Preparation and application of a self-assembled nanoformulation for initiating cancer immunity cycle

Abstract

The present invention belongs to the technical field of immunotherapy preparations, and specifically discloses a self-assembled nanoformulation for initiating a cancer immune cycle, the main body of which is spherical particles composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6, and is prepared by a "one-pot method". The nanoformulation of the present invention has the ability to consume lactic acid and produce oxygen and reactive oxygen species. In the tumor microenvironment, the nanoformulation combined with photodynamic therapy effectively induces immunogenic death of tumor cells, promotes activation and presentation of antigen-presenting cells, recruits a large number of immune effector cells in the tumor, and reduces the accumulation of immunosuppressive cells. Moreover, the nanoformulation of the present invention can be enriched in large quantities at the tumor site within 2 hours in vivo, and has a significant effect in inhibiting the growth of breast tumor cells in synergistic photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy preparations, and specifically relates to the preparation and application of a self-assembled nanoformulation for initiating cancer immune circulation. Background Art

[0002] Effective activation of each event in the cancer immunity cycle (CIC) is a prerequisite for achieving significant results in cancer immunotherapy. Insufficient presentation of tumor-associated antigens, limited tumor infiltration, and immune escape, influenced by the immunosuppressive tumor microenvironment, contribute to the failure of CIC. In clinical practice, immune checkpoint blockade has demonstrated exciting clinical value and strong therapeutic potential, but response rates are relatively low due to the stagnation of CIC. Therefore, there is an urgent need to develop strategies that target the suppressive tumor microenvironment to reactivate CIC and improve anti-tumor efficacy.

[0003] Inadequate presentation of tumor-associated antigens is a hallmark of the tumor microenvironment. Immunogenic cell death is a specialized form of cell death that promotes the massive release of tumor-associated antigens from dying cells. Photodynamic therapy, a common approach to inducing immunogenic cell death, has garnered widespread attention in cancer treatment. However, due to the immunosuppressive effects of the tumor microenvironment, photodynamic therapy alone often has low tumor clearance rates. Intratumoral vascular abnormalities and hypoxia severely impede T cell activation and infiltration, indirectly leading to the failure of CICs. Vascular endothelial growth factor (VEGF) inhibitors have been reported to induce tumor vascular normalization and significantly enhance immune cell infiltration within the tumor, thereby improving the efficacy of immunotherapy. Furthermore, the combination of VEGF inhibitors and immune checkpoint blockade has been shown to improve clinical outcomes in various cancers. Furthermore, lactate is a ubiquitous immunosuppressive molecule in the tumor microenvironment, significantly impairing antitumor immunity by suppressing effector T cells, promoting immune escape, and maintaining an acidic environment. Downregulating lactate production, interfering with lactate transporters, or directly depleting lactate can reprogram the immunosuppressive tumor microenvironment to enhance antitumor immunity. Lactate transporter inhibitors have been reported to significantly enhance the therapeutic effects of immune checkpoint blockade therapy in a mouse breast cancer model. Lactate oxidase-based nanomedicines have been reported to reverse tumor immunosuppression and enhance immunotherapy efficacy by reducing lactate concentrations in tumors. Although these strategies can alleviate immunosuppression to some extent, the anti-tumor immune response is a cyclical process, and limitations at each step may limit the optimal immunotherapy effect. Therefore, targeting multiple key steps in CIC is needed to provide more powerful strategies that can reverse the immunosuppressive tumor microenvironment and trigger effective anti-tumor immunity. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention proposes a self-assembled nanoformulation for initiating a cancer immune cycle. The main body of the self-assembled nanoformulation is spherical particles composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6, and is prepared by a "one-pot method". The prepared nanoformulation has significant anti-tumor effects.

[0005] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0006] On the one hand, the present invention provides a self-assembled nanoformulation for initiating a cancer immunity cycle, wherein the nanoformulation is a spherical particle composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6.

[0007] In a second aspect, the present invention provides a method for preparing the self-assembled nanoformulation for initiating cancer immunity cycle, comprising the following steps:

[0008] The catalase solution was added to a sterile tube and placed in an ice bath. Lactate oxidase solution, dihydrochlorin e6 solution, and sorafenib solution were added under ultrasound. After thorough mixing, the nanoformulation was obtained by centrifugation, washing, and drying.

[0009] Preferably, the mass ratio of catalase, lactate oxidase, sorafenib and dihydrochlorin e6 is 6:1:0.43:0.33.

[0010] Preferably, the catalase solution, lactate oxidase solution, sorafenib solution and dihydrochlorin solution are mixed and reacted under ultrasonic conditions for 15 minutes, and then the mixed solution is centrifuged at 4°C and a centrifugal force of 15,000×g for 10 minutes, washed twice with ultrapure water and then freeze-dried.

[0011] In a third aspect, the present invention provides the use of the nanoformulation prepared by the above preparation method in the preparation of anti-tumor drugs.

[0012] The beneficial effects of the present invention are:

[0013] Compared with the prior art, the nanoformulation of the present invention is a spherical particle composed of catalase, lactate oxidase, sorafenib, and dihydrochlorin e6, which has the ability to consume lactate and produce oxygen and reactive oxygen species. In the tumor microenvironment, the nanoformulation combined with photodynamic therapy effectively induces immunogenic death of tumor cells, promotes the activation and presentation of antigen-presenting cells, recruits a large number of immune effector cells in the tumor, and reduces the accumulation of immunosuppressive cells. Moreover, the nanoformulation of the present invention can be enriched in the tumor site in large quantities within 2 hours in vivo, and synergistically with photodynamic therapy has a significant effect in inhibiting the growth of breast tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 The morphology characterization of the nanoformulation CLSC, where a) is the TEM morphology of CLSC, scale: 100 nm; b) is the particle size distribution diagram of CLSC.

[0016] Figure 2 The general performance characterization results of nanoformulation CLSC, where a) is the distribution diagram of the main elements of CLSC, scale: 20nm; b) is the UV-visible absorption spectrum of Ce6, SF and CLSC; c) is the consumption of lactic acid by CLSC and LOD; d) is the catalytic production of O2 by CLSC and CAT in the presence of H2O2; e) is the production of CLSC and Ce6 under 660nm laser irradiation 1 O2.

[0017] Figure 3 Schematic diagram of the in vivo biodistribution of CLSCs, where a) is the real-time fluorescence image of 4T1 tumor-bearing mice at different time points after intravenous injection of CLSCs; b) is the quantitative analysis of the fluorescence signal of CLSCs at the tumor site at different time points; c) is the in vitro fluorescence image of the main organs and tumors of 4T1 subcutaneous tumor-bearing mice 12 hours after administration.

[0018] Figure 4 Schematic diagram of the effects of CLSC-assisted photodynamic therapy on breast cancer. (a) Changes in tumor volume in mice (n=5); b) Tumor weight on day 18 (n=5); c) Images of tumors on day 18; d) Survival curves of tumor-bearing mice in different treatment groups (n=8). Results are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0019] Figure 5 Schematic diagram of CLSC-photodynamic therapy-induced anti-tumor immune response, where a) is a representative flow cytometry plot and quantitative analysis of immunoregulatory T cells; b) is a representative flow cytometry plot and quantitative analysis of CD8+ T cells; the results are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0021] Catalase (CAT), lactate oxidase (LOD), sorafenib (SF), and chlorin (Ce6).

[0022] Example 1

[0023] Preparation and characterization of nanoformulated CLSC

[0024] 1) CLSCs were prepared using a one-pot method: 0.3 ml of CAT solution (7 mg / mL) was added to a 15 mL sterile tube and placed on ice. Then, 70 μL of LOD solution (5 mg / mL), 5.04 μL of Ce6 solution (38.8 mM), and 16.80 μL of SF solution (19.4 mM) were slowly added under sonication. The reaction mixture was sonicated for 15 min. After 15 min, the mixture was centrifuged (4°C, 10 min, 15,000 × g). The CLSC precipitate was washed twice with ultrapure water, lyophilized, and weighed for subsequent experiments.

[0025] 2) Particle size determination: A 1.0 mg / mL CLSC solution was prepared and the particle size of the nanoformulation CLSC was measured using a Zetasizer Pro (Malvern).

[0026] 3) Morphology measurement: 10 μL of a 10.0 mg / mL CLSC solution was dropped onto a 200-mesh carbon-coated copper grid. After standing for 1 min, the sample was removed from the edge of the grid with filter paper. 10 μL of ultrapure water was then added. After standing for 1 min, the sample was removed from the edge of the grid with filter paper. The morphology was observed using a 120 kV transmission electron microscope (FEI Tecnai G2 F30, America).

[0027] The experimental results are as follows Figure 1 shown. Figure 1 a shows that the nanoformulation CLSC is uniformly dispersed with a particle size of approximately 223 nm; Figure 1 b shows the morphology of the nanoformulated CLSC, which is a spherical structure with a diameter of approximately 150 nm. In summary, the one-pot method successfully prepared CLSCs; under ultrasonication, CLSCs assembled into a stable and uniform spherical structure.

[0028] Example 2

[0029] Determination of encapsulation efficiency and drug loading capacity of CLSC

[0030] 1) During the preparation of CLSCs, all centrifugation supernatants were saved and used to determine the encapsulation efficiency and drug loading of SF and Ce6 in CLSCs.

[0031] a. The free SF content in the supernatant was determined by high-performance liquid chromatography (HPLC, Waters e2695, USA). HPLC elution conditions: XBridge C18 column (5 μm, 4.6 × 250 mm); 60% B isocratic elution (Phase A: 100% water + 0.1% trifluoroacetic acid; Phase B: 20% water / 80% acetonitrile + 0.1% trifluoroacetic acid).

[0032] b. Measure the free Ce6 content in the supernatant at a wavelength of 400 nm using an enzyme-labeled instrument

[0033] d. Calculation formula:

[0034] LE(%)=We / Wm×100%

[0035] Where LE represents the percentage of drug loading in the preparation; We represents the amount of drug encapsulated in the nanoformulation; and Wm represents the total weight of the drug-loaded nanoformulation.

[0036] EN%=(1-Cf / Ct)×100%

[0037] Where EN represents the encapsulation efficiency of the drug in the preparation. Its expression is: where Cf is the amount of free drug; Ct is the total amount of nanoformulation.

[0038] The experimental results are shown in Table 1. In CLSCs, the encapsulation efficiency of SF was 92.64%, and the drug loading rate was 5.23%; the encapsulation efficiency of Ce6 was 84.82%, and the drug loading rate was 3.69%.

[0039] Table 1. Encapsulation efficiency and drug loading of sorafenib and chlorin in CLSCs

[0040]

[0041] Example 3

[0042] General performance characterization of nanoformulated CLSC

[0043] 1) Mapping analysis and UV absorption spectroscopy were used to determine that SF and Ce6 were successfully encapsulated in CLSCs. 2) A lactic acid content assay kit was used to investigate the ability of CLSCs to consume lactic acid. 3) A dissolved oxygen meter was used to monitor oxygen levels to investigate the ability of CLSCs to catalyze oxygen production. 4) A fluorescence spectrophotometer was used to investigate the ability of CLSCs to promote the production of reactive oxygen species under 660 nm light.

[0044] 1) Mapping analysis

[0045] 10 μL of 10.0 mg / mL CLSC solution was dropped onto a 200-mesh carbon-coated copper grid. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. Then 10 μL of ultrapure water was added. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. The elemental morphologies of C, N, O, S, and F were collected using a transmission electron microscope.

[0046] 2) Ultraviolet absorption spectrum

[0047] Ce6, SF, and CLSC solutions were prepared at 9.23, 13.08, and 250 μg / mL, respectively, and their absorption spectra were measured using a UV spectrophotometer (UKO T-U7S). The optimal detection wavelengths for Ce6 and SF were approximately 400 nm and 260 nm, respectively.

[0048] 3) Lactic acid consumption

[0049] 26 μg of free LOD and CLSC containing an equal amount of LOD were dissolved in 2 mL of PBS and placed in a dialysis bag sealed at both ends. The bag was placed in a 15 mL centrifuge tube containing 6 mL of sodium lactate solution (5 mg / mL) and incubated in a shaker at 37°C for 2 hours. Subsequently, 20 μL of the sodium lactate solution was collected from the centrifuge tube at 0, 10, 20, 30, 40, 60, and 90 minutes, and the lactate content was measured using a lactate assay kit.

[0050] 4) Oxygen production

[0051] 182 μg of free CAT and CLSC containing an equal amount of CAT were dispersed in a hypoxic H2O2 solution (250 μM, 10 mL) and stirred at room temperature for 15 min. The dissolved oxygen concentrations in the CAT and CLSC solutions were monitored using a dissolved oxygen meter (Orion Star A213) with readings every 5 seconds.

[0052] 5) Production of singlet oxygen

[0053] The fluorescence intensity change of the SOSG fluorescent probe at a detection wavelength of 530 nm was used as an indicator for the determination of singlet oxygen generation. The procedure was as follows: first, 2 mL of 100 μg / mL CLSC solution and 2 mL of 3.69 μg / mL Ce6 solution were prepared. 5 μL of SOSG fluorescent probe (500 μM) and 5 μL of H2O2 solution (100 mM) were added to the solutions respectively. The samples were then irradiated with a near-infrared laser (660 nm, 20 mW / cm 2 ), and the fluorescence intensity of SOSG in CLSC and Ce6 solutions was measured using a fluorescence spectrophotometer (PerkinElmer LS55) every 30 s.

[0054] The experimental results are as follows Figure 2 shown. Figure 2 a shows the overlapping distribution of elements C, N, O, F, and S in CLSC, indicating that CAT, LOD, SF, and Ce6 are encapsulated in CLSC; Figure 2 b shows that CLSC has characteristic absorption peaks of SF and Ce6 at wavelengths of 285 nm and 400 nm, respectively, indicating that SF and Ce6 are encapsulated in CLSC; Figure 2 c shows that CLSCs continuously consumed lactate, with the concentration decreasing from 57.2 mM to 28 mM within 90 min, which is similar to the free LOD; Figure 2 d shows that after adding CLSC, O2 in the H2O2 solution was slowly released, and the maximum O2 release amount was equal to that of free CAT, indicating that CLSC retained the catalytic performance of CAT; Figure 2 e shows that CLSC catalyzes the conversion of oxygen into 1 O2, indicating that CLSC retains the photosensitizer properties of Ce6.

[0055] Example 4

[0056] In vivo biodistribution of CLSCs

[0057] Small animal in vivo imaging was used to detect the enrichment of CLSCs in tumor sites and other organs after CLSCs were administered to mice via the tail vein.

[0058] The specific experimental methods are as follows:

[0059] 1) Three 5-6 week old female Balb / c mice were selected and injected subcutaneously on the left side of the back of each mouse with 2 x 10 5 After the inoculation of 4T1 breast cancer cells, the mice's condition and tumor growth were observed every 2 days.

[0060] 2) When the left tumor volume reaches 250mm 3 Around 2 h, mice were injected with CLSC (25 mg / kg) via tail vein. Then, at 0, 2, 4, 6, 8, and 12 h, the images were taken using a small animal in vivo imaging system ( Ce6 fluorescence signals were collected using a Lumina III, PerkinElmer (excitation: 670 nm; emission: 720 nm). At 12 h post-mortem, mice were euthanized, and liver, kidney, spleen, lung, and tumor tissues were collected. Ce6 fluorescence signals were also collected using a small animal in vivo imager (excitation: 670 nm; emission: 720 nm).

[0061] The experimental results are as follows Figure 3 shown. Figure 3Figures a and 3b show the accumulation of CLSCs in mouse tumors after administration. Quantitative analysis of the fluorescence signal reveals that the CLSC fluorescence signal first increases and then decreases over time. Ce6 fluorescence intensity reaches its maximum at 2 hours, indicating that 2 hours after administration is the optimal time for 660 nm illumination treatment. Figure 3 Figure c shows that CLSCs were clearly observed to accumulate in the tumor 12 hours after administration. This result suggests that CLSCs are significantly retained in the tumor, which is conducive to their anti-tumor effect.

[0062] Example 5

[0063] Effects of CLSC-assisted photodynamic therapy on breast cancer

[0064] A breast cancer model was used to evaluate the effect of CLSC treatment on tumor growth. First, a breast cancer mouse tumor model was established. Then, the tumor-bearing mice were treated with CLSCs. The inhibitory effect of CLSCs on breast cancer growth was evaluated by monitoring tumor volume and survival time in the mice.

[0065] The specific experimental methods are as follows:

[0066] 1) Twenty 5-6 week old female Balb / c mice were selected and housed in a conventional barrier environment.

[0067] 2) In healthy Balb / c mice, 2 x 10 5 4T1 breast cancer cells were used to observe the tumor growth of mice every 2 days.

[0068] 3) Wait until the tumor volume grows to 100mm 3 Mice were randomly divided into 4 groups: PBS, SF, CLSC, and CLSC(+). On days 0, 2, and 4 of the experimental period, mice in each group were injected with SF (1.30 mg / kg) and CLSC (25 mg / kg) via tail vein. 2 hours after administration, mice in the CLSC(+) group were illuminated with a 660 nm laser (0.5 W / cm 2 ) irradiate the tumor site for 5 minutes. Starting from day 0 of the experiment, the survival status of mice in each group was recorded every 2 days, and the tumor volume was calculated (tumor volume = 1 / 2 x the longest side of the tumor x the shortest side of the tumor x the shortest side of the tumor). At the same time, the tumor volume exceeded 1500mm 3 The end point of the experiment.

[0069] 4) After the experiment, the tumor tissues of each group of mice were collected, photographed and weighed

[0070] The experimental results are as follows Figure 4 shown. Figure 4a shows that compared with the PBS group, SF treatment had no significant inhibitory effect on mouse tumor growth, CLSC treatment had a slight inhibitory effect on tumor growth, and CLSC(+) treatment significantly inhibited the growth of breast cancer. Figure 4 bc showed that 18 days after administration, the average tumor weight of the CLSC(+) group was only 246.4 mg, which was 6.78, 5.70 and 4.34 times less than that of the PBS, SF and CLSC-treated groups, respectively. Figure 4 Figure d shows that CLSC(+) significantly prolonged the survival of tumor-bearing mice, with a median survival of 25.5 days after CLSC(+) treatment. In contrast, mice in the PBS, SF, and CLSC groups all reached the humane endpoint within 20 days. In summary, CLSCs synergized with photodynamic therapy to effectively inhibit breast cancer growth and achieved significant results.

[0071] Example 6

[0072] CLSCs synergistically induce anti-tumor immune responses through photodynamic therapy

[0073] Based on the significant inhibitory effect of CLSC synergistic photodynamic therapy on breast cancer growth, the mechanism of CLSC synergistic photodynamic therapy against tumor growth was further studied.

[0074] The specific experimental methods are as follows:

[0075] 1) Twelve 5-6 week old Balb / c female mice were selected and housed in a conventional barrier environment.

[0076] 2) In healthy Balb / c mice, 2 x 10 5 4T1 breast cancer cells were used to observe the tumor growth of mice every 2 days.

[0077] 3) Wait until the tumor volume grows to 100mm 3 Mice were randomly divided into 4 groups: PBS, SF, CLSC, and CLSC(+). On days 0, 2, and 4 of the experimental period, mice in each group were injected with SF (1.30 mg / kg) and CLSC (25 mg / kg) via tail vein. 2 hours after administration, mice in the CLSC(+) group were illuminated with a 660 nm laser (0.5 W / cm 2 ) irradiate the tumor site for 5 minutes.

[0078] 4) On day 5 of the experimental cycle, all mice were killed by dislocation, and the tumors were harvested and minced into 2-4 mm pieces. Single-cell suspensions of the tumors were obtained as follows:

[0079] a) Tumors were disaggregated into single-cell suspensions using a tumor dissociation kit (RWD DHTE-5001) and a single-cell suspension preparation instrument (RWD DSC-400).

[0080] b) Filter the cell suspension through a 70 μm cell sieve and centrifuge (450 g, 5 min).

[0081] c) Resuspend the cells in red blood cell lysis buffer, incubate at room temperature for 5 minutes, and centrifuge (450g, 5 minutes).

[0082] d) Stain with the live / dead dye FVS 510 (1x PBS, dilution ratio 1:1000) at room temperature in the dark for 15 minutes. Gently wash the cells three times with 1% FBS / PBS and centrifuge (450g, 5 minutes).

[0083] e) The cells were resuspended with rabbit anti-mouse CD16 / 32 antibody (diluted with 1% FBS / PBS, dilution ratio 1:100), incubated on ice for 10 min in the dark, and centrifuged (450 g, 5 min).

[0084] f) Cells were resuspended in rabbit anti-mouse CD45 (diluted with 1% FBS / PBS at a dilution ratio of 1:200), rabbit anti-mouse CD3 (diluted with 1% FBS / PBS at a dilution ratio of 1:200), rabbit anti-mouse CD4 (diluted with 1% FBS / PBS at a dilution ratio of 1:200), rabbit anti-mouse CD8 (diluted with 1% FBS / PBS at a dilution ratio of 1:200), and rabbit anti-mouse CD25 (diluted with 1% FBS / PBS at a dilution ratio of 1:200) antibodies, incubated on ice for 10 min in the dark, and centrifuged (450g, 5 min).

[0085] g) The cells were gently washed three times with 1% FBS / PBS, resuspended in 1% FBS / PBS, and analyzed by flow cytometry (BD FACS Aria II). + CD25 + ; effector cells (CD8 + T):CD3 + CD8 + .

[0086] The experimental results are as follows Figure 5 shown. Figure 5 a shows that the number of Treg cells in the tumor of the CLSC(+) group was significantly reduced, and the number of Treg cells decreased by 49.3% compared with the PBS group. Figure 5b shows a significant increase in the proportion of CD8+ T cells in CLSC(+) tumors, which was 10.6, 5.51, and 3.79 times higher than in PBS, SF, and CLSC tumors, respectively. Taken together, CLSC-assisted photodynamic therapy reduced the infiltration of immunosuppressive cells and increased the infiltration of immune effector cells. These findings support the concept that CLSC-assisted photodynamic therapy can reverse the tumor immunosuppressive microenvironment and activate antitumor immunity.

[0087] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification.

Claims

1. A method for preparing a self-assembled nanoformulation for initiating cancer immunity cycle, characterized in that: The steps include: The catalase solution was added to a sterile tube and placed in an ice bath. Lactate oxidase solution, dihydrochlorin e6 solution, and sorafenib solution were added under ultrasound. After thorough mixing, the mixture was centrifuged, washed, and dried to obtain a spherical nanoformulation.

2. The method for preparing the self-assembled nanoformulation for initiating cancer immunity cycle according to claim 1, characterized in that: The mass ratio of catalase, lactate oxidase, sorafenib and dihydrochlorin e6 is 6:1:0.43:0.

33.

3. The method for preparing the self-assembled nanoformulation for initiating cancer immunity cycle according to claim 2, characterized in that: The catalase solution, lactate oxidase solution, sorafenib solution and dihydrochlorin e6 solution were mixed and reacted under ultrasonic conditions for 15 minutes, and then the mixed solution was centrifuged at 4° C. and a centrifugal force of 15,000×g for 10 minutes, washed twice with ultrapure water and then freeze-dried.

4. Use of the self-assembled nanoformulation for initiating cancer immune cycle prepared according to any one of the preparation methods of claims 1 to 3 in the preparation of anti-tumor drugs.

Images