NANOPARTICLES AND PREPARATION METHOD

Abstract

The present invention relates to nanoparticles of a metal chosen from among platinum, bismuth or a mixture thereof, which are functionalised at their surface by functionalised polyethylene glycol, comprising in particular at least one OH, COOH, NH.sub.2 or SH functional group, and to their method of preparation, which comprises of the following steps: a) Mixing a precursor of nanoparticles with a functionalised polyethylene glycol, in particular comprising at least one OH, COOH, NH.sub.2 or SH functional group, in water; b) Exposing the mixture to ionising radiation.

Claims

1. Nanoparticles of a metal chosen from among platinum, gold or bismuth, or one of the mixtures thereof, which are functionalised at their surface with functionalised polyethylene glycol.

2. A nanoparticle preparation method for preparing nanoparticles of a metal chosen from among platinum, gold or bismuth, or a mixture thereof, the method comprising: a) mixing a precursor of nanoparticles with a functionalised polyethylene glycol comprising at least one OH, COOH, NH.sub.2 or SH functional group, in water; and b) exposing the mixture to ionizing radiation.

3. A method according to claim 2, wherein the ionizing radiation is chosen from gamma radiation deriving from a source of Cobalt 60 or Cesium 137, electrons or accelerated ions.

4. A method according to claim 2, i wherein the step of exposing extends over a period of time between 1 and 20 hours for a dose of 10 kGy applied with a source of gamma radiation from a Co.sup.60 source having a dose rate of about 95.5 Gy/min in order to completely reduce 10.sup.−3 mol/L.sup.−1 of platinum complex.

5. A method according to claim 2, wherein the functionalised polyethylene is chosen from among Hydroxyl poly(ethylene glycol) [PEG-OH], Poly(ethylene glycol) diamine [PEG-2NH.sub.2], α-hydroxyl-ω-carboxyl poly(ethylene glycol) [HO-PEG-COOH], and PEG-thiol [PEG-SH].

6. A method according to claim 2, wherein the precursor is used in implementation in an amount ranging from 10.sup.−4 to 10.sup.−2 mol/L, in terms of molar concentration of the total mixture of step a) and/or the functionalised polyethylene glycol is used in implementation in an amount ranging from 10.sup.−3 to 10.sup.−1 mol/L, in terms of molar concentration of the total mixture of step a).

7. A method according to claim 2, wherein the molar ratio between the precursor and the functionalised polyethylene glycol is between 10 and 100.

8. A method according to claim 2, that is carried out in the absence of any solvent other than water.

9. (canceled)

10. (canceled)

11. The method according to claim 4, wherein the period of time is between 1 and 2 hours.

12. A nanoparticle according to claim 1, wherein the functionalised polyethylenes are chosen from among Hydroxyl poly(ethylene glycol) [PEG-OH], Poly(ethylene glycol) diamine [PEG-2NH.sub.2], α-hydroxyl-ω-carboxyl poly(ethylene glycol) [HO-PEG-COOH], and PEG-thiol [PEG-SH].

13. Method for the treatment of cancers and tumours and/or for amplifying the effects of medical radiation used for the treatment of cancers or tumours, comprising administering to a patient in need thereof a therapeutically effective amount of a nanoparticle according to claim 1.

14. Method for the treatment of cancers and tumours and/or for amplifying the effects of medical radiation used for the treatment of cancers or tumours, comprising administering to a patient in need thereof a therapeutically effective amount of a nanoparticle obtainable by the method of claim 2.

15. Method for medical imaging for diagnostics in a subject in need thereof, comprising administering the nanoparticle of claim 1 to the subject, and performing the medical imaging on the subject.

16. Method for medical imaging for diagnostics in a subject in need thereof, comprising administering to the subject a nanoparticle obtainable by the method of claim 2, and performing the medical imaging on the subject.

17. The method of claim 15, wherein the medical imaging is performed by computed tomography (CT).

18. The method of claim 15, wherein the medical imaging is performed by computed tomography (CT).

19. The nanoparticles of claim 1, wherein the functionalised polyethylene glycol comprises at least one OH, COOH, NH.sub.2 or SH functional group.

Description

[0045] FIG. 1 is an image of the platinum nanoparticles functionalised by PEG-OH as viewed by High Resolution Transmission Electron Microscopy (HRTEM).

[0046] FIG. 2 is an image of the platinum nanoparticles functionalised by PEG-2NH2 as viewed by High Resolution Transmission Electron Microscopy (HRTEM).

[0047] FIG. 3 shows the internalisations of particles by the HeLa cells.

[0048] FIG. 4 shows the mitotic survival of HeLa cells in the presence or absence of platinum nanoparticles (PtNPs) (6 hours of incubation at 0.5 mM and 1 mM).

[0049] FIG. 5 shows the survival of HeLa cells treated with radiation of C.sup.6+ ions or Cs-137 gamma photons, with and without platinum nanoparticles.

[0050] FIG. 6 shows an image of the platinum and bismuth nanoparticles functionalised by PEG-2NH.sub.2 as viewed by High Resolution Transmission Electron Microscopy (HRTEM).

EXAMPLE 1: PREPARATION OF THE NANOPARTICLES

[0051] The platinum nanoparticles (PtNPs) are synthesised by using a platinum precursor, tetraamineplatinum(II) chloride (Pt(NH.sub.3).sub.4Cl.sub.2, 2H.sub.2O). This precursor is diluted in water in a proportion of 50 mg in 10 mL. A sample of this solution (6.67 mL) is mixed with 2 mL of polyethylene glycol 1000 (PEG-OH) (5 M) or polyethylene diamine 2000 (PEG-2NH.sub.2), the mixture is diluted in water (1.33 mL). This solution is degassed under nitrogen. It is then exposed for a period of approximately 17.5 hours, to radiation deriving from a cobalt 60 gamma source (dose rate: 95.5 Gy/min). This results in a colloidal solution that is black in colour, sterile, and composed exclusively of homogeneous platinum nanoparticles functionalised with PEG (—OH or -2NH.sub.2).

[0052] In the case of PEG-OH, the platinum core has a spherical shaped form and with a diameter equal to 3.2 nm (FIG. 1). In the case of PEG-2NH.sub.2, aggregates are obtained in flower-shaped forms, that measure 14.6 nm in size and contain nanoparticles (NPs) measuring 3.2 nm in diameter (FIG. 2).

[0053] The hydrodynamic diameter of the nanoparticles is obtained by means of Dynamic Light Scattering (DLS) measurements. It is 8.8 nm in the case of PEG-OH and 16.1 nm in the case of PEG-2NH.sub.2. The platinum nanoparticles functionalised with PEG-OH have a mean zeta potential of −17 mV. Additional measurements by X-ray Photoelectron Spectrometry (XPS) confirmed the absence of platinum precursor in the solution of platinum nanoparticles (PtNPs) and shows that all of the platinum has been reduced in its entirety (zero oxidation number), which proves that this method of synthesis provides a yield of 100%.

[0054] The bismuth-platinum nanoparticles (Bi/PtNPs) are synthesised by using a platinum precursor, tetraamineplatinum(II) chloride (Pt(NH.sub.3).sub.4Cl.sub.2, 2H.sub.2O) and a bismuth precursor, Ammonium Bismuth Citrate (ABC, C.sub.6H.sub.8BiNO.sub.7). The platinum precursor is diluted in water in a proportion of 50 mg in 10 mL, while the bismuth precursor is diluted in water in a proportion of 41.5 mg in 10 mL.

[0055] A sample of the platinum solution (1.38 mL) is mixed with a sample of the bismuth solution (0.5 mL), and with 2.5 mL of 100 mM polyethylene diamine 2000 (PEG-2NH.sub.2); thereafter this mixture is diluted in water (1.3 mL). A solution is obtained with a Bi/Pt atomic ratio of 1:3.7 and a Bi/PEG-2NH.sub.2 ratio of 1:50 mol/mol %.

[0056] This solution is degassed under nitrogen. It is then exposed for a period of approximately 4 hours to radiation deriving from a cobalt 60 gamma source (dose rate: 37 Gy/min). This results in a colloidal solution that is black in colour, sterile, and composed exclusively of homogeneous Bismuth-Platinum nanoparticles functionalised with PEG-2NH.sub.2. According to the TEM images, the platinum core has a spherical shaped form and a diameter equal to 21.2+/−12.9 nm.

[0057] The hydrodynamic diameter of the nanoparticles, which is obtained by means of Dynamic Light Scattering (DLS) measurements, is 35 nm. Additional measurements by X-ray Photoelectron Spectrometry (XPS) confirmed the absence of platinum and bismuth precursor in the solution of nanoparticles, given that it shows that all of the metals have been reduced in their entirety (zero oxidation number), which proves that this method of synthesis provides a yield of 100%.

Example 2: Implementation and Use of the Nanoparticles

[0058] After incubation for a period of 6 hours of the cells (HeLa) with a solution of PtNPs functionalised with PEG-OH containing 0.5 mM of platinum (obtained in Example 1), the latter are internalised by HeLa cells (FIG. 3) at a rate of 1.6 pg per cell, which corresponds to 49×10.sup.5 PtNPs per cell (quantification carried out by Inductively Coupled Plasma Mass Spectrometry or ICP-MS).

[0059] The PtNPs functionalised with PEG-OH or PEG-2NH.sub.2 exhibit a low level of cytotoxicity on the human cell lines. In fact, the HeLa cells incubated for a period of 6 hours with a solution of PtNPs (platinum nanoparticles) containing 0.5 mM or 1 mM of platinum, respectively, exhibit low mitotic death (<10%+/−5%) (FIG. 4).

[0060] When the nanoparticles are activated by ionising radiation (gamma radiation from Cobalt 60 or Cesium 137, X-rays, or indeed even medical radiation of carbon ions as used in hadrontherapy), they exhibit the property of amplifying molecular damage and radiation-induced cell death. For example, preliminary studies on nano-bioprobes show that the presence of platinum nanoparticles functionalised with PEG-OH amplifies by a factor of 2 the number of instances of molecular damage of nanometric dimensions induced by ion irradiation.

[0061] Studies on human tumour cells (HeLa) show that the presence of platinum nanoparticles functionalised with PEG-OH amplifies the radiotoxicity (cell death) of a medical radiation beam such as carbon ions or gamma photons. In fact, when HeLa cells are incubated for a period of 6 hours with a solution of PtNPs containing 0.5 mM of platinum, they exhibit cell death (as measured by clonogenic survival) induced by irradiation (by accelerated carbon ions or gamma photons from Cs 137), at a greater level than the cell death in the control cells (not comprising NPs) (FIG. 5).

Example 3: Implementation and Use of the Nanoparticles

[0062] In vivo experiments on healthy mice were carried out by injecting intravenously, into the tail of the mouse, 200 μl of PEG-OH functionalised nanoparticles with 10 g/l of Pt obtained according to the protocol of I Example 1, lyophilised and re-suspended in sterile water. A dose of 0.1 g of nanoparticles per kg were injected into the mice.

[0063] The tests did not show any toxicity thus confirming the biocompatibility of this product, a slight enhancement of the contrast in computed tomography (CT) was clearly evidenced in the bladder. This enhancement shows that if the nanoparticles get accumulated in the bladder, it signifies that the renal system is capable of excreting the nanoparticles that would not have been internalised by the tumour, and which would thus lead to little to no accumulation in healthy organs.

Example 4: Functionalisation of the Nanoparticles with Rhodamine

[0064] The platinum nanoparticles (PtNPs) are synthesised as described in Example 1 by using a platinum precursor, tetraamineplatinum(II) chloride (Pt(NH.sub.3).sub.4Cl.sub.2, 2H.sub.2O), and polyethylene glycol diamine 2000 (PEG-2NH.sub.2). Thereafter, these NPs have grafted on to them, a fluorescent marker or label: rhodamine B isothiocyanate or RB ITC (C.sub.29H.sub.30ClN.sub.3O.sub.3S). Thus, the amine functional group (NH.sub.2) of the PEG reacts with the isothiocyanate of the marker (formation of thiourea). For this, it is necessary to prepare a mixture in water of 4 ml containing these nanoparticles at a concentration of 5 mM of platinum and containing the RB ITC at a concentration of 0.5 mM. This mixture is then agitated at ambient temperature for a period of 24 hours. The labelled nanoparticles that are obtained are then ultrafiltered in water by centrifugation until such time as all the free marker is removed and only labelled nanoparticles are remaining in the solution, this is followed by UV-visible spectrophotometry. The rinsing by ultrafiltration is continued until such time as no more change occurs in the intensity of this spectrum; in fact it diminishes when the free marker is removed, when there is no more of it and the spectrum remains unchanged in intensity, this signifies that there is no longer any marker other than the marker bound to the labelled nanoparticles. In addition, the fluorescent marker has a very specific absorption spectrum. In the case of the labelled nanoparticles, the absorption peak is at 580 nm, therefore slightly offset from that of the marker alone, which is at 585 nm, which thus demonstrates the formation of a bond between the label and the nanoparticle.

[0065] This labelling makes it possible to follow the nanoparticles in the cells by fluorescence microscopy such as confocal microscopy for example.