Use of nanodiamonds for generating free radicals for therapeutic purposes under radiation

Abstract

The present invention relates to the use of nanodiamonds as drugs generating free radicals, in particular for treating tumors. The invention is based on generating free radicals on the surface of the nanodiamonds when they are exposed to radiation, for example ionizing radiation. In order to increase the effectiveness of the nanodiamonds, the nanodiamonds can be complexed with a radiosensitizing agent, such as a chemical molecule or an interfering RNA targeting a repairing gene.

Claims

1. A method of generating free radicals for therapeutic purposes comprising a step of exposing nanodiamonds to radiation, wherein the nanodiamonds are beforehand subjected to a graphitization, or a graphitization and a hydrogenation to have a surface that has been at least partially graphitized, or graphitized and hydrogenated, respectively.

2. The method as claimed in claim 1, wherein the generation of free radicals is coupled to heat generation.

3. The method as claimed in claim 1, wherein the nanodiamond has an average diameter that is less than 10 nm.

4. The method as claimed in claim 1, wherein oxygen-containing free radicals are generated.

5. The method as claimed in claim 1, wherein nitrogenous free radicals are generated.

6. The method as claimed in claim 1, wherein the radiation is electromagnetic radiation.

7. The method as claimed in claim 6, wherein the electromagnetic radiation consists of X-rays.

8. The method as claimed in claim 6, wherein the electromagnetic radiation consists of gamma-rays.

9. The method as claimed in claim 6, wherein the electromagnetic radiation consists of ultraviolet rays.

10. The method as claimed in claim 1, wherein the radiation is particulate radiation.

11. The method as claimed in claim 10, wherein the particulate radiation consists of protons.

12. The method as claimed in claim 10, wherein the particulate radiation consists of hadrons.

13. The method as claimed in claim 1, for use as a medicament intended for the destruction of target cells.

14. The method as claimed in claim 13, the target cells are cancer cells.

15. The method as claimed in claim 1 for use in treating a solid tumor.

16. The method as claimed in claim 1, wherein the nanodiamond is functionalized.

17. The method as claimed in claim 16, wherein the nanodiamond is bonded to a targeting molecule.

18. The method as claimed in claim 17, wherein the targeting molecule is a biological ligand recognized by a receptor overexpressed at the surface of certain cells.

19. The method as claimed in claim 18, wherein the biological ligand is chosen from the group consisting of a peptide, a protein, an antibody, a sugar, an oligonucleotide, an organic molecule, and an organometallic complex.

20. The method as claimed in claim 19, wherein the biological ligand is chosen from the group consisting of peptides comprising the RGD motif or the NGR motif.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following examples and the appended figures illustrate the invention without, however, limiting the scope thereof.

Figure Legends

(2) FIG. 1: Diagram of the scheme for hydrogenation of the nanodiamonds (NDs) by microwave-assisted hydrogen (H.sub.2) plasma.

(3) FIG. 2: High-resolution transmission electron microscopy (HRTEM) images of initial NDs (a), and NDs after 1 h (b) and 8h (c) of annealing under vacuum at 750 C. The diamond planes (111) and graphitic planes (001) are indicated by the white and gray lines, respectively. The graphitic surface reconstructions are indicated by white arrows. The scale bar is 5 nm.

(4) FIG. 3: X-ray photoelectron spectroscopy (XPS) spectra of the carbon core level (C1s) of the initial NDs (ND-initial), and NDs after 1 h (ND-1 h), 5 h (ND-5 h) and 8 h (ND-8 h) of annealing under vacuum at 750 C.

(5) FIG. 4: Evolution of the Zeta potential of the ND-1 h, ND-5 h, ND-8 h and hydrogenated NDs (ND-H) in ultrapure water as a function of the pH.

(6) FIG. 5: Size distribution of the proteins of the DMEM medium+10% fetal calf serum (FCS) (black), and of the NDs-5 h in deionized water (blue) and in MEM just after addition (green) and after 5 h (red). The measurements are carried out at 37 C.

(7) FIG. 6: HRTEM image of nanodiamonds which are hydrogenated (a) and graphitized at the surface (b). The planes (111) of the diamond are highlighted in the plane and the surface graphitic reconstructions are indicated by the white arrows. The scale bar is 5 nm.

(8) FIG. 7: XPS spectra of the carbon core level (C1s) of hydrogenated nanodiamonds after dispersion in water (a) and after 1 hour of annealing under vacuum at 400 C. (b).

(9) FIG. 8: Fourier transform infrared (FTIR) spectroscopy spectra of initial NDs (ND-initial), and NDs-G after 1 h (ND-1 h) and 8 h (ND-8 h) of annealing under vacuum.

(10) FIG. 9: Cell response of the Caki-1 line after exposure to NDs-COOH without irradiation.

(11) FIG. 10: Cell response of the Caki-1 line after exposure to NDs-COOH after an irradiation of 4 Gy.

(12) FIG. 11: Cell response of the Caki-1 line after exposure to NDs-H without irradiation.

(13) FIG. 12: Cell response of the Caki-1 line after exposure to NDs-H after an irradiation of 4 Gy.

(14) FIG. 13: Evolution of the Caki-1 cells after exposure to NDs-COOH and NDs-H, without irradiation.

(15) FIG. 14: Evolution of the Caki-1 cells after exposure to NDs-COOH and NDs-H, after an irradiation of 4 Gy.

(16) FIG. 15: Oxidative stress induced by the NDs-H with or without irradiation. The control without probe illustrates the background of luminescence which is not linked to the free radicals. The other control is exposed to the fluorescent probe but not to the NDs-H. The gray bars represent the intensity of the oxidative stress in the nonirradiated cells, and the white bars the intensity of the oxidative stress in the cells having undergone a radiation of 4 Gy, one hour after this irradiation.

(17) FIG. 16: Internalization of a peptide nucleic acid probe labeled with the fluorophore Cy3 (PNA-Cy3), adsorbed at the surface of ND-H. The observations were made after 24 h (A) or 72 h (B) of incubation of Caki cells in the presence of an ND-H/PNA-Cy3 mixture. The cell nuclei were labeled with bisbenzimide Hoechst 33342.

(18) FIG. 17: Diamond nanoparticle/radiosensitizing agent complex.

(19) FIG. 18: Operating principle. a) Incorporation of the nanodiamond (NP)/radiosensitizing agent complex into a cell, b) release of the radiosensitizing agents and inhibition of the defenses of the cell, c) generation of electrons and formation of free radicals under irradiation.

DETAILED DESCRIPTION

Examples

Example 1: Production of Nanodiamonds (NDs) Having Graphitic Surface Reconstructions or Surface Hydrogenated Functions

(20) The NDs having properties that are of use for the radiosensitization of tumor cells were modified using particular treatments allowing the formation of graphitic reconstructions (graphitization) or of hydrogenated functions of CH.sub.x type with x=1, 2 or 3 (hydrogenation). NDs comprising a combination of these two types of surface end groups can also be used in the context of the present invention. The methods described below are those used by the inventors for the hydrogenation (by microwave-assisted hydrogen plasma) and the graphitization (by annealing under vacuum at high temperature or by exposure to microwaves, under vacuum, of hydrogenated NDs), but these particular surface end groups can, a priori, also be obtained by other methods. It should be noted that the conditions set out are to be adapted according to the initial surface chemistry of the NDs, which can vary from one nanodiamond supplier to another. The treatments described herein were optimized for detonation NDs produced by the Nanocarbon Institute in Japan (Professor Eiji Osawa).

(21) 1.1. Hydrogenation by Microwave-Assisted Hydrogen Plasma

(22) 1.1.1. Procedure

(23) The method used to confer hydrogenated end groups on the NDs is described in the reference Girard et al., 2010. The NDs (approximately 50-100 mg) are introduced, via the dry route, into a quartz cartridge, or else directly into a quartz tube, which is inserted perpendicularly into a waveguide connected to a 2.45 GHz microwave generator (Sairem), as represented in FIG. 1. The waveguide is cooled with water and the tube is cooled with compressed air. This tube is connected to a device for primary pumping and for supplying high purity N9.0 hydrogen and argon gas.

(24) Firstly, a series of purges are carried out via primary pumping in the tube (pressure<0.1 mbar) and repressurization with high purity hydrogen, then the high purity hydrogen is injected until a pressure stabilized at 12 mbar is reached. This pressure is either maintained throughout the hydrogenation process by isolation of the tube (static mode), or maintained by the combination of a continuous stream of hydrogen and a valve for pressure regulation under instruction (dynamic mode). A microwave power of 300 W is used to induce the creation of a plasma in the tube. The geometry of the microwaves in the waveguide is adjusted so as to obtain a maximum power absorbed by the plasma and a zero reflected power at the level of the generator. The tube is regularly manually turned and moved translationally in order to ensure that the majority of the NDs are exposed to the plasma. The normal exposure time is 20-30 min. In order to obtain complete hydrogenation, it is important to perform a purge after 5 min of treatment in order to discharge oxidized species desorbed from the surface of the NDs; after interruption of the microwaves, the tube undergoes primary vacuum pumping, and then pure hydrogen is reintroduced into the tube in order to again initiate the formation of a plasma. This intermediate purge is not needed in the case of a hydrogenation under a dynamic hydrogen stream. At the end of the treatment, the tube is cooled under hydrogen until it is at ambient temperature, and then the residual gas is pumped. The tube is placed at ambient temperature again by introducing argon, then the NDs can be recovered.

(25) 1.1.2. Characterization

(26) Detailed characterizations of the surface properties of the hydrogenated NDs prepared in this way have been published (Girard et al., 2010; Girard et al., 2011; Arnault et al., 2011). The surface chemistry is studied therein by electron (XPS), infrared (FTIR) and Raman spectroscopies. In addition, three graftings, the selectivity of which on hydrogenated diamond films is known, were applied to these hydrogenated nanodiamonds; an equivalent selectivity with respect to the presence of the hydrogenated end groups of the nanodiamonds was demonstrated. This shows in particular that these NDs have negative electron affinity properties (Girard et al., 2011). These properties are responsible for their use to generate free radicals in water (see example 2).

(27) 1.2. Graphitization of Hydrogenated NDs by Microwave Exposure

(28) The NDs hydrogenated according to the process described above can be graphitized following their hydrogenation, in situ, by simple reexposure to microwaves under primary vacuum. This is because inventors have observed that the hydrogenated NDs have the ability to absorb microwaves under vacuum. Thus, by adjusting the geometry of the microwave cavity, most of the microwave power (the inventors used 300 W for 100 mg of NDs) is absorbed by the NDs and is converted into heat. An exposure of a few seconds is sufficient to allow a very rapid increase in the temperature of the NDs, inducing the formation of surface graphitic reconstructions, as occurs in a conventional graphitization process by high-temperature annealing (see below). An exposure of more than one minute, on the other hand, results in the formation of entirely graphitic nanoparticles where the diamond core has completely disappeared. This method can be an alternative to high-temperature annealings under vacuum, the experimental protocol of which is described in detail in the section which follows.

(29) 1.3. Surface Graphitization of the Nanodiamonds by Annealing Under Vacuum

(30) 1.3.1. Procedure

(31) The surface of the nanodiamonds can be graphitized by annealing under vacuum at high temperature (between 700 C. and 900 C.) (Petit et al., 2011). These annealings under vacuum are carried out in a dedicated metal-walled chamber equipped with a silicon carbide heating element which makes it possible to achieve temperatures above 1000 C. and a combined system of primary and turbomolecular pumping which makes it possible to obtain a secondary vacuum in the chamber (of about 10.sup.7 mbar).

(32) Between 50 and 100 mg of dry-route NDs are placed in an alumina crucible with a lid made of the same material, which is then placed on the heating element inside the chamber. During the annealing, the temperature of the crucible is measured using an infrared camera (FLIR SC300) precalibrated according to the emissivity of the crucible, while the temperature of the heating element is estimated with a thermocouple. The chamber is then pumped at ambient temperature until a pressure of less than 510.sup.7 mbar is obtained, then the temperature of the heating element is gradually increased up to 1000 C. (corresponding to 750 C. for the crucible), while maintaining the pressure in the chamber below 510.sup.6 mbar. Once the temperature has stabilized, the crucible is left at constant temperature for a predetermined time, then the temperature of the heating element is gradually reduced to ambient temperature. The crucible is then cooled under vacuum. Once it has been brought back to ambient temperature, the chamber of the reactor is again placed under atmospheric pressure under air, making it possible to remove the crucible. The NDs can then be recovered so as to be resuspended.

(33) 1.3.2. Characterizations

(34) Typically, an annealing at 750 C. for one hour is sufficient to obtain the formation of surface graphitic reconstructions, but longer annealings can be used to increase the degree of coverage of the surface with these graphitic reconstructions. Temperatures above 900 C. induce graphitization of the diamond core, limited graphitization at the surface of the NDs is therefore difficult to control above 900 C.

(35) The graphitization of the NDs is validated by high-resolution transmission electronmicroscopy (HRTEM) after 1 h and 8 h of annealing under vacuum at 750 C., corresponding to the temperature of the crucible (FIG. 2). The images make it possible to observe the modifications of the atomic structure that are induced by the annealings. This graphitization is also validated by the analysis of the surface chemistry by X-ray photoelectron spectroscopy (XPS). Indeed, a component bonded to the sp.sup.2 hybridized carbon appears after annealing under vacuum at low bonding energy compared with the sp.sup.3 hybridized carbon (FIG. 3).

Example 2: Suspending of the Modified NDs in Water

(36) The hydrogenated and/or graphitized NDs are then placed in colloidal suspension in ultrapure water (18.2 M.Math.cm at 25 C.) using a 300 W sonification immersion probe (Hielscher UP400S) operating at a frequency of 24 kHz. The NDs are initially placed in a solution of ultrapure water at a concentration of about 5 to 10 mg/ml and are then exposed to ultrasound for a minimum of 2 h. Following the sonification process, and in order to separate the largest nondispersible aggregates from the suspension, the suspensions are centrifuged at 4800 rpm for 1 h. Only the supernatant is recovered. The hydrodynamic diameter of the NDs in suspension is measured by dynamic light scattering (DLS) using dedicated equipment. The measurement of the Zeta potential characteristic of the surface charge of the nanodiamonds in solution is carried out on the same equipment (Nanosizer ZS, Malvern) with an added automatic titration module (MPT-2, Malvern) in order to carry out measurements as a function of the pH.

(37) The resulting suspensions consist of aggregates of NDs of which the hydrodynamic diameter is less than 50 nm and which have a positive Zeta potential in ultrapure water over a wide pH range, as indicated for NDs annealed under vacuum for 1 h (ND-1 h), 5 h (ND-5 h) and 8 h (ND-8 h) in FIG. 4. A similar evolution of the surface charge is observed on the hydrogenated NDs. In particular, the high Zeta potential at physiological pH makes it possible to ensure good colloidal stability of the modified NDs in this pH range.

(38) These NDs are stable for several months in water, but also in biological medium, as illustrated by the evolution in the hydrodynamic diameter of the NDs-5 h in a medium consisting of MEM (minimum essential medium) and 10% fetal calf serum, measured by DLS (FIG. 5). After more than 6 months in water, an average diameter of 35 nm is detected for the NDs-5 h. After addition to the [MEM+serum] medium at a concentration of approximately 0.5 mg/ml, the diameter increases to 144 nm, which is attributed to the adsorption of negatively charged serum proteins on the positive surface of the NDs. After incubation for 5 h at 37 C., the diameter is reduced to 121 nm, which shows that there is no significant effect of aggregation over time in biological medium.

Example 3: Generation of Free Radicals from the Hydrogenated (NDs-H)/Graphitized (NDs-G) Nanodiamonds (NDs)

(39) The effect of amplification of the generation of free radicals in the vicinity of the NDs-H/G is based on two physical properties: the high density of carbon atoms (about 10 000 atoms for a nanodiamond 5 nm in diameter) in the NDs, making it possible to efficiently absorb radiation, and their ability to efficiently transfer the electrons from the diamond core to oxygen-containing species attached at the periphery of the NDs.

(40) The absorption of ionizing radiation is much greater in the NDs than in the surrounding tissues because of the high atomic density of diamond (1.810.sup.23 at.Math.cm.sup.3). Indeed, the distance between two atomic planes of orientation (111) of the diamond mesh is 0.206 nm, as illustrated by the high-resolution transmission electron microscopy (HRTEM) image presented in FIG. 6. Under radiation, a high concentration of secondary electrons and photoelectrons is created and they are released locally at the surface of the NDs. Indeed, the surface of the NDs-H and NDs-G behaves respectively like the surface of hydrogenated diamond films or that of a graphene plane. These two surfaces are known to allow very efficient electron transfer to surface-adsorbed molecules (Chakrapani et al., 2007; Ryu et al., 2010).

(41) In parallel, the NDs-H and NDs-G have the possibility of efficiently adsorbing oxygen-containing species at their surface. Thus, a high concentration of oxygen was measured at the surface of the NDs-H and NDs-G, representing up to 6 atm. % according to the XPS spectra, after dispersion in ultrapure water. The oxygen comes from adsorption, via noncovalent bonds, of water (H.sub.2O) and dioxygen (O.sub.2) molecules and also from single-bond CO covalent bonds which can be bonded to hydroxyl, ether, epoxide or endoperoxide functions. This oxygen, covalently bonded to the surface of the NDs, is characterized by the presence of a high-energy shoulder of bonding on the C1s carbon core level spectra using X-ray electron spectroscopy (XPS) presented in FIG. 7a. On the other hand, this oxygen is weakly bonded since annealing under vacuum at 400 C. makes it possible to desorb most of this oxygen (FIG. 7b). Using infrared spectroscopy (FTIR), after desorption of the species adsorbed noncovalently by annealing under vacuum at 200 C., a significant band at 1100 cm.sup.1 was observed, which may be linked to functions of ether, epoxide or endoperoxide type (FIG. 8), validating the results obtained by XPS.

(42) Thus, the electrons generated by irradiation are transferred to these molecules adsorbed onto the surface of the NDs. Since these molecules are precursors of oxygen-containing free radicals (O.sub.2, HO, H.sub.2O.sub.2, etc.), the transfer of electrons coming from the NDs induces a strong production of free radicals at the surface of the NDs. It should be noted that nitrogen was also measured by XPS; it is therefore possible that nitrogenous molecules are also adsorbed at the surface of the NDs, implying the generation of nitrogenous free radicals.

(43) The adsorption of oxygen on the surface induces a positive Zeta potential of the NDs-H and NDs-G, ensuring good colloidal stability by electrostatic stabilization, even in biological medium. The biological environment which contains the NDs is therefore directly exposed to the free radicals generated at the surface of the NDs.

Example 4: Cell Index and Oxidative Stress Measured in the Caki-1 Tumor Line, Under Gamma-Irradiation in the Presence of NDs-COOH and NDs-H

(44) The radiosensitizing effect of the nanodiamonds was studied on a kidney tumor line Caki-1, known to be particularly radioresistant. Cells exposed to NDs-COOH and NDs-H at three concentrations (10, 100 and 500 g/ml), and also cells without NDs, were subjected to a radiation of 4 Gray (Gy).

(45) The evolution of the cell index, characteristic of the overall response of the cells (morphology, adhesion, viability, etc.), was monitored in real time over the course of 120 h after irradiation by impedancemetry using the xCELLigence system (Roche).

(46) The oxidative stress was then evaluated by observing the cells by optical microscopy and quantified by flow cytometry.

(47) 4.1. Results on the NDs-COOH

(48) After exposure to the NDs-COOH, the cell index evolves in an equivalent manner up to 48 h for the concentrations of 10 and 100 g/ml (FIG. 9). A very small decrease is observed at 100 g/ml for longer times. On the other hand, the cell index is greatly decreased for the 500 g/ml concentration.

(49) These results show that the NDs-COOH are not toxic for concentrations below 100 g/ml, but that a certain toxicity can be observed at higher concentration. The toxicity is therefore dose-dependent.

(50) After irradiation of 4 Gy the increase in the cell index of the control shows that this irradiation is too weak to create significant toxicity without nanoparticles (FIG. 10). On the other hand, the cell index is halved compared with the control after an exposure to the NDs-COOH at a concentration of 10 g/ml, or even further reduced for the higher concentrations.

(51) The NDs-COOH therefore clearly have a radiosensitizing effect, which is dependent on the dose of NDs-COOH injected into the cells. Furthermore, these NDs are not toxic at concentrations below 100 g/ml.

(52) 4.2. Results on the NDs-H

(53) The same protocol was applied with NDs-H (FIGS. 11 and 12). It should be noted that the toxicity of the NDs-H is even lower than the NDs-COOH since no toxicity is detected even for the concentration of 500 g/ml, which would be reflected by a decrease in the cell index. The decrease observed after 90 h is probably due to a saturation of the signal detected by impedancemetry, due to the high concentration of NDs-H used. A significant increase in the cell index is on the other hand observed, which may result, for example, from an increase in cell size after incorporation of the NDs-H.

(54) After irradiation, the control follows the same increase as in the previous case. On the other hand, with the presence of NDs-H, the toxicity is very significant. The cell index is thus divided by 3.4 for a concentration of 10 g/ml. The toxicity does not appear to be dependent on the dose of NDs-H since a similar evolution of the cell index is observed at higher concentrations. This result is coherent with toxicity induced by an oxidative stress at a very low concentration of NDs-H, only under irradiation.

(55) 4.3. Results Linked to Oxidative Stress

(56) Firstly, the oxidative stress was evaluated by observing the morphology of the cells by optical microscopy.

(57) There is no particular evolution at the level of the control cells without/with irradiation. After the addition of nanodiamonds, the formation of vacuoles (appearing with a blue contrast), characteristic of toxicity induced by oxidative stress, is observed (FIG. 13). The number of vacuoles increases over time.

(58) The concentration of vacuoles significantly increases after irradiation in the cells exposed to the NDs (FIG. 14), which is in agreement with the previous results.

(59) Quantitative oxidative stress measurements were carried out by measuring the fluorescence of a probe sensitive to oxygen-containing free radicals (2, 7-dichlorofluorescein) by flow cytometry. Once exposed to the various experimental conditions (NDs-H, irradiation, NDs-H+irradiation), the cells were detached from their culture support, resuspended, and then incubated for 10 minutes in the presence of this probe. Once it has entered the cells, the probe can remain in nonfluorescent reduced form or can become oxidized and therefore emit a fluorescent signal. The intensity of fluorescence is directly linked to the amount of oxygen-containing free radicals, which allows a relative quantification regarding oxidative stress (Chen et al., 2010). This method makes it possible to measure total intracellular oxygen-containing free radicals, contrary to the measurement of oxidized proteins for example.

(60) The results obtained in FIG. 15 show that: For the cells exposed to the NDs-H and not irradiated, the oxidative stress induced depends on the concentration. It is doubled for an NDs-H concentration of 100 g/ml. For the cells irradiated and not exposed to the NDs-H, the oxidative stress is, at 1 h after irradiation, identical to that of the cells not irradiated and not exposed to the NDs-H. For the cells exposed to the nanoparticles and irradiated, the oxidative stress is tripled compared with the reference without NDs-H, but this increase is not dependent on the dose of NDs-H.

(61) There is therefore a provision of free radicals in the cells after incorporation of the NDs-H, but these free radicals induce significant toxicity due to oxidative stress only after irradiation according to the evolution of the cell index. The generation of free radicals is maintained by the NDs-H since, even one hour after irradiation, the oxidative stress is higher than without irradiation, which is not the case with the control.

(62) 4.4. Conclusions

(63) The nanodiamonds therefore have a radiosensitizing effect which makes it possible to amplify the effect of the radiation by generating a greater creation of free radicals. Simple exposure to a dose that is normally insufficient to induce the death of tumor cells makes it possible to obtain death due to oxidative stress when the cells have been preexposed to the NDs. The cells exposed to the NDs can therefore be selectively treated. The NDs are particularly advantageous since they do not generate toxicity in the absence of radiation and the nonirradiated cells will not be affected by the presence of NDs. The initial toxicity is low and the radiosensitizing effect is amplified for NDs-H, which are therefore particularly advantageous.

Example 5: Use of Hydrogenated Nanodiamonds for Vectorizing Biological Molecules into Cells

(64) In order to verify the capacity of the hydrogenated nanodiamonds (ND-H) to bind and transport molecules of biological interest into cells, ND-H particles were mixed with an equal volume of a telomeric probe consisting of peptide nucleic acid analog (PNA), labeled with the fluorophore Cy3. The final concentrations in the mixture were 64.52 g/cm.sup.3 for the NDs-H and 0.5645 M for the PNA-Cy3 probe. The PNA-Cy3 probe was denatured by heating at 80 C. for 5 minutes, before being mixed with the NDs-H.

(65) After incubation for 10 minutes at ambient temperature, the mixture of NDs-H and PNA-Cy3 probe was exposed to Caki-1 cells in culture (in Labtek 8-well plates). The cells were maintained in the presence of the mixture for 24 h and 72 h, under standard cell culture conditions. The cell nuclei were then labeled with the fluorescent label (Hoechst 33342), and the plates were observed directly using an inverted fluorescence microscope. For the nuclear labeling, the excitation/emission filter was 350 nm/460 nm, and for the PNA-Cy3 labeling, it was 550 nm/570 nm.

(66) The internalization of the PNA-Cy3 probe was observed only in the case where the ND-H particles had been mixed with this probe. In the absence of ND-H, the PNA-Cy3 probe was not internalized (FIG. 16).

(67) These results demonstrate the capacity of the NDs-H to bind a PNA-Cy3 probe and to transport it into the cells. The molecule internalized here has no major cytotoxic activity, but it is chemically similar to cytotoxic molecules such as that used in example 6 below, which will make it possible to obtain synergy with the cytotoxic activity of the NDs-H subjected to radiation. In addition, this probe is less (negatively) charged than the cytotoxic molecules of therapeutic interest which may be used clinically. These more negatively charged molecules adsorb more easily at the surface of the NDs-H and will be more efficiently vectorized into the cells. These results therefore demonstrate the capacity of the NDs-H to vectorize, into cells, cytotoxic molecules of therapeutic interest such as nucleic acids or PNAs.

Example 6: Use of Radiosensitizing Nanodiamond/Interfering RNA Complexes for Treating Tumors

(68) Nanodiamonds (primary size of 5 nm) are prepared so as to have a positive Zeta potential according to the processes described above. For the hydrogenated and/or graphitized NDS, a step of sonification in water makes it possible both to disperse the nanodiamonds and to efficiently adsorb molecules (H.sub.2O, O.sub.2 and NO.sub.2 mainly) onto their surface, thus giving them a positive surface charge.

(69) POLQ interfering RNAs, having the capacity to inhibit the messenger RNAs encoding polymerase theta, enabling DNA repair in certain tumor cells (the POLQ gene is overexpressed in the most aggressive breast cancers, for example), are adsorbed onto the surface of the nanodiamonds. The RNAs, which have a negative surface charge, can be adsorbed by electrostatic interaction onto the surface of the positively charged nanodiamonds by simple addition of the RNAs to the nanodiamond suspension.

(70) These nanodiamond/RNA complexes are then injected into tumor cells, where they preferentially enter due to the increased permeability of tumor cell membranes. The POLQ interfering RNAs are gradually released in the tumor cells and inhibit polymerase theta synthesis. The tumor cells are made more sensitive to radiation.

(71) The tumor cells are irradiated with X-rays, which leads to the release of free radicals in the cells that have internalized the nanodiamonds.

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