RU2347563C1 - Method for selective destruction of melanoma - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: method includes introduction of solution of chemical compound of amino acid L-phenylalanine with gold or silver nanoparticles intravenously prior to radiation. At that minimum concentration of nanoparticles is 10⁸ cm^-3, and maximum concentration is 10¹² cm^-3. Plasma resonance of nanoparticles has spectral maximum in the area of biotissues transparency at the wave length of 750-1200 nm. These nanoparticles are gold or silver nanoshells with core of silicon dioxide or nanorods. Radiation of melanoma is performed at least after 1 hour and not more than after 4 hours after solution introduction, laser beam with length of radiation wave that coincides with spectral maximum of nanoparticles plasma resonance absorption. At that radiation is done by sequence of laser impulses with duration of laser impulse in the range of 10 microseconds - 100 ns at minimum porosity of three and higher, at energy density of at least 20 J/cm², but not more than 200 J/cm².

EFFECT: local performance of melanoma destruction with minimum destruction of surrounding healthy cells.

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Description

The invention relates to the field of biomedical technologies, in particular to the creation of non-invasive selective laser photothermolysis of malignant melanoma cells based on plasmon resonance gold or silver nanoparticle technology. In the treatment of neoplasms such as melanoma in warm-blooded animals or people by traditional methods of surgery, it is not always possible due to the peculiarities of tumor localization (for example, in the area of the hard palate or pharynx), and also, even if more than three centimeters of healthy tissue are excised from the border of the tumor, metastases occur in most cases, and many complications arise during chemotherapy (Guidelines for the Chemotherapy of Tumor Diseases, edited by N.I. Perevodchikova. M. Practical Medicine. 2005, p. 581-657; White R. Cancer Evan small pets. M .: Aquarium, 2003).

There is a method of short-focus x-ray therapy with a total focal dose of 100-120 Gy and remote gamma-ray therapy for radiation destruction of malignant melanoma cells with a total focal dose of 30-40 Gy (Sh.Kh. Gantsev. Oncology. M.: Medical information agency. 2004, p. .190-204; Stephen J., Withrow E., MacEwen G. Smal animal clinical oncology - 2001, p.305-308).

However, this method, despite the prevalence, has the following disadvantages. In the treatment of melanoma with the help of remote gamma therapy, even in combination with immunotherapy, as experience shows, leads to a 75-90% tumor recurrence, and metastases occur after 2-6 months.

A known method of photodynamic destruction of tumors, including intravenous administration of a photosensitizer and irradiation of the tumor with continuous laser radiation with a wavelength that matches the absorption band of the photosensitizer (Photodynamic therapy / Ed. TJ Dougherty / J. Clin. Laser Med Surg. 1996, Vol.14, P. 219-348; RF Patent No. 2184578, IPC A61N 5/06; Stranadko. Analysis of the direct results of PDT of malignant neoplasms. New advances in laser medicine. - M. - St. Petersburg., 1993, p. 218). The selective photodynamic mechanism of destruction of cancer cells is based on a higher density (contrast) of photosensitizer accumulation in tumor cells compared to ordinary cells, which is associated with a higher density of blood vessels in the tumor compared to healthy biological tissue.

However, this contrast for various tumors does not exceed two to three times. When laser radiation is absorbed by a photosensitizer, the dye molecules transform into an excited electronic state and, when they collide with oxygen molecules dissolved in biological tissues, transfer it from an unexcited to an excited electronic singlet state with a typical lifetime of several microseconds. During this time, the molecules of singlet oxygen, having passed a characteristic path commensurate with the size of the cells when interacting with the plasma membrane of the cell, damage it, and the cell dies due to necrosis. Thus, cell destruction occurs only during exposure to laser radiation in the spatial region of laser beam irradiation.

The photodynamic method for the destruction of melanoma cells has several disadvantages. The absorption coefficient of melanin reaches 10 ^4-3 cm ^-1 in the ultraviolet and violet region and drops to 10 ² cm ^-1 in the red region, so the depth of laser radiation penetration is less than hundreds of microns. The photosensitizers used in practice - phthalacins, porphyrins, chlorins have absorption bands in the ultraviolet or visible spectral range of the photosensitizers, and the lasers used cannot penetrate the melanoma. In addition, the photodynamic method has a low contrast accumulation of photosensitizers in cancer cells.

Closest to the proposed method is a neutron capture method for the selective destruction of melanoma (V. N. Mitin, N. G. Kozlovskaya, AM Arnopol. Neutron capture therapy of oral tumors in dogs. All-Russian Veterinary Journal. 2006. No. 1, p. 9-10).

The method involves intravenous administration of L-borphenylalanine, which selectively accumulates in a particular melanoma tumor, since L-phenylalanine is an essential amino acid from which melanin is produced, which forms melanocytes contained in melanoma cells. Thus, selective accumulation of L-borphenylalanine in melanoma cells occurs. Upon irradiation of a spatial zone commensurate with a tumor containing L-borphenylalan by a beam of slow neutrons obtained by a neutron guide from a nuclear reactor, melanoma cells are destroyed due to the induced secondary local radiation of boron.

However, this method has the following disadvantages.

1. Radiation exposure of patients, which is only partially reduced when using a lithium protective apron.

2. A complex and very expensive installation, including a compact nuclear reactor, requiring qualified non-medical specialists, in particular nuclear physicists, to serve.

3. A long exposure time of patients for an hour when monitoring the cardiovascular system.

4. The use of general anesthesia.

The objective of the present invention is to increase the locality of the method of selective destruction of melanoma with minimal destruction of the surrounding healthy biological tissue cells.

The technical result consists in the effectiveness of damage to malignant melanoma cells during surgery without general anesthesia.

The problem is solved in that in a method for the selective destruction of melanoma, including intravenous administration of a solution of the amino acid L-phenylalanine and irradiation of melanoma, according to the solution, the chemical compound of the amino acid L-phenylalanine with gold or silver nanoparticles representing gold or silver nanoblocks or nanorods is used as a solution at a minimum concentration of 10 ⁸ cm ^-3 and a maximum concentration of 10 ¹² / cm ^-3 with plasmon resonance, having a spectral maximum in transparency biological tissue at a wavelength of 750-1200 nm, irradiation of melanoma is carried out with a laser beam with a radiation wavelength coinciding with the spectral maximum absorption of the plasma resonance of nanoparticles, while for local destruction of melanoma cells, irradiation is carried out by a sequence of laser pulses with a laser pulse duration in the range of 10 μs - 100 ns with a minimum duty cycle of three or more with an energy density of at least 20 J / cm ² but not more than 200 J / cm ² .

The invention is illustrated by drawings.

Figure 1 shows a block diagram of an experiment in the destruction of laboratory rat melanoma in vivo based on pulsed laser photothermolysis using plasmon resonance gold or silver nanoparticle technology, where 1 is a laboratory rat, 2 is a chemical compound of L-phenylalanine with gold or silver plasmon -resonant nanoparticles consisting of nanoshells or nanorods; 3 - malignant tumor - melanoma; 4 - pulsed infrared (750-1200 nm) semiconductor laser with a fiber optic fiber.

Figure 2 - image of gold nanoparticles obtained using an electron microscope: (a) - nanospheres with a diameter of 5 nm, (b) - gold nanoshells with a diameter of 90 nm with a silicon dioxide core, (c) - gold nanorods with a diameter of 15 nm and 50 nm long.

Figure 3 - dependence of the extinction coefficient (absorption and scattering of light) on the wavelength (nm) when changing the geometric parameters of the nanoparticles: (a) gold nanospheres, (b) gold nanorods with a ratio of length to diameter from 1 to 6; (c) - gold shells with a thickness of 2 nm with a diameter of the core of silicon dioxide from 10 to 90 nm.

Figure 4 is a thermogram of the heating efficiency of gold plasmon resonance nanoshells when exposed to continuous and pulsed laser radiation with a wavelength of 810 nm with the same average optical power of 2 W and an irradiation time of 1 minute.

Figure 5 - experimental thermogram of uneven heating along the depth of the cuvette when the laser beam irradiates plasmon resonance gold nanoshells with different concentrations: (a) N = 5 · 10 ⁹ ml ^-1 , (b) N / 8 = 0.61 · 10 ⁹ ml ^-1 .

Figure 6 presents a photograph of a laboratory rat when exposed to radiation from a semiconductor laser used to heat plasmon resonance nanoparticles using continuous (crosses) and pulsed mode (points) at the same average laser energy density.

Figure 7 presents a thermogram of the effectiveness of laser photothermolysis of biological tissues with gold plasmon resonance nanoparticles introduced at different depths: (a) control without nanoparticles; (b) intramuscular injection of a volume of 0.1 ml SiO ₂ / Au to a depth of 5 mm; (c) subcutaneous administration; (d) intradermal administration.

On Fig - histological section of the biological tissue of a laboratory rat after intramuscular injection of plasmon resonance nanoshells to a depth of 5 mm with subsequent exposure to radiation of a semiconductor laser with a wavelength of 810 nm for 30 seconds, a power of 2 W in continuous mode, (a) - picture in dark field, (b) - a picture in transmitted light.

Figure 9 is a histological section from the epidermis to muscle tissue after extra-muscular injection of gold plasmon resonance nanoshells to a depth of 5 mm when exposed to radiation from a semiconductor laser with a wavelength of 810 nm for 10 seconds: (a) pulse mode, pulse duration 1 ms , duty cycle 5; (b) - continuous mode with the same laser energy of 20 J.

Figure 10 - destruction of the melanoma of the oral mucosa in a dog during laser photothermolysis based on plasmon resonance nanoparticle technology: (a) photograph (b) thermogram.

The method is as follows.

To a person or animal, for example, laboratory rat 1, a solution consisting of chemical compound 2 L-phenylalanine with gold or silver plasmon resonance nanoshells or nanorods (bioconjugate) at a concentration of not less than 10 ⁸ cm ^-3 is injected into the tail vein using an insulin syringe and not more than 10 ¹² cm ^-3, dissolved in saline, is not less than 1 hour and no more than 4 hours respective optimal accumulation of nanoparticles in cancer - melanoma 3, produce irradiation melanomas pulsed semiconductor laser 4 having a wavelength coinciding with the maximum plasmon resonance nanoparticles lying in the region of transparency of tissues (750-1200 nm) for a time which is determined by the appropriate level of energy density incident laser beam is not less than 20 J / cm ² and not more than 200 J / cm ² . It should be noted that solid state lasers based on yttrium aluminum garnet with neodymium with a wavelength of 960 and 1064 nm can be used as a pulsed laser with a micro- and nanosecond duration.

The lower limit of the energy density of the used lasers is determined by the heating temperature of the biological tissue containing nanoparticles, which causes necrosis of the tumor cells, and the upper limit is determined by the level of laser energy density, which does not cause pathological changes in the biological tissue, which does not contain plasmon resonance nanoparticles.

In the laboratory of dimensional nanosensors of the IBFRM RAS, the authors of the application developed a technology and actually produced plasmon resonance nanoparticles with the ability to control the spectral position of the plasmon resonance of gold or silver nanoparticles by changing the geometric parameters of the nanoparticles as shown in Fig. 4 (N.G. Khlebtsov, V.A. Bogatyrev, L. A. Dykman, B. N. Khlebtsov. Gold nanostructures with plasma resonance for biomedical research // Russian Nanotechnologies. 2007. V.2. No. 3-4. P.69-86; Khiebtsov NG, Trachuk LA, Melnikov AG Proc. SPIE, 2004. V.5 475, 1-11).

From a comparison of laser heating of a cuvette with plasmon-resonant gold nanoshells with a volume of 2 ml (Fig. 5) at continuous (maximum heating temperature 57.4 ° C) and pulsed (maximum heating temperature 40.7 ° C) (laser pulse duration 2 ms and time interval between pulses 14 ms, duty cycle 7) irradiation with the same average laser energy of 150 J (in continuous operation, laser power 2.5 W for 1 minute). It is easy to see that under pulsed exposure, the cuvette heats up by 17 ° C less than continuous heating, which experimentally proves the local heating of plasmon resonance gold nanoshells, due to the finite time it takes to establish the temperature.

The concentration of plasmon resonance gold or silver nanoparticles should be in a certain range. The maximum concentration of plasmon resonance gold or silver nanoparticles is determined by the processes of cluster formation and broadening of plasma resonance. The lower limit of the concentration of plasmon resonance gold or silver nanoparticles is determined by the efficiency of laser heating. In Fig.5. An experimental thermogram is shown of the uneven heating of plasmon resonance gold nanoshells in depth when irradiated with a laser beam at various concentrations. Eppendorf type tube with gold plasmon resonance nanoshells dissolved in a 2 ml saline solution with different nanoparticle concentrations of 5 · 10 ⁹ ml ^-1 (a) and 0.61 · 10 ⁹ ml (8 times different) when exposed to continuous laser radiation with a wavelength 810 nm, which coincides with the maximum absorption of nanoparticles at the same average laser power of 2 W and an irradiation time of 2 minutes.

Moreover, as shown by control experiments conducted on the irradiation of biological tissues of laboratory rats with gold plasmon resonance nanoparticles introduced into the tumor (Fig. 6) and subsequent histological analysis (Fig. 8), the modes of laser irradiation were determined, in particular, the duration of optical pulses, the time distance between them and the laser energy density that causes local necrosis of melanoma cells during photothermolysis of biological tissues (see article ILMaksimova, GGAkchurin, BNKhiebtsov, GSTerentyuk, GGAkchurin, IAErmolaev, AASkaptsov, EPSoboleva, NGKhiebtsov and n. Near-infrared laser photothermal therapy of cancer by using gold nanoparticles: computer simulations and experiment. Medical Laser Application, 2007, V.22).

As shown in Fig. 8, as a result of laser photothermolysis based on plasmon resonance nanoparticle technology according to histological data, destruction and fragmentation of the striated muscle, slight edema are observed. In the image in a dark field, nanoparticles located in the striated area of the striated muscle are clearly visible.

The results of comparing laser photothermolysis with pulsed and continuous laser irradiation are presented in Fig. 9, which shows a histological section from the epidermis to muscle tissue after intramuscular injection of gold plasmon resonance nanoshells to a depth of 5 mm. When exposed to radiation from a semiconductor laser with a wavelength of 810 nm for 10 seconds: (a) pulsed mode, pulse duration 1 ms, duty cycle 5; (b) - a continuous mode with the same laser energy of 20 J, it follows that when pulsed exposure is observed only swelling and loosening of the connective tissue, cell infiltration, indicating an inflammatory process. Under continuous exposure (b), as a result of laser photothermolysis, foci of destruction in the dermis appear in the form of homogenization and swelling of the main substance and collagen fibers.

During pulsed heating of gold nanoparticles, the duration of the laser pulse determines the locality of heating of the nanoparticles of the surrounding biological tissue. Estimates show that when irradiated with nanosecond pulses, the effective heating radius of the surrounding biological tissue is no more than 1 micron (Khlebtsov BN, Zharov VP, Melnikov AG, Tuchin VV, Khlebtsov NG Optical amplification of photothermal therapy with gold nanoparticles and nanoclusters. Nanotechnology, 2006; 17, 5267-5179).

However, the detected effect of defragmentation of plasmon resonance gold nanoshells at an energy of a single laser pulse of more than 2 mJ and a duration of 4 ns, causing the destruction of nanoshells or local melting of nanorods, limits the laser radiation intensity from above and its minimum duration. (G. Akchurin, B. Khlebtsov, G. Akchurin, V. Tuchin, V. Zharov, N. Khiebtsov. Laser-induced phonodestruction of silica / gold nanoshells under single nanosecond pulses. Nanothechnology, 2007, V.18).

Thus, the parameters of laser pulsed exposure were determined experimentally on phantoms of biological tissue and in vivo on spontaneous animal tumors, with subsequent histological analysis and the minimum duration of the laser pulse was limited by the condition specified in the claims.

The laser photothermolysis carried out by the patented technique based on the technology of plasmon resonance gold nanoshells of the melanoma of the oral mucosa in dogs and cats caused tissue scarring without remission and metastasis for several months to one year.

Claims (1)

A method for the selective destruction of melanoma, including irradiating melanoma, characterized in that, additionally, prior to irradiation, a solution of the chemical compound of the amino acid L-phenylalanine with gold or silver nanoparticles, which are gold or silver nanoshells with a core of silicon dioxide or a nanorod, is introduced at a minimum concentration of 10 ⁸ cm ^-3 and a maximum concentration of 10 ¹² cm ^-3 with a plasma resonance having a spectral maximum in the transparency region of biological tissues at a wavelength of 750-1200 nm, is irradiated melanomas are carried out not less than 1 hour and not more than 4 hours after the injection of the solution by a laser beam with a radiation wavelength coinciding with the spectral maximum of absorption of the plasma resonance of the nanoparticles, while irradiation is carried out by a sequence of laser pulses with a laser pulse duration in the range of 10 μs - 100 ns with a minimum duty cycle of three or more, with an energy density of at least 20 J / cm ² but not more than 200 J / cm ² .

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