RU2629390C2 - Photosensibile on the basis of semiconductor quantum points and chlorine e6 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a photosensitizer based on semiconductor quantum dots and chlorin e6 containing covalently bound complexes of semiconductor quantum dots and chlorin e6, where the complexes consist of chiral semiconductor quantum dots of the A2B6 group and chlorine e6 molecules in monomeric form with the possibility of selective photosensitizer accumulation in transformed cells.

EFFECT: increased selectivity of photosensitizer accumulation in transformed cells.

2 cl, 11 dwg, 3 ex

Description

The invention relates to the field of antitumor drugs, in particular to drugs combining inorganic and organic active ingredients that can be used for the diagnosis and treatment of cancer.

A wide range of drugs based on organic photosensitizers for the treatment and diagnosis of cancer is known: “Photosensitizer and method for its production” (RF Patent No. 2183956 C1, IPC A61K 31/409, A61P 35/00, application 2001108397/14, publication date 06/27/2002 , filing date March 30, 2001, “The drug for the photodynamic therapy of malignant neoplasms” (RF Patent No. 2145221, IPC A61K 31/40, A61K 47/42, application 96115788/14, publication date 02.10.2000, filing date 30.07.1996 ), "The drug for photodynamic therapy" (RF Patent No. 2146144, IPC A61K 31/675, A61K 31/40, application 97102 060/14, publication date 10.03.2000, filing date 02/11/1997), "Means for photodynamic diagnosis and treatment of cancer" (RF Patent No. 2152790, IPC G01N 33/52, A61K 31/79, application 99110071/14, publication date 07/20/2000, application submission date 05/12/1999) The main component of these preparations are organic photosensitizers, the principle of which is based on their ability to accumulate in transformed cells, effectively absorb light of a certain wavelength and transfer photoexcitation energy to oxygen molecules surrounding them. As a result, oxygen molecules are in an excited singlet state and are able to effectively oxidize the surrounding tissues, which leads to the destruction of transformed cells. It should be noted that organic photosensitizers have a number of disadvantages:

1. The complexity of the synthesis and purification of compounds;

2. The low selectivity of photosensitizer accumulation in transformed cells (the so-called oncotropy);

3. Dark and light cytotoxicity;

4. Fast penetration and elimination from cells;

5. Slow elimination from the body.

In this regard, the urgent task is to search for new approaches to the creation of drugs for photodynamic therapy (PDT), which would improve its effectiveness and reduce the effects of side effects.

One of the modern approaches in the development of new drugs for PDT is the creation of complexes with the participation of organic photosensitizers and nanoparticles. This approach uses the principle of increasing the concentration of the photosensitizer in the tumor through the use of nanoparticles as a carrier. The patent “Anticancer nanoparticles with enhanced target specificity and method for their preparation” is known (US Patent No. US 20150290332 A1, IPC A61K 31/555, A61K 47/36, A61P 35/00, A61K 9/16, application US 14 / 436,276, date publication 10/15/2015, priority date 05/02/2013), in which serum albumin is used as the basis for nanoparticles, to the surface of which porphyrin molecules are non-covalently attached. Thus, in the invention, an increase in the concentration of the photosensitizer in the cells is achieved through the use of serum albumin as a delivery agent for several porphyrin molecules to cancer cells simultaneously. The disadvantages of this approach are: 1) the selectivity of the accumulation of porphyrin molecules in transformed cells is determined by the properties of porphyrin itself, i.e. has no advantages over the use of free porphyrin molecules; 2) there is a high probability of desorption of porphyrin molecules from the surface of serum albumin during the transportation of these nanoparticles to transformed cells; 3) the conditions of photoexcitation are strictly determined by the properties of only porphyrin molecules. The patent “Composition and method of its manufacture for photosensitizer delivery” is known (US Patent No. US 20040047913 A1, IPC A61K 41/00, application US 10 / 440,505, publication date 11.03.2004, priority date 05.16.2002). According to the invention, polymer nanoparticles of about 200 nm in size are used to deliver hydrophobic porphyrin photosensitizers to transformed cells. In the presence of serum albumin, the photosensitizer molecules are released from the polymer nanoparticles, and the photosensitizer molecules are further delivered to the transformed cells with the participation of serum albumin. The present invention solves the problem of using hydrophobic photosensitizers for PDT, however, it has all the disadvantages of the invention set forth in the patent "Anticancer nanoparticles with enhanced target specificity and method for their preparation."

Another widespread approach in the manufacture of new drugs for PDT based on organic photosensitizers is the use of their complexes with nanoparticles, which, in addition to delivering the photosensitizer to the tumor, can serve as an effective donor of photoexcitation energy. Colloidal semiconductor quantum dots (QDs) are today the most common type of such nanoparticles (Jun Yao et al. Chem. Rev. 2014, 114 6130). A feature of QDs is the dependence of the wavelength of their luminescence on the size of the nanocrystal, the ability to absorb light in a wide spectral range, high photostability, and a high quantum yield of luminescence, which makes them effective donors of photoexcitation energy (Efros, AL, DJ Lockwood, et al. Semiconductor Nanocrystals: From Basic Principles to Applications, Springer, 2003). A number of studies show that quantum dot complexes with photosensitizers can significantly increase the photodynamic effect leading to the death of cancer cells, both due to efficient energy transfer and due to an increase in the intracellular concentration of the photosensitizer. A known method of using CT to increase the efficiency of photosensitizers "The use of photoluminescent nanoparticles for photodynamic therapy" (US Patent No. US 20020127224 A1, IPC A61K 39/44, A61K 41/00, application US 10/091144, publication date 09/12/2002, priority date 2.03 .2001), in which quantum dots are excited by an external light source that penetrates well into the tissue, and then CT emits light that activates the photosensitizer. At the same time, the authors proposed the use of CT of several sizes, which allows you to excite various photosensitizers and allows you to combine them during treatment. However, with this approach, the efficiency of excitation of the photosensitizer by quantum dots can be quite low, because, firstly, when QDs are introduced into the human body, the quantum yield of QD luminescence can be significantly reduced, and secondly, for effective photoexcitation of the photosensitizer, it is necessary that the CT and the photosensitizer turned out to be as close to each other as possible, which entails the use of high therapeutic doses of CT and photosensitizers.

Closest to the proposed invention and adopted as a prototype "Photosensitizer containing covalently bound complexes of quantum dots and chlorine derivatives for the treatment and diagnosis of cancer and photodynamic therapy" (Patent No. WO 2010151074 A2, A61K 33/24, A61K 31/409, A61K 49 / 06, А61Р 35/00, application PCT / KR 2010/004134, publication date 05/19/2011, priority date 06/26/2010). As described in the patent, the photosensitizer is a quantum dot covalently linked to a complex of chlorin e6 derivatives. This complex implements non-radiative resonant energy transfer (FRET) from CT to molecules derived from chlorin, which leads to sensitization of the generation of singlet oxygen by them. Such a photosensitizer has several advantages:

1. The use of covalent binding of CT and chlorin e6 to the complex ensures the stability of the complexes and the small distances between the CT and chlorin e6. This allows you to reduce the therapeutic dose of the drug due to the sensitization of the photodynamic effect of chlorin e6 quantum dots;

2. Significantly expanded the spectral range of radiation that activates the complexes;

3. Due to the large absorption cross sections at semiconductor quantum dots, two- and three-photon excitations can be effectively used to activate complexes, which spectrally correspond to the transparency region of biological tissues;

4. The surface of the CT can be used to attach special molecules of carriers, which increase the selectivity of the accumulation of complexes in transformed cells.

However, the prototype has the following disadvantages:

1. The formation of complexes is carried out in aqueous media in which organic photosensitizers and, in particular, chlorin e6, are characterized by poor stability and are prone to the formation of aggregates. This will lead to a significant decrease in the activation efficiency of oxygen molecules and, as a result, to a decrease in the PDT effect and to an increase in therapeutic doses of the drug. It should be noted that the aggregation of the photosensitizer can lead to a complete loss of the ability of the photosensitizer to activate oxygen molecules, i.e. to a complete loss of functionality of the drug. It is noteworthy that practically none of the examined patents pays attention to the issues of aggregation of organic photosensitizers in complexes with nanoparticles, and in particular with CT.

2. To increase the selectivity of the accumulation of complexes in the tumor, it is proposed to use special molecules. With this approach, we are essentially talking about creating a complex, three-component structure, the complication of the methods of formation and purification of which will lead to a sharp increase in the cost of the final drug based on such complexes. It should also be noted that in the case of using antibodies as agents for targeted delivery, certain difficulties arise that are due to their strict specific interaction with a particular antigen. First of all, the disadvantages of using antibodies are associated with a high degree of heterogeneity of real tumors. This makes it difficult to select a single antigen that is sufficiently well expressed for the entire tumor. It should also be noted that antibodies are large protein molecules that are difficult to penetrate both deep into the tumor and inside transformed cells. In the latter case, when the antibody remains on the surface of the cell, the probability of activation of the immune system and, as a result, the development of antibodies to these antibodies increases sharply.

The objective of the invention is to increase the efficiency of the photodynamic effect in transformed cells by maintaining the photophysical properties of the monomeric form of chlorin e6 in complexes with quantum dots and the presence on the surface of the CT of enantiomers of chiral stabilizer molecules, which increases the selectivity of the proposed photosensitizer in transformed cells.

The task is achieved in that a photosensitizer is formed on the basis of semiconductor quantum dots and chlorin e6, consisting of covalently linked complexes of chiral semiconductor quantum dots of the A2B6 group and chlorin e6 molecules in monomeric form, characterized by the possibility of selective accumulation in transformed cells due to the presence of semiconductor quantum points of the enantiomer of a chiral molecule.

The essence of the invention lies in the fact that the formation of complexes is carried out as a result of covalent binding to a complex of chiral semiconductor quantum dots with chlorin e6 molecules in monomeric form in dimethyl sulfoxide. After the formation of the complexes, they are purified and concentrated, then the resulting concentrated solution of the complexes is introduced into the aqueous solution. As a result of irradiation of the QD / Ce6 complexes with light with a wavelength that is effectively absorbed by them, the complexes go into an excited state, in which, when interacting with oxygen molecules, they can transform them into an active singlet form.

The selectivity of accumulation of the proposed photosensitizer in transformed cells is achieved due to the stereospecific interaction of the enantiomer of the chiral molecule on the surface of the CT, which will enhance the oncotropic properties of Ce6 molecules that make up these complexes.

The invention has the following advantages:

1. The formation of complexes of semiconductor quantum dots with chlorin e6 molecules is carried out in dimethyl sulfoxide, which ensures the presence of only the monomeric form of chlorin e6 in the complexes and, as a result, its maximum efficiency as a singlet oxygen generator when using the proposed photosensitizer in PDT.

2. The formation of complexes in which several chlorin e6 molecules are coupled to a quantum dot in monomeric form can significantly increase the PDT effect and, as a result, reduce the therapeutic photosensitizer.

3. The use of chiral quantum dots in complexes allows one to increase the selectivity of the accumulation of complexes in transformed cells without technical complication of the method of formation and purification of complexes in comparison with the use of special molecules acting as targeted delivery agents. This technical solution allows to significantly simplify the procedure for the formation of the proposed photosensitizer, which, in turn, increases its stability and reduces cost.

The essence of the invention is illustrated in FIG. 1-11, on which are presented:

FIG. 1. Spectra of circular dichroism of CdSe / ZnS quantum dots stabilized by L-, D-histidine and TOPO (curves 1, 2, and 3, respectively), as well as the absorption spectrum of CdSe / ZnS quantum dots.

FIG. 2. Absorption spectra of chlorin e6 activated by 1,3-dicyclohexylcarbodiimide chlorine e6 (1), CdSe / ZnS CT stabilized by L-histidine, (2) and their complexes (3) in equimolar concentrations in DMSO.

FIG. 3. Spectra of circular dichroism of Ce6 (1), CdSe / ZnS CT stabilized by L-histidine, (2) and their complexes (3) in DMSO.

FIG. 4. Luminescence spectra of CdSe / ZnS CT (1), Ce6 (2) and their complexes (3) in DMSO, the wavelength of the exciting light is 450 nm.

FIG. 5. Luminescence excitation spectra of Ce6 (1), CdSe / ZnS complexes of quantum dots with 1, 2, and 5 Ce6 molecules in complexes (2-4) and the absorption spectrum of quantum dots (5) in the distillate. The wavelength of registration of luminescence is 680 nm.

FIG. 6. Dependence of the luminescence intensity at the maximum of the luminescence band of a chemical sensor on SOSG singlet oxygen (525 nm) on the time of exposure to light with a wavelength of 450 nm in the presence of equimolar concentrations of Ce6 (1), CdSe / ZnS CT (2) and their complexes (3) .

FIG. 7. Dependence of the luminescence intensity at the maximum of the SOSG luminescence band (525 nm) on the time of exposure to light with a wavelength of 450 nm in the presence of QD / Ce6 complexes in which the QD surface is stabilized by different molecules.

FIG. 8. Dependence of the intracellular Ce6 concentration on the time of incubation of live AKE cells with CT / Ce6 complexes in which the surface of the CT is stabilized with L-histidine or D-histidine.

FIG. 9. Dependence of the intracellular Ce6 concentration on the time of incubation of live AKE cells with CT / Ce6 complexes, in which the surface of the CT is stabilized with L-cysteine or D-cysteine.

FIG. 10. Results of a PDT test on live AKE cells incubated with CT / Ce6 complexes in which the surface of the CT is stabilized with L-histidine or D-histidine.

FIG. 11. Results of a PDT test on live AKE cells incubated with CT / Ce6 complexes in which the surface of the CT is stabilized with L-cysteine or D-cysteine.

Example 1. In complexes of colloidal water-soluble semiconductor quantum dots with chlorin e6 molecules (CT / Ce6) formed as a result of electrostatic interaction or covalent bonding, dipole-dipole energy transfer from CdSe / ZnS quantum dots to chlorine e6 is observed (IV Martynenko, A. O. Orlova, VG Maslov, AV Baranov, AV Fedorov and M. Artemyev. Energy transfer in complexes of water-soluble quantum dots and chlorin eb molecules in different environments // Beilstein J. Nanotechnol. 2013, 4, 895-902 and IV Martynenko, VA Kuznetsova, AO Orlova, PA Kanaev, VG Maslov, A Loudon, V Zaharov, P Parfenov, Yu K. Gun'ko, AV Baranov and AV Fedorov. Chlorin e-ZnSe / ZnS quantum dots based system as reagent for photodynamics- ic therapy // Nanotechnology 26 (2015) 055102 (9pp)). However, in aqueous solutions in the QD / Ce6 complexes, with an increase in the Ce6 concentration, a decrease in the efficiency of the photoexcitation energy transfer from QDs to Ce6 molecules and a decrease in the luminescence quantum yield of Ce6 molecules are observed, which, as a rule, indicates a decrease in the efficiency of singlet oxygen generation by molecules. This circumstance noticeably reduces the effectiveness of the use of such complexes in PDT. The most probable reason for the decrease in the functioning efficiency of the CT / Ce6 complexes formed in aqueous solutions is the well-known aggregation of molecules. At the same time, it is known that in dimethyl sulfoxide (DMSO), chlorin e6 molecules are in monomeric form (DA Schwartz, DR Gamelin // Optical Science and Technology, SPIE's 48th Annual Meeting. 2003, International Society for Optics and Photonics. P. 1- 7.), which is characterized by the maximum quantum yield of singlet oxygen generation and, accordingly, is preferable when using the CT / Ce6 complexes in PDT. DMSO has high penetration in biological tissues and, when added in a small percentage (up to 10% by volume), does not show cytotoxicity to living cells (T.M. Rippin, VJ Bykov, SM Freund, G. Selivanova, K. Wiman , AR Fersht // Oncogene. 2002. V. 21. No. 14. P. 2119-2129). The existing restriction on the volume of DMSO additives is by no means an obstacle to the realization of the idea of forming a photosensitizer based on quantum-sized nanostructures suitable for PDT in this solvent. This is due to the fact that on the surface of the QD there are molecules that ensure the solubility of the QD and, correspondingly, the QD / Ce6 complexes both in DMSO and in aqueous solutions. Therefore, the complexes formed in DMSO can be converted into an aqueous solution as a result of microaddition of a highly concentrated solution of complexes in DMSO into it.

To demonstrate the operability of the present invention, complexes of chiral semiconductor QDs with chlorin e6 molecules (CT / Ce6) in dimethyl sulfoxide were formed. A typical representative of A2 B6 colloidal semiconductor quantum dots are core-shell type cadmium selenide quantum dots coated with several layers of zinc sulfide semiconductor obtained by hot injection (V.O. Dabbousi, J. Rodriguez-Viejo, FV Mikulec, JR Heine, H Mattoussi, R. Ober, KF Jensen, and MG Bawendi. (CdSe) ZnS Core-Shell Quantum Dots: Synthesis and Characterization of a Size Series of Highly Luminescent Nanocrystallites // J. Phys. Chem. B, 1997, 101 (46 ), pp. 9463-9475). To obtain hydrophilic QDs capable of forming colloidal solutions in aqueous media and in dimethyl sulfoxide, a standard procedure was carried out to replace the initial QD surface stabilizer with enantiomers of chiral molecules: L-, D-cysteine, L-, D-penicillamine, L-, D-arginine , L-, D-histidine. To analyze the effect of CT chirality on the efficiency of intracellular penetration, CdSe / ZnS CTs stabilized by achiral thioglycolic acid molecules (THC) were used as a reference sample. As a result of the replacement of the surface stabilizer, QDs from the organic phase were transferred to dimethyl sulfoxide. As an example in FIG. Figure 1 shows the spectra of circular dichroism (CD) of CdSe / ZnS quantum dots with a core diameter of 3.5 nm stabilized initially by trioctylphosphine oxide (TORO) molecules, which were converted from toluene to DMSO using L- and D-histidine. It is seen that CdSe / ZnS QDs stabilized by TOPO molecules do not exhibit optical activity, as evidenced by the absence of bands in their CD spectrum (curve 3). At the same time, stabilization of QDs by the L- and D-enantiomers of histidine (curves 1 and 2, respectively) led to the appearance of characteristic bands in their CD spectra, the spectral position of which corresponds to electronic transitions in the absorption spectrum of QD data (curve 4). When a similar replacement of the TOPO molecules on the surface of CdSe / ZnS QDs with enantiomers of arginine, cysteine, and penicillamine was carried out in the CD spectra of the samples, a similar picture was observed.

The standard linker 1,3-dicyclohexylcarbodiimide (DCC) (Ulrich, N. Chemistry and technology of carbodiimides. John Wiley & Sons, 2008), which forms a peptide bond between the amino group of the surface stabilizer of the QD (histidine, arginine, penicillamine and cysteine) and the Ce6 carboxyl group. To stabilize DCC, N-Hydroxysuccinimide (NHS) was used; these compounds were added to a solution of Ce6 in DMSO (C _Ce6 ~ 5⋅10 ^-5 M) in an equimolar ratio. The resulting mixture was actively mixed on a shaker for 150 minutes at a speed of 600 rpm. Activated Ce6 was added as microadditives (50 μl) to a solution of CdSe / ZnS CT in DMSO (3 ml), after which the solutions were kept in the dark at room temperature with continuous stirring on a magnetic stirrer for 12 hours.

In FIG. Figure 2 shows the absorption spectra of activated Ce6, CT, and their mixtures in an equimolar ratio (curves 1, 2, and 3, respectively). The position of the first absorption band of Ce6 (~ 660 nm) indicates that Ce6 is in monomeric form (Parkhots M.V., Galievsky V.A., Stashevsky A.S., Trukhacheva T.V., Dzhagarov B.M. Dynamics and effectiveness of photosensitized formation of singlet oxygen by chlorine e6: the effect of the pH of the solution and polyvinylpyrrolidone. Optics and Spectroscopy, 2009, V. 107, No. 6, P. 1026-1032.). An analysis of the absorption spectrum of the mixed solution of QD and Ce6 (curve 3) showed that in the presence of QD the absorption spectrum of Ce6 does not change, i.e. Ce6 mixed with CT is in monomeric form. The formation of CT / Ce6 complexes is evidenced by a change in the spectrum of Ce6 CD in a mixture with CT, shown in FIG. 3. It can be seen that the amplitude of the long-wavelength Ce6 band (~ 660 nm) increases by a factor of ~ 2, and the CD band in the region of the Soret band (~ 400 nm) decreases by a factor of 2.5. In FIG. Figure 4 shows the luminescence spectra of activated Ce6, CT, and their mixtures in an equimolar ratio in DMSO (curves 1, 2, and 3, respectively). It is seen that the addition of a Ce6 solution (50 μl) to a QD solution (3 ml) leads to a noticeable quenching of the luminescence of quantum dots and to the appearance of Ce6 luminescence in the sample (~ 670 nm). If the absorption spectrum of Ce6 in the mixture with QDs remains unchanged, changes in the spectrum of Ce6 CDs, a twofold increase in the Ce6 luminescence intensity indicates the formation of complexes with efficient energy transfer from quantum dots to Ce6 molecules.

At the next stage, the obtained complexes were concentrated using Amicon Ultra-10 Centrifugal Filter Units (10 kDa) concentrators, which allow separating the complexes from the free components of the mixture. The obtained CT / Ce6 complexes with different concentration ratios were separated from unreacted components by centrifuging the samples in concentrators for 10 minutes at a speed of 3000 rpm. The isolated QD / Ce6 complexes were immediately transferred to aqueous solutions with a physiological pH level and their absorption, luminescence, and luminescence excitation spectra were studied, which practically did not differ from similar spectra of complexes in DMSO. The most representative and informative for CT / Ce6 complexes are the excitation spectra of Се6 luminescence in complexes with quantum dots, since the presence of the contribution of the absorption of quantum dots to these spectra indicates an intracomplete energy transfer from quantum dots to Ce6. In FIG. Figure 5 shows the luminescence excitation spectra of Ce6 in complexes with quantum dots in aqueous solutions for complexes with different numbers of Ce6 molecules (from 1 to 5). The luminescence excitation spectra shown are normalized to the Ce6 concentration. This makes it possible to directly estimate the contribution of energy transfer from QDs to the total luminescence intensity of Ce6 directly from the Ce6 luminescence excitation spectra. The increase in the contribution of QD absorption in the region of 300–530 nm to the Ce6 luminescence excitation spectrum indicates that the energy transfer efficiency in these complexes can be doubled with an increase in the average number of Ce6 molecules in complexes with QD from 1 to 5. Thus, It has been demonstrated that in the QD / Ce6 complexes covalently bound in DMSO, an effective transfer of photoexcitation energy from QDs to the monomeric form of Ce6 is observed, and an increase in the average number of Ce6 molecules in complexes from 1 to 5 allows one to double the efficiency The nature of intracomplete energy transfer.

Example 2. The ability of CT complexes stabilized by the L- and D-enantiomers of penicillamine, cysteine, arginine, histidine, as well as achiral molecules of thioglycolic acid, with Ce6 molecules to generate a singlet form of oxygen was tested using a special chemical sensor based on a complex of fluorescein and anthracene ( Singlet Oxygen Sensor Green, SOSG) (S. Kim, M. Fujitsuka, and T. Majima. Photochemistry of Singlet Oxygen Sensor Green // J. Phys. Chem. B, 2013, 117 (45), pp 13985-13992). For this, mixed aqueous solutions of SOSG (C _SOSG = 10 ^-6 M) and complexes of CT / Ce6 (C _complexes = 10 ^-7 M) were prepared, in which from 1 to 5 Ce6 molecules averaged 1 CT. The solutions were repeatedly illuminated with light with a wavelength of 450 nm (power density incident on the samples, 4 mlW / cm), the time of each exposure was 20 seconds. During illumination, the solutions were continuously mixed on a magnetic stirrer. Singlet oxygen was detected by the appearance and increase in the intensity of the luminescence band with a maximum at 525 nm. In FIG. Figure 6 shows the dependences of the SOSG luminescence intensity at a wavelength of 525 nm for samples containing equimolar concentrations of CdSe CT (C _KT = 10 ^-7 M), Ce6, and CT / Ce6 complexes (1, 2, and 3, respectively). Since the luminescence intensity of SOSG is directly proportional to the concentration of singlet oxygen in the samples, at the same concentrations of complexes and individual components (CT and Ce6) under the same experimental conditions, one can compare the efficiency of generation of singlet oxygen by the complexes of CT / Ce6 and individual components. It can be seen that without Ce6, the QDs practically do not generate singlet oxygen, which is in good agreement with the data on the quantum yield of generation of water-soluble QDs (~ 1%), and the efficiency of the generation of singlet oxygen by complexes is more than two times higher than the monomeric form of Се6 with the same concentration. In FIG. Figure 7 shows the dependences of the SOSG luminescence intensity at a wavelength of 525 nm when light is illuminated with 450 nm of samples containing QD / Ce6 complexes (5 Ce6 molecules per 1 QD), in which the QD surface is stabilized by enantiomers of various chiral molecules: D-cysteine, L-arginine, D-penicillamine, L- and D-histidine, as well as achiral molecules of THC. It can be seen that the efficiency of generation of singlet oxygen by complexes containing QDs with various surface stabilizers does not depend much on the type of stabilizer. This result is quite expected, since the efficiency of generation of singlet oxygen is determined by the photophysical properties of the complexes, which, in turn, are determined by the conditions of their formation, the type of binding of the components to the complex, and the photochemical properties of Ce6 in the complexes, which were the same in this case and did not depend on the used type of surface stabilizer CT. Thus, it was demonstrated that the formed CT / Ce6 complexes are capable of efficiently generating singlet oxygen in aqueous media, and the efficiency of the generation of singlet oxygen by these complexes does not depend on the type of surface stabilizer of the QD.

Example 3. To demonstrate the operability of the invention, a PDT test was performed for complexes of chiral CdSe / ZnS CT with Ce6 molecules on living cells of Ehrlich ascites carcinoma (AKE), which was injected subcutaneously in suspension to white laboratory mice of both sexes at the age of eight weeks and weighing about 20 grams. Ascites were grown for 10 days. For studies, cell suspensions were obtained from the abdominal cavity of mice on day 10 after injection, which were purified from red blood cells in a buffer solution (~ 10 minutes) and centrifuged twice at a speed of 1500 rpm for 10 minutes in Hanks solution. The precipitate was dissolved in Hanks brine. For each sample, 10 ⁶ cells per 1 ml of Hanks saline were placed in the cells of a 24-well cell culture plate. Samples were incubated in the dark at 37 ° C in a 5% atmosphere of CO ₂ for 1, 2, 4, and 7 hours, the concentration of C _{KT / Ce6} complexes was 10 ^-6 M, and free C _Ce6 = 5 =10 ^-6 M .

The magnitude of the intracellular photodynamic effect, which leads to the destruction of transformed cells, is affected by both the efficiency of singlet oxygen generation by chlorin e6 in complexes with chiral QDs and the intracellular concentration of complexes, which depends on the chiral properties of QDs. An assessment was made of the intracellular photodynamic effect of CdSe / ZnS QD complexes stabilized by both chiral and achiral molecules with Ce6 molecules in which 5 Ce6 molecules were covalently bound to one CT. As reference samples, AKB cell samples incubated with free Ce6 were used, the concentration of which corresponded to the concentration of Ce6 in complexes with CT. To determine the intracellular concentration of Ce6 and complexes, cell samples were precipitated by centrifugation at a speed of 1500 rpm for 5 minutes. The resulting precipitate was placed in a 1 mm thick quartz cuvette, the cuvette being filled so that the cells formed an almost dense package. This ensured the absence of effects associated with sample unevenness. To minimize scattering, the absorption spectra were recorded on a UV Probe - 3600 spectrophotometer (Shimazu, Japan) with an integrated integrating sphere. The intracellular Ce6 concentration was determined from the absorption spectra of cell samples using the formula D = ε⋅c⋅l, where D is the optical density at the maximum of the Cooret band (405 nm), ε is the extinction coefficient of Ce6 in the Cooret band, l is the optical path length (thickness cuvettes - 1 mm). In FIG. Figure 8 shows, as an example, data on the concentration of Ce6 in AKE cells at different incubation times for samples with CT / Ce6 complexes in which the surface of the CT is stabilized by L- and D-histidine molecules and free Ce6. It can be seen that, irrespective of the incubation time, the intracellular Ce6 concentration is higher in complexes with QD, the surface of which is stabilized by L-histidine. Moreover, the use of QD complexes stabilized by D-histidine molecules leads to the fact that the intracellular concentration of Ce6 is slightly lower than when using the achiral stabilizer of the surface of QD - thioglycolic acid. To demonstrate that the effect of an increase in the intracellular concentration of Ce6 is due to enantioselective accumulation of chiral CT complexes with Ce6, FIG. Figure 9 shows data on the intracellular concentration of Ce6 in the composition of QD / Ce6 complexes in which the surface of the QD is stabilized by enantiomers of another chiral molecule, cysteine. It can be seen that, as in the case of using L-histidine as a surface stabilizer, for the CT / Ce6 complexes in which the surface is stabilized by L-cysteine, the Ce6 concentration is significantly higher than in the case of free Ce6. This indicates that the presence of enantiomers of chiral molecules on the surface of QDs leads to a noticeable increase in the intracellular Ce6 concentration in the QD / Ce6 complexes in comparison with its free form, i.e. increases the selectivity of its accumulation in cells.

Assessment of the intracellular photodynamic effect was performed on samples of AKE cells incubated for 7 hours with complexes of CT / Ce6 (10 ^-6 M, ratio of CT: Ce6 = 1: 5) and Ce6 (5⋅10 ^-6 M), when irradiated with light with a wavelength of 450 nm (power density incident on the samples, 25 mW / cm ² ) for 5 and 10 minutes. The number of dead cells was estimated using a 0.4% trypan blue solution, which stained the cells for 5 minutes. A feature of this dye is that it is able to penetrate and accumulate only in dead cells. To estimate the number of living and dead cells, an automated TC10 cell counter (Bio-Rad) was used. In FIG. Figure 10 shows the survival of AKE cells incubated with CT / Ce6 complexes, in which the surface of the CT is stabilized by L- and D-histidine molecules, and with individual components of the corresponding concentrations. It is seen that QDs have neither dark nor light cytotoxicity. At the same time, the photodynamic effect in samples incubated with QD / Ce6 complexes in which the surface of the QD is stabilized by L-histidine (C _set = 10 ^-6 M, QD: Ge6 = 1: 5) is almost ~ 8 times higher than at cell incubation with Ce6 molecules (C _Ce6 = 5⋅10 ^-6 M). In FIG. 11 shows data on the survival of AKE cells incubated with CT / Ce6 complexes, in which the surface of the CT is stabilized by L- and D-cysteine molecules, and with individual components of the corresponding concentrations. It is seen that, as in the case of the use of histidine enantiomers for samples incubated with CT / Ce6 complexes in which the surface of the CT is stabilized by L-cysteine molecules, (C _set = 10 ^-6 M, CT: Ce6 = 1: 5), the PDT value the effect is 7.5 times higher than when cells were incubated with Ce6 molecules (C _Ce6 = 5⋅10 ^-6 M). The obtained data clearly demonstrate that the increase in the PDT effect in AKE cells is due to the enantioselective accumulation of chiral QD complexes with Ce6 molecules, which is due to the presence of the enantiomer of the chiral molecule on the surface of the QD.

Thus, the above examples clearly demonstrate that a photosensitizer based on chiral semiconductor quantum dots and e6 chlorin molecules exhibits a multiple increase in the intracellular photodynamic effect on living AKE cells compared to free e6 chlorin molecules of the same concentration due to a synergistic effect due to enantioselective accumulation of chira complexes QD and efficient intracomplete energy transfer from QD to Ce6 molecules, which are in complex in mon dimensional form.

Claims (2)

1. A photosensitizer based on semiconductor quantum dots and chlorin e6, containing covalently bound complexes of semiconductor quantum dots and chlorin e6, characterized in that the complexes consist of chiral semiconductor quantum dots of the A2B6 group and chlorin e6 molecules in monomeric form with the possibility of selective accumulation of the photosensitizer in the transform cells. 2. The photosensitizer according to claim 1, characterized in that enantiomers of the chiral molecule are adsorbed on the surface of the chiral semiconductor quantum dots.

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