RU2607579C2 - Biocompatible nanomaterial for photosensitivity singlet oxygen and method for production thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, in particular to oncology, and describes a biocompatible nanomaterial and method of its production. Proposed biocompatible nanomaterial is a hybrid associates of colloidal quantum dots CdS with average size of 2–4 nm with cations of methylene blue (MB+) in concentration 10^-1–10^-4 (ν_paints/ν_CdS). Method involves double jet merging of 0.6–5 % solution of sodium sulphide and 0.8–7 % solution of cadmium bromide with melt of gelatin with producing of colloidal solution, containing colloidal quantum dots of CdS, solution is held at temperature of 4–10 °C, produced gelatine jelly is crushed to grain with size 5–10 mm, washed in distilled water at temperature from 7 to 13 °C for 30 minutes, decant excess water and granules are heated to temperature above 40°C. Nanomaterial is highly efficient generation of singlet oxygen and satisfactory parameters of cytotoxicity, testifies to its biocompatibility.

EFFECT: invention can be used in medicine and biology for photodynamic therapy of oncological and other human diseases.

2 cl, 6 dwg

Description

The invention relates to the fields of medicine and biology and can be used in the method of photodynamic therapy (PDT) of cancer.

In modern oncology, along with surgery, various radioisotope methods, chemotherapy, etc. The direction of photodynamic therapy (PDT) was recognized as promising. In PDT, the starting active agent is highly active singlet oxygen ¹ O ₂ . Its effect is detrimental to cancer cells [Zakharov SD Oxygen effect in cells and the prospects for its use in the treatment of tumors / S.D. Zakharov, A.V. Ivanov // Quantum Electronics. 1999.Vol. 29, No. 3. P. 192-197], and also positively for detoxification of the body, stimulation of metabolic and regenerative processes in tissues and photobactericidal purposes [A.A. Krasnovsky, Photodynamic regulation of biological processes. Problems of regulation in biological systems, Under the general ed. A.B. Ruby. M .: Izhevsk, (2006)]. The production of singlet oxygen by light in diseased tissues and cells is possible only in the presence of photosensitizers, since the direct ³ O ₂ → ¹ O ₂ phototransition is prohibited by the orbital and spin selection rules [Shinkarenko N.V. Singlet oxygen, methods of obtaining and detection / N.V. Shinkarenko, V.B. Aleskovsky // Advances in Chemistry. 1981. TL B.3. S. 406-427; Zakharov, S.D. Oxygen effect in cells and the prospects for its use in the treatment of tumors / S.D. Zakharov, A.V. Ivanov // Quantum Electronics. 1999.Vol. 29, No. 3. S. 192-197].

As photosensitizers, heterocyclic organic dyes with a high triplet yield have so far been most widely used. However, their successful use is hindered by low-threshold photodegradation, often irreversible, as well as a tendency to intra- and intermolecular interactions, leading to the formation of new forms of the dye. As a result, the spectral conditions of photosensitization are violated [A.A. Krasnovsky, Photodynamic regulation of biological processes. Problems of regulation in biological systems, Under the general ed. A.B. Ruby. M .: Izhevsk, (2006)]. Consequently, the development of new singlet oxygen photosensitizers more resistant to the environment and the action of light for the treatment of cancer is an extremely important and necessary subject of research in the field of integrated nano- and biotechnologies [S.D. Zakharov, A.V. Ivanov, Quantum Electronics. 29, No. 3. 192-197, (1999); A.A. Krasnovsky, Photodynamic regulation of biological processes. Problems of regulation in biological systems, Under the general ed. A.B. Ruby. M .: Izhevsk, (2006); A. Fernandez-Fernandez, R. Manchanda, A.J. McGoron, Appl. Biochem. Biotechnol, 165, No. 7-8, 1628-1651, (2011)].

To date, the construction of singlet oxygen photosensitizers based on fullerenes [US 5866316, 02.02.1999], silicon nanocrystals [JP 20020176515, 06/18/2002; RU 2329061, 07.20.2008], metalloporphyrins, organic dyes (methylene blue, thionine, etc.) and their derivatives [US 20120302557, 11.29.2012], as well as composites based on light-emitting nanoparticles in combination with molecules of organic photosensitizers [US 20020127224, 09/12/2002].

So, in the patent US 5866316, 02.02.1999, a method for producing a singlet oxygen photosensitizer based on C ₆₀ fullerenes in solution, which are characterized by a quantum yield of singlet oxygen sensitization close to unity and have low toxicity, is proposed. However, a significant drawback of the fullerenes obtained by the described method is hydrophobicity, as well as a tendency to aggregate at concentrations necessary for sensitization of singlet oxygen, which imposes restrictions on the use of such a photosensitizer in PDT.

Part of the drawbacks of the photosensitizer proposed in patent US 5866316, 02.02.1999, eliminated in patent RU 2329061, 07.20.2008, where the authors propose the use of nanocomposites based on silicon nanocrystals of 2-100 nm in size with fullerene molecules adsorbed on the surface with a relative fullerene content from 0.3 to 0.3 up to 4% by weight. The disadvantage of this invention is the use of toxic solvents (for example, carbon tetrachloride) in the synthesis of silicon nanocrystals. There are no satisfactory methods for the purification of synthesized nanocrystals that allow them to be actively used in medicine and biology.

The use of organic dyes as photosensitizers of singlet oxygen is well known [Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications / Joao Paulo Tardivo, Auro Del Giglio, Carla Santos de Oliveira, Dino Santesso Gabrielli, Helena Couto Junqueira, Dayane Batista Tada, Divinomarar Severino, Rozane de Fatima Turchiello, Mauricio S. Baptista Ph.D. // Photodiagnosis and Photodynamic Therapy (2005) 2, 175-191; Bashtanov M.E. The effect of solvents on the quantum efficiency of the delayed fluorescence of phthalocyanine induced by singlet oxygen under laser excitation / M.E. Bashtanov, A.A. Krasnovsky // Quantum Electronics. 1999. T. 26. No. 2. S. 163-167]. So, in patent US 20120302557, 11.29.2012, describes a method for using solutions of methylene blue and its derivatives for PDT. The use of methylene blue solutions for PDT has a number of disadvantages. For example, due to the high solubility of methylene blue molecules when it is introduced into the human body, distribution outside the affected organs and tissues is possible. In addition, the use of methylene blue solutions for PDT is hindered by difficulties associated with both low threshold photodegradation, often irreversible, and intra- and intermolecular interactions, leading to the formation of new forms of dye and violation of the spectral conditions of sensitization [Krasnovsky A.A. (ml.) Photodynamic regulation of biological processes // Problems of regulation in biological systems / Ed. A.B. Ruby. M .: Izhevsk. 2006. 480 p.]. To avoid undesirable effects, it is necessary to pair the dye with various agents.

The closest analogue of the invention is proposed in patent US 20020127224, published September 12, 2002, material for use in photodynamic therapy based on aggregates of various light-emitting semiconductor nanoparticles (nanocrystals, quantum dots, quantum rods) and organic photosensitizers. When a light quantum is absorbed, nanoparticles are excited, followed by the transfer of excitation energy from nanoparticles to photosensitizer molecules. The photosensitizer gives energy to triplet oxygen molecules and they go into an excited singlet state. It is proposed to use CdSe / ZnS as nanoparticles or their mixtures with TiO ₂ nanoparticles, and as photosensitizers various drugs already used for the treatment of cancer, including protein inhibitors (troponin, angiostatin, etc.), various enzymes, various dyes (methylene blue (methylene blue), indocyanin green, etc.), etc.

This material has several disadvantages: there is no data on the possibility of generating singlet oxygen by such systems, it is assumed to use quantum dots synthesized using highly toxic trioctylphosphine oxide (TOPO), the hydrophobicity of the colloidal solutions obtained in this case, to overcome which it is necessary to introduce additional technological procedures, for example, pairing nanoparticles with substances of the mercapto group (mercaptoacetic acid, etc.), which leads to a complication of technology and ozhaniyu final product, there is no information about the specific implementation of the proposed principle.

The objective of the invention is to develop a biocompatible nanomaterial with the ability to photosensitize the process of formation of singlet oxygen and a method for its production.

The technical result of the present invention is to expand the arsenal of singlet oxygen photosensitizers for photodynamic therapy.

The technical result is achieved in that the biocompatible nanomaterial for photosensitization of singlet oxygen is a low-toxic hybrid associates of luminescent colloidal quantum dots CdS with cations of methylene blue (MV ⁺ ) in a ratio of 10 ^-1 -10 ^-4 (ν _color / ν _CdS ), and its method the preparation includes two-jet draining of a 0.6-5% solution of sodium sulfide and a 0.8-7% solution of cadmium bromide in a temperature-controlled reactor, with a gelatin melt at a constant temperature of 40 ° C, according to the invention, is introduced into the resulting colloid solution was containing colloidal quantum dots, CdS, quantum dots in the final crystallization step MV ⁺ cations dissolved in the ratio of 10 ^-1 -10 ^-4 (ν _colors / ν _CdS), followed by cooling the obtained sol to a temperature of from 4 to 10 ° C, the solution is kept at this temperature for 24 hours, after which the gelatin gel obtained is crushed to a granule size of 5-10 mm, washed in distilled water at a temperature of 7 to 13 ° C for 30 minutes, after which excess water is decanted and the granules are heated to a temperature over 40 ° C.

The result is a uniform melt.

Water-soluble colloidal cadmium sulfide quantum dots are used, synthesized using the low-temperature sol-gel method using low-toxic synthesis components in a special thermostatic reactor (Utility Model Patent of the Russian Federation 134445, November 20, 2013). The conjugation of methylene blue (MV +) cations with colloidal CdS QDs increases the duration of the process of photosensitization of singlet oxygen by blocking the photodegradation mechanisms of methylene blue, as well as preventing the dye from transitioning to other protolytic and aggregate forms that are not suitable for photosensitizing singlet oxygen.

In FIG. Figure 1 shows a model of photophysical processes that occur during sensitization of the production of singlet oxygen by MB ⁺ cations coupled with CdS quantum dots.

In FIG. 2 shows the spectra: absorption of colloidal CT CdS - 1; luminescence of colloidal CT CdS - 2; luminescence of associates of CdS colloidal QDs with methylene blue cations (MV ⁺ ), in the ratio of 10 ^-2 (ν _color / ν _CdS ) - 3. The inset shows: electronic photograph of colloidal CdS QDs, size distribution of colloidal CdS QDs, and X-ray diffraction of colloidal QDs Cds.

In FIG. _Figure 3 shows the luminescence spectra of singlet oxygen in solutions: MV ⁺ cations at λ _exc = 660 nm - 1; associates of colloidal CdS QDs with MB ⁺ cations in a ratio of 10 ^-2 (ν _color / ν _CdS ) at λ _exc = 660 nm - 2; cations MV ⁺ at λ _exc = 405 nm - 3; associates of colloidal CdS QDs with MB ⁺ cations in a ratio of 10 ^-2 (ν _color / ν _CdS ) at λ _exc = 405 nm - 4. The inset shows the dependence of ¹ O ₂ production ^on time in solutions of MV ⁺ - 1 cations and colloidal CT associates CdS with MB ⁺ cations in a ratio of 10 ^-2 (ν _color / ν _CdS ).

In FIG. Figure 4 shows a table of the respiratory rate of rats Rattus norvegicus depending on the presence of CdS CT, MB ⁺ cations, and hybrid associates of colloidal CdS CT with MB ⁺ cations.

In FIG. Figure 5 shows the time diagrams of oxygen uptake by isolated rat liver mitochondria in the presence of CdS QDs, MB ⁺ cations and hybrid associates of colloidal CdS QDs with MB ⁺ cations.

In FIG. Figure 6 shows a histogram showing the effect of CdS QDs, MB ⁺ cations, and hybrid associates of colloidal CdS QDs with MB ⁺ cations on the generation of hydrogen peroxide by rat liver mitochondria.

The physical principle of functioning of the proposed biocompatible nanomaterial for the production of singlet oxygen is as follows. The proposed nanomaterial provides the possibility of obtaining singlet oxygen under the action of radiation from two ranges (Fig. 1): radiation with a wavelength of 600-750 nm, which corresponds to the absorption region of methylene blue cations (MV ⁺ ), and 380-450 nm per absorption region quantum dots of CdS. In the first case, when a light quantum is directly absorbed by methylene blue cations (MV ⁺ ) (600-750 nm), the energy from the dye molecule is transferred to oxygen, transferring it from the ground triplet state to one of the excited singlet states, and dye luminescence quenching in the triplet band is observed . The energy of the triplet level of excitation of methylene blue 1.47 eV and when it is quenched, only a transition to the first singlet state of the oxygen molecule can occur [Krasnovsky, A.A. ml Photosensitized luminescence of singlet oxygen in solution. / A.A. Krasnovsky // Biophysics. 1976.V.21. N.4. S. 748-749, Krasnovsky, A.A. Jr. Photoluminescence of singlet oxygen in pigment solutions / AA Krasnovsky Jr .: Photochem. Photobiol. 1979. V. 29. No. 1. P. 29-36]. From the singlet state, the oxygen molecule passes with radiation (λ = 1270 nm) into its main triplet state. The presence of glow in this area allows you to control the formation of singlet oxygen. In the second case, shorter wavelength radiation (380-450 nm) is absorbed by the CdS quantum dot, the excitation energy of which is transferred to methylene blue cations (MV ⁺ ). Further, the energy stored in the cations of methylene blue (MV ⁺ ) is transferred to oxygen molecules, transferring it from the ground triplet state to one of the excited singlet states.

To obtain a biocompatible nanomaterial for photosensitization of singlet oxygen, the following sequence of operations is performed. For the synthesis of semiconductor colloidal quantum dots, a 1-5% solution of sodium sulfide and a 1.1-7% solution of cadmium bromide in distilled water with a temperature of 7 to 13 ° C are used; A 2.5-5% polymer solution in distilled water is loaded into the reactor once until the solutions of sodium sulfide and cadmium bromide are drained at a temperature of 20-30 ° C, and the volume of the solutions is sodium sulfide: cadmium bromide: polymer 1: 1: 4, the gelatin solution is heated to 40-90 ° C, 96% ethanol 2.5% of the volume of gelatin solution is poured into the reactor, after which two solutions of sodium sulfide and cadmium bromide are poured into the reactor with constant stirring at a speed of 100-600 rpm, at the end of the mixing, they are mixed for 1 -300 min

At the final stage of crystallization of colloidal CdS quantum dots dispersed in gelatin, a methylene blue solution is added in a ratio of 10 ^-l ÷ 10 ^-4 (ν _color / ν _CdS )

After these procedures, the solution is cooled to a temperature of 4 to 10 ° C and the solution is maintained at this temperature for 24 hours, after which the cooled solution is crushed to a granule size of 5-10 mm, washing is carried out by immersion in distilled water at a temperature of 7 to 13 ° C for 30 minutes, after which excess water is decanted and the granules are heated to a temperature above 40 ° C to obtain a uniform melt.

The result is a biocompatible nanomaterial for photosensitization of singlet oxygen, which is a hybrid associates of colloidal CdS QDs 2-4 nm in size with methylene blue MB ⁺ cations in a ratio of 10 ^-1 -10 ^-4 (ν _color / ν _CdS ).

The formation of colloidal CdS QDs is confirmed by X-ray diffraction and transmission electron microscopy (Fig. 2, inset). The presence of a hybrid association of CdS QDs with MB ⁺ cations is confirmed by the presence of a dip in the luminescence spectrum of the associates of CdS QDs with MB ⁺ cations, which falls on the dye absorption region, which was not observed in the luminescence spectrum of pure CdS QDs (Fig. 2).

The photosensitization of singlet oxygen by the proposed biocompatible nanomaterial was confirmed by the presence of a band in the region of 1270 nm, corresponding to the luminescence of singlet oxygen (Fig. 3).

To establish the biocompatibility of the proposed nanomaterial, an assessment was made of the level and rate of oxygen consumption in rats Rattus norvegicus L. (Wistar) 5 months old, since a change in respiration rate indicates not only effective, but also safe intensification of the electron transport chain (ETC).

The oral route was chosen to introduce nanomaterial into the animal organism. In vivo respiration rate was measured using a Vernier LabQuest oximeter (Vernier, USA) equipped with oxygen and carbon dioxide sensors. Animals received 100 mg of the therapeutic composition; after 24 hours, the first measurement was performed. The selected dose was not toxic to animals. No mortality was observed among rats during the duration of the experiment. Body weight also remained unchanged. The animals were kept at a standard temperature of 22 ° C, a 12-hour light cycle in a closed sealed volume, and before the start of experiments, the oxygen content in this space reached 18-20%, as in a normal atmosphere.

The data on the assessment of the level and rate of oxygen gas consumption by laboratory rats of Rattus norvegicus of the Wistar strain are summarized in Table 1. The decrease in the rate of oxygen consumption under quantum dot therapy conditions seems insignificant and is within the limits of statistical error.

CT CdS and methylene blue have a stimulating effect on the respiration of isolated rat liver mitochondria. In this case, the form of the respiratory curve does not deviate from the standard one, which indicates the normal reaction of mitochondria to the presence of the quantum dot preparation in the recording medium (Fig. 5). The absorption of oxygen by isolated rat liver mitochondria in the presence of quantum dots, methylene blue, and hybrid associates of CdS CT with MB ⁺ cations demonstrates the absence of the toxic effect of the nanomaterial (oppression of cellular respiration).

A study on isolated mitochondria of rat liver in the presence of methylene blue dye, CdS CT and hybrid CdS CT associates with MB ⁺ cations generating hydrogen peroxide as the main representative of the group of reactive oxygen species formed by mitochondria showed an increase in the production of reactive oxygen species in the presence of nanomaterial (Fig. 6 ) This fact indicates the photosensitized formation of reactive oxygen species in mitochondria in the presence of methylene blue dye, CdS quantum dots and hybrid associates of CdS quantum dots with MB ⁺ cations. Moreover, further additions to mitochondria of rotenone, which induces a reverse current of electrons and antimycin, which blocks the Q-cycle, showed that mitochondria in each case responded to them with a similar pattern of change in the rate of peroxide production, which corresponds to that normal. This fact indicates the preservation of the respiratory activity of mitochondria, their molecular organization and, thus, the biocompatibility of the nanomaterial.

Claims (2)

1. A biocompatible nanomaterial for photosensitization of singlet oxygen, is a low-toxic hybrid associates of luminescent colloidal CdS quantum dots with a size of 2-4 nm with cations of methylene blue MB ⁺ in a ratio of 10 ^-1 -10 ^-4 (ν _color / ν _CdS ). 2. The method for producing the biocompatible nanomaterial according to claim 1 for photosensitizing singlet oxygen includes two-jet draining of a 0.6-5% solution of sodium sulfide and 0.8-7% solution of cadmium bromide in a temperature-controlled reactor with a gelatin melt at a constant temperature of 40 ° C, characterized by injecting a solution of MV ⁺ cations in a ratio of 10 ^-1 -10 ^-4 (ν _color / ν _CdS ) into the resulting colloidal solution containing colloidal quantum dots CdS, at the final stage of crystallization of quantum dots, followed by cooling of the obtained sol to t temperatures from 4 to 10 ° C, the solution is kept at this temperature for 24 hours, after which the gelatin gel obtained is crushed to a granule size of 5-10 mm, washed in distilled water at a temperature of 7 to 13 ° C for 30 minutes, decanted water and granules are heated to temperatures above 40 ° C.

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