CN101601607A - A kind of tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously - Google Patents
A kind of tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously Download PDFInfo
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Abstract
It is a kind of that tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously is how to heat cell as the nanometer heater after entering cell at magnetic nano-particle in the thermotherapy in the tumor cell, how the local temperature that each cell stands problem such as measures, proposed a kind ofly to realize tumor cell is carried out thermometric method in magnetic induction heating, imaging and the cell simultaneously, with the development of further promotion tumor magnetic induction thermotherapy technology by the multifunctional nano microsphere.Its method is that the magnetic nano-particle that will have the magnetic induction heating function is compound in macromolecule or the inorganic nanometer microballoon simultaneously with the fluorescence quantum with imaging and temp sensing function, under action of alternating magnetic field, utilize the Nano microsphere pair cell that is attached on the tumor cell to heat, under fluorescence microscope, utilize the fluorescence quantum pair cell to carry out imaging and thermometric simultaneously.
Description
Technical field
The present invention proposes and a kind ofly be used for the multi-functional nanometer material of thermotherapy in the tumor cell and carry out thermometric method in magnetic induction heating, imaging and the cell simultaneously, belong to biological and medical science nano material and technical field.
Background technology
The magnetic induction thermotherapy is a kind of of tumor thermotherapy, is magnetic medium is positioned tumor locus and produces heat under the effect of alternating magnetic field, and the temperature in tumor district is reached more than 42 ℃, thereby the kill tumor cell reaches the purpose of treatment.
1971, Burton has early studied matter thermotherapy (magnetic interstitialhyperthermia between the tissue of ferromagnetic hot seed, MIH), they are one group of about 1mm of diameter, be about 1.5cm alloyed iron magnet and (be called " hot seed ", thermoseed) by certain spacing, evenly in the implantation tumour, provide the alternation induced field by external coil, hot seed (produces heat owing to eddy-current loss and magnetic hystersis loss under the effect of general 50k～2MHz) at electromagnetic field, and conduct to tumor tissues, make it to heat up.Hot seed generally adopts the alloy material of Curie-point temperature lower (45～60 ℃) to constitute, as Ni-Si (4%), Ni-Ca (29.6%), Ni-Pd (73%) etc.Because hot seed do not need external cable, implant and place and can repeatedly use, this is for the thermotherapy of deep tumor highly significant.The shortcoming of this " hot seed " method needs operation that hot seed is implanted exactly, and because the distribution of hot seed certainly exists distance, can not and have cold spot to tumor body even heating.
Gordon in 1979 etc. proposed the magnetic liquid thermotherapy (magnetic fluid hyperthermia, MFH) or cell in thermotherapy (intracellular hyperthermia, viewpoint IH).(magnetic fluid is MF) as the heat medium of magnetic induction thermotherapy promptly to adopt magnetic liquid.The Jordan seminar of Humboldt-Universitaet zu Berlin's medical college of Germany just carried out the research of magnetic liquid thermotherapy from 1993.They are topmost backers of thermotherapy in the cell.They think magnetic liquid are injected in target (focus or the tissue) position, because engulfing and fusion of cell, magnetic particle can enter into cell, and along with the division of cell, magnetic particle in the blast cell can enter daughter cell, again target is placed power enough greatly, the sufficiently high alternating magnetic field of frequency, along with the heat production of the magnetic particle of cell interior, target cell can be dead because of partial heat effect, thereby reach therapeutic effect.In this research field, the seminar of Jordan has carried out a series of work, comprises the cytologic experiment of thermotherapy in the cell and the magnetic liquid thermotherapy zoopery that the tumor model of meiofauna (mice) is carried out.The Kobayashi seminar of Japan Nagoya university has also carried out the meiofauna experimentation of a series of magnetic liquid thermotherapy, also is one of main supporter of thermotherapy in the cell.Mitsumori etc. also combine the magnetic thermotherapy with the arterial thrombosis therapy, so-called arterial thrombosis thermotherapy (arterial embolization hyperthermia has been proposed, AEH) therapy, they with magnetic liquid through the rabbit kidney arterial perfusion, but thromboembolism renal artery not only, the magnetic field that applies simultaneously make the kidney district produce high temperature.
The magnetic liquid thermotherapy is as a kind of new treatment tumor technology, because the focus that advantages such as its distinctive targeting, Wicresoft, low toxic and side effects are subjected to people's attention just gradually and become research.At present, Jordan seminar is on the basis of experiment with the development of alternating magnetic field heater, gone out MFH300F type magnetic induction heat therapy machine with Berlin, Germany Magforce company joint research and development, now be installed in Berlin Xia Lite medical centre, it is reported and utilize this equipment combining nano magnetic fluid successfully to treat 25 routine patients.Units such as domestic in recent years Tsing-Hua University, Southeast China University, Shanghai Communications University, Fudan University, chemistry institute of the Chinese Academy of Sciences and the refined hospital in Hunan are also carrying out number of research projects aspect the magnetic induction thermotherapy, comprise equipment development, the exploitation of magnetic heat medium, zoopery etc., form certain influence in the world, and actively to clinical propelling.
However, biomedical mechanism, temperature survey and the hot meterological problem on some bases of magnetic liquid thermotherapy and advanced magnetic heat medium exploitation, remaining needs urgent basis and the bottleneck problem that solves.Though thermotherapy has obtained some experimental evidence supports in the cell, how magnetic nano-particle heats cell as the nanometer heater after entering cell, and how the local temperature that each cell stands is measured, and it is unknown to remain us.For this reason, this patent has proposed by advanced multi-function magnetic heat medium design, realizes temperature survey in the heating of pair cell and the cell simultaneously, and gives the probe function that material can be estimated as imageology.The multifunctional nano microsphere of design is to comprise magnetic nano-particle simultaneously (to be used for heating and nuclear magnetic resonance, can to select Fe
3O
4, Fe
2O
3Etc. the magnetic nanoparticle) and the temperature-sensitive fluorescence quantum (be used for thermometric and fluorescence imaging, can select CdTe, doped with Mn
2+Semi-conductor nano particles such as ZnS, their fluorescence intensity has the response of reversible linear temperature) macromolecule or inorganic nanometer microballoon.Magnetic nano-particle is owing to hysteresis effect, Neel relaxation and Brownian relaxation have good AC magnetism heat effect, than submicron or more the large scale microsphere can absorb and more many magnetic field energy, and because nano magnetic particle is easy to disperse and administration, can reduce adding the requirement of alternating magnetic field output, perhaps reduce dosage, thereby obtained broad research and application.Simultaneously, magnetic nano-particle has gone through to use clinically as nuclear magnetic resonance (MRI) contrast-enhancing agent, and U.S. AdvancedMagnetics company and German Schering company have developed series of products.Semiconductor-quantum-point has been widely used in biomedical research and application as fluorescent labeling.Utilize the temperature-sensitive fluorescence quantum to carry out temperature sensing and developed into a new research field " Nanoparticle Luminescence Thermometry ", some fluorescence quantums constantly are designed with synthetic, and they have stable responding with reversible linear temperature.By nanometer composite technology magnetic nano-particle and temperature-sensitive fluorescence quantum are carried out ingenious combination, thereby realize tumor cell is carried out thermometric in magnetic induction heating, imaging and the cell simultaneously, formed the innovation and the characteristic of this patent maximum.
Summary of the invention
Technical problem: how to heat cell as the nanometer heater after the objective of the invention is to enter cell at magnetic nano-particle in the thermotherapy in the tumor cell, how the local temperature that each cell stands problem such as measures, proposed a kind ofly to realize tumor cell is carried out thermometric method in magnetic induction heating, imaging and the cell simultaneously, with the development of further promotion tumor magnetic induction thermotherapy technology by the multifunctional nano microsphere.
Technical scheme: of the present inventionly tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously be: the fluorescence quantum that will have the magnetic nano-particle of magnetic induction heating function and have imaging and a temp sensing function is compound in macromolecule or the inorganic nanometer microballoon simultaneously, under action of alternating magnetic field, utilize the Nano microsphere pair cell that is attached on the tumor cell to heat, under fluorescence microscope, utilize the fluorescence quantum pair cell to carry out imaging and thermometric simultaneously.
Described magnetic nano-particle is selected Fe
3O
4Or Fe
2O
3Magnetic nano-particle has the function of magnetic induction heating cell.
Described fluorescence quantum is thermal sensitivity CdTe, doped with Mn
2+The ZnS semi-conductor nano particles, their fluorescence intensity has the response of reversible linear temperature, the temperature when being used to measure magnetic nano-particle magnetic induction heating cell and be used for pair cell and carry out fluorescence imaging.
Described macromolecule or inorganic nanometer microballoon are selected macromolecule or SiO such as polystyrene, polylactic acid, sodium alginate, chitosan
2Inorganic material is as the carrier of magnetic nano-particle and fluorescence quantum.
Described Nano microsphere forms stable water slurry system after the organic molecule finishing, have biocompatibility, the microsphere size in tens nanometers to 1 micrometer range.
When being adsorbed onto tumor cell surface or internalization after described Nano microsphere and tumor cell are cultivated altogether and entering in the cell, the magnetic nano-particle that imposes in the alternating magnetic field driving Nano microsphere heats, the fluorescence intensity of the temperature-sensitive fluorescence quantum in the Nano microsphere changes with the temperature of monitoring heating simultaneously, fluorescence intensity changes and can measure or carry out direct imaging with fluorescence microscope by fiber spectrometer, thus heating when realizing, thermometric and imaging to tumor cell.
Described when under action of alternating magnetic field, carrying out fluorescence imaging and thermometric, adopt nonmetal excitation light path system and nonmetal detection light path system, to avoid metal easy heated problem in alternating magnetic field.
Beneficial effect:
(1) at thermotherapy research in the magnetic induction tumor cell, the proposition of novelty is by making up the multifunctional nano microsphere system, the temperature-sensitive fluorescence quantum that will have thermometric and imaging function carries out ingenious the combination with the magnetic nano-particle with magnetic induction heating and nuclear magnetic resonance function, with provide one can heat simultaneously, nanotechnology solution that thermometric and imageology are estimated, this is significant to magnetic induction thermotherapy and applied research.
(2) the quantum dot fluorescence temp sensing function can reflect the temperature of nanosphere heater microcell, and fluorescent labeling can be carried out the imaging observation simultaneously, and this micromechanism to thermotherapy in the research cell is significant.
Description of drawings
Fig. 1 is the structure of multifunctional nuclear shell composite nano-microsphere.Comprising: the magnetic nano-particle 1 with magnetic induction heating function, fluorescence quantum 2 with imaging and temp sensing function, the macromolecule or the inorganic nano kernel 3 that contain magnetic nano-particle contain the macromolecule or the inorganic shell 4 of fluorescence quantum, organic molecule finishing coat 5.
The specific embodiment
The multifunctional nano microsphere of design is to comprise magnetic nano-particle simultaneously (to be used for heating and nuclear magnetic resonance, can to select Fe
3O
4, Fe
2O
3Etc. the magnetic nanoparticle) and the temperature-sensitive fluorescence quantum (be used for thermometric and fluorescence imaging, can select CdTe, doped with Mn
2+Semi-conductor nano particles such as ZnS, their fluorescence intensity has the response of reversible linear temperature) macromolecule or inorganic nanometer microballoon (, can select macromolecule or SiO such as polystyrene, polylactic acid, sodium alginate, chitosan as carrier
2Etc. inorganic material).This Nano microsphere can form stable water solution system after the organic molecule finishing, by surperficial coupling specificities tumor cell identification molecule, can also realize the targeting to tumor cell simultaneously.Being targeted to tumor cell surface or internalization after Nano microsphere and the co-culture of cells enters in the cell, at this moment imposing the magnetic nano-particle that the alternating magnetic field of certain frequency and intensity drives in the Nano microsphere heats, the fluorescence intensity of the temperature-sensitive fluorescence quantum in the Nano microsphere changes with the temperature of monitoring heating simultaneously, fluorescence intensity changes and can measure or carry out direct imaging with fluorescence microscope by fiber spectrometer, thus heating when realizing, thermometric and imaging to tumor cell.
(1) the multifunctional nano microsphere has nucleocapsid structure, and kernel is macromolecule or inorganic (polylactic acid, alginate, the silicon dioxide etc.) microsphere that comprises magnetic nano-particle, by selecting the Fe of suitable dimension
3O
4Or Fe
2O
3Can effectively strengthen the magnetic induction heats etc. magnetic nanoparticle (10-30 nanometer) and increase magnetic loading amount; Shell is macromolecule (polyelectrolyte, polyvinyl alcohol etc.) or the SiO that comprises the temperature-sensitive fluorescence quantum
2Shell, with protection with intercept contacting of quantum dot and magnetic nano-particle, avoid taking place fluorescent quenching.This nucleocapsid composite nano-microsphere after poly-D-lysine, chitosan or albumin finishing, can form stable and biocompatibility water solution system, it has the enhanced ability that enters cell, direct heating tumor cell under action of alternating magnetic field, and the temperature of the method pair cell internal heating of available fluorescence measurement is measured, carry out direct observation with fluorescence microscope, thus heating when realizing, thermometric and iconography research to tumor cell.
Specific requirement to the multifunctional nano microsphere:
1) to 1 micrometer range, the surface has biocompatiblity molecules to be modified the Nano microsphere size, can form stable water slurry in tens nanometers;
2) macromolecule or the inorganic material of formation Nano microsphere have biocompatibility, and magnetic nano-particle and fluorescence quantum are carried out making whole Nano microsphere have enough magnetic loading amount and fluorescence intensity after the embedding to have low cytotoxicity simultaneously;
3) magnetic nano-particle is selected the Fe of good biocompatibility
3O
4, Fe
2O
3Deng material, size Control has strong intensification effect in the 10-30 nanometer in alternating magnetic field;
4) temperature-sensitive fluorescent quantum point selection CdTe, doped with Mn
2+Semi-conductor nano particles such as ZnS, its fluorescence intensity has the response of reversible linear temperature.
(2) can carry out the method for alternating magnetic field heating and fluorescence measurement simultaneously.Magnetic induction heater (frequency 50-200KHz takes place to adopt in alternating magnetic field, intensity 0-15KA/m, planar induction coil), fluorescence excitation (as 488nm) and emission light (as the 567nm emission light of 3nm diameter CdTe nanoparticle) are measured and are adopted fibre-optical probe and nonmetal collection light path, to avoid metal in alternating magnetic field, to be heated even to melt, spectral measurement employing fiber spectrometer.In addition, quantum dot temperature-sensitive fluorescence can also be observed by inverted fluorescence microscope under the action of alternating magnetic field, needs to use the nonmetal object lens that mate with microscope in the experiment.Embodiment one, be nuclear, make up the method for the polymer shell that contains the temperature-sensitive fluorescence quantum by self-assembling method layer by layer with magnetic alginic acid microsphere:
1) adopt chemical coprecipitation to prepare Fe
3O
4Magnetic nano-particle, direct oxidation prepares γ-Fe under acid condition
2O
3Magnetic nano-particle, and carry out finishing to form stable and water solution system biocompatibility with dimercaptosuccinic acid (DMSA).
2) adopting electrochemical process is that the presoma that the tellurium source generates has synthesized the water soluble cadmium telluride quantum point (CdTe QDs) that mercaptopropionic acid (MPA) is modified with the negative electrode tellurium, obtains the quantum dot of different-grain diameter and fluorescent emission wavelength by the control presoma heating in water bath time.
3) γ-Fe that adopts microemulsion method to modify with sodium alginate and DMSA
2O
3The magnetic nano-particle mixed solution is a water, and isobutyltrimethylmethane. is an oil phase, and polysorbate85 and sorbester p37 are that surfactant forms water in oil micelle, add CaCl again
2Aqueous solution carries out crosslinked preparation composite magnetic alginic acid microsphere, and by controlling mass ratio, hydrophile-lipophile balance value (HLB), mixing speed and the CaCl of magnetic-particle and sodium alginate
2Rate of addition obtain the microsphere of different size and magnetic loading amount.The size Control of whole complex microsphere is in hundreds of nanometer to 1 micron.
4) by the nucleocapsid structure of self-assembling method (layer-by-layer) preparation layer by layer complex microsphere.Principle is to utilize composite magnetic alginic acid microsphere surface electronegative, by alternately adsorbing cationic polyelectrolyte and anionic polyelectrolyte, form the polymer transition zone, the CdTe quantum dot that alternately adsorbs cationic polyelectrolyte and electronegative surface again forms the polymer shell layer that contains the temperature-sensitive fluorescence quantum.
5) with organic molecules such as poly-D-lysine, chitosan or albumin the nucleocapsid structure complex microsphere surface that obtains is modified, obtained stable aqueous suspension system.
Embodiment two, be nuclear, make up the method for the polymer shell that contains the temperature-sensitive fluorescence quantum by the chemical coupling method with the magnetic polylactic acid microsphere:
1) adopt chemical coprecipitation to prepare Fe
3O
4Magnetic nano-particle, the black nano particle adds oleic acid as coating material in case form the back, obtains oil-soluble Fe after washing and alcohol are washed
3O
4Magnetic nano-particle is scattered in it in normal hexane.
2) adopting electrochemical process is that the presoma that the tellurium source generates has synthesized the water soluble cadmium telluride quantum point (CdTe QDs) that mercaptopropionic acid (MPA) is modified with the negative electrode tellurium, obtains the quantum dot of different-grain diameter and fluorescent emission wavelength by the control presoma heating in water bath time.
3) adopt solvent evaporated method to prepare the magnetic polylactic acid microsphere: with polylactic acid and oil-soluble Fe
3O
4Magnetic nano-particle is scattered in the mixed solution (ratio is 7: 3) of dichloromethane and acetone, and fully ultra-sonic dispersion is made oil phase; The polyvinyl alcohol water solution that disposes 1% concentration is as water; Oil phase is mixed with aqueous phase solution, with 3 each 7min of homogenizing separating apparatus (12000rpm room temperature) emulsifying; The emulsion that emulsifying is obtained changes 40 degrees centigrade of constant temperature stirring at low speed volatilization 2h in the 400ml secondary water over to; With the magnetic sample separating, washing three times of volatilization gained, fixed molten with secondary water to 50ml, obtain the magnetic polylactic acid microsphere.
4) coupling of fluorescence quantum on the magnetic polylactic acid microsphere surface: at first coat obtaining the magnetic polylactic acid microsphere surface, on microsphere surface, introduce amino with containing amino silane coupler; Reuse carbonization imidodicarbonic diamide and N-hydroxy-succinamide are as coupling agent, and the cadmium telluride quantum dot that the surface is contained carboxyl is coupled to microsphere surface.
5) with organic molecules such as poly-D-lysine, chitosan or albumin the nucleocapsid structure complex microsphere surface that obtains is modified, obtained stable aqueous suspension system.
Claims (7)
1. one kind is carried out magnetic induction heating, imaging and thermometric method simultaneously to tumor cell, it is characterized in that the fluorescence quantum that will have the magnetic nano-particle of magnetic induction heating function and have imaging and temp sensing function is compound in macromolecule or the inorganic nanometer microballoon simultaneously, under action of alternating magnetic field, utilize the Nano microsphere pair cell that is attached on the tumor cell to heat, under fluorescence microscope, utilize the fluorescence quantum pair cell to carry out imaging and thermometric simultaneously.
2. according to claim 1 tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously, it is characterized in that described magnetic nano-particle selects Fe
3O
4Or Fe
2O
3Magnetic nano-particle has the function of magnetic induction heating cell.
3. according to claim 1 tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously, it is characterized in that described fluorescence quantum is thermal sensitivity CdTe, doped with Mn
2+The ZnS semi-conductor nano particles, their fluorescence intensity has the response of reversible linear temperature, the temperature when being used to measure magnetic nano-particle magnetic induction heating cell and be used for pair cell and carry out fluorescence imaging.
4. according to claim 1 tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously, it is characterized in that described macromolecule or inorganic nanometer microballoon select macromolecule or SiO such as polystyrene, polylactic acid, sodium alginate, chitosan
2Inorganic material is as the carrier of magnetic nano-particle and fluorescence quantum.
5. according to claim 1 tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously, it is characterized in that described Nano microsphere forms stable water slurry system after the organic molecule finishing, have biocompatibility, the microsphere size in tens nanometers to 1 micrometer range.
6. according to claim 1 tumor cell is carried out magnetic induction heating simultaneously, imaging and thermometric method, when it is characterized in that being adsorbed onto after described Nano microsphere and tumor cell are cultivated altogether tumor cell surface or internalization and entering in the cell, the magnetic nano-particle that imposes in the alternating magnetic field driving Nano microsphere heats, the fluorescence intensity of the temperature-sensitive fluorescence quantum in the Nano microsphere changes with the temperature of monitoring heating simultaneously, fluorescence intensity changes and can measure or carry out direct imaging with fluorescence microscope by fiber spectrometer, thus heating when realizing to tumor cell, thermometric and imaging.
7. according to claim 6 tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously, it is characterized in that described when under action of alternating magnetic field, carrying out fluorescence imaging and thermometric, adopt nonmetal excitation light path system and nonmetal detection light path system, to avoid metal easy heated problem in alternating magnetic field.
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| CN112587812B (en) * | 2020-12-14 | 2022-11-25 | 哈尔滨工程大学 | Heat seed containing self-temperature-control magnetic nanowires for magnetic thermal therapy and manufacturing method thereof |
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