NANOPARTICLES SEQUENTIALLY EXPOSED TO LOW INTENSITY ACOUSTIC WAVES FOR MEDICAL OR COSMETIC APPLICATIONS
20200000914 ยท 2020-01-02
Assignee
Inventors
Cpc classification
A61K41/0033
HUMAN NECESSITIES
A61K9/0009
HUMAN NECESSITIES
A61K41/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K41/00
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
An acoustic wave medical treatment of a body part of an individual in which nanoparticles are administered to the body part of the individual and the acoustic wave is applied on the body part. The acoustic wave is sequentially applied on the body part, and/or the nanoparticles are magnetosomes. Also, compositions that include these nanoparticles.
Claims
1-15. (canceled)
16. A method of medically treating a body part of an individual with an acoustic wave, comprising applying the acoustic wave on the body part, wherein the acoustic wave is sequentially applied and/or is unfocused.
17. The method according to claim 16, wherein the sequential application of the acoustic wave on the body part comprises at least one sequence, which is: i) the application of the acoustic wave during a time t.sub.1 followed by the non-application of the acoustic wave or radiation during a time t.sub.2, or ii) the application of an acoustic wave during a time t.sub.1 followed by the application of another acoustic wave during a time t.sub.3, wherein the intensity, power, energy, or frequency of the acoustic wave applied during the time t.sub.3 is lower than the intensity, power, energy, or frequency of the acoustic wave applied during the time t.sub.1.
18. The method according to claim 17, wherein t.sub.1, t.sub.2, and/or t.sub.3 is between 10.sup.3 seconds and 10.sup.20 minutes.
19. The method according to claim 17, wherein a sequence is repeated at least 2 times.
20. The method according to claim 17, comprising at least one session, wherein a session consists of several sequences separated by a lapse of time that is longer than t.sub.1, t.sub.2, t.sub.3, t.sub.1+t.sub.2, or t.sub.1+t.sub.3, preferentially by a factor of more or less than 1.001.
21. The method according to claim 16, wherein the unfocused acoustic wave has at least one property selected from the group consisting of: i) the unfocused acoustic wave covers more than 10.sup.5% by volume of the body part, using less than 10.sup.3 application spots, ii) the unfocused acoustic wave is applied over an acoustic wave volume that is larger than 10.sup.10 cm.sup.3, iii) the unfocused acoustic wave is applied over an acoustic wave volume that is larger than the nanoparticle region, body part, healthy site, or pathological site, either by a factor larger or equal to than 1.1 or by more than 1 cm.sup.3, and iv) the unfocused acoustic wave is applied over an acoustic wave volume that is larger, either by a factor larger or equal to than 1.1 or by more than 1 cm.sup.3, than the acoustic wave volume that is reached by or exists with or results from a focused acoustic wave or the application of a focused acoustic wave, wherein an application spot is defined as the acoustic volume that is covered during a single application of acoustic wave or during one sequence, wherein the acoustic wave volume is the volume, which is exposed to the acoustic wave or which receives the acoustic wave energy or which undergoes the effects of the acoustic wave, wherein the nanoparticle region is: i) the portion of the body part comprising the nanoparticles, ii) the volume occupied by the nanoparticles in the body part, or iii) the volume occupied by the nanoparticles outside of the body part, wherein the healthy site is a site that comprises healthy cell(s), wherein the pathological site is a site that comprises pathological cell(s), wherein the acoustic volume of a focused acoustic wave is the volume that covers less than 10.sup.5% by volume of the body part, using more than 1 application spot or is the volume covered by a high intensity focused ultrasound.
22. The method according to claim 16, wherein the acoustic wave has at least one property selected from the group consisting of: a power or power density lower than 1000 W (Watt), a power or power density lower than 1000 W per cm, cm.sup.2, or W per cm.sup.3, a power or power density lower than 1000 W per cm of body part, W per cm.sup.2 of body part, or W per cm.sup.3 of body part, a power or power density lower than 1000 W per cm of transducer, W per cm.sup.2 of transducer, or W per cm.sup.3 of transducer, a power or power density lower than 1000 W per gram of nanoparticle, an energy or energy density lower than 10.sup.5 W.Math.sec, an energy or energy density lower 10.sup.5 W.Math.sec per cm, W.Math.sec per cm.sup.2, or W.Math.sec per cm.sup.3, an energy or energy density lower than 10.sup.5 W.Math.sec per cm of body part, W.Math.sec per cm.sup.2 of body part, or W.Math.sec per cm.sup.3 of body part, an energy or energy density lower than 10.sup.5 W.Math.sec per cm of transducer, W.Math.sec per cm.sup.2 of transducer, W.Math.sec per cm.sup.3 of transducer, an energy or energy density lower than 10.sup.5 J (Joule), an energy or energy density lower than 10.sup.5 J per cm, J per cm.sup.2, or J per cm.sup.3, an energy or energy density lower than 10.sup.5 J per cm of body part, J per cm.sup.2 of body part, or J per cm.sup.3 of body part, an energy or energy density lower than 10.sup.5 J per cm of transducer, J per cm.sup.2 of transducer, J per cm.sup.3 of transducer, an energy or energy density lower than 10.sup.5 J per gram of nanoparticle, a frequency lower than 10.sup.5 MHz, and a penetration depth in the body part larger than 10.sup.5 cm.
23. The method according to claim 16, wherein the acoustic wave is not a focused ultrasound or is not a high intensity focused ultrasound.
24. The method according to claim 16, wherein the acoustic wave is an ultrasound.
25. The method according to claim 16, further comprising administering a concentration of nanoparticles to the body part before applying the acoustic wave.
26. The method according to claim 25, wherein the nanoparticles are sono-sensitizers.
27. The method according to claim 16, wherein the nanoparticles are magnetosomes.
28. The method according to claim 16, wherein the nanoparticles are chemical analogues of magnetosomes.
29. The method according to claim 16, wherein the concentration of nanoparticles, exposed to the acoustic wave or radiation or onto which the acoustic wave or radiation is applied, located in the body part or nanoparticle region, is lower than 10 g of nanoparticles per cm.sup.3 of body part or of g iron comprised in nanoparticles per cm.sup.3 of body part.
30. The method according to claim 16, wherein the method has or results in at least one of the following properties: i) the production of a slope of the initial variation, preferentially increase, of temperature with time, which is larger than 10.sup.9 or 10.sup.50 C. per second as measured per gram of nanoparticle or per cm.sup.3 of body part or gram of nanoparticle per cm.sup.3 of body part. ii) a specific absorption rate that is larger than 10.sup.9 Watt per gram of nanoparticle or Watt per cm.sup.3 of body part, and/or iii) a specific absorption rate, which increases with increasing power of the acoustic wave applied on the nanoparticles at a rate that can increase with decreasing nanoparticle concentration.
31. The method according claim 16, wherein the medical treatment is the treatment of a disease or disorder selected from the group consisting of: a disease associated with a proliferation of cells that is different from the cellular proliferation in a healthy individual, a disease associated with the presence of pathological cells in the body part, a disease associated with the presence of a pathological site in an individual or body part, a disease or disorder or malfunction of the body part, a disease associated with the presence of radio-resistant or acoustic-resistant cells, an infectious disease, an auto-immune disease, a neuropathology, a cancer, a tumor, a disease comprising or due to at least one cancer or tumor cell, a cutaneous condition, an endocrine disease, an eye disease or disorder, an intestinal disease, a communication disorder, a genetic disorder, a neurological disorder, a voice disorder, a vulvovaginal disorder, a liver disorder, a heart disorder, a heating disorder, a mood disorder, anemia, preferentially iron anemia, a personality disorder, a disease or disorder belonging to the individual or body part, the disease or disorder from which the individual is suffering, and a cancer or tumor.
32. The method according to claim 31, wherein the cancer or tumor is selected from the group consisting of: cancer of an organ, cancer of blood, cancer of a system of a living organism, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, heart cancer, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma cancer, ovarian cancer, pancreatic cancer, pancreatic penile cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma cancer, vaginal cancer, vulvar cancer, waldenstrom macroglobulinemia wilms tumor, castleman disease ewing family of tumor, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, myelodysplastic syndrome pituitary tumor, and a cancerous disease such as gestational trophoblastic disease, Hodgkin disease, kaposi sarcoma, malignant mesothelioma, and multiple myeloma.
33. The method accord to claim 31, wherein anemia disease is selected from the group consisting of: Iron deficiency anemia, Vitamin deficiency anemia, Anemia of chronic disease, Aplastic anemia, Anemia associated with bone marrow disease, Hemolytic anemia, Sickle cell anemia, Thalassaemia, Pernicious anaemia, Fanconi anaemia, Sideroblastic Anemia, Congenital Dyserythropoietic Anemia (CDA), Diamond-Blackfan Anemia, Megaloblastic Anemia, a decrease in the total amount of red blood cells (RBCs) or hemoglobin in the blood, and a lowered ability of the blood to carry oxygen.
34. The method according to claim 16, wherein the application of the acoustic wave on the body part induces a temperature increase of the body part, which is between 10.sup.10 and 10.sup.10 C.
35. The method according to claim 16, wherein a compound is attached to the nanoparticles and the application of the acoustic wave on the nanoparticles induces the dissociation of the compound from the nanoparticles.
36. The method according to claim 16, wherein the sequential application of the acoustic wave on the body part or nanoparticle induces: i) a series of temperature increases of the body part followed by temperature decreases of the body part, and/or ii) a series of dissociations of the compound from the nanoparticles followed by non-dissociation of the compound from the nanoparticles.
37. The method according to claim 16, wherein the sequential application of the acoustic wave on the body part prevents a decrease of the temperature of the body part.
38. The method according to claim 36, wherein the application of the acoustic wave on the nanoparticles produces or generates radical or reactive species.
39. A composition comprising nanoparticles treated by the method according to claim 16 that are magnetosomes or chemical analogs of magnetosomes.
40. A medical device, drug, or cosmetic composition comprising nanoparticles treated by the method according to claim 16 that are magnetosomes or chemical analogs of magnetosomes.
41. The method according to claim 19, wherein the nanoparticles are magnetosomes synthesized by, originating from, extracted from, or isolated from magnetotactic bacteria.
42. The method according to claim 19, wherein the application of the acoustic wave on the nanoparticles induces a temperature increase of the nanoparticles or the body part, which is between 10.sup.10 and 10.sup.10 C.
43. The method according to claim 19, wherein the sequential application of the acoustic wave on the nanoparticles prevents a decrease of the temperature of the body part or nanoparticles.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLE 1
[0670] Materials and Methods:
[0671] Nanoparticles:
[0672] We used magnetosomes extracted from magnetotactic bacteria and further purified to remove most organic material from magnetotactic bacteria, which were composed of: i) a core or mineral of maghemite with a percentage in mass of organic material originating from magnetotactic bacteria of 0.3%, and ii) a coating made of carboxy-methyl-dextran surrounding the core. Magnetosomes formed chains and were prepared using an adapted and improved protocol described in patent PCT/FR2016/000095 (Pub. Number WO2016/203121A1) incorporated in reference (example 8). These magnetosomes are designated as M-CMD. We also used i) nanoparticles composed of iron oxide of sizes 3513 nm purchased from Sigma designated as Sigma nanoparticles (Ref: 637106-25G, Lot #MKBK2270V), ii) superparamagnetic nanoparticles composed of iron oxide of 20 nm purchased from Micromod designated as SPION20 (Nanomag-D-spio 20, Ref: 79-02-201), iii) superparamagnetic nanoparticles composed of iron oxide of 50 nm purchased from Micromod designated as SPION50 (synomag-D50, Ref: 104-000-501), iv), superparamagnetic nanoparticles composed of iron oxide of 100 nm purchased from Micromod designated as SPION100 (Nanomag-D-spio 100, Ref: 79-00-102).
[0673] Preparation of Samples Containing Nanoparticles Inserted in Tissue or Dispersed in Water:
[0674] For heating experiments in tissues, 10 l of suspensions containing water alone or 204 g in iron of nanoparticles (Magnetosome(s) and Sigma or Sigma nanoparticle(s)) were inserted homogenously in 4.5 cm.sup.3 of liver tissue leading to a concentration of 45 lag in iron of nanoparticles per cm.sup.3 of liver tissue. For heating experiments in aqueous conditions, 100 l of water alone or 100 l of water mixed with 100 g in iron of nanoparticles (Magnetosome, Sigma, SPION20, SPION50, SPION100) were dispersed in a 200 l Eppendorf.
[0675] Heating Apparatus Generating Ultrasound:
[0676] Samples made of tissues with/without nanoparticles or water with/without nanoparticles were exposed to ultrasound of intensity 0.5, 1, or 1.5 W/cm.sup.3, and frequency 3 MHz, during 10 minutes. The intensity corresponds to that red on the apparatus and it is possible that there is a difference between the ultrasound intensity in the body part and the ultrasound intensity that the phyaction 190i indicates. To apply the ultrasound, we used a phyaction 190i ultrasound generator with a transducer of surface area 4 cm.sup.2. The ultrasound power indicated in the example corresponds to that read on the 190i ultrasound generator and not to an ultrasound power measured with an external probe. We used an ultrasound gel (Winelec, Ref: 1741/WINELEC) located between the transducer and the samples to favor the transmission of the ultrasounds.
[0677] Measurement of Temperature:
[0678] We used an infrared camera (EasIR-2, Optophase) positioned 13 cm above the transducer to measure the spatial distribution in temperature as a function of time during the experiments. We measured the temperature distribution at the following time points: 0 sec., 30 sec., 1 min., 2 min., 3 min., 4 min., 5 min., 6 min., 7 min., 8 min., 9 min., and 10 min. We only considered the maximum temperature recorded at each time point.
[0679] Results and Discussion:
[0680] a) Non-Sequential Heating Experiment in Tissues:
[0681]
[0682] For the magnetosomes mixed in tissue and exposed to different ultrasound powers of 0.5, 1, or 1.5 W/cm.sup.2, we have also estimated the values of T.sub.10minreal(M), which is equal to T.sub.10min(M)T.sub.10min(W), where T.sub.10min(M) and T.sub.10min(W) are the temperature increases observed after 10 minutes of ultrasound application for the samples containing tissue with the magnetosomes and tissue without the magnetosomes, respectively. We observed that T.sub.10minreal(M) increases from 6 C. at 0.5 W/cm.sup.2 to 28 C. at 1.5 W/cm.sup.2 (table 1). We also estimated the percentage in temperature rise, Temperature rise .sub.(M), expressed using the formula Temperature rise.sub.(M)=(T.sub.10min(M)/T.sub.10min(W)1).Math.100. It increases from 37% at 0.5 W/cm.sup.2 to 100% at 1.5 W/cm.sup.2 (table 1). We also estimated the value of the specific absorption rate of the magnetosomes inserted in tissue, SAR.sub.real(M), expressed in watt per gram of magnetosomes in iron (W/g.sub.Fe), using the formula SAR.sub.real(M)=Slope.sub.real(M).Math.C.sub.v/C.sub.nano, where Slope.sub.real(M)=Slope.sub.(M)Slope.sub.(W), with Slope.sub.(M) and Slope.sub.(W) representing the initial slopes of the temperature variations with time deduced from the plots of
[0683] For Sigma nanoparticles mixed in tissue and exposed to different ultrasound powers of 0.5, 1, or 1.5 W/cm.sup.2, we have also estimated the values of T.sub.10minreal(S), which is equal to T.sub.10min(S)T.sub.10min(W), where T.sub.10min(S) and T.sub.10min(W) are the temperature increases observed after 10 minutes of ultrasound application for the samples containing tissue with the Sigma nanoparticles and tissue without the Sigma nanoparticles, respectively. We observed that T.sub.10minreal(S) decreases from 14 C. at 0.5 W/cm.sup.2 to 6-7 C. at 1-1.5 W/cm.sup.2 (table 1). We also estimated the percentage in temperature rise, Temperature rise .sub.(S), expressed using the formula Temperature rise .sub.(S)=(T.sub.10min(S)/T.sub.10min(W)1).Math.100. It decreases from 90% at 0.5 W/cm.sup.2 to 17-26% at 1-1.5 W/cm.sup.2 (table 1). We also estimated the value of the specific absorption rate of the Sigma nanoparticles inserted in tissue, SAR.sub.real(S), expressed in watt per gram of Sigma nanoparticles in iron (W/g.sub.Fe), using the formula SAR.sub.real(S)=Slope.sub.real(S).Math.C.sub.v/C.sub.nano, where Slope.sub.real(S) represent the initial slopes of the temperature variations with time deduced from the plots of
[0684] b) Non-Sequential Heating Experiments in Water:
[0685]
[0686] For the magnetosomes mixed in water and exposed to different ultrasound powers of 0.5, 1, or 1.5 W/cm.sup.2, we have estimated the values of T.sub.10minreal(M), which is equal to T.sub.10min(M)T.sub.10min(W), where T.sub.10min(M) and T.sub.10min(W) are the temperature increases observed after 10 minutes of ultrasound application for the samples containing tissue with the magnetosomes and tissue without the magnetosomes, respectively. We observed that T.sub.10minreal(M) remains at 3 to 9 C. between 0.5 W/cm.sup.2 and 1.5 W/cm.sup.2 (table 2), smaller values of T.sub.10minreal(M) than those observed in tissue at 1.5 W/cm.sup.2 (table 1). We also estimated the percentage in temperature rise, Temperature rise .sub.(M), expressed using the formula Temperature rise .sub.(M)=(T.sub.10min(M)/T.sub.10min(W)1).Math.100. It decreases from 37-43% at 0.5-1 W/cm.sup.2 down to 10% at 1.5 W/cm.sup.2 (table 2) and is also smaller than Temperature rise .sub.(M) measured in tissue at 1.5 W/cm.sup.2 (table 1). We also estimated the value of the specific absorption rate of the magnetosomes dispersed in water, SAR.sub.real(M), expressed in watt per gram of magnetosomes in iron (W/g.sub.Fe), using the formula SAR.sub.real(M)=Slope.sub.real(M).Math.C.sub.v/C.sub.nano, where Slope.sub.real(M) represents the initial slopes of the temperature variations with time deduced from the plots of
[0687] For Sigma nanoparticles dispersed in water and exposed to different ultrasound powers of 0.5, 1, or 1.5 W/cm.sup.2, we have also estimated the values of T.sub.10minreal(S), which is equal to T.sub.10min(S)T.sub.10min(W), where T.sub.10min(S) and T.sub.10min(W) are the temperature increases observed after 10 minutes of ultrasound application for the samples containing Sigma nanoparticles dispersed in water and water without the Sigma nanoparticles, respectively. We observed that T.sub.10minreal(S) remains at 6-12 C. for ultrasound energies of 0.5-1.5 W/cm.sup.2 (table 2). We also estimated the percentage in temperature rise, Temperature rise .sub.(S), expressed using the formula Temperature rise .sub.(S)=(T.sub.10min(S)/T.sub.10min(W)1).Math.100. It remains at 31-60% for powers of 0.5-1.5 W/cm.sup.2 (table 2). We also estimated the value of the specific absorption rate of the Sigma nanoparticles inserted in tissue, SAR.sub.real(S), expressed in watt per gram of Sigma nanoparticles in iron (W/g.sub.Fe), using the formula SAR.sub.real(S)=Slope.sub.real(S).Math.C.sub.v/C.sub.nano, where Slope.sub.real(S) represents the initial slopes of the temperature variations with time deduced from the plots of
[0688] For SPION50, SPION100, and SPION20 nanoparticles dispersed in water and exposed to different ultrasound powers of 0.5, 1, or 1.5 W/cm.sup.2, we have also estimated the values of T.sub.10minreal(S50), T.sub.10minreal(S100), and T.sub.10minreal(S20), which are equal to T.sub.10min(S50)T.sub.10min(W), T.sub.10min(S100)T.sub.10min(W) and T.sub.10min(S20)T.sub.10min(W), respectively. T.sub.10min(S50), T.sub.10min(S100), T.sub.10min(S20) and T.sub.10min(W) are the temperature increases observed after 10 minutes of ultrasound application for the samples containing SPION50, SPION100, and SPION20 nanoparticles dispersed in water and water without nanoparticles, respectively. We observed that T.sub.10minreal(S50), T.sub.10minreal(S100), and T.sub.10minreal(S20), remain at 0-7 C. for ultrasound energies of 0.5-1.5 W/cm.sup.2 (table 2). We also estimated the percentage in temperature rise, Temperature rise .sub.(S), expressed using the formula Temperature rise .sub.(S50)=(T.sub.10min(S50)/T.sub.10min(W)1).Math.100 for SPION50, Temperature rise .sub.(S100)=(T.sub.10min(S100)/T.sub.10min(W)1).Math.100 for SPION100, Temperature rise .sub.(S20)=(T.sub.10min(S20)/T.sub.10min(W)1).Math.100 for SPION20. It remains at 1-35% for powers of 0.5-1.5 W/cm.sup.2 for the different SPION (table 2). We also estimated the value of the specific absorption rate of the SPION50, SPION100, and SPION20 nanoparticles inserted in tissue, SAR.sub.real(S50), SAR.sub.real(S100), SAR.sub.real(S20), expressed in watt per gram of SPION50, SPION100, and SPION20 nanoparticles in iron (W/g.sub.Fe). For that, we used the formula SAR.sub.real(S50)=Slope.sub.real(S50).Math.C.sub.v/C.sub.nano, SAR.sub.real(S100)=Slope.sub.real(S100).Math.C.sub.v/C.sub.nano, SAR.sub.real(S20)=Slope.sub.real(S20).Math.C.sub.v/C.sub.nano for SPION50, SPION100, and SPION20 nanoparticles, respectively. Slope.sub.real(S50), Slope.sub.real(S100), Slope.sub.real(S20) represent the initial slopes of the temperature variations with time deduced from the plots of
[0689] c) Sequential Heating Experiments in Water:
[0690] Eppendorf containing 500 g of Magnetosomes dispersed in 100 l of water were exposed sequentially to ultrasounds.
Conclusion
[0691] We can draw the following conclusion from this example:
[0692] (i) The values of T, the difference in temperature between the temperature of nanoparticles in tissues or water exposed to ultrasound and the temperature of tissue or water alone exposed to ultrasound, is always positive, indicating that the different nanoparticles (Magnetosome, Sigma, SPION20, SPION50, SPION100) enhance the heating efficacy of ultrasound in the tested conditions (ultrasound frequency=3 MHz, ultrasound power=0.5-1.5 W/cm.sup.2, nanoparticle concentration varied between 60 g/mL and 8 mg/mL and nanoparticles either inserted in tissue or dispersed in water).
[0693] (ii) For water suspensions, lower SAR values observed for magnetosomes than for other nanoparticles (table 2) may be explained by more aggregation for the magnetosomes than for other nanoparticles following application of the ultrasound (as was observed by eyes).
[0694] (iii) in some cases, the SAR may have been underestimated due to the heat produced by the transducer generating the ultrasound that can heat the tissue and interfere with the heat produced by nanoparticles exposed to ultrasound. This may be the reason why some of the SAR values are reported to be 0 W/g for example.
[0695] (iv) When SAR.sub.real is zero, the value of T.sub.10min real is non-zero (table 2), indicating that nanoparticles increase the quantity of heat generated by the acoustic wave but that SAR.sub.real is underestimated, possibly due to the interference with the heat generated by the transducer.
[0696] (v) Nanoparticle SAR values estimated by applying ultrasound in tissue comprising the various nanoparticles reach the largest value for the the magnetosomes.
[0697] (vi) Using magnetosomes, we can produce sequences consisting in heating steps (application of ultrasound on magnetosomes) followed by cooling steps (non-application of ultrasound on magnetosomes), with enhanced magnitudes of heating and cooling compared with heating and cooling steps obtained without the magnetosomes (
EXAMPLE 2
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EXAMPLE 3: CELLULAR TOXICITY AND TEMPERATURE MEASUREMENT OF CELLS BROUGHT INTO CONTACT WITH DIFFERENT CONCENTRATIONS OF MAGNETOSOMES AND SUBJECTED (OR NOT) TO THE SEQUENTIAL OR CONTINUOUS APPLICATION OF ULTRASOUNDS
[0699] Materials and Methods:
[0700] The magnetosomes used in its example are M-CMD. U87-MG glioblastoma cells were purchased from ATCC (ATCC HTB-14) and cultivated in High-Glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 1 mM pyruvate, 10% fetal calf serum, 100 units/mL of penicillin and 100 g/mL of streptomycin. The cells were seeded in a T175 flask with culture medium. When 80-90% confluence was reached, the supernatant was removed and replaced with PBS to rinse the cells. Subsequently, the PBS solution was removed and replaced with a volume of 5 mL of 0.25% trypsin-EDTA. The cells were incubated for 5 minutes at 37 C. with 5% carbon dioxide in an incubator with a humidity of 90-95%. The cells were then harvested. A volume of 10 ml of culture medium was added to deactivate the action of trypsin and the cells were homogenized. A volume of 30 L of cells was collected and mixed with 30 L of 4% trypan blue to count the cells using a cell counter (Countess II FL Automated Cell Counter (Thermo Fisher scientific)) and thus to determine the cell concentration of the initial suspension. A volume of 2 mL of 250 000 cells was deposited per petri dish and then incubated at 37 C. with 5% CO2 for 24 hours so that the cells adhere at the surface of the petri dish. The cell medium was then removed and replaced either by a new medium without magnetosomes or by a new medium comprising magnetosomes at a concentration of 100 g/mL or 500 g/mL in iron. The cells were then either continuously exposed to ultrasounds or sequentially exposed to ultrasounds. The power of the ultrasounds used was 0 mW/cm.sup.2, 100 mW/cm.sup.2, or 500 mW/cm.sup.2, and the frequency of the ultrasound used was 1 MHz.
[0701] The ultrasound was applied as follows: the surface of the transducer was oriented upward, a gel-pad coated with ultrasound gel was deposited on the surface of the transducer to reduce the heat released by the transducer. Then above the gel-pad, the petri dishes were deposited. The petri-dishes were maintained above the gel-pad for 5 minutes, so that ultrasounds can cross the different surfaces.
[0702] For the continuous application of ultrasounds, ultrasounds were applied continuously during 5 minutes to petri dishes containing cells with or without magnetosomes.
[0703] For the sequential application of ultrasounds, the ultrasounds were sequentially applied to petri dishes containing the cells with or without magnetosomes in the following way: first application of ultrasound for 1 min, no application of ultrasound during 1 min, second application of ultrasound for 1 min 24 s, no application of ultrasound during 1 min 24 s, third application of ultrasound for 1 min, no application of ultrasound during 1 min 30 s, fourth application of ultrasound for 1 min, no application of ultrasound during 1 min 18 s, fifth application of ultrasound for 1 min 12 s, no application of ultrasound during 1 min 18 s. The total time of application of the ultrasound was 5 min 36 seconds, close to the duration of 5 minutes during which the ultrasounds were continuously applied.
[0704] During the application of ultrasounds, the heating temperature was measured using the infra-red camera EasylR-2 from the company Guide Infrared, which was positioned 20 cm above the petri dishes.
[0705] 24 hours after the treatments, the medium with and without magnetosomes was removed and then replaced with a PBS buffer solution. The cells were washed twice with PBS buffer solution and then 2 ml of a solution of bromide of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium at 1 mg/ml was brought into contact with the cells during 4 hours, the tetrazolium salt was removed and then replaced with 2 mL of isopropanol. After gentle stirring, a volume of 100 L of each petri dish was transferred to a 96-well plate. Absorbance was measured at 620 nm. The percentage of living cells was determined by measuring the ratio between the optical density measured for the cells treated with ultrasounds with/without magnetosomes and the optical density measured for the cells treated alone without magnetosomes without the application of the ultrasounds, and the ratio was multiplied by 100.
[0706] Results:
[0707]
[0708] On the one hand, it is observed that when the magnetosome concentration increases from 0 to 500 g/mL, the percentage of living cells decreases: i) from 100% to 35% in the absence of application of the ultrasounds, and ii) from 87% to 10% in the presence of the application of the ultrasounds of power 500 mW/cm.sup.2.
[0709] On the other hand, it is observed that when the power of the ultrasounds increases from 0 mW/cm.sup.2 to 500 mW/cm.sup.2, the percentage of living cells decreases: i) from 100% to 87% in the absence of magnetosomes and ii) from 35% to 10% in the presence of 500 g/mL of magnetosomes.
[0710] It is observed that the percentage of living cells decreases when the power of the ultrasounds and the magnetosome concentration are increased.
[0711]
[0712]
[0713] We can deduce from these results that:
[0714] i) In order to obtain a temperature increase by continuously applying ultrasounds on magnetosomes, it is necessary to use a sufficiently large magnetosome concentration (500 g/mL) and a sufficiently large power of the ultrasounds of 500 mW/cm.sup.2 (
[0715] ii) The percentage of living cells resulting from the treatment which consists in applying ultrasounds of 100 mW/cm.sup.2 and 500 mW/cm.sup.2 on magnetosomes is similar for a magnetosome concentration of 100 and 500 g/mL, at 10% for a power of the ultrasounds of 500 mW/cm.sup.2 and at 32-40% for a power of the ultrasounds of 100 mW/cm.sup.2, indicating that the magnetosome concentration has a limited impact on the efficacy of cellular destruction for this range of concentration (
[0716] iii) When ultrasounds of power 500 mW/cm.sup.2 are applied on magnetosomes of concentrations of 100 g/mL and 500 g/mL, it results in a similar percentage of living cells (
[0717]
[0718]
[0719]
[0720] We have shown the possibility of efficiently destroying U87 tumor cells by applying ultrasounds on these cells in the presence of magnetosomes under conditions in terms of ultrasounds power and frequency that are such that the sole application of the ultrasounds without the magnetosomes induces limited or no cellular toxicity.
[0721] When the quantity of magnetosomes continuously exposed to ultrasounds of 500 mW/cm.sup.2 is increased from 100 g/mL to 500 g/mL, the treatment results either in the absence of additional heating at 100 g/mL or to an additional temperature increase of 11 C. (15-4 C.) at 500 g/mL, compared with the condition of ultrasound application of 500 W/cm.sup.2 without magnetosomes (
[0722] These results pave the way to effective treatment obtained at low magnetosome concentration and/or in conditions of limited or no temperature increase, thus potentially reducing the toxicity of nanoparticle-based treatment often combining high nanoparticle concentrations with strong heating such as those using magnetic hyperthermia currently tested in the clinic.
EXAMPLE 4: CELLULAR TOXICITY AND TEMPERATURE MEASUREMENT OF CELLS BROUGHT INTO CONTACT WITH MAGNETOSOMES AND SUBJECTED (OR NOT) TO THE CONTINUOUS OR SEQUENTIAL APPLICATION OF THE LASER
[0723] Materials and Methods:
[0724] Magnetosomes used in this example ate M-CMD. U87-MG glioblastoma cells were purchased from ATCC (ATCC HTB-14) and cultivated in High-Glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 1 mM pyruvate, 10% fetal calf serum, 100 units/mL of penicillin and 100 g/mL of streptomycin. The cells were seeded in a T175 flask with culture medium. When 80-90% confluence was reached, the supernatant was removed and replaced with PBS to rinse the cells. Subsequently, the PBS solution was removed and replaced with a volume of 5 mL of 0.25% trypsin-EDTA. The cells were incubated for 5 minutes at 37 C. with 5% carbon dioxide in an incubator with a humidity of 90-95%. The cells were then harvested. A volume of 10 ml of culture medium was added to deactivate the action of trypsin and the cells were homogenized. A volume of 30 L of cells was collected and mixed with 30 L of 4% trypan blue to count the cells using a cell counter (Countess II FL Automated Cell Counter (Thermo Fisher scientific)) and thus to determine the cell concentration of the initial suspension. A volume of 100 L of 10.sup.4 cells was inserted in each well of a 96 well plate and the cells were incubated at 37 C. with 5% CO.sup.2 for 24 hours so that the cells adhere at the surface of well. The cell medium was then removed and replaced either by a new medium without magnetosomes or a new medium containing magnetosomes at a concentration of 1 mg/mL in iron of magnetosomes.
[0725] BALB/3T3 clone A31 fibroblast cells were purchased from ATCC (ATCCCCL-163)) and cultivated in High-Glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 1 mM pyruvate, 10% bovine calf serum, 100 units/mL of penicillin and 100 g/mL of streptomycin. The cells were seeded in a T175 flask with culture medium. When 80-90% confluence was reached, the supernatant was removed and replaced with PBS to rinse the cells. Subsequently, the PBS solution was removed and replaced with a volume of 5 mL of 0.25% trypsin-EDTA. The cells were incubated for 5 minutes at 37 C. with 5% carbon dioxide in an incubator with a humidity of 90-95%. The cells were then harvested. A volume of 10 ml of culture medium was added to deactivate the action of trypsin and the cells were homogenized. A volume of 30 L of cells was collected and mixed with 30 L of 4% trypan blue to count the cells using a cell counter (Countess II FL Automated Cell Counter (Thermo Fisher scientific)) and thus to determine the cell concentration of the initial suspension. A volume of 100 L of 10.sup.4 cells was deposited in each well of a 96 well plate and the cells were incubated at 37 C. with 5% CO.sub.2 for 24 hours so that the cells adhere at the surface of well. The cell medium was then removed and replaced either by a new medium without magnetosomes or a new medium containing magnetosomes at a concentration of 1 mg/mL in iron of magnetosomes.
[0726] U87-MG or 3T3 cells, treated as described above, were then either continuously exposed to a laser of average power 3 W/cm.sup.2 during 6 minutes or sequentially exposed to the laser. The power of the laser used was 3 W/cm.sup.2, where the power is the ratio between the laser power at the end of the fiber and the exposed surface (the surface of the well). The wavelength of the laser was 808 nm. The beam of laser light was focused at the bottom of the well containing cells with/without magnetosomes.
[0727] The laser light was applied as follows:
[0728] For the continuous application of the laser, the laser was applied continuously during 6 minutes. For the sequential application of the laser, two conditions were tested. In condition 1, the cells were brought into contact with 1 mg/mL of magnetosomes and exposed sequentially to a laser in the following way: (a) for U87-MG: First sequence: i) application of the laser of average power 3 W/cm.sup.2 during 60 seconds until the temperature reaches 45 C., ii) non-application of the laser during 18 seconds resulting in a temperature decrease from 45 C. to 37 C.; Second sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; Third sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fourth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fifth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22 seconds resulting in a temperature decrease from 45 C. to 37 C.; ninth sequence: i) application of the laser of average power at 3 W/cm.sup.2 during 13 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; tenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; eleventh sequence: i) application of the laser of average power at 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; twelfth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 25 seconds resulting in a temperature decrease from 45 C. to 37 C.; thirteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fourteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; fifteenth sequence: i) application of the laser of average power 3 W/cm2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventeenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; nineteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; twentieth sequence: i) application of the laser an average power at 3 W/cm.sup.2 during 14 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; twenty first sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; twenty second sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 16 seconds resulting in a temperature decrease from 45 C. to 37 C. The total duration of the application of the laser is 6 min 2 sec. (b), for 3T3: First sequence: i) application of the laser of average power 3 W/cm.sup.2 during 90 seconds until the temperature reaches 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; Second sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22 seconds resulting in a temperature decrease from 45 C. to 37 C.; Third sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fourth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fifth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; ninth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 20 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; tenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eleventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; twelfth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; thirteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fourteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fifteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 19.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventeenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; nineteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 19 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C. In condition 2, the cells were not brought into contact with the magnetosomes and sequentially exposed to the laser using the same sequence durations those of condition 1.
[0729] During the application of the laser, the heating temperature was measured using the infra-red camera EasylR-2 from the company Guide Infrared, which was positioned 20 cm above the well.
[0730] 24 hours after the treatments, the medium with and without magnetosomes was removed and then replaced with a PBS buffer solution. The cells were washed twice with this buffer solution and then 100 l of a solution of bromide of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium at 1 mg/ml was brought into contact with the cells during 4 hours, the tetrazolium salt was removed and then replaced with 100 L of isopropanol. After gentle stirring, absorbance was measured at 620 nm using a microplate spectrophotometer system. The percentage of living cells was determined by measuring the ratio between the optical density for the cells treated with laser and magnetosomes and the optical density measured for the cells treated alone without magnetosomes without the application of the laser, and the ratio was multiplied by 100.
[0731] Results:
[0732]
[0733]
[0734] The details of the sequences are as follows for U87-MG cells: First sequence: i) application of the laser of average power 3 W/cm.sup.2 during 60 seconds until the temperature reaches 45 C., ii) non-application of the laser during 18 seconds resulting in a temperature decrease from 45 C. to 37 C.; Second sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; Third sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fourth sequence: i) application of the laser of average power at 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during resulting in a temperature decrease from 45 C. to 37 C. during 20.5 seconds; Fifth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during resulting in a temperature decrease from 45 C. to 37 C. during 21.5 seconds; sixth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22 seconds resulting in a temperature decrease from 45 C. to 37 C.; ninth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; tenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; eleventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; twelfth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 25 seconds resulting in a temperature decrease from 45 C. to 37 C.; thirteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fourteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; fifteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventeenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 12.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 24 seconds resulting in a temperature decrease from 45 C. to 37 C.; nineteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 13.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; twentieth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; twenty first sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 23 seconds resulting in a temperature decrease from 45 C. to 37 C.; twenty second sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 16 seconds resulting in a temperature decrease from 45 C. to 37 C. The total duration of laser application is 6 min 2 sec.
[0735] The details of the sequences are as follows for 3T3 cells. First sequence: i) application of the laser an average power at 3 W/cm.sup.2 during 90 seconds until the temperature reaches 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; Second sequence: i) application of the laser of average power at 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 22 seconds resulting in a temperature decrease from 45 C. to 37 C.; Third sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fourth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 14.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; Fifth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixth sequence: i) application of the laser of average power at 3 W/cm.sup.2 during 15.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21 seconds resulting in a temperature decrease from 45 C. to 37 C.; ninth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 20 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; tenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eleventh sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; twelfth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; thirteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fourteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; fifteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 19.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 21.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; sixteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 18 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; seventeenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 19.5 seconds resulting in a temperature decrease from 45 C. to 37 C.; eighteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 17.5 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 20 seconds resulting in a temperature decrease from 45 C. to 37 C.; nineteenth sequence: i) application of the laser of average power 3 W/cm.sup.2 during 19 seconds resulting in a temperature increase from 37 C. to 45 C., ii) non-application of the laser during 18.5 seconds resulting in a temperature decrease from 45 C. to 37 C.
[0736]
[0737]
[0738]
[0739] In conclusion, we have shown that:
[0740] i) It was possible to carry out regular or periodic sequences of temperature increase up to 45 C. of average duration 16 seconds by applying the laser of an average power 3 W/cm2 followed by temperature decreases from 45 C. to 37 C. of average duration 22 seconds by not applying the laser.
[0741] ii) The sequential application of the laser enables destroying more cells than the continuous application of the laser for the two studied cell lines (U87-Luc and 3T3 cells).
EXAMPLE 5: ROS PRODUCTION
[0742] Materials and Methods:
[0743] Magnetosomes M-CMD were used. U87-MG glioblastoma cells and CAL-33 were purchased from ATCC and cultivated in High-Glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 1 mM pyruvate, 10% fetal calf serum, 100 units/mL of penicillin and 100 g/mL of streptomycin. The cells were seeded in a T175 flask with culture medium. When 80-90% confluence was reached, the supernatant was removed and replaced with PBS to rinse the cells. Subsequently, the PBS solution was removed and replaced with a volume of 5 mL of 0.25% trypsin-EDTA. The cells were incubated for 5 minutes at 37 C. with 5% carbon dioxide in an incubator with a humidity of 90-95%. The cells were then harvested. A volume of 10 ml of culture medium was added to deactivate the action of trypsin and the cells were homogenized. A volume of 30 L of cells was collected and mixed with 30 L of 4% trypan blue to count the cells using a cell counter (Countess II FL Automated Cell Counter (Thermo Fisher scientific)) and thus to determine the cell concentration of the initial suspension. A volume of 100 L of 104 cells was deposited per well in 96 plate well and then incubated at 37 C. with 5% CO2 for 24 hours so that the cells adhere at the surface of well. The cell medium was then removed and replaced by a new medium containing 2,7 dichlorofluoresceine diacetate (DCFH-DA) at a concentration of 100 M. The cells were then incubated during 45 minutes at 37 C. with 5% CO2, and the medium was removed and replaced by PBS to rinse the cells and measure the production of intracellular ROS. Then PBS was replaced by a new medium without magnetosomes or a new medium containing magnetosomes at a concentration of: i) 1 mg/mL in iron of magnetosomes for U87-MG exposed to the AMF or laser or ii) 1000, 500, 250 and 16 g/mL in iron of magnetosomes for cal33 cells exposed to gamma radiation. BALB/3T3 clone A31 fibroblast cells were purchased from ATCC (ATCCCCL-163)) and cultivated in High-Glucose Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 1 mM pyruvate, 10% bovine calf serum, 100 units/mL of penicillin and 100 g/mL of streptomycin. The cells were seeded in a T175 flask with culture medium. When 80-90% confluence was reached, the supernatant was removed and replaced with PBS to rinse the cells. Subsequently, the PBS solution was removed and replaced with a volume of 5 mL of 0.25% trypsin-EDTA. The cells were incubated for 5 minutes at 37 C. with 5% carbon dioxide in an incubator with a humidity of 90-95%. The cells were then harvested. A volume of 10 ml of culture medium was added to deactivate the action of trypsin and the cells were homogenized. A volume of 30 L of cells was collected and mixed with 30 L of 4% trypan blue to count the cells using a cell counter (Countess II FL Automated Cell Counter (Thermo Fisher scientific)) and thus to determine the cell concentration of the initial suspension. A volume of 100 L of 104 cells was deposited in each well of a 96 plate well and then incubated at 37 C. with 5% CO2 for 24 hours so that the cells adhere at the surface of the well. The cell medium was then removed and replaced by a new medium containing 2,7 dichlorofluoresceine diacetate (DCFH-DA) at a concentration of 100 M. Cells were then incubated during 45 minutes at 37 C. with 5% CO2 and the medium was then removed and replaced by PBS to rinse the cells and measure the intracellular production of ROS. Then, PBS was replaced by: i) a new medium without magnetosomes, ii) a new medium containing magnetosomes at a concentration of 1 mg/mL in iron of magnetosomes for the AMF or laser treatment, or iii) 1000, 500, 250 and 16 g/mL in iron of magnetosomes when cells are irradiated by gamma radiation. U87-MG or 3T3 cells, treated as described above, were then either continuously exposed to a laser of an average power 3 W/cm2 during 6 minutes or sequentially exposed to the laser. The power of the laser used was 3 W/cm2 and the wavelength of the laser was 808 nm. The beam of laser light was focused at the bottom of the well containing cells with/without magnetosomes.
[0744] The laser light was applied as follows: For the continuous application of the laser, the laser was applied continuously during 6 minutes. For the sequential application of the laser, the sequences of application of the laser are described in the legend of
[0745] The AMF was applied as follows: For the continuous application of AMF, the well containing cells with/without magnetosomes was positioned at the center of the coil and exposed to an AMF of strength of 34-47 mT and frequency 198 KHz pendant 30 minutes. For the sequential application of the laser, the details of the sequences used are given in the legend of
[0746] During the application of the laser and AMF, the heating temperature was measured using the infra-red camera EasylR-2 from the company Guide Infrared, which was positioned 20 cm above the well.
[0747] For gamma irradiation, the wells were placed at the center of a plate inside a GSR_D1 irradiator containing 4 sources of Cesium 137 (GSR Cs 137/ C.) of total activity 190 TBq, purchased from Gamma Service Medical GmbH. The irradiation dose was determined in real time by the time of exposure of the surface of the wells and therefore of the cells to the irradiations, whose time was comprised between 5 minutes and 1.7 hours, and irradiation doses were comprised between 5 and 80 Gy.
[0748] After 30 minutes of the treatment, the medium with and without magnetosomes was removed and then replaced with a PBS buffer solution. Fluorescence was measured at 530 nm with an excitation at 485 nm using a microplate fluorometer system. The rate of ROS production was determined by subtraction between the intensity of fluorescence measured for the cells treated with laser or AMF or gamma radiation and magnetosomes and the intensity of fluorescence measured for the cells treated alone without magnetosomes without the application of the laser or AMF or gamma radiation, and this ratio was multiplied by 100.
[0749] For MTT assay, 72 hours after the treatments, the medium with and without magnetosomes was removed and then replaced with a PBS buffer solution. The cells were washed twice with this buffer solution and then 100 l of a solution of bromide of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium at 1 mg/ml was brought into contact with the cells during 4 hours, the tetrazolium salt was removed and then replaced with 100 L of isopropanol. After gentle stirring, absorbance was measured at 620 nm. The percentage of living cells was determined by measuring the ratio between the optical density measured for the cells treated with laser or AMF or gamma radiation and magnetosomes and the optical density measured for the cells treated alone without magnetosomes without the application of the laser or AMF or gamma radiation, and the ratio was multiplied by 100.
[0750] Results:
[0751] The rate of ROS production corresponds to the percentage of free radical produced such as singlet oxygen, peroxides, anion superoxide or hydroxyl that have reacted with DCFH-DA, in the presence of cells brought (or not) into contact with the magnetosomes, and continuously or sequentially exposed to the laser, AMF, or gamma radiation.
[0752]
[0753] For 3T3 cells, in the absence of the magnetosomes, cells continuously or sequentially exposed to the laser yield a similar low percentage of ROS production of 30%. In the presence of the magnetosomes, this rate of 30% increases: i) moderately by a factor of 3 without laser excitation up to 100%, ii) more importantly by a factor of 15 for the continuous laser excitation up to 450%, and iii) strongly by a factor of 22 for the sequential laser excitation up to 650% (
[0754] A similar behavior is observed with U87-MG cells. In the absence of the magnetosomes, U87-MG cells continuously or sequentially exposed to the laser yield a similar low percentage of ROS production of 50%. In the presence of the magnetosomes, this rate of 50% increases: i) moderately by a factor of 3 without laser excitation up to 150%, ii) more importantly by a factor of 11 for continuous laser excitation up to 550%, and iii) strongly by a factor 16 for the sequential application up to 800% (
[0755]
[0756] For 3T3 cells, in the absence of the magnetosomes, cells continuously or sequentially exposed to the AMF yield a low percentage of ROS of 30-50%. In the presence of the magnetosomes, this rate of 30-50% increases: i) moderately up to 100% in the absence of AMF application, ii) more importantly up to 810% in the presence of a continuous AMF application, iii) strongly up to 1100% in the presence of a sequential AMF application (
[0757] A similar behavior is observed with U87-MG cells. In the absence of the magnetosomes, U87-MG cells continuously or sequentially exposed to the AMF yield a low percentage of ROS production of 50-80%. In the presence of the magnetosomes, this rate of 50-80% increases: i) moderately up to 200% in the absence of AMF application, ii) more importantly up to 1450% in the presence of the continuous AMF application, iii) strongly up to 1700% in the presence of a sequential AMF application.
[0758]
[0759]
[0760]
[0761]
[0762] We can draw the following conclusions from this example:
[0763] i) Magnetosome brought into contact with different cell lines (Cal-33, 3T3, U87-MG) produce ROS at a concentration of 1 mg/mL. For the cell lines Cal-33 and 3T3, the production of ROS is observed at 250, 500, and 1000 g/mL, but not at 16 g/mL, indicating that the amount of ROS produced in the absence of excitation source can be adjusted by varying the magnetosome concentration.
[0764] ii) For the excitation sources (laser and AMF), the rate of ROS production is increased moderately by continuously applying the excitation on the magnetosomes and strongly increased by sequentially applying the excitation on the magnetosomes, both on 3T3 and U87-MG cells.
[0765] iii) When 3T3 cells are irradiated with gamma radiation in the presence of magnetosomes (magnetosome concentration larger than 500 g/mL), the rate of ROS production increases with increasing irradiation dose, while the cell viability does not strongly decrease with increasing irradiation dose. In the case of the healthy cells, the production of ROS does not seem to strongly affect cellular viability under gamma irradiation.
[0766] Table 1.
[0767] For 210 g in iron of nanoparticles Magnetosome inserted in 4.6 cm.sup.3 of tissue exposed to ultrasounds of frequency 3 MHz and power 0.5 W/cm.sup.2, 1 W/cm.sup.2, 1.5 W/cm.sup.2, (Slope.sub.(M)) designs the slope at the origin of the temperature variation with time of magnetosomes mixed with tissue (Slope.sub.real(M)) designing the difference between slope at the origin of the temperature variation with time of magnetomes mixed with tissue (Slope.sub.(M)) and the slope at the origin of the temperature variation with time of the tissue without the nanoparticles (Slope.sub.(w)). Slope.sub.realN(M) is Slope.sub.real(M) divided by the magnetosome concentration in gram of iron comprised in magnetosomes per mL. Slope rise (Slope rise (M)) designates the percentage in slope rise estimated using the formula for magnetosomes: Slope rise .sub.(M) (%)=((Slope.sub.(M)/Slope.sub.(W))1)*100. The specific absorption rate of magnetosomes (SAR.sub.(M)), estimated in watt per gram of magnetosomes is deduced from the values of Slope .sub.(M), using the formula: SAR.sub.(M)=C.sub.v.Math.Slope.sub.(M)/C.sub.nano, where C.sub.v=4.2 J.Math.K.sup.1 g.sup.1 is the specific heat of water and C.sub.nano is the magnetosome concentration in gram of magnetosomes per cm.sup.3 of tissue. The variation in temperature between the initial temperature measured before the application of the ultrasound and the temperature measured after 10 minutes of application of the ultrasound is designated as T.sub.10min(M) for magnetosomes. The difference between T.sub.10min(M) and T.sub.10min(w) is designated as T.sub.10min,real(M). The percentage in temperature rise is estimated for Magnetosomes using the formula: Temperature rise .sub.(M) (%)=((T.sub.10min(M)/T.sub.10min(W))1)*100 for Magnetosomes. For 210 g in iron of Sigma nanoparticles inserted in 4.6 cm.sup.3 of tissue exposed to ultrasounds of frequency 3 MHz and power 0.5 W/cm.sup.2, 1 W/cm.sup.2, 1.5 W/cm.sup.2, (Slope.sub.(S)) designs the slope at the origin of the temperature variation with time of Sigma nanoparticles mixed with tissue (Slope.sub.real(S)) designs the difference between slope at the origin of the temperature variation with time of Sigma nanoparticles mixed with tissue (Slope.sub.(S)) and the slope at the origin of the temperature variation with time of the tissue without the nanoparticles (Slope.sub.(w)). Slope.sub.realN(S) is Slope.sub.real(S) divided by the Sigma nanoparticle concentration in gram of iron comprised in Sigma nanoparticles per mL. Slope rise (Slope rise (S)) designates the percentage in slope rise estimated using the formula for magnetosomes: Slope rise .sub.(S) (%)=((Slope.sub.(S)/Slope.sub.(W))1)*100. The specific absorption rate of Sigma nanoparticles (SAR.sub.(S)), estimated in watt per gram of Sigma nanoparticles is deduced from the values of Slope (s), using the formula: SAR.sub.(S)=C.sub.v.Math.Slope.sub.(S)/C.sub.nano, where C.sub.v=4.2 J.Math.K.sup.11 g.sup.1 is the specific heat of water and C.sub.nano is the Sigma nanoparticle concentration in gram of Sigma nanoparticles per cm.sup.3 of tissue. The variation in temperature between the initial temperature measured before the application of the ultrasound and the temperature measured after 10 minutes of application of the ultrasound is designated as T.sub.10min(S) for Sigma nanoparticles. The difference between T.sub.10min(S) and T.sub.10min(w) is designated as T.sub.10min,real(S). The percentage in temperature rise is estimated for Sigma nanoparticles using the formula: Temperature rise .sub.(S) (%)=((T.sub.10min(S)/T.sub.10min(W))1)*100 for Sigma nanoparticles.
TABLE-US-00001 TABLE 1 Heating on tissue Water Magnetosome Sigma 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 W/cm.sup.2 Slope.sub.(w) 0.063 0.145 0.142 Slope.sub.(M) ( C./sec) 0.092 0.158 0.318 Slope.sub.(S) ( C./sec) 0.137 0.197 0.184 ( C./sec) Slope.sub.real (M) ( C./sec) 0.029 0.013 0.177 Slope.sub.real (S) ( C./sec) 0.074 0.052 0.042 Slope.sub.real N(M) 3 1 17 Slope.sub.real N(S) 7 5 4 (mL. C./sec/g.sub.Fe) ( C./sec/g.sub.Fe) Slope rise.sub.(M) (%) 47 9 124 Slope rise.sub.(S) (%) 118 36 30 SAR.sub.(M) (W/g.sub.Fe) 37 64 128 SAR.sub.(S) (W/g.sub.Fe) 52 75 70 SAR.sub.real (M) (W/g.sub.Fe) 12 5 71 SAR.sub.real (S) (W/g.sub.Fe) 28 20 16 T.sub.10 min (w) 16 32 28 T.sub.10 min (M) ( C.) 22 40 56 T.sub.10 min (S) ( C.) 30 38 35 ( C.) T.sub.10 min real (M) ( C.) 6 8 28 T.sub.10 min real (S) ( C.) 14 6 7 Temperature rise.sub.(M) 37 25 100 Temperature rise.sub.(S) 90 17 26 (%) (%)
[0768] Table 2.
[0769] For 100 g in iron of nanoparticles (Magnetosome, Sigma, SPION20, SPION50, SPION100) dispersed in 100 l of water exposed to ultrasounds of frequency 3 MHz and power 0.5 W/cm.sup.2, 1 W/cm.sup.2, 1.5 W/cm.sup.2, slopes at the origin of the temperature variation with time of the different nanoparticles dispersed in water, designated as Slope .sub.(M) for Magnetosome, Slope .sub.(S) for Sigma nanoparticles, Slope .sub.(S20) for SPION20, Slope .sub.(S50) for SPION50, Slope .sub.(S100) for SPION100. The difference between the slope at the origin of the temperature variation with time of nanoparticles dispersed in water and the slope at the origin of the temperature variation with time of water without the nanoparticles is designated by Slope.sub.real(M) for Magnetosome, Slope.sub.real(S) for Sigma nanoparticles, Slope.sub.real(S20) for SPION20, Slope.sub.real(S50) for SPION50, Slope.sub.real(S100) for SPION100. Values of slope rise in percentage estimated using the formula: Sloperise.sub.(M)=((Slope.sub.(M)/Slope.sub.(W)1)*100 for magnetosomes; Sloperise.sub.(S20)=((Slope.sub.(S20)/Slope.sub.(W))1)*100 for SPION20; Sloperise.sub.(S50)=((Slope.sub.(S50)/Slope.sub.(W)1)*100 for SPION50; Sloperise.sub.(S100)=((Slope.sub.(S100)/Slope.sub.(W)1)*100 for SPION100. The specific absorption rate (SAR), measured in watt per gram of nanoparticles, deduced from the values of Slope, using the formula: SAR.sub.(M)=C.sub.v.Math.Slope.sub.(M)/C.sub.nano for Magnetosome, SAR.sub.(S)=C.sub.v.Math.Slope.sub.(S)/C.sub.nano for Sigma nanoparticles, SAR.sub.(S20)=C.sub.v.Math.Slope.sub.(S20)/C.sub.nano for SPION20, SAR.sub.(S50)=C.sub.v.Math.Slope.sub.(S50)/C.sub.nano for SPION50, and SAR.sub.(S100)=C.sub.v.Math.Slope.sub.(S100)/C.sub.nano for SPION100, where C.sub.v=4.2 J.Math.K.sup.1g.sup.1 is the specific heat of water, C.sub.nano is the nanoparticle (Magnetosome, Sigma, SPION20, SPION50, or SPION100) concentration in gram of nanoparticles per mL of water. Slope.sub.(M), Slope.sub.(S), Slope.sub.(S20), Slope.sub.(S50), Slope.sub.(S100) are the initial slopes of the temperature variation for Magnetosome, Sigma nanoparticles, SPION20, SPION50, and SPION100. The real specific absorption rate (SAR.sub.real), measured in watt per gram of nanoparticles is deduced from the values of Slope.sub.real, using the formula: SAR.sub.real(M)=C.sub.v.Math.Slope.sub.real(M)/C.sub.nano for magnetosomes, SAR.sub.real(S)=C.sub.v.Math.Slope.sub.real(S)/C.sub.nano for Sigma nanoparticles, SAR.sub.real(S20)=C.sub.v.Math.Slope.sub.real(S20)/C.sub.nano for SPION20, SAR.sub.real(S50)=C.sub.v.Math.Slope.sub.real(S50)/C.sub.nano for SPION50, SAR.sub.real(S100)=C.sub.v.Math.Slope.sub.real(S100)/C.sub.nano for SPION100, where C.sub.v=4.2 J.Math.K.sup.1g.sup.1 is the specific heat of water and C.sub.nano is the nanoparticle (Magnetosome, Sigma, SPION20, SPION50, or SPION100) concentration in gram of nanoparticles per mL of water. Slope.sub.real(M), Slope.sub.real(S), Slope.sub.real(S20), Slope.sub.real(S50) Slope.sub.real(S100) designate the difference between the initial slope of the temperature variation with time of nanoparticles dispersed in water and the initial slope of the temperature variation with time of water without nanoparticles. For nanoparticles dispersed in water, the variation in temperature between the initial temperature measured before the application of the ultrasound and the temperature measured after 10 minutes of application of the ultrasound is designated as T.sub.10min(W) for water alone, T.sub.10min(M) for Magnetosomes, T.sub.10min(S) for Sigma nanoparticles, T.sub.10min(S20) for SPION20, T.sub.10min(S50) for SPION50, T.sub.10min(S100) for SPION100. The differences between T.sub.10min(M) and T.sub.10min(W), T.sub.10min(S) and T.sub.10min(W), T.sub.10min(S20) and T.sub.10min(W), T.sub.10min(S50) and T.sub.10min(W), T.sub.10min(S100) and T.sub.10min(W), are designated as T.sub.10minreal(M), T.sub.10minreal(S), T.sub.10minreal(S20), T.sub.10minreal(S50), T.sub.10minreal(S100) for magnetosomes, Sigma nanoparticles, SPION20, SPION50, SPION100, respectively. The percentage in temperature rise, estimated for Sigma using the formula: Temperature rise (%)=((T.sub.10min(S)/T.sub.10min(W))1)*100, for Magnetosomes using the formula: Temperature rise (%)=((T.sub.10min(S)/T.sub.10min(W))1)*100, for SPION20 using the formula: Temperature rise (%)=((T.sub.10min(S20)/T.sub.10min(W))1)*100, for SPION50 using the formula: Temperature rise (%)=((T.sub.10min(S50)/T.sub.10min(W))1)*100, for SPION100 using the formula: Temperature rise (%)=((T.sub.10min(S10)/T.sub.10min(W))1)*100.
TABLE-US-00002 TABLE 2 Heating on aqueous solutions Water Magnetosome 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 Slope.sub.(w) 0.291 0.467 0.645 Slope.sub.(M) 0.361 0.571 0.747 ( C./sec) ( C./sec) Slope.sub.real (M) 0.070 0.104 0.101 ( C./sec) Slope.sub.real N (M) 70 104 101 (mL. C./sec/g.sub.Fe) Slope rise.sub.(M) 24 22 16 (%) SAR.sub.(M) (W/g.sub.Fe) 1511.3 2389.5 3124.0 SAR.sub.real (M) 294.5 437.0 424.3 (W/g.sub.Fe) T.sub.10 min (w) ( C.) 13 20 30 T.sub.10 min (M) ( C.) 18 29 33 T.sub.10 min real (M) 5 9 3 ( C.) Temperature 37 43 10 rise.sub.(M) (%) Sigma SPION 50 nm 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 Slope.sub.(S) 0.275 0.503 1.287 Slope.sub.(S50) 0.274 0.481 1.313 ( C./sec) ( C./sec) Slope.sub.real (S) 0 0.036 0.642 Slope.sub.real (S50) 0 0.015 0.668 ( C./sec) ( C./sec) Slope.sub.real N (S) 0 36 642 Slope.sub.real N (S50) 0 15 668 (mL. C./sec/g.sub.Fe) (mL. C./sec/g.sub.Fe) Slope rise.sub.(S) 0 8 99 Slope rise.sub.(S50) 0 3 104 (%) (%) SAR.sub.(S) (W/g.sub.Fe) 1150.1 2104.7 5385.6 SAR.sub.(S50)(W/gFe) 1145.5 2014.5 5495.0 SAR.sub.real (S) 0 152.1 2685.9 SAR.sub.real (S50) 0 62.0 2795.3 (W/g.sub.Fe) (W/gFe) T.sub.10 min (S) ( C.) 19 32 39 T.sub.10 min (S50) ( C.) 15 26 34 T.sub.10 min real (S) 6 12 9 T.sub.10 min real (S50) 2 5 4 ( C.) ( C.) Temperature 49 60 31 Temperature 18 27 14 rise.sub.(S) (%) rise.sub.(S50) (%) SPION 100 nm SPION 20 nm 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 0.5 W/cm.sup.2 1 W/cm.sup.2 1.5 W/cm.sup.2 Slope.sub.(S100) 0.300 0.446 0.992 Slope.sub.(S20) 0.453 0.439 0833 ( C./sec) ( C./sec) Slope.sub.real (S100) 0.009 0 0.346 Slope.sub.real (S20) 0.162 0 0.188 ( C./sec) ( C./sec) Slope.sub.real N (S100) 0 0 346 Slope.sub.real N (S20) 162 0 188 (mL. C./sec/g.sub.Fe) (mL. C./sec/g.sub.Fe) Slope rise.sub.(S100) 3 0 54 Slope rise.sub.(S20) 56 0 29 (%) (%) SAR.sub.(S100) (W/gFe) 1253.2 1864.4 4149.1 SAR.sub.(S20) (W/gFe) 1894.2 1837.7 3486.8 SAR.sub.real (S100) 36.4 0 1449.4 SAR.sub.real (S20) 677.4 0 787.1 (W/gFe) (W/gFe) T.sub.10 min (S100) ( C.) 17 27 36 T.sub.10 min (S20) ( C.) 16 26 30 T.sub.10 min real (S100) 4 7 6 T.sub.10 min real (S20) 3 6 0 ( C.) ( C.) Temperature 34 35 20 Temperature 24 28 1 rise.sub.(S100) (%) rise.sub.(S20) (%)
[0770] Table 3.
[0771] For 500 g of magnetosomes dispersed in 100 l of water, exposed sequentially to ultrasounds, time t.sub.1 necessary to reach the desired temperature of 431.5 C. during the heating step (application of an ultrasound of frequency 3 MHz and power 1.5 W/cm.sup.2), time t.sub.2 necessary to reach 34.50.5 C. during the cooling step (non-application of ultrasound) during each of the 13 sequences, frequency of each sequence in mHz, 1/t.sub.1+t.sub.2.
TABLE-US-00003 TABLE 3 Time of non- Time of application application of the of the ultrasound, t.sub.1 = ultrasound, t.sub.2 = Utrasound heating step cooling step sequences (minuntes) (minutes) f (mHz) 1 0.43 0.2 26 2 0.26 0.36 27 3 0.22 0.27 34 4 0.21 0.27 35 5 0.23 0.23 36 6 0.22 0.27 34 7 0.24 0.26 33 8 0.2 0.24 38 9 0.2 0.21 41 10 0.24 0.3 31 11 0.2 0.25 37 12 0.24 0.28 32 13 0.25 0.29 31 Mean 0.24 0.26 33