WO2020217231A2

WO2020217231A2 - Magnetic nanosystem and method to produce the nanosystem

Info

Publication number: WO2020217231A2
Application number: PCT/IB2020/053947
Authority: WO
Inventors: Ana Rita OLIVEIRA RODRIGUES; Daniela Sofia MARQUES PEREIRA; Beatriz DIAS CARDOSO; Elisabete Maria DOS SANTOS CASTANHEIRA COUTINHO; Paulo José GOMES COUTINHO
Original assignee: Universidade do Minho
Current assignee: Universidade do Minho
Priority date: 2019-04-26
Filing date: 2020-04-27
Publication date: 2020-10-29
Anticipated expiration: 2021-10-26
Also published as: EP3958904A2; WO2020217231A3; PT115474B; PT115474A

Abstract

The present application discloses a magnetic nanosystem comprising magnetoliposomes and a method to produce the magnetoliposomes. Magnetoliposomes are nanocarriers based on liposomes entrapping magnetic nanoparticles. They consist in an inner magnetic core, either a cluster of nanoparticles or a magnetic fluid, covered by a lipid bilayer. The magnetoliposomes can be loaded with one type, or more than one type of molecules, particularly therapeutic drugs that will be released according to the specific disease target, particularly a cancer. In this sense the lipid composition of the bilayer can be manipulated according to the cancer family type to be treated and microenvironment specific requirements.

Description

DESCRIPTION

"MAGNETIC NANOSYSTEM AND METHOD TO PRODUCE THE NANOSYSTEM"

Technical Field

The present application relates to a magnetic nanosystem and method to produce the nanosystem.

Background of the invention

The words cancer, tumor and neoplasia are generic terms that define a broad range of diseases characterized by the uncontrolled proliferation of cells of a given tissue or organ as a result of genetic or epigenetic changes in somatic cells. Cancer is one of the main causes of death in the world, with an estimated 7.6 million deaths per year and a predicted increase to 13.1 million by 2030 (Singh, 2008) . The main obstacles in the fight against cancer include (i) the absence of effective biomarkers in diagnosis and prognosis; (ii) invasive diagnostic and treatment methods; (iii) reduced epigenetic profile; (iv) lack of individualized treatments; (v) limitations associated with existing chemotherapeutic agents and (vi) the occurrence of metastases.

Even though there is no standard treatment regimen adopted, most patients are typically submitted to surgery, chemotherapy (QT) and radiation therapy (RT) . These treatments do not have enough therapeutic effectiveness and are associated with severe side effects that may decrease the patient's quality of life and, in some cases, lead to the suspension of the treatment. Most therapeutic drug molecules have a limited pharmacokinetics and no selectivity, which induces toxicity in non-target regions and cause severe side effects. In order to overcome some of the obstacles of conventional treatments, new approaches that integrate biological principles at the nanoscale have been explored. Controlled drug delivery systems to target specific sites has become increasingly important as an approach to overcome the lack of specificity of the conventional cytotoxic agents (Caban, 2014; Cho, 2008) . Such systems can improve the properties of the therapeutic drug molecules and their pharmacokinetics by increase half time circulation (acting as a stealth nanocarrier to the immune system) and to overcome certain biological barriers, such as the blood-brain barrier (Pradhan, 2010; Caban, 2014) . The application of these nanomaterials in the treatment of cancer has already shown to improve drugs behavior. The first success in nanomedicine research was the approval of liposomal doxorubicin for clinical use (Doxil^® approved in 1995) . Since then, several liposome-based products have been approved for cancer therapy, such as DaunoXome^® approved in 1996, Depocyt^® approved in 1999, Myocet^® approved in 2000, Mepact^® approved in 2004, Marqibo^® approved in 2012 and Onivyde™ approved in 2015.

Although liposome-based products have shown an improvement in therapeutic drug molecules pharmacokinetics, active targeting approaches has been exploited in order to enhance a specific interaction with cancer cells. Consequently, specific ligands and surface moieties have been investigated for the efficient nanocarriers internalization by active targeting, namely, folate or transferrin receptors, glycoproteins, EGFR (epidermal growth factor receptor) and ssDNA or RNA aptamers (single-stranded nucleic acid oligonucleotides which bind to their targets with high specificity and affinity) . However, the clinical validation of active targeting is limited and it has not been easily achieved. Despite the improved biodistribution and therapeutic outcomes of ligand-targeted liposomes in a number of preclinical studies, the advantages have so far been negligible in the clinical research phase. Another mechanism to improve liposomes efficacy is the stimuli-sensitive based on the physiological differences between the normal and tumor tissues. For instance, hypoxia (deprivation of oxygen) , low pH, high temperature, elevated redox potential and up-regulated protein/enzyme levels. In order to enhance drug release at target sites, a new generation of liposomes focusing on technologies that make use of exogenous triggers (such as electric pulse/high energy radiation, ultrasound and light) have been exploited. However, all present limitations and none allows the physical control over the nanocarriers localization. Magnetic nanoparticles location can be controlled by the use of an external magnetic field gradient and heat can be produced by these nanoparticles under an alternate magnetic field. Particularly, nanoparticles with superparamagnetic behavior are of major interest because they only present magnetization in the presence of an external magnetic field. The fact that they do not present permanent magnetism shows itself as an added value, since it prevents their aggregation (in the absence of a magnetic field) . Based on this, magnetic nanoparticles are very desirable for target drug delivery and hyperthermia. Magnetic fluid hyperthermia is a therapeutic approach in which the patient is subjected to electromagnetic waves with frequencies in the order of 100 to 1000 kHz and fields between 150 and 250 Gauss. This stimulus incites the heat production by the nanoparticles and increase tumor tissue temperature (between 43 ° C and 46 ° C) in order to interrupt the processes of regulation and growth of tumor cells. At these temperatures, heat causes structural damage to tumor cells leading to apoptosis. In addition to direct cell damage, hyperthermia may induce a synergistic effect with chemotherapy and radiotherapy, as cells become more sensitive to these treatments.

Despite being promising for cancer treatment (target drug delivery and hyperthermia) , the FDA approved magnetic nanoparticles are mainly for magnetic resonance imaging (Feridex^® and Lumirem^®) and chronic kidney disease (Venofer^®, Ferrlecit^®, INFeD^® and Dexlron^®) . More recently, the Nanotherm^® therapy was the first magnetic nanoparticle-based therapy approved (2010) and uses biocompatible aminosilane-coated superparamagnetic iron oxide nanoparticles in for Glioblastoma treatment. Magnetic nanoparticles present some issues related to their accumulation, degradation and clearance. In fact, some of the FDA approved products (Feridex^® and Lumirem^®), both based on SPIONs (superparamagnetic iron oxide nanoparticles) are no longer commercially available. Recent studies have demonstrated that these iron oxide nanoparticles exhibit peroxidase-like activity, leading to the production of reactive oxygen species (ROS), thus showing some toxicity.

In order to overcome the problems of liposomes and magnetic nanoparticles, magneto-sensitive liposomes have been proposed, combining the advantages of both. Magnetoliposomes are known for their potential in the field of cancer diagnosis and therapy. Therapeutic drug molecules can be encapsulated into liposomes by either passive loading or active loading. In passive loading the drugs are encapsulated during the formation of liposomes, in which lipophilic drugs are entrapped in the lipid bilayer and hydrophilic drugs in the aqueous compartment. In active loading, drug molecules are loaded into preformed liposomes. This process is driven by an electrochemical potential created by the pH or ion gradients established across the lipid bilayer of the liposomes, which are created during liposomes preparation by using a buffer of specified pH and ion concentration. This nanosystem has the capacity to encapsulate, transport and release drugs due to its ability to generate heat (when subjected to an alternating magnetic field) combined with the thermo-sensibility of lipid bilayer composition. Comparing to liposomes, magnetoliposomes offer several advantages in oncology, such as guiding capabilities (using a permanent magnetic field) , magnetic stimulus controlled release and hyperthermia. Therefore, magnetoliposomes promote improved release of the drug in a tissue of interest, offer the possibility of combined therapy (chemotherapy and hyperthermia) and deeper intracellular penetration of drugs in the tumor environment. Currently, the major drawback regarding this type of nanosystems remains on the final magnetic behavior of the magnetoliposomes. This occurs since the ratio between the magnetic and the non magnetic components decrease (after magnetic nanoparticle encapsulation into the liposomes), compromising the magnetic nanosystems overall magnetic behavior.

Regarding the magnetic-based products developed (US2003/0211045 Al, US 2008/0260648 Al, US 2013/0302408 Al, US 2017/0151174 Al), they comprise liposomes and magnetic nanoparticles. The above-mentioned provide nanosystems containing a magnetic core, typically of metal oxide consisting of various transition metal elements. However, iron oxide nanoparticles have shown peroxidase-like activity that may cause severe damage to cell components and induce inflammatory responses. In comparison with the cited documents, the presently described solution provides iron oxide nanoparticles based on biocompatible elements. This product is based on ferrite and mixed ferrite nanoparticles of alkaline earth metals (magnesium, calcium and strontium, ) , being strong, highly biocompatible candidates due to their decreased oxidation rate and, consequently, less radical oxygen species (ROS) production.

The existing methods for the synthesis of magnetoliposomes are not simple or expedite and the resulting insufficient magnetic properties compromise their therapeutic efficacy. New approaches, in which coating occurs only after nanoparticles synthesis, have been drawn to improve the magnetism of the magnetoliposomes. Yet, these routes are either long in time or complex. For instance, a dialysis procedure can take up to two days and does not have the proper control of magnetoliposomes diameters and size distribution. Thin film hydration has been one of the most used protocols, however, it is reported that it can form multilamellar structures, display low drug encapsulation efficiency and cannot control the diameter and size distribution of the synthesized structures. In the first elaborated synthesis methods of magnetoliposomes, nanoparticles synthesis and lipid bilayer are formed at the same time. As a result, the calcination step (an additional procedure that improve magnetic properties of nanoparticles) is not allowed since the high temperatures destroy the lipid bilayer. Moreover, the nanoparticles used in this type of nanosystem typically possess a spherical structure. Recent studies have proven that non-spherical structures (e.g. prisms, rods, cubes) enhance the total magnetic properties due to increased nanoparticles shape anisotropy.

The present solution describes a route for the synthesis of a new generation of magnetoliposomes for better therapeutic effect. Here, the synthesized magnetic nanoparticles present, preferably, inherent higher shape anisotropy, which translate in a better magnetic response and heating capabilities. Furthermore, as the calcination step is independent of nanoparticles encapsulation into liposomes, the nanosystems overall magnetic behavior is ensured by this additional procedure. Also, the present solution for nanoparticles encapsulation ensure that the final product possess a well- defined morphology, size distribution and that the magnetic behavior is similar to net nanoparticles, by increasing the ratio between the magnetic and non-magnetic components of the final structure.

Summary

The present application relates to magnetic nanosystem characterized by magnetoliposomes comprising:

a lipid bilayer comprising dipalmitoylphosphatidylcholine lipids, surrounding an

inner magnetic core; and

therapeutic drug molecules.

In one embodiment the magnetoliposomes have a size between 100 and 200 nm.

In one embodiment the inner magnetic core comprises superparamagnetic nanoparticles with particle sizes between 1 and 100 nm.

In another embodiment the superparamagnetic nanoparticles have a spherical shape, cubic shape or flower shape. In yet another embodiment the superparamagnetic nanoparticles are made of metal oxide composed by alkaline earth metals.

In one embodiment the superparamagnetic nanoparticles are ferrite magnetic particles composed by the metals of magnesium and calcium.

In another embodiment the ferrite magnetic nanoparticles further comprise alkaline earth metals of strontium (Sr), Calcium (Ca) and Magnesium (Mg), alone or in combination.

In yet another embodiment the lipid bilayer is composed by a lipid formulation with transition temperatures between 40 and 45°C .

In one embodiment the lipid bilayer formulation further comprises phospholipid molecules such as phosphatidylcholine, phosphatidylethanolamine , phosphatidylserine , phosphatidylglycerol , phosphatidic acid, phosphatidylinositol , sphingomyelin, alone or in combination.

In one embodiment the lipid bilayer formulation further comprises cholesterol.

In another embodiment the lipid bilayer formulation further comprises pH sensitive lipids such as cholesteryl hemisuccinate, N- ( 4-carboxybenzyl ) -N, N-dimethyl-2 , 3- bis (oleoyloxy) propan-l-aminium, 1, 2-dipalmitoyl-sn-glycero- 3-succinate, 1 , 2-dioleoyl-sn-glycero-3-succinate and N- palmitoyl homocysteine. In yet another embodiment the surface of the lipid bilayer is functionalized with polyethylene glycol.

In one embodiment the surface of the lipid bilayer is further functionalized with permeability inducing ligands such as bradykinin, vascular permeability factor/endothelial growth factor, Prostaglandins, Collagenase, Peroxynitrite, Tumor necrosis factor-a.

In one embodiment the therapeutic drug molecules are located inside the lipid bilayer if the drug molecules are hydrophobic .

In another embodiment the therapeutic drug molecules are located in the hydrophilic compartment of the magnetoliposome if the drug molecules are hydrophilic.

In one embodiment the surface of the lipid bilayer is functionalized with active targeting molecules.

The present application further relates to a method of producing the magnetic nanosystem, comprising the following steps :

Synthesis of the superparamagnetic nanoparticles;

Encapsulation of the nanoparticles into liposomes with the following steps:

synthesis of a first lipid layer by the preparation of :

a thin film of a lipid and/or surfactant of concentration between 0.01 mM and 2 mM under nitrogen flow; - a nonpolar solvent, pre-heated between 43 and 46°C is added to the thin film and ultrasonicated for a time interval between of 15 min and 60 min;

- the superparamagnetic nanoparticles are added to the lipid formulation, in an iron concentration between 20 mg/ml and 180 mg/ml;

- the solution is ultrasonicated for a time between 15 min and 60 min;

- purification of the solution by magnetic decantation;

encapsulation of the therapeutic drug molecules by the addition of an ethanolic solution of a concentration between lxl0 ⁶ M and 5xl0⁵ M;

- addition of a second lipid layer, prepared in the same manner as the first layer, to the solution previously prepared;

- the resulting magnetoliposomes are washed and purified with ultrapure water by magnetic decantation.

In one embodiment the superparamagnetic nanoparticles are added to the lipid formulation in a concentration between 5xlCh⁴ M and 2xlCh⁵ M with a percentage of water between 0.005% and 1 % .

In another embodiment the superparamagnetic nanoparticles are added to the lipid formulation in a concentration between 5xl0⁴ M and 2xl0⁵ M with a percentage of water between 1 and 5% .

In yet another embodiment the surfactant is selected from Triton X-100, AOT , SDS and CTAB . In one embodiment the nonpolar solvent is selected from hexane, heptane, octane, decane, dodecane, chloroform, cyclohexane, toluene, benzene.

In another embodiment the first lipid formulation has a concentration between 0.01 mg/mL and 10 mg/mL.

In yet another embodiment the second lipid formulation has a concentration between 0.05 mM and 1.5 mM.

In one embodiment cholesterol and/or active targeting molecules and/or polyethylene glycol are added to the lipid formulations .

The present application further relates to a method to prepare cubic shaped superparamagnetic nanoparticles of the nanosystem, comprising the following steps:

50 mmol of octadecylamine is heated until reaching its melting point;

a solution of 0.5 mmol of magnesium acetate tetrahydrate , 0.5 mmol of calcium acetate hydrate, 2 mmol of iron (iii) citrate tribasic monohydrate and 3.1 mmol of oleic acid is added to the octadecylamine solution;

the mixture is heated, with 10°C per minute, until reaching 200 °C and then left 90 minutes at this temperature; nanoparticles are washed with an ethanol solution, by several cycles of centrifugation and aqueous redispersion; the nanoparticles are calcined between 300°C and 800°C for a time range between 1 h and 4 h. The present application also relates to a method to prepare flower shaped superpara agnetic nanoparticles of the nanosystem, comprising the following steps:

a solution containing 0.05 mol of potassium oxalate, 0.05 mol of sodium hydroxide and 2.5 mmol of agarose is added to 25 mL of ultrapure water under nitrogen flow and magnetic stirring;

a solution containing 2.5 mmol of iron (iii) citrate tribasic monohydrate is added drop by drop to the previous solution;

the mixture is heated to 90°C for a time range between 2 and 4 h;

the final product is washed with a basic solution of 1 M sodium hydroxide, by several cycles of centrifugation, aqueous redispersion and magnetic decantation;

the nanoparticles are calcined preferably between 300°C and 800°C for a time range between 1 h and 4 h.

General Description

The present application relates to a magnetic nanosystem comprising :

- liposomes comprising a lipid bilayer that can be sensitive to temperature, pH, among other properties,

and an inner magnetic core comprising nanoparticles of alkaline earth metal mixed ferrite, either composed of a magnetic fluid or a nanoparticle cluster;

- and therapeutic drug molecules loaded into the liposomes by either passive or active loading. These therapeutic drug molecules can be located inside the bilayer of phospholipids (if the drug molecules are hydrophobic) or in the hydrophilic compartment of the nanosystems (if the drug molecules are hydrophilic) . Magnetoliposomes are nanocarriers based on liposomes entrapping magnetic nanoparticles. They consist of an inner magnetic nanoparticles part, either a cluster of nanoparticles or a magnetic fluid, covered by a lipid bilayer. Lipid composition can be manipulated according to the cancer family type and microenvironment specific requirements.

In the present technology, aqueous magnetoliposomes (AMLs) are described, which consist in an aqueous magnetic nanoparticles solution entrapped in the liposomes hydrophilic compartment, which result from self-assembling, an inherent behaviour of the phospholipids when in contact with nanoparticles aqueous solutions. AMLs are especially useful to encapsulate hydrophilic drug molecules.

AML's are produced when the inner core of the magnetoliposomes is formed by magnetic nanoparticles and a high percentage of water, between 1% and 5%.

Additionally, solid magnetoliposomes (SMLs) are also described, which consist in a magnetic nanoparticles cluster covered with a lipid bilayer, as a result of the interaction of the polar head group of the phospholipids with the magnetic nanoparticles cluster. This interaction can either be covalent bonding, electrostatic attraction, Van der Waals interactions or by the balance between the hydrophilic and hydrophobic forces. SMLs present a pronounced magnetic behaviour and a better therapeutic effect. They are more easily guided, present a higher temperature range, provide more drug release and better hyperthermia effect. SMLs are produced when the inner core of the magnetoliposomes is formed by magnetic nanoparticles and a reduced percentage of water, between 0.005% and 1%, or no presence of water.

Magnetoliposomes can be loaded with one type, or more than one type of molecules, particularly therapeutic drugs, including but not limited to chemotherapeutic drugs, prodrugs, DNA, proteins, antibiotics, anti-inflammatory and inflammatory agents, diagnostic and theranostic compounds. Upon administration, the nanosystem has the ability to protect the encapsulated drugs, preserving their bioactive action. Also, it improves the pharmacokinetics and enables magnetic guidance to the site of interest through a magnetic field gradient (physical active targeting) . Furthermore, the surface of the nanosystem can be functionalized in order to mask it from the reticuloendothelial system (RES) for increased circulation time and make the nanosystem sensitive to trigger release. After reaching the target area, the nanosystem interacts with the cancer cells, by specific ligand-receptor interaction, it is internalized through endocytosis or membrane fusion. Here, the tumor microenvironment physiological conditions (such as acidic pH) combined with the thermo-sensibility of the nanosystems, destabilize the nanosystem lipid bilayer leading to the controlled payload release, thus increasing drug concentration on target site.

Brief descriptions of the drawings

For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein. Figure 1 illustrates four representations of the nanosystem of the present technology, wherein the reference numbers mean: A - Magnetic nanosystem comprising a lipid formulation, encapsulating a therapeutic drug molecule;

B- Magnetic nanosystem comprising a lipid formulation based with cholesterol, encapsulating a therapeutic drug molecule; C- Magnetic nanosystem comprising a lipid formulation with cholesterol and PEG-lipid conjugates, encapsulating a therapeutic drug molecule and functionalized with an active targeting ligand;

D- Magnetic nanosystem comprising a lipid formulation with cholesterol, encapsulating a therapeutic drug molecule and functionalized with an active targeting ligand;

wherein the reference numbers are: 1 - Inner magnetic core; 2 - Therapeutic drug molecule; 3 - Lipid; 4 - Cholesterol molecule; 5 - pH-sensitive lipid; 6 - Active targeting molecule; 7 - PEG-lipid conjugates.

Figure 2 illustrates a solid magnetoliposome with flower-like nanoparticles .

Figure 3 illustrates a solid magnetoliposome with cubic nanoparticles .

Detailed description

The present application describes a magnetic nanosystem based on superparamagnetic nanoparticles and the method to produce the nanosystems. High shape-anisotropic superparamagnetic nanoparticles, which is the preferred magnetic component of the magnetic nanosystems, have an improved magnetic response and heating capabilities compared with conventional spherical ones. This method ensures that the superpara agnetic nanoparticles incorporation does not affect the nanosystems overall magnetic behavior, keeping it slightly the same as the net superparamagnetic nanoparticles. It also guarantees the synthesis of magnetic nanosystems with a size range between 100 and 200 nanometers and a reduced polydispersity index .

Considering cancer therapy, thermo-sensible magnetoliposomes are a very promising multifunctional nanosystem. Additionally to the magnetic guidance ability, the superparamagnetic nanoparticles heating capabilities induces drug release and synergistic cytotoxic effect in cancer cells (combined chemotherapy and hyperthermia) .

According to Figures 1, 2 and 3, the present application relates to a nanosystem comprising:

an inner magnetic core (1), composed by a cluster of superparamagnetic nanoparticles, regarding SMLs, or an aqueous magnetic solution of superparamagnetic nanoparticles, regarding AMLs;

- a thermo-sensible lipid bilayer (3), composed by a lipid formulation with transition temperatures between 40°C and 45°C, preferably between 41°C and 43°C, that surrounds the magnetic core in both AMLs or SMLs;

the thermo-sensible lipid bilayer formulation can optionally include pH-sensitive lipids (5), in order to make pH-sensitive nanosystems that are stable at physiological pH but are destabilized at acidic cancer microenvironment, inducing the release of drugs in situ;

- encapsulated therapeutic drug molecules (2), that can be located inside the lipid bilayer (if the drug molecules are hydrophobic) or in the hydrophilic component of the nanosystem (if the drug molecules are hydrophilic);

optionally the surface is functionalized with active targeting molecules (6);

- optionally the surface is functionalized with polyethylene glycol (PEG) molecules in a process in which PEG molecules are covalently attached to the surface of preformed magnetoliposomes or by including PEG-lipid conjugates (7) in the mixture of lipids in magnetoliposomes preparation.

The terms "payload" means the segment of content localized in the lipid bilayer of the nanosystem. Herein, the terms "drug", "compound", designates a polymeric or non-polymeric organic chemical, a nucleic acid or an oligonucleotide, a peptide or a peptidomimetic or a protein, antibody, growth fragment or a fragment thereof presenting a linear or cyclic conformation, or a non-naturally occurring compound.

The terms "ligand" designate molecules that are attached to the lipid bilayer surface in order to promote the effective active targeting to a specific subset cell tissue.

The terms "tumor", "cancer" and "neoplasia" are generic terms that define a broad range of diseases characterized by the uncontrolled proliferation of cells of a given tissue or organ as a result of genetic or epigenetic changes in somatic cells. As used herein, the terms "nanosystem", "nanocarrier" and "drug delivery system", represents the combination of the single components (liposomes and superparamagnetic nanoparticles) in an integrated whole that can interact with the target tissue.

The intrinsic guiding capabilities of the present technology rely on the active physical targeting (magnetic) due to the presence of the magnetic nanoparticles and on the passive targeting because of the enhanced retention and permeability (EPR) effect. Therefore, the absence of chemical active targeting molecules does not compromise guiding capabilities. Yet, optionally, these active targeting molecules can be linked through covalent bonding to the lipid bilayer surface in order to improve the therapeutic effect in specific microenvironments .

According to the specific type of cancer, several lipid formulations can be used, alone or in combination, including but not limited to, phospholipid molecules such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl serine (PS), phosphatidylglycerol (PG) , phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM) . The phospholipids can be either synthetic or derived from natural sources such as egg or soy.

In a preferred embodiment, the liposome formulations comprise dipalmitoylphosphatidylcholine (DPPC) , which has an ideal transition temperature for physical drug release.

Other lipids are added to the liposome formulation in order to modulate, for instance, content release, prolong circulation time, etc.

In one embodiment, the thermo-sensitive liposome formulations are originally composed by dipalmitoylphosphatidylcholine (DPPC) and 1 , 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), in a molar ratio of 3:1.

In order to modulate thermo-sensibility of the magnetic nanosystem for therapeutic applications, the lipid bilayer formulation is preferably composed by a lipid or a lipid mixture with a global lipid formulation transition temperature between 40 and 45°C.

DPPC, is used as a major lipid component in thermo-sensitive liposome formulations. As a result, in a preferred embodiment, DPPC lipid presence is ubiquitous in all possible lipid-mixture formulations, as its transition temperature is above body temperature. A slightly increase in liposomes transition temperature (which can be necessary to avoid unwanted drug leakage at body temperature) can be achieved by mixing small percentages of other phospholipids with higher transition temperatures, as the final transition temperature of the formulation depends on the composition of miscible phospholipids .

This mixture can include phospholipids such as 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) , Hydrogenated soybean phosphatidylcholine (HSPC) , l-tetradecanoyl-2- octadecanoyl-sn-glycero-3-phosphocholine (MSPC) , 1,2- dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) , among others .

The choice of DPPC for the main constitution of magnetoliposomes formulations is based on the fact that this lipid contains a choline group and phospholipids containing choline groups are the most abundant class in eukaryotic cells. Furthermore, since DPPC presents a transition temperature at 41.3°C, being in a solid-like gel state at a temperature between 20 to 25°C, when the heating generation by the superparamagnetic nanoparticles reaches higher temperatures, above 41.3°C, DPPC behaves in a fluid like liquid crystalline state because the hydrophobic interaction between the lipid chains decreases. For in vivo application, thermo-sensitive nanocarriers should retain the drug at the biological temperature, approximately 37°C, and release it when the tumor environment warms to temperatures between 40- 42 °C. Thus, in choosing the lipid composition of the magnetoliposomes, it is possible to model the transition temperature of the nanosystem itself and, as such, to model its permeability.

This feature assures that DPPC-based magnetoliposomes are thermo-sensitive nanosystems and can be used as thermo- responsive drug delivery systems, that is a process dependent on the transition temperature of the lipids that make up the lipid formulation of the nanosystem.

Supplementation of DPPC-based liposomes with other lipids and molecules has an effect in the response of the magnetic nanosystems in the amount and rate of drug release at specific microenvironments, temperature conditions and drug molecules bioavailability .

The inclusion of molecules such as cholesterol and polyethylene glycol into the DPPC-based lipid bilayer, can prolong the inherent circulation time of the nanosystems and enhance the controlled drug molecules release upon heating.

The inclusion of cholesterol (4) as shown in Figures 1 to 3, in magnetoliposomes lipid formulation improves the nanosystems resistance to aggregation, makes them more rigid by decreasing their fluidity, protecting the magnetoliposomes in severe shear stress. It also reduces bilayer permeability to non-electrolyte and electrolyte solutes. In another embodiment the formulation is composed by DPPC and cholesterol in the molar ratio preferably between 7:3 and 9:1, more preferably of 8:2, respectively.

In order to overcome the magnetic nanosystems rapid elimination by the reticuloendothelial system (RES), responsible for the decreased of the encapsulated drug molecules bioavailability, and prolong the circulation half- life by increasing its hydrophilicity, the supplementation of DPPC-based liposomes with PEG is proposed. PEG molecules can be added by either covalent attachment to the surface of preformed magnetoliposomes or by including ligand-PEG lipids in the mixture of lipids in the magnetoliposomes preparation.

Magnetoliposomes surface can also be functionalized with ligands that aim permeability enhancement, including, but not limited to: bradykinin, vascular permeability factor/endothelial growth factor (VPF / VEGF) , Prostaglandins, Collagenase, Peroxynitrite , Tumor necrosis factor (TNF) -a.

In another embodiment the formulation is composed by DPPC, cholesterol and 1, 2 Distearoyl-sn-glycero-3- phosphoethanolamine-Polyethylene glycol (DSPE-PEG) in a molar ratio of 55:15:2.

Additionally, DPPC-based nanosystems may include pH-sensitive lipids, resulting in pH-responsive drug delivery systems with combination of the thermo-sensitive response. These formulations are designed to trigger drug molecules release into the cytoplasm of cells, via the endocytotic pathway, as a response to the pH change. Drug molecules are specifically released in tumors microenvironments since the proton concentration in pathological conditions are enhanced (acidic pH) comparably to the normal physiological conditions, as a result of higher endosome processing, eschemia, tumor growth and inflammation.

In one embodiment the lipid formulations include pH-sensitive lipids such as cholesteryl hemisuccinate (CHEMS), N-(4- carboxybenzyl ) -N, N-dimethyl-2 , 3-bis (oleoyloxy) propan- 1- aminium (DOBAQ) , 1 , 2-dipalmitoyl-sn-glycero-3-succinate

(16:0 DGS), 1 , 2-dioleoyl-sn-glycero-3-succinate (18:1 DGS ) and N-palmitoyl homocysteine (PHC) ) , allow to obtain stable nanosystems at a physiological range of pH 7 to 9, as found in blood and plasma. The destabilization of these pH- sensitive nanosystems and consequent release of drugs is induced under acidic conditions, preferably below pH 6, such as those present in the tumor microenvironment. This type of pH-sensitive magnetoliposomes is an alternative and / or complementary route to thermo-sensitive magnetoliposomes in the effective deliver of therapeutic drug molecules to target specific cells.

In another embodiment the formulation is composed by a mixture of DPPC, CHEMS and DSPE-PEG in a molar ratio of 6:3:1, respectively. More preferably, by a mixture of DPPC cholesterol and CHEMS in the molar ratio of 6:3:1, respectively .

The magnetic nanoparticles (MNPs) in the present nanosystem are metal oxide nanoparticles composed by alkaline earth metals, localized in the hydrophilic compartment of the liposome. In one embodiment ferrite magnetic nanoparticles are composed by the metals of magnesium and calcium (Ca_xMgi- _xFe₂C>₄, where x is preferably between 0.1 and 0.9, more preferably between 0.3 and 0.7) . In another embodiment ferrite magnetic nanoparticles could comprise, single or in combination, other alkaline earth metals of calcium (Ca) strontium (Sr) and Magnesium (Mg) .

If the particle size decreases beyond the nanoparticles inherent critical size, will be reached a diameter where magnetic moments do not cancel each other out. This diameter is defined as superparamagnetic diameter (D_SPM) at which ferromagnetic particles exhibit superparamagnetic behavior. In this state, the magnetization of the nanoparticles is approximated as one giant moment, by adding the individual magnetic moments (m) of each constituent atom which form the nanoparticle. This approximation is called the "macro-spin approximation" and is responsible for the strong magnetic moment in superparamagnetic nanoparticles.

Nanoparticles ideal size diameter should be between 1 and 100 nm, preferably between 5 and 70 nm and more preferably 10 and 50 nm, making sure that they are suitable for biomedical applications and possess superparamagnetic behavior.

The nanoparticles used in the magnetoliposomes formulations consist on a single domain structure, consequently, heat production per unit mass is much higher than larger multi- domain ferrite particles of similar composition. In the superparamagnetic nanoparticles, the magnetization disappears once the external magnetic field is removed, avoiding particle agglomeration and hence the possible embolization of the capillary vessels. They also exhibit remarkable magnetic heating properties that can be finely tuned by adjusting the composition, mean size, structure and magnetic anisotropy.

Heating mechanisms of superparamagnetic nanoparticles are based on Neel and Brownian losses. Low amplitudes of alternating magnetic fields, almost "transparent" to the human body, are being extremely useful in hyperthermia treatment methods. Superparamagnetic nanoparticles should exhibit high specific absorption rate (SAR) in order to reach temperatures with therapeutic effect with minimal particle concentration. SAR values are highly dependent on the particle mean size, the alternating magnetic field amplitude (Hmax) and frequency (f), saturation magnetization (M_s) and magnetic anisotropy (K) . Magnetic anisotropy can be controlled by changing nanoparticles shape. Cubic nanoparticles possess higher hyperthermia performance as a result of higher surface magnetic anisotropy and the facilitated tendency towards aggregation into nano-chains by the cubic shape. Flower-like nanoparticles architectures are associated with higher SAR values since these particles magneto-structures are composed of highly ordered nanocrystals that do not behave like isolated grains.

At lower field intensity, small MNPs are supposed to produce more heat than the larger ones, since triggering the magnetic losses of large particles requires a threshold field amplitude stronger than their coercivity field. Heat generation is optimal in the frequency ranges of 100 and 1000 kHz and fields of 150 and 250 Gauss. For a safe treatment, experiments have shown that the product of field amplitude and frequency must be lower than 5 ^c 10⁹ Am^_1s^_1. The pathophysiology associated to the tumor microenvironment allows to explore passive targeting strategies. This approach allows nanocarriers such as liposomes, magnetoliposomes and nanospheres to accumulate at the target site, without necessarily interacting at a cellular level. The process that allows passive bioaccumulation of drug nanosystems into solid tumors is known as EPR effect. This effect results from the accelerated angiogenesis associated with tumors as they reach about 2-3 mm as a result of the increase of nutritional and oxygen necessity. The newly generated vasculature presents irregular shape and with wide fenestrations, making it permeable to macromolecules and nanometric systems such as liposomes, magnetoliposomes and polymeric micelles. The increased permeability in the tumor is the result of the absence of an efficient lymphatic drainage that allows the retention of these nanosystems in the tumor.

Although the EPR effect is currently the clinical base of nanosystem functioning and delivery of existing drugs, it presents limitations that need to be rethought when formulating new approaches. Interstitial pressure associated within the tumor, the extent of macrophage infiltration into the tumor and the heterogeneity in the pore dimensions of the vasculature, highly dependent of type, size and location of the tumor, are factors that may hinder the accumulation of drugs in the interstitial tumor space. Considering that the pore diameter of the tumor vasculature varies between 100 and 1200 nm and the diameter of the magnetoliposomes being around 100-200 nm, it is possible to preferentially accumulate these nanosystems in tumor tissues without affecting healthy tissues by the EPR effect (passive targeting) . Additionally, nanoparticles heat generation can be used in order to increase the permeability of the tumor vasculature.

The duration and respective bioaccumulation of magnetoliposomes in the tumor can be increased by the use of an external magnetic field gradient, in a process known as physical active targeting.

Given that there are differences in the receptors expression in the cell surface between healthy and tumor cells, it is possible to functionalize the surface of the magnetoliposomes with specific active targeting molecules for cancer cell surface receptors, included but not limited to folate, transferrin and EGFR receptors.

After targeting and retaining the magnetoliposomes in the tumor microenvironment, the magnetoliposomes thermo sensitivity is exploited. Under the action of an external alternate magnetic field, temperature gradients are generated and the thermo-sensible lipid bilayer is destabilized becoming more permeable for therapeutic drug molecules release .

According to the chemical structure presented by the chemotherapy drugs and their interaction with other types of drugs, they can be classified into different types, including but not limited to alkylating agents, antimetabolites, anti tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors and corticosteroids. Some examples of clinical available chemotherapeutic drugs, which can be encapsulated into magnetoliposomes, include but are not limited to: Revlimid®, Avastin®, Herceptin®, Rituxan®, Opdivo®, Gleevec®, Imbruvica®, Velcade®, Zytiga®, Xtandi®, Alimta®, Gardasil®, Ibrance®, Perj eta®, Tasigna®, Xgeva®, Afinitor®, Jakafi®, Tarceva®, Keytruda®, Sutent®, Yervoy®, Cytoxan®, Gemzar®, Nexavar®, Zoladex®, Erbitux®, Darzalex®, Xeloda®, Gazyva®, Venclexta® and Tecentriq®.

The administration route of the magnetoliposomes depends on the intended use. For therapeutic delivery, the administration of the systems may be carried out in several ways ( intravascularly, intralymphatically, parenterally, subcutaneously, intramuscularly, intranasally, intrarectally, intraperitoneally, interstitially, into the airways via nebulizer, hyperbarically, orally, topically, or intratumorly) , using different dosages. The preferred route of administration is intravascularly, where the delivery system is generally injected intravenously, but may be injected intra arterially as well. The magnetoliposomes may also be injected interstitially or into any body cavity.

Preparation of magnetoliposomes

A new route for the synthesis of magnetoliposomes with better magnetic properties is here presented. This method was developed taking into account the problems that compromise the therapeutic effect of previously developed magnetoliposomes, which include: (i) nanoparticles biocompatibility; (ii) nanoparticles magnetic behavior and heating efficiency; (iii) drugs and nanoparticles encapsulation efficiency; (iv) magnetoliposomes magnetic behavior and heating efficiency; and (v) magnetoliposomes size polydispersity index.

Each step of the superparamagnetic nanoparticles synthesis was designed and tested to accomplish the best structural and magnetic properties, as the magnetic component is a functional key factor (for the targeting, controlled drug release and hyperthermia) . Intrinsic characteristics as size, shape anisotropy and chemical composition were adapted to overcome the current difficulties in biological applications, ensuring the best behaviour of the magnetoliposomes and their heating capabilities at physiological conditions. In this context, we focused on the preparation of shape-anisotropic superparamagnetic nanoparticles with improved biocompatibility .

The approach for the encapsulation of the nanoparticles into the liposomes was carefully thought to keep almost the same magnetic properties as net nanoparticles, one of the main drawbacks of the current magnetic based nanosystem solutions. It was also investigated a new approach that ensures optimal size and size distribution. Furthermore, the liposomal component of the magnetoliposomes was designed to ensure a sensitive composition that supports a safe encapsulation and transport of the encapsulated drugs with a trigger release at the target site.

The MNPs of the present technology present a spherical shape, a cubic shape, such as represented in Figure 2, flower-like, such as represented in Figure 3, and a combination between them. In a preferred embodiment the magnetic nanoparticles of the present technology are shape-anisotropic, such as cubic shaped or flower shaped.

In one embodiment, spherical mixed ferrite nanoparticles can be prepared by the coprecipitation method. First, an aqueous solution containing 50 mmol of alkaline earth metals precursors, 53 mmol of iron (II) sulphate heptahydrate and a 10% sulfuric acid solution are heated at 75°C, under magnetic stirring, until a clear solution is obtained. Then, 55 mmol of potassium oxalate monohydrate is dissolved in warm deionized water. The two solutions are then mixed, under vigorous stirring, at 90°C. After 15 min, the solution is cooled to temperature between 20 and 25°C. The precipitated nanoparticles are washed by several cycles of centrifugation and redispersion in water. Finally, the nanoparticles are calcined preferably between 300°C and 800°C, more preferably between 400°C and 600°C for a time range between 1 h and 4 h.

In one embodiment, shape-anisotropic cubic superparamagnetic nanoparticles of magnesium and calcium mixed ferrites can be synthetized by co-precipitation method. 50 mmol of octadecylamine is heated until reaching its melting point (50-52°C) in continuously magnetic stirring. After that, a solution containing 0.5 mmol of magnesium acetate tetrahydrate , 0.5 mmol of calcium acetate hydrate, 2 mmol of iron (iii) citrate tribasic monohydrate and 3.1 mmol of oleic acid is added to the pre-heated 50 mmol octadecylamine solution. The mixture is heated (10°C per minute) until reaching 200 °C and then left 90 minutes at this temperature. The resulting nanoparticles are washed with an ethanol solution, by several cycles of centrifugation and aqueous redispersion. Finally, the nanoparticles are calcined preferably between 300°C and 800°C, more preferably between 400°C and 600°C for a time range between 1 h and 4 h.

In order to create nanoparticles with a higher shape anisotropy, flower-like superparamagnetic nanoparticles were also synthetized. In one embodiment, a solution (solution A) containing 0.05 mol of potassium oxalate, 0.05 mol of sodium hydroxide and 2.5 mmol of agarose is added to 25 mL of ultrapure water (Milli-Q grade) under nitrogen flow and magnetic stirring. A solution containing 2.5 mmol of iron (iii) citrate tribasic monohydrate is added drop by drop to solution A. The mixture is heated to 90 °C for a time range between 2 and 4 h, resulting in the precipitation of Fe(OH)2. The final product is washed with a basic solution of sodium hydroxide (1 M), by several cycles of centrifugation, aqueous redispersion and magnetic decantation. Finally, the nanoparticles are calcined preferably between 300°C and 800°C, more preferably between 400°C and 600°C for a time range between 1 h and 4 h.

Encapsulation of the nanoparticles into liposomes magnetoliposomes

The encapsulation of the synthesized nanoparticles into the liposomes was prepared fall back on water in oil micelles. For that, a thin film (for the first lipid layer synthesis) of a lipid and/or surfactant, included but not limited the lipids DPPC, DOPG, DPPE, Egg-PC, soy lecithin and the surfactants Triton X-100, aerosol-OT (AOT), sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB), of concentration above critical micelle concentration, preferably between 0.01 mM and 2 mM and ideally between 0.05 mM and 1.5 mM, was prepared under nitrogen flow.

A nonpolar solvent, such as, but not limited to hexane, heptane, octane, decane, dodecane, chloroform, cyclohexane, toluene, benzene, was pre-heated at 41°C and 48°C, preferably between 43°C and 46°C, added to the thin film and ultrasonicated at a power range between 180 W and 220 W, preferably between 185 W and 200W, for a time interval between of 15 min and 60 min, preferably between 25 min and 45 min.

Then, the superparamagnetic nanoparticles are added to the lipid formulation prepared previously in a total lipid concentration preferably of 0.01 mM and 2 mM and ideally between 0.05 mM and 1.5 mM.

For AMLs synthesis, in one embodiment the concentration of magnetic nanoparticles is added in aqueous solution in a molar concentration between 5xl0⁴ M and 2xl0⁵ M, and ideally between lxl0⁴ and lxl0⁵ M, with a percentage of water between l%-5% (v/v) .

For SMLs, in another embodiment the concentration of magnetic nanoparticles is added in aqueous solution in a molar concentration between 5xlCh⁴ M and 2xlCh⁵ M, and ideally between lxlCh⁴ M and lxlCh⁵ M, with a percentage of water between 0.005%-l% (v/v) .

Next, the solution is ultrasonicated at a power range between 180 W and 220 W, preferably between 185 W and 200 W, for a time interval between of 15 min and 60 min, preferably between 25 min and 45 min. Magnetic decantation was used to purify the solution containing the reversed micelles with the magnetic nanoparticles.

Then, a second lipid layer is prepared according to the same steps for the first lipid layer, in a total lipid concentration preferably of 0.01 mM and 2 mM and ideally between 0.05 mM and 1.5 mM, and is added to the aqueous solution prepared previously which is at a temperature range between 41°C and 48°C, preferably between 43°C and 46°C. In one embodiment, in this step, the total DPPC lipid formulation can include cholesterol in a DPPC : cholesterol molar ratio of 8:2, respectively.

In one embodiment for the lipid formulation containing DPPC :CHEMS: PEG, CHEM-lipid conjugates and PEG-Lipid conjugates are included in the total lipid concentration in a molar ratio of 6:3:1, respectively.

In another embodiment for the lipid formulation containing DPPC : Choi : CHEMS , cholesterol and CHEM-lipid conjugates are included in the total lipid concentration in a molar ratio of 6:3:1, respectively, for co-ethanolic injection.

Magnetoliposomes functionalization with specific molecules for active targeting is done by the incorporation of a ligand, for instance, lipid-ligand conjugate into the liposome formulation step, at a ligand/total lipid molar ratio ranging between 1:1000 and 1:100.

Encapsulation of the therapeutic drug molecules

Immediately before the addition of the second lipid layer, the therapeutic drug molecules are added in an ethanolic solution for passive loading into magnetoliposomes, in a preferably concentration between lxlCh⁶ M and 5xlCh⁵ M.

The resulting magnetoliposomes, were then washed and purified with ultrapure water by magnetic decantation.

Example :

Preferably magnetoliposomes comprise a lipid bilayer of DPPC and cholesterol, in a molar ratio of 8:2, encapsulating drug molecules of Methotrexate as a chemotherapeutic agent for cancer therapy. The magnetic component is composed of high anisotropic cubic shaped mixed ferrites of magnesium and calcium (Cao.5Mgo.5Fe204) .

References

1. Caban, S., Aytekin, E., Sahin, A., Capan, Y., "Nanosystems for drug delivery," Drug Design and Delivery, 2014, vol . 2, p. 1-7.

2. Cho, K., Wang, X., Nie, S., Chen, Z., Shin, D. M., "Therapeutic nanoparticles for drug delivery in cancer", Clinical Cancer Research, 2008, vol. 14, pp . 1310-1316.

3. Pradhan, P., Giri, J., Rieken, F., Koch, C., Mykhaylyk, 0., Dbblinger, M. , Banerjee, R., Bahadur, D., Plank, C., "Targeted temperature sensitive magnetic liposomes for thermochemotherapy", Journal of Controlled Release, 2010, vol. 142, pp . 108-12

4. Cardoso, B. D., Rio, I. S. R. , Rodrigues, A.R. 0., Fernandes, F. C. T., Almeida, B. G., Pires, A., Pereira, A. M., Araiijo, J. P., Castanheira, E. M. S., Coutinho, P.

5. J. G., "Magnetoliposomes containing magnesium ferrite nanoparticles as nanocarriers for the model drug curcumin"

Royal Society Open Science, 2019 vol. 5, pp . 181017-181032.

Claims

1. Magnetic nanosystem characterized by magnetoliposomes comprising :

inner magnetic core; and

therapeutic drug molecules.

2. Magnetic nanosystem according to the previous claim, wherein the magnetoliposomes have a size between 100 and 200 nm.

3. Magnetic nanosystem according to any of the previous claims, wherein the inner magnetic core comprises superparamagnetic nanoparticles with particle sizes between 1 and 100 nm.

4. Magnetic nanosystem according to any of the previous claims, wherein the superparamagnetic nanoparticles have a spherical shape, cubic shape or flower shape.

5. Magnetic nanosystem according to any of the previous claims, wherein the superparamagnetic nanoparticles are made of metal oxide composed by alkaline earth metals.

6. Magnetic nanosystem according to any of the previous claims, wherein the superparamagnetic nanoparticles are ferrite magnetic particles composed by the metals of magnesium and calcium.

7. Magnetic nanosystem according to any of the claim 1 to 5, wherein the ferrite magnetic nanoparticles further comprise alkaline earth metals of magnesium (Mg) , strontium (Sr) and calcium (Ca), alone or in combination.

8. Magnetic nanosystem according to any of the previous claims, wherein the lipid bilayer is composed by a lipid formulation with transition temperatures between 40 and 45°C.

9. Magnetic nanosystem according to any of the previous claims, wherein the lipid bilayer formulation further comprises phospholipid molecules such as phosphatidylcholine, phosphatidylethanolamine , phosphatidylserine , phosphatidylglycerol , phosphatidic acid, phosphatidylinositol , sphingomyelin, alone or in combination.

10. Magnetic nanosystem according to any of the previous claims, wherein the lipid bilayer formulation further comprises cholesterol.

11. Magnetic nanosystem according to any of the previous claims, wherein the lipid bilayer formulation further comprises pH sensitive lipids such as cholesteryl hemisuccinate, N- ( 4-carboxybenzyl ) -N, N-dimethyl-2 , 3- bis (oleoyloxy) propan-l-aminium, 1, 2-dipalmitoyl-sn-glycero- 3-succinate, 1 , 2-dioleoyl-sn-glycero-3-succinate and N- palmitoyl homocysteine.

12. Magnetic nanosystem according to any of the previous claims, wherein the surface of the lipid bilayer is functionalized with polyethylene glycol.

13. Magnetic nanosystem according to any of the previous claims, wherein the surface of the lipid bilayer is further functionalized with permeability inducing ligands such as bradykinin, vascular permeability factor/endothelial growth factor, Prostaglandins, Collagenase, Peroxynitrite, Tumor necrosis factor-a.

14. Magnetic nanosystem according to any of the previous claims, wherein the therapeutic drug molecules are located inside the lipid bilayer if the drug molecules are hydrophobic .

15. Magnetic nanosystem according to any of the claims 1 to 13, wherein the therapeutic drug molecules are located in the hydrophilic compartment of the magnetoliposome if the drug molecules are hydrophilic.

16. Magnetic nanosystem according to any of the previous claims, wherein the surface of the lipid bilayer is functionalized with active targeting molecules.

17. Method of producing the magnetic nanosystem described in any of the claims 1 to 16, comprising the following steps:

Synthesis of the superparamagnetic nanoparticles;

Encapsulation of the nanoparticles into liposomes with the following steps:

synthesis of a first lipid layer by the preparation of :

- the superparamagnetic nanoparticles are added to the lipid formulation;

- the solution is ultrasonicated for a time between 15 min and 60 min;

- purification of the solution by magnetic decantation;

18. Method according to the previous claim, wherein the superparamagnetic nanoparticles are added to the lipid formulation in a concentration between 5xlCh⁴ M and 2xl0⁵ M with a percentage of water between 0.005% and 1%.

19. Method according to claim 17, wherein the superparamagnetic nanoparticles are added to the lipid formulation in a concentration between 5xl0⁴ M and 2xl0⁵ M with a percentage of water between 1 and 5%.

20. Method according to any of the claims 17 to 19, wherein the surfactant is selected from Triton X-100, AOT, SDS and

CTAB .

21. Method according to any of the claims 17 to 20, wherein the nonpolar solvent is selected from hexane, heptane, octane, decane, dodecane, chloroform, cyclohexane, toluene, benzene .

22. Method according to any of the claims 17 to 21, wherein the first lipid formulation has a concentration between 0.01 mg/mL and 10 mg/mL .

23. Method according to any of the claims 17 to 22, wherein the second lipid formulation has a concentration between 0.05 mM and 1.5 mM .

24. Method according to any of the claims 17 to 23, wherein cholesterol and/or active targeting molecules and/or polyethylene glycol are added to the lipid formulations.

25. Method to prepare cubic shaped superparamagnetic nanoparticles of the nanosystem described in any of the claims 1 to 16, comprising the following steps:

50 mmol of octadecylamine is heated until reaching its melting point;

the mixture is heated, with 10°C per minute, until reaching 200 °C and then left 90 minutes at this temperature; nanoparticles are washed with an ethanol solution, by several cycles of centrifugation and aqueous redispersion; the nanoparticles are calcined between 300°C and 800°C for a time range between 1 h and 4 h.

26. Method to prepare flower shaped superparamagnetic nanoparticles of the nanosystem described in any of the claims

1 to 16, comprising the following steps:

the mixture is heated to 90°C for a time range between

2 and 4 h;