NANOMATERIAL AND METHOD OF PRODUCTION OF A NANOMATERIAL FOR MEDICAL APPLICATIONS, SUCH AS MRI OR SERS

Abstract

A method for producing nanomaterial product which comprises at least one hybrid nanoparticle Gold-metal-Polymer, the polymer comprising at least one biopolymer, the atoms of metal being linked with the atom of Gold, the metal being chosen among: Gd, Co, Eu, Tb, Ce, Mn, Fe, Zn, Cu, the method being realized in an aqueous solvent, without reactive or stabilizer agent, and presenting a step of reducing: tetrachloroauric acid (HAuCl.sub.4) and metal ions, in the presence of the biopolymer, the biopolymer being used as a stabilizer agent.

Claims

1. A method for the synthesis of nanomaterial product which comprises at least one hybrid nanoparticle of gold-metal-biopolymer, the biopolymer comprising at least one biopolymer being chosen among: collagen, chitosan, polyethylene glycol diacid, alginate, or theirs derivatives, the metal being chosen among: Gd, Co, Eu, Tb, Ce, Mn, Fe, Zn, Cu, the method being realized in an aqueous solvent and presenting: Preparation steps (Ai-Aii, Bi-Bii) of mixing the gold ions, the metal ions and the biopolymer, for obtaining a resulting solution with gold-metal-biopolymer complex, One single step (Aiii, Biii) of synthesizing nanomaterial product which is the reduction of gold ions and metal ions in the presence of the biopolymer, by introducing a reducing agent in the resulting solution, the biopolymer being used as a stabilizer agent, the method being realized without chemical linker.

2. The method according to claim 1, wherein said preparation steps, and said step of reducing the gold-metal-biopolymer complex, present each a stirring which lasts less than fifteen minutes, the method for synthesizing nanomaterial product lasts less than 2 hours.

3. A method according to claim 1, wherein the steps of the method are conducted at room temperature.

4. The method according to claim 1, comprising the following preparation steps: (Ai) introduction of metal ions in the aqueous solvent which comprises AuCl.sub.4.sup., for realizing complexation between metal ions and gold ions conducted to form gold-metal complex, during a first period; (Aii) stacking, by mixing said gold-metal complex with a first biopolymer introduced in the aqueous solvent, for obtaining a gold-metal-biopolymer complex, during a second period.

5. The method according to claim 1, comprising the following preparation steps: (Bi) staking by mixing tetrachloroauric acid HAuCl.sub.4 with a first biopolymer for obtaining a gold-polymer complex, during a first time period, (Bii) complexation by addition of metal ions to the gold-polymer complex during a second period, for obtaining a gold-metal-biopolymer complex.

6. The method according to claim 4, wherein the method comprises a step of adding a second biopolymer: between the step (Aii) and the step of reduction (Aiii), or between the step (Bi) and the step (Bii).

7. A nanomaterial product, comprising at least one hybrid nanoparticle of gold-metal-biopolymer, wherein: The metal is selected from: Gd, Co, Eu, Th, Ce, Mn, Fe, Zn, Cu, The biopolymer is selected from: collagen, chitosan, polyethylene glycol diacid, alginate, hydroxyapatite (HAPA), and theirs derivatives, the atom(s) of gold and the atoms of metal being linked by ionic bonds.

8. Nanomaterial product synthetized as described in claim 7, with at least one hybrid nanoparticle gold-metal-biopolymers, the metal is chosen among: Gd, Co, Eu, Th, Ce, Mn, Fe, Zn, Cu, the hybrid nanoparticle presenting at least two different biopolymers.

9. Nanomaterial product according to claim 7, wherein the hybrid nanoparticle of gold-metal-biopolymer comprises: an inner core with one or several atoms of gold, and atoms of metal surrounding the atom of gold; and an external shell with the biopolymer.

10. Nanomaterial product according to claim 7, wherein the hybrid nanoparticle of gold-metal-biopolymer comprises: an inner core with one or several atoms of gold; and an external shell with atoms of metal and the biopolymer; the atom(s) of gold and the atoms of metal being linked by ionic bonds.

11. The nanomaterial product according to claim 7, wherein the metal is Gd, and the at least one biopolymer is collagen.

12. The nanomaterial product according to claim 7, comprising two biopolymers, wherein the metal is Gd, and the biopolymers are collagen and PEG.

13. A nanomaterial product of claim 7, for use as a therapeutic agent or a theranostic agent, such as: A thermo-therapy agent, or A phototherapy agent.

14. Use of the nanomaterial product according to claim 7, as: A substrate for Enhanced Raman Spectroscopy; A contrast agent for Magnetic Resonance Imaging; A radio sensitive agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The objects, advantages and other features of the present invention will become more apparent from the following disclosure and claims. The following non-restrictive description of preferred embodiments is given for the purpose of exemplification only with reference to the accompanying drawings in which:

[0085] FIGS. 1a, 1b and 1c are views of nanomaterial according to some embodiments of the present invention;

[0086] FIG. 2 is an EDX spectrum performed on AuGd-polymer nanomaterial;

[0087] FIG. 3 is a UV-VIS spectra of AuGd-polymer nanomaterials;

[0088] FIG. 4 and FIG. 5 are Raman spectra of AuGd-polymer nanomaterials;

[0089] FIG. 6 is a UV-VIS spectra of AuMn-polymer nanomaterials;

[0090] FIG. 7 are Raman spectra of AuMn-polymer nanomaterials;

[0091] FIG. 8 is a schematic of proposed mechanism of GdCl.sub.3AuCl.sub.4.sup. reduction by complexation and particle formation in the presence of PEG, Col, ALG, CHIT as surfactants;

[0092] FIG. 9 is a schematic of proposed mechanism of GdCl.sub.3AuCl.sub.4.sup. reduction and particle formation in the presence of PEG, Col, ALG, CHIT;

[0093] FIG. 10a is a Dark field and FIG. 10b is a bright field schematic view (20, 0.25 NA objective) of MIAPACA-2 cell lines treated with the GdAuNPs; the same sample region is monitored above and below;

[0094] FIG. 11 is a schematic view of GdAuNps internalization into skin cells;

[0095] FIG. 12 is UV-Vis Spectra of Cu AuNPs;

[0096] FIG. 13 is a Raman Spectra of CuAuNPs; experimental conditions: exc=785 nm; laser power 20 mW; accumulation time 180 s;

[0097] FIG. 14 is a UV-Vis Spectra of HAuCl.sub.4 (full line), CuCl.sub.2 (dashed line) and HAuCl.sub.4CuCl.sub.2 (dot-dashed line) in the range 200-900 nm;

[0098] FIGS. 15 a and 15 b are UV-Vis Spectras of HAuCl.sub.4 (full line), CuCl.sub.2 (dashed line) and HAuCl.sub.4CuCl.sub.2 (dot-dashed line) in the range 200-900 nm at different pH;

[0099] FIG. 16 is a Raman Spectra of CuAuCl complex; experimental conditions: exc=785 nm; laser power 20 mW; accumulation time 180 s;

[0100] FIG. 17 is a Raman Spectra of CuAuCl complex at different pH; experimental conditions: exc=785 nm; laser power 20 mW; accumulation time 180 s.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Abbreviation Used

[0101] ALG: sodium alginate; [0102] AuNPs: nanoparticles comprising gold; [0103] CHIT: chitosan (deacetylated chitin, poly(D-glucosamine)); [0104] Col: collagen type I from calf skin; [0105] COSY: correlation spectroscopy; [0106] GdAuNPs: hybrid nanoparticles comprising gold and gadolinium; [0107] MnAuNPs: hybrid nanoparticles comprising gold and manganese; [0108] HSQC: heteronuclear single quantum coherence; [0109] IEFPCM polarizable continuum model using the integral equation formalism variant; [0110] PBS: phosphate buffer solution; [0111] PEG: dicarboxylic polyethylene glycol (PEG)-600; [0112] SEM-EDX scanning electron microscopy-energy dispersive x-ray analysis; [0113] TEM: transmission electron microscopy.

[0114] Materials

[0115] Tetrachloroauric acid (HAuCl.sub.4), gadolinium chloride hexahydrate (GdCl.sub.3, 6H.sub.2O), sodium borohydride (NaBH.sub.4), PBS (pH 7.2-9.0), sodium chloride (NaCl), PEG, Col, CHIT, ALG were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

[0116] All chemicals were used as such, without further purification.

[0117] Milli Q water was used throughout the experiments.

[0118] All solvents were used without any further purification.

[0119] Experiments were carried out at room temperature, if not specified otherwise.

[0120] Physico-Chemical Characterization

[0121] All the measurements were performed in triplicate, in order to validate the reproducibility of the synthetic and analytical procedures.

[0122] UV-Vis Absorption Spectroscopy

[0123] Absorption spectroscopy measurements were carried out by means of a double-beam Varian Cary 500 UV-Vis spectrophotometer (Agilent, France).

[0124] Absorption spectra were recorded in the 350-900 nm spectral range in water, at AuNPs concentration equal to 10.sup.4 M. For stability studies, Gd-AuNPs were dispersed in PBS (0.1 M; pH 4.0 or pH 7.0), and absorption spectra collected over three months.

[0125] TEM

[0126] TEM images were acquired with a JEOL JEM 1011 microscope (JEOL, USA) at an accelerating voltage of 100 kV.

[0127] TEM specimens were prepared after separating the PEG from the metal particles by centrifugation. Typically, 1 ml of Gd-AuNPs was centrifuged for 20 min at a speed of 14000 rpm. The upper part of the colorless solution was removed and the solid portion was re-dispersed in 1 ml of water. 2 L of this re-dispersed particle suspension was placed on a carbon coated copper grid (manufactured by Smethurst High-Light Ltd and marketed by Agar Scientific) and dried at room temperature.

[0128] SEM-EDX

[0129] Scanning electron microscopy (SEM) investigation was performed on an environmental SEM (ESEM, Quanta 200 FEG, FEI Company Hillsboro, Oreg.) equipped with an (EDX) spectrometer (Genesis 2000, XMS System 60 with a Sapphire Si/Li Detector from EDAX Inc., Mahwah, N.J.

[0130] Raman Spectroscopy

[0131] The Raman experiments have been performed on an Xplora spectrometer (Horiba Scientifics-France).

[0132] The Raman spectra have been recorded using an excitation wavelength of 785 nm (laser diode) at room temperature.

[0133] For measurements in solution, a macro-objective with a focal length of 40 mm (NA=0.18) was used in backscattering configuration. The achieved spectral resolution is close to 2 cm.sup.1.

[0134] .sup.1H-NMR

[0135] Experiments were conducted on a AVANCE III spectrometer (Bruker) operating at 500 MHz with a 5 mm gradient indirect detection probe, at a probe temperature of 300 K.

[0136] 64 scans were acquired to generate ID proton spectra of GdCl.sub.3 (5 mM), PEG (1 mM) and Col, CHIT ALG products (1 mM), whereas 2048 scans were acquired for AuGd NP1-AuGd NP5 (1 mM).

[0137] The data acquisition size was 32 K and the spectral width was 5000 Hz.

[0138] Typical 9.5 s pulse length and relaxation delay of 2 s were used. Water signal was suppressed by applying a secondary irradiation field at the water resonance frequency (pre-saturation sequence). For each sample 500 L aliquots were directly mixed with 100 L Deuterium Oxide (D20) 99.96% (Eurisotop) providing an internal field-frequency lock. Samples were placed in 5 mm diameter tubes for .sup.1H-NMR analysis. For .sup.1H resonance assignment, COSY spectra were acquired for DOX with 4 K data points and 32 transients for each of the 128 increments. Moreover, 2D NMR HSQC experiments were used for .sup.13C resonance assignment. Chemical shifts (8, in ppm) were compared to the NMR solvent signal D.sub.20 (4.73 ppm at 300K).

[0139] Zeta Potential Measurements

[0140] The zeta potential of Gd-AuNPs dispersed in water was measured using the electrophoretic mode of a Zetasizer NANOZS (Malvern Instruments Ltd, UK).

[0141] Preparation of Collagen Solution (Col)

[0142] Collagen solution (Col) was diluted in PBS (pH 7.2) for 1 h at room temperature.

[0143] Preparation of Alginate Solution (ALG)

[0144] 10 mg of ALG was dissolved in 10 mL of PBS (pH 9.0) under ionic strength conditions (NaCl: 0.5 mM) for 1 h at room temperature until homogeneous solution.

[0145] Preparation of Chitosane Solution (CHIT)

[0146] 10 mg of CHIT was dissolved in 10 mL of PBS (pH 9.0) under ionic strength conditions (NaCl: 0.5 mM) for 1 h at room temperature until homogeneous solution.

[0147] Preparation of HAuCl.sub.4 6H.sub.2O Solution

[0148] 16 mg of HAuCl.sub.4 6H.sub.2O was dissolved in 50 mL of MilliQ water at room temperature until homogenous solution.

[0149] Preparation of GdCl.sub.3 (Gd) Solution

[0150] Solution a) 50 mg of GdCl.sub.3.6H.sub.2O was dissolved in 50 mL of HAuCl.sub.4 solution (9.4*10.sup.4M) at room temperature until homogeneous solution. Solution b) 16 mg of GdCl.sub.3.6H.sub.2O was dissolved in 50 mL of MilliQ water at room temperature until homogeneous solution.

[0151] Preparation of MnCl.sub.2 Solution

[0152] 16 mg of MnCl.sub.2.6H.sub.2O was dissolved in 50 mL of MilliQ water at room temperature until homogeneous solution.

[0153] Synthesis Procedures

[0154] Procedures are described know with reference to the protocol represented on FIG. 8.

[0155] Synthesis of GdAuNP1 (GdAu-ColNPs), See FIG. 1a

[0156] GdAu-ColNPs were prepared by a fast steps chemical process.

[0157] 1 ml of Gd solution b) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. After this time, 1 ml of Col solution was added in the resulting solution for 10 min. Then 6 ml (and/or 1.2 ml) of 0.01M NaBH.sub.4 was added drop-wise to the resulting solution, followed by rapid stirring. After 15 min without agitation, the solution became brune-pale. The product was centrifuged at 9000 rpm for 25 min and the pellet was recovered and purified by washing with milli Q water for three times. The entire synthesis takes less than two hours.

[0158] Synthesis of GdAuNP2 (GdAu-PEG-ColNPs), See FIG. 1b

[0159] 2 ml of Col solution was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. Then 250 l of PEG solution was mixed in the resulting solution under stirring at room temperature. To the resulting solution 6 ml (and/or 1.2 ml) of NaBH.sub.4 (0.01M) was added dropwise followed by rapid stirring. After 15 min without agitation, the solution became red-violet. The product was centrifuged and purified at the same conditions. The entire synthesis takes less than two hours.

[0160] Synthesis of GdAuNP3 (GdAu-PEGNPs) See FIG. 1c

[0161] GdAu-PEGNPs were prepared under two followed protocol.

1) 1 ml of Gd (solution b) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. After this time, 250 l of PEG solution was added under stirring at room temperature in the resulting solution. After 10 min, 3 mL (and/or 1.2 ml) of NaBH.sub.4 (0.01 M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions.
2) Gd solution a) was dissolved in 50 mL of HAuCl.sub.4 solution (9.4*10.sup.4 M) at room temperature until homogeneous solution. After this time, 250 l of PEG solution was added under stirring at room temperature in the resulting solution. After 10 min, 3 mL (and/or 1.2 ml) of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 15 min. The resulting red-rose solution was centrifuged and purified at the same conditions.

[0162] Stability of Gd-AuNPs as a Function of pH

[0163] The stability of hybrid nanoparticles was detected by UV VIS. All hybrid nanoparticles were dissolved in PBS solution 0.1M at different pH (pH 1, 6, 7, 8, 12) during 18 h.

[0164] Synthesis of MnAuNP1 (MnAu-PEG-ColNPs)

[0165] 5 ml of Mn solution was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. After this time, 250 l of Polyethylene glycol 600 Diacid (PEG) was added in the resulting solution under stirring for 5 min. Then 500 l of Collagen was added under stirring at room temperature. After 10 min, 1.2 ml of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0166] Synthesis of MnAuNP2 (MnAu-PEG-Col-ALGNPs)

[0167] 5 ml of Mn solution was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. After this time, 0.5 ml of Collagen and 1 ml of ALG solutions were added in the resulting solution under stirring at room temperature. After 10 min, 1.2 ml of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0168] Synthesis of MnAuNP2 (MnAu-PEG-Col-CHITNPs)

[0169] 5 ml of Mn solution was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution for 10 min. After this time, 1 ml of Collagen and 1 ml of CHIT solutions were added in the resulting solution under stirring at room temperature. After 10 min, 1.2 ml of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0170] Procedures are described know with reference to the protocol represented on FIG. 9.

[0171] Synthesis of GdAuNP1 (GdAu-PEG-ColNPs)

[0172] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 500 l of Collagen and 1 ml of Gd (protocol b) were added in the resulting solution under stirring at room temperature. After 10 min, 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0173] Synthesis of GdAuNP2(GdAu-PEG-Col-ALGNPs)

[0174] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 50 l of Collagen, 1 mL of ALG solution and 1 ml of Gd solutions were added in the resulting solution under stirring at room temperature. After 10 min, 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0175] Synthesis of GdAuNP3(GdAu-PEG-Col-CHITNPs)

[0176] GdAu-PEG-Col-CHITNPs were prepared under same protocol of GdAuNP2.

[0177] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 500 l of Collagen, 1 mL of CHIT solution and 1 ml of Gd solutions were added in the resulting solution under stirring at room temperature. After 10 min, 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0178] Synthesis of MnAuNP1 (MnAu-PEG-ColNPs)

[0179] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 500 l of Collagen, and 2 ml of Mn solutions were added in the resulting solution under stirring at room temperature. After 10 min. 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions.

[0180] Synthesis of MnAuNP2 (MnAu-PEG-Col-ALGNPs)

[0181] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 50 l of Collagen, 1 mL of ALG solution and 2 ml of Mn solutions were added in the resulting solution under stirring at room temperature. After 10 min, 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions

[0182] Synthesis of MnAuNP2 (MnAu-PEG-Col-CHITNPs)

[0183] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 500 l of Collagen, 1 mL of CHIT solution and 2 ml of Mn solutions were added in the resulting solution under stirring at room temperature. After 10 min, 3 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions.

[0184] Results and Discussion

[0185] Change in the surface charge (z-potential) for each AuGdNP in my (+ standard deviation) are as follows

AuGdNP1 12.8 (+0.2)

AuGdNP2 14.8 (+2.0)

AuGdNP3 13.8 (+1.0)

[0186] Formation mechanism of hybrid GdAu NPs and role of polymers as stabilizer agent.

[0187] The synthesis of Gd-AuNPs (NP1-NP3) was carried out by reducing tetraclororoauric acid (HAuCl.sub.4) in the presence of GdCl.sub.3 and different biopolymers (PEG, Col, ALG, CHIT) using sodium borohydride (NaBH.sub.4) as a reducing agent in a resulting solution by using two protocols in order to show the critical importance of order of reagents and concentrations for the shape, organization, morphology of the nanoproduct and the hybrid nanoparticles of the nanoproduct. Particles formation and growth were controlled by the amphiphilic (dual nature) character of the polymers.

[0188] In the case of the process represented on FIG. 8, GdAu-polymer modification onto the surface of AuNPs, solutions was prepared to this end. Particles formation and growth were controlled by the amphiphilic character of the polymers and includes three steps: complexation between GdCl.sub.3 and AuCl.sup.4 to form gold-gadolinium clusters; adsorption (stacking) of COOH-terminated polymer molecules onto GdAu complex; reduction of metal ions in that vicinity, growth of gadolinium-gold particles and colloidal stabilization.

[0189] In the latter case, GdAu complex molecules are expected to be involved in the nucleation process and may, thus, influence the final shape and size of hybrid nanoparticles.

[0190] In the case of the process represented on FIG. 9, the formation of golf hybrid nanoparticles from AuCl.sup.4 can be summarized in three steps: formation of polymer mixture (i.e. PEG, Col, CHIT, ALG); initial reduction of metal ions facilitated by dicarboxylic acid-terminated PEG and/or Col to form gold clusters; adsorption (stacking) of polymers molecules on the surface of gold clusters and reduction of metals in that proximity and growth of gold particles and colloidal stabilization by molecules of polymers.

[0191] As can be seen on FIGS. 8 and 9, depending on the process used (complexation/stacking/reduction or stacking/complexation/reduction), the metal ions (Gd, Eu, Tb, Ce, Mn, Fe, Zn, Cu) are not at the same distance to the atom of gold.

[0192] Gd (or the other metals: Co, Eu, Tb, Ce, Mn, Fe, Zn, Cu when used instead of Gd) is close to Au in the core of the hybrid nanoparticle the outer shell (or a matrix) being essentially based on polymer (FIG. 8). Gd being not covalent linked with the gold atom on FIG. 8.

[0193] On the contrary, Gd is placed at a larger distance to Au in the outer polymer shell (or a matrix) of the hybrid nanoparticle (FIG. 9), Gd is more far from Au in the FIG. 9 than in the FIG. 8. Gd being not covalent linked with the gold atom on FIG. 9.

[0194] In the present method, reduction of the salts is made in an aqueous solvent, salts of metal being chosen from a list comprising Gd, Eu, Tb, Ce, Mn, Fe, Zn, Cu, in the presence of a polymer, to obtain complexes, before obtaining hybrid nanoparticles realized after reduction. This method forms ionic bonds (i.e. electrostatic weak bonds) between the atom of gold and the atoms of metal in the created hybrid nanoparticle for the two mechanisms illustrated on FIG. 8 and on FIG. 9, whereas covalent bonds (strong bonds) are forms in prior art methods between the atom of gold and the atom of metal in view of the use of linkers or the like, for obtaining gold hybrid nanoparticles.

[0195] Interactions between AuCl.sub.4.sup. ions and mixtures of polymers molecules (attractive ion-dipole interactions versus repulsive interactions due to hydrophobicity) are important in controlling the competition between the reactivity of AuCl.sub.4.sup. reduction both in the bulk solution, which causes the nucleation of gold seeds.

[0196] The addiction of GdCl.sub.3 to the gold-polymer complex induce a migration of Gd.sup.3+ ions inside polymer chains and consequent reduction to Gd.sup.2+.

[0197] Reduction with sodium borohydride NaBH.sub.4 to obtain final hybrid nanoparticles

[0198] This result is likely due to the strong competition between PEG diacid and other polymers during reduction process of HAuCl.sub.4

[0199] Characterization of GdAu NPs

[0200] FIG. 3 report absorption spectra of hybrid Gd AuNPs, all characterized by a small peak at 300 nm and a surface plasmon band in the range 530-550 nm. The slow shift of the band position depends on the ratio of the gold salt and the capping materials during the reaction processes.

[0201] GdAuNP1 (GdAu-Col) shows a plasmon peak at 532 nm. This peak is generally ascribed to collective oscillation, known as the surface plasmon oscillation of the metal electrons in the conduction band, due to interaction of electrons with light of that wavelength. Col can be used as stabilizing polymers for AuNPs because of the dispersed solutions could be obtained due to the formation of coordination bands between Au and Gd ions with the amine or carboxylic groups respectively.

[0202] The absorption peak due at GdAuNP2 (GdAu-PEG-Col) is centered at 545 nm. This chelation evenly better dispersed Au ions and Gd which were reduced to form single AuNPs of relatively uniform size.

[0203] GdAuNP3 (GdAu-PEG) shows a strong resonance band at around 300 nm and a weaker one at 520 nm.

[0204] The shape of the nanomaterial can be modified depending on the concentrations of reagents and protocol used in the synthesis. As an example, NP1 obtained by the process of FIG. 8 shows hybrid gold spheres with a diameter of around 50 nm (see FIG. 1a), whereas NP3 obtained by the process of FIG. 8 shows snows shape particles of about 80 nm (see FIG. 1c), possibly due to the presence of CHIT in the growth process.

[0205] The presence of Gd ions enhance the Raman signal of biomolecules, e.g. protein or natural polymer capped onto gold hybrid nanoparticles.

[0206] Raman spectra for powder GdCl.sub.3 and GdAuNPs at 4.5 10.sup.4 M are show in FIGS. 4 and 5. The region from 1200 to 1550 cm.sup.1 corresponds to vibrations of NH bending and CN stretching, while the region between 1550 and 1750 cm.sup.1 corresponds to the CO stretching mode. The band observed near 836 cm.sup.1 corresponds to vibrations of the aromatic ring. This enhancement is the result of the electric field around the gold hybrid nanoparticles interacting with collagen molecules in the presence of gadolinium. The vibration mode for the amine group is located at 1630 cm.sup.1, this band showing a signal enhancement factor of around 40 times higher than that of free collagen. This increment in the Raman signal of collagen could be due to a combination of three factors: [0207] formation of well-defined hot spots where Gd.sup.2+ ions work as spacers between gold hybrid nanoparticles; [0208] the refractive index contrast produced by the presence of Gd.sup.2+ ions causing an increment in the electric field around the hybrid nanoparticle and producing a higher enhancement factor; [0209] a charge transfer effect between gold hybrid nanoparticles and Gd.sup.2+ ions.

[0210] The invention provides inter alia synthesis of Gd-AuNPs in an aqueous solvent by reducing tetraclororoauric acid (HAuCl.sub.4) in the presence of GdCl.sub.3 and different polymers (PEG-diacid, Col, ALG, CHIT) using sodium borohydride (NaBH.sub.4) as a reducing agent by using two protocols. Particles formation and growth were controlled by the amphiphilic (dual nature) character of the polymers. Synthesis are easy and brief.

[0211] The invention provides nanomaterial product that can be used as a substrate for Enhanced Raman Spectroscopy, or as a contrast agent for Magnetic Resonance Imaging, or as a therapeutic agent, a theranostic agent, a radio sensitive agent, a thermo-therapy agent, a radioactive agent, or a phototherapy agent.

[0212] The nanomaterial product has a low toxicity. Biopolymers such as PEG, Col, CHIT. ALG are used as stabilizer agent during the production of the nanomaterial and also as support for fixing biomolecules (e.g. DNA, medical product), enabling the use of the nanomaterial for the delivery of drugs for cancer treatment, by passively and actively targeting to tumor cells.

The method presents very fast previous (or extratemporeanous) steps (10 minutes), before the step of reduction, to obtain hybrid nanoparticles. In this sense, the method is a single step method, the single step (Aiii, Biii) being the step of reduction of the gold ions and metal ions by introducing a reducing agent, in the presence of the biopolymer, the biopolymer being used as a stabilizer agent, for obtaining a gold-metal-polymer nanoparticle.

[0213] These fast previous steps can be realized consecutively in the same receptacle.

[0214] In one first embodiment, the gold ions and the metal ions are put together (resulting solution), and ten minutes after the polymer is added in the resulting solution.

[0215] In a second embodiment, the gold ions and polymer are put together (resulting solution), and ten minutes after the metal ions are added in the resulting solution. These fast steps can be realized consecutively in the same receptacle.

[0216] Photothermia Properties

[0217] Photothermia Experiments

[0218] A 660 nm laser diode is focused on the cells and the gold nanoparticles using a 80 objective (numerical aperture=0.75) for 10 s using the optical microscope of an Xplora Raman microspectrometer (Horiba Scientifics) with a laser irradiation of 2.2 mW/m.sup.2.

[0219] Cell Treatment

[0220] Miapaca-2 cells (pancreatic cells) were exposed to a series of Gd-AuNPs dilutions in complete media (concentrations ranging from 0 to 17 M of nano) for 48 h in 12 well plates. Untreated cells were also included in the experimental design. The cell were then washed, detached from the well using Trypsin-EDTA (0.25%) and resuspended in complete media. A staining with propidium iodide was added to the cell suspension at a 3 M final concentration and incubated for 15 min in the dark at room temperature. After staining with PI, cell were counted by means of BD Accuri C6 flow cytometer, keeping constant the counting time among samples. For each sample 10 000 cells were acquired and analyzed by flow cytometry (BD Accuri C6 Cytometer) where FSC, SSC and FL3 data was collected.

[0221] Measurements for each sample were carried out in triplicate and are presented as median.

[0222] Photothermal Effect into PDAC Cells

[0223] Cell internalization of the synthesized colloids (Gd-AuNPs) was checked with a confocal microscope under bright and dark field illumination. For comparison purpose, the glass slide contained regions with identically grown cells which were not exposed to the colloid solution. The images reported in FIGS. 10a and 10b are from the treated MIAPACA-2 cells, the same sample region is seen in dark field (FIG. 10a) and in bright field conditions (FIG. 10b). The dark field image shows a high density of bright, small scattering centers dispersed all over the glass slide. These dots are individual colloids or small aggregates resting on the glass slide (i.e., outside the large, microns sized cells); they are not seen under bright field due to their very small, submicrometric dimensions. Such a background of bright dots is not seen when observing the non-treated cell. Furthermore, the density of these bright spots clearly decreases if the original concentration of the incubated colloids is decreased tenfold. Within the cells, larger, bright regions are also seen (some have been marked with arrows). It appears that the colloids have a tendency to accumulate inside the cells: most of them present a central bright region. In the bright field image, dark patches, indicating lower transmission of the incident white light are seen at the corresponding positions for the larger aggregates of nanoparticles. Very similar results were obtained when treating MIAPACA-2 cells with the same batch of colloids (see FIGS. 10a and 10b for the relative dark field and bright field images). Individual cells were observed with a higher magnification objective (100) to gain a better insight into the distribution of the colloidal particles.

[0224] Similar experiments were carried out after internalization of Gd AuNPs into skin cells. FIG. 11 shows an example of a good internalization and homogenization in the cells. A red point confirm the presence of Gd NPs

[0225] Synthesis CuAu-PEG AuNPs and Description of CUAuCL.sub.2 Complex

[0226] Materials and Methods

[0227] Preparation of HAuCl.sub.4 6H2O (Au) Solution

[0228] 16 mg of HAuCl.sub.4*6H2O was dissolved in 50 mL of MilliQ water at room temperature until homogeneous solution.

[0229] Preparation of CuCl.sub.2 (Cu) Solution

[0230] 16 mg of CuCl.sub.2*6H2O was dissolved in 50 mL of MilliQ water at room temperature until homogeneous solution.

[0231] Preparation of AuCuCl.sub.2 (AuCu) Solution

[0232] 5 mL of Cu solution was added to 5 mL of Au solution and stirring during 20 min

[0233] This solution was also carried out at different pH (4, 7, 9)

[0234] Synthses of CuAu-NPs (CuAu-PEG-NPs)

[0235] 250 l of Polyethylene glycol 600 Diacid (PEG) was mixed with 20 ml of 0.0001M aqueous HAuCl.sub.4 solution under stirring for 10 min. After this time, 5 ml of Cu solutions were added under stirring at room temperature. After 10 min, 1.2 mL of NaBH.sub.4 (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2 h. The resulting red-rose solution was centrifuged and purified at the same conditions.

[0236] Characterization of Cu-AuNPs

[0237] FIG. 12 report absorption spectra of hybrid Cu-AuNPs, all characterized by a surface plasmon band in the range 530-560 nm and a small peak at 720 nm. The slow shift of the band position depends on the ratio of the gold salt and the capping materials during the reaction processes. CuAuNP1 (picture on the right) (FIG. 12) shows a plasmon peak at 560 nm. This peak is generally ascribed to collective oscillation, known as the surface plasmon oscillation of the metal electrons in the conduction band, due to interaction of electrons with light of that wavelength. PEG can be used as stabilizing polymers for AuNPs because of the dispersed solutions could be obtained due to the formation of coordination bands between Au and Cu ions with the carboxylic group. This chelation evenly better dispersed Au ions and Cu which were reduced to form single AuNPs of relatively uniform size.

[0238] FIG. 13 showed Raman spectra of Cu-AuNPs. In detail, Raman spectra of Cu-AuNPs exhibit many bands in the region 500-2000 cm-1 (FIG. 13). The wide band observed around 1600 cm-1 on the Raman spectra is assigned to the water. Only few bands remain as the two bands around 1000 cm-1 and the one around 1620 cm-1. For instance, the strong band at 1712 cm-1 was assigned to CO carbonyl stretching of PEG diacid. Raman spectra new bands also appear as an intense doublet at 720-760 cm-1 due to CH plane deformation and a strong peak at 1439 cm-1 assigned to v CC stretching. These bands are due to variation of the steric conformation of the PEG diacid, and become more prominent upon complexation with CuAuCl.sub.2. As matter of fact, when CO and hydroxyl groups of PEG diacid interacts with a metal, the sterical conformation becomes more tilted in respect to the planarone. Focusing our attention on the spectral range 200-500 cm-1 (FIG. 13), one can detect several spectral changes which confirms a chemical and steric modification of each drug (PTX; DTX) after complexation with gold ions and PEG-diacid molecules. One of the Raman fingerprint of the Cu-PEG-AuNPs is the presence of a band around 263 cm.sup.1 and a double peak at 235-285 cm.sup.1 was observed. These bands can be assigned to the gold chloride stretches, v (AuCl), and (OAuO) and (CuAuO) is a clear evidence of the formation of a complex between AuCl.sub.2.sub. and Cu and PEG diacid in solution. The common peak at 430 cm.sup.1 is due to the vibrations (OH . . . O), v(OH . . . O) of the PEG. The bands exhibited in the region 3000 cm.sup.1 can be assigned to aromatic CH stretching (FIG. 13). However, the vibrational frequencies exhibited at 2946-2952 cm.sup.1, are considered to be due to CH stretching vibration of the compounds. A broad band composed of several peaks appears in the spectral range 2850-2930 cm.sup.1 is due to the symmetric CH.sub.2CH.sub.3 stretch vibration of PEG-diacid molecules confirming the main role of the polymer in the synthesis of the nanoparticle.

[0239] Mechanism of AuCu Complex in the Growth of Hybrid Pegylated Nanoparticles

[0240] The formation of complex AuCu in the growth of Nanoparticles was monitored by UV-Vis and Raman.

[0241] FIG. 14 showed the UV-Vis fingerprint of a solution of HAuCl.sub.4, CuCl.sub.2 and the mixed of them (HAuCl.sub.4+CuCl.sub.2).

[0242] The UV-Vis Spectra showed a typical spectra of HAuCl.sub.4 with two prominent peaks at 256 nm and 290 nm (full line in the FIG. 14).

[0243] The UV-Vis spectra of CuCl.sub.2 solution showed a peak at 256 nm, a small peak at 280 nm and a broadened peak at 800 nm (dashed line in the FIG. 14).

[0244] When CuCl.sub.2 was added to HAuCl.sub.4 solutions, a chemical-physical modification appeared, showed in the UV Vis Spectra (dot-dashed line in FIG. 14).

[0245] The principal optical modification in the spectra is due to the increase and shift of the peak from 280 nm to 320 nm due to the electronic transition. The dramatic decrease of the peat at 800 nm, confirm that the hybrid system (AuCu) was done.

[0246] FIGS. 15a and 15b show LSP resonance spectra before and after incubation of HAuCl.sub.4 mixed to CuCl.sub.2 under specific conditions (pH: 4.0-7.0-9.0; time: 96 h).

[0247] At pH 4, we observe an increase of the peak intensity at 256 nm probably due to CuCl.sub.2 fingerprint associated to AuCl.sub.2 ions upon complexation was observed. The increase of the peak at 800 nm, confirming the reaction under acidic conditions.

[0248] A different behavior is observed in the case of pH 7 and pH 9 (FIG. 15 b), in which after incubation at pH 7 and pH 9 for 96 h, we observe a disappearance of LSP bands. This spectroscopical behavior during pH-release gives evidence of the change of conformation of the reagents when it is encapsulated into gold nanoparticles.

[0249] The complex CuAuCl was confirmed by Raman Spectroscopy (FIG. 16), the peak at 254 cm-1 due to CuAu Cl and CuOH stretching confirm the successful of reaction

[0250] Similar experimental results were confirmed at different pH (FIG. 17), in order to understand the chemical behavior of Cu and Au during complex formation in the synthesis of final nanoparticle.