BACKGROUND OF THE INVENTION

To the present, much has been said about the benefits of the polyunsaturated fatty acids n-3 in order to prevent Inflammatory disorders, neurodegenerative processes and other diseases. Polyunsaturated fatty acids are essential In the sense of human body cannot synthesize them de novo and therefore must be obtained from the diet. Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids. Modern habits In westernized diets have produced an imbalance between the n-3 and n-6 series of fatty acids In favor of the latter. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane composition, receptor signaling and gene expression. The predominant n-6 fatty acid Is arachidonic acid, which Is converted to prostaglandins, leukotrlenes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with Inflammatory, atherogenic and prothrombotlc effects. Typical n-3 fatty acids are docosahexaenolc acid (DHA) and elcosapentaenoic acid (EPA), which are competitive substrates for the enzymes and receptors of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived elcosanoids antagonize the pro-inflammatory effects of n-6 fatty acids, n-3 and n-6 fatty acids are llgands/modulators for the nuclear receptors NFKB, PPAR and SREBP-lc, which control various genes of inflammatory signaling and lipid metabolism, n-3 fatty acids down-regulate Inflammatory genes and lipid synthesis, and stimulate fatty acid degradation, In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane strongly Influences membrane function and numerous cellular processes such as cell death and survival.

In 2002, the American Heart Association released a scientific statement, "Fish Consumption, Fish Oil, Omega-3 Fatty Acids and Cardiovascular Disease," on the effects of omega-3 fatty acids on heart function (including antiarrhythmic effects), hemodynamics (cardiac mechanics) and arterial endothelial function, The link between omega-3 fatty acids and CVD risk reduction are still being studied, but research has shown that omega-3 fatty acids decrease risk of arrhythmias, which can lead to sudden cardiac death, decrease triglyceride levels, decrease growth rate of atherosclerotic plaque and lower blood pressure (slightly).

Currently there Is a huge variety of nutraceutical supplements based on n-3 polyunsaturated fatty acids in the market, supported by a lot of scientific evidence on the benefits of polyunsaturated fatty acids. However these products present the inconvenience of bioavailability of the n-3 fatty acids ingested, and serious concerns arose concerning the absorption of such supplements.

There is also a prescription drug available in the market under commercial name Omacor/Lovaza which active ingredient is a combination between EPA and DHA n-3 fatty acids.

The traditional method of administering bioactive agents to treat diseases of the internal organs and vasculature has been by systemic delivery. Systemic delivery involves administering a bioactive agent at a discrete location followed by the agent migrating throughout the patient's body including of course, to the afflicted organ or area of the vasculature.

Systemic delivery introduces the bioactive agent in two ways: into the digestive tract (enteral administration) or in the vascular system (parenteral administration), either directly, such as injection into a vein or an artery, or indirectly, such as injection into a muscle or into the bone marrow. The ADMET factors (absorption, distribution, metabolism, excretion and toxicology) strongly influence delivery by each of these routes.

Magnetic nanoparticles have awakened great expectations on their nanomedical applications due to the potentiality of its magnetic properties on therapeutic (hyperthermia) or diagnostic uses (M I contrast agents).

Nanoparticles have been intensively used as injectable drug delivery systems. A significant obstacle to the use of these injectable drug delivery materials was the rapid clearance of the materials from the blood stream by the macrophages of the reticuloendothelial system.

Two types of MRI contrast agents have been developed during the last two decades: one based on the paramagnetic nature of the Gadolinium, and the other on the superparamagnetic nature of ferrite nanocristals. Superparamagnetic nanoparticles high transverse relaxivity has triggered the development of many interesting candidates to innovative MRI contrast agents, as a promising alternative to the Gadolinium based contrast agents (Neuwelt EA, 2008). These developments on superparamagnetic contrast agents has come up with several commercial products as ferumoxides or ferucarbotran, based on superparamagnetic iron oxide (SPIOs) nanoparticles, extensively used on imaging diagnosis of liver disorders. These commercial contrast agents consist on oligosaccharide matrixes (dextran or carboxidextran) wrapping up hundreds of nanometrical (2-15 nm) SPIOs. Other common synthetic strategy of stabilization and control of size and shape on the SPIOs production have been the peptization of the particles with help of surfactants as a fatty acid, usually oleic or lauric acid (Lan Q, 2007). We use this strategy of coating with fatty acids but instead of oleic or lauric we use high concentrates of EPA or DHA acids. As far as we know, no previous coatings of SPIOs with 80 omega-3 fatty acids have been reported.

A very promising approach from systemic delivery is local delivery, which comprises administering the bioactive agent directly to the afflicted site. With localized delivery, the ADMET factors abovementioned tend to be less important that with systemic administration. 85 Localized delivery of bioactive agents is currently considered state-of-the-art approach to the treatment of many diseases such as cancer or atherosclerosis. However, administering locally nanoparticles without losing a substantial fraction of them is quite challenging.

A novel method described in patent application US20090047318A1 is an implantable medical 90 device that includes a coating containing a plurality of nanoparticles, wherein the nanoparticles include one or more bioactive agents encapsulated within, adhered to a surface of or integrated into the structure of the nanoparticles and further include one or more contrast enhancing agents encapsulated within, adhered to a surface or integrated into the structure of the nanoparticles. However an implantable medical device is too invasive for the 95 patient.

An emerging trend on the MRI field consists on the use of these magnetic particles as intracellular tags that enables the visualization of cells by MRI (Cell Imaging) (Huang H, 2009). Macrophage is a phagocytic cell present in the conjunctive tissue of mammals and produced

100 by the differentiation of monocytes. It is a cell genuinely versatile as plays a role in the antigens processing, in the production of molecules with biological activity such as cytokines and in the lipid metabolism. When a monocyte enters damaged tissue through the endothelium of a blood vessel (a process known as the leukocyte extravasation), it undergoes a series of changes to become a macrophage. Monocytes are attracted to a damaged site by

105 chemical substances through chemotaxis, triggered by a range of stimuli including damaged cells, pathogens and cytokines released by macrophages already at the site.

The present application intends to conjugate the MRI technique with the therapeutic effect of the n-3 polyunsaturated fatty acids, using the macrophages as vehicles to target the 110 inflammation site. Macrophages carrying the "magnetoliposome", (thus the liposome with magnetic nanoparticles incorporated), herein described with nanoparticles and the n-3 polyunsaturated fatty acid would go to the inflammation site in response to the chemotaxis. They will also deliver the polyunsaturated fatty acid to the inflammation site in order to compete with the proinflammatory metabolic cascade. The superparamagnetic nanoparticle or 115 group of nanoparticles will allow imaging the inflammation site in order to see the performance of the polyunsaturated fatty acid in situ.

The role of macrophages on immunity response makes them to be present on many diseases and disorders. Of special interest is their role on inflammation processes, being responsible of the initiation and maintenance of inflammation, through production of different cytokines and 120 growth factors. Macrophages travel to where there is a damaged tissue and start a healing process that comprises an inflammatory response. Macrophage is clearly, therefore, a very interesting target for cell therapy with the additional advantage of a longer survival than neutrophils in the body that can take up to several months.

125 The product hereby described takes advantage of the strategy of macrophages transfection with magnetic particles of different nature. Among the several methods of transfection that can be used for cellular internalization of nanoparticles, the methodology applied is lipofection (liposome transfection) because it makes use of liposomes, whose nature can be conveniently modified as explained below. Liposomes incorporating a magnetic load have been also

130 extensively studied and are known by the term "magnetoliposomes", (De Cuyper M, 2007).

Those Magnetoliposomes have been proposed for several uses on nanomedicine, generally as drug delivery agents (J Cocquyt, 2008) or lipofection vehicles for magnetic particles (Soenen SJ, 2009).

135 Usually, the magnetoliposomes used for lipofection are formed by cationic phospholipids which help on the interaction with the cellular membrane (with negative net charge) (P L Feigner, 1987). There are tens of synthetic formulations of cationic phospholipids, usually proposed for lipofection with DNA, as DOTAP, DODAP or DOTMA. However, we use a mixture of omega-3 (EPA/DHA) enriched neutral and cationic phospholipids or just enriched

140 phospholipids, in order to lipofect the macrophages, not only with magnetic nanoparticules but also with omega-3 enriched phospholipids. The production of such omega-3 enriched phospholipids can be achieved by chemical or enzymatic procedures and has been described on several publications and patents as (WO/2005/038037) "Methods for preparing phospholipids containing omega-3 and omega-6 moieties" licensed from Enzymotec LTD (Piatt,

145 Laouz, Pelled, & Shulman N, 2005). Magnetically tagged monocytes and macrophages have been previously used on cell imaging. Sometimes the SPIOs are directly administered to the bloodstream and the Macrophages uptake the particles in vivo, in others the monocytes/macrophages are previously tagged and then administered (Beduneau A et al, 2009 " Facilitated Monocyte-Macrophage Uptake and

150 Tissue Distribution of Superparmagnetic Iron-Oxide Nanoparticles. PLoS ONE 4(2): e4343. doi:10.1371/journal. pone.0004343). Less commonly, has been proposed that magnetically tagged macrophages can be used with therapeutic purpose (Santhi Gorantia et al, 2006 Quantitative magnetic resonance and SPECT imaging for macrophage tissue migration and nanoformulated drug delivery). Never before, macrophages lipofected by omega-3 enriched

155 phospholipids and magnetic nanoparticles have been reported with therapeutic purposes.

Macrophages are white blood cells derived from monocytes, and their role on immunity response makes them to be present on many diseases and disorders and therefore highly suitable as targets.

160 DETAILED DESCRIPTION OF THE INVENTION

There is no method known in the prior art that uses macrophages isolated from a small volumen of blood from the patient in order to load them with the magnetoliposome described herein that allow detection, imaging and therapy at the same time.

165

One aspect of this invention is to provide with a method to design a magnetoliposome, consisting in a nanoparticle with magnetic properties and a polyunsaturated fatty acid from the group of n-3 or one derivative from them, linked to it and the complex being embedded by a liposome. The liposome therefore shall bear properties for diagnosis and detection and 170 simultaneously a therapeutic one derived from the presence of the polyunsaturated fatty acid or one of its derivatives.

More particularly the nanoparticle or group of nanoparticles will have magnetic properties and will be selected from iron oxide, cobalt ferrite, manganese ferrite or magnesium ferrite or 175 combinations thereof and such nanoparticle or group of nanoparticles could or not contain and outer coating consisting of metals, polymers, proteins, oxides or combinations thereof.

The fatty acid present in the magnetoliposome will be a polyunsaturated fatty acid of marine origin, preferably eicosapentaenoic acid, docosahexaenoic acid or one of its derivatives or a 180 conjugated fatty acid in the form of a free fatty acid, an ester, a salt, a solvate or any other pharmaceutical acceptable complex.

The phospholipid embedding the nanoparticle and the fatty acid will be natural or synthetic.

185 It is a further object of the present invention to describe a method to lipofect the previously isolated macrophage from a blood sample from the patient with the magnetoliposome described in the present invention.

It is a further object of the present invention to add another complementary complex agent in 190 combination with the polyunsaturated fatty acid selected from the group of HMG-CoA reductase inhibitors, thiazolidinediones, fibrates, niacin, fenofibrates, phytosterols or tocopherols.

EXAMPLES:

195 The following examples described below are intended to be only descriptive and must not be understood as limits to the present invention.

Example 1

Synthesis of SPIOs labeled macrophages via EPA enriched liposome lipofection.

200 Materials

Iron (III) chloride hexahydrate (FeCI ₃ .6H ₂ 0) 99%, Iron (II) chloride tetrahydrate (FeCI ₂ .4H ₂ 0) 99%, 2-(2 ethanoic acid)-2-hydroxybutanedioate (citrate), ammonium hydroxide (5 M), EPA enriched phosphatidylcholine, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), ( TES buffer (5mM, pH 7.0), PBS buffer (5mM, pH 7.0).

205 Synthesis of citrate stabilized superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide (SPIOs) nanoparticles were prepared as previously described by Bee et al ¹ . In short, 5 ml of a 50% NH ₃ solution was dropped into 20 ml of an aqueous solution containing Fe(lll) chloride (7.5 mmol) and Fe(ll) chloride (5 mmol) and citric acid (2.9 mmol). Larger particle aggregates were removed by centrifugation, the resulting ferrofluid had a

210 magnetite content of 14.3 mg Fe ₃ 0 ₄ /ml and a core diameter of approximately 4 nm. The pH was adjusted to 7.0 by HCI (1M). Magnetoliposome preparation

A mixture of 250 mg of a synthetically enriched phosphatidylcholine in which at least 90% of the fatty acids present are EPA, and 15 mg of the cationic phospholipid l,2-dioleoyl-3-

215 trimethylammonium-propane (DOTAP) were dissolved in 15 ml of a chloroform/methanol (3:1, v/v) solution. The solvent was then evaporated with a rotatory evaporator and the resulting powder was resuspended on 15 ml of the iron oxide nanoparticles solution and sonicated for 5 min. The mixture was then cooled in ice-cold water and repeatedly (at least 10 times) extruded through a polycarbonate membrane (200nm pore diameter, LiposoFast system, Avestin,

220 Mannheim, Germany) in batches of 1 ml. The fractions were pooled and concentrated in a dialysis bag immersed in Spectra/Gel Absorbent until a final volume of 5 ml was obtained. Macrophages Labeling

Macrophages were isolated and purified from spleens of BN rats, then maintained in culture. 500 ΘΙ of the magnetoliposome solution were added into 10 ml of macrophages culture (1 x 225 10 ⁷ cells per ml) for overnight incubation. After proper washing the macrophages were resuspended in PBS to desirable cell densities (1 x 10 ⁷ cells per ml).

Example 2

230 Synthesis of macrophages labeled with EPA stabilized SPIOS via EPA enriched liposome lipofection.

Materials

Iron (III) chloride hexahydrate (FeCI ₃ .6H ₂ 0) 99%, Iron (II) chloride tetrahydrate (FeCI ₂ .4H ₂ 0) 99%, ammonium hydroxide (5 M), eicosapentanoic acid (EPA) 80 wt %, EPA enriched

235 phosphatidylcholine, TES buffer (5mM, pH 7.0), PBS buffer (5mM, pH 7.0).

Synthesis of superparamagnetic iron oxide nanoparticles stabilized by 80 wt % EPA

An aqueous mixture of 1 M Fe(lll) chloride (50 ml) and 1 M Fe(ll) chloride (25 ml) was treated with 5 ml of 5M ammonium hydroxide drop wise added under mechanical stirring. After 20 min of stirring at 10 ⁵ C under nitrogen-gas atmosphere the solution was heated to 80 ⁵ C for 30

240 min to evaporate the ammonia, and then cooled down to room temperature.

The particles obtained were washed twice with 15 ml of deionized water using ultracentrifigation (30.000 rpm for 30 min at 10 ⁵ C), decanted and finally resuspended on 50 ml of eicosapentanoic acid (EPA) 80 wt %, (20 mM). A turbid supernatant appeared due to the excess of EPA that formed an emulsion, being removed during the washing steps.

245 Magnetoliposome preparation Small unilamellar vesicles (SUVs) were prepared by mixing synthetically enriched phosphatidylcholine (250 mg), in which at least 80% of the fatty acids present are EPA, with 20 ml of TES buffer (5 mM, pH 7,0), followed by a 15 minutes sonication at 25 ^Q C.

1 ml of the magnetic nanoparticles stabilized by EPA aqueous solution were added to the previously prepared SUVs in TES buffer. The mixture was then dialyzed for 2 days at 37 ^Q C (SpectraPor no. 2 dialysis membrane, 12.000-14.000 MWCO; Spectrum Laboratories) against 2 I of PBS buffer (5 mM, pH 7.0) with a buffer exchange every 8 h. SUVs loaded with magnetic nanoparticles were then separated from excess vesicles by high-gradient magnetophoresis, with a final concentration of 400 Eg Fe/ml.

Macrophages Labeling

Macrophages were isolated and purified from spleens of BN rats, then maintained in culture. 500 ΘΙ of the SUVs PBS solution were added into 10 ml of macrophages culture (1 x 10 ⁷ cells per ml) for overnight incubation. After proper washing the macrophages were resuspended in PBS to desirable cell densities (1 x 10 ⁷ cells per ml).

Example 3

Synthesis ofSPIOs labeled macrophages via DHA enriched liposome lipofection.

Materials

Iron (III) chloride hexahydrate (FeCI ₃ .6H ₂ 0) 99%, Iron (II) chloride tetrahydrate (FeCI ₂ .4H ₂ 0) 99%, ammonium hydroxide (5 M), docosahexaenoico (DHA) 85 wt %, enriched phosphatidylcholine, PBS buffer (5mM, pH 7.0), acetone, paraffin.

Synthesis of superparamagnetic iron oxide nanoparticles stabilized by 85 wt % DHA

Following the procedures previously described by Reimers et af, 0,2 mole of FeCI ₃ .6H ₂ 0 and 0,1 mole FeCI ₂ .4H ₂ 0 were dissolved in 50ml of water. Rapid precipitation of ferrous oxide was prompted by the addition of 10 ml of 5M ammonium hydroxide. Then a mixture of 20 ml of DHA and 40 ml of paraffin was added while stirring of the aqueous solution. The mixture was then heated, under argon current to evaporate the water, then heated up till 120 ^Q C and held at this point for five minutes and finally cool to room temperature.

Organic solution was then transferred to a flask and 250 ml of acetone were added with the consequent flocculation of the magnetic particles. A magnet was placed at the bottom of the flask, keeping the magnetic material and pouring off the acetone. A second addition of 200 ml of acetone was used to wash the flocculate. Acetone traces were evaporated by moderate heating on a stove. 280

Magnetoliposome preparation

A mixture of 250 mg of a synthetically enriched phosphatidylcholine in which at least 85% of the fatty acids present are DHA were dissolved in 15 ml of a chloroform/methanol (3:1, v/v) solution. The solvent was then evaporated with a rotatory evaporator and the resulting

285 powder was resuspended on 15 ml of PBS buffer (5 mM, pH 7.0) and then added to the iron oxide nanoparticles stabilized by 85 wt % DHA, and finally sonicated for 5 min. The mixture was then cooled in ice-cold water and repeatedly (at least 10 times) extruded through a polycarbonate membrane (200nm pore diameter, LiposoFast system, Avestin, Mannheim, Germany) in batches of 1 ml. The fractions were pooled and concentrated in a dialysis bag

290 immersed in Spectra/Gel Absorbent until a final volume of 5 ml was obtained.

Macrophages Labeling

Macrophages were isolated and purified from spleens of BN rats, then maintained in culture. 500 ΘΙ of the magnetoliposome solution were added into 10 ml of macrophages culture (1 x 10 ⁷ cells per ml) for overnight incubation. After proper washing the macrophages were 295 resuspended in PBS to desirable cell densities (1 x 10 ⁷ cells per ml).

Example 4

Synthesis of macrophages labeled with cobalt ferrite nanoparticles via DHA enriched liposome lipofection.

300 Materials

Iron (III) chloride hexahydrate (FeCI ₃ .6H ₂ 0) 99%, Iron (II) chloride tetrahydrate (FeCI ₂ .4H ₂ 0), sodium hydroxide (1M), docosahexaenoico (DHA) 90 wt %, enriched phosphatidylcholine, PBS buffer (5mM, pH 7.0).

305 Synthesis of cobalt ferrite nanoparticles stabilized by 85 wt % DHA

5ml of a 2M solution of CoCI ₂ .6H ₂ 0 were mixed under stirring with 40ml of an aqueous solution of FeCI ₃ -6H ₂ 0 0.5M and heated to 505C. 100 ml of a solution of 1M sodium hydroxide was prepared and slowly added to the salt solution dropwise. The reactants were constantly stirred using a magnetic stirrer until a pH level of 11-12 was reached. 100ml of 310 docosahexaenoic acid (DHA) 90 wt %, were added to the solution as a surfactant and coating material. The liquid precipitate was then brought to a reaction temperature of 80 ⁵ C and stirred for one hour. The product was cooled to room temperature and then washed twice with distilled water. Magnetoliposome preparation

Small unilamellar vesicles (SUVs) were prepared by mixing synthetically enriched phosphatidylcholine (250 mg), in which at least 85% of the fatty acids present are DHA, with 20 ml of TES buffer (5 mM, pH 7,0), followed by a 15 minutes sonication at 25 ⁵ C.

2 gr of the magnetic nanoparticles were added to the previously prepared SUVs in TES buffer. The mixture was then dialyzed for 2 days at 37 ⁵ C (SpectraPor no. 2 dialysis membrane, 12.000-14.000 MWCO; Spectrum Laboratories) against 2 I of PBS buffer (5 mM, pH 7.0) with a buffer exchange every 8 h. SUVs loaded with magnetic nanoparticles were then separated from excess vesicles by high-gradient magnetophoresis.

Macrophages Labeling

Macrophages were isolated and purified from spleens of BN rats, then maintained in culture. 500 μΙ of the magnetoliposome solution were added into 10 ml of macrophages culture (1 x 10 ⁷ cells per ml) for overnight incubation. After proper washing the macrophages were resuspended in PBS to desirable cell densities (1 x 10 ⁷ cells per ml).

Example 5

One pot synthesis of 90% EPA coated nanoparticles.

Magnetic nanoparticles were synthesized according to the method reported in the literature (Sun et al). 1,4 g of Iron(lll) acetylacetonate, 5 g of 1,2-hexadecanedio, 2,2g of oleylamine, and 3,6 g of EPA acid (90%), were dissolved on 50 ml of benzyl ether and mixed under a flow of argon by magnetic stirring. The mixture was heated to 200 °C for 3 h. The black mixture was cooled to room temperature and ethanol was added producing nanoparticles peptization. Supernatant was through up and the remaining precipitate was redissolved on hexane. Centrifugation at 5000 rpm for 10 min was used to remove any undispersed residue and the product was then precipitated with ethanol.