METHOD FOR INCREASING THE RELEASE OF MEDICAL COMPOUNDS FROM NANOPARTICLES BY AN ALTERATION STEP AND A PHYSICO-CHEMICAL DISTURBANCE STEP

Abstract

A method for increasing the release of at least one compound, the compound being initially an initial compound bound to at least one initial nanoparticle, the initial compound bound to the initial nanoparticle forming an initial particle, wherein the initial particle includes at least one active ingredient, the method including at least one step of alteration of the initial particle and at least one step of physico-chemical disturbance of an altered particle resulting from the alteration.

Claims

1-15. (canceled)

16. A method for increasing the release of at least one compound, said compound being initially an initial compound bound to at least one initial nanoparticle, said initial compound bound to said initial nanoparticle forming at least one initial particle, and wherein said initial particle comprises at least one active ingredient, and wherein said method comprises the following two steps a) and b): a) altering said initial particle, wherein said altering is associated with modification of at least one property of said initial particle, said altering resulting in formation of an altered particle composed of at least one altered nanoparticle and at least one altered compound, and wherein said altered particle comprises at least one active ingredient, and wherein said altering is defined as at least one step selected in the group consisting of steps i) to xii): i) decreasing particle size from the size of the initial particle down to the size of the altered particle, where this decrease is such that S.sub.A/S.sub.I or (S.sub.I−S.sub.A)/S.sub.I is between 10.sup.−3% and 99.99%, where S.sub.A and S.sub.i are the sizes of the altered and initial particles, respectively, ii) decreasing a number of compounds bound to the nanoparticle, from a number n.sub.i of initial compounds bound to the initial nanoparticle down to a number n.sub.a of altered compounds bound to the altered nanoparticle, where n.sub.i/n.sub.a is between 1 and 10.sup.10, iii) decreasing a binding strength of least one bond between the compound and the nanoparticle, from a binding strength S.sub.i of at least one initial bond between the initial compound and the initial nanoparticle to a binding strength S.sub.a of at least one altered bond between the altered compound and the altered nanoparticle, iv) breaking at least one bond between the altered compound and the altered nanoparticle, v) decreasing a bond-dissociation energy between the compound and the nanoparticle, from a bond-dissociation energy E.sub.di between the initial compound and the initial nanoparticle down to a bond-dissociation energy E.sub.da between the altered compound and the altered nanoparticle, vi) decreasing a coating thickness of the nanoparticle, from a coating thickness CT.sub.i of the initial nanoparticle down to a coating thickness CT.sub.a of the altered nanoparticle, vii) decreasing a percentage in mass of organic material or carbon or carbonaceous material of the altered particle, compared with the percentage in mass of organic material or carbon or carbonaceous of the initial particle, viii) decreasing cluttering of the compound bound to the nanoparticle, from a large cluttering of the initial compound bound to the initial nanoparticle down to a small cluttering of the altered compound bound to the altered nanoparticle, ix) decreasing a number or a concentration of compounds N.sub.1 that prevent the release of compounds N.sub.2 from the nanoparticle, from a number of initial compounds N.sub.1i that prevent the release of initial compounds N.sub.2i from the initial nanoparticle down to a number of altered compounds N.sub.1a that prevent the release of altered compounds N.sub.2a from the altered nanoparticle, x) inactivating, attenuating, destroying a cell, part of a cell, a virus, part of a virus, a bacterium, and/or part of a bacterium, from an initial cell, part of an initial cell, an initial virus, part of an initial virus, an initial bacterium, and/or part of an initial bacterium that is/are not inactivated, not attenuated, and/or not destroyed by or in the presence of the initial nanoparticle to an altered cell, part of an altered cell, an altered virus, part of an altered virus, an altered bacterium, and/or part of an altered bacterium that is/are inactivated, attenuated, and/or destroyed by or in the presence of the altered nanoparticle or of the nanoparticle that transforms itself from the initial to the altered nanoparticle, xi) presenting, processing and/or exposing an antigen or part of an antigen such as an epitope by or in the presence of the altered nanoparticle, from an initial antigen or part of an initial antigen that is not presented, not processed and/or not exposed by or in the presence of the initial nanoparticle to an altered antigen or part of an altered antigen that is presented, processed and/or exposed by or in the presence of the altered nanoparticle, and xii) coating, binding, and/or assembling a nanoparticle by or with a cell, part of a cell, a virus, part of a virus, a bacterium, part of a bacterium, an antigen, and/or part of an antigen, from an initial nanoparticle that is not coated, bound, and/or assembled by or with an initial cell, part of an initial cell, an initial virus, part of an initial virus, an initial bacterium, part of an initial bacterium, an initial antigen, and/or part of an initial antigen to an altered nanoparticle that is coated, bound, and/or assembled by or with an altered cell, part of an altered cell, an altered virus, part of an altered virus, an altered bacterium, part of an altered bacterium, an altered antigen, and/or part of an altered antigen, and wherein said alteration results in xiii) and xiv): xiii) a first partial release generating a first part of altered compounds released from the altered nanoparticle, where the first partial release is due to the complete breaking of the bond between said altered nanoparticle and the first part of said altered compound, and xiv) an absence of release generating a second part of altered compounds that remain bound to the altered nanoparticle, where the absence of release is due to the absence of breaking of the altered bond between said altered nanoparticle and said second part of altered compound, and wherein the first part and second part of altered compounds originate from the initial particle, and the sum of said first part and second part of said altered compounds represent the total number of initial compounds bound to said initial nanoparticle, and wherein said alteration, which is applied on the first transforming particle transforming from the initial particle to the altered particle, is carried out in at least one of the following conditions among xv) to xx): xv) by a first internalization of the first transforming particle in a cell, a virus, a bacterium, xvi) by a first variation of the pH of the first transforming particle or of its environment, xvii) by a first variation of temperature of the first transforming particle or of its environment, xviii) by bringing the first transforming particle in the presence of altering biological or chemical material, xix) by applying a first radiation on the first transforming particle, and xx) by a first variation the environment of the first transforming particle, b) by applying a physico-chemical disturbance on said altered particle, resulting in the formation of an altered and disturbed particle, and wherein said altered and disturbed particle comprises at least one active ingredient, and wherein step b) is associated with xxv), xxvi), or xxvii): xxv) an absence of release generating non-released altered and disturbed compounds belonging to group 1 of the second part, which originates from the second part of altered compounds not released by alteration at step a)xiii), wherein the absence of release is due to the absence of breaking of the altered and disturbed bond between the altered and disturbed nanoparticle and the group 1 of the second part of altered and disturbed compounds, xxvi) a second partial release generating released altered and disturbed compounds belonging to group 2 of the second part, which originates from the second part of altered compounds not released by alteration at step a)xiii), wherein said second partial release is due to the complete breaking of the bond between the group 2 of the second part of the altered and disturbed compounds and the altered and disturbed nanoparticle, or xxvii) a second total release of altered and disturbed compounds, which originate from the second part of altered compounds not released by alteration at step a)xi), wherein said second total release is due to the complete breaking of the bond between all said altered and disturbed compounds and said altered and disturbed nanoparticle, and wherein said physico-chemical disturbance which is applied on the second transforming particle transforming from the altered particle to the altered and disturbed particle, is carried out in at least one of the following conditions among xxviii) to xxxiv): xxviii) by a second internalization of the second transforming particle in a cell, a virus, a bacterium, xxix) by a second variation of the pH of the second transforming particle or of its environment, xxx) by a second variation of temperature of the second transforming particle or of its environment, xxxi) by bringing the second transforming particle in the presence of an altering biological or chemical material, xxxii) by applying a second radiation on the second transforming particle, and xxxiii) by a second variation the environment of the second transforming particle.

17. The method according to claim 16, wherein the alteration of step a) is repeated a number of time N.sub.a, wherein each alteration lasts for a time t.sub.A, wherein the physico-chemical disturbance of step b) is repeated a number of time N.sub.b, wherein each physico-chemical disturbance lasts for a time t.sub.b, wherein two different alterations are separated by a length of time t.sub.aa, wherein two physico-chemical disturbances are separated by a length of time t.sub.bb, wherein N.sub.a N.sub.b, t.sub.a, t.sub.b, t.sub.aa, and t.sub.bb have at least one property selected in the group consisting of: i) N.sub.a is smaller than N.sub.b, ii) N.sub.a is equal to one, iii) N.sub.b is larger than one, iv) t.sub.a is larger than t.sub.b, and v) t.sub.bb is larger than t.sub.aa.

18. The method according to claim 16, wherein the alteration, which is applied on the first transforming particle transforming from the initial particle to the altered particle, and the physico-chemical disturbance, which is applied on the second transforming particle transforming from the altered particle to the altered and disturbed particle, have at least one property selected from the group consisting of: i) The alteration is or is due to a first variation of pH of the first transforming particle or of its environment, which is larger than 10-3 pH units, ii) The physico-chemical disturbance is or is due to a second variation of pH of the second transforming particle or of its environment, which is larger than 10.sup.−3 pH units, iii) The alteration is or is due to a first variation of temperature of the first transforming particle or of its environment, which is larger than 10.sup.−3° C., iv) The physico-chemical disturbance is or is due to a second variation of temperature of the second transforming particle or of its environment, which is larger than 10.sup.−3° C., v) The physico-chemical disturbance is associated with a second internalization of the second transforming particle, which is an extension a first internalization of the first transforming particle due to alteration, vi) The alteration is associated with the first transforming particle being brought in the presence of altering chemical or biological material, vii) The physico-chemical disturbance is associated with the second transforming particle being brought in the presence of altering chemical or biological material, viii) The alteration is due to a first radiation or to the application of a first radiation on the first transforming particle, ix) The physico-chemical disturbance is due to a second radiation or to the application of a second radiation on the second transforming particle, and x) The physico-chemical disturbance is a second radiation that has a strength, power, frequency, and/or intensity that is/are larger than the strength, power, frequency, and/or intensity of the first radiation being the alteration, and wherein the altering chemical or biological material is selected in the group consisting of: a) at least one denaturing material, where a denaturing material can be selected from a first material that induces a loss in crystallinity, activity or a reduction in size of a second material or a first material that induces unfolding such as protein unfolding or a loss in quaternary, ternary, secondary, first structure of a second material such as an enzyme or protein or a first material that induces a loss in sheet, preferentially β sheet, or helix, preferentially a helix, structures of a second material, b) at least one cell, cell organelle, protein, peptide, enzyme, DNA, RNA, DNA strand or base, RNA strand or base, part of any of these substances, preferentially denaturing, c) at least one detergent, d) at least one acid such as HCl, e) at least one base such as NaOH, f) at least one chaotropic agent, g) a compound with at least one chemical function selected in the group consisting of: carboxylic acids, phosphoric acids, sulfonic acids, esters, amides, ketones, alcohols, phenols, thiols, amines, ether, sulfides, acid anhydrides, acyl halides, amidines, nitriles, hydroperoxides, imines, aldehydes, and peroxides, h) Acetic acid, i) Alcohol, j) DMSO (Dimethylsulfoxyde), k) Ethanol, l) Formaldehyde, m) Formamide, n) Guanidine, o) Glutaraldehyde, p) Guanidinium chloride, q) Guanidine Thiocyanate, r) HCl, s) Lithium perchlorate, t) NaOH, u) Nitric Acid, v) Picric acid, w) Propylene glycol, x) Sodium bicarbonate, y) Sodium dodecyl sulfate, z) Sodium salicylate, aa) Sulfosalicylic acid, bb) Trichloroacetic acid, cc) Urea, dd) Polar solvent, ee) Apolar solvent, ff) an acidic, basic, oxidized, reduced, neutral, positively charged, negatively charged derivative of these compounds, and gg) a combination of several of these compounds or derivatives, and wherein the first and/or second radiation(s) is/are selected in the group consisting of: a) electromagnetic radiation, b) acoustic radiation forces, c) radiation forces, d) radiation pressures, e) irradiation, preferentially of the body part, f) a source of radiation, g) a magnetic or electric field, h) an alternating magnetic or electric field, i) a magnetic or electric field gradient, j) light or laser light, k) light produced by a lamp, l) light emitted at a single wavelength, m) light emitted at multiple wavelengths, n) a ionizing radiation, o) microwave, p) radiofrequencies, q) acoustic wave, r) alpha, beta, gamma, X-ray, neutron, proton, electron, ion, neutrino, muon, meson, photon particles or radiation, s) infrasound, sound, ultra-sound, or hypersound, t) particle with a non-zero weight, and u) oscillating waves with a zero-weight.

19. A method for obtaining an altered and disturbed particle comprising at least one step selected from the group consisting of: a) applying an alteration on at least one initial particle comprising at least one initial nanoparticle and at least one releasable initial compound, which is initially bound to said initial nanoparticle via an initial bond, b) obtaining at least one altered particle, comprising at least one altered nanoparticle and at least one releasable altered compound, where a first partial release generates the release of a first part of altered compound during the alteration, said altered compounds being divided between i) and ii): i) a first part of altered compounds comprising altered compounds released from the altered nanoparticle, and ii) a second part of altered compounds comprising altered compounds bound to the altered nanoparticle via an altered bond, c) applying a physico-chemical disturbance on said altered particle, d) obtaining at least one altered and disturbed particle, comprising at least one altered and disturbed nanoparticle and at least one releasable altered and disturbed compound, where α second partial release generates the release of a second part of altered and disturbed compounds during physico-chemical disturbance, said altered and disturbed compounds being divided between i) and ii): i) group 1 of second part of altered and disturbed compounds comprising altered and disturbed compounds bound to the altered and disturbed nanoparticle via an altered and disturbed bond, and ii) group 2 of second part of altered and disturbed compounds comprising altered and disturbed compounds released from the altered and disturbed nanoparticle, wherein the said initial particle, altered particle, and/or altered and disturbed particle comprise at least one active ingredient.

20. An altered and disturbed particle obtainable by the method of claim 19, said altered particle comprising at least one altered nanoparticle and at least one releasable altered compound, said altered compounds being divided between a) and b): a) a first part of altered compounds being released altered compounds from the altered nanoparticle, and b) a second part of altered compounds being altered compounds bound via an altered bound to the altered nanoparticle, wherein said altered particle comprises at least one active ingredient, wherein said altered particle comprises at least one of the properties selected from the group consisting of i) to xii): i) a size of the altered particle that is smaller than the size of the initial particle, by a percentage between 10.sup.−3% and 99.99%, where this percentage is S.sub.A/S.sub.I or (S.sub.I−S.sub.A)/S.sub.I, where S.sub.A and S.sub.I are the sizes of the altered and initial particles, respectively, ii) a number of altered compounds bound to the altered nanoparticle, n.sub.a, that is smaller than the number of compounds bound to the initial nanoparticle, n.sub.i, where n.sub.i/n.sub.a is between 1 and 10.sup.10, iii) a binding strength of least one bond between the altered compound and the altered nanoparticle, S.sub.a, that is smaller than the binding strength of at least one bond between the initial compound and the initial nanoparticle, S.sub.i, iv) a breaking of at least one bond between the altered compound and the altered nanoparticle, v) a bond-dissociation energy between the altered compound and the altered nanoparticle, E.sub.da, that is smaller than the bond-dissociation energy between the initial compound and the initial nanoparticle, E.sub.di, vi) a coating thickness of the altered nanoparticle, CT.sub.a, that is smaller than the coating thickness of the initial nanoparticle, CT.sub.i, vii) a percentage in mass of organic material or carbon or carbonaceous material of the altered particle that is smaller than the percentage in mass of organic material or carbon or carbonaceous material of the initial particle, viii) a cluttering of the altered compound bound to the altered nanoparticle that is smaller than the cluttering of the initial compound bound to the initial nanoparticle, ix) a number of altered compounds N.sub.1a that prevent the release of altered compounds N.sub.2a from the altered nanoparticle that is smaller than the number of initial compounds N.sub.1i that prevent the release of initial compounds N.sub.2i from the initial nanoparticle, x) at least one altered compound that is an inactivated, attenuated, or destroyed cell, part of a cell, virus, part of a virus, bacterium, and/or part of a bacterium, xi) at least one altered compound that is a presented, processed, and/or exposed antigen or part of an antigen such as an epitope, and xii) at least one altered compound that is a virus, part of virus, a bacterium, part of a bacterium, an antigen, and/or part of an antigen, which is/are bound, assembled, and/or coated with, to or on top of the altered nanoparticle.

21. The altered and disturbed particle obtainable by the method of claim 19, said altered and disturbed particle comprising at least one altered and disturbed nanoparticle and at least one altered and disturbed compound, wherein said altered and disturbed particle comprises at least one active ingredient, where the altered and disturbed compound is divided into one or more of the three following categories of compounds: category A: altered and disturbed compounds, originating from the first part of the altered compound that is released in the first partial release and is not further released by physico-chemical disturbance from the altered and disturbed nanoparticle, category B: a group 2 of a second part of altered and disturbed compounds, originating from the second partial release of the altered compound that is not released by alteration from the altered nanoparticle, and said group 2 of the second part of altered and disturbed compounds is further released by physico-chemical disturbance from the altered and disturbed nanoparticle, category C: a group 1 of a second part of altered and disturbed compounds, originating from the second part of the altered compound that is not released by alteration from the altered nanoparticle, and is further not released by physico-chemical disturbance from the altered and disturbed nanoparticle, where said group 1 does not exist when all altered and disturbed compounds are released from the altered and disturbed nanoparticle.

22. A method of treating a disease, an infectious disease, a cancer, a tumor, an infection, a virus infection, or a bacterial infection in a subject, comprising administering to a subject in need thereof the altered and disturbed particle according to claim 20.

23. A pharmaceutical a composition comprising the altered and disturbed particle as defined in claim 20 and a pharmaceutically acceptable carrier, wherein the active ingredient is a therapeutically effective amount of a medicament.

24. The pharmaceutical composition according to claim 23, wherein said active ingredient is selected from the group comprising: i) a contrast agent, ii) a luminescent compound, iii) a drug or medicament, iv) a medical device, v) a cosmetic compound, vi) a therapeutic compound, vii) a medical compound, viii) a biological compound, ix) a diagnostic compound, x) a medical equipment or apparatus, xi) a composition, xii) a suspension, xiii) an excipient, xiv) an adjuvant, xv) a cytotoxic compound, xvi) a non-cytotoxic compound, xvii) an immunogenic compound, xviii) a non-immunogenic compound, xix) a pharmacological compound, xx) a non-pharmacological compound, xxi) a metabolic compound, xxii) a non-metabolic compound, xxiii) an antigen, xxiv) an antibody, xxv) a vaccine, xxvi) a virus, preferentially an attenuated or inactivated virus, xxvii) a metal, preferentially a non-toxic metal, iron, silver, or gold, xxviii) an antibiotic, xxix) a compound that is activated by being released from the nanoparticle, xxx) a compound that is activated more than once my being released more than once from the nanoparticle, and xxxi) a compound that is activated at least once by being released at least once from the nanoparticle by at least one alteration and/or physico-chemical disturbance.

25. The pharmaceutical composition according to claim 23, wherein: the released compound such as the released altered compound or the released altered and disturbed compound is at least one active ingredient or behaves like at least one active ingredient, and/or the non-released compound such as the initial compound, the non-released altered compound or the non-released altered and disturbed compound is not or does not behave like at least one active ingredient, and/or the nanoparticle such as the initial nanoparticle, the altered nanoparticle, or the altered and disturbed nanoparticle is not or does not behave like at least one active ingredient, and/or the bond such as the initial bond between initial compound and initial nanoparticle, the altered bond between altered nanoparticle and altered compound, the altered and disturbed bond between altered and disturbed compound and altered and disturbed nanoparticle is not or does not behave like at least one active ingredient.

26. The pharmaceutical composition according to claim 23, wherein the nanoparticle is a magnetosome.

27. A kit comprising at least one particle of the method according to claim 16 and further comprising a magnet or a gel.

28. The kit according to claim 27, wherein the magnet or gel keeps the at least one initial nanoparticle at an injection site and the compound is released over time.

29. The altered and disturbed particle according to claim 20, wherein said active ingredient is selected from the group comprising: i) a contrast agent, ii) a luminescent compound, iii) a drug or medicament, iv) a medical device, v) a cosmetic compound, vi) a therapeutic compound, vii) a medical compound, viii) a biological compound, ix) a diagnostic compound, x) a medical equipment or apparatus, xi) a composition, xii) a suspension, xiii) an excipient, xiv) an adjuvant, xv) a cytotoxic compound, xvi) a non-cytotoxic compound, xvii) an immunogenic compound, xviii) a non-immunogenic compound, xix) a pharmacological compound, xx) a non-pharmacological compound, xxi) a metabolic compound, xxii) a non-metabolic compound, xxiii) an antigen, xxiv) an antibody, xxv) a vaccine, xxvi) a virus, preferentially an attenuated or inactivated virus, xxvii) a metal, preferentially a non-toxic metal, iron, silver, or gold, xxviii) an antibiotic, xxix) a compound that is activated by being released from the nanoparticle, xxx) a compound that is activated more than once my being released more than once from the nanoparticle, and xxxi) a compound that is activated at least once by being released at least once from the nanoparticle by at least one alteration and/or physico-chemical disturbance.

30. The altered and disturbed particle according to claim 20, wherein: the released compound such as the released altered compound or the released altered and disturbed compound is at least one active ingredient or behaves like at least one active ingredient, and/or the non-released compound such as the initial compound, the non-released altered compound or the non-released altered and disturbed compound is not or does not behave like at least one active ingredient, and/or the nanoparticle such as the initial nanoparticle, the altered nanoparticle, or the altered and disturbed nanoparticle is not or does not behave like at least one active ingredient, and/or the bond such as the initial bond between initial compound and initial nanoparticle, the altered bond between altered nanoparticle and altered compound, the altered and disturbed bond between altered and disturbed compound and altered and disturbed nanoparticle is not or does not behave like at least one active ingredient.

31. The method according to claim 16, wherein the nanoparticle is a magnetosome.

32. The altered and disturbed particle according to claim 20, wherein the nanoparticle is a magnetosome.

Description

DESCRIPTION OF THE FIGURES

[0849] FIG. 1: For chains of magnetosomes extracted from magnetotactic bacteria (CM), size histogram, (a), TEM microscopic image, (b), and percentage of endotoxin released following one MS, (c). For HCl treated magnetosomes, size histogram, (d), TEM image, (e), and percentage of endotoxin released following one MS, (f). For chemically synthesized iron oxide nanoparticles (IONP), size histogram, (g), TEM image (h), and percentage of endotoxins released following one MS, (i). The MS consisted in the application of an AMF of 200 kHz and 27 mT during 30 minutes.

[0850] FIG. 2: (a), (b), TEM images and associated size histogram of U87-Luc cells incubated with magnetosomes during 24 hours. In (b), magnetosomes are internalized in a cell, within an intracellular vesicle. (c), Percentage of iron internalized in U87-Luc cells, when these cells were brought into contact with 700 μg of CM or IONP during 24 hours, and either not subjected to one MS (W/o MS) or subjected to one MS (with MS). (d), Percentage of living cells measured following a treatment in which U87-Luc cells were brought into contact with 700 μg/mL of IONP or CM during 24 hours and either no exposed to one MS or exposed to one MS, MS parameters are the same as those of the legend of FIG. 1.

[0851] FIG. 3: For mice having received glucose without MS, or with 3 or 15 MS, variations of tumor BLI following the day of tumor cell implantation (0 corresponding to D-8), (a), temperature variation measured during each MS, (b), survival rate following the day of tumor cell implantation, (c). For mice having received IONP without MS or with 3 or 15 MS, variations of tumor BLI following the day of tumor cell implantation, (a), temperature variation measured during each MS, (b), survival rate following the day of tumor cell implantation, (c).

[0852] FIG. 4: For mice having received CM without MS, or with 3 or 15 MS, variations of tumor BLI following the day of tumor cell implantation, (a), temperature variation measured during each MS, (b), survival rate following the day of tumor cell implantation, (c). In the inset of (c), two representative histological images of a brain slide of a mouse euthanized 250 days following tumor cell implantation. Both show an absence of tumor. One image shows the presence of CM while the other one lacks CM.

[0853] FIG. 5: (a), Scanning electron microscopic images of a brain section collected at D30 from a mouse treated by CM administration, showing cells and CM. (b), Magnetosome size distribution deduced from (a).

[0854] FIG. 6: (a), Scanning electron microscopic images of a brain section collected from a mouse treated by CM administration followed by 15 MS at D30, where each MS consisted in the application of an AMF of 200 kHz and 27 mT applied during 30 minutes, showing cells and CM. (b), Magnetosome size distribution deduced from (a).

[0855] FIG. 7: Schematic diagram showing a method for increasing the release of at least one compound initially bound to at least one nanoparticle, wherein said compound and/or said nanoparticle contain at least one active ingredient (M), said method comprising the steps of:

alteration of said nanoparticle and said compound leading to a size reduction of said nanoparticle, preferentially without reducing the size of said compound, and leading to the weakening or breaking of said bond between said compound and said nanoparticle,
first partial release of a first part of said compound(s) from said nanoparticle, for which said bond between the said compound and the said nanoparticle is entirely broken, the second part of said altered compound is the compound(s) still weakly bound to said nanoparticle,
irradiation of said degraded and size-reduced nanoparticle and of said second part of the degraded compound(s) remaining at the surface of said degraded and size-reduced nanoparticle,
second partial release of said second part of the degraded and irradiated compound(s),
the total percentage of release of the compounds being higher when performing steps 1) and 2), preferentially successively, than only performing step 3).

[0856] In FIG. 7) before step 1) or 3) the compounds are strongly, preferentially covalently, bound to the nanoparticle being preferentially a magnetosome (e.g. Fe.sub.3O.sub.4 or Fe.sub.3S.sub.4). This strong, preferentially covalent, binding is shown with 3 bonds.

[0857] In FIG. 7) the compounds are either released from the nanoparticle at step 1) (first partial release) or the compounds are still linked to the nanoparticle via a week link at step 3) (shown with one bond instead of three bonds).

[0858] In FIG. 7) at step 2) the remaining linked compounds are either released from the nanoparticle (second partial release) or a minority of said remaining linked compounds are still linked to the nanoparticle via a week link (shown with one bond instead of three bonds).

[0859] The technical effect of the alteration (step 1) is to weaken the bond between the altered nanoparticle and the altered compound in order to render the irradiation more efficient at step 2, i.e. increase the release of the compounds containing the active ingredient from the nanoparticles.

[0860] The radiation step 3 leads to a low release of compounds, while the successive combination of steps 1 and 2 leads to a high release of compounds.

[0861] FIG. 8: Schematic diagram showing the method for obtaining an altered particle comprising at least one of the following steps: α) applying an alteration on at least one initial particle comprising at least one initial nanoparticle and at least one releasable initial compound, which is initially bound to said initial nanoparticle via an initial bond, β) obtaining at least one altered particle, comprising at least one altered nanoparticle and at least one releasable altered compound, where a first partial release generates the release of a first part of altered compound during the alteration, said altered compounds being divided between: i) a first part of altered compounds comprising altered compounds released from the altered nanoparticle, and ii) a second part of altered compounds comprising altered compounds bound to the altered nanoparticle via an altered bond, wherein the said initial particle, altered particle, and/or altered and disturbed particle comprise at least one active ingredient (M). The initial bond is represented by three bonds and the altered bond is represented by two bonds only.

[0862] FIG. 9: Schematic diagram showing the method for obtaining an altered and disturbed particle comprising at least one of the following steps: γ) applying a physico-chemical disturbance on said altered particle, η) obtaining at least one altered and disturbed particle, comprising at least one altered and disturbed nanoparticle and at least one releasable altered and disturbed compound, where a second partial release generates the release of a second part of altered and disturbed compounds during physico-chemical disturbance, said altered and disturbed compounds being divided between: i) group 1 of second part of altered and disturbed compounds comprising altered and disturbed compounds bound to the altered and disturbed nanoparticle via an altered and disturbed bond, and ii) group 2 of second part of altered and disturbed compounds comprising altered and disturbed compounds released from the altered and disturbed nanoparticle, wherein the said initial particle, altered particle, and/or altered and disturbed particle comprise at least one active ingredient (M). The altered bond is represented by two bonds and the altered and disturbed bond is represented by one bond only.

[0863] FIG. 10: Schematic diagram showing the method for obtaining an altered and disturbed particle comprising at least one of the following steps: γ) applying a physico-chemical disturbance on said altered particle, η) obtaining at least one altered and disturbed particle, comprising at least one altered and disturbed nanoparticle and 100% of released altered and disturbed compound, when all altered and disturbed compounds are released from the altered and disturbed nanoparticle (i.e. the group 1 does not exist in this embodiment). The altered bond is represented by two bonds and the bound was entirely broken (no more shown) for released altered and disturbed compounds divided in two categories: a first category of altered compounds, originating from the first partial release of the altered compound and is not further released by physico-chemical disturbance from the altered and disturbed nanoparticle, and a second category of altered and disturbed compounds, originating from the second total release of the altered and disturbed compounds. (M) being the active ingredient. The altered bond is represented by two bonds. The released altered and disturbed compounds do no have any more bond associated to nanoparticle. The second total release represents 100% of the second part of altered compounds not released during the alteration step but which were released during/after the physico-chemical disturbance step.

EXAMPLES

[0864] Nano-therapies against cancer led to encouraging results, notably in the treatment of glioblastoma by magnetic hyperthermia. In order to be optimal, such treatments necessitate that nanoparticles remain for a sufficiently long time in the tumor to induce strong and persistent anti-tumor activity until full tumor disappearance. At the same time, nanoparticles also need to be eliminated. Their long term accumulation in a specific part of the organism should be avoided. Such a fine adjustment of nanoparticle bio-distribution properties can be obtained by using nanoparticles that are progressively captured and degraded by the organism. However, such behavior is often associated with a reduction in size, crystallinity, heating power, and anti-tumor efficacy of nanoparticles. To the author knowledge, it has not yet been shown that nanoparticles could maintain efficient anti-tumor activity under altering and/or size-reduced conditions.

[0865] Here, we introduce nanoparticles that remain efficient against the tumor even after their degradation in vitro and in vivo by a mechanism of release of immune-stimulant substances under the application of an alternating magnetic field (AMF). Due to the use of gram negative magnetotactic bacteria to synthesize them, these nanoparticles, called magnetosomes, are made of a mineral iron oxide core surrounded by a layer consisting of biological material, mainly consisting of lipids, proteins, and endotoxins. To determine if endotoxin release is modified under conditions of alteration, such mechanism was studied for two types of magnetosome suspensions, which were either untreated or partly dissolved by being mixed with a solution of HCl. Furthermore, to examine the effect of cellular internalization on magnetosome sizes and cytotoxicity, magnetosomes were brought into contact with U87-Luc glioblastoma tumor cells, followed (or not) by one magnetic session (MS). The cell viability and the size of the magnetosomes resulting from such treatment were then measured. In vivo studies were also carried out on mice bearing implanted intracranial U87-Luc glioblastoma tumors of 2 mm3, which received 40 μg of a suspension of magnetosomes followed by 3 or 15 MS. While a moderate increase in temperature was observed during the first 3 MS (0.5 to 3° C.), the temperature did not vary during the following ones. These conditions of mild temperature increase didn't prevent antitumor activity to remain persistent during the various MS, leading to total tumor disappearance among 50% of treated mice after 15 MS. To examine the possible presence of an immune response acting against the tumor and the kinetic of magnetosome degradation and/or alteration and/or size-reduction, histological and structural analyzes were carried out by optical and scanning electron microscopies on brain slices originating from mice treated with magnetosome injection followed (or not) by magnetic hyperthermia. Finally, in order to confirm or refute the hypothetic role of endotoxins in the antitumor activity, the results obtained with the magnetosome were compared with those collected under the same conditions but using chemically synthesized nanoparticles (BNF-Starch) without endotoxins instead of the magnetosomes.

[0866] Results and Discussion

[0867] Synthesis and characterization of nanoparticles (magnetosomes) with a large endotoxin concentration. The preparation of the suspension of magnetosomes involved the following steps: i), growth of AMB-1 Magnetospirillum Magnetotacticum magnetotactic bacteria (ATCC 700264) during 7 days, ii), harvesting of a concentrated pellet of these bacteria, iii), lysis of these bacteria under sonication at 0° C. during 2 hours at 30 W, iv), isolation of magnetosome chains (CM) from cellular organic debris using a magnet, v), re-suspension of CM in a sterile injectable solution containing 5% of glucose, and vi), partial sterilization of the CM suspension by exposing CM suspension to UV irradiation for 12 h. To determine the composition of CM, infra-red absorption measurements were first carried out on a lyophilized suspension of CM, which was denatured and solubilized with KBr. The infra-red absorption spectrum of CM displays the following features: i), Amide I and Amide II bands due to protein absorption at 1650 cm.sup.−1 and 1530 cm.sup.−1, ii), absorption bands due to lipopolysaccharide (LPS) or phospholipids contained in the magnetosome membrane at 1050 cm.sup.−1 and 1250 cm.sup.−1, iii), a peak at 580 cm.sup.−1 attributed to maghemite or magnetite. These results suggest a magnetosome composition consisting of an iron oxide mineral core, composed of maghemite and/or magnetite, surrounded by biological material, containing endotoxins (LPS), which binds magnetosomes together, in agreement with magnetosome composition reported elsewhere. A CHNS analysis of CM further confirmed the presence of a percentage of 12% of carbonaceous material surrounding the magnetosome mineral core. Furthermore, as can be observed in the TEM image of a dried suspension of CM deposited on top of a carbon grid, magnetosomes are characterized on the one hand by an organization in chains that prevents their aggregation, and on the other hand by a bimodal size distribution with two peaks centered at 22 and 40 nm (FIG. 1(a)), resulting in nanoparticles of sufficiently large sizes to yield a ferrimagnetic behavior at room temperature, i.e. with HC (coercivity) ˜20 mT and Mr/MS (ratio between remanent and saturating magnetization) ˜0.3. The stability of CM in suspension, which is required for efficient administration, is revealed, firstly by the behavior of the potential zeta variation of this suspension as a function of pH that displays a well-defined and repeatable behavior, i.e. a decrease from 20 mV at pH 2 to −35 mV at pH 12, and secondly by the absorption of this suspension, measured at 480 nm, which decreases moderately, i.e. by less than 30%, within 20 minutes following homogenization of this suspension (data not shown). Given the high endotoxin concentration of a CM suspension, i.e. 2000 EU per mg per mL, as well as the strong magnetosome heating power, we have studied if CM could both release endotoxins and produce heat following a MS. For the heating study, 2 μl containing 40 μg of CM mixed in water were introduced in a caliper and exposed during 600 seconds to an AMF of 200 kHz and strength 20 mT to approach in vivo conditions of treatment. Interestingly, while such treatment resulted in a significant heating power, characterized by temperature increase over the whole MS (ΔT) and specific absorption rate (SAR) of ΔT˜33° C. and SAR˜57 W/gFe (table 1), it produced a moderate release of endotoxins, estimated as QD/Qi˜0.5% (FIG. 1(c) and table 2), where QD is the quantity of endotoxins contained in the supernate of a CM suspension following MS, and Qi is the quantity of endotoxin contained in a CM suspension.

[0868] Magnetosomes treated in conditions mimicking in vivo degradation and/or alteration and/or size-reduction display an enhanced faculty to release endotoxins.

[0869] In an attempt to approach in vivo conditions of magnetosome degradation and/or alteration and/or size-reduction, we have introduced 3 mg in iron of CM in a solution containing 50 mL of 10 mM HCl at pH 1. We have then sonicated this suspension during 5 minutes at 10 W, using a sonicating finger. After having left the mixture over night at 50° C., we have collected the partly dissolved magnetosomes with a magnet. As can be seen in the TEM image of FIG. 1(e) and its corresponding size histogram (FIG. 1(d)), these magnetosomes appear to have lost their chain arrangement, to be more rounded and less faceted than untreated ones, and to possess a size distribution centered at 37 nm, which is mono-modal, less broad, and contains a lower percentage of small magnetosomes as compared with untreated magnetosomes of FIG. 1(a). Magnetosomes seem to be attacked by HCl in a different way depending on their size. While HCl treatment leads to the disappearance of the smallest magnetosomes, it does not induce a large variation in size of the largest ones, but instead seems to modify their surface, as deduced from the differences in magnetosome shapes observed between FIGS. 1(b) and 1(e). Most interestingly, when magnetosomes treated with HCl are heated by the AMF in the same conditions as for CM in section 2.1, the percentage of endotoxins that they release in the supernate following one MS is estimated as QAD/QA1˜7% (FIG. 1(f) and table 2), where QAD is the quantity of endotoxins contained in the supernate of the suspension of altered and disturbed particle and QA1 is the quantity of endotoxins contained in the suspension of altered particles. QAD/QA1 is larger than the value of QD/Qi˜0.5% measured for untreated magnetosomes (FIG. 1(c)). HCl treatment may have favored the release of endotoxins from the magnetosomes. Before such treatment, endotoxins may possibly be trapped in the biologic membrane surrounding the magnetosome mineral core, while after it the membrane could be partly denatured, letting endotoxins escape more easily.

[0870] Synthesis and characterization of nanoparticles (IONP) with a low endotoxin concentration.

[0871] IONP, which are chemically synthesized nanoparticles purchased from Micromod (BNF-Starch, reference: 10-00-102), are characterized by a series of different features compared with CM. They are composed of an iron oxide core surrounded by hydroxy-methyl-starch, as deduced from the analysis of the FT-IR spectrum of IONP, which shows a peak at 607 cm.sup.−1 attributed to iron oxide and two peaks at 1022 cm.sup.−1 and 1150 cm.sup.−1 due to starch polymer. Compared with CM, the percentage of carbonaceous material in IONP is lower at 8.5%, as revealed by CHNS measurements. Furthermore, as deduced from the TEM image of FIG. 1(g) and associated histogram (FIG. 1(h)), the majority of IONP are smaller than CM, i.e. IONP average size is 20 nm, and IONP organize in well dispersed small aggregates that differ from CM organization in chains. Despite their difference in organization and zeta potential values that are larger than those of CM for 4<pH<12, IONP appear to be sufficiently stable to be administered to mice or used for in vitro studies. Furthermore, although IONP behave ferrimagnetically at room temperature, their values of He˜10 mT and Mr/Ms˜0.15, are significantly smaller by a factor of ˜2 than those of CM. Possibly due to the low values of their hysteretic parameters, IONP heat less efficiently than CM, producing smaller temperature increase, ΔT˜4±2° C., and specific absorption rate, SAR 10±2 W/gFe (table 1), under the same heating conditions as for CM. IONP were chosen since they originate from an endotoxin free synthesis, leading to a nanoparticle suspension with a much lower endotoxin concentration than CM (0.1 EU/mg for IONP compared with 40 EU/mg for CM). In addition, less endotoxins were released from IONP than from CM following one MS, i.e. QD˜10.sup.−5 EU for IONP compared with QD˜7.7 10.sup.−3 EU for CM (table 2), and QD/Qi˜0.25% for IONP (FIG. 1(i)).

[0872] Magnetosomes brought into contact with U87-Luc cells internalize inside these cells, decrease in sizes, while maintaining a certain heating power and yielding cytotoxicity.

[0873] To study cellular interactions of magnetosomes in vitro, CM were incubated with U87-Luc cells during 24 hours, the cells were cut with a microtone in 80 nm thick slices, the latter were deposited on top of a carbon grid, and examined by transmission electron microscopy (TEM). Under these conditions of treatment, the TEM images of FIGS. 2(a) and 2(b), show that magnetosomes are localized inside a cell, more specifically within a cellular vesicle, which is most probably a lysosome. Compared with magnetosomes of FIG. 1(a) that are not in contact with cells, those of FIGS. 2(a) and 2(b) appear to have lost their organization in chains, to have acquired a different cubic shape, as highlighted by the black arrows designating cubic magnetosomes in FIGS. 2(a) and 2(b), and to have become smaller. Most interestingly, between before and after cellular internalization, the nanoparticle size distribution is shifted from a majority of large magnetosomes (˜40 nm, FIG. 1(a)) to a majority of small ones (˜11 nm, FIG. 2(a)). These observations indicate magnetosome cellular degradation and/or alteration and/or size-reduction, in agreement with the observations made on HCl treated magnetosomes (FIGS. 1(d) and 1(e)). Furthermore, cellular internalization appears to be enhanced following one MS. Indeed, the quantity of magnetosomes internalized inside U87-Luc cells following 24 hours of incubation increasing from 0.8% before MS to 9.2% after one MS (FIG. 2(c)). This behavior is further accompanied by a decrease in the percentage of living cells from 55% without MS to 35% with one MS (FIG. 2(d)). To understand if the efficacy of magnetosomes to destroy cells comes from intracellular heating, we have compared the properties of CM with those of IONP, where both types of nanoparticles have similar heating properties in vitro, i.e. SAR=57±11 W/gFe for CM compared with SAR=51±8 W/gFe for IONP and ΔT=7.4±0.7° C. for CM compared with ΔT=6.2±2° C. for IONP, but different internalization rates, i.e. the percentage of internalized nanoparticles varies between 0.2% (without MS) and 0.8% (with MS) for IONP and between 1.5% (without MS) and 9.2% (with MS) for CM. This last trend was confirmed by optical microscopy observations of U87-Luc cells incubated with IONP and CM during 24 hours, which were further exposed to one MS and stained with Prussian blue. Indeed, optical microscopy observations of these cells presented in the inset of FIG. 2(c) show a more persistent cyan coloration at cell location for CM than IONP, revealing the presence of a larger quantity of iron originating from nanoparticles in these cells for CM than IONP. Although these results support the idea that the cellular killing power of the magnetosomes stems from their ability to generate intracellular heating, the release of endotoxins can't be fully ruled out as another parameter responsible for in vitro magnetosome anti-tumor efficacy.

[0874] Persistent anti-tumor activity leading to full glioblastoma tumor disappearance by intra-tumor tumor administration of magnetosomes followed by AMF application even in the absence of a measured temperature increase.

[0875] Cancer thermotherapies currently in use in hospital such as high intensity focused ultrasound necessitate high heating temperatures (typically 80-90° C.) to be efficient, resulting in a number of side effects. To prevent them, the treatment can be carried out at more moderate temperatures under controlled conditions by using an external source of energy such as an AMF applied on nanoparticles contained in a tumor. Here, we therefore examine in vivo if glioblastoma can be efficiently treated when the tumor is only slightly heated during three first MS or not heated during twelve additional MS. We compare the behavior of CM with that of IONP to examine the potential roles of initial heating, endotoxin release, and cellular internalization in the anti-tumor activity.

[0876] For that, we have set-up an in vivo protocol of treatment in which 90 nude mice were divided into 9 different groups of 10 mice. We first administered 105 U87-Luc cells inside the brains of mice at the injection site (0.2.0) using a stereotactic helmet. We waited for 1 week that the tumor reached 2 mm3 and started the treatment at D0. The groups were treated as follows (Table 4):

Group 1 received at D0 2 μl of a solution of 5% of glucose at (0.2.0);
Group 2 received at D0 2 μl of 5% of glucose at (0.2.0) followed by 3 MS at D0, D1, and D2;
Group 3 received at D0 2 μl of 5% glucose at (0.2.0) followed by 12 MS at D0, D1, D2, D7, D8, D9, D14, D15, D16, D21, D22, D23;
Group 4 received at D0 2 μl of CM at (0.2.0);
Group 5 received at D0 2 μl of CM at (0.2.0) followed by 3 MS at D0, D1, D2;
Group 6 received at D0 2 μl of CM at (0.2.0) followed by 15 MS at D0, D1, D2, D7, D8, D9, D14, D15, D16, D21, D22, D23, D28, D29, D30;
Group 7 received at D0 2 μl of IONP at (0.2.0);
Group 8 received at D0 2 μl of IONP at (0.2.0) followed by 3 MS at D0, D1, D2;
Group 9 received at D0 2 μl of IONP at (0.2.0) followed by 12 MS at D0, D1, D2, D7, D8, D9, D14, D15, D16, D21, D22, D23.

[0877] Each MS (magnetic session) consisted in the application of an AMF (alternating magnetic field) of average strength 27 mT and frequency 200 kHz during 30 minutes. During the course of each MS, the temperature of the mouse brain was monitored with an infra-red camera. The size of the tumor, which was shown to be proportional to the tumor bioluminescence intensity BLI, was followed by measuring the BLI of the mouse brains during the day preceding each MS.

[0878] We first consider the control groups, which were not treated by nanoparticle injection followed by AMF application, i.e. those receiving glucose with/without MS (groups 1, 2, 3), or IONP/CM without MS (groups 4 and 7). In these groups, tumor BLI increased exponentially from 0 at D0 to 109-7.109 at D28 (FIGS. 3(a), 3(b), and 4(a)) and the temperature of the mouse brain remained constant during the course of each MS (FIGS. 3(b) and 3(e)). Antitumor activity did not take place and mice belonging to these groups were euthanized at D29 to D36 (FIGS. 3(c), 3(f), 4(c) and table 5), when the tumor volume exceeded 1000 mm3. Mice belonging to groups 8 and 9, which received IONP followed by 3 or 12 MS, displayed a similar behavior with an absence of any sign of efficacy or temperature increase (FIGS. 3(d) and 3(f)).

[0879] By contrast to the behaviors described above, mice treated by CM administration followed by 3 MS were prone on the one hand to a temperature increase, which was moderate and decreasing with increasing number of MS, i.e. 4, 2, and 0.5° C. during the first MS at D0, second MS at D1, and third MS at D3, respectively (FIG. 4(b)), and to a partial anti-tumor activity highlighted by the BLI decrease observed between D3 and D10 in a mouse belonging to group 5. Such partial effect was insufficient to prevent tumor re-growth after D10, which is highlighted by an exponential increase of tumor BLI between D10 and D17. Mice needed to be euthanized at D34 (FIG. 4(c) and table 5), without any improvement in median survival day compared with control groups (FIG. 4(c) and Table 5).

[0880] In order to further enhance therapeutic activity, mice belonging to group 6 were injected with CM and exposed to an additional 12 MS as compared with group 5. Under these conditions, the improvement of therapeutic activity is revealed firstly by the BLI averaged over all mice that does not increase between D0 and D242 (FIG. 4(a)), secondly by the BLI of a mouse of group 6, which continuously decreases between D4 (second MS) and D30 (fifteenth MS) and remains almost undetectable after D30, thirdly by the full tumor disappearance in 50% of mice belonging to this group, which are still alive at D242 (FIG. 4(c)), and fourthly by a mouse median survival day (MSD) above D242, which is much larger than the MSD of D27 to D38 estimated for the other groups (table 5). Furthermore, mice, which were still alive at D242, were euthanized for histological analysis. Optical micrographs of two representative brain sections of these mice are presented in FIG. 4(c), showing either some remains of magnetosomes or no sign of these nanoparticles. They are further characterized by an absence of tumor cells, lesion and edema, supporting the idea that the treatment leads to full tumor disappearance without inducing severe side effects. We concluded that although the tumor temperature stopped to increase following the third MS (FIG. 4(b)), anti-tumor activity remained strong. Furthermore, the presence of anti-tumor activity for CM and not for IONP might suggest that anti-tumor activity is only triggered in the presence of a minimum of initial temperature increase, such as that observed during the three first MS, nanoparticle cellular internalization, and/or endotoxin release. We therefore decided to study if such behavior could be due to the activation of the immune system against the tumor, possibly triggered by endotoxin release under conditions of magnetosome degradation and/or alteration and/or size-reduction.

[0881] Immune system activation by magnetosomes exposed to a magnetic session.

[0882] To examine the potential involvement of the immune system in the anti-tumor activity, we have analyzed histologically slides of mouse brains belonging to groups 1, 2, 4, 5, 7 and 8, which were collected at 6 and 72 hour following nanoparticle or glucose administration. In mice treated by injection of IONP or glucose followed (or not) by MS, the presence of other cells than tumor or healthy ones was not observed, suggesting that the immune system may not have been activated in this case. By contrast, when CM were administered in the mouse brain without MS (group 4), poly-nuclear neutrophils (PNN) were initially observed 6 hours following injection within the same region as that of CM. This behavior may be due to the release of endotoxins by magnetosomes, as observed for CM in suspension, which could attract PNN towards the magnetosomes. At 72 hours, PNN seemed to have left this region since they were not observed by histological analysis in a slide of mouse brain belonging to this group. The most interesting behavior was observed in group 5, in mice having received magnetosomes followed by MS. Indeed, the co-localization of magnetosomes and PNN was observed both at 6 hour following one MS and at 72 hours following three MS. After MS, PNN were observed to be in the proximity of the magnetosomes, suggesting that AMF application triggers a mechanism of PNN re-attraction towards magnetosomes after PNN have left the magnetosome region between 6 and 72 hours. Thus, we have highlighted a system of repeatable immune system activation by exposing nanoparticles releasing endotoxins to several AMF applications. The involvement of PNN in the destruction of the tumor is an assumption that can't be dismissed although its firm proof is difficult to establish and previous studies suggested that pro-tumor and anti-tumor activities can both be triggered by PNN.

[0883] Magnetosomes reduce in size following intra-tumor administration without losing their faculty to trigger antitumor activity.

[0884] We have established that partly dissolved magnetosomes efficiently released endotoxins under AMF application. We therefore examined if such magnetosome degradation and/or alteration and/or size-reduction was taking place in vivo, since it could yield a strong immune response triggered by endotoxins. For that, slides of brains of mice bearing U87-Luc tumors treated by CM administration followed (or not) by MS were analyzed by scanning electron microscopy (SEM). In the absence of MS, the SEM image of FIG. 5(a) shows that magnetosomes are localized in the same region as cells, and are characterized by an average size of 43 nm and a mono-modal size distribution (FIG. 5(b)). Interestingly, between before and after CM administration, the magnetosome size distribution switches from bimodal to mono-modal and the magnetosomes of the smallest sizes disappear (FIGS. 1(a) and 5(b)), possibly due to magnetosomes cellular degradation and/or alteration and/or size-reduction, which could lead to full dissolution of the smallest magnetosomes of ˜22 nm (FIG. 1(a)). This behavior is consistent with that observed for magnetosomes treated with HCl. Following 15 MS, FIGS. 6(a) and 6(b) show that magnetosomes are located in the same region as cells with an average size that has decreased down to ˜29 nm, suggesting that magnetosomes have partly dissolved following the various MS, but still have a sufficiently large size to potentially induce cytotoxicity. Indeed, their size is not smaller than that of internalized magnetosomes (FIG. 2(a)) that induced efficient cellular destruction. While the decrease in size of magnetosomes leads to a loss of magnetosome heating power, i.e. the tumor temperature does not increase between the third and fifteenth MS (FIG. 4(b)), such behavior is not associated with a loss of antitumor activity, i.e. the tumor decreases in size until full disappearance between the third and fifteenth MS. Such interesting observations could be attributed to an increase of endotoxin release in degraded and/or altered and/or size-reduced magnetosomes of smaller sizes, which could activate the immune system against the tumor following AMF application.

[0885] Conclusion

[0886] Cancer nano-therapies have raised a surge of interest in the medical field due to their potential larger benefit to risk ratio compared with conventional treatments. To achieve optimal treatment outcome, nanoparticle distribution needs to be precisely controlled. On the one hand, nanoparticles should be degraded and/or altered and/or size-reduced to enable their elimination by the organism. On the other hand, such mechanism should not prevent persistent antitumor activity until full tumor disappearance. Here, we have studied nanoparticles with such desired properties, which are called magnetosome, are synthesized by magnetotactic bacteria, and are extracted from these cells for their use. Magnetosome composition consists of a ferrimagnetic iron oxide mineral core surrounded by a layer containing endotoxins, enabling both a favorable coupling between the magnetic moment of these nanoparticles and the external magnetic field and the release of endotoxins, which can potentially trigger an immunogenic reaction against the tumor. The different conditions of magnetosome degradation and/or alteration and/or size-reduction and the behaviors resulting from them were as follows:

i) When magnetosomes were treated by being mixed in solution with a HCl solution, the smallest magnetosomes were dissolved and the surface of the largest ones were degraded and/or altered and/or size-reduced, leading to a percentage of endotoxins released from magnetosomes, which increased from 0.5% to 7% between before and after treatment.
ii) Magnetosomes brought into contact with U87-Luc tumor cells internalize inside these cells, yielding a decrease of the average size of the majority of magnetosomes from ˜40 nm to ˜11 nm. Despite this decrease in sizes, magnetosomes are still able to induce ˜20% of cellular death when they are exposed to an AMF of 200 kHz and 27 mT during 20 minutes in the presence of these cells.
iii) Between before and after their administration to U87-Luc mouse tumor, the smallest magnetosomes disappear from the magnetosome size distribution that switches from bimodal (two peaks centered at ˜22 nm and ˜40 nm) before administration to mono-modal (one peak centered at ˜43 nm) after administration. An additional mouse treatment involving 15 MS, where each MS consisted in the application of an AMF of 200 kHz and 27 mT for 30 minutes, led to a further reduction in magnetosome sizes down to ˜29 nm. Despite this reduction in sizes, which resulted in a moderate temperature increase in the tumor, i.e. ˜4° C., ˜2° C., and ˜0.5° C. during the first, second, and third MS, respectively, and an absence of temperature increase between the fourth and fifteen MS, magnetosomes remained active against the tumor during the various MS until full tumor disappearance. Efficient magnetic hyperthermia in the absence of a strong temperature increase was previously reported. Here, such behavior could be explained by an immune reaction against the tumor, as suggested by the presence of poly-nuclear-neutrophils observed after one or three MS, or the release of endotoxins that is enhanced when magnetosomes are degraded and/or altered and/or size-reduced. Table 6 summarizes the effect of the different conditions of degradation on nanoparticle sizes.

Experimental Section

[0887] Preparation of magnetosomes. To synthetize CM, we purchased magnetospirillum magneticum strain AMB-1 magnetotactic bacteria from the ATCC (700264). 4.107 of these bacteria were introduced into one liter of sterile 1653 ATCC culture medium. The media containing the bacteria were then placed in an incubator at 30° C. for 7 days to enable bacterial growth and magnetosome production. After 7 days, the media were centrifuged at 4000 g for 45 minutes. The bacterial pellet was washed using 1 ml of sterile water. Magnetotactic bacteria were concentrated using a strong Neodinium magnet (0.6 Tesla), resuspended in 0.05 M TRIS and sonicated continuously with finger at 0° C. during 2 hours at 30 W. The suspension of magnetosomes was washed several times with sterile water using a magnet to isolate magnetosome chains from the supernatant containing cellular debris and residual bacteria until cellular debris have disappeared from the supernate. Between each wash, sonication was carried out at 30 W by a series of three pulses of 2 seconds. Magnetosome chains were then resuspended in 1 mL of sterile water. For intracranial injections, magnetosome chains were resuspended in a sterile injectable solution containing 5% of glucose and exposed to irradiation of a UV lamp (UV) for 12 h for partial sterilization.

[0888] Preparation of chemically synthesized iron oxide nanoparticles (IONP). Chemically synthesized ferrimagnetic iron oxide nanoparticles (IONP) were purchased from Micromod (BNF-Starch, reference: 10-00-102. They were then centrifuged at 14,000 rpm (12×4 g) for 30 min and washed 3 times with a sterile injectable solution of 5% glucose.

[0889] TEM. To determine nanoparticle sizes, shapes, and organization, 7 μl of nanoparticle suspension were deposited on top of a carbon grid, left to dry, and then nanoparticles were then imaged using a transmission electron microscope (JEM-2100, JEOL, Japan). To obtain TEM micrographs of assemblies of cells and nanoparticles, we prepared the samples in the following manner: i), removal of culture medium from the sample containing U87 cells incubated with magnetosomes for 24 hours, ii) washing of cells with 0.2 M sodium cacodylate buffer, iii) fixing cells for 1 hour at room temperature with 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer, iv) washing 2 times the cells with 0.2 M cacodylate buffer, v) storage of cells at 4° C., vi) post-fixing of cells with osmium tetraoxide 1% and passing cells through uranyl acetate, vii) cell dehydration in an ethanol series (30%-100%), viii) embedment of cells in epoxy medium (EPON 812; Shell Chemical, San Francisco, Calif.). Ultrathin sections (80 nm) were stained by lead citrate and were examined by using a ZEISS EM902 TEM operated at 80 kV (Carl Zeiss-France, MJMA2 Microscopy Platform, UR1196, INRA, Jouy en Josas, France). Images were acquired with a charge-coupled device camera (Megaview III) and analyzed with ITEM Software (Eloise, France).

[0890] Nanoparticle characterization by absorption, CHNS, FTIR, DLS, magnetic measurements. The stability of nanoparticles in suspension was estimated by measuring the variation of the optical density of nanoparticle suspensions at 1 mg/mL in iron, measured at 480 nm, within 15 min following the homogenization of the suspension. Zeta potential of the different nanoparticles in suspension was measured by Dynamic light scattering, DLS (ZEN 3600, Malvern Instruments, UK) whose pH was adjusted between a pH 2 and 12 by using HCl and NaOH solutions. Nanoparticle FTIR spectra were recorded with a FTIR spectrometer (Vertex 70, Bruker, USA) on lyophilized nanoparticle suspensions mixed with KBr. The percentage in mass of organic material at nanoparticle surface was estimated using an elemental CHNS analyzer (Flash EA 1112, Thermo Fisher Scientific, USA). Magnetic properties of the nanoparticles were determined by measuring nanoparticle magnetization curves at room temperature between −1 and +1 T, using a vibrating sample magnetometer (VSM3900, Princeton Measurements Corporation, USA).

[0891] Measurement of the quantity of endotoxins contained in nanoparticle suspensions with the LAL assay. The endotoxin concentration was measured with the LAL assay. The latter was carried out under sterile conditions using the 88282 ThermoScientific kit called “Pierce LAL Chromogenic Endotoxin Quantitation Kit”.

[0892] Measurement of the percentage of endotoxins released by nanoparticles in suspension. 2 μl of suspensions containing 28 μg in iron of nanoparticles were introduced at the bottom of a small caliper mimicking in vivo conditions and exposed (or not) to 1 MS, during which an AMF of 202 kHz and strength 27 mT was applied during 30 minutes. The supernate was then isolated from the nanoparticles using a magnet and its endotoxin concentration was measured using the LAL assay, as described above. The percentage of endotoxins released corresponded to the ratio QAD/Qi, QAD/QA1, QD/Qi. Determination of the nanoparticle specific absorption rate. The variations of temperatures as a function of time were measured in the various treatments, during which nanoparticles in suspension, in contact with cells or in tumor brain were exposed to the AMF. The specific absorption rate was measured using the relation: SAR=(ΔT/δt).Math.(Cv/XFe), where Cv=4.2 J/gK is the specific heat capacity of water, XFe is the nanoparticle concentration in iron expressed in g/mL, (ΔT/δt) is the initial temperature variation with time expressed in K/sec.

[0893] Cell cultivation. Human GBM cell lines (U87-MG Luc) transduced with a Luciferase gene were cultivated in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37° C. in the presence of 5% CO2. After reaching confluence, culture medium was removed using Hank's Balanced Salt Solution (HBSS). Following trypsinization at 37° C. during 5 minutes, cells were detached, FBS was then added to stop the action of trypsin, and cellular concentration was measured using a Malassez counting cell.

[0894] In vitro treatment of cells. 5.10.sup.5 U87-Luc cells were seeded at the bottom of Petri dishes of 35 mm diameter for 24 hours. Nanoparticles of concentration in iron of 700 μg/mL were added (or not) and exposed (or not) to on MS, during which an AMF of 27 mT and 200 kHz was applied for 30 minutes. The treated assemblies of cells and nanoparticles, hence obtained, were incubated at 37° C. for an additional day. The medium containing (or not) nanoparticles was then removed and the cells were washed twice with cold PBS. Following the in vitro treatment, the percentages of living and apoptotic cells were measured using the FITC Annexin V/Dead Cell Apoptosis Kit (ThermoFisher scientific, reference: V13242). For that, 10 μL of the washed cells were loaded into the sample slide and were inserted completely into a Countess™ II FL Automated Cell Counter (Thermo Fisher scientific, reference: 15307812), which was able to detect Annexin and Propidium Iodide fluorescence emission. Following the in vitro treatments, the number of cells in the assemblies was counted by a Countess™ II FL Automated Cell Counter. Assemblies of washed cells were centrifuged, the supernatant was removed and replaced by 286 μl of HNO.sub.3 (70%). The treated assemblies were kept at 4° C. during 24 hours to lyse cells and dissolve nanoparticles into free iron. Finally, 10 mL of filtered water were added to all treated mixtures and iron concentration was then determined using ICP-AES measurements. We deduced the average quantity of iron coming from the magnetosomes, which was internalized in each cell, using the following formula: Iron internalization (%)=100*(Q/Q°), where Q and Q° correspond to the quantity of iron internalized per cells after and before treatment, respectively. After in vitro treatment described above, cells were also stained with Prussian blue, and observed under optical microscope to examine the presence of iron, which appeared in blue color. In vivo mouse treatments. The in vivo protocol was approved by the local animal ethics committee of the University Pierre-et-Marie-Curie (Paris, France). 6 weeks old CD-1 female nude mice of average weight 20 g were purchased from Charles River. All mice were treated and kept in an environment complying with ethical guidelines and surgery was carried out following the guidelines of the Institutional Animal Care and Use Committee (“Ethic committee Charles Darwin N.sup.o 5”). Mice were fed and watered according to these guidelines and we used cervical dislocation to euthanize them when their weight had decreased by more than 20% or when signs of pain, unusual posture or prostration were observed. Mice were divided in 9 groups of 10 mice. For the various treatments, the mice were anesthetized with a mixture of Ketamine (100 mg/kg) and Xylazine (8 mg/kg) in isotonic solution (0.9% of NaCl). To administer the tumor cells at D-8 and the various treatments at D0 (glucose, IONP, CM), a surgical procedure was carried out. For that, the mouse heads were fixed in a stereotactic frame, a craniotomy was realized at coordinates (0.2.0) mm and the cell suspension or various treatments (glucose, IONP, CM) were administered at (0.2.2.) mm. To follow tumor size evolution, Bioluminescence intensity (BLI) emitted by living tumor cells was measured during the day preceding each MS. We estimated that BLI maximum signal was reached 10 minutes following luciferin administration and we therefore measured BLI at that time in each mouse. A relation between tumor volume and tumor BLI was established by measuring histologically tumor volumes in a series of mice euthanized at different days following tumor cell implantation and tumor BLI in living mice at the same days as those of the euthanasia. The spatial temperature distribution in the tumor was recorded during each MS with an infrared camera (EasIRTM-2, Optophase) positioned 20 cm above the coil generating the AMF. We verified that the maximum temperature measured with the infrared camera was the same as that of the temperature measured with a thermocouple microprobe (IT-18, Physitemp, Clifton, USA) positioned at tumor center and we plotted the maximum temperature as a function of time during each MS. Mouse body weights were measured every day and mice were euthanized when losses in mouse body weights exceeded 20%. Mice, which were still alive at D250, were euthanized and brain sections were collected for further histological examination by the hematoxylin and eosin (H&E) staining to determine if the tumor had fully disappeared. Mouse survival times were plotted according to the Kaplan-Meier method. Statistical significance of survival time between the different groups was evaluated using the log rank test. Parameters were expressed as median and p-values, relative to control group.

[0895] Scanning electron microscopy (SEM) analysis of slides of mouse brains. Mouse brains were washed with 10% of sucrose, embedded in OTC (TissueTek), and kept at −80° C. in bath of isopentane cooled by liquid nitrogen. Frozen sections of their brains were obtained by cryocut (10 μm), deposited on a stub, covered by a carbon layer and analyzed by scanning electron microscope (SEM-FEG Zeiss Ultra55). We obtained surface images of cells with nanoparticles from which we could measure magnetosome sizes.

[0896] Histological analysis of slides of mouse brain. Brains were extracted from euthanized mice, fixed with a 4% solution of formaldehyde for 24 hours, cut into 2 mm thick transverse slices, washed in ethanol (70%) bath for 12 hours, and embedded in paraffin. Sections of 4 μm thick paraffin blocks were deposited on glass slides and stained with hematoxylin-eosin (H&E) to distinguish between healthy and tumor area, to determine nanoparticle location, and to examine the presence of poly-nuclear neutrophils (PNN).

[0897] Summary of experimental results:

[0898] First type of alteration: introduction of magnetosomes in a 10 mM HCl solution of pH 1.

[0899] Effect of alteration on magnetosome properties (observed for at least one magnetosome):

Organization of magnetosomes changes from an arrangement in chains before alteration to an arrangement not in chains after alteration (FIGS. 1(b) and 1(e));
Magnetosome morphology changes from cubo-octahedric before alteration to round after alteration (FIGS. 1(b) and 1(e));
Size distribution of magnetosomes changes from bimodal before alteration to mono-modal after alteration (FIGS. 1(a) and 1(d)). Magnetosomes smaller than 15 nm disappear after alteration (FIG. 1(d)).

[0900] Quantity of compounds (endotoxins) released from magnetosomes by application of a radiation, which is an AMF, increases from ˜0.5% without alteration to ˜7% with alteration (FIGS. 1(c) and 1(f)). Increase by a factor 14 of the faculty to release a compound from the magnetosomes under radiation by the alteration of the magnetosomes.

[0901] Second type of alteration: Magnetosomes brought into contact with U87-Luc cells.

[0902] Effect of alteration on magnetosome properties:

Organization of magnetosomes changes from an arrangement in chains before alteration to an arrangement not in chains after alteration (FIGS. 1(b) and 2(b));
Magnetosome morphology changes from cubo-octahedric before alteration to cubic after alteration (FIGS. 1(b) and 2(b));
Size distribution of magnetosomes changes from a distribution with a majority of large magnetosomes of average sizes 40 nm before alteration to a distribution with a majority of magnetosomes of average sizes 11 nm after alteration (FIGS. 1(a) and 2(a)).

[0903] Magnetosome anti-tumor activity is maintained after alteration. The percentage of dead cells decreases from ˜55% for magnetosomes incubated with U87-Luc cells in the absence of magnetic session to ˜35% for magnetosomes incubated with U87-Luc cells followed by one magnetic session (FIG. 2(d)).

[0904] Third type of alteration: Magnetosome administration to tumors with (or without) magnetic sessions. In the absence of magnetic session, the magnetosome size distribution changes from bimodal (two peaks centered at 22 nm and at 40 nm) before magnetosome administration to mono-modal (1 peak centered at 43 nm) after magnetosome administration (FIGS. 1(a) and 5(b)).

[0905] In the presence of magnetic sessions, the magnetosome size distribution changes from mono-modal with 1 peak centered at 43 nm 30 days after magnetosome administration (D30) without magnetic sessions to mono-modal with 1 peak centered at 29 nm at D30 following 15 magnetic sessions (FIGS. 5(b) and 6(b)). The large magnetosomes disappear following the magnetic sessions.

[0906] Despite magnetosome alteration, anti-tumor activity is persistent, which could be explained by the release of the compound (endotoxin) under altering conditions that can attract and activate the cells of the immune system against the tumor in vivo, under AMF application.

TABLE-US-00001 TABLE 1 Table 1: Specific absorption rate (SAR), and temperature increase over the whole MS, ΔT, measured for: i), 2 μl or 100 μl of water containing 20 mg/mL of CM and IONP exposed to one MS, ii), 700 μg/mL of CM and IONP incubated with U87-Luc cells during 24 hours and exposed to one MS, iii), 2 μl containing 20 mg/mL of CM or IONP administered to 2 mm.sup.3 U87-Luc tumors and exposed to 1 MS, 2 MS, 3 MS, 4 MS, 5 MS, 6 MS to 15 MS. To comment the values of i), when the SAR and ΔT are measured in a volume as small as 2 μl, which corresponds to the maximum volume that can be administered in a mouse brain, SAR and ΔT values are clearly underestimated compared SAR and ΔT values measured in a larger volume (100 μl). Each MS consisted in the application of an AMF of 200 kHz and 27 mT during 30 minutes: Nanoparticle heating properties Nanoparticle Type/condition SAR (W/g.sub.Fe) ΔT (° C.) CM (in 2 μL suspension) 57 ± 6 33 ± 3  20 mg/mL in iron IONP (in 2 μL suspension) 10 ± 2 4 ± 2 20 mg/mL in iron CM (in 100 μL suspension) 1234 ± 307 95 ± 8  20 mg/mL in iron IONP (in 100 μL suspension) 58 ± 3 62 ± 5  20 mg/mL in iron CM (in vitro, 2 mL)  57 ± 11 7.4 ± 0.7 700 μg/mL in iron IONP (in vitro, 2 mL) 51 ± 8 6.6 ± 2.sup.  700 μg/mL in iron CM (in vivo) 1 MS  4.7 ± 1.5 4 ± 1 2 μL, 20 mg/mL in iron CM (in vivo) 2 MS 2.5 ± 1  1.7 ± 1.sup.  2 μL, 20 mg/mL in iron CM (in vivo) 3 MS  2 10.sup.−3 ± 1 10.sup.−4 0.4 ± 0.4 2 μL, 20 mg/mL in iron CM (in vivo) 4 MS  2 10.sup.−3 ± 1 10.sup.−4 0.4 ± 0.4 2 μL, 20 mg/mL in iron CM (in vivo) 5 MS  2 10.sup.−3 ± 1 10.sup.−4 0.4 ± 0.4 2 μL, 20 mg/mL in iron CM (in vivo) 6 MS to 15 MS 0 0 2 μL, 20 mg/mL in iron IONP (in vivo) 1 MS to 12 MS 0 0 2 μL, 20 mg/mL in iron

TABLE-US-00002 TABLE 2 For a suspension of CM (chains of magnetosomes extracted from magnetotactic bacteria) or IONP (chemically synthesized nanoparticles), quantity of endotoxins in initial nanoparticle suspension before alteration by HCl treatment, Qi, quantity of endotoxins in suspensions of altered nanoparticles after alteration by HCl treatment, QA1, quantity of endotoxins removed from initial nanoparticles by alteration, QA2 = Qi − QA1, quantity of endotoxins in supernate of suspension of disturbed nanoparticle, QD, quantity of endotoxins in supernate of suspension of altered and disturbed nanoparticles, QAD, where the application of the physico-chemical disturbance consists in the application of one MS. Percentage of endotoxin release for CM exposed to alteration and/or physico- chemical disturbance, QA2/Qi (alteration step), QAD/Q1 (physico-chemical disturbance step). Percentage of endotoxin release for CM exposed to physico-chemical disturbance, QD/Qi. Percentage of endotoxin release for IONP exposed to physico-chemical disturbance, QD/Qi. The MS consists in the application of an AMF of 200 kHz and 27 mT during 30 minutes. Percentage of endotoxins released by nanoparticles in suspension following a magnetic hyperthermia session Quantity of Quantity of Quanity of endotoxins in Quantity of Quantity of endotoxins endoxotins in supernate of Steps of the endotoxins endotoxins in removed from supernate of suspension of method in initial suspension intial suspension of altered and followed nanoparticle of altered nanoparticle disturbed disturbed Percentage of (alteration: A) suspension nanoparticle by alteration nanoparticle nanoparticle endotoxin (Disturbance: D) (Q ) (QA.sub.2) (QA.sub.2) (Q ) (Q ) release A + D (CM) 1.6 EU  EU ( ) = N.A 6.6 10.sup.−3 EU  = 94% 1.5 EU (A step)  = 7% (D step) D (CM) 1.6 EU N.A. N.A. 7.7 10  EU N.A. Q /Q  = 0.5% D (IONP) 4.10.sup.−3 EU   N.A. N.A. 1.0 10  EU N.A. Q /Q  = 0.25% indicates data missing or illegible when filed
Endotoxins=compounds
QA2/Qi: For CM, percentage of endotoxin release after the alteration step relatively to the quantity of endotoxins in the initial particle;
QAD/QA1: For CM, percentage of endotoxin release after alteration and physico-chemical disturbance steps relatively to the quantity of endotoxins in the altered particle;
QD/Qi: For CM or IONP, percentage of endotoxin release after physico-chemical disturbance step relatively to the quantity of endotoxins in the initial particle (no alteration step is performed in this case).

[0907] The method of the present invention shows that:

For CM exposed to the altered and physico-chemical disturbance steps, a release percentage of endotoxins (altered compound) from the altered nanoparticle of 94% when performing the alteration step, followed by a release percentage of endotoxins from the altered and disturbed nanoparticle of 7% of the 94% when performing the physico-chemical disturbance step, as illustrated in FIGS. 7 to 10. For CM exposed to the physico-chemical disturbance step, a release percentage of endotoxins (compounds) of 0.5% when performing the physico-chemical disturbance step (one MS) in the absence of alteration.

[0908] For IONP exposed to the physico-chemical disturbance step, a release percentage of endotoxins (compounds) of 0.25% when performing the physico-chemical disturbance step (one MS) in the absence of alteration.

TABLE-US-00003 TABLE 3 Table 3: Percentages of stability of 1 mg/mL of CM and IONP mixed in water, measured by estimating the decrease in absorption at 480 nm of these suspensions within 20 minutes. Thickness of the coating of CM and IONP. Size of IONP and CM. Iselectric point of CM and IONP suspension. CM IONP % Stability on water 70   100 ([Fe] = 1 mg/mL) Coating thickness (nm) 1-5 1-4 Size (nm) 22 and 40 20 Isoelectric point (pH) 4.2 9.5

TABLE-US-00004 TABLE 4 Table 4: Treatment conditions of the different groups of mice. D −8 0 1 2 7 8 9 14 15 16 21 22 23 28 29 30 Days 0 8 9 10 15 16 17 22 23 24 29 30 31 36 37 38 Group 1 Injection G5 euthanasia of mice The end by Group 2 of G5 +H +H +H euthanasia of mice hyperthermia Group 3 U87-Luc G5 +H +H +H +H +H +H +H +H +H +H +H +H euthanaisa of mice treatment Group 4 cells CM euthanaisa of mice Group 5 CM +H +H +H euthanaisa of mice Group 6 CM +H +H +H +H +H +H +H +H +H +H +H +H +H +H +H Group 7 IONP Group 8 IONP +H +H +H euthanaisa of mice Group 9 IONP +H +H +H +H +H +H +H +H +H +H +H +H euthanaisa of mice +H => Applications of alternating magnetic field (27 mT, 202 kHz, 30 min) CM => Injection of chain of magnetosome IONP => Injection of IONP G5 => Infection of isotonic solution (5% of glucose) Euthanasia of mice (weight decreased of 20%) Bioluminescence measurement days: 7, 14, 21, 28, 35, 39, 45, 51, 59, 150 and 250

TABLE-US-00005 TABLE 5 Table 5: Median survival day and associated p-value estimated for the different groups of treated mice. Treatment Median Survival day p-value Group 1 G5 37 (D 29) Group 2 G5 + 3 MS 37 (D 29) 0.591 Group 3 G5 + 15 MS 42 (D 36) 0.263 Group 4 CM 36 (D 28) 0.552 Group 5 CM + 3 MS 42 (D 34) 0.069 Group 6 CM + 15 MS 250 (D 242) 0.001 Group 7 IONP 39 (D 31) 0.072 Group 8 IONP + 3 MS 35 (D 27) 0.480 Group 9 IONP + 12 MS 48 (D 38) 0.005

TABLE-US-00006 TABLE 6 Properties of magnetosomes after and before degradation, where the properties are: i) the average size measured over the whole size distribution (Pt) or over different peaks of the size distribution (P1 or P2), ii) the FWHM of the whole size distribution (Pt) or of the different peaks of the size distribution (P1 or P2), iii) minimum nanoparticle size of the whole size distribution (Min), and iv) maximum nanoparticle size of the whole size distribution (Max). The magnetosomes are either not degraded (condition before degradation), or degraded (condition after degradation) in the following conditions: i) the suspensions of magnetosomes are mixed with a 10 mH HCl solution of pH 1, ii) the magnetosomes are brought into contact with U87-Luc cells, iii) the magnetosomes are administered to mouse tumors without AMF application, iv) the magnetosomes are administered to mouse tumors with AMF applications. BEFORE DEGRADATION AFTER DEGRADATION Distri. Av FWHM Min Max Condition of Distrib. Av FWHM Min Max type (nm) (nm) (nm) (nm) degradation type (nm) (nm) (nm) (nm) Bi- 17.5 (P1) 20 (P1) 2.5 5.5 Suspension of Mono- 37 20 15 55 model 37.5 (P2 15 (P2) magnetosomes model dominant) 35 (Pt) mixed with HCl 27.5 (Pt) Magnetosomes Bi- 11 (P1 9 (P1) 2.5 50 brought into contact model dominant) 17.5 (P2) with U87-Luc cells 36 (P2) 26.5 (Pt) 23.5 (Pt) Magnetosomes Mono- 43 30 5 60 administered to model mouse tumors Without AMF application Magnetosomes Mono- 29   17.5 5 50 administered to model mouse tumours with AMF application Nanoparticle average sized of the nanoparticle size distrubtion = Av Full width half maximum of the nanoparticle size distribution = FWHM The Av and FWHM measured for each individual peak of the size distribution (P1, P2 . . .) as well as for the whole size distribution (Pt) Maximum size of the nanoparticle whole size distribution = Max Minimum size of the nanoparticle whole size distribution = Min

[0909] Conclusion: The previous examples show an unexpected and surprising increase of compounds that are able to treat cancer by nano-therapy.