WO2015026252A1

WO2015026252A1 - Multifunctional superparamagnetic nanosystem as contrast agent for magnetic resonance imaging and its production method

Info

Publication number: WO2015026252A1
Application number: PCT/PT2014/000054
Authority: WO
Inventors: MARIA Clara Henriques Baptista GONCALVES; Luis Miguel Amante FORTES; Maria Barbara Dos Anjos Figueira MARTINS; Alexandra Maria Fonseca Castelo Dias De CARVALHO; Antonio Gabriel Malagueta FEIO
Original assignee: Instituto Superior Tecnico; Faculdade De Ciencias E Tecnologia Da Universidade Nova De Lisboa; Universidade De Lisboa
Priority date: 2013-08-23
Filing date: 2014-08-20
Publication date: 2015-02-26
Also published as: PT107125A; PT107125B

Abstract

The present invention concerns a multifunctional superparamagnetic nanosystem and its production method for nano-biomedical imaging, human and animal, intended to be used as contrast agent (CA) for Magnetic Resonance Imaging (MRI). Furthermore, it can be associated with therapies such as hyperthermia, and to the transport and targeted release of selected drugs, by conjugation with biomolecules. The multifunctional superparamagnetic nanosystem can be used as negative contrast agent, reducing the T2 value of tissues in MRI, found on the extremely high magnetic moment, under the action of a magnetic field. It can also be used as positive contrast agent. Its efficiency as MRI contrast agent allows for the usage of clinical dosages that are much lower than the ones currently administered to patients who undergo this diagnosis technique. The multifunctional superparamagnetic nanosystem provides a technological platform for several applications in nano- biomedical imaging. It is biocompatible and biodegradable, and its production is free of organic compounds and surfactants.

Description

DESCRIPTION

Multifunctional Superparamagnetic Nanosystem as Contrast Agent for Magnetic Resonance Imaging and Its Production Method

Field of the invention Technical field of the invention

The present invention concerns a multifunctional superparamagnetic nanosystem, and its production method for nano-biomedical imaging , human and animal, intended to be used as contrast agent (CA) for Magnetic Resonance Imaging (MRI) . Furthermore, it can be associated to therapies such as hyperthermia, and to the transport and targeted release of selected drugs by conjugation with biomolecules.

The multifunctional superparamagnetic nanosystem provides a technological platform for several applications in nano- biomedical imaging. It is biocompatible and biodegradable, and its production is free of organic compounds and surfactants.

The aforementioned investigation action is inscribed in the technical domain of Nanotechnologies for medical application, aimed to attain an integrated solution for theragnostic (therapy and diagnostic) , combining MRI medical imaging, as negative contrast agent, with the possibility of being associated with therapies such as hyperthermia, as well as the transport and targeted release of selected drugs by conjugation with biomolecules . The present invention relates to the technical domain of Nanomaterials/Pharmacy/Physics . State of the Art

MRI is a non-invasive imaging technique used in clinical diagnosis without the use of ionizing radiation, both for the patient and the equipment operator. Its spatial resolution is equal or lower than 1 mm, which equals or outperforms the spatial resolution of the computerized axial tomography (CAT) , though it has a thousand-fold higher sensitivity [1], what makes MRI a very important technique for the current clinical diagnosis, particularly in pathologies of the central nervous system.

The anatomical and physiological tissue analysis through MRI uses the nuclear magnetic resonance (NMR) signal of the two hydrogen nucleus present in water to generate the image. Besides any local differences in water content, the fundamental contrast in MRI is due to intrinsic regional relaxation times, longitudinal and transverse (Tl and T2, respectively) , which can be selected independently to control the contrast of the image [1] . When the contrast between adjacent tissues does not allow for a diagnosis to be performed, the use of contrast agents may be recommended, which puts some restrictions on the non-invasive character of MRI procedures. The typical dosage is 0.1 to 0.3 mmol of Gadolinium/Kg and 15 pmol Fe/Kg per patient. Of the 27.5 millions of MRI exams performed in 2008, in the USA, 43% resorted to the usage of contrast agents as part of the diagnosis procedure [2] .

The magnetic materials most commonly used as contrast agents have been the paramagnetic gadolinium (Gd) compounds [3, 4], used without major restrictions until their association with nephrogenic systemic fibrosis (NSF) [5-8], a rare yet severe collateral damage in patients with renal problems. The study of other metals such as non-rare earths has been directed towards manganese (Mn) [9], due to the intensification in the positive contrast of the image, and towards superparamagnetic iron oxide nanoparticles (Fe) (SPIONs) [10-12].

The utilization of SPIONs as negative contrast agents in MRI, reducing the T2 values of tissues, is found on the very high permanent magnetic moment in these nanoparticles, under the action of a magnetic field, in comparison with gadolinium chelates [13] . SPIONs have also been successfully used as positive contrast agents [14] .

The intense R&D work that has been developed in this area, has allowed to put a reasonable number of MRI contrast agents in the market. However, the relatively high dosages to be administered to a patient - 12.5 to 25 mol/Kg of Primovist, 15 umol Fe/Kg of Endorem, and 0.9 rriL (for patients whose weight is ≤ 60Kg) and 1.4 mL (for patients whose weight is > 60 Kg) of Resovist, of concentration 0.5 mol Fe/L - associated with their respective biochemical drawbacks, gives us a major opportunity in the multifunctional superparamagnetic nanosystem market, providing a technological platform for several applications in nano- biomedical imaging, human and animal. The contrast agents already developed and commercialized have^' been the cornerstones of the multifunctional superparamagnetic nanosystem development - composed of a core-shell nanostructure, where the core comprises one or more superparamagnetic nanoparticles of iron oxide (SPIONs) or of other mixed spinels, such as CoFe₂04, MnFe₂04, or others, and the shell comprises silica or hybrid silica. Besides the high biocompatibility and hydrophilic character, the silica shell presents a high versatility for conjugation, chemical and/or physical, allowing high charge and increasing the circulation time in the bloodstream, even allowing a diversified conjugation range with biomolecules and polymers which is adequate for medicinal and pharmaceutical applications [15-20] . The introduction of non-hydrolysable functional organic groups during the in situ colloidal synthesis of the shell leads to hybrid silica shells, also called ORMOSIL (from the Anglo-Saxon ORganically Modified SILica) , where non-hydrolysable functional organic groups such as methyl-, vinyl-, amine-, glycidoxypropyl-, methyldiethyl-, or others, are present and bonded to the tridimensional silica matrix through the covalent bond between silicon and carbon (-Si-C-) , silicon and nitrogen (-Si-N-) , or others. These hybrid silica shells display even more conjugation versatility with biomolecules relative to the silica shells: the presence of non-hydrolysable functional organic groups facilitates the conjunction of biomolecules, the higher concentration in pharmaceuticals, hydrophilic or hydrophobic, in addition to the possibility of fine control of the surface wettability, through the judicious choice of the monomers of synthesis. Silica and hybrid silica are ecofriendly; note that silica is part of the human diet.

The document WO2011/156895A2 presents a nanostructured contrast agent, where one or more ^■ Fe₃0₄ superparamagnetic cores, chemically stabilized, are silica-coated. This coating occurs in a second step of the synthesis, in an inverse microemulsion process, in the presence of surfactants. A few hours after the beginning of the silica polymerization reactions, alkoxide precursors with non-hydrolysable amine groups are added, whereby the amine groups remain only present and available at the surface of the nanostructured contrast agent.

Patent US 6,203777 BI proposes a negative contrast agent for MRI, SPIONs coated by carbohydrates or carbohydrate compounds.

The work Int J Epidemiol Genet 2011; 2 (4 ): 367-390 makes a reference to silica-coated SPIONs chemically stabilized.

Document WO 2010/060209 Al proposes a signal intensifier for Raman spectroscopy, with a core-shell nanostructure, where SPIONs are silica-coated, used for the detection of pathogenic agents.

Patent WO 2009/038659A2 proposes a nanosystem composed of organically modified silica nanoparticles, with photoactive molecules for photodynamic therapy (PDT) conjugated in their surface. The patent also mentions the possibility of conjugating other medical imaging agents, such as magnetic resonance, radionuclides or fluorescence in the surface of the nanoparticle . The presence of photoactive molecules and imaging agents in the organically modified nanoparticles surface aims to make the photodynamic therapy and medical imaging processes more efficient. Organically modified silica nanoparticles are synthetized in an inverse microemulsion reaction medium in the presence of surfactant Tween80. The multifunctional superparamagnetic nanosystem, subject of the present invention, provides a technological platform for several applications in nano-biomedical imaging, human and animal, and presents an integrated solution for theragnostic (therapy and diagnostic) , combining MRI medical imaging, as a negative contrast agent, with the possibility of being associated with therapies such as hyperthermia, as well as the transport and targeted release of selected drugs conjugated with biomolecules. The multifunctional superparamagnetic nanosystem, subject of the present invention, protects the superparamagnetic nanoparticles that constitute the core of a direct contact with biologic fluids after the insertion in human or animal organisms. The present invention is also biocompatible and biodegradable, and its production is free of organic compounds and surfactants.

The multifunctional superparamagnetic nanosystem, object of the present invention, is composed of a core-shell nanostructure, where the core is composed of one or more superparamagnetic nanoparticles of iron oxide (SPIONs) or of other mixed spinels, such as Co_xFe₂__x0₄, Mn_xFe₂__x0₄, (Co,Mn)_xFe₂-_x0₄, or others, and the shell comprises silica or hybrid silica.

In the silica/hybrid silica shell synthesis, through colloidal chemistry, non-chemically stabilized SPIONs and other superparamagnetic mixed spinels nanoparticles, such as Co_xFe₂-_x0₄, Mn_xFe₂__x0₄, (Co, Mn) _xFe₂-_x0₄, or others, behave as nucleating agents for the growth of silica/hybrid silica shells. All the alkoxide precursors, inorganic (e.g., tetraethyl orthosilicate (TEOS) , Si(OC₂H₅)₄) and/or hybrid (e.g., methyltriethoxysilane, (MTES) , Si (OCH₂CH₃) ₃CH₃, vinyltriethoxysilane, (VTES) , Si (OC₂H₅) ₃CH=CH₂; 3- aminopropyltriethoxysilane (APTES) , Si (OC₃H₇) ₃NH₂; (3-

Glycidoxypropyl) methyldiethoxysilane (GPTMS) , Si (OCH₃) ₂C₉Hi₈0₂/ among others) are added in only one step, in situ, at controlled temperature, making the process more efficient, reproducible and free of organic solvents and surfactants, required in the silica/hybrid silica synthesis through inverse microemulsion method. The obtained shells present a spherical geometry and a unimodal distribution with linear thickness between 5 and 500 nm. In the synthesis through inverse microemulsion the organic solvents/surfactants elimination process is not 100% efficient, whereby it is not free of risk in biologic applications, comprising also environmental risks. The hybrid silica shell is chemically and structurally homogeneous. The possibility of combining several non-hydrolysable organic groups in varying proportions in · the same shell adds new possibilities to the multifunctional superparamagnetic nanosystem.

The present invention proposes a multifunctional superparamagnetic nanosystem with a core-shell nanostructure, where the core is composed of one or more superparamagnetic nanoparticles of iron oxide (SPIONs) or of other mixed spinels, such as Co_xFe₂-x0_4/ Mn_xFe₂-x0₄, (Co, Mn) _xFe₂-_x0 , or others, and the shell comprises silica or hybrid silica.

The proposed shell allows the MRI image contrast adjustment through the combination of the composition, diversity and proportionality of different non-hydrolysable functional organic groups, with the thickness of the silica/hybrid silica shell.

Summary of the Invention

The present invention concerns a multifunctional superparamagnetic nanosystem as MRI contrast agent and its production method. The present invention can be used as platform for nano-biomedical imaging, human and animal, and presents an integrated solution for theragnostic (therapy and diagnostic) , combining MRI medical imaging, as negative contrast agent, with the possibility of being associated with therapies such as hyperthermia, as well as the transport and targeted release of selected drugs by conjugation with biomolecules.

The multifunctional superparamagnetic nanosystem can be used as negative contrast agent, reducing the T2 value of tissues in MRI, found on the extremely high magnetic moment, under the action of a magnetic field. It can also be used as positive contrast agent. Its efficiency as MRI contrast agent allows for the usage of clinical dosages that are much lower than the ones currently administered to patients who undergo this diagnosis technique.

The efficiency of a CA is quantified by the longitudinal and transversal relaxivities, rl and r2 respectively. In negative contrast agents, a higher efficiency is obtained for higher values of r2/rl. The multifunctional superparamagnetic nanosystem is composed of a core-shell nanostructure, where the core comprises iron oxide or mixed spinels nanoparticles, characterized by having a silica/hybrid silica shell, which thickness varies between 5 and 500 nru, with interconnected pores of varying size, forming a three dimensional network, permeable to water molecules, being conditioned by the ratio between the non-hydrolysable functional organic and the inorganic groups, in bulk and at its surface, displaying ratios r2/rl higher than 100.

The multifunctional superparamagnetic nanosystem presents a range of possible ratios between the non-hydrolysable precursors with Si-C bonds and the (hydrolysable) precursors only with Si- OR bonds that varies between 0 and 1.

The multifunctional superparamagnetic nanosystem can even be conjugated with biomolecules, such as proteins, peptides, enzymes, antibodies, and polymers, such as polyethylene glycol linear or branched polymer chains.

The multifunctional superparamagnetic nanosystem provides a technological platform for several applications in nano- biomedical imaging. It can be conjugated with biomolecules and polymers establishing chemical or physical bonds between the non-hydrolysable functional organic and inorganic groups present in the silica/hybrid silica shells and the accessible functional organic groups in the biomolecules that do not alter their biologic activity. The hydrophilic/hydrophobic character as well as the surface roughness of the shells can be easily controlled. The multifunctional superparamagnetic nanosystem is biocompatible and biodegradable, and its production is free of organic compounds and surfactants.

"Hybrid silica" implies a tridimensional silica network where non-hydrolysable functional organic group such as, for example, methyl-, vinyl-, amine-, glycidoxypropyl-, methyldiethyl-, and their mixtures, are present, in a defined concentration, and covalently bonded to silicon (e.g., ≡Si-C≡, ≡Si-N-, or others). "Conjugation with biomolecules" implies a chemical (covalent or of second degree) or physical (adsorption) bond of a reactive group of a biomolecule and an inorganic or functional group of silica or hybrid silica.

Detailed description of the invention

The object of the present invention is the development of a multifunctional superparamagnetic nanosystem as MRI contrast agent, and its production method. This nanosystem provides a technological platform for several applications in nano- biomedical imaging and as MRI contrast agent. It is biocompatible and biodegradable, and its production is free of organic compounds and surfactants.

The multifunctional superparamagnetic nanosystem provides a technological platform for several applications in nano- biomedical imaging. It can be conjugated with biomolecules and polymers establishing chemical or physical bonds between the non-hydrolysable functional organic and the inorganic groups present in the silica/hybrid silica shells and the accessible functional organic groups in the biomolecules that do not alter their biologic activity. The hydrophilic/hydrophobic character as well as the surface roughness of the shell can be easily controlled. The multifunctional superparamagnetic nanosystem is biocompatible and biodegradable, and its production is free of organic compounds and surfactants .

• PRODUCTION METHOD OF THE MULTIFUNCTIONAL SUPERPARAMAGNETIC NANOSYSTEM TO USE AS PLATFORM FOR NANO-BIOMEDICAL IMAGING, HUMAN AND ANIMAL, AN INTEGRATED SOLUTION FOR THERAGNOSTICS

The present invention proposes a multifunctional superparamagnetic nanosystem with a core-shell nanostructure, where the core comprises one or more superparamagnetic nanoparticles of iron oxide (SPIONs) or of other mixed spinels, such as Co_xFe₂-_x0_4/ Mn_xFe₂__x0₄, (Co,Mn) _xFe₂-_x0₄, or others, and the shell comprises silica or hybrid silica. One of the main goals of the development of the multifunctional superparamagnetic nanosystem is the core-shell nanostructure production process development .

SPIONs are synthetized via wet chemistry, by reduction precipitation or co-precipitation. Mixed spinels defined by the general formula A_xFe₂-_x0₄, where A represents divalent cations, such as cobalt, manganese, ruthenium or others and their mixtures, are synthetized through organometallic synthesis at high temperature, or via wet chemistry, by the co-precipitation of di- and trivalent ions.

Regarding the silica/hybrid silica shell developed around the superparamegnetic cores based on iron oxide, SPIONs or mixed spinels, a colloidal synthesis method was developed, modifying the LaMer method, which revealed itself to be reliant and efficient in the obtainment of spherical and unimodal size distribution nanosystems. The thickness control of the aforementioned shells and, therefore, the multifunctional superparamagnetic nanosystem dimension revealed itself to be reliable and reproducible.

The synthesis time in that method is lower than 15 minutes.

This method utilizes neither organic solvents nor surfactants. Besides "ecofriendly", this method leads to the synthesis of nanosystems without the risk of any chemical residues, which is not the case in the alternative synthesis method through inverse naicroemulsion .

The multifunctional superparamagnetic nanosystem production method as MRI contrast agent comprises the following steps: a) Synthesis of superparamagnetic iron oxide nanoparticles, establishing the core of the nanosystem, through iron (II) and iron (III) ions co-precipitation, or through iron (III) ions reduction-precipitation, in the presence of aqueous solution of ammonium hydroxide, under ultrasound or magnetic stirring, or alternatively;

b) Synthesis of superparamagnetic mixed spinels nanoparticles, such as Co_xFe₂-_x0₄, Mn_xFe₂-_x0₄, (Co,Mn)_x Fe₂__x0₄ or others, establishing the core of the nanosystem, through reduction- precipitation, co-precipitation of divalent cations and Fe (III) in basic medium, or through organometallic synthesis, under ultrasound or magnetic stirring;

c) Coating of the superparamagnetic iron oxide or mixed spinels nanoparticles by silica/hybrid silica nanoparticles, constituting the shell of the nanosystem obtained in steps a) and b) with an ethanol, ammonia and water solution mixture, under strong stirring and with the addition of silica organometallic precursors in adequate proportions, for a period of time lower than 15 minutes; d) A modification of the LaMer method was utilized, where:

SPIONs are added to an ethanol, distilled water and ammonia solution, in an ultrasonic bath at controlled temperature; a precursors mixture is rapidly added to the aforementioned solution, in ultrasonic bath; the superparamagnetic nanoparticles are finally separated by centrifugation.

UTILIZATION OF THE MULTIFUNCTIONAL SUPERPARAMAGNETIC NANOSYSTEM

The multifunctional superparamagnetic nanosystem proposed in the present invention allows for the improvement of the NMR image contrast, when it is obtained by T2 weighting, efficiently and economically advantageous and securely.

The negative contrast agent efficiency is controlled by the thickness of the silica/hybrid silica shell, by the nature and concentration of the non-hydrolysable functional inorganic and organic groups and by the magnetic moment of the core. The multifunctional superparamagnetic nanosystem can even be conjugated with biomolecules, such as proteins, peptides, enzymes, antibodies and polymers, such as polyethylene glycol linear or branched polymer chains. The multifunctional superparamagnetic nanosystem provides a technological platform for several applications in nano- biomedical imaging, allowing the association of a nano-bio- imaging system with a therapy such as, for example, hyperthermia, gene therapy, controlled/gradual drug release, among others.

The present invention comprises a core-shell nanostructure, which production process occurs in two steps: in the first the superparamagnetic iron oxide or mixed spinels cores are synthetized via wet chemistry or by organometallic synthesis at high temperature; in a second step, the coating of the superparamagnetic iron oxide or mixed spinels core is produced, promoting the growth of silica/hybrid silica shells on the superparamagnetic iron oxide or mixed spinels cores, in situ, at controlled temperature, whereby the non-hydrolysable functional organic groups are present inside and at the surface of the shell.

The multifunctional superparamagnetic nanosystem is composed of a core-shell nanostructure, where the core comprises iron oxide or mixed spinels nanoparticles, characterized by having a silica/hybrid silica shell, which thickness varies between 5 and 500 nm, with interconnected pores of varying size, forming a three dimensional network, permeable to water molecules, being conditioned by the ratio between non-hydrolysable functional organic and inorganic groups, in bulk and at its surface, displaying ratios r2/rl higher than 100.

The multifunctional superparamagnetic nanosystem presents a ratio between the non-hydrolysable precursors with Si-C bonds and the hydrolysable precursors only with Si-OR bonds that varies between 0 and 1.

The colloidal synthesis method of the silica/hybrid silica shell proposed in the present invention, avoids the utilization of surfactants or other organic chemicals used in the inverse microemulsion method - by which the multifunctional superparamagnetic nanosystem does not show traces of any type of organic residues. The surfactants elimination process is not 100% effective, whereby in biological/biomedical applications it has toxicity risks for humans and animals, besides environmental risks, both during the synthesis process and after human or animal excretion, after being administered as a MRI contrast agent .

Different chemical compositions were tested for the shell, i.e., different ratios between the organic content and the silicon from the amorphous silica network, non-hydrolysable functional organic groups, different mixtures of distinctive non- hydrolysable functional organic groups, with the goal of improving the contrast of a T2 weighted MRI image, as it can be seen by the relaxivities values (rl and r2 and the ratio r2/rl) displayed in Table 1.

Rl is the longitudinal relaxivity of CA, i.e., its efficiency to alter the Tl values of tissues.

R2 is the longitudinal relaxivity of CA, i.e., its efficiency to alter the T2 values of tissues.

The ratio, r2/rl measures the efficiency of a negative contrast agent.

Surprisingly, the presence of inorganic groups such as ≡Si-OH, ≡Si-0-, ≡Si-0-Si≡, and of one or more non-hydrolysable functional organic groups, such as methyl-, vinyl-, amine-, glycidoxypropyl-, methyldiethyl-, and their mixtures, in the shell, around the superparamagnetic iron oxide or mixed spinels cores, revealed high efficiency as a contrast agent.

In Figures 5a) and b) , 6a) and b) and 7a) and b) where the superparamagnetic behavior of the iron oxide cores and of the multifunctional superparamagnetic nanosystem comprising a core- shell nanostructure is displayed, it is clear a decrease in the saturation magnetization value when going from the cores to the core-shell systems, from 65 Am²/kg to nearly one-third of this value. This allowed concluding that the relaxivity of the nanosystem is a function of not only the superparamagnetic core magnetic properties but also of its surrounding shell. These two components have to be considered for the construction of a high efficiency multifunctional superparamagnetic manosystem as a MRI contrast agent. There is the possibility of combining several inorganic and non- hydrolysable functional organic groups in the same shell, which adds new possibilities to the tailor-made multifunctional superparamagnetic nanosystem, which constitutes a clear advantage in its utilization, since it provides a technological platform for several applications in nano-biomedical imaging.

There is the possibility of conjugating the silica/hybrid silica shell with distinctive biomolecules in the same multifunctional superparamagnetic nanosystem. Included within this scope are the multif nctional superparamagnetic nanosystems where the silica/hybrid silica shell possesses groups with chemical reactivity that allow the conjugation with biomolecules, specifically the bond of a protein, a peptide, an antibody, or other molecule that allows for the targeting of a nanoparticle to specific sites in the organism, in particular to specific cellular receptors. The conjugation of silica/hybrid silica of the multifunctional superparamagnetic nanosystem with proteins, peptides or antibodies is performed through chemical or physical bond between the organic and inorganic functional groups that are available and accessible at the surface and in the porosity of the shell of the multifunctional superparamagnetic nanosystem and the reactive groups that are available and accessible at the surface of the conjugating biomolecules . Conjugation with biomolecules may occur simultaneously or stepwise.

There is also the possibility of conjugation between silica/hybrid silica of the shell with several polymers, such as polyethylene glycol (PEG) of different sizes in the same multifunctional superparamagnetic nanosystem. Included within this scope are the multifunctional superparamagnetic nanosystems where the silica/hybrid silica has groups with chemical reactivity that allow for the conjugation of biomolecules with polymers able to increase the residence time of the nanosystems in circulation in the organism of an animal or a human. There is even the possibility of conjugation between the silica/hybrid silica of the shell and several biomolecules in the same multifunctional superparamagnetic nanosystem using a bifunctional polymer as spacer. Included within this scope are the multifunctional superparamagnetic nanosytems where the silica/hybrid silica shell is conjugated to polymers having a reactive group in the polymer chain terminus, for binding one or more biomolecules, such as a protein, a peptide, an antibody, or other molecule, allowing both the targeting of the nanosystem to specific sites in the organism, and the increase in the residence time of the nanosystems in circulation in the organism of an animal or a human.

For illustrative but not limitative purposes, we subsequently present some examples of biomolecules, which can be utilized in the conjugation with the multifunctional superparamagnetic nanosystem. Proteins with catalytic activity (enzymes) :

L-asparaginase; plasminogen activator: tPA (tissue plasminogen activator); urokinase; streptokinase, among others.

Proteins with immunomodulatory activity (cytokines) :

Interferon alpha, Interferon beta, Interferon gamma, Interleukin-2, Initerleukin-11, among others.

Antibodies : Trastuzumab; Rituximab; Alemtuzumab; Cetuximab; Bevacizumab; Panitumumab; Canakinumab; Ofatunumab; Denosumab; Ipilimumab; Pertuzumab, among others.

Peptides with affinity for cellular receptors : RGDSK or (H-Arg-Gly-Asp-Ser-Lys-OH) ;

c(RGDfK) or (cyclo (Arg-Gly-Asp-D-Phe-Lys) ) ;

c(RGDfC) or (cyclo (Arg-Gly-Asp-D-Phe-Cys ) ) ;

c(RGDfE) or (cyclo (Arg-Gly-Asp-D-Phe-Glu) ) ;

c(RGDyK) or (cyclo (Arg-Gly-Asp-D-Tyr-Lys) ) ;

H-E- [c (RGDyK) ] 2 or (H-Glu [cyclo (Arg-Gly-Asp-D-Tyr-Lys) ] 2 ;

c (RGDfK (PEG-PEG) ) or (cyclo [Arg-Gly-Asp-D-Phe-Lys (PEG-PEG) ] ;

c [RGDfK (AC-SCH2C0) ] or. cyclo (Arg-Gly-Asp-D-Phe-Lys (Ac-SCH2CO) ] ;

E- [c (RGDfK) 2] or (H-Glu [cyclo (Arg-Gly-Asp-D-Phe-Lys )] 2 ;

c(RGDfV) or (cyclo (Arg-Gly-Asp-D-Phe-Val) , among others. Family of polymers :

Reactive functional PEG in which n is equal to 8, 12 or 24:

Succinimidyl- ( [N-methyl] - (n) -ethyleneglycol) ester or NHS-PEGn- Methyl; Maleimidepropionamide- [ [N-methyl] - (n) -ethylene glycol] or

Maleimide-PEGn-Methyl .

Reactive homobifunctional PEG in which n is equal to 3 or 5:

Bis (succinimidyl) - (n) - (ethylene glycol) or BS(PEG)n

Bis (maleimide) - (n) -ethylene glycol or BM(PEG) .

Reactive heterofunctional PEG where n equals 4, 6, 8, 12 or 24 :

Succinimidyl- ( [N-maleimidepropionamide] - (n) , ethyleneglycol) ester or NHS-PEGn-maleimide . The production method of the multifunctional superparamagnetic nanosystem conjugated with biomolecules comprises the following steps : a) Activation of the multifunctional nanosystem, through treatment with succinic anhydride under constant gentle stirring;

b) Dialysis of the multifunctional nanosystem in water;

c) Solubilization of the biomolecule followed by its addition to the suspension comprising the multifunctional nanosystem;

d) Incubation of the reaction mixture under gentle stirring; e) Separation of the multifunctional nanosystem from the reaction medium. Table 1 presents the values of longitudinal (rl) and transversal (r2) relaxivities and the respective r2/rl ratios for several SPION-dextran T10 (sodium citrate), SPION- CARBOXILMETHYL- DEX RAN T10, SPION- CARBOXIDEXTRANE, SPION- CITRATE commercial contrast agents, and the proposed multifunctional superparamagnetic nanosystem, with silica/hybrid silica shell, functionalized with vinyl- groups, which precursor is VTES and glycidoxypropyl- methyldiethoxysilane-, which precursor is GPTMS, presented in two molar TEOS:GPTMS ratios, of 9:1 and 5:5. Table 1: Values of the longitudinal (rl) and transversal (r2) relaxivities .

It is possible to verify that the value of the r2/rl ratio of the presented multifunctional superparamagnetic nanosystem is at least one order of magnitude higher than the proposed substitute commercial drugs, which allows the contrast agent to be administered to the patient in considerably lower dosages, being obtained values of the ratio r2/rl higher than 100, to a 7T magnetic field.

There . is the tendency of the clinical equipment to work in higher magnetic fields, 3 or even 7T, since it improves the signal-to-noise ratio and with it the quality of the image. There already is commercial equipment working in 3T and even 7T fields, hence the logic to obtain our measures in high magnetic fields .

The colloidal methodology developed revealed itself to be reliable and effective from the perspective of the morphology and diameter control, and in the alteration of the intensity of the signal in MRI, in the proposed multifunctional superparamagnetic nanosystem, as it can be seen in the Figures 5a) and b) , 6a) and b) , 7a) and b) of the isothermal hysteresis curves and the magnetization behavior when the samples are field-cooled (FC) or zero-field-cooled (ZFC) , the obtained cores and core-shell nanostructures are superparamagnetic.

Description of the figures Figure 1 depicts the scheme of the multifunctional superparamagnetic nanosystem with silica shell. The north (N) and south (S) magnetic polarity is indicated in the center. Oxygen (0) and silicon (Si) atoms.

Figure 2 depicts the scheme of the multifunctional superparamagnetic nanosystem with hybrid silica shell. The north

(N) and south (S) magnetic polarity is indicated in the center.

Oxygen (0), silicon (Si) and functional groups, such as methyl-, vinyl-, amine- or others, covalently bonded to silicon (e.g., ≡Si-C≡, ≡Si-N-, or others) .

Figure 3 depicts the diffraction pattern (a) and images of transmission electron microscopy (TEM) of quasi-spherical iron oxide precipitates, corresponding to the core (5-6 nm of diameter), with x50.000 magnification, (b) and xlOO.000 magnification, (c) .

Figures 4a) and 4b) depict TEM images of the multifunctional superparamagnetic nanosystem with iron oxide core and hybrid silica shell, with x200.000 and xlOO.000 magnification, respectively.

Figure 5 depicts the results of studies of the magnetization referring to iron oxide superparamgnetic nanoparticles, cores in the present multifunctional superparamagnetic nanosystem: a) depicts the magnetic hysteresis curve for different temperatures where the Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis identified by μ₀Η refers to the magnetic field, in T; Figure 5b) depicts the magnetization curve for a magnetic field (H) of 5mT, where the Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis refers to temperature values, in K degrees, for situations where the sample was field-cooled (FC) and zero-field-cooled (ZFC) .

Figure 6 depicts the results of studies of the magnetization referring to the multifunctional superparamagnetic nanosystem with silica shell: a) depicts the magnetic hysteresis curve for different temperatures where the Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis identified by μ₀Η refers to the magnetic field, in T; Figure 6b) depicts the magnetization curve for a magnetic field (H) of 5mT, where the

Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis refers to temperature values, in K degrees, for situations where the sample was field-cooled (FC) and zero- field-cooled (ZFC) .

Figure 7 depicts the results of studies of the magnetization referring to the multifunctional superparamagnetic nanosystem with hybrid silica shell with vinyl group: a) depicts the magnetic hysteresis curve for different temperatures where the Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis identified by μ₀Η refers to the magnetic field, in T; Figure 7b) depicts the magnetization curve for a magnetic field (H) of 5mT, where the Y-axis, identified by M refers to the magnetization in Am²/kg and the X-axis refers to temperature values, in K degrees, for situations where the sample was field- cooled (FC) and zero-field-cooled (ZFC) . Figure 8 depicts magnetic resonance micro-images of agar gel phantom, in the bottom line, and of the agar gel phantom with the multifunctional superparamagnetic nanosystem with hybrid silica shell, functionalized with (3-

Glycidoxypropyl) methyldiethoxysilane (GPTMS) , in the upper line, obtained with different echo times. Echo time is half the interval time between one excitation and acquiring the signal in a Spin-echo sequence, broadly used in NMR.

Figure 9 depicts a magnetic resonance micro-image obtained in a 7T magnetic field of 0.5 mm axial cuts of 4 NMR tubes of 5 mm each with: clockwise, tube in the left, agar-agar aqueous solution (0.5% p/p) , agar-agar aqueous solution with 0.17 nM of Fe (Fe(II) and FE(III)) in ORMOSIL NPs with GPTMS (1:9); agar- agar aqueous solution with 0.17 nM of Fe (Fe(II) and FE(III)) in ORMOSIL NPs with APTES (1:9) and agar-agar aqueous solution with 0.17 nM of Fe (Fe(II) and FE(III)) in silica NPs.

Figure 10 graphically depicts the normalized contrast for an agar gel phantom and a gel phantom with the multifunctional superparamagnetic nanosystem with hybrid silica shell, functionalized with (3-Glycidoxypropyl) methyldiethoxysilane (GPTMS), where the Y-axis refers to the normalized contrast in arbitrary units and the X-axis identified by t refers to time, in milliseconds. Contrast is the difference in signal intensity in two different regions of the image (group of pixel) . In MRI and NMR the signal is always a frequency-radio signal and the intensity it refers to is this one.

Figure 11 depicts the efficiency of the CA of a MR image depending on the composition of the shell, in animal model, in this case in zebrafish (Danio rerio) injected with core-shell nanosystem: a) non injected animal (white, or reference) ; b) animal injected with CA - silica shell (TEOS) ; c) animal injected with CA - APTES (1:9) shell and d) animal injected with CA - GPTMS (1:9) shell. For a better understanding of the invention and for illustrative but not limitative purposes, the following is a description of application examples of the present invention.

Examples 1.1 Synthesis of superparamagnetic iron oxide nanoparticles by co-precipitation - core

The synthesis of superparamagnetic iron oxide nanoparticles, cores in the present multifunctional superparamagnetic nanosystem, is performed via wet chemistry, through an iron (II) and iron (III) ions co-precipitation method.

0.3975g of FeCl₂.4H₂0 are added to lmL of HCI 2M, forming iron (II) chloride solutions (A) and 0.2702g of FeCl₃.6H₂0 are added to 4mL of HCI 2M, forming the iron (III) chloride solution (B) . Solutions (A) and (B) form, under strong magnetic stirring, solution (C) . 50mL of ammonia aqueous solution (NH₄0H, 0.7 M) are added drop wise to C, maintaining the magnetic stirring. The precipitation of iron oxide (of black color) starts with the presence of ammonia aqueous solution. The precipitation of the iron compound is facilitated by the magnetic field. The supernatant liquid is removed and the precipitate washed 2-3 times with alcohol, in the end, the washing liquid is eliminated through evaporation in an oven, at 40°C.

1.2 Synthesis of superparamagnetic iron oxide nanoparticles by reduction-precipitation - core in the present invention

The synthesis of superparamagnetic iron oxide nanoparticles, cores in the present multifunctional superparamagnetic nanosystem, is performed via wet chemistry, by an iron (III) ions reduction-precipitation method.

2.703g of FeCl₃.6H₂0 (D) are added to 2.5mL of HCI, 2M and are maintained under strong magnetic stirring and are added to a sodium sulfite solution, in aqueous medium (E) .

8.46mL of ammonia aqueous solution (NH₄OH, 0.7 M) are added drop wise to E, maintaining the magnetic stirring. The precipitation of iron oxide (of black color) starts with the presence of ammonia aqueous solution. The supernatant liquid is removed and the precipitate washed 2-3 times with alcohol. In the end, the washing liquid is eliminated through evaporation in an oven, at 40°C.

1.3 Synthesis of superparamagnetic mixed spinels nanoparticles by co-precipitation or organometallic synthesis at high temperature - core

The synthesis of superparamagnetic mixed spinels nanoparticles, cores in the present multifunctional superparamagnetic nanosystem, can be performed via wet chemistry, by an iron (III) ions and divalent cation co-precipitation method.

25 mL of 0.4 M iron (III) chloride aqueous solution and 25 mL of 0.2 M cobalt(II) chloride solution are prepared (F) . 25 mL of 3 M sodium hydroxide (NaOH) solution (G) are added drop wise to the solution F and maintained under strong magnetic stirring. A pH value of 11-12 is kept constant through the addition of NaOH. The precipitate is kept at 80°C under strong magnetic stirring for lh. The precipitate is separated through centrifugation and the washing liquid is eliminated through evaporation in an oven, at 100°C. Subsequently, the precipitate of CoFe₂0₄ is heat- treated at 600°C, for lOh.

The organometallic synthesis at high temperature of unimodal non-aggregated nanoparticles of MnFe₂0₄ mixed spinels occurs through the thermal decomposition of Fe(CO)₅ and Mn₂(CO)io, from which results the formation of FeMn, oxidized in a second step in the presence of trimethylamine oxide.

1.4 Coating of iron oxide or mixed spinels nanoparticles with silica/hybrid silica - shell

The coating of superparamagnetic iron oxide nanoparticles with silica/hybrid silica is performed through a colloidal chemistry process .

An ethanol, ammonia and water solution (H) is prepared and maintained under strong stirring at a temperature kept constant in the interval 10 and 100°C, more specifically between 0 and 60°C.

The iron oxide cores are added to solution H, kept under strong stirring. The silica organometallic precursors are added to the aforementioned solution and the multifunctional superparamagnetic nanosystems separated by the presence of a magnetic field.

2. Conjugation of proteins , peptides , antibodies with the multifunctional superparamagnetic nanosystem

The conjugation of proteins, peptides or antibodies with the silica/hybrid silica multifunctional superparamagnetic nanosystem is performed through the covalent bond between the functional organic and inorganic groups available and accessible at the surface and in the porosity of the shell of the multifunctional superparamagnetic nanosystem and the reactive groups available and accessible at the surface of the conjugating biomolecules.

The conjugation reaction medium is an aqueous medium or a heterogeneous medium by the presence of micelles which allow the minimization of the exposure of the biomolecule to organic molecules in the case, they are needed to the conjugation reaction, minimizing the presence of solvents that can alter the tridimensional structure of the conjugating biomolecules. The procedure may include a prior activation of the functional groups available at the surface of the shell of the multifunctional nanosystem, followed by protection of the activated groups, followed by a separation process for the extraction of the reagents in excess, followed by the deprotection of the activated group, followed by gentle stirring for homogenization.

Alternatively the procedure can include the utilization of bifunctional reagents adequate to the direct establishment of the chemical reaction between the functional groups available at the surface and in the open porosity of the shell, and the functional groups available at the surface of the biomolecule.

In this case, gentle stirring of the suspension of the multifunctional superparamagnetic nanosystem in aqueous medium is performed, followed by the addition of the bifunctional reagent maintaining the gentle stirring for homogenization.

In any of the cases after the homogenization, shall be immediately added the solution comprising the biomolecule to conjugate namely a protein, a peptide, an enzyme, an antibody, or other biomolecule that allows for the targeting of the nanoparticle to specific sites in the organism, namely to specific cellular receptors. The suspension to an adequate pH and a concentration of a biomolecule to be determined case by case is then incubated under gentle stirring at room temperature for a period of 2 hours. After this reaction time, the multifunctional superparamagnetic nanosystems undergo conventional separation processes for removal of reaction products, excess reagents and unconjugated biomolecules. The conjugation of the shell of the multifunctional superparamagnetic nanosystems with biomolecules is proven by characterization methods and by biological activity assessment methods specific of the different biomolecules.

2.1 Conjugation of a protein (enzyme) with the multifunctional superparamagnetic nanosystem

Considering as example the conjugation of the L-asparaginase enzyme to the silica/hybrid silica multifunctional superparamagnetic nanosystem, proceeds the solubilization of the L-asparaginase enzyme, in the concentration of 1.2X10^"6 M in a 50mM carbonate buffer solution, pH 9.4. A volume of 1 mL of this solution is added to a volume of 5 mL of multifunctional superparamagnetic nanosystem suspension with free amino groups in the shell, which were previously activated through treatment with succinic anhydride in a N, N-dimethylformamide solution, in the 5 to 12% range, for 6 hours under constant gentle stirring. The multifunctional superparamagnetic nanosystems are then dialyzed against water for 18 hours, time by which the multifunctional superparamagnetic nanosystems are removed from the dialysis sleeve. Thereafter, the procedure is the addition of 1 mL of 0.1M N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide solution per 5 mL of multifunctional superparamagnetic nanosystem suspension followed by gentle stirring for 30 minutes. After this multifunctional superparamagnetic nanosystem activation procedure continues with the immediate addition of the solution comprising the L-asparaginase enzyme in the volume and concentration already described. The reaction mixture is placed under gentle stirring for 2 hours at 4°C. The multifunctional superparamagnetic nanosystems obtained are separated from the reaction medium through size-exclusion chromatography. The percentage of functional groups conjugated to the L-asparaginase in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 40 to 50%. The retention of biological activity of the L- asparaginase in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 60 to 80%.

2.2 Conjugation of a protein (cytokine) wi'th the multifunctional superparamagnetic nanosystem

Considering as example the conjugation of the interferon-alpha protein to the silica/hybrid silica multifunctional superparamagnetic nanosystems, the procedure is the solubilization of interferon-alpha, in a 5 mg/mL concentration in a 50 mM borate buffer solution, pH 9. A volume of 1 mL of this solution is added to a volume of 5 mL of multifunctional superparamagnetic nanosystem suspension with free amino groups in the shell, which were previously activated through treatment with succinic anhydride in a N, -dimethylformamide solution, in the 5 to 12% range, for 6 hours under constant gentle stirring. The multifunctional superparamagnetic nanosystems are then dialyzed against water for 18 hours, time by which the multifunctional superparamagnetic nanosystems are removed from the dialysis sleeve. Thereafter, proceed with the addition of 1 mL of 0.1M N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide solution per 5 mL of multifunctional superparamagnetic nanosystem suspension followed by gentle stirring for 30 minutes. After this multifunctional superparamagnetic nanosystem activation step proceed with the immediate addition of the solution comprising the interferon-alpha in the volume and concentration already described. The reaction mixture is placed under gentle stirring for 2 hours at 4°C. The multifunctional superparamagnetic nanosystems obtained are separated from the reaction medium through size-exclusion chromatography. The percentage of functional groups conjugated to the interferon- alpha in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 10 to 30%. The retention of biological activity of the interferon-alpha in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 30 to 40%.

2.3 Conjugation of an antibody with the multifunctional superparamagnetic nanosystem

Considering as example the conjugation of the trastuzumab antibody to the silica/hybrid silica multifunctional superparamagnetic nanosystems, the procedure is the solubilization of the trastuzumab antibody, in the 5 mg/mL final concentration in a 0.1 M bicarbonate buffer solution, pH 8.5. A volume of 1 mL of this solution is added to a volume of 5 mL of multifunctional superparamagnetic nanosystem suspension with free amino groups in the shell, which were previously activated through treatment with succinic anhydride in a N,N- dimethylformamide solution, in the 5 to 12% range, for 6 hours under constant gentle stirring. The multifunctional superparamagnetic nanosystems are then dialyzed against water for 18 hours, time by which the multifunctional superparamagnetic nanosystems are removed from the dialysis sleeve. Thereafter, proceed with the addition of 1 mL of 0.1M N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide solution per 5 mL of multifunctional superparamagnetic nanosystem suspension followed by gentle stirring for 30 minutes. After this multifunctional superparamagnetic nanosystem activation procedure continues with the immediate addition of the solution comprising the trastuzumab antibody in the volume and concentration already described. The reaction mixture is placed under gentle stirring for 2 hours at 4°C. The multifunctional superparamagnetic nanosystems obtained are separated from the reaction medium through size-exclusion chromatography. The percentage of functional groups conjugated to the trastuzumab antibody in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 20 to 40%. The retention of biological activity of the trastuzumab antibody in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 50 to 80%.

2.4 Conjugation of a peptide with the multifunctional superparamagnetic nanosystem

Considering as example the conjugation of the c(RGDfK) or (cyclo (Arg-Gly-Asp-D-Phe-Lys) ) peptide to the silica/hybrid silica multifunctional superparamagnetic nanosystems, the procedure is the solubilization of the peptide, in the 0.1 mg/mL final concentration in a 0.1 M bicarbonate buffer solution, pH 8.5. A volume of 0.2 mL of this solution is added to a volume of 5 mL of multifunctional superparamagnetic nanosystem suspension with free amino groups in the shell, which were previously activated through treatment with succinic anhydride in a N,N- dimethylformamide solution, in the 5 to 12% range, for 6 hours under constant gentle stirring. The multifunctional superparamagnetic nanosystems are then dialyzed against water for 18 hours, time by which the multifunctional superparamagnetic nanosystems are removed from the dialysis sleeve. Thereafter, continues with the addition of .1 mL of 0.1M N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide solution per 5 mL of multifunctional superparamagnetic nanosystem suspension followed by gentle stirring for 30 minutes. After this multifunctional superparamagnetic nanosystem activation procedure continues with the immediate addition of the solution comprising the peptide in the volume and concentration already described. The reaction mixture is placed under gentle stirring for 2 hours at 4°C. The multifunctional superparamagnetic nanosystems obtained are separated from the reaction medium through size-exclusion chromatography. The percentage of functional groups conjugated to the peptide in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 10 to 30%. The retention of biological activity of the peptide in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 30 to 40%.

3. Conjugation of a polymer with the multifunctional superparamagnetic nanosystem

Silica/hybrid silica multifunctional superparamagnetic nanosystems with free amino groups are suspended in a buffer solution of pH in the range of 8 to 9. To a certain multifunctional superparamagnetic nanosystem suspension volume is added an equal volume of 5 mg/mL methoxy polyethylene glycol succinimidyl dissolved in a buffer of pH in the range of 8 to 9. As an example, the conjugation of the polyethylene glycol polymer of average molecular weight 400 Dalton has one of the chain-ends reactive owing to the presence of the succinimidyl valerate, as mentioned before. The mixture is prepared under gentle stirring at room temperature, remaining under stirring for 4 hours. The nanosystems are separated from the excess reagent through adequate separation processes. The silica/hybrid silica multifunctional superparamagnetic nanosystems conjugated with polyethylene glycol polymer chains of average molecular weight 400 Dalton are characterized in comparison with the initial multifunctional superparamagnetic nanosystems and quantified the amino groups that remain free after the conjugation utilizing appropriate analytical techniques. The length of the polymer chain utilized may vary in a range of average molecular weight from 40 to 6000 Dalton. The polyethylene glycol polymer can be constituted by a linear or by a branched chain with two or more arms where the length of the chains may vary in the ranges already mentioned. The polymer chain comprises reactive groups to which one or more biomolecules are bonded.

Linear polyethylene glycol polymer chains of average molecular mass in the ranges already mentioned comprising functional groups in the two chain-ends may be utilized, enabling the bonding of the silica/hybrid silica multifunctional superparamagnetic nanosystem, as mentioned before, and where the other chain-end comprises a functional group, an amino group for example .

3.1 Conjugation of proteins (enzymes) with the polymer in the multifunctional superparamagnetic nanosystem

Considering as example the bonding of the L-asparaginase enzyme to an amino group situated in the terminus of the polymer that is bonded to the silica/hybrid silica multifunctional superparamagnetic nanosystems, the procedure is the treatment with succinic anhydride in a N, -dimethylformamide solution, in the 5 to 12% range, for 6 hours under constant gentle stirring.

The multifunctional superparamagnetic nanosystems are then dialyzed against water for 18 hours, time by which the multifunctional superparamagnetic nanosystems are removed from the dialysis sleeve. Thereafter, continues with the addition of 1 mL of 0.1 M N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide solution per 5 mL of multifunctional superparamagnetic nanosystem suspension to conjugate with the biomolecule under gentle stirring for 30 minutes.

Then proceeds to the conjugation of the L-asparaginase enzyme to the silica/hybrid silica multifunctional superparamagnetic nanosystem with polyethylene glycol chains with the chain-end activated, the procedure is the solubilization of the L- asparaginase enzyme, in the concentration of 1.2X10^"6 M in a 50 mM carbonate buffer solution, pH 9.4. A volume of 1 mL of this solution is added to a volume of 5 mL of multifunctional superparamagnetic nanosystem suspension previously activated, as aforementioned. The reaction mixture is placed under gentle stirring for 2 hours at 4°C. The multifunctional superparamagnetic nanosystems obtained are separated from the reaction medium through size-exclusion chromatography. The percentage of functional groups conjugated to the L-asparaginase in the polyethylene glycol chain-ends at the surface of silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 35 to 45%. The retention of biological activity of the L-asparaginase in the silica/hybrid silica multifunctional superparamagnetic nanosystems is in the range of 60 to 80%.

References

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[2] M. L. Matson, L. J. Wilson, Nanotechnology and MRI contrast enhancement, Future Med. C em. (2010) 2 (3), 491-502. [3]) P. Caravan, Strategies for increasing the sensitivity of gadolinium based MRI contrast agents, Chem. Soc. Rev. (2006) 35, 512-523.

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[5] Mark A. Perazella, "Gadolinium-Contrast Toxicity in Patients with Kidney Disease: Nephro-toxicity and Nephrogenic Systemic Fibrosis", Current Drug Safety (2008) 3, 67-75.

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[9] Dipanjan Pan, Anne H. Schmieder, Samuel A. Wickline, Gregory M. Lanza, "Manganese-based MRI contrast agents: past, present, and future", Tetrahedron 67 (2011) 8431-8444.

[10] A.G. Roca, R. Costo, A.F. Rebolledo, S. Veintemillas- Verdaguer, P. Tartaj , T. Gonzalez-Carreno, M.P. Morales, C.J. Serna, Progress in the preparation of magnetic nanoparticles for applications in biomedicine, J. Phys. D: Appl . Phys . 42 (2009) 224002.

[11] Enzo Terreno, Daniela Delli Castelli, Alessandra Viale, and Silvio Aime, "Challenges for Molecular Magnetic Resonance Imaging", Chemical Reviews 2010 110 (5), 3019-3042.

[12] Aaron Joseph L. Villaraza, Ambika Bumb, and Martin . Brechbiel, "Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics", Chemical Reviews 2010 110 (5), 2921-2959.

[13] Q. A: Pankhurst, N.K.T. Thanh, S. K. Jones, J. Dobson, "Progress in applications of magnetic nanoparticles in biomedicine" J. Phys . D: Appl. Phys . (2009) 42.

[14] Kenneth E. Kellar, Dennis K. Fujii, Wolfgang H.H. Gunther, Karen Briley-Seeb0, Atle Bj0rnerud, Marga Spiller, Seymour H. Koenig, "NC100150 Injection, a Preparation of Optimized Iron Oxide Nanoparticles for Positive-Contrast MR".

[15] Wang, L.; Zhao, W. J.; Tan, W. H., Bioconjugated Silica Nanoparticles: Development and Applications. Nano Res. 2008, 1, 99-115.

[16] Couleaud, P.; Morosini, V.; Frochot, C; Richeter, S . ; Raehm, L.; Durand, J. 0., Silica-based nanoparticles for photodynamic therapy applications. Nanoscale 2010, 2, 1083-1095.

[17] Kumar, R.; Roy, I.; Hulchanskyy, T. Y. / Goswami, L. . ; Bonoiu, A. C; Bergey, E. J.; Tramposch, K. M.; Maitra, A. ; Prasad, P. N., Covalently dye-linked, surface-controlled, and bioconjugated organically modified silica nanoparticles as targeted probes for optical imaging. ACS Nano 2008, 2, 449-456.

[18] Dash, S.; Mishra, S . ; Patel, S . ; Mishra, B. K. , Organically modified silica: Synthesis and applications due to its surface interaction with organic molecules. Adv. Colloid Interface Sci. 2008, 140, 77-94. [19] Burns, A.; Ow, H.; Wiesner, U., Fluorescent core-shell silica nanoparticles: towards "Lab on a Particle" architectures for nanobiotechnology . Chem. Soc. Rev. 2006, 35, 1028-1042.

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Date: August 20^th, 2014

Claims

Multifunctional superparamagnetic nanosystem as contrast agent for magnetic resonance imaging formed by a core-shell structure where cores are formed by iron oxide or mixed spinels nanoparticles, characterized by being coated by a hybrid silica shell, with linear thickness between 5 and 500 nm, with interconnected pores of varying size, forming a three dimensional network, permeable to water molecules, being conditioned by the ratio between non-hydrolysable functional organic and inorganic groups, in bulk and at its surface, displaying ratios r2/rl higher than 100.

Multifunctional superparamagnetic nanosystem according to claim 1, characterized by a ratio between the precursors with non-hydrolysable Si-C bonds and the precursors only with hydrolysable Si-OR bonds that varies between 0 and 1.

Multifunctional superparamagnetic nanosystem, according to claims 1 and 2, characterized by the non-hydrolysable functional organic groups being chosen among the following: methyl-, vinyl-, amine-, glycidoxypropyl-, methyldiethyl-, and their mixtures.

Multifunctional superparamagnetic nanosystem according to claims 1 and 2, characterized by the functional inorganic groups being chosen among the following: ≡Si-OH, ≡Si-0, ≡Si- 0-Si≡.

Multifunctional superparamagnetic nanosystem according to the previous claims, characterized by, in alternative, also being able to be- conjugated with biomolecules and polymers.

Multifunctional superparamagnetic nanosystem according to claim 5, characterized by being combined with biomolecules chosen from one of the following groups: proteins, peptides, enzymes, antibodies.

7. Multifunctional superparamagnetic nanosystem according to claim 5, characterized by being combined with linear or branched polyethyleneglycol polymer chains.

8. Multifunctional superparamagnetic nanosystem according to claim 7, characterized by the polymer chain comprises reactive groups to which one or more biomolecules are bonded.

9. Production method of the multifunctional superparamagnetic nanosystem as contract agent in magnetic resonance imaging defined in claims 1 to 8, comprising the following steps: a) Synthesis of superparamagnetic iron oxide nanoparticles, establishing the core of the nanosystem, through iron (II) and iron (III) ions co-precipitation, or through iron (III) ions reduction-precipitation, in the presence of aqueous solution of ammonium hydroxide, under ultrasound or magnetic stirring, or in alternative;

b) Synthesis of superparamagnetic mixed spinels nanoparticles, such as Co_xFe2-_x0₄, Mn_xFe2-_x0₄, (Co, Mn) _xFe2-_x0₄ or others, establishing the core of the nanosystem, through reduction-precipitation, co-precipitation of divalent cations and Fe (III) in basic medium, or through organometallic synthesis, under ultrasound · or magnetic stirring;

characterized by also comprising the following step:

c) Coating of the superparamagnetic iron oxide or mixed spinels nanoparticles by silica/hybrid silica nanoparticles, constituting the shell of the nanosystem obtained in steps a) or b) with an ethanol, ammonia and water solution mixture, under strong stirring and with the addition of silica organometallic precursors in adequate proportions, for a period of time lower than 15 minutes.

10. Production method of the multifunctional superparamagnetic nanosystem, according to claim 9, characterized by comprising, in alternative, the conjugation of biomolecules

-2- through chemical or physical bond between the functional organic and inorganic groups, present in the shell of the multifunctional superparamagnetic and the reactive groups available and accessible at the surface of the biomolecules .

11. Production method of the multifunctional superparamagnetic nanosystem, according to claim 10, characterized by the biomolecules being chosen among the following: proteins, peptides, enzymes, antibodies.

12. Production method of the multifunctional superparamagnetic nanosystem, according to claims 10 and 11, characterized by comprising the following steps:

a) Activation of the multifunctional nanosystem, through treatment with succinic anhydride under constant gentle agitation;

b) Dialysis of the multifunctional nanosystem in water;

d) Incubation of the reaction mixture under gentle stirring; e) Separation of the multifunctional nanosystem from the reaction medium.

13. Production method of the multifunctional superparamagnetic nanosystem, according to claims 10 to 12, characterized by the conjugation with biomolecules occurs simultaneously or stepwise .

14. Use of the multifunctional superparamagnetic nanosystem as defined in claims 1 to 8 and produced through the method defined in claims 9 to 13, characterized by its application as negative contrast agent in magnetic resonance imaging, in association with therapies, such as hyperthermia, transport, targeted release of drugs, by conjugation with biomolecules.

Date: August 20 , 2014

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