of molecules expressed by pathological cells in particular ligands of tumor antigens, hormone receptors, cytokine receptors or growth factor receptors.
  • Said targeted agents can be selected for example in the group consisting in LHRH, EGF, a folate, anti-B-FN antibody, E-selectin/P-selectin, anti-IL-2R ⁇ antibody, GHRH, etc.
  • the coating can also contain different functional groups (or linker segments), allowing any molecule of interest to bind to the surface of the liposome, such as a surface component enabling specific targeting of biological tissues or cells.
  • nanoparticle refers to a particle or aggregate of particles, said nanoparticle comprising a core (or central core) and a coating, the largest dimension of the core being less than about 100 nm.
  • the largest dimension of the core of the nanoparticle is the diameter of a nanoparticle of round or spherical shape, or the longest length of a nanoparticle of ovoid or oval shape.
  • size of the nanoparticle and “largest size of the nanoparticle” herein refers to the “largest dimension of the core of the nanoparticle”.
  • the “core” can designate a single particle (crystal or crystallite) or an aggregate of particles (aggregate of crystal or crystallites).
  • TEM Transmission Electron Microscopy
  • cryoTEM can be advantageously used to measure the size of the core of the nanoparticle especially when the core consists in a single particle (see FIG. 2 ).
  • DLS Dynamic Light Scattering
  • these two methods may further be used one after each other to compare size measures and confirm said size.
  • the material is an inorganic material.
  • the electronic surface charge of the herein used nanoparticles is below ⁇ 15 mV or above +15 mV, for example between ⁇ 15 mV and ⁇ 20 mV or between +15 mV and +20 mV, typically below ⁇ 20 mV or above +20 mV, when determined by zeta potential measurements, performed on nanoparticles suspensions with concentration varying between 0.2 and 8 g/L, nanoparticles being suspended in aqueous medium at pH comprised between 6 and 8.
  • the nanoparticle's shape can be for example round, flat, elongated, spherical, ovoid or oval, and the like.
  • the shape can be determined or controlled by the method of production, and adapted by the person of the art according to the desired applications.
  • particles having a quite homogeneous shape are preferred.
  • nanoparticles being essentially spherical, round or ovoid in shape are thus preferred.
  • Spherical or round shape is particularly preferred.
  • the largest size of the nanoparticles i.e. the largest dimension of the core of the nanoparticles, used in the context of the present invention is typically comprised between 1 and 50 nm.
  • the nanoparticle used in the context of the present invention comprise a core and a coating, said coating being responsible for the presence of an electrostatic surface charge below ⁇ 20 mV or above +20 mV when measured in an aqueous medium at physiological pH.
  • the electrostatic coating is advantageously a “full coating” (complete monolayer). This implies the presence of a very high density of molecules creating an appropriate and homogeneous charge on the all surface of the nanoparticle.
  • the coating can be an inorganic or organic surface coating.
  • the coating may be selected from the group consisting of an oxide, an hydroxide, and an oxyhydroxide.
  • the inorganic coating may comprise for example silicium, aluminium, calcium and/or magnesium.
  • An inorganic agent selected from the group consisting of, for example magnesium and calcium, will bring a positive charge (above +20 mV) to the surface of the nanoparticle at pH 7.
  • a silicium group may be used to bring a negative charge (below ⁇ 20 mV) to the surface of the nanoparticle at pH 7.
  • the coating is prepared with a molecule capable of interacting, through covalent binding or electrostatic binding, with the nanoparticle surface and of giving surface properties to said nanoparticle.
  • the surface coating organic molecule has two groups, R and X.
  • the function of X is to interact with the nanoparticle surface and the function of R is to give the nanoparticle's surface its specific properties.
  • X may be selected for example from a carboxylate (R—COO ⁇ ), a silane (R—Si(OR) 3 ), a phosphonic (R—PO(OH) 2 ), a phosphoric (R—O—PO(OH) 2 ), a phosphate (R—PO 4 3 ⁇ ) and a thiol (R—SH) group.
  • R brings at least the electronic surface charge to the nanoparticle in aqueous suspension at a physiological pH.
  • R When R brings a positive charge to the nanoparticle's surface, R may be an amine (NH 2 —X).
  • R When R brings a negative charge to the nanoparticle's surface, R may be phosphate (PO 4 3 ⁇ —X) or a carboxylate (COO ⁇ —X).
  • Organic coating conferring a positive charge (above +20 mV) to the nanoparticles surface may be selected from for example aminopropyltriethoxisilane, polylysine or 2-aminoethanethiol.
  • Organic coating conferring a negative charge (below ⁇ 20 mV) to the nanoparticles surface may be selected from for example a polyphosphate, a metaphosphate, a pyrophosphate, etc., or for example from citrate or dicarboxylic acid, in particular succinic acid.
  • the electrostatic coating is advantageously a “full coating”.
  • This electrostatic coating and especially amino or carboxylic moieties can further be used to link any group or agent on the nanoparticle's surface.
  • the nanoparticle surface can be functionalized using a steric group.
  • a group may be selected from polyethylene glycol (PEG), polyethylenoxide, Polyvinylalcohol, Polyacrylate, Polyacrylamide (poly(N-isopropylacrylamide)), Polycarbamide, a biopolymer or polysaccharide such as Dextran, Xylan, cellulose, collagene, and a switterionic compound such as polysulfobetain.
  • This steric group increases the nanoparticles stability in a biocompatible suspension, such as a physiological fluid (blood, plasma, serum, etc.), any isotonic media or physiological media, for example media comprising glucose (5%) and/or NaCl (0.9%).
  • a physiological fluid blood, plasma, serum, etc.
  • any isotonic media or physiological media for example media comprising glucose (5%) and/or NaCl (0.9%).
  • the inorganic material constituting the core is a superparamagnetic material.
  • Superparamagnetic materials include for example iron, nickel, cobalt, gadolinium, samarium, neodymium, preferably in the form of an oxide, an hydroxide or a metal, and any mixture thereof.
  • the material forming the core is selected from the group consisting of ferrous oxide and ferric oxide.
  • the oxide nanoparticles are made of magnetite or maghemite.
  • Mixed material can also be used to optimize interactions between a magnetic field and nanoparticles.
  • Solid solution forms (well known by the man skilled in the art as random mixtures of several materials) such as CoFe 2 O 4 for example can be used as a mixed material.
  • Solid solution form in demixed phases, such as Fe 2 O 3 /Co for example, can further be used.
  • Products of interest in the context of the present invention include any compound (for example a drug) with therapeutic or prophylactic effects. It can be a compound that affects or participates in tissue growth, cell growth, cell differentiation, a compound that is able to invoke a biological action such as an immune response, or a compound that can play any other role in one or more biological processes.
  • antimicrobial agents including antibacterial, in particular antibiotics, antiviral agents and anti-fungal agents
  • anti-tumor agents in particular anticancer chemotherapeutic agents such as cytostatic(s), cytotoxic(s), and any other biological or inorganic product intended to treat cancer
  • a therapeutic nucleic acid in particular a micro RNA (miRNA), a short-hairpin RNA (shRNA) and/or a small interfering RNA (siRNA).
  • miRNA micro RNA
  • shRNA short-hairpin RNA
  • siRNA small interfering RNA
  • Drugs in the present invention can also be prodrugs.
  • the compound(s) of interest may be present in the inner, the outer, or both of the compartments of the carrier, e.g. in the cavity and/or in the shell of a liposome.
  • kits comprising any one or more of the herein-described thermosensitive liposomes comprising superparamagnetic nanoparticles and a compound of interest to be delivered and released on a target site, or compositions comprising such liposomes.
  • the kit comprises at least one thermosensitive liposome or population of thermosensitive liposomes as herein described and also one or more containers filled with one or more of the ingredients of the diagnostic compositions herein described.
  • a labelling notice providing instructions for using the products can further be provided for using the thermosensitive liposome, population of thermosensitive liposomes, or compositions according to the present methods of the invention.
  • Iron oxide nanoparticles with a size distribution centered on 5 nm are synthesized by coprecipitation of ferrous and ferric ions adapted from (U.S. Pat. No. 4,329,241; Bacri et al., J. Magn. Magn. Mat., 1986; 62:36-46).
  • the reacting medium consists in a solution of sodium nitrate 3M maintained at pH 12.
  • a 3M sodium nitrate solution of ferrous and ferric ions in a molar ratio Fe(III)/Fe(II) equal 2 is prepared and slowly added to the reacting medium under mixing. It rapidly turns black. The whole solution is then aged during one night at ambient temperature under mixing.
  • Ferrite nanoparticles are then sedimented on a magnet and the supernatant is removed in order to eliminate the reacting medium. Then peptization (acidification) and oxidation of the nanoparticles surface is performed by diluting the pellet in a solution of nitric acid HNO 3 2 M at room temperature under vigorous mixing.
  • Oxidation of the nanoparticles core is performed by incubation of the pellet in a solution of ferric nitrate at elevated temperature (>90° C.) under vigorous mixing.
  • the nanoparticles are then washed by centrifugation.
  • the pellet is finally diluted in acidic water in order to reach a concentration of 150 g/L in iron oxide.
  • the solution is homogenized by sonication and pH is then adjusted to pH 2.
  • the morphology (size and shape) of nanoparticles was observed by Transmission Electron Microscopy ( FIG. 1 ).
  • the crystalline structure of iron oxide nanoparticles was confirmed by X-ray diffraction analysis.
  • Suspension of sodium hexametaphosphate is added to the suspension of iron oxide nanoparticles from example 1 (the amount of sodium hexametaphosphate added being below LD50/5) and the pH of the suspension is adjusted to a pH comprised between 6 and 8
  • Electronic surface charge ⁇ 20 mV is determined by zeta potential measurements on a Zetasizer NanoZS (Malvern Instruments), using a 633 nm HeNe laser, performed on nanoparticles suspensions with concentrations varying between 0.2 and 2 g/L, nanoparticles being suspended in aqueous medium at pH comprised between 6 and 8.
  • thermosensitive liposomes encapsulating iron oxide nanoparticles are prepared using the lipidic film re-hydration method (Bangham et al., J. Mol. Bio., 1965; 13:238-252; Martina et al., J. Am. Chem. Soc., 2005; 127:10676-10685):
  • Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow. Re-hydration of the lipidic film is performed at 55° C. with 2 mL of the iron oxide solution described in Example 2, so that the lipidic concentration is 50 mM.
  • lipidic composition dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol) and pegylated distearylphosphatidylethanolamine (DSPE-PEG2000) in the molar ratio 100:33:27:7 (DPPC:HSPC:Chol:DSPE-PEG2000).
  • Freeze-thaws cycles are then performed 20 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 55° C.
  • thermobarrel extruder (LIPEXTM Extruder, Northern Lipids) was used to calibrate the size of the nanoparticles-containing liposomes under controlled temperature and pressure. In all cases, extrusion was performed at 55° C., under a pressure ranging from 2 to 20 bars.
  • Elution profile is determined by quantification of magnetic nanoparticles by UV-visible spectroscopy (Cary 100 Varian spectrometer) via a ferrous ions/phenanthroline colorimetric reaction ( FIG. 2 ) adapted from Che et al., Journal of Chromatography B, 1995; 669:45-51, and Nigo et al., Talanta, 1981; 28:669-674.
  • Liposomes-containing fractions are collected ( FIG. 2 , first peak).
  • the concentration of iron oxide in liposomes ranges from 1 to 2.5 g/L.
  • T m of the composition is of 43° C.
  • doxorubicin DOX
  • pH gradient Once trapped in the internal aqueous core of liposome, doxorubicin leakage is prevented by complexation of the anthracycline molecule with an ammonium salt:
  • Iron oxide-containing liposomes (prepared as explained in example 3.1) are incubated in a solution of ammonium hydrogen phosphate (NH 4 ) 2 HPO 4 120 mM, at pH around 5 during 1 hour.
  • NH 4 ammonium hydrogen phosphate
  • HEPES buffer saline HEPES 20 mM, NaCl 140 mM, pH 7.4 to eliminate external (NH 4 ) 2 HPO 4 : once over 1 night at 4° C., then twice during 3 h at ambient temperature (final dilution factor of 250*250*250).
  • Non-encapsulated doxorubicin is then eliminated by size-exclusion chromatography on a Sephadex G50 filtration gel (column height: 5 cm; eluant: HEPES buffer saline).
  • the volume of doxorubicin loaded liposomes deposited onto the gel is 700 ⁇ L. Fractions of 500 ⁇ L are collected.
  • Elution profile is performed by quantifying doxorubicin concentration in each fraction (by measurement of absorbance at 480 nm). Liposomes/doxorubicin-containing fractions are collected.
  • NSL Non-Thermosensitive Liposomes
  • step a) the following lipidic composition was used: hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol) and pegylated distearylphosphatidylethanolamine (DSPE-PEG2000) in the molar ratio 100:65:7 (HSPC:Chol:DSPE-PEG2000).
  • HSPC hydrogenated soybean phosphatidylcholine
  • Chol cholesterol
  • DSPE-PEG2000 pegylated distearylphosphatidylethanolamine
  • steps a), b) and c) re-hydration of the lipidic film, thaw cycles and extrusion process are performed at 62° C.
  • FIG. 3 shows the release kinetics profile of doxorubicin (DOX)-loaded iron oxide-containing thermosensitive liposomes (TSL) as prepared in example 3.2.
  • Doxorubicin release is determined by fluorescence measurements.
  • PBS phosphate buffer saline
  • FBS fetal bovine serum
  • the diluted samples are plunged in a water bath to reach temperature T (37° C., 41° C., 43° C., 45° C.).
  • T o initial temperature
  • the fluorescence intensity I released is then measured.
  • 0.1% volume of Triton TX100 (10% vol in distilled water) is then added in order to break apart the lipidic membrane to obtain I total corresponding to the total amount of doxorubicin.
  • thermosensitive liposomes solution As shown in FIG. 3 , release of doxorubicin is maximal when the thermosensitive liposomes solution is heated above 43° C. (this corresponds to Tm which is the main gel-to-liquid crystalline phase transition temperature of this liposome composition). At this temperature the lipidic membrane is in a fluid state and its permeabilization allows for the release of the drug out of the liposome.
  • r 1 and r 2* relaxivities were done thanks to measurements of relaxation times T1 and T2* performed on a 1.5 Tesla clinical MRI Philips Achieva, using a head SENSE coil, for thermosensitive liposomes (TSL) (example 3.1) and non-thermosensitive liposomes (NTSL) (example 3.3), both loaded with 5 nm-sized iron oxide nanoparticles, and for non-encapsulated iron oxide nanoparticles (free NP) (example 1).
  • TSL thermosensitive liposomes
  • NTSL non-thermosensitive liposomes
  • free NP free NP
  • a range of dilution for each sample was performed corresponding to 40, 20, 10 and 5 ⁇ g/mL in iron oxide respectively. Dilutions were performed in agarose gel (4%). Samples were heated using a water bath first at 37° C. and then at 45° C., and relaxation times T1 and T2* of each dilution were measured at 37° C. and
  • FIG. 4 shows the modification of relaxivity values r 1 ( FIG. 4A ) and r 2* ( FIG. 4B ) upon heating at 45° C. corresponding to a temperature above the main gel-to-liquid crystalline phase transition temperature Tm (at this temperature the lipidic membrane is then in a fluid state).
  • Non-encapsulated iron oxide nanoparticles (free NP) and iron oxide-loaded NTSL demonstrated no significant evolution of the longitudinal relaxivity r 1 before and after heating at 37° C. and 45° C. respectively.
  • Non-encapsulated iron oxide nanoparticles (free NP) and iron oxide-loaded NTSL demonstrated no significant evolution of the transverse relaxivity r 2* before and after heating at 37° C. and 45° C. respectively.
  • FIG. 5 shows MR imaging of iron oxide-containing samples (TSL, NTSL and free nanoparticles) for different dilutions factors (40, 20, 10 and 5 ⁇ g/mL in iron oxide).
  • TSL thermosensitive liposome
  • This major modification of the ratio r 2* /r 1 upon heating at a temperature above Tm constitutes a highly sensitive “probe” of the permeabilization of the lipidic membrane of liposomes.
  • the subsequent drug release that occurs when a drug is co-encapsulated with the iron oxide nanoparticles inside the liposome can be monitored via detection of this marked MRI signal modification.
  • thermosensitive liposomes solution may be monitored by a T2*-mapping prior and after heating of the thermosensitive liposomes solution at or above Tm.
  • ultrasound experiments were performed with an in-house-designed, single-channel focused ultrasound transducer (Imasonic SA) incorporated in the bed of the 1.5-T MRI system.
  • the transducer has a focal length of 80 mm, with sinusoidal signal of 1.5 MHz. Heating was performed with acoustic power of 20 W during 60 seconds.
  • thermosensitive liposomes Upon heating, thermosensitive liposomes showed punctual MR signal and T 2 * relaxation time enhancement, with variations clearly visible at the HIFU focal point (red arrow). On average, the positive enhancement was of about 51%. Before heating, T 2* was 4.9 ⁇ 0.4 ms, and increased to 54 ⁇ 31.4 ms after HIFU heating (red arrow).

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