WO2005030329A1 - Procede d'endommagement de biomolecules et appareil d'endommagement de biomolecules - Google Patents

Procede d'endommagement de biomolecules et appareil d'endommagement de biomolecules Download PDF

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Publication number
WO2005030329A1
WO2005030329A1 PCT/JP2004/004472 JP2004004472W WO2005030329A1 WO 2005030329 A1 WO2005030329 A1 WO 2005030329A1 JP 2004004472 W JP2004004472 W JP 2004004472W WO 2005030329 A1 WO2005030329 A1 WO 2005030329A1
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photosensitizer
light beam
biomolecule
light
dna
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PCT/JP2004/004472
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English (en)
Japanese (ja)
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Tetsuro Majima
Kiyohiko Kawai
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Japan Science And Technology Agency
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention provides direct or indirect clarification by photoexciting a photosensitizer.
  • a method and an apparatus for damaging (cutting, etc.) a body molecule More specifically, a method for damaging a biomolecule by a multi-step photoexcitation method and a method for writing the method are described.
  • the present invention relates to an apparatus for performing the operations.
  • PDT photodynamic therapy
  • a cancer patient is ingested by intravenous injection of a tumor-affinity chemical substance (such as a photosensitizer) that easily causes a photochemical reaction (photosensitization reaction).
  • a tumor-affinity chemical substance such as a photosensitizer
  • photochemical reaction photosensitization reaction
  • the chemical is not easily taken up by normal cells and excreted quickly, but is easily taken up by cancer cells and remains for a long time. Therefore, by irradiating the cancer tissue with a laser beam, the cancer cells are locally and selectively destroyed by a photochemical reaction.
  • Figure 2 (a) shows the mechanism I of DNA damage caused by reactive oxygen species generated by the reaction between an excited state photosensitizer and oxygen molecules.
  • S photosensitizer
  • S * photosensitizers excited state
  • S * molecular oxygen
  • FIG. 2 (b) shows the mechanism II of DNA damage caused by photosensitization and electron oxidation.
  • the photosensitizer (S) accumulated in the affected area is irradiated with laser light (hv), so that the photosensitizer is excited (S *). Up to this point, it is the same as mechanism I.
  • S * photosensitizer excited state
  • S * photosensitizer excited state
  • D NA * + a radical cation
  • S e - photosensitizer radical Anion
  • Treatment with PDT requires a high-intensity pulsed laser. For this reason, a complex reaction of two or more active species, or the light of the generated active intermediate The reaction is thought to be important for DNA damage. Further, when the target of the photosensitization reaction is DNA, the above mechanism II is considered to be more important.
  • the present invention has been made in view of the above problems, and has as its object to provide a biomolecule damage method that enhances the efficiency of biomolecule damage (cleavage, rupture, etc.) by photosensitization-electron oxidation, and Providing a device for performing the method can reduce the burden on the patient and the physician's work in a short time. It may be necessary to realize a method of PDT that can achieve highly efficient therapeutic effects. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, after irradiating the photosensitizer with a laser to excite the photosensitizer (first stage photoexcitation),
  • the photosensitizer radical anion generated by the one-electron oxidation reaction between the photosensitizer and DNA is irradiated with a laser to excite the photosensitizer radical anion (second-step photoexcitation),
  • Release of the sensitizer into the solvent to regenerate the sensitizer can prevent the charge recombination between the radical cation and the radical cation generated in the DNA.
  • the present invention has been completed in that DNA damage due to an electron oxidation reaction can be generated with high efficiency.
  • a biomolecule damage method is a biomolecule damage method for irradiating a photosensitizer with a light beam to damage a biomolecule in order to solve the above-mentioned problems.
  • the method is characterized by irradiating the sensitizer with light beams of different wavelengths at least once to excite the photosensitizer a plurality of times.
  • the biomolecule damage method according to the present invention includes a first photoexcitation step of exciting a photosensitizer by irradiating a light beam, and a photosensitizer and a biomolecule excited by the first photoexcitation step. Irradiating the radical anion of the photosensitizer generated by the reaction with the light beam with a light beam to further excite the radical anion of the photosensitizer to emit electrons, thereby including a second photoexcitation step. preferable.
  • the emitted electrons react with oxygen molecules to generate active oxygen species.
  • the light beam used in the second light excitation step is a light beam having a longer wavelength than the light beam used in the first light excitation step.
  • an incident angle of the light beam irradiated in the second light excitation step is different from an incident angle of the light beam irradiated in the first light excitation step.
  • the irradiation of the light beam in the second photoexcitation step may be delayed from the irradiation of the light beam in the first photoexcitation step on the order of nanoseconds to microseconds. It is preferably performed in time.
  • the biomolecule damage method according to the present invention is a biomolecule damage method for damaging a biomolecule by irradiating a photosensitizer with a light beam of the same wavelength a plurality of times to solve the problems described above. Therefore, a photosensitizer capable of two-photon excitation is used as the photosensitizer, and a light beam having a wavelength approximately twice the absorption wavelength of the photosensitizer is applied to the photosensitizer.
  • the biomolecule is preferably DNA or RNA.
  • the biomolecule damage device according to the present invention is characterized by including a light beam irradiation means capable of irradiating at least two kinds of light beams having different wavelengths.
  • the light beam irradiation means may include a control means capable of controlling the delay time of irradiation of at least two kinds of light beams having different wavelengths in the order of nanoseconds to the order of microphone mouth seconds. preferable.
  • the biomolecule damage device includes light beam irradiation means capable of irradiating a single-wavelength light beam, and the light beam irradiation means performs irradiation of the single-wavelength light beam. It is characterized by further comprising control means capable of controlling the delay time on the order of nanoseconds to microseconds.
  • the light beam irradiating means is formed so that the incident angles of the at least two kinds of light beams can be changed.
  • ADVANTAGE OF THE INVENTION According to the biomolecule damage method or biomolecule damage apparatus according to the present invention, there is an effect that the efficiency of biomolecule damage by photosensitization-electron oxidation reaction can be increased. Therefore, it can be used for the treatment of tumors such as cancer by new PDT by causing biomolecule damage more efficiently. Further objects, features, and advantages of the present invention will be made clear by the description below. Also, the advantages of the present invention will become apparent in the following description with reference to the accompanying drawings. Brief Description of Drawings
  • FIG. 1 is a diagram schematically showing the basic mechanism of the biomolecule damage method according to the present invention.
  • Fig. 2 (a) and Fig. 2 (b) show the mechanism of cell death by PDT
  • Fig. 2 (a) shows the mechanism when DNA is damaged by reactive oxygen species.
  • FIG. 2 (b) is a diagram showing the mechanism when DNA is damaged by a photosensitization-electron oxidation reaction.
  • FIG. 3 is a diagram schematically showing an experiment performed in an example according to the present invention.
  • FIG. 4 is a diagram schematically showing a water-soluble NDI used in Examples according to the present invention and a DNA to which the water-soluble NDI is bound.
  • FIG. 5 is a diagram schematically showing that the cutting efficiency of the DNA is changed by changing the timing of irradiating the light beam in the second light excitation step.
  • FIG. 6 is a diagram showing the results of an experiment performed in an example according to the present invention. More specifically, FIG. 6 contains oligodoxynucleotide (ODN s) damaged by a photosensitization reaction.
  • FIG. 7 is a view showing the results of an experiment performed in an example according to the present invention. More specifically, FIG. 7 shows a pulse obtained by using NDI combined with oligo godetoxynucleotide. After generating NDI radical anion by radiolysis, two microseconds later, electron emission from NDI radical anion excited by a pulsed laser of 52 nm was shown. The figure shows the results of observing the generation and decay of solvated electrons at 63 nm.
  • FIG. 6 contains oligodoxynucleotide (ODN s) damaged by a photosensitization reaction.
  • FIG. 7 is a view showing the results of an experiment performed in an example according to the present invention. More specifically, FIG. 7 shows a pulse obtained by
  • FIG. 8 is a diagram showing the results of experiments performed in Examples according to the present invention. More specifically, NDI radicals generated by a photosensitized electron transfer reaction by irradiating a pulse laser of 365 nm Investigate anion production and decay
  • FIG. 9 is a diagram showing the results of an investigation and the effect of the delay time between two pulsed lasers on the destruction of guanine.
  • FIG. 9 is a diagram showing the results of examining the effect of the irradiation time of the 52nd second laser beam on DNA damage.
  • FIG. 10 is a diagram showing the result of examining the effect of the irradiation intensity of the 52nd 2nd laser on the damage of DNA.
  • a biomolecule damaging method is a biomolecule damaging method for irradiating a photosensitizer with a light beam to damage a biomolecule, wherein the photosensitizer has different wavelengths of light. Any method may be used as long as the method irradiates the beam at least once to excite the photosensitizing substance a plurality of times, and other specific conditions and steps are not particularly limited. That is, the present invention can be referred to as a biomolecule damage method by multi-step light excitation.
  • the “method of damaging biomolecules using a photosensitizer” refers to irradiating the photosensitizer with a light beam (for example, a pulsed laser) to excite the photosensitizer, and then generating the excited photosensitizer.
  • a light beam for example, a pulsed laser
  • a method by which a substance directly or indirectly damages a biomolecule More specifically, as shown in Fig. 2 (a), when the excited photosensitizer generates reactive oxygen species, and this reactive oxygen species damages biomolecules, and Fig. 2 (b) ),
  • the excited photosensitizer is Includes cases where electrons are extracted from biomolecules and the biomolecules are directly damaged.
  • the latter excited photosensitizer extracts one electron from the base (particularly guanine) that constitutes DNA, and generates DNA radical cation (photosensitization-electron oxidation reaction) to convert DNA.
  • the method of damaging is preferred.
  • biomolecule refers to various intracellular molecules such as DNA, RNA, protein, and lipid, but DNA and RNA are particularly preferred.
  • photosensitizer refers to a substance that undergoes a photochemical reaction that absorbs photons and generates an electronically excited state, and is sometimes referred to as a photoreactive substance.
  • a conventionally known photosensitizer used for PDT or the like can be used. Specific examples include hematoporphyrin, HpD, porphyrin ⁇ chlorin ⁇ purpurin ⁇ ALA, and naphthaldiimide (NDI) used in Examples described later.
  • the term "damaging a biomolecule” means damaging a biomolecule in a cell to such an extent that the cell is killed.
  • the biomolecule is DNA or RNA
  • the genetic information is accurately reproduced.
  • DNA or RNA is damaged to such an extent that it cannot be transcribed, and includes cases where DNA or RNA is cleaved.
  • “DNA” basically refers to double-stranded DNA, but also includes single-stranded DNA. It also includes genomic DNA and cDNA present in living organisms, and DNA produced by chemical synthesis techniques.
  • the chain length of the DNA is not particularly limited, and it is sufficient that the DNA has a chain length that allows the photosensitizer to interact.
  • FIG. 1 shows the basic part of the biomolecule damage method according to the present invention.
  • the present invention selectively incorporates a photosensitizer into a site where a biomolecule is to be damaged, for example, a disease site (such as a site where a cancer occurs), and then selectively irradiates the light with the photosensitizer. It can be used for PDT, which can cause biomolecule damage and treat the disease without damaging the normal site around the diseased site.
  • DNA will be mainly described as a biomolecule, but the present invention is not limited to this.
  • the photosensitizer S may be excited to 1 S * after absorbing light and then converted to 3 S *.
  • the photosensitizer S * which is bound to DNA and excited by light irradiation, is a base constituting DNA, for example, by extracting electrons from guanine to form a photosensitizer, Radical anion (s Generates a nin radical cation (G * + ) to create a charge separation state.
  • Radical anion s Generates a nin radical cation (G * + ) to create a charge separation state.
  • G * + nin radical cation
  • the photosensitizer S is excited in two stages. That is, first, the photosensitizer S is excited by the first-stage light irradiation, and electrons are extracted from, for example, guanine in DNA. It generates photosensitizer radical anion (S *) and guanine radical force thione (G * +). Then, the second stage light irradiation is performed on the generated photosensitizer radical anion. The second stage of light irradiation excites the photosensitizer radical A-one and emits electrons. As a result, the photosensitizer radical-one can be eliminated, so that charge recombination with radical cations in DNA can be prevented, and efficient DNA damage can be caused.
  • S * photosensitizer radical anion
  • G * + guanine radical force thione
  • the biomolecule damage method according to the present invention preferably includes a first photoexcitation step for performing the first-stage photoexcitation and a second photoexcitation step for performing the second-stage photoexcitation.
  • the first light excitation step and the second light excitation step may be repeatedly performed.
  • the first light excitation step and the second light excitation step will be described separately in detail.
  • the first photoexcitation step according to the present invention may be any step as long as the photosensitizer is excited by irradiating a light beam, and other specific conditions and materials are not particularly limited. That is, it can be said that the first photoexcitation step is a step of exciting the photosensitizer by irradiation with a light beam to generate an excited photosensitizer.
  • the photosensitizer in the excited state generated by this process for example, directly extracts electrons from base molecules (any of the four nucleic acid bases) constituting DNA to generate DNA radical cations, Or, it reacts with oxygen molecules to generate reactive oxygen species, thereby damaging biomolecules such as DNA.
  • the “light beam” a conventionally known light beam irradiation device such as a pulse laser can be used. This makes it easy and efficient to increase light Sensitive substance can be excited.
  • “exciting the photosensitizer” preferably means, but not limited to, selectively exciting the photosensitizer by irradiation with a light beam.
  • the wavelength of the light beam irradiated in the first photoexcitation step is a wavelength that can excite the photosensitizer used, and can be appropriately set according to the type and properties of the photosensitizer. Needless to say, there is.
  • the irradiation time, irradiation intensity, etc. of the light beam irradiated in the first light excitation step can be appropriately set according to the photosensitizer used and other conditions.
  • the photosensitizer excited by the first photoexcitation step reacts with a biomolecule to irradiate a light beam to a radical anion of the photosensitizer generated by reacting with a light beam, Any steps may be used as long as the step further excites the photosensitizer radical anion and emits electrons from the photosensitizer radical anion, and other specific conditions and materials are not particularly limited.
  • the photosensitizer in the excited state generated in the first photoexcitation step extracts one electron from a base molecule (any of four nucleic acid bases) constituting DNA
  • the photosensitizer radical radical Nions and DNA radical cations are generated, which usually recombine by electron transfer reactions.
  • the efficiency of DNA damage by DNA radical cations decreases.
  • the generated photosensitizer radical anion is again excited to emit one electron, thereby extinguishing the photosensitizer radical anion and converting the photosensitizer into a photosensitizer. Can be played. Therefore, the photosensitizer radical anion and DNA radiation It is possible to prevent a decrease in DNA damage efficiency due to charge recombination with a calcation.
  • the biomolecules can be efficiently damaged.
  • the light beam irradiation in the second photoexcitation step is performed on the order of nanoseconds (1) with respect to the light beam irradiation in the first photoexcitation step.
  • the delay is preferably in the order of 0 to 19 seconds to microseconds (10 to 6 seconds).
  • the delay time of the light beam irradiation in the second light excitation step with respect to the light beam irradiation in the first light excitation step depends on the type and properties of the photosensitizer used, the bonding state and the bonding position between the photosensitizer and the biomolecule. It can be set as appropriate according to the conditions. More specifically, as shown in FIG.
  • the radiation generated in the DNA for example, guanine It is preferable to irradiate the light beam in the second photoexcitation step before the charge of the calcation is recombined. This is because when the light beam is irradiated in the second light excitation step at such a timing, the DNA cutting efficiency is the highest.
  • the absorption wavelength often shifts to longer wavelengths due to the reduction of photosensitizer molecules to radical anions.
  • the light beam used in the second light excitation step has a longer wavelength than the light beam used in the first light excitation step.
  • the photosensitizer radical anion can be efficiently excited to emit electrons.
  • the photosensitizer in the excited state generated by the first photoexcitation step is provided.
  • Transient species generated by one-electron reduction of a substance from a biomolecule (eg, DNA) are further excited by the second photoexcitation step, so that the The light beam used in the second light pumping step requires a higher output than the light beam used in the first light pumping step.
  • a long-wavelength light beam has high cell permeability, so that when the present invention is applied to PDT, it can be used for treatment of deep cancer.
  • long-wavelength light beams have the advantage of less damage to normal cells, and are superior to high-intensity short-wavelength light beams used in the first light excitation step. .
  • the wavelength of the light beam used in the second photoexcitation step is a wavelength that can excite the photosensitizer in the excited state in the first photoexcitation step again. It goes without saying that it can be set as appropriate according to the type and nature.
  • the electrons emitted from the photosensitizer radical anion are preferably emitted to a solvent (eg, water).
  • a solvent eg, water
  • the released electrons react with oxygen molecules in the solvent to generate active oxygen species.
  • the generated reactive oxygen species damages the biomolecules, so that the biomolecule damage efficiency is further enhanced.
  • reactive oxygen species used herein is used in medicine and biology, and refers to an oxygen compound (singlet oxygen) that exhibits higher reactivity in vivo than normal oxygen molecules. , Superoxydion, hydrogen peroxide, hydroxyradical, etc.).
  • the incident angle of the light beam irradiated in the second light excitation step is It is preferable that the angle of incidence is different from the incident angle of the light beam irradiated in the one light excitation step.
  • the irradiation time and intensity of the light beam in the second light excitation step can be appropriately set, and are not particularly limited.
  • the efficiency of biomolecule damage increases in proportion to the irradiation time and intensity of the light beam (see FIGS. 9 and 10). It is preferable to appropriately set according to the condition of the patient, treatment time, patient's condition, and the like.
  • an appropriate photosensitizer it is possible to generate a radical anion via electron transfer reaction by single-wavelength two-photon excitation and to emit electrons via photoexcitation of the radical ayon by excitation of the third photon. That is, when a high-intensity laser beam having a wavelength twice as long as the absorption wavelength of the sensitizer is irradiated, photoexcitation of the sensitizer may occur and an excited state of the sensitizer may be generated. In this way, electron transfer occurs between the excited state of the sensitizer and a biomolecule (for example, DNA), and a radical anion and a DNA radical cation of the sensitizer can be generated.
  • a biomolecule for example, DNA
  • the radical anion of the sensitizer is Electron emission occurs by absorbing the same laser light.
  • the charge recombination between the radical cation of the radical anion and the DNA radical cation is suppressed, so that the efficiency of DNA damage is dramatically improved as in the case of the two-wavelength two-laser irradiation method.
  • This method involving a three-photon excitation process with a single laser can also occur within one laser pulse.
  • two-photon excitation of the photosensitizer causes electron transfer, radical cations of the sensitizer and DNA radical cations are generated, and the radical cations of the sensitizer are continuously excited within the laser pulse. Electron emission from the radical anion of the sensitizer occurs, and DNA damage can be caused efficiently.
  • This single-wavelength, one-laser method is particularly important in terms of practicality.
  • the photosensitizer when a photosensitizer capable of two-photon excitation is used as the photosensitizer, the photosensitizer is irradiated with the light beam a plurality of times (at least twice), and the light is irradiated.
  • a method of exciting the sensitizer a plurality of times (at least twice) is also included in the present invention. In this case, first, a predetermined light beam is irradiated to bring the photosensitized substance into a first excited state. The photosensitizer in the excited state extracts one electron from the biomolecule, generating a photosensitized radical anion and a radical cation in the biomolecule.
  • the two-photon excitation includes continuous two-photon excitation and stepwise two-photon excitation.
  • the former is a nonlinear optical phenomenon caused by high intensity short pulse laser light irradiation at wavelength; L when the sensitizer has absorption at half the wavelength (LZ 2) of the wavelength (e) at which there is no absorption.
  • L when the sensitizer has very weak absorption at wavelength ⁇ , and the excited state generated by one-photon excitation has relatively strong absorption at wavelength ⁇ , the excitation of the intermediate excited state (excitation of the second photon) It is a phenomenon that occurs.
  • Many organic compounds that can be sensitizers can more or less cause such two-photon excitation, and it is possible to use various molecules as sensitizers in irradiation methods using one wavelength. It is possible.
  • the biomolecule damage device according to the present invention only needs to be provided with a light beam irradiation means capable of irradiating at least two types of light beams having different wavelengths. That is, the biomolecule damage device according to the present invention can be said to be a device for performing the biomolecule damage method according to the present invention described in the above section (II).
  • Examples of the light beam irradiation means include a laser irradiation device having two or more light sources having different wavelengths.
  • a configuration of a conventionally known laser irradiation device can be used.
  • the light beam irradiation means is formed so as to be able to change the incident angles of the at least two types of light beams. According to the above configuration, for example, it is possible to irradiate two or more light beams at different incident angles, thereby enabling spatial control of the reaction point, and treatment of a tumor whose space is controlled. Is possible It works.
  • a biomolecule damage device provided with an incident angle changing means capable of changing the incident angles of at least two kinds of light beams
  • the incident angle changing means can control and change the incident angle of light beam irradiation manually or automatically by the user in accordance with the affected part.
  • the light beam irradiation means may include a control means capable of controlling the delay time of irradiation of at least two kinds of light beams having different wavelengths in the order of nanoseconds to the order of microphone mouth seconds.
  • a control means capable of controlling the delay time of irradiation of at least two kinds of light beams having different wavelengths in the order of nanoseconds to the order of microphone mouth seconds.
  • the photosensitizer radical anion to be irradiated with the light beam in the second photoexcitation step has a very short life, and therefore, two kinds of light beams having different wavelengths are very different. Irradiation with a short delay time (substantially at the same time) is possible. Note that the delay time of light irradiation can be appropriately changed depending on the bonding position and the bonding state between the photosensitizer and the DNA.
  • the biomolecule damage device includes light beam irradiation means capable of irradiating a single-wavelength light beam, and the light beam irradiation means includes a single-wavelength light beam irradiation. Also includes those further provided with control means capable of controlling the delay time on the order of nanoseconds to microseconds.
  • the method and the device for damaging DNA by photosensitization-electro-oxidation reaction can be used for the treatment of tumors such as cancer by the new PDT or the therapeutic device. That is, the present invention can be used and applied to medical treatment.
  • PDT is an effective treatment for patients who are difficult to apply surgical treatment immediately after treatment of elderly patients, heart failure, or other diseases.
  • the biomolecule damage method and biomolecule damage device according to the present invention it is sufficient to use a smaller amount of the photosensitizer, and the treatment time can be reduced.
  • the number of patients that can be treated in a certain period of time is not limited, and it can be said that this is an epoch-making invention that can simultaneously reduce the burden on patients and the labor of doctors.
  • the present invention can be said to be an invention having a very high social impact.
  • NDI naphthalimide
  • water-soluble NDI which was easier to use was used.
  • NDI radical anion (NDI-) is generated by pulse radiolysis using this water-soluble NDI
  • the water-soluble NDI has a maximum at 495 nm in the aqueous solution.
  • NDI oligodeoxynucleotide
  • ODN oligodeoxynucleotide
  • NDI-ODN introduced by a covalent bond was synthesized from a DNA synthesizer (see Fig. 4).
  • NDI-ODN NDI was bound to 5, terminal adenine (NDI-AAAAAGTGCGCGTC / TTTTTTCACGCG).
  • NDI radical anion (NDI *) is generated in NDI-ODN by pulse radiolysis.
  • ⁇ DI * NDI radical anion
  • the line shown by gray, ND I - ⁇ DN the NDI ⁇ which is generated by pulse radiolysis - indicates Deruta_ ⁇ D 49 5, the black line electronic pulse 2 mu sec after 5 3 ⁇ ⁇ when irradiating 2 nm light to excite NDI
  • NDI Destruction of guanine in ODN and NDI — HC 1 (N, N, —bis— [3 -— (N—dimethyl) propyl] —1,4,5,8 -
  • the degree of guanine destruction in ODN-G and ODN-GG in the presence of naphthaldiimide dichloridite) was examined.
  • NDI-ODN was used at a chain concentration of 40 ⁇ in 20 mM sodium phosphate buffer at pH 7.0.
  • 40 ⁇ M of NDI_HCl was added to the ODN-G solution and the ODN-GG solution (chain concentration: 40 ⁇ ).
  • a 52-nm laser (20 mJ / pulse) is synchronized with a 35-55 ⁇ m pulse laser (1.6 mj / pulse), and the delay time is 10 nanoseconds.
  • the photosensitized ⁇ DNs is enzymatically degraded to 2'-deoxymononucleotide by the snake venom phosphodiesterase nuclease P1Z alfa phosphatase, and the amount of guanine destruction is determined by product analysis using HPLC. did.
  • the light irradiation time for NDI-ODN was 5 minutes, and the light irradiation time for ODN-G and ODN-GG was 10 minutes.
  • Figure 6 shows the results.
  • the nucleotide sequences of NDI-ODN, ODN-G, and ODN-GG were as shown in the table, respectively.
  • guanine blasting was performed by using a 350-nm laser in the first stage and a 52-32 nm laser in the second stage, compared to the case of irradiating only 355 nm. Increased when irradiated. In addition, there was no damage to the DNA when irradiated only with a 52-32 nm laser. In addition, the amount of guanine ruptured in ODN-GG was higher when compared to ⁇ DN_G. This suggests that the destruction of guanine is based on the photosensitization-electro-oxidation reaction caused by the first stage of light irradiation. In addition, the irradiation of the second laser promoted the destruction of guanine in the ODN to which the NDI was covalently bonded. From the above, it was found that DNA can be cut with high efficiency by using two wavelengths and two lasers.
  • NDI-ODN time coordinate of NDI-ODN in one-wavelength laser photolysis of NDI-ODN
  • Fig. 8 The left axis in the figure shows the results of the observation of the temporary absorption of NDI ⁇ -at 495 nm after excitation of 355 nm.
  • the right axis in the figure shows the amount of guanine destruction as a function of the delay time of the 532 nm pulse laser with respect to the 3.55 nm pulse laser.
  • the circles in the figure are the measurement results.
  • the dotted line in the figure shows the amount of guanine destruction when the laser beam irradiation is not performed.
  • NDI-ODN NDI was covalently bonded to thymine at the 5 'end (NDI-TTTCCGCGTT / AAAGCGCGAA).
  • the ODN-GG solution is irradiated with light for 20 minutes, and then enzymatically decomposed into 2,1-deoxymononucleotide by snake venom phosphodiesterase / nuclease P1 / alternative phosphatase, and guanine
  • the amount of rupture was determined by product analysis using HPLC.
  • Figure 9 shows the results.
  • open circles ( ⁇ ) indicate the amount of guanine destruction when only 355 nm was irradiated
  • closed circles ( ⁇ ) indicate the case where 355 nm laser and 532 nm laser were irradiated in two steps. Shows the amount of guanine rupture.
  • the amount of guanine destruction increased in proportion to the irradiation time when the laser was irradiated in two stages with the 355111111 laser and the 532111111 laser.
  • the results are shown in FIG.
  • the white circle ( ⁇ ) in the figure indicates that the laser is a 3.55 nm laser.
  • the figure shows the amount of guanine destruction when two stages of irradiation were performed with 5 and 32 n lasers.
  • the irradiation intensity was proportional to the irradiation intensity of the second laser, the 52-nm laser. It was found that the amount of destruction of GCCn increased. From the results of the above examples, it is considered that when the present invention was implemented, the phenomenon shown in FIG. 3 occurred.
  • the photosensitizer NDI is selectively excited by the irradiation of the first laser (1st laser).
  • the excited NDI * withdraws electrons from a nearby base (guanine), generating an NDI radical anion (NDI *-) and a guanine radical cation (G * + ).
  • irradiation with the laser (2nd laser) in the second stage excites the NDI radical anion, releases electrons, and regenerates NDI.
  • the emitted electrons and oxygen react with each other to generate singlet oxygen ( ⁇ 2 ⁇ ), and the singlet oxygen and guanine radical force oxidize and damage DN ⁇ .
  • the present invention can be used for PDT, which has recently attracted attention as one of cancer treatment methods for skin cancer, stomach cancer and the like. Therefore, it can be used in fields such as clinical medicine and medical device manufacturing. It can be an effective cancer treatment, especially for elderly patients, heart failure, and patients who have difficulty in surgery immediately after other illnesses.

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Abstract

La présente invention se rapporte à un procédé d'endommagement de biomolécules qui améliore l'efficacité d'endommagement des biomolécules (clivage) par l'intermédiaire d'une réaction d'oxydation à un électron avec photosensibilisation. L'invention concerne également un appareil permettant de mettre en oeuvre ledit procédé. Ainsi, il est possible de mettre en oeuvre un procédé de thérapie photodynamique permettant de réduire les contraintes imposées aux patients et la charge de travail des médecins, ainsi que d'obtenir des effets curatifs avec une grande efficacité et en un laps de temps réduit. En particulier, l'invention a trait à un procédé d'endommagement de biomolécules, qui consiste à effectuer l'endommagement des biomolécules par l'irradiation d'une substance photosensibilisée à l'aide de faisceaux optiques, ladite substance photosensibilisée étant exposée à chacun des faisceaux optiques de longueurs d'ondes différentes au moins une fois, afin que la substance photosensibilisée reçoive de multiples excitations. Ce procédé permet de décomposer efficacement des biomolécules.
PCT/JP2004/004472 2003-09-29 2004-03-29 Procede d'endommagement de biomolecules et appareil d'endommagement de biomolecules WO2005030329A1 (fr)

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JP2003338082A JP2005102831A (ja) 2003-09-29 2003-09-29 生体分子損傷方法、および生体分子損傷装置
JP2003-338082 2003-09-29

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JP4623515B2 (ja) * 2005-03-14 2011-02-02 国立大学法人京都大学 オリゴチオフェンを用いた光記憶媒体

Citations (2)

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Publication number Priority date Publication date Assignee Title
JP2000189527A (ja) * 1998-12-28 2000-07-11 Ishikawajima Harima Heavy Ind Co Ltd レ―ザ診断・治療方法及び装置
JP2002253571A (ja) * 2001-02-28 2002-09-10 Nidek Co Ltd レーザ治療装置

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Publication number Priority date Publication date Assignee Title
DE68919328T2 (de) * 1988-10-28 1995-05-24 Ibm Ultraviolette Laserablation und Ätzen von organischen Feststoffen.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000189527A (ja) * 1998-12-28 2000-07-11 Ishikawajima Harima Heavy Ind Co Ltd レ―ザ診断・治療方法及び装置
JP2002253571A (ja) * 2001-02-28 2002-09-10 Nidek Co Ltd レーザ治療装置

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