Coated nanoparticles for use for modulating electrical polarization of neurons

Abstract

The present invention relates to the medical field, in particular to the modulation of electrical polarization of neurons. More specifically the present invention relates to a nanoparticle or nanoparticles' aggregate for use for modulating electrical polarization of neurons in a subject, for example for use in prevention or treatment of a neuronal disease in a subject, typically by modulating electrical polarization of neurons in the subject, wherein i) when the nanoparticle or nanoparticles' aggregate is exposed to a light source, the nanoparticle's or nanoparticles' aggregate's material is selected from a material enabling opto-electric transduction, opto-thermal transduction or opto-optical transduction, ii) when the nanoparticle or nanoparticles' aggregate is exposed to a magnetic field, the nanoparticle's or nanoparticles' aggregate's material is selected from a material enabling magneto-electric transduction or magneto-thermal transduction, iii) when the nanoparticle or nanoparticles' aggregate's surface is exposed to an ultrasound source, the nanoparticle's or nanoparticles' aggregate's material is a material enabling acousto-electric transduction, and wherein the nanoparticle or nanoparticles' aggregate is either neutrally charged in the absence of any coating or is coated with a hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticles' aggregate. It further relates to compositions and kits comprising such nanoparticles and/or nanoparticles' aggregates as well as to uses thereof.

Claims

1. A method for treating a neuronal disease in a subject by modulating electrical polarization of neurons in the subject, wherein the method comprises a) administering a composition of nanoparticle or nanoparticle aggregate to the subject, wherein i) when the nanoparticle or nanoparticle aggregate is exposed to a light source, the nanoparticle or nanoparticle aggregate material is selected from a material enabling opto-electric transduction, opto-thermal transduction or opto-optical transduction, or ii) when the nanoparticle or nanoparticle aggregate is exposed to a magnetic field, the nanoparticle or nanoparticle aggregate material is a material enabling magneto-electric transduction or magneto-thermal transduction, or iii) when the nanoparticle or nanoparticle aggregate is exposed to an ultrasound source, the nanoparticle or nanoparticle aggregate material is a material enabling acousto-electric transduction, and wherein the nanoparticle or nanoparticle aggregate surface is either neutrally charged in the absence of any coating or coated with a hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticle aggregate, the neutral charge being of about −10 mV to +10 mV, and b) exposing the subject to a light source, a magnetic field, or an ultrasound source, thereby modulating electrical polarization of neurons in the subject, and wherein the nanoparticle or nanoparticle aggregate of step a) is at least two distinct nanoparticles and/or nanoparticle aggregates, each nanoparticle or nanoparticle aggregate consisting of a distinct material selected from a material enabling opto-electric transduction, opto-thermal transduction, opto-optical transduction, magneto-electric transduction, magneto-thermal transduction or acousto-electric transduction, and the nanoparticle or nanoparticle aggregate surface being optionally coated with a hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticle aggregate.

2. The method according to claim 1, wherein the material enabling opto-electric transduction is a semiconductor material with a band gap Eg below 3.0 eV.

3. The method according to claim 1, wherein the material enabling opto-thermal transduction is a plasmonic metal material.

4. The method according to claim 1, wherein the material enabling opto-optical transduction is a lanthanide element-doped material selected from a lanthanide-doped oxide, a lanthanide-doped mixed-oxide, a lanthanide-doped metal-phosphate, and a lanthanide-doped metal-vanadate.

5. The method according to claim 1, wherein the material enabling magneto-thermal transduction is a superparamagnetic material.

6. The method according to claim 1, wherein the material enabling acousto-electric transduction is a piezoelectric material.

7. The method according to claim 1, wherein the hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticle aggregate displays a functional group selected from an alcohol (R—OH), an aldehyde (R—COH), a ketone (R—CO—R), an ester (R—COOR), an acid (R—COOH), a thiol (R—SH), a saccharide, an anhydride (RCOOOC—R), and a pyrrole.

8. The method according to claim 1, wherein the hydrophilic agent is selected from a poly(lactic acid), a polyhydroxyalkanoic acid, a polyether, a polyethylene oxide, a polyethylene glycol, a polyvinylalcohol, a polycaprolactone, a polyvinylpyrrolidone, a polysaccharide, a polypyrrole, a cyclodextrin, a thioglucose, a 2-mercaptoethanol, a 1-thioglycerol, a thiodiglycol, a hydroxybutyric acid, a hydroxymethyltriethoxysilane, a fructose 6-phosphate and a glucose 6-phosphate.

9. The method according to claim 1, wherein the subject is a human being.

10. A method for treating a neuronal disease in a subject by modulating electrical polarization of neurons in the subject, wherein the method comprises a) administering a composition to the subject, the composition comprising nanoparticles and/or nanoparticle aggregates and a pharmaceutically acceptable support, wherein i) when the nanoparticle or nanoparticle aggregate is exposed to a light source, the nanoparticle or nanoparticle aggregate material is selected from a material enabling opto-electric transduction, opto-thermal transduction or opto-optical transduction, or ii) when the nanoparticle or nanoparticle aggregate is exposed to a magnetic field, the nanoparticle or nanoparticle aggregate material is a material enabling magneto-electric transduction or magneto-thermal transduction, or iii) when the nanoparticle or nanoparticle aggregate is exposed to an ultrasound source, the nanoparticle or nanoparticle aggregate material is a material enabling acousto-electric transduction, and wherein the nanoparticle or nanoparticle aggregate surface is either neutrally charged in the absence of any coating or coated with a hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticle aggregate, the neutral charge being of about −10 mV to +10 mV, and b) exposing the subject to a light source, a magnetic field, or an ultrasound source, thereby modulating electrical polarization of neurons in the subject, and wherein the composition comprises at least two distinct nanoparticles and/or nanoparticle aggregates, each nanoparticle or nanoparticle aggregate consisting of a distinct material selected from a material enabling opto-electric transduction, opto-thermal transduction, opto-optical transduction, magneto-electric transduction, magneto-thermal transduction or acousto-electric transduction, and the nanoparticle or nanoparticle aggregate surface being optionally coated with a hydrophilic agent conferring a neutral surface charge to the nanoparticle or nanoparticle aggregate.

11. The method according to claim 10, wherein the subject is a human being.

12. The method according to claim 1, wherein the material enabling magneto-electric transduction is CoFe.sub.2O.sub.4@BaTiO.sub.3.

Description

FIGURES

(1) FIG. 1. The electromagnetic spectrum.

(2) FIG. 2. Schematic representation of the brain (sagittal plane).

(3) FIG. 3. Metabolic activity assessment using the MTT assay on neuron cells treated with gold nanoparticles coated with a coating conferring (i) a neutral surface charge of −3.4 mV to nanoparticles from example 1, (ii) a negative surface charge of −27.0 mV to nanoparticles from example 2, and (iii) a positive surface charge of +26.1 mV to nanoparticles from example 3. The absorbance is measured at 570 nm. The cellular health is correlated with the Absorbance value at 570 nm: the higher the Absorbance value, the higher the cellular health. The absorbance value is higher for gold nanoparticles from example 1 than for gold nanoparticles from example 2 (about a 2-fold increase) or gold nanoparticles from example 3 (about a 3-fold increase).

(4) FIG. 4. Metabolic activity assessment using the MTT assay on neuron cells treated with silicon nanoparticles coated with a coating conferring (i) a neutral surface charge of −4.5 mV to nanoparticles from example 6, and (ii) a positive surface charge of +16.0 mV to nanoparticles from example 7. The absorbance is measured at 570 nm. The cellular health is correlated with the Absorbance value at 570 nm: the higher the Absorbance value, the higher the cellular health. The absorbance value is higher for Si nanoparticles from example 6 than for Si nanoparticles from example 7 (about a 1.5-fold increase).

(5) FIG. 5. Metabolic activity assessment using the MTT assay on neuron cells treated with iron oxide nanoparticles coated with a coating conferring (i) a neutral surface charge of +8.5 mV to nanoparticles from example 4, and (ii) a negative surface charge of −36.4 mV to nanoparticles from example 5. The absorbance is measured at 570 nm. The cellular health is correlated with the Absorbance value at 570 nm: the higher the Absorbance value, the higher the cellular health. The absorbance value is higher for Fe.sub.2O.sub.3 nanoparticles from example 4 than for Fe.sub.2O.sub.3 nanoparticles from example 5 (about a 1.5-fold increase).

EXAMPLES

(6) In Vitro Studies of Neurons

(7) At the neuron level, patch clamp technique is very useful for detecting action potentials, as it allows simultaneous direct measurement and control of membrane potential of a neuron.

(8) This technique is used to assess the effects of nanoparticles on a single neuron.

(9) In Vitro Studies of a Network of Neurons

(10) Multi-electrode arrays (MEAs) permit stimulation and recording of a large number of neurons (neuronal network). Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with stimulation sequences through the array's multiple electrodes. This technique is very useful to assess physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

(11) Cellular health can also typically be monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT cytotoxicity assay uses metabolic activity as a readout to describe cellular health.

(12) This technique is used typically to assess the functional effect of nanoparticles on neuronal network(s).

Example 1

Nanoparticles Enabling Opto-Thermal Transduction: Synthesis of Gold Nanoparticles Coated with a Biocompatible Coating Conferring a Neutral Surface Charge to Gold Nanoparticles

(13) Gold nanoparticles were synthesized by reducing a gold chloride salt (HAuCl.sub.4) with a capping agent (sodium citrate) (protocol was adapted from G. Frens Nature Physical Science 241 (1973) 21). In a typical experiment, HAuCl.sub.4 solution was heated to boiling. Subsequently, sodium citrate solution was added. The resulting solution was maintained under boiling for an additional period of 5 minutes.

(14) A 0.22 μm filtration (filter membrane: poly(ether sulfone) (PES)) of the nanoparticles' suspension was performed and gold concentration in suspension was determined by a UV-visible spectroscopy assay at 530 nm.

(15) A surface coating was performed using α-methoxy-ω-mercaptopoly(ethylene glycol) 20 kDa (“thiol-PEG20 kDa”). A sufficient amount of “thiol-PEG 20 kDa” was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the gold nanoparticle surface. pH was adjusted between 7 and 7.2, and the nanoparticles' suspension was stirred overnight.

(16) The hydrodynamic diameter (measure in intensity) was determined by Dynamic Light Scattering (DLS) with a Nano-Zetasizer (Malvern) at a scattering angle of 173° with a laser emitting at 633 nm, by diluting the nanoparticles' suspension in water (final concentration: 0.1 g/L). The hydrodynamic diameter of the so obtained biocompatible gold nanoparticles in suspension was found equal to 122.5 nm, with a polydispersity index (dispersion of the nanoparticles' population in size) of 0.15.

(17) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to −3.4 mV.

Example 2

Nanoparticles Enabling Opto-Thermal Transduction: Synthesis of Gold Nanoparticles Coated with a Biocompatible Coating Conferring a Negative Surface Charge to Gold Nanoparticles

(18) Gold nanoparticles were prepared as described in example 1 (same gold inorganic core).

(19) A 0.22 μm filtration on PES membrane filter was performed and gold concentration in suspension was determined by a UV-visible spectroscopy assay at 530 nm.

(20) A surface coating was performed using meso-2, 3-dimercaptosuccinic acid (DMSA). A sufficient amount of DMSA was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the surface of gold nanoparticles. pH was adjusted between 7 and 7.2, and the nanoparticles' suspension was stirred overnight.

(21) The hydrodynamic diameter (measure in intensity) was determined by Dynamic Light Scattering (DLS) with a Nano-Zetasizer (Malvern) at a scattering angle of 173° with a laser emitting at 633 nm, by diluting the nanoparticles' suspension in water (final concentration: 0.1 g/L). The hydrodynamic diameter of the so obtained nanoparticles in suspension was equal to 127.4 nm, with a polydispersity index (dispersion of the nanoparticles' population in size) of 0.54.

(22) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to −27.0 mV.

Example 3

Nanoparticles Enabling Opto-Thermal Transduction: Synthesis of Gold Nanoparticles Coated with a Biocompatible Coating Conferring a Positive Surface Charge to Gold Nanoparticles

(23) Gold nanoparticles were prepared as described in example 1 (same gold inorganic core).

(24) A 0.22 μm filtration on PES membrane filter was performed and gold concentration in suspension was determined by a UV-visible spectroscopy assay at 530 nm.

(25) A surface coating was performed using poly(diallyldimethylammonium) chloride (PDADAC). A sufficient amount of PDADAC was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the surface of gold nanoparticles. pH was adjusted between 7 and 7.2, and the nanoparticles' suspension was stirred overnight.

(26) The hydrodynamic diameter (measure in intensity) was determined by Dynamic Light Scattering (DLS) with a Nano-Zetasizer (Malvern) at a scattering angle of 173° with a laser emitting at 633 nm, by diluting the nanoparticles' suspension in water (final concentration: 0.1 g/L). The hydrodynamic diameter of the so obtained nanoparticles in suspension was equal to 94.1 nm, with a polydispersity index (dispersion of the nanoparticles' population in size) of 0.51.

(27) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to +26.1 mV.

Example 4

Nanoparticles Enabling Magneto-Thermal Transduction: Synthesis of Iron Oxide Nanoparticles Coated with a Biocompatible Coating Conferring a Neutral Surface Charge to Iron Oxide Nanoparticles

(28) Iron oxide (Fe.sub.2O.sub.3) nanoparticles were synthesized by co-precipitation of iron (III) nitrate (Fe(NO.sub.3).sub.3) and iron (II) chloride (FeCl.sub.2) with sodium hydroxide (NaOH) at a basic pH, in a reacting medium with a high ionic strength. The resulting nanoparticles' suspension was washed three times with water by centrifugation.

(29) A 0.22 μm filtration on PES membrane filter was performed and (Fe.sub.2O.sub.3) nanoparticles' concentration was determined by a colorimetric assay in UV-visible spectroscopy.

(30) A coating was prepared using silane-poly(ethylene) glycol 20 kDa (“Si-PEG 20 kDa”). A sufficient amount of “Si-PEG 20 kDa” was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the surface of iron oxide nanoparticles. The nanoparticles' suspension was stirred overnight and subsequently the pH was adjusted to 7.

(31) The hydrodynamic diameter (measure in intensity) was determined by Dynamic Light Scattering (DLS) with a Nano-Zetasizer (Malvern) at a scattering angle of 173° with a laser emitting at 633 nm, by diluting the nanoparticles' suspension in water (final concentration: 0.1 g/L). The nanoparticles' hydrodynamic diameter was found equal to 102.5 nm, with a polydispersity index (dispersion of the nanoparticles' population in size) of 0.11.

(32) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to +8.5 mV.

Example 5

Nanoparticles Enabling Magneto-Thermal Transduction: Synthesis of Iron Oxide Nanoparticles Coated with a Biocompatible Coating Conferring a Negative Surface Charge to Iron Oxide Nanoparticles

(33) Iron oxide (Fe.sub.2O.sub.3) nanoparticles were prepared as described in example 4 (same Fe.sub.2O.sub.3 inorganic core).

(34) A 0.22 μm filtration on PES membrane filter was performed and (Fe.sub.2O.sub.3) nanoparticles' concentration was determined by a colorimetric assay in UV-visible spectroscopy.

(35) A coating was prepared using sodium hexametaphosphate. A sufficient amount of sodium hexametaphosphate was added to the nanoparticles' suspension to reach at least 1 molecule of sodium hexametaphosphate/nm.sup.2 on the surface of iron oxide nanoparticles. The nanoparticles' suspension was stirred overnight and subsequently the pH was adjusted to 7.

(36) The hydrodynamic diameter (measure in intensity) was determined by Dynamic Light Scattering (DLS) with a Nano-Zetasizer (Malvern) at a scattering angle of 173° with a laser emitting at 633 nm, by diluting the nanoparticles' suspension in water (final concentration: 0.1 g/L). The nanoparticles' hydrodynamic diameter was found equal to 81.5 nm, with a polydispersity index (dispersion of the nanoparticles' population in size) of 0.16.

(37) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to −36.4 mV.

Example 6

Nanoparticles Enabling Opto-Electric Transduction: Silicon Nanoparticles Coated with a Biocompatible Coating Conferring a Neutral Surface Charge to Silicon Nanoparticles

(38) Silicon (Si) nanoparticles' suspension (50 g/L) with 5 nm size were obtained from Meliorum Technologies Inc.

(39) A 0.22 μm filtration on PES membrane filter was performed and the (Si) nanoparticles' concentration was determined by drying the suspension to a powder and weighing the as-obtained mass.

(40) A coating was prepared using silane-poly(ethylene) glycol 20 kDa (“Si-PEG 20 kDa”). A sufficient amount of “Si-PEG 20 kDa” was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the surface of silicon nanoparticles. The nanoparticles' suspension was stirred overnight and subsequently the pH was adjusted to 7.

(41) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to −4.5 mV.

Example 7

Nanoparticles Enabling Opto-Electric Transduction: Silicon Nanoparticles Coated with a Biocompatible Coating Conferring a Positive Surface Charge to Silicon Nanoparticles

(42) Silicon (Si) nanoparticles were obtained from Meliorum Technologies Inc. as described in example 6 (same Si inorganic core).

(43) A 0.22 μm filtration on PES membrane filter was performed and the (Si) nanoparticles' concentration was determined by drying the suspension to a powder and weighing the as-obtained mass.

(44) A coating was prepared using 3-aminopropyltriethoxysilane (APS). A sufficient amount of APS was added to the nanoparticles' suspension to reach at least half a monolayer coverage (2.5 molecules/nm.sup.2) on the surface of silicon nanoparticles. The nanoparticles' suspension was stirred overnight and subsequently the pH was adjusted to 7.

(45) The zeta potential was determined by measuring the electrophoretic mobility of the nanoparticles (Nano-Zetasizer, Malvern) by diluting the nanoparticles' suspension in a NaCl solution at 1 mM at pH 7 (final concentration: 0.1 g/L). The zeta potential at pH 7 was found equal to +16.0 mV.

Example 8

Assessment of Cytotoxicity in Midbrain/Frontal Cortex Mouse Neurons Induced by Nanoparticles from Examples 1, 2 and 3 Via the MTT Assay

(46) The MTT cytotoxicity assay uses metabolic activity as a readout to describe cellular health. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. The conversion of the tetrazolium salt to its formazan product is a marker reflecting viable cell metabolism.

(47) Material and Methods

(48) Primary Cell Culture

(49) Midbrain/frontal cortex tissue was harvested from embryonic day 14 chr:NMRI mice (Charles River). Mice were sacrificed by cervical dislocation. Tissue was dissociated by enzymatic digestion (133,3 Kunitz units/ml DNase; 10 Units/ml Papain) and mechanical trituration, counted, vitality controlled, and plated in a 20 μl drop of DMEM containing laminin (10 μg/ml), 10% fetal bovine serum and 10% horse serum on 48-wells microelectrode array neurochips (Axion Biosystems Inc.) coated for 1 hour with Polyethyleneimine (PEI, 50% in Borate buffer), washed and air-dried. Cultures were incubated at 37° C. in a 10% CO.sub.2 atmosphere until ready for use. Culture media were replenished two times a week with DMEM containing 10% horse serum.

(50) MTT Assay

(51) The mouse midbrain/frontal cortex neuronal cell cultures were cultured for 4 weeks. After 4 weeks, the nanoparticles' suspensions from examples 1, 2 and 3 (800 μM) were added to the wells. Twenty-four (24 h) post nanoparticles' suspensions treatment, the medium was changed to avoid direct interaction of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reagent with the nanoparticles. Forty-eight hours (48 h) post nanoparticles' suspensions treatment, cells were treated with the MTT reagent at 50 μg/ml, then incubated for 120 minutes at 37° C. and 10% CO.sub.2, and subsequently lysed in 200 μl lysis buffer (DMSO, 0.4 M acetic acid). Lysates were transferred into 96-well plates and optical density was recorded at 570 nm to quantify the MTT-specific absorbance. Values were plotted after blank value (lysis buffer) substraction.

(52) FIG. 3 presents the Absorbance measured at 570 nm for neuron cells treated with gold nanoparticles coated with a coating conferring (i) a neutral surface charge of −3.4 mV to nanoparticles from example 1, (ii) a negative surface charge of −27.0 mV to nanoparticles from example 2, and (iii) a positive surface charge of +26.1 mV to nanoparticles from example 3. The absorbance value is higher for gold nanoparticles from example 1 than for gold nanoparticles from example 2 (about a 2-fold increase) or gold nanoparticles from example 3 (about a 3-fold increase).

(53) These results demonstrate that the neuron health is enhanced in neuron cultures treated with the nanoparticles described in the present application when said nanoparticles are coated with an hydrophilic coating agent conferring them a neutral surface charge, typically a neutral charge above −10 mV and below +10 mV, rather than a surface charge above +10 mV or below −10 mV.

(54) Therefore, an enhanced safety profile and an increased therapeutic effect is expected when using the nanoparticles described in the present application provided that they are coated with an hydrophilic coating agent conferring them a neutral surface charge.

Example 9

Assessment of Long-Term Cytotoxicity in Midbrain/Frontal Cortex Mouse Neurons Induced by Nanoparticles from Examples 6 and 7 Via the MTT Assay

(55) Material and Methods

(56) Primary Cell Culture

(57) Midbrain/frontal cortex tissue was harvested from embryonic day 14 chr:NMRI mice (Charles River). Mice were sacrificed by cervical dislocation. Tissue was dissociated by enzymatic digestion (133,3 Kunitz units/ml DNase; 10 Units/ml Papain) and mechanical trituration, counted, vitality controlled, and plated in a 20 μl drop of DMEM containing laminin (10 μg/ml), 10% fetal bovine serum and 10% horse serum on 48-wells microelectrode array neurochips (Axion Biosystems Inc.) coated for 1 hour with Polyethyleneimine (PEI, 50% in Borate buffer), washed and air-dried. Cultures were incubated at 37° C. in a 10% CO.sub.2 atmosphere until ready for use. Culture media were replenished two times a week with DMEM containing 10% horse serum.

(58) MTT Assay

(59) The mouse midbrain/frontal cortex neuronal cell cultures were cultured for 4 weeks. After 4 weeks, the nanoparticles' suspensions from examples 6 and 7 (200 μM) were added to the wells. Twenty-four (24 h) post nanoparticles' suspensions treatment, the medium was changed to avoid direct interaction of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reagent with the nanoparticles. Fourteen (14) days post nanoparticles' suspensions treatment, cells were treated with the MTT reagent at 50 μg/ml, then incubated for 120 minutes at 37° C. and 10% CO.sub.2, and subsequently lysed in 200 μl lysis buffer (DMSO, 0.4 M acetic acid). Lysates were transferred into 96-wells plates and optical density was recorded at 570 nm to quantify the MTT-specific absorbance. Values were plotted after blank value (lysis buffer) substraction.

(60) FIG. 4 presents the Absorbance measured at 570 nm for neuron cells treated with Si nanoparticles coated with a coating conferring (i) a neutral surface charge of −4.5 mV to nanoparticles from example 6, and (ii) a positive surface charge of +16.0 mV to nanoparticles from example 7. The absorbance value is higher for Si nanoparticles from example 6 than for Si nanoparticles from example 7 (about a 1.5-fold increase).

(61) These results demonstrate that the neuron health is enhanced in the neuron cultures treated with the nanoparticles described in the present application when they are coated with an hydrophilic coating agent conferring them a neutral surface charge, typically a neutral surface charge above −10 mV and below +10 mV, rather than a surface charge above +10 mV or below −10 mV.

(62) Therefore, an enhanced safety profile and an increased therapeutic effect is expected when using the nanoparticles described in the present application provided that they are coated with an hydrophilic coating agent conferring them a neutral surface charge.

Example 10

Assessment of Long-Term Cytotoxicity in Midbrain/Frontal Cortex Mouse Neurons Induced by Nanoparticles from Examples 4 and 5 Via the MTT Assay

(63) Material and Methods

(64) Primary Cell Culture

(65) Midbrain/frontal cortex tissue was harvested from embryonic day 14 chr:NMRI mice (Charles River).

(66) Mice were sacrificed by cervical dislocation. Tissue was dissociated by enzymatic digestion (133,3 Kunitz units/ml DNase; 10 Units/ml Papain) and mechanical trituration, counted, vitality controlled, and plated in a 20 μl drop of DMEM containing laminin (10 μg/ml), 10% fetal bovine serum and 10% horse serum on 48-wells microelectrode array neurochips (Axion Biosystems Inc.) coated for 1 hour with Polyethyleneimine (PEI, 50% in Borate buffer), washed and air-dried. Cultures were incubated at 37° C. in a 10% CO.sub.2 atmosphere until ready for use. Culture media were replenished two times a week with DMEM containing 10% horse serum.

(67) MTT Assay

(68) The mouse midbrain/frontal cortex neuronal cell cultures were cultured for 4 weeks. After 4 weeks, the nanoparticles' suspensions from examples 4 and 5 (200 μM) were added to the wells. Twenty-four (24 h) post nanoparticles' suspensions treatment, the medium was changed to avoid direct interaction of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reagent with the nanoparticles. Fourteen (14) days post nanoparticles' suspensions treatment, cells were treated with the MTT reagent at 50 μg/ml, then incubated for 120 minutes at 37° C. and 10% CO.sub.2, and subsequently lysed in 200 μl lysis buffer (DMSO, 0.4 M acetic acid). Lysates were transferred into 96-wells plates and optical density was recorded at 570 nm to quantify the MTT-specific absorbance. Values were plotted after blank value (lysis buffer) substraction.

(69) FIG. 5 presents the Absorbance measured at 570 nm for neuron cells treated with iron oxide nanoparticles coated with a coating conferring (i) a neutral surface charge of +8.5 mV to nanoparticles from example 4, and (ii) a negative surface charge of −36.4 mV to nanoparticles from example 5. The absorbance value is higher for Fe.sub.2O.sub.3 nanoparticles from example 4 than for Fe.sub.2O.sub.3 nanoparticles from example 5 (about a 1.5-fold increase).

(70) These results demonstrate that the neuron health is enhanced in the neuron cultures treated with the nanoparticles described in the present application when they are coated with an hydrophilic coating agent conferring them a neutral surface charge, typically a neutral surface charge above −10 mV and below +10 mV, rather than a surface charge above +10 mV or below −10 mV.

(71) Therefore, an enhanced safety profile and an increased therapeutic effect is expected when using the nanoparticles described in the present application provided that they are coated with an hydrophilic coating agent conferring them a neutral surface charge.