WO2018154165A1 - Nanoparticules modifiées comprenant des dérivés alcoxi-silanes - Google Patents

Nanoparticules modifiées comprenant des dérivés alcoxi-silanes Download PDF

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WO2018154165A1
WO2018154165A1 PCT/ES2018/070130 ES2018070130W WO2018154165A1 WO 2018154165 A1 WO2018154165 A1 WO 2018154165A1 ES 2018070130 W ES2018070130 W ES 2018070130W WO 2018154165 A1 WO2018154165 A1 WO 2018154165A1
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aptes
teos
nps
spion
nanoparticle
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Spanish (es)
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Guillermo DE LA CUEVA MÉNDEZ
Manuel CANO LUNA
Rebeca NÚÑEZ LOZANO
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Fundación Pública Andaluza Progreso Y Salud
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention is within the field of medicine (Nanomedicine), chemistry and biochemistry, and refers to a superparamagnetic nanoparticle, preferably iron oxide (SPION) obtained by thermal decomposition, superficially modified by an exchange of linking with two alkoxy silane derivatives simultaneously.
  • SPION iron oxide
  • the present invention also relates to the compositions, the method of modifying the nanoparticle and its uses.
  • NMR nuclear magnetic resonance
  • Hydrogen is one of the most appropriate elements for the phenomenon of nuclear magnetic resonance, and is the most common element contained in the human body.
  • magnetic resonance imaging is capable of providing high resolution images of soft tissues with detailed anatomical information.
  • the images are obtained by placing the subject in a magnetic field and observing the interactions between the magnetic spins of the subject's water protons and the radiofrequency of radiation applied.
  • the image is solved by applying orthogonal magnetic field gradients that ultimately encode spatially the three coordinates of each pixel in the image.
  • T1 longitudinal relaxation time
  • T2 transverse relaxation time
  • T1 or spin-net relaxation time represents the transfer of energy between the spins of the observed proton and the surrounding network
  • T2 or spin-spin relaxation time is the transfer of energy between different spins or protons.
  • T2 * is also necessary to properly describe the total decay of the magnetic induction.
  • the detected magnetic resonance (MR) signal includes, by both, a combination of relaxation times T1, T2 and T2 *, as well as the contribution of proton density.
  • An advantage of this technique is that it does not use ionizing radiation, providing high quality images without exposing the patient to any type of harmful radiation.
  • endogenous and inherent NMR contrasts are, in many cases, insufficient to adequately resolve small anatomical lesions or properly characterize tissue physiology.
  • specific series of exogenous agents have been developed to enhance the T1, T2 or T2 * components of the image, respectively.
  • T1 and T2 enhancing agents much less is known about the investigation of T2 * potentiation, which could make possible the image of tissue and tumor perfusion with greatly increased resolution and sensitivity.
  • the contrast agents for magnetic resonance imaging are divided into two general classes of magnetically active materials: Paramagnetic and superparamagnetic or ferromagnetic materials.
  • Paramagnetic contrast agents include substances based on small gadolinium (III) chelates (Gd-DTPA, Gd-DTPA-BMA, Gd-DOTA, Gd-D03A) [E. Toth et al. , 2001.
  • NPs nanoparticles
  • Fe 3 0 4 , Fe 2 0 3 iron oxide core
  • SPIO-superparamagnetic ⁇ ron oxide very small ( ⁇ 10 nm, USPIO-ultrasmall superparamagnetic ⁇ ron oxide particles) or reduced ( ⁇ 100 nm, SPIO-superparamagnetic ⁇ ron oxide)
  • Paramagnetic agents induce an increase in MR image intensity in T1-weighted sequences (positive contrast enhancement), and superparamagnetic agents induce a decrease in the magnetic resonance signal in T2-weighted sequences (negative contrast enhancement).
  • the sensitivity and specificity of both types of agents is very different. While gadolinium chelates have a relaxivity that requires millimolar concentrations of the compound in the target tissue, superparamagnetic NPs, due to their greater molecular weight, are effective in micromolar or nanomolar ranges.
  • Superparamagnetic nanostructured materials were developed as a contrast agent for MRI since their nanoscale structure profoundly modified the relaxation time of protons, thereby enhancing the sensitivity of MRI diagnosis. Furthermore, by modifications on the surface of the NPs with specific biologically active vectors, such as monoclonal or polyclonal antibodies or avidin-biotin systems, the specificity of the MRI diagnosis can also be increased.
  • the quality of the particles used as an MRI contrast agent is determined by the magnetic properties of the core of the material, the particle size distribution, the particle's loading surface, stability in almost neutral solvents or physiological serum, as well. as the chemical and functional properties of immobilized molecules on the surface. In addition, pharmacokinetic behavior is an important determinant in magnetic resonance imaging applications, since the agent should ideally remain in the target tissue only during the MRI exam, and be quickly removed afterwards, without accumulating anywhere in the body.
  • dextran or carboxydextran coatings result in a significant and non-specific binding by absorption of these particles to vascular and tissue surfaces, limiting the effective removal of these particles once the imaging study has been performed being required. relatively long waiting times until complete elimination and eventual readministration. For these reasons, the production and characterization of magnetic NPs with poor tissue and vascular adhesion that favor rapid elimination and low tissue accumulation are currently of great relevance.
  • An appropriate protocol for producing magnetic iron oxide NPs comprises coprecipitation of ferric and ferrous salts in an alkaline medium in the absence or presence of surfactants.
  • the NPs thus obtained have a core with a diameter between 1 and 50 nm.
  • NPs with biocompatible polymers or copolymers have a hydrodynamic diameter between 1 and 150 nm.
  • M Fe, Co, Mn
  • the efficient obtaining of this type of inorganic NPs with an adequate size, shape, homogeneity and crystallinity is achieved using synthesis methods based on the thermal decomposition of their organic iron precursors, and require the use of organic solvents, surfactants and elevated temperatures.
  • the superparamagnetic iron oxide NPs (SPIONs, Superparamagnetic ron oxide nanoparticles) generated by these methods are hydrophobic, being coated with surfactant ligands, and therefore stable in apolar solvents.
  • hydrophobic SPIONs In order to use these hydrophobic SPIONs in any industrial and clinical application it is necessary to make them hydrophilic, especially in those cases in which their use necessarily takes place in aqueous media (such as those oriented to biology, biotechnology and biomedicine).
  • One of the most used methods to change the solubility of these NPs are those of "Ligand Exchange", which are based on the replacement of hydrophobic ligands on their surface with other ligands that have a reactive end with the surface of the particle and another extreme with hydrophilic groups [De Palma et al., 2007. Chem. Mater. 19, 1821-1831].
  • this method is carried out by adding an excess of the new ligand to a very dilute solution of the NPs in a certain organic solvent, which causes a displacement of the original hydrophobic ligand by concentration gradient.
  • Some of the most commonly used ligand exchange ligands usually contain carboxylic acid groups (eg Citrate) [Lattuada M. et al., 2007. Langmuir 23, 2158-2168], phosphonate or bisphosphonate [Sandiford L. et al., 2013. ACS Nano 7, 500-512], alcohol (eg Dextrano) [López-Cruz A. et al., 2009, J. Mater. Chem.
  • the SPIONs obtained in this way can lose any functionalization to which they have been subjected during the synthesis process, since this type of non-covalent binding can be affected (compromised) by the conditions in which the reaction takes place and / or in the processes required for purification (especially in the case of the use of Sephadex® columns), which induce the separation of molecules associated with the particle, necessary for its correct function (clinical, biotechnological, industrial, etc.).
  • the surface modification of the SPIONs obtained by thermal decomposition was proposed with the minus two alkoxy silane derivatives simultaneously.
  • the present invention describes how the use of a combination of 60% Si (OCH2CH3) 4 (TEOS) and 40% NH2- (CH2) 3-Si (OCH2CH3) 3 (APTES), for surface modification of These hydrophobic SPIONs, and their subsequent PEGylation, allow to generate particles with better T2-MRI contrast properties than their predecessors obtained only with APTES (100%).
  • PEGylated SPIONs produced by this method maintain good properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization.
  • Figure 1 Schematic illustration of the synthesis of structural and colloidal stable SPIONs. Both molecules and NPs are not drawn to scale.
  • Figure 3 Colloidal stability of SPION-TEOS / APTES NPs in MES buffer solution at pH 6.0
  • A and SPION-TEOS / APTES-PEG in water
  • C SPION-TEOS / APTES-PEG in water
  • B Variation of the hydrodynamic size of the SPION-TEOS / APTES NPs in water or 0.25 M PBS (pH 7.4) or 0.05 M MES (pH 6.0) or 30 mg / mL BSA at 0.25 M PBS (pH 7.4) over time For 5 days.
  • D Same as (B) for SPION-TEOS / APTES-PEG NPs over time for 7 days.
  • FIG. 4 (A) FTIR and (B) TGA of the SPION-OA (black), SPION-TEOS / APTES (blue) and SPION-TEOS / APTES-PEG NPs (red). (C) XPS of NPs SPION-APTES (black) and SPION-TEOS / APTES (red). (D) Zeta-potential variation as a function of pH for NPs SPION-APTES-PEG (black) and SPION-TEOS / APTES-PEG (red). Figure 5. (A) VSM analysis of the SPION-OA (black), SPION-APTES (blue) and SPION-APTES-PEG (red) NPs.
  • (B) MRI phantom images showing the positive (T1) and negative (T2) contrast produced by the SPION-TEOS / APTES-PEG NPs and / or the SPION-APTES-PEG at the indicated concentrations at a magnetic field of 9.4 T.
  • FIG. 6 Cytotoxicity of HepG2 cells treated with SPION-APTES / TEOS-PEG NPs.
  • B Total number of cells in culture treated with the indicated concentrations of NPs for 24 and 48 hours.
  • C Total number of dead cells in culture analyzed in B.
  • D Percentage of dead cells in the same cells analyzed in B.
  • FIG. 7 Representative T2W images obtained from mice before and after intravenous administration of 9-11 mg Fe Kg-1 of SPION-APTES-PEG (AF) and SPION-APTES / TEOS-PEG (GL ) at the indicated times. The two transverse images of the animal at each time show certain organs.
  • MN T2 relaxation time ratios in the different tissues measured from the MRI maps after intravenous injection of NPs SPION-APTES-PEG (M) and SPION-APTES / TEOS-PEG (N).
  • A UV-visible spectrum of NPs SPION-TEOS / APTES-PEG-Cy5 (pink), SPION-TEOS / APTES-PEG (red), SPION-TEOS / APTES (blue) and a blank as control sample (black) treated with ninhydrin.
  • B Schematic illustration of the functionalization of the SPION-TEOS / APTES-PEG NPs with Cy5-NHS ester.
  • C UV-visible absorption spectrum of the SPION-TEOS / APTES-PEG-Cy5 NPs (pink), SPION-TEOS / APTES-PEG (red) and free Cy5 (green).
  • Figure 10 Representation of some alkoxy silane derivatives that can be used for chemical modification of different surfaces.
  • Nano particles preferably SPIONs obtained by thermal decomposition, which is based on an exchange of ligand with at least two alkoxy silane derivatives simultaneously in a determined proportion such as for example , but without representing a limitation, 60% TEOS and 40% APTES, and its preferable subsequent PEGylation by an amidation reaction with a polyethylene glycol derivative (a-Methoxy-w-carboxy PEG).
  • Nano particles, preferably PEGylated, generated in this way have better T2-MRI contrast properties and longer circulation time than their predecessors obtained only with APTES (100%).
  • the NPs, preferably PEGylated, produced by this method maintain excellent properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization. These results open the door to a new strategy for surface modification of nano particles, with thin layers of silica, which allows the development and optimization of these nano particles for certain applications, both in the diagnosis of cancer and in its therapy, as well as for other cosmetic, industrial, biotechnological, etc. applications, such as but not limited to, catalysis, detoxification, decontamination, purification of biomolecules, cells, etc. NANOPARTICLE OF THE INVENTION
  • a first aspect of the invention relates to a nanoparticle (NP), preferably SPIONs obtained by thermal decomposition, comprising at least two different alkoxy silane derivatives.
  • the NPs are SPIONs.
  • thermal decomposition is understood as the process of synthesis of inorganic nanocrystals or NPs with a controlled size and a homogeneous distribution of NPs through the thermal decomposition of organometallic compounds, in high boiling organic solvents containing surfactants stabilizers
  • the thermal decomposition allows a very high degree of control in the size and difference of sizes in the NPs, which gives rise to a very homogeneous behavior in terms of their physicochemical characteristics. This is important to transfer NPs to clinical practice.
  • alkoxy silane derivatives means chemical compounds derived from silicon that are characterized by the presence of Si-O-C radicals (alkoxy).
  • alkoxy silanes derivatives are understood as any compound having at least one hydrolyzable silicon group, OR, which is crosslinked by "silane polycondensation” in the presence of moisture. More particularly, the present invention is illustrated by the use of the following two “alkoxy silane derivatives” simultaneously: Si (OCH2CH3) 4 (TEOS) and NH2- (CH2) 3-Si (OCH2CH3) 3 (APTES).
  • R is an amino group attached to a C3 group or an alkoxy of the methoxy- or ethoxy type; where m can be 0, 1, 2 or 3; where n represents a natural number between 0 and 3; and where X represents an alkoxide group, preferably a methoxy- or an ethoxk
  • organofunctional or “functional” group is understood as the site where most chemical reactions take place.
  • the double bond in the alkenes and the triple bond in the aiquino are also considered as functional groups.
  • the main functional groups are selected from the list consisting of aliens, alkenes, alkynes, alcohols, ethers, aldehydes, ketones, carboxylic acids, esters, amines, amides and any combination thereof,
  • the NP of the invention is superficially modified with two alkoxy silane derivatives by a ligand exchange process.
  • ligand exchange is understood as the chemical change that occurs when one ligand X is displaced by another Y, a ligand being a chemically linked molecule to an NP.
  • the NP of the invention is magnetic, and more preferably super-paramagnetic.
  • the magnetic NP consists of one or more of the following components: i) an inorganic core containing one or more of the elements selected from transition metals, including but not limited to iron, cobalt, manganese, copper and magnesium; or ii) an inorganic core composed of an alloy containing elements selected from transition metals, including but not limited to iron, cobalt, manganese, copper and magnesium.
  • the inorganic core of the magnetic NP is selected from the group consisting of iron oxide, cobalt ferrite, manganese ferrite, magnesium ferrite and combinations thereof.
  • the inorganic core of the magnetic NP is iron oxide.
  • the NP obtained by thermal decomposition that is hydrophobic is converted into hydrophilic by a surface modification process based on an exchange of ligand with two alkoxy silane derivatives simultaneously. Therefore, in a preferred embodiment of this aspect of the invention, the NP of the invention is hydrophilic.
  • the alkoxy silane derivatives are selected from the list consisting of: Tetraethylorthosilicate (TEOS), 3-aminopropyl-triethoxysilane (APTES), 3-aminopropyl-trimethoxysilane (APTMS), 3- mercaptopropyl triethoxysilane (MPTES), 3- mercaptopropyl trimethoxysilane (MPTMS), 3-Glycidoxypropyl triethoxysilane, 3-Glycidoxypropyl trimethoxysilane, 3-Cyanatopropyl triethoxysilane (CPTES), 3-Cianatropropyl trimethoxysilane (CPTMS), 3-Azianapropyl trimethoxypropyl and silylpropyl silylpropyl silylpropyl simethoxyethylpropyl triethoxyethylene w-methoxy polyethylene glycol, or derivatives or analogs
  • the NP of the invention comprises two alkoxy silane derivatives that are selected from: Tetraethylorthosilicate (TEOS) and 3- aminopropyl triethoxysilane (APTES), and more preferably in a proportion of 60% TEOS and 40% APTES or in other proportions close to said proportion 60/40 TEOS / APTES as they are any +/- 20% proportion of said proportion 60/40, such as 40/60 or 80/20 proportions and any other intermediate proportion Between these two values.
  • TEOS Tetraethylorthosilicate
  • APTES 3- aminopropyl triethoxysilane
  • the NPs of the invention are characterized by having an average particle size of less than 50 nm, more preferably less than 30 nm, preferably having an average size between 5 and 25 nm, and even more preferably between 10 and 20 nm.
  • average size or “average diameter” is meant the average diameter of the population of NPs dispersed in an aqueous medium.
  • the average diameter of these systems can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the examples below.
  • the NPs of the invention are PEGylated. More preferably, with the polyethylene glycol derivative a-Methoxy-w-carboxy PEG. More preferably, PEGylation is performed by an amidation reaction.
  • a second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the NP of the invention.
  • the composition of the invention is a pharmaceutical composition, more particularly for medical diagnosis in vivo.
  • the composition is a cosmetic composition.
  • the NPs of the invention can have multiple applications, such as, but not limited to, industrial, biotechnological applications, etc.
  • compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art.
  • the pharmaceutical compositions of the invention include, but are not limited to, any liquid composition (suspension of the system including NPs in water or in water with additives such as viscosizers, pH buffers, etc.) or solid (the system including Lyophilized or atomized NPs forming a powder that can be used to make granules, tablets or capsules) for administration either orally, orally or sublingually, topically, or in liquid or semi-solid form for administration transdermally, ocularly, nasal, vaginal or parenteral.
  • these systems offer the possibility of modulating the in vivo distribution of associated drugs or molecules. They may also be suspensions in biological fluids, such as serum. Aqueous suspensions may be buffered or unbuffered and may have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives, including, but not limited to, microbial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals.
  • compositions may be combined with various inert vehicles or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch, etc.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch, etc.
  • the composition of the invention further comprises at least one biologically active compound or molecule, a therapeutic agent or drug, or a labeling agent.
  • biologically active molecule has a broad meaning and comprises molecules such as high, or more preferably, low molecular weight drugs, polysaccharides, proteins, peptides, lipids, oligonucleotides and nucleic acids, as well as combinations thereof.
  • the biologically active molecule has the function of preventing, alleviating, curing or diagnosing diseases.
  • the biologically active molecule has a cosmetic function.
  • biologically active molecule also includes the terms “active substance”, “active substance”, “pharmaceutically active substance”, “ingredient active, "” therapeutic agent, “” drug, “” agent or molecule for diagnostic purposes in vivo, “or” pharmaceutically active ingredient, “meaning any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.It also includes cosmetics, diagnostics as well as industrial ones (enzymes, chelating molecules, etc.).
  • the NPs of the invention may also comprise other biologically active molecules and other agents, for industrial uses, such as enzymes and chelating agents
  • the NP of the invention may comprise more than one distinct biologically active molecule.
  • Said additional active principle or principles may be included in the system for the transport of biologically active molecules of the invention or be part of the pharmaceutical composition of the invention without being part of the transport system.
  • composition of the invention further comprises a pharmaceutically acceptable excipient.
  • the properties of the NP can vary, with different levels of diagnostic capacity in vivo and therapeutic, as well as with different biotechnological, industrial, etc. applications. It is noted that both the nanoparticle of the invention and the pharmaceutical composition of the invention are useful for diagnosis and therapy, in particular for in vivo diagnosis.
  • a third aspect of the invention relates to the use of the NP of the invention or of the composition of the invention, in the preparation of a medicament, or alternatively, to the nanoparticle of the invention or the composition of the invention for its use as a medicine or as a useful agent in diagnosis in vivo.
  • NPs of the invention can serve as active ingredient vehicles.
  • Another preferred embodiment of this aspect of the invention relates to the use of the NP or the composition of the invention in the administration of active ingredients.
  • tissue ischemia or other systems, such as neurodegeneration, inflammation, edema or cancer, in animals or humans by means of magnetic resonance imaging.
  • another preferred embodiment of this aspect of the invention relates to the use of the nanoparticle or the composition of the invention in the in vivo diagnosis by nuclear magnetic resonance.
  • a fourth aspect of the invention relates to a method for the synthesis of NPs of the present invention comprising: 1. Synthesis of hydrophobic NPs, preferably hydrophobic SPIONs.
  • said SPIONs of, for example 10 nm, spherical, monodispersed and hydrophobic can be produced by the "thermal decomposition” method described by Park et al. [Park J. et al. , 2004. Nat. Mater. 3, 891-895]. It is noted that there are different variants of the method of synthesis of SPIONs by "Thermal Decomposition” and that they are based primarily on the use of other organic iron precursors. Although the solvent and the surfactants used can also be changed.
  • NPs preferably SPIONs-derived silicon alkoxysilanes from the NPs of step 1 or other identical or similar NPs with two or more alkoxy- silanes, preferably through a ligand exchange procedure executed simultaneously.
  • the combination of alkoxy silanes of interest is added to the SPIONs suspended in an oily medium.
  • 0.15% (v / v) of TEOS (300 ⁇ _), 0.10% (v / v) APTES (200 ⁇ _), and 0.01% (v / v) acetic acid (20 ⁇ _) were added to 40 mg of SPION-OA suspended in 200 mL of n-hexane. This mixture was stirred orbitally for 72 h at room temperature to allow progressive displacement of oleic acid molecules in the SPION-OA through the 2 alkoxy silane derivatives used (TEOS and APTES).
  • NPs SPION-TEOS / APTES
  • SPION-TEOS / APTES NP-hexane
  • APTES n-hexane
  • NPs were separated with the help of a neodynium magnet and washed three times with 50 mL of n-hexane to remove excess TEOS and APTES and any other unreacted reagents. Finally, the particles were dried at 60 ° C and under vacuum for 48 h, to allow complete condensation of the alkoxy silanes bound to the surface of the NP. 3.
  • a PEGylation of the NPs obtained in step 2) is carried out.
  • step 2 PEGylation of said NPs is carried out.
  • this step can be carried out by suspending the SPION-TEOS / APTES (30 mg) of step 2) in 10 mL of ultrapure water.
  • 2 kDa CH30-PEG-COOH (180 mg), EDC HCI (20 mg) and NHS (12 mg) were mixed in 10 mL of 0.1 M MONTH MONTH (pH 6), and incubated at room temperature to allow the activation of the carboxylic acid groups of the PEG. After the activation time had elapsed, both solutions were mixed and allowed to react for 2-3 hours at room temperature and with magnetic stirring.
  • the NPs were purified by dialysis against ultrapure water using a 12-14 KDa (cut-off) membrane; and optionally 4. Functionalization of the NPs of step 3) or step 2).
  • the SPIONs of step 3 are functionalized, for example, but not limited to cyanine-5.
  • Cy5-NHS ester (2 mg) was dissolved in 200 of DMSO and slowly added to 14 mg of SPION-TEOS / APTES-PEG dissolved in 800 L of 0.25 M PBS pH 8.0. The mixture was allowed to react in the absence of light and with orbital stirring for 4 h at 4 o C to allow the binding of Cy5 to the amino groups on the surface of the NPs.
  • the SPION-TEOS / APTES-PEG-Cy5 NPs were purified using a Sephadex G-25 column and a solution of PBS pH 7.4 as the mobile phase.
  • PEGylated SPION generated by the method described above have better T2-MRI contrast properties and longer circulation time than their predecessors. obtained only with APTES (100%).
  • PEGylated SPIONs produced by this method maintain good properties of structural and colloidal stability, low cytotoxicity and ease of subsequent functionalization.
  • nanosystems such as the NPs of the invention, allow the nanotechnological development of new, promising and intriguing possibilities in diagnosis and medical treatment.
  • the nanodevices used as contrast agents in medical imaging diagnosis (especially in IMR, ultrasound and tomography) have clear advantages over traditional agents in relation to better optical dispersion, better biocompatibility, a decrease in the probability of denaturation and , especially, their ability to bind ligands, which converts them into devices with multiple functions that bind to the target cells, allowing to obtain an image for diagnosis and at the same time transporting medications, allowing a more specific and efficient treatment.
  • Nanotechnology allows the design of multifunctional nanomaterials with a prolonged plasma half-life (thanks to the superficial incorporation of hydrophilic polymer chains), necessary to safely achieve its objective and specifically release the dose of drug vehicle in the desired place, increasing at the same time the bioavailability of the active agent in the target tissue.
  • these NPs can reach the tumor region simply by accumulation or retention (passive transport). This phenomenon is called the increased permeation and retention effect ("EPR" effect), and it is due to the differential characteristics of the tumor environment, where the vascular tissue is altered. This explains why there is a greater accumulation of NPs in the tumor mass, compared to a healthy tissue [Mart ⁇ nez-Soler G.l. et al., 2010. ARS Pharmaceutica 51, Supplement 3, 113-116; N ⁇ ez-Lozano R. et al., 2015. Current Opinion in Biotechnology 35, 135-140].
  • NPs characterized by an extensive plasma half-life can pass through the abnormal structure of blood vessels of pathological tissues (cancer, inflammation, infection).
  • the biological fate of any drug transport colloid can be significantly improved if it has: i) a very small particle size ( ⁇ 100 nm) and a spherical morphology; and, ii) adequate surface electrical and thermodynamic properties (very low electrical charge, and hydrophilicity).
  • the composition of the invention may additionally comprise a detectable label.
  • a "detectable label” refers to any label that can be used to locate the composition in vivo or in vitro. Examples of markers, but not limited to these, would be fluorophores (for example, Cyanine-5), chemical or protein markers that allow the visualization of a polypeptide.
  • the visualization can be done with the naked eye or by an apparatus (such as, but not limited to a microscope) and may involve a source of energy or light. Therefore, another aspect of the invention relates to a diagnostic kit or device, comprising at least one nanoparticle of the invention, or the pharmaceutical composition of the invention.
  • biologically active molecule drug or therapeutic agent, or detectable label
  • biologically active molecules have similar meanings and are used interchangeably in the memory of this invention. They refer to any substance that provides pharmacological activity and is used in the treatment, cure, prevention, mitigation or diagnosis of a disease or that affects the structure or function of the body of man or other animals.
  • biologically active molecules can include from low molecular weight drugs to molecules of the polysaccharide type, proteins, peptides, lipids, oligonucleotides and nucleic acids and combinations thereof. These molecules are well known to the person skilled in the art and include the meaning of a compound that has the characteristics that make it acceptable for use in medicine, for example and without being limited to the active ingredient in a medicine.
  • these molecules can be synthesized by different organic chemistry techniques, or molecular and biochemical biology techniques.
  • the terms used herein are understood as any compound that is administered to a patient for the treatment of a condition and that can more efficiently reach the target tissue when it is attached to the nanoparticle of the invention than when it is administered without the nanoparticle of the invention.
  • the term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
  • the therapeutic agent includes, but is not limited to, hydrophilic and hydrophobic compounds. Accordingly, the therapeutic agents contemplated in this invention include but are not limited to drug-like molecules, nucleic acids, proteins, peptides, antibodies, antibody fragments, aptamers and small molecules.
  • a protein therapeutic agent includes but is not limited to peptides, enzymes, structural proteins, receptors, and other circulating or cellular proteins as well as fragments and derivatives thereof whose aberrant expression gives rise to one or more medical conditions.
  • a therapeutic agent also includes chemotherapeutic compounds and radioactive materials. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, etc. of the mammal.
  • the “therapeutically effective amount” refers to the amount of active substance, or its salts, pro-drugs, derivatives or analogs or their combinations, which produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said pro-drugs, derivatives or analogs and the therapeutic effect to be achieved.
  • the “adjuvants” and “pharmaceutically acceptable carriers” that can be used in said compositions are the vehicles known to those skilled in the art.
  • excipient refers to a substance that aids the absorption or distribution or action of any of the active ingredients of the present invention, which stabilizes said active substance or aids in the preparation of the medicament in the sense of giving it consistency or providing flavors that make it more pleasant.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine without excluding other types of excipients not mentioned in this paragraph.
  • pharmaceutically acceptable excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
  • the excipient must be pharmaceutically suitable, that is, a dossier that allows the activity of the active substance or of the active ingredients, that is, that is compatible with the active substance, in this case, the active substance is any of the compounds of the present invention.
  • a “pharmaceutically acceptable carrier” refers to those substances, or combination of substances, known in the pharmaceutical sector used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.
  • the vehicle like the excipient, is a substance that is used in the medicament to dilute any of the compounds of the present invention to a certain volume or weight.
  • the pharmaceutically acceptable carrier is an inert substance or action analogous to the active ingredients of the present invention.
  • the function of the vehicle is to facilitate the incorporation of other compounds, allow greater dosage and administration or give consistency and form to the pharmaceutical composition.
  • the pharmaceutically acceptable carrier is the diluent.
  • small molecule refers to a chemical compound, for example a peptidomimetic that can be derivatized or any other organic compound of low molecular weight, natural or synthetic. Said small molecules may be therapeutically transported substances or they may be derivatized to facilitate transport.
  • Low molecular weight means compounds whose molecular weight is less than 1000 Daltons, usually between 300 and 700 Daltons.
  • Figure 1 shows a summary scheme of this procedure.
  • the concrete manufacturing protocol of these SPION-TEOS / APTES-PEG NPs is described in the section entitled "METHOD OF THE INVENTION”.
  • First, hydrophobic, spherical and monodispersed 10 nm SPION-OA NPs were synthesized by the method of thermal decomposition of iron oleate [Park J. et al., 2004. Nat. Mater. 3, 891-895].
  • Figure 2A shows an image of transmission electron microscopy (TEM) obtained for a SPION-OA preparation, where it can be seen that they have the desired size, shape and homogeneity.
  • TEM transmission electron microscopy
  • the hydrophobic SPION-OA NPs obtained were converted to hydrophilic by means of a ligand exchange using two alkoxy silane derivatives simultaneously (60% TEOS and 40% APTES).
  • This method displaces the oleic acid ligands of the SPION-OA and covers them with a very thin layer of silicon oxide, generating hydrophilic SPION-TEOS / APTES NPs.
  • this ligand exchange process does not produce apparent morphological changes, nor does it affect the size, or the monodispersity of the resulting particles.
  • the SPION-TEOS / APTES NPs obtained are water soluble. However, the progressive protonation of the amino groups on the surface of these NPs entails the establishment of hydrogen bonds between the particles, causing the progressive formation of aggregates. Although the use of a pH 6.0 buffer solution can slow this phenomenon, its aggregation is inevitable ( Figure 3A and 3B). To solve this problem, partial PEGylation of the SPION-TEOS / APTES NPs was carried out. For this, the covalent binding of a-methoxy-w-carboxylate PEG (2 KDa) molecules to the surface amino groups of these particles was induced by an amidation reaction. The resulting PEGylated SPIONs were easily purified using simple dialysis.
  • the FTIR spectrum of the PEGylated SPIONs had a peak (-1650 cm “1 ) that can be assigned to the carbonyl groups of the amide bond formed with the PEG and the typical bands from the polymer (between 1718 and 837 cm “1 ) covalently bonded to the surface of the SPION-TEOS / APTES NPs ( Figure 4A).
  • the TGA curve obtained for the NPs obtained by thermal decomposition (SPION-OA) showed a weight loss of 69 .5%, corresponding to the considerable amount of oleic acid ligands that line the core of the particle ( Figure 4B).
  • the TGA curve obtained for SPION-TEOS / APTES NPs has a weight loss of 30.9%, much smaller than that obtained with SPION-OA, and usual in particles subjected to silane ligand exchange processes [Cano M. et al., 2017. Nanoscale 9, 812-822; Cano M. et al., 2016. RSC Advances 6, 70374-70382] ( Figure 4B).
  • the TGA curve obtained for PEGylated SPIONs showed a weight loss of 70.8%, which means that there is a 39.9% increase generated by PEG molecules covalently linked through the amines of the precursor SPION-TEOS / APTES NPs ( Figure 4B).
  • FIG. 4C shows the general spectra of XPS or Survey obtained for NPs produced by ligand exchange with 2 alkoxy silane ligands (SPION-TEOS / APTES) and with a single ligand (SPION-APTES).
  • SPION-TEOS / APTES 2 alkoxy silane ligands
  • SPION-APTES single ligand
  • the average thickness (f) can be estimated using the Lambert-Beer equation, described in the following publications for very thin layers [Kallury KMR et al., 1995. Anal. Chem. 67, 3362-3370; Wong AKY et al., 2005. Anal. Bioanal Chem. 383, 187-200; XPS Applications in Thin Films Research. Geng S. et al., 2002. Materials Technology 17 (4)]:
  • / is the Si2p (eV) signal strength obtained for the solid support with silane layer
  • l 0 is the Si2p (eV) signal strength obtained for the solid support without silane layer
  • / is the exhaust depth of the Si2p electrons through the Silane layer (taken as 20 Armstrong in this work) and ⁇ the angle of escape at which electrons are expelled from the surface.
  • the thickness value obtained from this equation for the surfaces of NPs SPION-TEOS / APTES and SPION-APTES were 1.15 and 0.52 nm, respectively.
  • T1 and T2 relaxation times were measured at 0.5 mM Fe (measured by ICP-AES).
  • the average cross-sectional relaxivity (r2) of our PEGylated SPIONs was 156.3 mM- 1 s-1 at 1.5 T and 1 10.7 mM-1 s-1 at 9.4 T.
  • the measure of the longitudinal relaxivity (r1) of the they were 4.76 mM-1 s-1 at 1.5 T and 0.28 mM-1 s-1 at 9.4 T, giving r2 / r1 ratios of 32.8 and 395.3 to said magnetic fields respectively.
  • NPs are retained in renal corpuscles based on their size, these being located in the renal cortex [Choi C.H.C. et al., 201 1. PNAS 108, 6556-6661; Pern ⁇ a M. et al., 2015. Nanoscale 7, 2050-2059] but there is no glomerular accumulation for PEGylated NPs less than 25 nm.
  • the SPION-TEOS / APTES-PEG NPs which have a hydrodynamic size around 25 nm in fetal bovine serum (medium very similar to blood), can be found both in the peritubular capillaries, which anatomically they are both in the medulla and in the renal cortex, and probably also slightly retained in the glomerulus, which is found only in the renal cortex. Therefore, only the decay of the signal observed in the renal medulla would be entirely due to the circulating NPs in blood that shorten the T2 signal when they pass through the peritubular capillaries and return to the circulatory system.
  • Example 4 Ease of subsequent functionalization for adaptation to specific applications.

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Abstract

La présente invention concerne une nanoparticule obtenue par décomposition thermique, comprenant au moins deux dérivés alcoxi-silanes. L'invention concerne également une composition, des utilisations et un procédé de synthèse.
PCT/ES2018/070130 2017-02-21 2018-02-21 Nanoparticules modifiées comprenant des dérivés alcoxi-silanes WO2018154165A1 (fr)

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