FR2952648A1 - In vitro poration of cells contained in substrate comprises contacting substrate with nanotubes and macromolecules of medicament including nucleic acids in cells and applying electromagnetic field - Google Patents

Abstract

Process of in vitro poration of cells contained in a substrate comprises: contacting the substrate with nanotubes providing hyperfrequency receptor function and with transferable macromolecules of medicament type including nucleic acids in cells; and applying electromagnetic field for generating electromagnetic excitation absorbed by nanotubes generating the poration of cells by electronic circulation within the nanotube. ACTIVITY : Cytostatic. MECHANISM OF ACTION : None given.

Description

Process for poration of cells comprised in a substrate comprising the use of nanotubes as a microwave receiver The invention lies in the field of nanoscale and microwave technologies applied to biology and medicine, and in particular to the diffusion of drugs through membranes. cell. It is recalled in general that the administration of ~ o drugs can be performed by different methods. Systemic (systemic) administration and topical (local) application are distinguished. Systemic forms of administration act on the whole organism, topical forms of application only where the drug is applied, especially on the skin. Systemic Forms of Administration: Drugs can be absorbed through the mouth. At the level of the gastrointestinal tract, the active substances enter the blood, which transports them to the organ where they must act. In the case of rectal administration, the active substances diffused by the suppositories are absorbed by the mucous membrane of the rectum. There is also parenteral administration, that is to say by a route other than the digestive tract. It is usually an injection. A substance can be injected intravenously (into the veins), intramuscularly (into the muscle) or subcutaneously (under the skin). When a solution is administered intravenously for a prolonged period, it is referred to as an infusion. Galenic forms, such as aerosols, vapors or gases, are used by inhalation. The transdermal system is a kind of patch - or "patch" - containing a certain amount of active substance that diffuses into the body continuously. Topical administration forms: In the case of a cutaneous application, the drug - an ointment, for example - is applied to the skin. Currently, physical methods of gene transfer (and / or drugs) allow the direct entry into cells of 35 non-permeable molecules, both large and small, that otherwise can not diffuse across the plasma membrane or be actively transported. through the plasma membrane. There are many methods of gene transfer or in vivo drugs, for example biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electropermeabilization (Villemejane, J. and Mir, L British Journal of Pharmacology 157, 207-219, 2009). Electropermeabilization (or electroporation) is a technique used to introduce macromolecules (cell transfection), including DNA, RNA, genes and proteins, and drugs in general, into cells (Neumann, E et al., Fundamentals of electroporative delivery of drugs and genes, Bioelectrochem Bioenerg., 48, 3016, 1999) by application of electric fields, generally pulsed, having an intensity and duration sufficient to induce a temporary increase in the permeability of the cell membrane. The high transmembrane potentials induced in this way cause the formation of very small pores (20-120 nm in diameter). Macromolecules introduced through the cell membrane pass through the pores by electrophoresis in the case of charged molecules (Neumann, E. et al., Calcium-mediated DNA adsorption to yeast cells and kinetics of this / transformation by electroporation, Biophys. J. 71, 868, 877, 1996), and by passive diffusion in the case of neutral molecules (Neumann, E. et al Mechanism of electroporative dye uptake by mouse B cells, Biophys J. 74, 98108, 1998). Currently, for in vivo electroporation, the pulsed electric field is transmitted by needle electrodes implanted in the tissue to be treated, or by flat electrodes applied in surface contact with the tissue. Electrodes and electrode arrays, for releasing electrical pulses for the purpose of achieving therapeutic effects, are described for example in patent application WO 98/47 562. While the method based on the use of Needle electrodes are invasive, the one based on the use of flat electrodes is not, and so is in principle preferable. However, it is limited only to surface application on small areas. This is correlated with available scientific data regarding the intensity of electric fields and the time required for efficient electroporation of cells, as described in "Guidelines for the limitation of electromagnetic fields and electromagnetic fields". (up to 300 GHz) "of the International Commission on Non-ionizing Radiation Protection (ICNIRP). Thus, if the threshold values recommended by these instructions are observed, the processing depth (that is to say the distance between the flat electrodes, or from the flat electrode) is only a few millimeters which means that the method is effective in vivo only in areas close to the body surface (for example the epidermis and the dermis). Other electroporation methods are well established, which make use of "mediators", including and in particular carbon nanotubes (CNTs). Carbon nanotubes are very thin tubular structures formed by one or more layers of graphite wound on themselves. The carbon nanotubes produced by an arc discharge between two graphite electrodes have been discovered and described for the first time by Sumio lijima ("Helical Microtubules of Graphitic Carbon", Nature, Vol 354, Nov. 7, 1991). , pages 56-58). In general, carbon nanotubes are classified into two categories: single-walled nanotubes, or SWNTs, composed of a tubular structure formed of a sheet of graphite wound on itself and closed at both ends by two hemispherical caps, and which differ from the multi-wall or MWNT nanotubes, which can be considered as consisting of several concentric SWNT nanotubes. The use of NTCs as electroporation mediators has already been proposed to reduce the voltage requirements for irreversible electroporation, with the aim of developing a low-power portable lab-on-a-chip ( Yantzi JD et al., Carbon Nanotube Enhanced Pulsed Electric Field Electroporation for Biomedical Applications, Proceedings of the IEEE International Conference on Mechatronics & Automation Niagara Falls, Canada, 2005), or to cause reversible permeabilization of bacterial cells by exposure to microparticles. Cells Contacted with NTCs (Rojas-Chapana, JA et al., Enhanced Introduction of Gold Nanoparticles into Vital Acidothiobacillus ferrooxidans by Carbon Nanotube-based Microwave Electroporation, Nano Letters, 4, 5, 985 -988, 2004), or for reversible electroporation by exploiting the dielectric properties of CNTs (PCT / IB 2007/05 47 54, November 22, 2004). 2007).

It is also worth mentioning the patents describing a device that exploits the property of SWCNTs to release heat in a radio frequency (RF) field for the purpose of producing thermal ablation of malignant cells (US Patent No. US 2006/01 90 063 Al, US 2005/02 51 12 33 A1, and US 2005/02 51 234 A1 and international application WO 2007/02 76 14). In this context, the present invention proposes a new cell permeabilization method, which is based on the application of an electromagnetic field in the vicinity of nano-antennas constituted by nanotubes, in order to perform electroporation of a cell membrane. . This action is intended to allow in particular the introduction into the body of nanotubes coated with a substance containing one or more types of macromolecules, it may be for example therapeutic agent, then to activate these nanotubes using a localized electromagnetic field allowing penetration into the cell and the release of said agents to generate a localized treatment. Thus this type of process opens up possibilities in terms of the fight against diseases such as cancer by limiting the treatment to the contaminated zone.

This method makes it possible in particular to transfer a gene and / or a drug into the cytoplasm of the target cells. The method is applicable to both in vitro and in vivo applications and allows reversible poration of cells without degradation of the cells involved in the treatment.

More specifically, the subject of the present invention is a process for poration of cells contained in a substrate, characterized in that it comprises: the placing in the presence of the substrate, with nanotubes providing a microwave receiver function and with macromolecules may be of the drug type including nucleic acids and transferable into cells; the application of an electromagnetic field for generating an electromagnetic excitation absorbed by said nanotubes generating the poration of said cells by circulating electrons within said nanotubes. According to a variant of the invention, the electromagnetic field emits in the Gigahertz frequency band. According to one variant of the invention, the electromagnetic field is applied continuously. According to a variant of the invention, the electromagnetic field is applied in a pulsed manner. According to one variant of the invention, the nanotubes are used in solution at a concentration of between approximately 0.5 and 500 μg / ml. According to a variant of the invention, the nanotubes are brought into direct contact with the substrate by direct mixing. According to one variant of the invention, the nanotubes are brought into contact with the substrate by systemic administration. According to one variant of the invention, the nanotubes are brought into contact with the substrate by parenteral injection, which can be intravenous. According to one variant of the invention, the nanotubes are coated with a binder containing transferable macromolecules in cells. According to a variant of the invention, the process comprises an operation of functionalization of the nanotubes with a surfactant.

The invention will be better understood and other advantages will become apparent on reading the following description, which is given in a nonlimiting manner and by virtue of the appended figures in which: FIG. 1 illustrates the structures of nanotubes; Figures 2a and 2b illustrate the functionalized NTC structure used in the invention and their behavior as nano-antennas; FIGS. 3a and 3b illustrate the efficiency of gene transfer into bacterial cells exposed to an electromagnetic field for different powers and different substrate temperature increases (exposure time 30 seconds), b) gene transfer efficiency in bacterial cells exposed to 5W EMC (exposure times 30 seconds and 5 seconds), Figures 4a to 4c illustrate linear temperature increase (R2 = 0.9992) as a function of power (a) which is due to the heating of the water (b) and is independent of the concentration of the nano-antennas (c).

In general, the following definitions are used in the following description: A substrate: any material containing living cells, in particular a part of a living being (plant, human or animal), or a substrate separated from the be alive and contained in a container (for example a culture of animal, plant or fungal cells). An electromagnetic field EM physical field produced by electrically charged objects. It is characterized by its amplitude, frequency (Hz) and power (W). A drug: any molecule to be transferred into cells, including nucleic acids.

The microwave operation on which the present invention is based is described below: First principle used: the wired antenna The principle of a wired antenna consists of resonating an electromagnetic wave in a linear metallic conductor subjected to an EM field. a given frequency. As with the principles of acoustic resonance, the resonance of an EM wave is subject to the dimensions of the metallic element supporting the EM wave. The associated wavelength must be of dimension adapted to the length of the metallic element. The direct consequence is that if one places a metal bar of length equal to or T, / 2 (at the wavelength defined by f f, c the speed of propagation of the wave and f the frequency of this wave. wave) then an EM resonance is obtained in the metallic conductor. The consequence of this resonance in the linear metallic conductor is the appearance of an alternating current in this conductor. This energy is partially dissipated by the Joule effect (depending on the resistivity of the conductor) and partially re-emitted into the medium.

Second principle used: Carbon Nanotubes (CNTs) ~ o CNTs are elements of nanometric size (typically their diameter, the length of a CNT up to a few centimeters.) Consisting of a carbon atom. The latter are organized as a graphene sheet (a) wound on itself to form a single-layer tube (b) (SWNT) but can also be found in concentric elements (c) (MWNT) as illustrated in FIG. The electromagnetic properties of these CNTs are very different from those of a macroscopically sized wire. Due to their structure, the speed of propagation of a wave is much lower (of the order of 1/50 to 1/1 oo) but their resistance is very important. All this is mainly due to their atomic scale size. They are still much more interesting than metal nanowires whose conductivity to these dimensions can be considered almost zero. According to the principles of electroporation explained above, one of the aims of the present invention is to allow the introduction of CNTs selectively in an area of the organism on which it is desired to work and then to expose this zone to microwaves. to generate electroporation of cell membranes in this area. According to one variant of the invention, the CNTs may advantageously be intermediates coated with a substance for fixing the molecules that it is desired to pass through the cells. Figures 2a and 2b illustrate for this purpose nanotubes functionalized and behaving as nano-antennas. CNTs are thus used as receiving nano-antennas which under the influence of an EM field will both create the electroporation necessary to allow the passage of macro-molecules through the cell membrane and the release of said macromolecules of the layer that coats the CNTs for the purpose of transporting these macromolecules.

According to principles demonstrated by experiments, CNTs act as microwave receivers having a length appropriate to the wavelength used to excite them (or the harmonics). They thus receive the energy of this microwave wave which generates in each of them a circulation of electrons. Since nanotubes have a relatively high absorption factor below 100 GHz, most of this received energy is dissipated by generating electroporation on the one hand and heat on the other hand, releasing the coating layer. and the macro-molecules it contains. According to the invention, the function of the cellular pore mediators, filled by the CNT, is achieved by exploiting their antenna properties. This happens when the CNTs are placed near the target cells. By exposing the substrate to the electromagnetic field, the nanotubes induce the phenomenon of permeabilization of the cell membrane, allowing the intracellular administration of therapeutic molecules. In large scale production of CNTs, the native product (as prepared) exists as agglomerates up to a hundred microns in size. These virgin NTCs are entirely hydrophobic and can not be used for biological purposes.

For any biomedical application, it is therefore essential that the CNTs are treated, so that they can be dispersed in water. The solution / dispersion of CNTs used in the process described by the present invention is carried out by procedures well established in the art, for example those described in T. Saito et al., Chemical treatment and modification of multi-walled carbon. nanotubes, Physica B 323: 280-283, 2002. The purpose of the treatment is to produce a cytocompatible solution of nanotubes coated / wrapped in polymers, surfactants, etc. The wrapping mechanism can be ionic, nonionic, covalent, etc. These functionalized nanotubes (CNT-f) are used at concentrations ranging from 0.1 to 500 μg / ml. Preferred concentrations are in the range of 1 to 10 μg / ml.

Exemplary embodiment of functionalization of nanotubes According to one example of the invention, MWNT type carbon nanotubes for "multi-wall nanotube" are manufactured in a known manner according to a conventional method of growth by chemical vapor deposition still commonly referred to as " CCVD "for" catalytic chemical vapor deposition "or" CVD "for" chemical vapor deposition "using hydrocarbon sources deposited on a type Al 2 O 3 oxide substrate impregnated with iron-based metal catalyst as described in the article from Kuravelou KB, Sotirchos SV, Verykios XE. Catalytic effects of carbon nanotube production in a thermogravimetric CVD reactor. Surf Coat Technol 2007; 201: 9226.

The coating of the carbon nanotubes is carried out in the following manner in a second step, by performing a functionalization of the nanotubes with a Pluronic F127 surfactant corresponding to a polyoxyethylene / polyoxypropylene block block copolymer marketed by the company Sigma, St. Louis, MO

More specifically, the nanotubes are dispersed in a solution of water-soluble surfactant, with a molecular weight of 12,600. Typically 5 mg of nanotubes are mixed with 10 ml of solution comprising Plutonic F127, in a buffered solution of phosphate. The mixture is placed on a hot plate heated at 70 ° C. for 4 hours and then sonicated with a Branson 2510 ultrasonic apparatus (sold by Brasonoc, CT, USA) at 20 W for 12 hours. . The mixture is finally centrifuged. The nanotubes thus treated retain their initial electronic properties as described in the article by Zhang J, Zou H, Qing Q, Yang Y, Li Q, Liu Z, et al. Effect of chemical oxidation on the structure of single-walled carbon nanotubes. J Phys Chem B 2003; 107: 3712. It should be noted that Pluronic in low concentration, as in the present case, does not present any particular problem of toxicity as described in the article by Monteiro-Riviere NA, Inman AO, Wang YY and Nemanich RJ. Surfactant effects on carbon nanotube interactions with human keratinocytes. Nanomed: Nanotechnol Biol Med 2005; 1: 293-9. The MWNT nanotubes thus obtained are treated with nitric acid to obtain nanotubes of the COOH-MWNT type.

The nanotubes are sonicated for 10 minutes in 65% HNO3 acid. The solution is stirred at 220 ° C for 20 minutes, cooled to temperature, filtered and finally dried at room temperature for a few hours. 100 μg of COOH-MWNT are dispersed in 20 ml of a mixture of dimethylformamide anhydride (DMFA) / dichloromethane anhydride (DCM) in the ratio 1: 2 and cooled. To this solution, 2 ml of hydroxybenzotriazole (HOBt) in DMFA (125 μg / ml, 37 eqv) is added and the mixture is stirred for 10 minutes. 1 ml of 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in DMFA (100 μg / l ml, 10 μg) are added slowly to react and then stirred for 30 minutes at a temperature of 0 ° C. then for 3 hours at room temperature. Thus, active nanotubes are obtained by filtration through a nylon 66 membrane (with pores 0.45 μm in diameter). The collected solution is washed in 200 ml of anhydrous DCM and redissolved in EDAC and HOBt. The nanotubes are then dispersed in 10 ml of anhydrous DCM and added slowly to a solution of PEI (MW = 25,000 gmol -1, 1.0 g) in 40 ml of anhydrous DCM at a temperature of 0 ° C. The reaction is obtained with stirring during 30 minutes at 0 ° C. and then overnight at room temperature The resulting nanotubes are filtered through a nylon 66 membrane and then washed in 30 ml of anhydrous DCM, 50 ml of ethanol, 100 ml of water and 20 ml. After drying under vacuum, 88.5 mg of functionalized nanotubes are obtained.The previously obtained CNT solution can be contacted with the cells and the substrate in various ways depending on the administration, for example by simple mixing, systemic administration or intravenous, intramuscular or direct injection into the injured tissues.

Exemplary embodiment of the method of the invention: The results of experimental tests carried out on E. coli bacterial cells are described below and illustrated in FIGS. 3a-3b and 4a-4c. In the existing orthodox bacterial transfection protocol, viable bacterial cells are placed on ice with DNA, then briefly heat-shocked (eg 42 ° C for 30 seconds), resulting in DNA to enter the cell. In the described experiments, bacterial cells are incubated on ice for 30 minutes with a solution prepared by mixing 2 μg of plasmid DNA with 0.25 μg of NTC-f functionalised nanotubes (NTC-f: pDNA complex). . This solution is then exposed to electromagnetic energy (wavelength about 3 cm, power range 0.1-5 Watt, frequency 10 GHz and exposure time 5-60 seconds at room temperature). In these experiments, the plasmid backbone contains a bacterial promoter expressing kanamycin resistance in E. coli. Thus, the plasmids used in these transfection experiments contain a gene conferring resistance to the antibiotic kanamycin. After the transfection performed by this new technique, the bacterial cells that acquired the pDNA were identified by plating a pretreated Petri dish with 30 μg / ml kanamycin because only the bacteria transfected with the resistance gene kanamycin are able to grow in the presence of this antibiotic, while untransfected bacterial cells do not grow. All tests are carried out in an Eppendorf tube with a sample volume of 50 μl. The temperature during exposure to electromagnetic field radiation is monitored using a thermocouple type k (sensitivity 0.1 ° C). In the absence of application of the electromagnetic field, the number of colonies growing in the Petri dishes is about 50 (control test). Different powers are thus applied for 30 seconds, while monitoring the corresponding increase in temperature and the number of colonies grown. As seen in FIG. 3a, the optimal efficiency of transfection of E. coli bacterial cells is carried out with 1 W. The increase of the transfection of the bacteria is, in the presence of the nano-antenna (NTC-f: DNA) much larger than in the presence of DNA alone (or of f: pDNA, data not shown). The observed efficiency of transsection decreases with increasing or decreasing power above or below 1.0 W. At 0.1 W (temperature increase: 2.5 ° C), the number of colonies is 50 (analogous to the control) and 100, for cells respectively in contact with the DNA and the NTC-f: DNA. At 0.5 W (temperature increase: 4.5 ° C), the number of colonies is 100 and 400, for cells respectively in contact with DNA and NTC-f: DNA. At 1 W (temperature increase: 7 ° C), the number of colonies is 350 and 800 for the cells respectively in contact with the DNA and the NTC-f: DNA. At 5 W (temperature increase: 35 ° C) all cells are killed due to the heating effect. The experimental data show that the results made at 1 W for 30 seconds are very similar to those obtained at 5 W for 5 seconds (Figure 3b), which reveals that the efficiency of the transfection depends on the energy released, rather than the applied power. It is also observed that the increase in temperature is linear (R2 = 0.9992) as a function of the power. This increase results from the heating of the water and is independent of the concentration of the nanotubes as illustrated in FIGS. 4a-4c. All experimental data are in agreement with the theory that nanotubes function as a receiving antenna, capable of inducing a local increase in permeabilization of the cell membrane. The cell poration of the present invention is suitable for both in vitro and in vivo applications, and the means for transposing the method into practice will vary depending on the intended purpose of cell poration.

For example, if it is a culture of cells fixed in a cell culture dish, it can be exposed to radiation emitted by an appropriate source. In the case of small animals (eg rat or mouse), the part of the animal (eg head, limb, etc.) can be placed in the drilled hole in a metal tube directly connected to the generator. wave. If cell poration is to involve larger parts of animals, or part of the human body, a suitable metal chamber could be developed. The cell poration method of the present invention can be used for (i) research and investigations and (ii) clinical therapeutic applications, for example, targeted drug delivery, including tumor electrochemotherapy, and therapy. gene for a range of clinical disorders. With this method, it is possible to introduce into target cells / tissues, using also, if necessary, a nanoparticle functionalized with substances or molecules of the type described above, bioactive molecules in general, nucleic acids and a genetic material, proteins, substances with pharmacological properties and chemotherapeutic agents, neuroregenerators, etc.

An important advantage of the cellular poration method according to the present invention lies in the fact that the use of nanotubes, as a localized treatment, allows targeted and selective poration, only cells treated with CNTs, which avoids an involuntary permeabilization or unwanted surrounding tissue, permeabilization that occurs with existing electroporation methods. The method described by the present invention can also be applied to the implementation of cell lysis by irreversible poration (or ablation) of the cells, by an increase in the power applied (for example for the treatment of cancer). 30

Claims (10)

REVENDICATIONS1. Technical process for in vitro poration of cells contained in a substrate, characterized in that it comprises: the placing in the presence of the substrate, with nanotubes providing a microwave receiver function and with macromolecules that can be of the medicinal type including nucleic acids and transferable into cells; The application of an electromagnetic field making it possible to generate an electromagnetic excitation absorbed by said nanotubes generating the poration of said cells by circulation of electrons within said nanotubes. 15 2. Technical process of in vitro poration according to claim 1, characterized in that the electromagnetic field emits in the frequency band Gigahertz. 3. Technical process of in vitro poration according to one of claims 1 or 2, characterized in that the electromagnetic field is applied continuously. 4. Technical poration process according to one of claims 1 or 2, characterized in that the electromagnetic field is applied in a pulsed manner. 5. Technical process of in vitro poration according to one of claims 1 to 4, characterized in that the nanotubes are used in solution at a concentration between about 0.5 tag / ml and 500 tag / ml. 6. Technical process of in vitro poration according to one of claims 1 to 5, characterized in that the nanotubes are brought into direct contact with the substrate by direct mixing. 30 7. Technical process of in vitro poration according to one of claims 1 to 6, characterized in that the nanotubes are coated with a binder containing the transferable macro-molecules. 8. Technical process of in vitro poration according to one of claims 1 to 7, characterized in that it comprises an operation of functionalization of the nanotubes with a surfactant. 10 9. Use of nanotubes for the preparation of a support for electroporation coated with a pharmaceutical composition capable of being introduced by electroporation through a cell membrane. 10. Use of nanotubes for the preparation of a support for electroporation coated with a pharmaceutical composition capable of being introduced by electroporation through a cell membrane, the nanotubes being coated with a binder containing macromolecules transferable. 20