CN101346149A - Ultrasonic cancer treatment enhancer and cell killer - Google Patents
Ultrasonic cancer treatment enhancer and cell killer Download PDFInfo
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- CN101346149A CN101346149A CNA2006800493695A CN200680049369A CN101346149A CN 101346149 A CN101346149 A CN 101346149A CN A2006800493695 A CNA2006800493695 A CN A2006800493695A CN 200680049369 A CN200680049369 A CN 200680049369A CN 101346149 A CN101346149 A CN 101346149A
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Abstract
To provide an ultrasonic cancer therapy accelerator, or cytotoxic agent, that while ensuring high safety, can strikingly enhance the efficacy of ultrasonic cancer therapy. There is provided an ultrasonic cancer therapy accelerator, or cytotoxic agent, comprising metal semiconductor particles. The ultrasonic cancer therapy accelerator or cytotoxic agent upon ultrasonic irradiation is activated so as to achieve annihilation or destruction of cancer cells.
Description
Technical field
The present invention relates to a kind of ultrasonic cancer treatment enhancer, it is used to promote by affected part is shone the ultrasonic cancer treatment that ultrasound wave carries out, the invention still further relates to a kind of cytocide, it is subjected to hyperacoustic irradiation, become cytotoxin by this irradiation, can kill and wound the cell that object is killed in conducts such as cancerous cell.
Background technology
In recent years, in cancer treatment,, proposed Noninvasive and can effectively treat the sound power chemotherapy of affected part for this two aspect of QOL (quality of life) that improves therapeutic effect and patient.This therapy is to activate by the antitumor that throw and medicament (ultrasound wave and use agent) are carried out in hyperacoustic irradiation, carries out tumor treatment.Known have use fullerene (fullerene) as ultrasonic therapy with and with the method for agent (for example, disclosing pamphlet No. 2002/66061) with reference to the TOHKEMY 2002-241307 communique and the world.In addition, also known use pigment as ultrasonic therapy with and with the method for agent (disclosing No. 98/1131 pamphlet and TOHKEMY 2003-226654 communique) with reference to TOHKEMY 2001-253836 communique, the world.In this method, the effect by the using ultrasound ripple excites Organic substance or the fullerene that produces free radical species (radical species) because of light, thereby treats.
On the one hand, also proposed in the cancer treatment, to use the scheme of the photocatalytic activity of titanium oxide.For example, titanium oxide has been proposed as the application (with reference to Japanese kokai publication hei 4-357941 communique) of medical instruments, as the scheme of the application (with reference to TOHKEMY 2001-200050 communique and No. 6677606 description of United States Patent (USP)) of the carrier of drug-supplying system (DDS).
And then, also proposed to carry out the ultrasonic irradiation of 35～42kHz and produce hydroxyl radical free radical and the technology (for example, with reference to TOHKEMY 2003-26406 communique) of decomposing organic matter by titanium oxide to 2～3mm granularity.
Summary of the invention
Specifically, the inventor obtains following opinion: by certain semiconductor grain of ultrasonic irradiation with characteristic frequency, significantly improve the therapeutic effect that uses hyperacoustic cancer when can guarantee high safety.And, the inventor also obtains following opinion: by using drug-supplying system, can further improve the therapeutic effect of cancer, wherein, this drug-supplying system throws above-mentioned semiconductor grain with in patient's body and arrive cancerous cell, thereafter to the ultrasound wave of affected part irradiation characteristic frequency.
Therefore, the purpose of this invention is to provide ultrasonic cancer treatment enhancer and the cytocide that the cell of object is killed in conducts such as can effectively killing and wounding cancerous cell when guaranteeing high security.
And ultrasonic cancer treatment enhancer of the present invention contains the metal semiconductor granule.
And cytocide of the present invention contains the metal semiconductor granule, is subjected to ultrasonic irradiation, becomes cytotoxin (cytotoxin) by this irradiation.
Further, the Therapeutic Method of cancer of the present invention is characterised in that, the animal that comprises the people is thrown and cytocide, throw with after, to cancerous cell irradiation ultrasound wave, make cytocide become cytotoxin by this irradiation, this cytotoxin kills and wounds cancerous cell.
And, the purposes that of the present invention, cytocide is used to make ultrasonic cancer treatment enhancer is that above-mentioned ultrasonic cancer treatment enhancer is used for following method: the animal that comprises the people is thrown and aforementioned ultrasonic cancer treatment enhancer, throw with after, to cancerous cell irradiation ultrasound wave, make cytocide become cytotoxin by this irradiation, this cytotoxin kills and wounds cancerous cell.
Description of drawings
Fig. 1 is the figure of kill rate that various granules are measured, the cell when shining 1 minute ultrasound wave in the expression example 4.
Fig. 2 is to the TiO that contains of various concentration in the expression example 5
2Granule (TiO
2The figure of the survival rate of the cell that/PAA) testing liquid measures.
Fig. 3 be in the expression example 6 to the generation of various granules superoxide anion that measure, when resulting from ultrasonic irradiation, be the figure that absorbance that colour reagent shows rises by reduction.
Fig. 4 is to the figure generation of various granules hydroxyl radical free radical that measure, when resulting from ultrasonic irradiation, detect the fluorescence intensity that shows with fluorometric reagent by active oxygen in the expression example 7.
Fig. 5 is the figure that in the expression example 8 absorbance generation, that show by the TPM-PS oxidation of various granules hydrogen peroxide that measure, when resulting from ultrasonic irradiation is risen.
Fig. 6 is to the figure generation of various granules singlet oxygen (Singlet oxygen) that measure, when resulting from ultrasonic irradiation, detect the fluorescence intensity that shows with fluorometric reagent by singlet oxygen in the expression example 9.
Fig. 7 is to TiO in the expression example 10
2(A) generation that measure, the superoxide anion when resulting from ultrasonic irradiation, be the figure that absorbance that colour reagent shows rises by reduction.
Fig. 8 is the figure of survival rate that various granules are measured, the cell when shining 1 minute ultrasound wave in the expression example 12.
Fig. 9 is to the figure generation of various granules hydroxyl radical free radical that measure, when resulting from ultrasonic irradiation, detect the fluorescence intensity that shows with fluorometric reagent by active oxygen in the expression example 13.
Figure 10 is to the TiO that contains of various concentration in the expression example 14
2The figure of particulate testing liquid survival rate that measure, cell.
Figure 11 is the figure of kill rate that various granules are measured, the cell when shining 1 minute ultrasound wave in the expression example 15.
Figure 12 is to TiO in the expression example 16
2That/PAA granule and PBS buffer solution are measured, irradiation is during 2 minutes ultrasound wave (ultrasound wave is arranged) and do not have the figure of the survival rate of the cell that shines (no ultrasound wave).
Figure 13 is the time dependent figure of relative tumor proliferation rate that measure, mice in the expression example 17.
The specific embodiment
Ultrasonic cancer treatment enhancer
The ultrasonic cancer treatment enhancer of the present invention's first scheme contains the metal semiconductor granule.The metal semiconductor granule that can use in the present invention can be various metal semiconductor granules so long as contain by ultrasonic irradiation activation can be killed or tumoricidal metal semiconductor forms granule then without limits.Though whole mechanism that cell was killed or destroyed to certain metal semiconductor granule by the ultrasonic irradiation activation are indeterminate, so long as can generate the metal semiconductor granule of free radical species at least by ultrasonic irradiation, then can expect above-mentioned effect.
That is the biological fragmentation effect that, the metal semiconductor granule brought can and obtain by irradiation ultrasound wave generation free radical species.That is, think that biological fragmentation effect that these semiconductor grains brought is the increase of the quality and quantity of free radical species.Its reason is inferred as follows, but following reason is hypothesis after all, and the present invention is not limited to following any explanation.That is, only then produce hydrogen peroxide and hydroxyl radical free radical by ultrasonic irradiation in system, still according to the inventor's opinion, in the presence of semiconductor grains such as titanium oxide, the generation of hydrogen peroxide and hydroxyl radical free radical obtains promoting.And, in the presence of these semiconductor grains, particularly under the existence of titanium oxide, see that the generation of superoxide anion and singlet oxygen obtains promoting.The specific generation of these free radical species is observed phenomenons when shining ultrasound wave under the situation of using nano level microgranule.Think that this effect is the special phenomenon that ultrasound wave produces in the presence of the metal semiconductor microgranule.
According to the preferred embodiment of the present invention, the preferred metal semiconductor granule with photoinduction characteristic that uses more preferably has the metal semiconductor granule and because the luminous quantum dot of quantum effect demonstration of photocatalytic activity as the metal semiconductor granule.Herein, the metal semiconductor granule with photocatalytic activity is meant and is subjected to the granule that luminous energy causes the metal semiconductor of separation of charge.In addition, quantum dot is meant the structure with following feature: because the Energy distribution of electronic carrier disperses, therefore mobility of charge carrier also takes place discontinuously existing under the such state of thermal excitation such as room temperature between quantum level (quantum level), the result demonstrates sharp-pointed luminous.Use this granule, can activate fully by hyperacoustic irradiation and kill cell.As the particulate object lesson of this metal semiconductor, can enumerate TiO
2, ZnO, WO
3, SnO
2, Fe
2O
3, In
2O
3, BaTiO
3, TiO
3, SrTiO
3, Nb
2O
5, Ta
2O
5Deng metal-oxide; CdS, MoS
2, metal sulfide such as ZnS; With composition metal quasiconductors such as SiC, CdSe, InGaP.These metal semiconductor granules have the photoinduction characteristic, but owing to be not photosensitizers such as fullerene or pigment, therefore the patient is thrown with after treatment stage in can not produce the problem of photoallergy, safety is very high.
According to the preferred embodiment of the present invention, can use metal semiconductor granule as the metal semiconductor granule with photocatalytic activity, can enumerate TiO as preference
2, ZnO, SnO
2, WO
3, In
2O
3, SrTiO
3, Nb
2O
5, Ta
2O
5, more preferably TiO
2
According to the preferred embodiment of the present invention, can use quantum dot as the metal semiconductor granule, can enumerate CdSe, CdS, CdTe, ZnS, ZnSe, InGaP, ZnTe and composition thereof as preference.
According to the preferred embodiment of the present invention, the metal semiconductor granule preferably has the particle diameter of 50～200nm, more preferably 50～150nm.In this particle size range, be that purpose is thrown with in patient's body the time to arrive tumor, can be as drug-supplying system, because EPR effect and arrive cancerous tissue effectively and accumulate.Therefore, can kill or destroy cancerous tissue expeditiously by hyperacoustic irradiation.
Other the preferred implementation according to the present invention, have at the metal semiconductor granule under the situation of particle diameter of not enough 50nm (for example count nm), also can will be between the metal semiconductor granule connect, strengthen the size on apparent, obtain the EPR effect with multifunctional connexon (linker).This embodiment is particularly using quantum dot effective under the particulate situation as metal semiconductor.That is,, assemble or be combined into the form of the second particle of particle diameter by making a plurality of quantum dots, thereby because the EPR effect can realize high cancer therapeutic effect with 50～150nm though quantum dot generally has the particle diameter of 2nm～10nm.Other the preferred implementation according to the present invention in order to utilize the EPR effect, can also be encapsulated into the metal semiconductor granule the such medicament of liposome and enclose in the body.In addition, if further give biochips such as antibody, also may produce the targeting that exceeds the EPR effect to tumor to the composite particles that obtains like this.
Can be used for metal semiconductor granule of the present invention and be not only the metal semiconductor granule of single kind, also comprise particulate mixture of multiple metal semiconductor or complex.Can enumerate the complex of complex, TiOx nano grain and platinum of TiOx nano granule and ferric oxide nanometer particle and coated with silica titanium oxide etc. as concrete example.
According to preferred implementation of the present invention, preferably at the particulate surface combination polymer of metal semiconductor or be derived from the macromolecule of organism and form.Thus, even originally be under the neutral so semi-conductive situation of solid, also can realize with the semiconductor grain being ultrasonic cancer treatment enhancer decentralized of main component, administration safely in the organism environment using.Here, as with the bonded polymer of metal semiconductor particle surface, so long as hydrophilic macromolecule gets final product.
According to preferred implementation of the present invention, as with the bonded polymer of metal semiconductor particle surface, the preferred polymer that uses with carboxyl, for example, carboxymethyl starch, Sensor Chip CM 5, carboxymethyl cellulose, polycarboxylic acid and have the unitary copolymer of carboxyl (copolymer), viewpoint from the water-disintegrable and dissolubility of hydrophilic macromolecule, more preferably polycarboxylic acid such as polyacrylic acid, poly and acrylic acid/maleic acid or acrylic acid/sulfonic acid are monomeric copolymer (copolymer), further optimization polypropylene acid.When the polymer with carboxyl combines with the metal semiconductor particle surface, granule is just electronegative, thereby high degree of dispersion and make the organism molecule easily or the medicament immobilization, thereby is applicable to by drop etc. and carries out the whole body administration.Therefore, surface combination polyacrylic metal semiconductor granule be suitable for the treatment of the cancer of internal organs inside, under will be with the affinity of specific cells high (organism) molecule or the immobilized situation of medicament, as the higher metal semiconductor granule of affinity, be specially adapted to targeted therapies to affected part.
According to preferred implementation of the present invention, as with the bonded polymer of metal semiconductor particle surface, the preferred cation property copolymer that uses, for example weight average molecular weight is at the amine of 1000～100000 scopes, more preferably polyamino acid, polypeptide, polyamine class and have the copolymer (copolymer) of amine unit, from the viewpoint of the water-disintegrable and dissolubility of water soluble polymer, further polyamine classes such as preferably polyethylene imines, polyvinylamine, PAH; Polymine most preferably.When cation property copolymer combines with the metal semiconductor particle surface, granule is just positively charged, thus high degree of dispersion and easily rapidly absorption enter in the cell, be applicable to by injection or smear the topical that carries out.Therefore, surface combination the metal semiconductor granule of polymine be particularly suitable for the treatment of the cancer on top layers such as skin carcinoma or early cancer.
According to preferred implementation of the present invention, as with the bonded polymer of metal semiconductor particle surface, preferably use the nonionic polymer, for example Polyethylene Glycol, polyvinyl alcohol, polyethylene glycol oxide, glucosan or their copolymer, more preferably Polyethylene Glycol.When the nonionic polymer combines with the metal semiconductor particle surface, granule is not charged just can be disperseed by hydration, thereby (in the blood) long-term stability in vivo, accumulates at cancerous tissue easily, also is applicable to the whole body administration.Therefore, be particularly suitable for the treatment of the cancer of the wide scope from the top layer to the deep layer with the metal semiconductor granule of coated with polyethylene glycol.
According to preferred implementation of the present invention, the preferable alloy semiconductor grain is dispersed in the solvent and forms.Thus, can be by drop, the whole bag of tricks such as inject, smear effectively to throwing and the metal semiconductor granule in patient's body.In addition, from the viewpoint of safety, dispersion liquid preferably has neutral liquid property, more preferably normal saline.
Cytocide
According to second embodiment of the invention, cytocide is provided, it contains the metal semiconductor granule, is subjected to ultrasonic irradiation, becomes cytotoxin by this irradiation.By this cytocide is thrown with in body, be subjected to ultrasonic irradiation, become cytotoxin by this irradiation, thereby can destroy or kill cell, but be not limited in the body, in test tube, also can destroy or kill as the cell of killing object.In this embodiment, there is no particular limitation to kill object, but be preferably cancerous cell.In the present embodiment, cytotoxin is preferably produced by the free radical species that ultrasonic irradiation produces by above-mentioned semiconductor grain.
According to preferred implementation of the present invention, the cytocide among the present invention comprises and is selected from TiO
2, SnO
2, at least a semiconductor grain in the group that ZnO and CdSe formed, be preferably TiO
2According to preferred implementation of the present invention, these semiconductor grains are subjected to the ultrasonic irradiation of 400kHz～20MHz, can become cytotoxin by this irradiation.This cytocide is thrown with in body, is subjected to ultrasonic irradiation, becomes cytotoxin by this irradiation, thereby can killer cell, but is not limited in the body, also can kill and wound in test tube as the cell of killing object.Among the present invention, there is no particular limitation to kill object, is preferably cancerous cell.That is, the cytocide in according to the present invention can kill and wound cancerous cell by the irradiation ultrasonic activation.In addition, these semiconductor grains are owing to be not photosensitizers such as fullerene or pigment, thus the patient is thrown with after treatment stage in can not produce the problem of photoallergy, safety is very high.
These semiconductor grains are the effect of killer cell by the irradiation ultrasonic activation, can obtain by producing the free radical species by ultrasonic irradiation.That is, think that biological fragmentation effect that these semiconductor grains brought is the increase of the quality and quantity of free radical species.Its reason is inferred as follows, but following reason is hypothesis after all, and the present invention is not limited to following any explanation.That is, only then produce hydrogen peroxide and hydroxyl radical free radical by ultrasonic irradiation in system, still according to the inventor's opinion, in the presence of semiconductor grains such as titanium oxide, the generation of hydrogen peroxide and hydroxyl radical free radical obtains promoting.And, in the presence of these semiconductor grains, particularly under the existence of titanium oxide, see that the generation of superoxide anion and singlet oxygen obtains promoting.The specific generation of these free radical species be under using the situation of nano level microgranule the frequency during the irradiation ultrasound wave the scope of 400kHz～20MHz, preferably in the scope of 600kHz～10MHz, more preferably in the remarkable observed phenomenon of scope of 1MHz～10MHz.Think that this effect is the special phenomenon that ultrasound wave produces in the presence of the metal semiconductor microgranule.
According to preferred implementation of the present invention, semiconductor grain has the particle diameter of 20～200nm, more preferably the particle diameter of 50～200nm, the further particle diameter of preferred 50～150nm.In this particle size range, be that purpose is thrown with in patient's body the time to arrive tumor, can be as drug-supplying system, because EPR effect and arrive cancerous tissue effectively and accumulate.And, as mentioned above, cause the specificity generation of free radical species by the ultrasonic irradiation of 400kHz～20MHz.Therefore, can kill and wound cancerous tissue expeditiously by the irradiation ultrasound wave.
Other the preferred implementation according to the present invention has at semiconductor grain under the situation of particle diameter of not enough 50nm (for example count nm), also can strengthen the size on apparent and obtains the EPR effect.That is, will have the form that particle diameter is the second particle of 50～150nm by being combined between the semiconductor grain with methods such as multifunctional connexon connections, thereby owing to the EPR effect realizes high cancer therapeutic effect.Other preferred embodiment in order to utilize the EPR effect, can also be encapsulated into semiconductor grain in the medicament inclosure body as liposome according to the present invention.
The particle diameter of the semiconductor grain among the present invention can pass through dynamic light scattering determination.Particularly, the represented value of Z-averagesize that can obtain using particle size distribution analyzer (Zetasizer Nano, malvern instruments company make), obtains with the cumulant analysis.
According to preferred implementation of the present invention,, then also may produce the targeting that exceeds the EPR effect to tumor if further semiconductor grain is given biochips such as antibody.
Can be used for semiconductor grain of the present invention and be not only the semiconductor grain of single kind, also comprise the mixture or the complex of multiple semiconductor grain.Can enumerate the complex of complex, TiOx nano granule and platinum of TiOx nano granule and ferric oxide nanometer particle and coated with silica titanium oxide etc. as concrete example.
According to preferred implementation of the present invention, preferably at the surface combination polymer of semiconductor grain and/or be derived from the macromolecule of organism and form.Thus, even originally be under the neutral so semi-conductive situation of solid, also can realize with the semiconductor grain being cytocide decentralized of main component, administration safely in the organism environment using.Here, as with the polymer of semiconductor grain surface combination, for hydrophilic macromolecule gets final product.From guaranteeing the viewpoint of blood retention, as semiconductor grain and polymer and/or be derived from the high molecular combining form of organism, so long as throw the combining form that to guarantee the degree of dispersibility with back in body after 24～72 hours, then do not do special qualification, but be based on the free and characteristics few to Normocellular injury that also do not have polymer behind dispersion stabilization excellence under physiological condition and the ultrasonic irradiation, hope is covalent bond.
According to preferred implementation of the present invention, as with the polymer of semiconductor grain surface combination, the preferred anionic property polymer that uses, for example carboxymethyl starch, Sensor Chip CM 5, carboxymethyl cellulose, polycarboxylic acid and have the polymer that unitary copolymer of carboxyl (copolymer) etc. has carboxyl, viewpoint from the water-disintegrable and dissolubility of hydrophilic macromolecule, more preferably polycarboxylic acid such as polyacrylic acid, poly and acrylic acid/maleic acid or acrylic acid/sulfonic acid are monomeric copolymer (copolymer), further optimization polypropylene acid.When anionic property polymer and semiconductor grain surface combination, granule is just electronegative, thereby high degree of dispersion and the functional group by this polymer make biomolecule or medicament immobilization, thereby is applicable to by drop etc. and carries out the whole body administration.Therefore, surface combination polyacrylic semiconductor grain be suitable for the treatment of the cancer of internal organs inside, under will be with the affinity of specific cells high (organism) molecule or the immobilized situation of medicament,, be specially adapted to targeted therapies to affected part as the higher semiconductor grain of affinity.The preferred zeta potential of having fixed the semiconductor grain complex of anionic property polymer is-50～-20mV.In the time of in this scope, owing to be easy to guarantee particulate dispersibility, be difficult for forming aggregation block, thereby needn't worry to cause after the administration secondary harm such as blood vessel blockage by the repulsion of negative charge.
According to preferred implementation of the present invention, as with the polymer of semiconductor grain surface combination, the preferred cation property copolymer that uses, for example weight average molecular weight is at the amine of 1000～100000 scopes, more preferably polyamino acid, polypeptide, polyamine class and have the copolymer (copolymer) of amine unit, from the viewpoint of the water-disintegrable and dissolubility of water soluble polymer, further polyamine classes such as preferably polyethylene imines, polyvinylamine, PAH, most preferably polymine.When cation property copolymer and semiconductor grain surface combination, granule is just positively charged, thereby enters in the cell also rapid easily absorption of high degree of dispersion, is applicable to by injecting or smear the topical that carries out.Therefore, surface combination the semiconductor grain of polymine be particularly suitable for the treatment of the cancer on top layers such as skin carcinoma or early cancer.The preferred zeta potential of having fixed the semiconductor grain complex of cation property copolymer is+20～+ 50mV.In the time of in this scope,, can be applicable to topical owing to be easy to guarantee particulate dispersibility by the repulsion of negative charge.
According to preferred implementation of the present invention, as with the polymer of semiconductor grain surface combination, the preferred polymer that uses the possess hydrophilic property group (hydroxyl and/or polyoxyalkylene) of nonionic, for example Polyethylene Glycol, polyvinyl alcohol, polyethylene glycol oxide, glucosan or their copolymer, more preferably Polyethylene Glycol.When nonionic polymer and semiconductor grain surface combination, granule is not charged just can be disperseed by hydration, thereby (in the blood) long-term stability in vivo, accumulates at cancerous tissue easily, also is applicable to the whole body administration.Therefore, be particularly suitable for the treatment of the cancer of the wide scope from the top layer to the deep layer with the semiconductor grain of coated with polyethylene glycol.The preferred zeta potential of semiconductor grain complex of polymer of having fixed the possess hydrophilic property group of nonionic is-20～+ 20mV.In this scope, because protein is difficult for Electrostatic Absorption in the blood that becomes, thereby be easy to avoid absorption to enter fine mesh endothelial tissue, renal excretion, be absorbed into liver etc., can guarantee can be enough to arrive the blood retention of target site (tumor).
According to preferred implementation of the present invention, preferably be dispersed in the solvent and form by semiconductor grain.Thus, can be by drop, the whole bag of tricks such as inject, smear semiconductor grain is thrown effectively with in patient's body.In addition, from the viewpoint of safety, dispersion liquid preferably has neutral liquid property, more preferably normal saline.Semiconductor grain preferably contains below 0.001～1 quality % with respect to dispersion, more preferably contains 0.001～0.1 quality %.In this scope, throw with after, can after 24～72 hours, make granule be accumulated in affected part (tumor) effectively.That is, be easy to accumulate granule density, but also can guarantee the particulate dispersibility in the blood, be difficult for to form aggregation block, needn't worry to cause after the administration secondary harm such as blood vessel blockage at affected part (tumor).
Therapeutic Method
The whole bag of tricks such as ultrasonic cancer treatment enhancer of the present invention and cytocide can be by drops, inject, smear are thrown with in patient's body.From utilizing EPR effect that granular size brings and blood retention and, especially preferably can use by vein or subcutaneous administration path by the viewpoint that so-called DDS treatment comes relieve patient ' s burden.And, is thrown and arrived intravital promoter or cytocide, as drug-supplying system, can arrive cancerous tissue and accumulate.Then, carry out ultrasonic Treatment to accumulating the cancerous tissue that promoter or cytocide are arranged.From the effect of killer cell and the viewpoint of safety, the preferred 20kHz～20MHz of employed frequency of ultrasonic, more preferably 400kHz～20MHz, further preferred 600kHz～10MHz, most preferably 1MHz～10MHz.Hyperacoustic irradiation time should be considered as the position of cancerous tissue of treatment target and size and come at discretionly, and there is no particular limitation.Thus, can by ultrasonic high-efficiency kill and wound patient's cancerous tissue, realize high cancer therapeutic effect.Ultrasound wave can arrive biological intravital deep from the outside, by promoter of the present invention or cytocide are used in combination, can realize under non-invasive state the affected part that is present in deep in the organism or the treatment of target site.And, the accumulating by promoter of the present invention or cytocide at affected part or target site, can be by the normal cell of periphery not being produced the faint ultrasound wave of pernicious effect, the only part generation effect that promoter of the present invention or cytocide are accumulated.
According to preferred implementation of the present invention, though as more preferably TiO of metal semiconductor granule
2, but also proposed in cancer treatment, to utilize the scheme of the particulate photocatalytic activity of this metal semiconductor.At this moment, can come affected part or target site are treated by using ultraviolet irradiation, the degree of depth that this irradiation arrives be apart from the organism top layer 2mm of portion.On the other hand, when using hyperacoustic irradiation to carry out biological intravital diagnosis etc., the inside more than the 2mm of portion of distance organism top layer also can be carried out.Therefore, can arrive the position, more deep of organism inside from the outside,, can under non-invasive state, realize being present in the such affected part in deep in the organism or the treatment of target site by being used in combination with promoter of the present invention by using ultrasound wave.
According to preferred implementation of the present invention, as the preferred position that is suitable for, can consider to be difficult to carry out the internal organs of resection operation, particularly can enumerate spleen adenocarcinoma, bladder cancer, the cerebral tumor.
Do not limit ultrasonic irradiation portion is special,,, ultrasonic irradiation portion can be arranged at utensil and the direct irradiation that endoscope or conduit etc. can arrive affected part or target site in order to obtain better effect according to the preferred embodiment of the present invention.In addition, for the internal organs that are difficult to carry out resection operation, perhaps can be from external with non-invasive state irradiation from the viewpoint of patient's QOL.Particularly,, can carry out ultrasonic irradiation, make ultrasound wave arrive affected part, kill and wound cancerous tissue from abdominal part for Alimentary top layer cancer.
Embodiment
Example 1:
Polyacrylic acid conjunction type titanium oxide (TiO 2 / PAA) particulate manufacturing
Mix 3.6g tetraisopropoxy titanium and 3.6g isopropyl alcohol, under ice-cooled, be added drop-wise in the 60ml ultra-pure water, be hydrolyzed.After the dropping, stirring at room 30 minutes.After the stirring, drip 1ml 12N nitric acid, stirred peptization 8 hours at 80 ℃.After peptization finishes, use the filter of 0.45 μ m to filter, using desalting column (PD-10, AmershamPharmacia Bioscience corporate system) to carry out the solution exchange and making solid constituent is 1% anatase-type titanium oxide colloidal sol.This dispersion liquid is packed in the bottle of capacity 100ml, with the ultrasonic Treatment of 200kHz 30 minutes.The average mark shot footpath of carrying out the ultrasonic Treatment front and back is respectively 36.4nm, 20.2nm.After the ultrasonic Treatment, concentrated solution is modulated into solid constituent and is 20% titanium oxide sol (Detitanium-ore-type).
The titanium oxide sol that 0.75ml is obtained is scattered in the dimethyl formamide (DMF) of 20ml, adds after 10ml dissolved the DMF of 0.2g polyacrylic acid (mean molecule quantity: 5000 and the pure medicine of light), mixes.Solution is moved in the hydro-thermal reaction container, and 150 ℃ were reacted 6 hours down.After reaction finished, being cooled to the reaction vessel temperature was below 50 ℃, behind the taking-up solution, added 80ml water, mixed.After using vaporizer to remove DMF and water, add 20ml water once more, make polyacrylic acid and modify the titanium oxide aqueous solution.Add 1ml 2N hydrochloric acid, the precipitation titan oxide particles separates unreacted polyacrylic acid thereby remove supernatant after centrifugal.Add entry once more and wash, centrifugal back is removed and is anhydrated.After adding 10ml 50mM phosphate buffer (pH7.0), the ultrasonic Treatment of usefulness 200kHz 30 minutes is disperseed titan oxide particles.After the ultrasonic Treatment, use the filter of 0.45 μ m to filter, make solid constituent and be 1.5% polyacrylic acid and modify titanium oxide sol.The polyacrylic acid that uses Zetasizer Nano ZS (sysmex corporate system) mensuration to make is modified the dispersion particle diameter of titanium oxide microparticle (Detitanium-ore-type), and drawing the average mark shot with the The Cumulant Method Using analysis directly is 45.5nm.This mensuration is following carries out: add in the zeta potential cell and contain the dispersion liquid 0.75ml of polymine in conjunction with titanium oxide microparticle, the various parameters of solvent are set to identical with the value of water, measure by dynamic light scattering method at 25 ℃.
Example 2:
Polymine conjunction type titanium oxide (TiO 2 / PEI) particulate manufacturing
Mix 3.6g tetraisopropoxy titanium and 3.6g isopropyl alcohol, under ice-cooled, be added drop-wise in the 60ml ultra-pure water, be hydrolyzed.After the dropping, stirring at room 30 minutes.After the stirring, drip 1ml 12N nitric acid, stirred peptization 8 hours at 80 ℃.After peptization finishes, use the filter of 0.45 μ m to filter, and then to use desalting column (PD-10, Amersham Pharmacia Bioscience corporate system) to carry out the solution exchange and make solid constituent be 1% acidic oxidation titanium colloidal sol.This dispersion liquid is packed in the bottle of capacity 100ml, with the ultrasonic Treatment of 200kHz 30 minutes.The average mark shot footpath of carrying out the ultrasonic Treatment front and back is respectively 36.4nm, 20.2nm.After the ultrasonic Treatment, concentrated solution is modulated into solid constituent and is 20% titanium oxide sol.
The resulting titanium oxide sol of 0.75ml is scattered in the dimethyl formamide (DMF) of 20ml, adds after 10ml dissolved the DMF of 450mg polymine (mean molecule quantity: 10000 and the pure medicine corporate system of light), mix.Solution is moved in the hydro-thermal reaction container (HU-50, three possess a fondness for science corporate system) 150 ℃ of reactions 6 hours down.After reaction finished, being cooled to the reaction vessel temperature was below 50 ℃, added the isopropyl alcohol of doubling dose, and polymine is precipitated in conjunction with titanium oxide microparticle, separated unreacted polymine thereby remove supernatant after centrifugal.Add 70% ethanol and wash, remove ethanol after centrifugal.After adding the 10ml distilled water, the ultrasonic Treatment of usefulness 200kHz 30 minutes, the dispersion polyethylene imines is in conjunction with titanium oxide microparticle.After the ultrasonic Treatment, use the filter of 0.45 μ m to filter, make solid constituent and be 1.5% polymine dispersion liquid in conjunction with titanium oxide microparticle.The polymine that use Zetasizer Nano ZS (sysmex corporate system) mensuration makes is in conjunction with the dispersion particle diameter of titanium oxide microparticle, and polymine is 67.7nm in conjunction with the mean diameter of titanium oxide microparticle.This mensuration is following carries out: add in the zeta potential cell and contain the dispersion liquid 0.75ml of polymine in conjunction with titanium oxide microparticle, the various parameters of solvent are set to identical with the value of water, measure by dynamic light scattering method at 25 ℃.
Example 3:
Polyethylene Glycol conjunction type titanium oxide (TiO 2 / PEG) particulate manufacturing
Mix 3.6g tetraisopropoxy titanium and 3.6g isopropyl alcohol, under ice-cooled, be added drop-wise in the 60ml ultra-pure water, be hydrolyzed.After the dropping, stirring at room 30 minutes.After the stirring, drip 1ml 12N nitric acid, stirred peptization 8 hours at 80 ℃.After peptization finishes, use the filter of 0.45 μ m to filter, and then to use desalting column (PD-10, AmershamPharmacia Bioscience corporate system) to carry out the solution exchange and make solid constituent be 1% acidic oxidation titanium colloidal sol.This acidic oxidation titanium colloidal sol is packed in the bottle of capacity 100ml, use supersonic generator MIDSONIC200 (KAIJO corporate system) with the ultrasonic Treatment of 200kHz 30 minutes.After 1000 times of the nitric acid of 12N dilutions, use Zetasizer Nano ZS (sysmex corporate system), in quartzy cell, add dispersion liquid 0.1ml, the various parameters of solvent are set to identical with the value of water, at 25 ℃ by the average mark shot after the dynamic light scattering determination ultrasonic Treatment directly.Consequently disperseing particle diameter is 20.2nm.Use evaporating dish that this titanium oxide sol is carried out concentrating of solution under 50 ℃, finally be modulated to solid constituent and be 20% acidic oxidation titanium colloidal sol.
Next, in the copolymer of polyoxyethylene-monoene propyl group-monomethyl ether and maleic anhydride (mean molecule quantity: 33659, Japanese oils and fats system) 1g, add 5ml water, carry out lyophilization after the hydrolysis.After reaction finishes, be dissolved in dimethyl formamide (DMF) solution of 5ml and modulate the 200mg/ml polyglycol solution.The polyglycol solution that 1.875ml is obtained is scattered in the DMF solution of 27.725ml, adds the anatase-type titanium oxide colloidal sol 0.9ml that makes in advance, mixes.Solution is moved among the hydro-thermal reaction container HU-50 (three possess a fondness for science corporate system) 150 ℃ of reactions 5 hours down.After reaction finished, being cooled to the reaction vessel temperature was below 50 ℃, after the use vaporizer is removed DMF, added the 10ml distilled water and made Polyethylene Glycol in conjunction with the titanium oxide aqueous solution.Further, with HPLC on the following condition, confirm that not keeping fraction has the UV absworption peak, reclaims this fraction.
HPLC:AKTA purifier, Amersham Pharmacia Bioscience corporate system chromatographic column: HiPrep 16/60Sephacryl S-300HR, Amersham PharmaciaBioscience corporate system mobile phase: phosphate buffer (pH7.4)
Flow velocity: 0.3ml/min
Is 0.01% aqueous solution with distilled water with this dispersion liquid dilution, disperses particle diameter and zeta potential by dynamic light scattering determination, and the dispersion particle diameter is 45.4nm, and zeta potential is 1.1mV.This mensuration is following carries out: use Zetasizer Nano ZS, add Polyethylene Glycol in conjunction with titanium oxide aqueous solution 0.75ml in the zeta potential cell, the various parameters of solvent are set to identical with the value of water, measure at 25 ℃.
Example 4:
Cell killing test by ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2/ PAA granule (granule of preparation in example 1)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
(neutral dispersion is disperseed particle diameter: 8nm) to the CdSe granule for Quantum Dot corporate system, Qdot655ProteinA labelling
Here, obtain above-mentioned various particulate dispersion particle diameter by the dynamic light scattering method shown in the example 1.
Next, above-mentioned semiconductor grain is scattered in the PBS buffer solution (pH6.8), is containing 1 * 10
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add this solution, be modulated into final concentration and be 0.05% testing liquid with 1/10 amount.
In addition, as a comparative example, below preparing is not the granule of metal semiconductor, modulates testing liquid with above-mentioned method.
SiO
2Granule (C.I. changes into system, Si MS 10WT%-G360, and neutral dispersion is disperseed particle diameter: 105nm)
The Au granule (ICN Biomedicals, the Inc system, ProteinA 20nm, Goldconjugate, neutral dispersion is disperseed particle diameter: 40nm)
Obtain above-mentioned various particulate dispersion particle diameter by the dynamic light scattering method shown in the example 1 herein.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 1 minute ultrasound wave of each testing liquid's irradiation that obtains, carry out the fragmentation test of cell.Its result as shown in Figure 1.As shown in Figure 1, all cells survival rates of solution of having added semiconductor grain are all low, i.e. kill rate height, thereby confirmed to pass through the ultrasonic irradiation killer cell.On the other hand, this fragmentation effect is not at the SiO that uses as a comparative example
2Be confirmed on granule or the Au granule.
Example 5:
The dependency of semiconductor grain concentration
Make the TiO that makes in the use-case 1
2Granule (TiO
2/ PAA) as semiconductor grain, carry out the test of cell killing concentration dependent by ultrasonic irradiation.The modulation titan oxide particles is containing 1 * 10 in PBS buffer (pH6.8)
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add, be modulated into final concentration and be 0.001%, 0.01%, 0.05% and 0.1% testing liquid with 1/10 amount.By ultrasonic unit (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to 1 minute ultrasound wave of this testing liquid's irradiation, carry out the fragmentation test of cell, the dependency of research semiconductor grain concentration.Its result as shown in Figure 2.Confirmed to have added in the solution concentration of titan oxide particles at all, the cells survival rate all reduces.Particularly the survival rate of cell reduces in final concentration 0.05% solution, and hence one can see that, and the high final concentration of cell killing effect is 0.05%.
Example 6:
The evaluation of the superoxide anion generative capacity during ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
Next, above-mentioned semiconductor grain is dispersed in the PBS buffer (pH6.8), the final concentration that makes solid constituent is 0.1%, will be the solution that colour reagent WST-1 (colleague's chemistry system) adds formation 0.5% in the aqueous solution as the reduction of superoxide anion generation reagent, modulation testing liquid.
In addition, as a comparative example, preparing is not the SiO of metal semiconductor
2Granule (C.I. changes into system, Si MS 10WT%-G360, and neutral dispersion is disperseed particle diameter: 105nm), modulate testing liquid with above-mentioned method.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to 5 minutes ultrasound wave of each testing liquid's irradiation of above-mentioned preparation, measure absorption under the 450nm wavelength by ultraviolet-visible spectrophotometer.Its result as shown in Figure 3.As shown in Figure 3, for all granules, generate yellow first along with the decomposition of WST-1 as semiconductor grain
(formazan), thus confirmed the rising of absorbance.That is, confirmed TiO
2Granule, SnO
2Granule and ZnO granule generate superoxide anion by ultrasonic irradiation.
Example 7:
The evaluation of the hydroxyl radical free radical generative capacity during ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
Next, above-mentioned semiconductor grain is dispersed in the PBS buffer (pH6.8), the final concentration that makes solid constituent is 0.1%, to generate active oxygen detection fluorometric reagent hydroxy phenyl fluorescein (hydroxyl the phenylfluorescein) (HPF of reagent as hydroxyl radical free radical, the first chemical drugs system) joins the solution that forms 5 μ M in the above-mentioned solution that contains metal oxide particle, modulation testing liquid.
In addition, as a comparative example, prepare SiO
2Granule (C.I. changes into system, Si MS10WT%-G360, and neutral dispersion is disperseed particle diameter: 105nm), modulate testing liquid with above-mentioned method.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 5 minutes ultrasound wave of each testing liquid's irradiation that obtain, by the wavelength under fluorescent spectrophotometer measuring Ex=490nm, the Em=515nm.Its result as shown in Figure 4.As shown in Figure 4, for all granules as semiconductor grain, the HPF reaction generates fluorescein, has confirmed the rising of fluorescence intensity.That is, confirmed TiO
2Granule, SnO
2Granule and ZnO granule generate hydroxyl radical free radical by ultrasonic irradiation.
Example 8:
The evaluation of the hydrogen peroxide generative capacity during ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
Next, above-mentioned semiconductor grain is dispersed in the PBS buffer (pH6.8), the final concentration that makes solid constituent is 0.1%, modulation testing liquid.
In addition, as a comparative example, prepare SiO
2Granule (C.I. changes into system, Si MS10WT%-G360, and neutral dispersion is disperseed particle diameter: 105nm), modulate testing liquid with above-mentioned method.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 5 minutes ultrasound wave of each testing liquid's irradiation that obtain, workbook according to Amplex RedHydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes corporate system), use this test kit, get the above-mentioned testing liquid of 100 μ l and measure.Its result as shown in Figure 5.As shown in Figure 5, confirmed TiO
2Granule can more effectively generate hydrogen peroxide by ultrasonic irradiation.
Example 9:
The evaluation of the singlet oxygen generative capacity during ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
TiO
2/ PAA (making) by example 1
TiO
2/ PEG (making) by example 3
Next, above-mentioned semiconductor grain is dispersed in the PBS buffer (pH6.8), the final concentration that makes solid constituent is 0.05%, modulation testing liquid.
In addition, as a comparative example, prepare SiO
2Granule (C.I. changes into system, Si MS10WT%-G360, and neutral dispersion is disperseed particle diameter: 105nm), modulate testing liquid with above-mentioned method.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 5 minutes ultrasound wave of each testing liquid's irradiation that obtain, workbook according to SingletOxygen Sensor Green Reagent (Molecular Probes company), use the test kit of this company, get the above-mentioned testing liquid of 100 μ l, result from the fluorescence intensity under Ex=488nm, Em=525nm of singlet oxygen by fluorescent spectrophotometer measuring.Its result as shown in Figure 6.As shown in Figure 6, confirmed TiO
2Granule can more effectively generate singlet oxygen by ultrasonic irradiation.
Example 10:
Dependency to frequency
Modulate in the microtubule of 2ml by the PBS buffer: 850 μ l, reduction are colour reagent WST-1 (colleague's chemistry system): 50 μ l, 0.1% titan oxide particles: the solution that 100 μ l form.Use TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm) as titan oxide particles.
The microtubule of gained is placed water-bath,, use multi-frequency ultrasonic generator (MODEL4021{KAIJYO}, output: 200W) with same intensity irradiation ultrasound wave in the distance of ultrasonic oscillator 3cm.Respectively at the sampling in 0,3,6 minute of irradiation back, get 200 μ l at every turn, with the assay method of example 6, measure superoxide anion.Respectively to 28,50,100,200 and each irradiation frequency of 600kHz measure.In addition, in contrast, the situation of not adding titan oxide particles is carried out and above-mentioned same mensuration.Its result as shown in Figure 7.As shown in Figure 7, owing to the existence of titanium produces superoxide anion, and then its effect is the most remarkable under the situation of the highest 600kHz of irradiation frequency.
Example 11:
Particulate stability
Following granule is scattered in water, PBS buffer (pH7.4) respectively and contains in the culture medium of RPMI1640 of 10% serum, obtain the granule ultimate density and be 0.01% sample.
Titanium oxide (C) (is scattered in P25 granule (Japanese Aerosil corporate system) in the PBS buffer (pH6.8) and makes, disperse particle diameter: 500nm)
The TiO that makes according to example 1
2/ PAA granule
The TiO that makes according to example 3
2/ PEG granule
(neutral dispersion is disperseed particle diameter: 52nm) to titan oxide particles (A) for STS-240, the former industry system of stone
Titan oxide particles (B) (TKS-203, the Tayca system, anatase-type titanium oxide, neutral dispersion is disperseed particle diameter: 120nm)
SnO
2Granule (C.I. changes into system, SNW15WT%-G02, and neutral dispersion is disperseed particle diameter: 39nm)
The ZnO granule (C.I. changes into system, Zn MS 15WT%-G01, and neutral dispersion is disperseed particle diameter: 67nm)
As the index of each particulate stability, measured the variation in average mark shot footpath in each dispersion liquid.This mensuration is following carries out: add dispersion liquid 0.1ml in quartzy cell, the various parameters of solvent are set to identical with the value of water, use ZetasizerNano ZS (sysmex corporate system), measure by dynamic light scattering method at 25 ℃ after 1 hour He after 24 hours.The average mark shot that each dispersion liquid is recorded directly is shown in Table 1.As shown in table 1, and relatively as the commercially available titanium oxide of neutral dispersion (A) or titanium oxide (B), the TiO that this example makes
2/ PAA and TiO
2The average mark shot of/PEG footpath with low uncertainty, hence one can see that has excellent stability.
Table 1
(unit: nm)
Example 12:
Cell killing test by ultrasonic irradiation
At first, with TiO
2(make in the example 1, neutral dispersion is disperseed particle diameter: 45.5nm) be scattered in the PBS buffer solution (pH6.8), containing 1 * 10 to/PAA granule
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add this solution, modulation TiO with 1/10 amount
2/ PAA granule final concentration is 0.01% and 0.001% testing liquid.
In addition, as a comparative example, prepare TiO
2Granule (C) (P25 granule (Japanese Aerosil corporate system) is scattered in the PBS buffer (pH6.8) makes, disperse particle diameter: 500nm), with above-mentioned method modulation testing liquid.Obtain above-mentioned various particulate dispersion particle diameter by the dynamic light scattering method shown in the example 1 herein.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, above-mentioned 1 minute ultrasound wave of each testing liquid's irradiation that obtains is carried out the cell killing test.Its result as shown in Figure 8.As shown in Figure 8, confirmed that all have added the dispersion particle diameter is the TiO of 45.5nm
2The tumor cytotoxicity effect of the particulate solution of/PAA is all high.On the other hand, at the TiO of the dispersion particle diameter 500nm that uses as a comparative example
2This cell killing effect is not confirmed in the granule (C).
Example 13:
The evaluation of the hydroxyl radical free radical generative capacity during ultrasonic irradiation
At first, prepare following semiconductor grain.
TiO
2Granule (A) (the former industry system of stone, anatase-type titanium oxide, STS-240, neutral dispersion is disperseed particle diameter: 52nm)
TiO
2Granule (D) (the former industry system of stone, anatase-type titanium oxide, STS-230, neutral dispersion is disperseed particle diameter: 15nm)
TiO
2Granule (C) (is scattered in P25 granule (Japanese Aerosil corporate system) in the PBS buffer (pH6.8) and makes, disperse particle diameter: 500nm)
Next, above-mentioned semiconductor grain is dispersed in the PBS buffer (pH6.8), the final concentration that makes solid constituent is 0.05%, to generate the active oxygen detection fluorometric reagent hydroxy phenyl fluorescein (HPF of reagent as hydroxyl radical free radical, the first chemical drugs system) joins the solution that forms 5 μ M in the above-mentioned solution that contains metal oxide particle, modulation testing liquid.
In addition, in contrast, prepare PBS buffer (pH6.8), modulate testing liquid with above-mentioned method.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 5 minutes ultrasound wave of each testing liquid's irradiation that obtain, result from the fluorescence intensity under Ex=490nm, Em=515nm of hydroxyl radical free radical by fluorescent spectrophotometer measuring.Its result as shown in Figure 9.As shown in Figure 9, be the TiO of 52nm for disperseing particle diameter
2Granule (A) and dispersion particle diameter are the TiO of 15nm
2Granule (D) has confirmed to generate comparison according to more significant hydroxyl radical free radical.Yet, be the TiO of 500nm disperseing particle diameter
2In the granule (C), generate hydroxyl radical free radical and be not confirmed.
Example 14:
Particulate safety
At first, with TiO
2(make in the example 1, neutral dispersion is disperseed particle diameter: 45.5nm), TiO to/PAA granule
2(make in the example 2, neutral dispersion is disperseed particle diameter: 67.7nm) and TiO to/PEI granule
2(make in the example 3, neutral dispersion is disperseed particle diameter: 45.4nm) be scattered in the PBS buffer solution (pH6.8), obtain various dispersion liquids to/PEG granule.Containing 1 * 10
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add these dispersion liquids, be modulated into TiO with 1/10 amount
2The final concentration that is composite particles is the test liquid of 0.1,0.01 and 0.001 quality %.In addition, PBS buffer modulation control sample liquid is equally only added in test in contrast.After the modulation, at CO
2Cultivated 24 hours for 37 ℃ in the couveuse, use CellTiterGro Kit (BIO RAD corporate system) to measure viable count.With the survival rate of control sample liquid as 100% with each experimental liquid result relatively as shown in figure 10.As shown in figure 10, for all test liquids of 0.1～0.001 quality %, shown high cells survival rate, i.e. high security.
Example 15:
Use is combined with the TiO of polymer 2 Particulate by ultrasonic irradiation The cell killing test
With TiO
2/ PAA granule (making in the example 1), TiO
2/ PEI granule (making in the example 2) and TiO
2/ PEG granule (making in the example 3) is scattered in the PBS buffer (pH6.8), is containing 1 * 10
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add these solution, be modulated into final concentration and be 0.05% testing liquid with 1/10 amount.
By ultrasonic irradiation device (the OG skill is ground corporate system, ULTRASONICAPPARATUS ES-2:1MHz), with 0.5W/cm
2, 50%duty cycle running, to above-mentioned 1 minute ultrasound wave of each testing liquid's irradiation that obtains, carry out the fragmentation test of cell.Its result as shown in figure 11.As shown in figure 11, added TiO
2/ PAA granule, TiO
2/ PEI granule and TiO
2The cells survival rate of the particulate any particulate solution of/PEG is low, i.e. the kill rate height.Particularly added TiO
2The cells survival rate of the particulate solution of/PEG is extremely low, therefore, confirms this TiO
2/ PEG granule can be high especially by the effect of ultrasonic irradiation killer cell.
Example 16:
Cell killing test 2 by ultrasonic irradiation
At first, with TiO
2(make in the example 1, neutral dispersion is disperseed particle diameter: 45.5nm) be scattered in the PBS buffer (pH6.8), containing 5 * 10 to/PAA granule
4In the RPMI1640 culture medium (Invitrogen company) of adding 10% serum of the Jurkat cell of cells/ml, add this solution, modulation 3mlTiO with 1/10 amount
2The particulate final concentration of/PAA is 0.05% testing liquid.
In addition, prepare PBS buffer solution (pH6.8) in contrast, the same method is modulated testing liquid.
By the ultrasonic irradiation device (the SUNX corporate system, EH2435:5MHz) to above-mentioned each testing liquid that obtains with 2 minutes ultrasound wave of peak power Continuous irradiation, carry out the fragmentation test of cell.Its result as shown in figure 12.The living cells quantity of adding PBS buffer solution and not carrying out ultrasonic irradiation as 100%, is represented with survival rate.As shown in figure 12, having confirmed to add the dispersion particle diameter is the TiO of 45.5nm
2The tumor cytotoxicity effect height of the particulate solution of/PAA.
Example 17:
The antitumous effect test
(T-24) to nude mice (nude mouse) (Balb/c, hero, 3 ages in week) subcutaneous vaccination and form tumor, form about 0.63mm with the establishment cell strain that is derived from the human bladder cancer (established cell line)
3About tumor.With the PAA-TiO that makes in the example 1
2Be diluted to 0.5% with the PBS buffer, to locally injected into tumor 100 μ l.Behind the self administration of medication 24 hours, (the OG skill is ground corporate system, ULTRASONIC APPARATUS ES-2:1MHz to use the ultrasonic irradiation device; Probe footpath: 10mm), with the ultrasonic irradiation of power 1W, 50% pulse, 1MHz 1 minute.On skin, smear the water soluble polymer gel, and closely shine behind the contact probe (ultrasonic irradiation portion) thereon.Every group of number of mice is 6, and the animal of throwing and PBS or non-processor is only organized in contrast.Behind the ultrasonic irradiation, measure each individual gross tumor volume, the PAA-TiO when obtaining with each individual ultrasonic irradiation construction day (0 day)
2Gross tumor volume before the administration is as the gross tumor volume (tumor proliferation rate relatively) of each minute point of 1 o'clock.
The results are shown among Figure 13.
As shown in figure 13, only when titanium oxide+ultrasonic irradiation exists, confirmed the significantly proliferation inhibiting effect of tumor.
Claims (37)
1. ultrasonic cancer treatment enhancer, it contains the metal semiconductor granule.
2. ultrasonic cancer treatment enhancer according to claim 1, above-mentioned metal semiconductor granule can generate the free radical species by ultrasonic irradiation.
3. ultrasonic cancer treatment enhancer according to claim 1 and 2, above-mentioned metal semiconductor granule has the photoinduction characteristic.
4. according to each described ultrasonic cancer treatment enhancer of claim 1～3, above-mentioned metal semiconductor granule has photocatalytic activity.
5. ultrasonic cancer treatment enhancer according to claim 4, above-mentioned metal semiconductor granule with photocatalytic activity is selected from TiO
2, ZnO, SnO
2, WO
3, In
2O
3, SrTiO
3, Nb
2O
5And Ta
2O
5The group of being formed.
6. according to each described ultrasonic cancer treatment enhancer of claim 1～5, above-mentioned metal semiconductor granule is TiO
2
7. according to each described ultrasonic cancer treatment enhancer of claim 1～6, above-mentioned metal semiconductor granule has the particle diameter of 50～200nm.
8. according to each described ultrasonic cancer treatment enhancer of claim 1～3, above-mentioned metal semiconductor granule is a quantum dot.
9. ultrasonic cancer treatment enhancer according to claim 8, above-mentioned quantum dot are to be selected from least a in the group that CdSe, CdS, CdTe, ZnS, ZnSe, InGaP and ZnTe form.
10. according to claim 1～6,8 and 9 each described ultrasonic cancer treatment enhancers, above-mentioned metal semiconductor granule has the form of second particle, and the form of this second particle forms by gathering or combination, and has the particle diameter of 50～200nm.
11. according to each described ultrasonic cancer treatment enhancer of claim 1～10, above-mentioned metal semiconductor granule is particulate mixture of multiple metal semiconductor or complex.
12. according to each described ultrasonic cancer treatment enhancer of claim 1～11, at the particulate surface combination polymer of above-mentioned metal semiconductor or be derived from the macromolecule of organism and form.
13. ultrasonic cancer treatment enhancer according to claim 12, above-mentioned polymer are the polymer with carboxyl.
14. ultrasonic cancer treatment enhancer according to claim 12, above-mentioned polymer is a cation property copolymer.
15. ultrasonic cancer treatment enhancer according to claim 12, above-mentioned polymer are non-ionic polymers.
16. according to each described ultrasonic cancer treatment enhancer of claim 1～15, by above-mentioned metal semiconductor Dispersion of Particles in solvent and form.
17. ultrasonic cancer treatment enhancer according to claim 16, above-mentioned dispersion liquid has neutral liquid property.
18. according to claim 16 or 17 described ultrasonic cancer treatment enhancers, above-mentioned dispersion liquid is a normal saline.
19. cytocide, it contains the metal semiconductor granule, is subjected to ultrasonic irradiation, becomes cytotoxin by this irradiation.
20. cytocide according to claim 19, it is thrown with in body, is subjected to ultrasonic irradiation, becomes cytotoxin by this irradiation.
21., kill to liking cancerous cell according to claim 19 or 20 described cytocides.
22. according to each described cytocide of claim 19～21, above-mentioned cytotoxin is produced by the free radical species that ultrasonic irradiation produces by above-mentioned semiconductor grain.
23. according to each described cytocide of claim 19～22, it contains and has 20～200nm particle diameter and be selected from as the particulate TiO of above-mentioned metal semiconductor
2, SnO
2, at least a semiconductor grain in the group that ZnO and CdSe formed, be subjected to the ultrasonic irradiation of 400kHz～20MHz, become cytotoxin by this irradiation.
24. cytocide according to claim 23, above-mentioned semiconductor grain is TiO
2
25. according to claim 23 or 24 described cytocides, above-mentioned semiconductor grain is to combine and form at this particle surface and polymer and/or the macromolecule that is derived from organism.
26. cytocide according to claim 25, above-mentioned polymer are the anionic property polymer.
27. cytocide according to claim 25, above-mentioned polymer is a cation property copolymer.
28. cytocide according to claim 25, above-mentioned polymer are the polymer of the possess hydrophilic property group of nonionic.
29., be dispersed in the solvent by above-mentioned semiconductor grain and form according to each described cytocide of claim 23～28.
30. cytocide according to claim 29, above-mentioned solvent has neutral liquid property.
31. according to claim 29 or 30 described cytocides, above-mentioned solvent is a normal saline.
32. according to each described cytocide of claim 29～31, it contains the above-mentioned semiconductor grain of 0.001～1 quality %.
33. according to each described cytocide of claim 23～28, above-mentioned semiconductor grain has and is frozen exsiccant powder morphology.
34. according to each described cytocide of claim 23～33, by throwing with in body via venous administration path.
35. according to each described cytocide of claim 23～33, by throwing with in body via subcutaneous administration path.
36. the Therapeutic Method of cancer is characterized in that, this method is for throwing and each described cytocide of claim 19～35 animal that comprises the people, throw with after, to cancerous cell irradiation ultrasound wave, make cytocide become cytotoxin by this irradiation, this cytotoxin kills and wounds cancerous cell.
37. each described cytocide of claim 19～35 is used to make the purposes of ultrasonic cancer treatment enhancer, wherein, above-mentioned ultrasonic cancer treatment enhancer is used to following method: the animal that comprises the people is thrown and above-mentioned ultrasonic cancer treatment enhancer, throw with after, to cancerous cell irradiation ultrasound wave, make cytocide become cytotoxin by this irradiation, this cytotoxin kills and wounds cancerous cell.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069491A (en) * | 2013-03-27 | 2014-10-01 | 中国科学院宁波材料技术与工程研究所 | Ternary nano composite drug, preparation method thereof, and application of the drug in preparation of pharmaceutical composition for treating tumors |
| CN104127873A (en) * | 2014-07-29 | 2014-11-05 | 中国科学院化学研究所 | Nano material and external radiation source-containing tumor treatment medicine for realizing tumor vascular occlusion |
| CN105497911A (en) * | 2016-01-08 | 2016-04-20 | 南京大学医学院附属鼓楼医院 | Preparation method of CdTe quantum dot nanometer medicine carrying system carrying daunorubicin and gambogic acid |
| CN110917350A (en) * | 2019-12-27 | 2020-03-27 | 广西师范大学 | Preparation and application of black tin dioxide nano photothermal material |
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2006
- 2006-10-26 CN CNA2006800493695A patent/CN101346149A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069491A (en) * | 2013-03-27 | 2014-10-01 | 中国科学院宁波材料技术与工程研究所 | Ternary nano composite drug, preparation method thereof, and application of the drug in preparation of pharmaceutical composition for treating tumors |
| CN104069491B (en) * | 2013-03-27 | 2018-01-02 | 中国科学院宁波材料技术与工程研究所 | Ternary nano combination drug, its preparation method and its purposes for being used to prepare the pharmaceutical compositions for the treatment of tumour |
| CN104127873A (en) * | 2014-07-29 | 2014-11-05 | 中国科学院化学研究所 | Nano material and external radiation source-containing tumor treatment medicine for realizing tumor vascular occlusion |
| CN105497911A (en) * | 2016-01-08 | 2016-04-20 | 南京大学医学院附属鼓楼医院 | Preparation method of CdTe quantum dot nanometer medicine carrying system carrying daunorubicin and gambogic acid |
| CN105497911B (en) * | 2016-01-08 | 2018-12-28 | 南京大学医学院附属鼓楼医院 | The preparation method of daunorubicin and gambogicacid CdTe quantum nanometer medicine-carried system is carried altogether |
| CN110917350A (en) * | 2019-12-27 | 2020-03-27 | 广西师范大学 | Preparation and application of black tin dioxide nano photothermal material |
| CN110917350B (en) * | 2019-12-27 | 2021-08-10 | 广西师范大学 | Preparation and application of black tin dioxide nano photothermal material |
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