A kind of doxorubicin-codonopsis acetic acid composite tumor-targeting micelle and preparation method thereof

Abstract

The present invention belongs to the field of drug manufacturing, and specifically relates to a doxorubicin-telobranch acetic acid composite tumor-targeting micelle and a preparation method thereof. The micelle is self-assembled by combining doxorubicin and telobranch acetic acid with a polypeptide-modified amphiphilic polymer and other one or more amphiphilic materials as carriers. The composite tumor-targeting micelle of the present invention simultaneously loads doxorubicin and telobranch acetic acid, giving full play to the synergistic anti-cancer effect of chemotherapy drugs and angiogenesis inhibition drugs; the polypeptide tLyp-1 in tLyp-1-TPGS targets the surface of the polymer micelle, and the carrier material TPGS can resist multidrug resistance, further improving the therapeutic effect; the composite micelle has good targeting, can significantly improve the anti-tumor effect of doxorubicin, and reduce toxic side effects, and has a long-term stable therapeutic effect in vivo.

Description

Technical Field

[0001] The invention belongs to the field of drug manufacturing, and particularly relates to an adriamycin-codonopsis thunbergii acetic acid composite tumor-targeting micelle and a preparation method thereof. Background Art

[0002] Doxorubicin (DOX) is an anthracycline antitumor antibiotic. Its hydrochloride salt is commonly used clinically to increase its water solubility. It has a broad antitumor spectrum and is commonly used in the treatment of breast, lung, and ovarian cancers. However, its poor tissue selectivity and significant side effects limit its clinical use. Currently, drug delivery systems are primarily used to modify the biodistribution of doxorubicin and thereby reduce its toxic side effects. Polymeric micelles, in particular, hold great promise for application in cancer therapy and drug delivery.

[0003] The formation of tumor angiogenesis is the basis of tumor growth and metastasis. It mainly provides nutrients and oxygen for tumor growth and provides a path for its metastasis to other sites. Inhibiting tumor angiogenesis and blocking the supply of nutrients and oxygen is one of the important and effective options for tumor treatment. Pseudolaric acid B (PAB) is a diterpene acid isolated from the root bark of Pseudolaric acid, which is used in traditional Chinese medicine to treat skin fungal infections. Studies have reported that PAB has good antifungal, antiviral, antiangiogenic and antifertility activities. In addition, PAB has anticancer activity against a variety of tumor cells, such as gastrointestinal cancer cells, respiratory tumor cells and gynecological tumor cells. It is worth mentioning that studies have found that PAB has a significant inhibitory effect on angiogenesis, and can inhibit cancer cell metastasis and reverse the multidrug resistance of cancer cells.

[0004] D-alpha-Tocopheryl polyethylene glycol succinate (TPGS) is formed by esterifying vitamin E succinate (VES) with polyethylene glycol (PEG). It is a novel nonionic surfactant that forms stable micelles in aqueous media. As a P-gp inhibitor, it can effectively overcome multidrug resistance in tumor cells and improve the oral bioavailability of anticancer drugs. TPGS has been approved by the FDA as a safe pharmaceutical excipient and is widely used in drug delivery systems.

[0005] Researchers have discovered a class of tumor-homing peptides that bind to receptors on tumor cells, thereby enhancing the ability of drug delivery systems to release drugs from blood vessels and penetrate solid tumors. tLyP-1, with the amino acid sequence CGNKRTR, is one such tumor-homing peptide. It binds to the neurofibromin-1 (NRP-1) receptor, which is highly expressed on tumor cells such as breast cancer and gliomas, allowing it to enter solid tumors and penetrate tumor blood vessels.

[0006] As a commonly used anti-tumor drug in clinical practice, doxorubicin preparations have significant toxic side effects and multidrug resistance problems that need to be addressed urgently. Summary of the Invention

[0007] The present invention aims to address the deficiencies of the prior art by providing a doxorubicin-tetraacetic acid composite tumor-targeting micelle and its preparation method, effectively enhancing the anti-tumor effect of doxorubicin while reducing drug resistance and toxic side effects. The present invention synthesizes tLyp-1-TPGS and combines it with TPGS to prepare polymer micelles, which are loaded with DOX and the angiogenesis inhibitor PAB for targeted cancer therapy. By combining DOX and PAB, leveraging tLyp-1's ability to actively recognize tumor cells, and utilizing TPGS to combat multidrug resistance, the micelles achieve a trinity effect: enhancing the efficacy of doxorubicin while reducing toxic side effects.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A doxorubicin-pyraclostrobin complex tumor-targeting micelle, the micelle is formed by combining doxorubicin (DOX) and pyraclostrobin (PAB) with a carrier self-assembly, the carrier comprising a polypeptide-modified amphiphilic polymer and one or more amphiphilic materials. A schematic diagram of micelle formation is shown in FIG. Figure 1 .

[0010] Preferably, the polypeptide is tLyp-1, and the polypeptide-modified amphiphilic polymer is tLyp-1-modified vitamin E polyethylene glycol succinate TPGS (tLyp-1-TPGS); more preferably, the polypeptide-modified amphiphilic polymer is tLyp-1-modified TPGS2000 or TPGS1000.

[0011] Preferably, the amphiphilic material is vitamin E polyethylene glycol succinate TPGS and a phospholipid compound; more preferably, the amphiphilic material is a mixture of TPGS2000 or TPGS1000 and phosphatidylcholine (PC).

[0012] The present invention also provides a method for preparing any one of the aforementioned DOX-PAB composite tumor-targeting micelles, the method comprising the following steps:

[0013] A certain amount of DOX and PAB are respectively dissolved in an organic solvent, and the two solutions are mixed evenly. A certain amount of carrier is then added to the mixed solution, and the solvent is removed to obtain a dry drug-containing film. Finally, an appropriate amount of purified water consistent with the hydration temperature is added, and the mixture is stirred at the hydration temperature for a certain time for hydration. The obtained micellar solution is centrifuged and the supernatant is passed through a membrane to obtain the doxorubicin-tlyptothecin composite tumor-targeting micelles (tLyp-1-DP-M).

[0014] Preferably, the organic solvent is methanol, and the amount of methanol used is sufficient to dissolve the drug.

[0015] Preferably, the weight ratio of DOX to PAB is 1:0.8.

[0016] Preferably, in the micelle, the proportions of the components in parts by weight are 1 part of DOX, 0.8 parts of PAB, 3 parts of tLyp-1-TPGS, 7-9 parts of TPGS and 1-3 parts of phospholipid compounds.

[0017] Preferably, the amount of purified water used is calculated as (8-12) mL of water per 10 mg of the sum of the weight of TPGS and phospholipid compounds, the hydration temperature is 40-50°C, and the hydration time is 1-2 hours; more preferably, the amount of purified water used is calculated as 10 mL of water per 10 mg of the sum of the weight of TPGS and phospholipid compounds, the hydration temperature is 40°C, and the hydration time is 2 hours.

[0018] Preferably, the centrifugal speed is 10000 r / min and the centrifugal time is 15 min.

[0019] Preferably, the filter membrane is a 0.22 μm microporous filter membrane.

[0020] Preferably, the supernatant is passed through a membrane and then freeze-dried to obtain tLyp-1-DP-M freeze-dried powder.

[0021] Preferably, the freeze-drying method is to place the prepared micelles in a -80°C low-temperature refrigerator for pre-freezing for 12 hours, then take out the sample and place it in a freeze dryer for drying for 24 hours.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a doxorubicin-telobraxane acetic acid composite tumor-targeting micelle and a preparation method thereof. The targeted micelle simultaneously carries two drugs, giving full play to the synergistic anti-cancer effects of the chemotherapy drug and the angiogenesis-inhibiting drug; tLyp-1 targets and modifies the surface of the polymer micelle, and the carrier material TPGS can resist multidrug resistance, further improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the formation of doxorubicin-codonopsis acetic acid composite tumor-targeting micelles;

[0025] Figure 2 The graph shows the changes in average particle size and PDI of DP-M and tLyp-1-DP-M when diluted with PBS (n=3);

[0026] Figure 3 This is a graph showing the uptake of each group in MCF-7 cells detected by flow cytometry;

[0027] Figure 4 Figure 2 is a graph showing the changes in tumor volume of tumor-bearing mice in each group during the drug administration period (n=5);

[0028] Figure 5 Figures are the anatomy and weight of the tumors in each group of tumor-bearing mice (n=5);

[0029] Figure 6 These are the pathological sections of tumors in the tumor-bearing mice in each group;

[0030] Figure 7 Figure 2 is the relative body weight changes of tumor-bearing mice in each group during the administration period (n=6);

[0031] Figure 8 Pathological sections of the heart, liver, spleen, lung, and kidney of tumor-bearing mice in each group;

[0032] Figure 9 Figure 2 is the expression of CD31 in tumors of tumor-bearing mice in each group (SP, ×400);

[0033] Figure 10 Statistical graph of MVD of tumors in tumor-bearing mice in each group (SP, ×400). DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0035] Example 1 Preparation of doxorubicin-codonopsis acetic acid composite tumor-targeting micelles

[0036] (2) Doxorubicin hydrochloride was desalted with triethanolamine to prepare a methanol solution containing 1 mg of doxorubicin and 0.8 mg of PAB. A methanol solution containing 8 mg of TPGS1000, 2 mg of PC, and 3 mg of tLyp-1-TPGS2000 was then added to the mixed methanol solution. The solvent was then removed by rotary evaporation in a 25°C water bath to obtain a dry drug-containing film. Finally, 10 mL of purified water at 40°C was added and the mixture was magnetically stirred for hydration at 40°C for 2 h. The resulting micelle solution was centrifuged at 10,000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain doxorubicin-tlyptic acid composite tumor-targeting micelles (tLyp-1-DP-M). The micelles were pre-frozen in a -80°C low-temperature refrigerator for 12 h, then taken out and placed in a freeze dryer for drying for 24 h to obtain tLyp-1-DP-M lyophilized powder.

[0037] The micelle size in aqueous solution was measured using a Malvern laser particle size analyzer to be (128.4±0.79) nm, with a PI value of 0.27 and a zeta potential of (22.8±0.15) nm. UV-light analysis of DOX content and HPLC analysis of PAB content revealed an encapsulation efficiency of 97.67% (DOX) and 96.89% (PAB) for tLyp-1-DP-M, and drug loadings of 7.02% (DOX) and 5.82% (PAB).

[0038] Example 2 Preparation of doxorubicin-codonopsis acetic acid composite tumor-targeting micelles

[0039] When preparing doxorubicin-codonopsis acetic acid composite tumor-targeting micelles, TPGS1000 was 7 mg, PC was 3 mg, the hydration temperature was 40° C., the hydration volume was 10 mL, the hydration time was 1 h, and the remaining operations were the same as in Example 1.

[0040] Example 3 Preparation of doxorubicin-codonopsis acetic acid composite tumor-targeting micelles

[0041] When preparing doxorubicin-codonopsis acetic acid composite tumor-targeting micelles, TPGS1000 was 9 mg, PC was 1 mg, the hydration temperature was 50° C., the hydration volume was 8 mL, the hydration time was 1.5 h, and the remaining operations were the same as in Example 1.

[0042] Example 4 Preparation of peptide-free doxorubicin-dapoxetine complex micelles (DP-M) The preparation was carried out according to the method of Example 1, except for the step of not adding tLyp-1-TPGS2000.

[0043] The present invention demonstrates the beneficial effects of the present invention through the following test examples:

[0044] Experimental Example 1 Physicochemical Properties of Doxorubicin-Acetic Acid Composite Tumor-Targeting Micelles of the Present Invention

[0045] (1) Particle size and potential

[0046] A solution of tLyp-1-DP-M and a solution of DP-M were placed in a cuvette, filling approximately two-thirds of the cuvette volume. Air bubbles were expelled, and the solution was placed in a laser particle size analyzer for particle size measurement. A solution of DP-M and a solution of tLyp-1-DP-M were placed in a potentiometric cup, ensuring that both sides of the solution were higher than the metal sheet within the U-shaped groove. Air bubbles were expelled, and the solution was placed in a laser particle size analyzer for potential measurement. The measured particle size and potential were as follows: The DP-M particle size was (120.3±0.54) nm, with uniform distribution, and the potential was (0.58±0.21) mV; the tLyp-1-DP-M particle size was (128.4±0.79) nm, with uniform distribution, and the potential was (22.8±0.15) mV. tLyp-1-DP-M undergoes a significant change in potential due to the modification of tLyp-1 peptide on the surface of the micelles. The positively charged properties of the micelles bind better to the negatively charged cell membrane, indicating that the cells have a higher uptake rate of the drug.

[0047] (2) Stability investigation

[0048] The prepared tLyp-1-DP-M and DP-M solutions were mixed with pH 7.4 PBS buffer at a volume ratio of 1:3, mixed evenly, and incubated in a 37°C constant temperature oscillator. Samples were taken at 0, 6, 12, 24, 36, and 48 hours, and the particle size and PDI value of the diluted micelles were measured. The results are shown in Figure 2 As can be seen from the figure, although the particle size of DP-M and tLyp-1-DP-M increased slightly, it remained almost unchanged, and the PDI of both was less than 0.3. The experimental results show that the prepared polymer micelles can be stably present in vivo.

[0049] Experimental Example 2 Tumor Cytotoxicity Experiment of Doxorubicin-Acetic Acid Composite Tumor Targeting Micelles of the Present Invention

[0050] (1) Preparation of drug concentrations in different prescription preparations

[0051] Accurately weigh tLyp-1-DP-M and DP-M micellar lyophilized powders, dissolve in purified water, and prepare a 1 mg / mL solution (calculated based on DOX content). Filter through a 0.22 μm sterile filter membrane. Dilute with RPMI-1640 culture medium to DOX concentrations of 12.5, 6.25, 1.25, 0.25, 0.05, and 0.01 μg / mL for toxicity testing in MCF-7 cells. The DOX API and PAB were first dissolved in a small amount of DMSO and then prepared in RPMI-1640 culture medium to the same concentration as the above-mentioned DOX concentration. The physical mixed solution of PAB and DOX (D+P) was prepared by mixing the two at a molar ratio of 1:1 and diluting them in RPMI-1640 culture medium to 12.5, 6.25, 1.25, 0.25, 0.05, and 0.01 μg / mL (calculated based on the DOX content), ensuring that the final solution contained less than 0.1% DMSO.

[0052] (2) MTT assay to detect toxicity

[0053] Take MCF-7 cells in the logarithmic growth phase, remove the old culture medium and rinse the cells 2-3 times with PBS buffer at pH 7.4. After trypsin digestion, terminate the digestion with RPMI-1640 culture medium containing 10% fetal bovine serum. Place the cell suspension after pipetting in a centrifuge at 1000 rpm for 5 minutes, then add an appropriate amount of culture medium and pipette evenly. Count the diluted cell suspension using a hemocytometer, and then count the MCF-7 cells at 4×10 3 Cells were seeded at a concentration of 100 μL cells / mL in a 96-well plate. 100 μL of PBS was added around the periphery of the plate to reduce experimental error. The plate was incubated at 37°C, 5% CO2, and saturated humidity for 24 hours to allow the cells to adhere. Then, 100 μL of the prepared drug solution was added to each well. Four replicates were set up for each concentration. A control group received 100 μL of blank culture medium, while a blank group received only 200 μL of cell-free culture medium. After 48 hours of incubation in the same incubator, 20 μL of 5 mg / mL MTT solution was added to each well in the dark, and the plate was incubated in the incubator for another 4 hours. After the incubation period, the supernatant was carefully removed from each well using a pipette tip, and the plate was inverted onto filter paper to remove any residual solvent. Then, 150 μL of DMSO was added to each well and the plate was shaken on a horizontal shaker for 15 minutes to completely dissolve the purple crystals. Finally, place the 96-well plate in a microplate reader, observe the absorbance of each well at a wavelength of 492 nm, and calculate the IC of each group. 50 , the results are shown in Table 1.

[0054] Table 1 Inhibitory effect of each group on the growth of MCF-7 cells (n=4)

[0055]

[0056] The results of cytotoxicity experiments showed that DP-M was more toxic than DOX, and it was speculated that the carrier material TPGS played an anti-drug resistance role. At the same time, tLyp-1-DP-M was the most toxic to MCF-7 cells. It was speculated that this might be due to the adsorption effect between the positive charge on the surface of tLyp-1-DP-M and the negative charge on the cell surface, which was more conducive to cell uptake. It might also be because tLyp-1 can actively recognize receptors on the surface of MCF-7 cells, thereby improving cell uptake.

[0057] Experimental Example 3 Tumor cell uptake experiment of the adriamycin-tauracenic acid composite tumor-targeting micelles of the present invention

[0058] Flow cytometry was used to quantitatively investigate the uptake of DOX, a physical mixture of DOX and PAB (D+P), DP-M, and tLyp-1-DP-M by tumor cells. MCF-7 cells in the logarithmic growth phase were trypsinized and counted, and the cell suspension was adjusted to 3×10 5 Cells were seeded into 6-well plates at a density of 10 cells / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. Once the cells had attached to the plate, the culture medium was removed with a pipette tip and rinsed twice with PBS (pH 7.4). 2 mL each of DOX, D+P, DP-M, and tLyp-1-DP-M solutions diluted in serum-free culture medium was added to each well (final DOX concentration: 10 μg / mL) and the plates were placed in the incubator for incubation. After 2 hours of incubation, the drug-containing culture medium was removed from each well and rinsed two to three times with PBS at 4°C. Then, 400 μL of 0.25% trypsin was added to each well. After digestion for 2 to 3 minutes, the cells gradually became spherical and began to float and detach from the plate under a microscope. Fresh culture medium supplemented with 10% serum was immediately added to terminate digestion. The cells were evenly transferred to a centrifuge tube by pipetting and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. Finally, resuspend the cells in 400 μL of 4°C PBS per well. The cell suspensions were then added to each well of a 96-well plate and analyzed using an analyzer. Signals were collected on the FL2 channel. DOX uptake was measured in 10,000 MCF-7 cells in triplicate (DOX excitation wavelength: 486 nm, emission wavelength: 593 nm).

[0059] The fluorescence intensity peak of cells without drug treatment was taken as blank control, and the corresponding fluorescence intensity peaks were obtained after treatment of each group of samples. Figure 3As shown in the figure. As can be seen from the figure, within the 2-hour uptake period, there was no statistical difference in the amount of doxorubicin taken up by cells in the DOX and D+P groups (p>0.05). There was a significant difference between the DP-M group and the D+P group (p<0.05). It is speculated that the DP-M group contains TPGS1000, which has an efflux inhibitory effect and can increase the uptake of doxorubicin. The uptake fluorescence of MCF-7 cells in the tLyp-1-DP-M group was significantly higher than that in the DP-M group (p<0.01), proving that the addition of tLyp-1 can specifically target MCF-7 cells and further increase the uptake of doxorubicin by MCF-7 cells.

[0060] Experimental Example 4: Study on the in vivo targeting and efficacy of doxorubicin-codonopsis acetic acid composite tumor-targeting micelles

[0061] In vivo imaging was used to investigate the tissue distribution and targeting of tLyp-1-DP-M in MCF-7 tumor-bearing mice. In vivo pharmacodynamic studies were then conducted, using tail vein injection to examine the inhibitory effects of DOX, D+P, DP-M, and tLyp-1-DP-M on nude mouse tumors. Changes in tumor volume and body weight were used as indicators, and immunohistochemistry and histopathological analysis of nude mouse tissues were used to evaluate the efficacy and side effects of the formulations.

[0062] Establishment of tumor model in tumor-bearing mice: The night before inoculation of MCF-7 cells, fresh culture medium was replaced overnight. When the cell density in the culture dish reached 80% to 90%, the culture medium was aspirated and the cells were rinsed 2 to 3 times with PBS buffer (pH 7.4). Then, 0.25% trypsin was added for digestion and counting. After centrifugation, the supernatant was discarded and the cells were resuspended in PBS solution at 4°C to adjust the cell density to 5×10 7 200 μL of inoculation solution was injected subcutaneously under the left forelimb arm of each nude mouse, that is, the number of cells inoculated per nude mouse was 10 7 After the injection, the needle was pulled out in a zigzag pattern to prevent leakage of cell fluid. Under normal feeding conditions, the tumor volume grew to 100-120 mm. 3 When the model is established, it can be considered successful and the next experiment can be started.

[0063] Investigation of the targeting properties of micelles in vivo:

[0064] (1) In vivo imaging

[0065] In vivo imaging was used to demonstrate the targeting ability of tLyp-1-DP-M micelles. Successfully established MCF-7 tumor-bearing nude mice were divided into three groups: DOX, DP-M, and tLyp-1-DP-M. Each group received a 200 μL tail vein injection of a 5 mg / kg DOX solution. In vivo imaging was performed at five time points: 1, 3, 6, 8, and 24 hours. Two minutes before imaging, the mice were rapidly anesthetized with fluoxetine and monitored using an imaging system at an emission wavelength of 593 nm. Autofluorescence was observed in the tumors in the DOX group 1 hour after administration. Systemic distribution of the drug was evident by 3 hours, but the fluorescence intensity significantly decreased by 24 hours, with irregular changes over time. Doxorubicin accumulated in the tumors in small amounts and remained there for a short time. It was then dispersed through the bloodstream to other tissues or excreted through metabolism, confirming the poor tissue distribution of the doxorubicin solution. In both the DP-M and tLyp-1-DP-M groups, after intravenous injection of the micelle solution, the fluorescence intensity at the tumor site of nude mice initially increased and then slowly decreased. In the DP-M group, the fluorescence intensity at the tumor site reached its peak at 6 hours and gradually decreased after 24 hours. However, in the tLyp-1-DP-M group, the fluorescence intensity at the tumor site remained strong after 24 hours. These results demonstrate that the tLyp-1-DP-M micelles are targeted after in vivo administration, effectively accumulating at the tumor site. Furthermore, the micelles slowly release the drug within the tumor, allowing the drug to remain active even after 24 hours.

[0066] (2) Fluorescence imaging of tissues and organs

[0067] After 24 hours of in vivo imaging, nude mice in the DOX, DP-M, and tLyp-1-DP-M groups were sacrificed by cervical dislocation and rapidly dissected in a cleanroom. Heart, liver, spleen, lung, kidney, and tumor tissues were removed. The removed tissues, organs, and tumors were then washed in saline, dried on filter paper, and placed sequentially on black plastic plates for imaging. An exposure time of 400 ms was maintained, ensuring consistent exposure time at each time point. The results showed that DOX was prominently distributed in the liver, with no fluorescence observed in the tumor. Fluorescence in the DP-M micelle group was observed 24 hours after administration, primarily in the liver, with some fluorescence also detected in the tumor site. No fluorescence was observed in other organs, indicating that DP-M also has toxic effects on the liver. However, in the tLyp-1-DP-M micelle group, fluorescence was concentrated in the tumor site, with no fluorescence detected in other organs. This phenomenon further confirms the efficient tumor targeting of the micelles.

[0068] In vivo pharmacodynamic study:

[0069] (1) Tumor growth inhibition

[0070] The MCF-7 tumor-bearing nude mice successfully modeled above were randomly divided into 5 groups: Saline, DOX, D+P, DP-M, and tLyp-1-DP-M groups, with 5 mice in each group. The drugs were administered once every three days at a concentration of DOX 10 mg / kg and PAB 7.5 mg / kg, for four consecutive times. At the same time, each nude mouse was weighed every other day, and the longest diameter (A) and shortest diameter (B) of the tumor were measured using a vernier caliper. Two hours after administration on the last day, the nude mice were killed by cervical dislocation, and the intact subcutaneous tumor was removed and weighed. The nude mouse tumor volume-time and body weight-time change curves were drawn. The calculation formulas for tumor volume and tumor inhibition rate are as follows:

[0071]

[0072] Where, V: tumor volume

[0073] A: Maximum diameter of the tumor

[0074] B: Minimum diameter of the tumor

[0075]

[0076] from Figure 4 It can be seen that the relative volume change of the tumor in the nude mice in the normal saline group was the most obvious, and the tumor growth rate was not affected. On the 21st day after inoculation, the tumor volume had grown to 6.2 times that of the 11th day. The growth rate of the tumor in the micelle group was slower, and the relative volume change of tLyp-1-DP-M was the smallest, and its tumor inhibition effect was the strongest. After the last administration of the experiment, the nude mice in each group were killed, and the tumor tissues were removed and weighed, and the tumor inhibition rates of the DOX group, D+P group, DP-M group and tLyp-1-DP-M group were calculated. The tumor inhibition rates of each group were 18.8%, 36.0%, 52.8% and 76.2%, respectively. Figure 5 It can be seen that the tumor volume in the saline group was the largest, while that in the tLyp-1-DP-M group was the smallest, indicating the most significant inhibitory effect. On day 21 after inoculation, i.e., day 11 after administration, the tumor weights in the tLyp-1-DP-M group were 23.7% (P < 0.001) of those in the saline group, 29.3% (P < 0.01) of those in the DOX group, 35.3% (P < 0.001) of those in the D+P group, and 50.4% (P < 0.05) of those in the DP-M group. Tumor growth in the DOX, D+P, and DP-M groups was also inhibited to a certain extent, reaching 81.2% (P > 0.05), 67.3% (P < 0.01), and 47.2% (P < 0.01) of those in the saline group, respectively. This indicates that the tLyp-1-DP-M group has the most significant inhibitory effect on nude mouse tumor growth. There was also a significant difference in tumor size between the D+P group and the DOX group (P < 0.05), indicating that doxorubicin and tricholoma acetic acid have a combined anti-tumor effect.

[0077] (2) Histological investigation

[0078] The histological pathological characteristics of the tumor were observed through HE staining sections. The method for making paraffin sections is as follows: after the nude mice were killed, the complete heart, liver, spleen, lung, kidney tissues and tumors were dissected out and placed in a 50mL centrifuge tube, and 4% paraformaldehyde was added for immersion and fixation. Take it out the next day, put it in a dehydration box, and put the dehydration box into a dehydrator for gradient alcohol dehydration, and then put the above-treated tumors and tissues into the embedding machine for embedding. After embedding, cut it into slices with a thickness of 4μm, and finally stain it with hematoxylin-eosin and seal it with neutral gum. Place the prepared tumor tissue slices under a microscope, observe the conditions of each tissue part and take pictures for record. From Figure 6 As can be seen in the saline group, tumor cells showed large, darkly stained nuclei, a high cell density, and a small amount of cytoplasm. In the DOX and D+P groups, tumor nuclei began to shrink and the cytoplasm increased. This phenomenon was also observed in the DP-M and tLyp-1-DP-M groups, but was even more pronounced. In the tLyp-1-DP-M group, nuclei further shrank, becoming almost invisible in the field of view and exhibiting large gaps. These results demonstrate that tLyp-1-DP-M has the strongest anti-tumor effect.

[0079] Toxic and side effect evaluation:

[0080] Within 11 days of administration, the body size and weight of the nude mice in the control group and the treated groups were observed, and they were weighed every other day. Their excrement and coat color were observed. At the same time, HE staining experiments were used to further evaluate the toxicity of tLyp-1-DP-M on various major organs.

[0081] The changes in body weight of nude mice in the blank control group and each drug-treated group during the drug-treated period are as follows: Figure 7 As shown. The body weight of the nude mice in the normal saline group changed most significantly, showing a continuous upward trend. At the end of the experiment, the body weight of the nude mice was 1.23±0.06 times the body weight on the first day. In the tLyp-1-DP-M group, the body weight first increased within the first 5 days and then showed a slight downward trend. The overall body weight showed a slight upward trend. Similarly, the relative body weight of the DP-M group increased slightly within the first 5 days, and then the change was not obvious. The tumor-bearing mice in the DOX and D+P groups lost a significant amount of body weight, and the fur color of the nude mice became dull and some died during the drug administration period. The above results all indicate that the raw material drug doxorubicin has great toxic side effects, and the preparation of tLyp-1-DP-M micelles can reduce the toxic side effects of doxorubicin.

[0082] Histopathological sections of nude mice in each drug-treated group and blank control group are shown in Figure 2. Figure 8. As can be seen from the figure, compared with normal saline, the cardiomyocytes of nude mice in the DOX group and the D+P group were damaged, the myocardial fibers were broken, and the intermuscular spaces were widened, while there was no obvious damage in the DP-M and tLyp-1-DP-M groups. Based on this result, it can be inferred that the raw material drug DOX encapsulated in micelles can reduce the cardiotoxicity of doxorubicin. In the liver pathological sections, granulomas were formed on the liver tissue of the DOX group, indicating that it is very toxic to the liver. A small amount of granulomas were also formed in the D+P group, indicating that the combined administration of doxorubicin and acetic acid can reduce the toxicity to the liver. In the spleen pathological sections, the tissue morphology of the DP-M, tLyp-1-DP-M groups was similar to that of the normal saline group, with clear tissue structure, clear red pulp, white pulp and edges, and no spleen toxicity was shown. The tissue sections of the DOX group and the D+P group were whitish, indicating that there were more apoptotic cells inside, indicating that doxorubicin also has certain toxicity to the spleen. Lung sections in the DP-M and tLyp-1-DP-M groups appeared identical to those in the saline group, with intact cells and complete staining, indicating no evidence of lung toxicity. However, tissue damage and incomplete staining were observed in the DOX and D+P groups. Renal pathology images showed no significant differences between the drug-treated and control groups, indicating no significant renal toxicity.

[0083] Immunohistochemical examination of tumor tissue:

[0084] (1) Experimental methods and steps

[0085] Tumor sections were processed using the conventional immunohistochemical SP method (streptavidin-peroxidase conjugate method) and developed with DAB. After dehydration at various levels of alcohol, the sections were mounted with neutral resin and then lightly counterstained with hematoxylin. The sections were then dehydrated, cleared, dried, and mounted. CD31 protein expression was examined under an optical microscope, with the intensity of the brownish-yellow color as the observation indicator. The results are shown in Figure 2. Figure 9 .

[0086] (2) Methods for microvessel counting

[0087] CD31, also known as platelet-endothelial cell adhesion molecule, is mainly present on the surface of platelets, neutrophils, monocytes and certain types of T cells, as well as in tight junctions between endothelial cells. It is likely involved in leukocyte migration, angiogenesis and integrin activation. In immunohistochemistry experiments, CD31 is mainly bound to the CD31 protein on vascular endothelial cells, and is brown in color. It is used to evaluate tumor angiogenesis in vascular endothelial tissue. The determination of MVD refers to the proofreading counting method of Weidner et al. First, observe the entire section under a low-power microscope to find three areas of high vascular density, namely "hot spots"; then count the number of blood vessels that are positively stained under a 200-fold light microscope. The identification of microvessels does not require a complete lumen and red blood cells. As long as there is obvious staining of vascular endothelial cells and it can be separated from adjacent blood vessels, tumor cells and interstitial components, it can be regarded as an independent blood vessel. Vascular vessels that are unclear and have a thicker muscle layer are not included in the count; the average number of blood vessels in the three fields of view is counted as the MVD value (pieces / HP). Results are shown in Figure 10 .

[0088] Tumor angiogenesis is an important condition for tumor growth, invasion and metastasis. Currently, the microdensity of tumor new blood vessels is mostly detected by using the CD31 vascular endothelial marker. Figure 9 It can be observed that CD31 protein is strongly expressed in both microvessels and macrovessels in the tumors of the saline group, appearing as brown-yellow lines, with an MVD value of 30.0±2.6. In the tumors of the DOX group, both macrovessels and microvessels are expressed, with an MVD value of 18.7±3.0, which is significantly lower than that of the saline group (P<0.05). However, in the D+P group, the tumor vascular density was significantly reduced compared with the DOX group (P<0.05), while there was no significant difference in microvessel density among the three groups of tumors, D+P, DP-M, and tLyp-1-DP-M. Expression was only observed in microvessels, and no obvious staining was observed in macrovessels. Based on the experimental results, it is speculated that PAB has an inhibitory effect on angiogenesis, thereby inhibiting tumor growth.

[0089] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. A doxorubicin-codonopsis acetic acid composite tumor-targeting micelle, characterized in that: The micelles are formed by combining doxorubicin (DOX) and arbutin acetate (PAB) and carrier self-assembly. The carrier includes a polypeptide-modified amphiphilic polymer, and the carrier also includes multiple amphiphilic materials. The polypeptide-modified amphiphilic polymer is tLyp-1-modified polyethylene glycol vitamin E succinate TPGS (tLyp-1-TPGS), and the amphiphilic materials are polyethylene glycol vitamin E succinate (TPGS) and lecithin. The amount of each component in the micelles is recorded by weight as 1 part of DOX, 0.8 part of PAB, 3 parts of tLyp-1-TPGS, 7-9 parts of TPGS, and 1-3 parts of lecithin. The preparation method of the micelles includes the following steps: Doxorubicin and tiliacetic acid are dissolved in organic solvents respectively, and the two solutions are mixed evenly. A certain amount of carrier is added to the mixed solution, and the solvent is removed to obtain a dry drug-containing film. Finally, an appropriate amount of purified water consistent with the hydration temperature is added, and the mixture is stirred at the hydration temperature for a certain time for hydration. After the obtained micelle solution is centrifuged, the supernatant is taken and passed through a membrane to obtain the doxorubicin-tiliacetic acid composite tumor-targeting micelles.

2. The doxorubicin-codonopsis acetic acid composite tumor-targeting micelle according to claim 1, characterized in that: The TPGS is TPGS1000 or TPGS2000.

3. A method for preparing the adriamycin-codonopsis acetic acid composite tumor-targeting micelles according to claim 1 or 2, characterized in that: The steps include: Doxorubicin and tiliacetic acid are dissolved in organic solvents respectively, the two solutions are mixed evenly, a certain amount of carrier is added to the mixed solution, the solvent is removed to obtain a dry drug-containing film, and finally an appropriate amount of purified water consistent with the hydration temperature is added, and the mixture is stirred at the hydration temperature for a certain time to hydrate. The obtained micelle solution is centrifuged and the supernatant is filtered through a membrane to obtain the doxorubicin-tiliacetic acid composite tumor-targeting micelles; The amounts of the components added are, in parts by weight, 1 part of DOX, 0.8 parts of PAB, 3 parts of tLyp-1-TPGS, 7-9 parts of TPGS, and 1-3 parts of lecithin.

4. The method for preparing a doxorubicin-codonopsis acetic acid composite tumor-targeting micelle according to claim 3, characterized in that: The amount of purified water used is calculated as 8 to 12 mL of water for every 10 mg of TPGS and lecithin, the hydration temperature is 40 to 50° C., and the hydration time is 1 to 2 hours.

5. The method for preparing the adriamycin-codonopsis acetic acid composite tumor-targeting micelles according to claim 3, characterized in that: The organic solvent is methanol.

6. The method for preparing the adriamycin-codonopsis acetic acid composite tumor-targeting micelles according to claim 3, characterized in that: The centrifugal speed is 10000 r / min, the centrifugal time is 15 min, and the membrane filter is a 0.22 μm microporous membrane.

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