Docetaxel-loaded retinoic acid derivative polymer micelle and preparation method thereof

Abstract

The invention discloses a docetaxel-loaded retinoic acid derivative polymer micelle and a preparation method thereof, and belongs to the technical field of biological medicines. The polymer micelle is prepared from docetaxel and a retinoic acid derivative polymer mPR. The preparation method comprises the following steps: mixing and dispersing the polymer mPR and docetaxel into a drug film, and hydrating with a proper medium to prepare the polymer micelle. The docetaxel polymer micelle disclosed by the invention is high in encapsulation efficiency, high in drug loading capacity, high in stability and relatively good in curative effect and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a docetaxel-loaded retinoic acid derivative polymer micelle and its preparation method. Background Technology

[0002] The extremely low water solubility of docetaxel (DTX) (<10 μg / mL) greatly limits its clinical application. To overcome the limitations caused by low water solubility, suitable dosage forms need to be developed. Currently, docetaxel dosage forms include liposomes, lipid nanoparticles, albumin nanoparticles, inorganic nanoparticles, and electrospun fibers. Furthermore, the structure of the docetaxel molecule can be modified to prepare prodrugs or conjugate micelles.

[0003] Traditional docetaxel injection requires polysorbate 80 and ethanol as solubilizers, which is one of the reasons for many adverse reactions. Liposome formulations, by encapsulating the drug in a phospholipid bilayer, fundamentally avoid the use of these excipients. Studies have shown that liposomes are superior to traditional formulations in terms of the incidence of adverse reactions such as peripheral neurotoxicity, dyspnea, and rash. Furthermore, with appropriate surface modification, they can achieve long circulation and active targeting effects.

[0004] Currently, common processes in liposome preparation include thin-film dispersion, reverse-phase evaporation, secondary emulsification, and ultrasonic dispersion. These traditional processes often introduce organic solvents, posing challenges to formulation safety. Furthermore, the resulting liposomes exhibit unstable particle size and encapsulation efficiency, potentially leading to instability in in vivo behavior and drug waste. Many methods that perform well in the laboratory stage face challenges in large-scale production due to poor process reproducibility, low yield, and high cost. Among the authorized nanoliposome patents, only paclitaxel and doxorubicin nanoliposomes have been successfully translated into clinical applications; most nanoliposomes have not yet achieved effective translation. Novel liposome preparation processes include hot or cold high-pressure homogenization, supercritical CO2 fluid technology, and microfluidic technology, but these involve high equipment investment costs; they may face poor process reproducibility during scale-up; and yields are relatively low. The complex phospholipid components in liposome formulations exacerbate the chemical instability of the formulation, posing challenges to the content determination of related substances, and existing liposomes have low drug loading capacities.

[0005] Albumin can be preferentially taken up by tumor or inflamed tissues and has low toxicity and low immunogenicity, making it an ideal carrier for drug delivery. Celgene's docetaxel albumin nanoparticles ABI-008 are prepared using the same method as the already marketed paclitaxel albumin nanoparticles Abraxane. The specific process involves dissolving high concentrations of docetaxel and albumin in organic solvents dichloromethane and water, respectively, as the oil phase and aqueous phase. The two phases are mixed and homogenized to form a coarse emulsion. The emulsification process is repeated several times using a high-pressure homogenizer to form an extremely fine oil-in-water emulsion. Subsequently, the dichloromethane is rapidly evaporated under reduced pressure in a rotary evaporator to obtain a colloidal suspension composed of nanoparticles. Further freeze-drying yields the lyophilized powder for injection. This preparation method is currently the most widely used, but it still has many limitations: It involves multiple operations such as homogenization emulsification, high-pressure homogenization emulsification, and vacuum evaporation, resulting in a long preparation cycle, complex process flow, and numerous process parameters requiring strict control. Furthermore, the equipment involved has a low throughput and is expensive, making it difficult to guarantee high batch-to-batch consistency in large-scale production. The introduced organic solvent dichloromethane residue must be strictly controlled, otherwise it can lead to severe liver, kidney, lung, and neurotoxicity. Currently, bovine serum albumin (BSA), widely used in albumin nanoparticle preparation, may trigger immunogenic reactions, and naturally derived albumin may exhibit significant batch-to-batch variability, posing challenges to quality control. Recombinant human serum albumin (rHSA), through genetic engineering, has an amino acid sequence highly consistent with natural human serum albumin, effectively reducing immunogenicity and batch-to-batch variability, but its cost is relatively high.

[0006] Polymer micelle technology has become a key solution due to its unique nanoscale drug delivery advantages. However, current polymer micelles generally have low drug loading capacity, wide particle size distribution, poor stability, and must be used within 4 hours after preparation. Furthermore, they cannot reverse multidrug resistance in tumors, and their efficacy has not been significantly improved. Based on this, this invention designs a method to prepare docetaxel polymer micelles using retinoic acid derivatives as a carrier. Summary of the Invention

[0007] One of the objectives of this invention is to provide docetaxel micelles with a narrow particle size distribution, high drug loading, good stability, and long retention time at the tumor site.

[0008] The second objective of this invention is to provide a method for preparing the above-mentioned polymer micelles, and to screen preparation process conditions including film-forming solvent, rotary evaporation temperature, and drug loading ratio.

[0009] A third objective of the present invention is to provide the drug delivery performance of the amphiphilic polymer micelles.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A docetaxel micelle nanomedicine is made of docetaxel and polymer mPR, wherein the mass ratio of docetaxel to polymer mPR is 1:2-1:3; The structural formula of the polymer mPR is as follows: n=1000.

[0011] Preferably, the mass ratio of docetaxel to polymer mPR is 1:2.4.

[0012] The preparation method of the above-mentioned docetaxel micelle nanomedicine is as follows: docetaxel and polymer mPR are mixed and dispersed into a drug film and then hydrated to obtain polymer micelles.

[0013] Preferably, the docetaxel and polymer mPR are mixed with a solvent to form a drug film, which is then rotary evaporated. The solvent is selected from acetonitrile, methanol, ethanol, and chloroform; the rotary evaporation temperature is 33-40°C.

[0014] Specifically, mPR compound and DTX are precisely weighed into a round-bottom flask according to a specific drug loading ratio, dissolved in an appropriate solvent, and the organic solvent is removed by rotary evaporation under reduced pressure at a specific temperature, resulting in a uniform drug film adhering to the flask wall. An appropriate hydration medium is then added to the round-bottom flask and gently shaken to obtain a polymer micelle dispersion with an opalescent appearance. The solvents include acetonitrile, methanol, ethanol, and chloroform; the drug loading ratios (w / w) include 1:3, 1:2.5, 1:2.4, 1:2.2, and 1:2; the rotary evaporation temperatures include 40, 37, 35, and 33°C; and the hydration media include ultrapure water, physiological saline, and PBS buffer (pH=7.4).

[0015] The above-mentioned application of docetaxel micelle nanomedicines in the preparation of tumor therapeutic drugs.

[0016] This invention utilizes retinoic acid derivative mPR and, through formulation process screening, determined the optimal formulation process: the film-forming solvent is ethanol, the drug carrier is 1:2.4 (w / w), the rotary evaporation temperature is 35℃, and the hydration medium is physiological saline. This process yields a docetaxel polymer micelle with high encapsulation efficiency, high drug loading, high stability, and good efficacy and safety.

[0017] The mPR-DTX polymer micelles prepared according to the formulation process selected in this invention have an average encapsulation efficiency of 99.67%, a drug loading of 29.42%, and an average particle size of 63.62 nm, with a relatively concentrated particle size distribution, indicating good reproducibility of the optimal preparation process. In in vitro release experiments, the drug is rapidly released within the first 6 hours, followed by slow release over the next 42 hours, with a cumulative release of approximately 70% within 48 hours. Within 12 hours of incubation at 25°C, the particle size of the prepared micelles fluctuated within a small range, showing a slight increase, but not significantly. Within 10 hours, the encapsulation efficiency of the formulation was above 95%, indicating good in vitro stability of the micelles prepared by the optimal formulation process. Furthermore, compared to the traditional DTX formulation Taxotere, mPR-DTX also exhibits better safety. The Taxotere formulation contains Tween 80, which has a strong hemolytic effect. The hemolysis rate reaches 38.6% at a low dose of 0.06 mg / mL, and as high as 63.2% at a high dose of 0.1 mg / mL. In contrast, the hemolysis rate of mPR-DTX polymer micelles is less than 3% at a low dose of 0.06 mg / mL, and only 18.3% at a high dose of 0.1 mg / mL. The blank carrier showed an IC50 value of 376.4 μg / mL for fibroblast 3T3 cells at 24 h and 366.2 μg / mL at 48 h, indicating a high safety profile. In vivo experiments in mice showed that the maximum tolerated dose of mPR-DTX was 20 mg / kg, twice that of the Taxotere group (10 mg / kg). In vivo pharmacodynamic experiments showed that, compared with the saline group, both the mPR-DTX polymer micelle group and the Taxostat group significantly inhibited tumor growth. When the dosage was 10 mg / kg, the mPR-DTX polymer micelle group had a better inhibitory effect on tumor growth than the Taxostat group (p < 0.0001 for both). Furthermore, the mPR-DTX-PM-5 mg / mL group had a better inhibitory effect on tumor growth than the Taxostat-10 mg / kg group (p < 0.01 for both). For the mPR-DTX polymer micelle group, the high-dose group (10 mg / kg) had a better tumor-suppressing effect than the low-dose group (5 mg / kg). Attached Figure Description

[0018] Figure 1 The micelle size distribution is obtained from the screening of the optimal process.

[0019] Figure 2 In vitro release curves of micelles prepared for the selected optimal process.

[0020] Figure 3 The particle size change of micelles prepared by the selected optimal process under constant temperature of 25°C.

[0021] Figure 4The changes in polydispersity index of micelles prepared by the optimal process were observed when the micelles were placed at a constant temperature of 25°C.

[0022] Figure 5 The changes in drug content in micelles prepared using the optimal process were observed when placed at a constant temperature of 25°C.

[0023] Figure 6 Transmission electron microscopy (TEM) images of micelles prepared using the selected optimal process.

[0024] Figure 7 The results are from an in vitro hemolysis experiment using polymer micelles. A is the blank control, B is Taxol, and C is mPR-DTX.

[0025] Figure 8 This is the result of an in vitro toxicity test of polymer micelles on breast cancer cell lines.

[0026] Figure 9 The change in body weight of mice at the maximum tolerated dose over time.

[0027] Figure 10 This shows the change in relative tumor volume over time in mice during in vivo pharmacodynamic experiments.

[0028] Figure 11 The change in mouse body weight over time after drug administration.

[0029] Figure 12 H&E staining images of pharmacodynamic mouse organs.

[0030] Figure 13 The distribution of polymer micelles in mice. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] In this invention, the polymer mPR is synthesized using the following route: , Where n=1000. Example 1

[0035] 1. Polymer micelles were prepared using acetonitrile, methanol, ethanol, and chloroform as film-forming solvents. mPR-DTX polymer micelles were prepared by thin-film dispersion. 60 mg of mPR polymer and 20 mg of DTX were accurately weighed and placed in a round-bottom flask. 3 mL of acetonitrile, methanol, ethanol and chloroform were added respectively and shaken until completely dissolved. The organic solvent was removed by rotary evaporation at 37°C to obtain a drug film containing the drug and carrier material. 5 mL of ultrapure water was added to dissolve and disperse the drug film. The hydrated solution was then filtered through a 0.22 μm filter membrane to obtain the mPR-DTX polymer micelle solution.

[0036] When ethanol is used as the solvent, the encapsulation efficiency is the highest, reaching 99.68%, while methanol has the lowest encapsulation efficiency at 98.30%. Acetonitrile and chloroform have encapsulation efficiencies of 99.14% and 98.58%, respectively. Considering both drug loading efficiency and safety, ethanol was chosen as the solvent.

[0037] 2. Preparation of polymer micelles with different drug loading ratios mPR-DTX polymer micelles were prepared by thin-film dispersion. 20 mg of DTX was accurately weighed and placed in a globular flask. The formulated amounts of mPR polymer were weighed and placed in the globular flask according to drug-to-carrier ratios of 1:3 (60 mg), 1:2.5 (50 mg), 1:2.4 (48 mg), 1:2.2 (44 mg), and 1:2 (40 mg). 3 mL of ethanol was added and shaken until completely dissolved. The organic solvent was removed by rotary evaporation at 37°C to obtain a drug-carrier membrane. 5 mL of ultrapure water was added to dissolve and disperse the drug membrane. The hydrated solution was then filtered through a 0.22 μm filter membrane to obtain the mPR-DTX polymer micelle solution.

[0038] As the drug loading ratio increases, the drug loading gradually decreases. When the drug loading ratio is 1:2 and 1:2.2, the encapsulation efficiency of the formulation is 90.83% and 95.75%, respectively. Among them, when the drug loading ratio is 1:2.4, 1:2.5, and 1:3, the encapsulation efficiency is greater than 99%. Considering the drug loading, 1:2.4 is selected as the optimal drug loading ratio.

[0039] 3. Preparation of polymer micelles at different rotary evaporation temperatures mPR-DTX polymer micelles were prepared by thin-film dispersion. 48 mg of mPR polymer and 20 mg of DTX were accurately weighed and placed in a flask. The organic solvent was removed by rotary evaporation at 40, 37, 35 and 33 °C, respectively, to obtain drug films containing the drug and carrier material. 5 mL of ultrapure water was added to dissolve and disperse the drug film. The hydrated solution was then filtered through a 0.22 μm filter membrane to obtain the mPR-DTX polymer micelle solution.

[0040] When the rotary evaporation temperature range is 33-40℃, the effect of rotary evaporation temperature on drug loading is not significant. When the rotary evaporation temperature is 40℃, the encapsulation efficiency is 99.00%, but the resulting drug film cannot be completely dispersed in a thin film form, and some parts are in a liquid state. This may be due to the low melting point of the mPR polymer, which causes the carrier to melt during rotary evaporation at 40℃. Taking all factors into consideration, 35℃ was selected as the rotary evaporation temperature.

[0041] 4. In vitro release behavior study The in vitro release behavior was investigated using dialysis. With a drug-to-capacity ratio of 1:2.4 and ethanol as the film-forming solvent, the mixture was rotary evaporated at 35°C, and the drug film was hydrated with 5 mL of physiological saline to obtain an mPR-DTX polymer micelle solution. The Taxotere solution was prepared according to the instructions for Taxotere injection. 1 mL of the above solution (1 mg / mL, based on DTX concentration) was placed in a dialysis bag (relative molecular weight cutoff 3500), tied at both ends with cotton thread, and placed in a 50 mL centrifuge tube. 40.0 mL of release medium (PBS 7.4, containing 0.5% Tween 80, v / v) was added, and the tube was placed in a 37°C constant temperature water bath shaker at 75 rpm / min. 1.0 mL of release medium was collected as samples at 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h, and an equal volume of fresh release medium was added simultaneously. The sample was filtered through a 0.22 μm organic filter membrane. 0.2 mL of the initial filtrate was discarded, and the subsequent filtrate was collected in a liquid chromatography vial. 20 μL of this vial was injected into the high-performance liquid chromatograph (HPLC), and the peak area was recorded. The release amount of DTX at each time point was calculated, release curves were plotted, and statistical difference analysis was performed.

[0042] like Figure 2 As shown, the drug is released rapidly within the first 6 hours, followed by a slow release over the next 42 hours, with a cumulative release of approximately 70% within 48 hours. This may be because at 37°C, some polymer micelles are unstable, and structural damage leads to the rapid release of DTX. Simultaneously, because both the polymer carrier and DTX contain benzene rings and double bonds, the strong attraction between conjugated double bonds and the π-π interactions between benzene rings create a strong binding force between the carrier and DTX. Consequently, some polymer micelles remain structurally intact, resulting in a slower release rate of DTX.

[0043] 5. Stability Study Based on a drug loading ratio of 1:2.4, with ethanol as the film-forming solvent, three parallel mPR-DTX polymer micelle solutions were prepared by rotary evaporation at 35℃ and hydration with 5 mL of physiological saline. Each solution was diluted with physiological saline to 1 mg / mL (based on DTX concentration) and placed in a 25℃ constant temperature water bath. Samples of 500 μL from each solution were taken at 0, 1, 2, 4, 6, 8, 10, and 12 h, filtered through a 0.22 μm organic filter, and 400 μL of the filtrate was collected. 800 μL of acetonitrile was added, the mixture was vortexed for 30 s, filtered through another 0.22 μm organic filter, and the initial filtrate was discarded. The subsequent filtrate was transferred to a liquid chromatography vial, and 20 μL was injected into the HPLC system. The peak area was recorded. The changes in DTX content within different time ranges were investigated, and statistical differences were analyzed.

[0044] Meanwhile, the particle size of each solution was measured at 0, 1, 2, 4, 6, 8, 10, and 12 hours to examine the changes in micelle size within the corresponding time range.

[0045] The changes in the polydispersity index of micelle size during storage are as follows: Figure 3 and 4 As shown, within 12 hours of placement, the micelle size fluctuated within a small range, showing a slight increase, but not significantly. The change in micelle encapsulation efficiency during this period is shown in the figure. Figure 5 As shown, the encapsulation efficiency of the formulation was above 95% within 10 hours. In summary, this indicates that the micelles prepared using the optimal formulation process exhibit good in vitro stability.

[0046] 6. Morphological characterization of micelles With a drug loading ratio of 1:2.4 and ethanol as the film-forming solvent, micelle solutions were prepared by rotary evaporation at 35°C and hydration with 5 mL of ultrapure water. The mPR-DTX polymer micelle solution was placed on a copper grid covered with a carbon film and allowed to stand at room temperature for about 3 minutes to dry. 2% phosphotungstic acid was dropped onto the copper grid for negative staining, and the solution was allowed to air dry at room temperature. After drying under an infrared lamp, the micelle structure was observed and photographed using a transmission electron microscope.

[0047] The morphology of micelles under an electron microscope is as follows: Figure 6 As shown, the mPR-DTX polymer micelles are spherical with a particle size of 50-80 nm and a relatively uniform distribution. Example 2

[0048] hemolytic studies A suitable volume of blood was collected from the orbital cavity of a healthy rat and placed in a beaker. The blood was gently stirred in one direction with a glass rod to remove fibrinogen, resulting in defibrinated blood. Ten times the volume of 0.9% sodium chloride injection was added, and the mixture was centrifuged at 3600 rpm for 10 minutes. The supernatant was discarded. This washing process was repeated several times until the supernatant was no longer red. The resulting red blood cells were then resuspended in 0.9% sodium chloride injection to prepare a 2% red blood cell suspension for later use.

[0049] Take centrifuge tubes and add 2% red blood cell suspension, 0.9% sodium chloride injection, 2% Triton, and mPR-DTX polymer micelle injection (DTX concentration: 1 mg / mL) or mPR polymer injection (2.4 mg / mL) or Taxotere injection (DTX concentration: 1 mg / mL) in sequence. Mix well and shake in a 37℃ constant temperature shaker for 3 hours, then carefully observe the solution phenomena. Centrifuge each tube at 3600 rpm / min for 10 min, and take the supernatant to measure the absorbance at 570 nm wavelength using an ELISA reader. For drug-loaded micelles and blank carriers, the background absorbance of the blank carrier needs to be subtracted. Calculate the hemolysis rate using the following formula:

[0050] In the formula, A s A is the absorbance value of the sample to be tested, and A0 is the absorbance value of the negative control group. i The absorbance value is for the positive control group.

[0051] Experimental Conclusion: Based on the principle that heme released from ruptured red blood cells has maximum absorption in the visible light wavelength range, the hemolysis of the test substance was determined by spectrophotometry, with an ultraviolet absorption detection wavelength of 570 nm. Within the specified time, the upper layer of liquid in tubes 1-5 of the blank carrier group was observed to be colorless and transparent, with red blood cells gradually settling to the bottom, indicating no hemolysis occurred. The Taxostat formula contains Tween 80, which has a strong hemolytic effect, with a hemolysis rate of 38.6% at a low dose of 0.06 mg / mL and as high as 63.2% at a high dose of 0.1 mg / mL. In contrast, the hemolysis rate of mPR-DTX polymer micelles was less than 3% at a low dose of 0.06 mg / mL and 18.3% at a high dose of 0.1 mg / mL. Considering that the polymer micelles are diluted in the blood after injection, resulting in a concentration far below 0.1 mg / mL, the possibility of hemolysis is relatively low. In summary, mPR-DTX polymeric micelle nanoparticles avoid the use of Tween 80 in formulations, greatly improving the safety of the formulation. Example 3

[0052] In vitro cytotoxicity studies Remove the cryovials from the liquid nitrogen tank and quickly transfer them to a 37°C constant temperature water bath to thaw. When the cell line has thawed to the point where only a small amount of ice residue remains, transfer the cell suspension from the cryovial to a 15mL sterile centrifuge tube using a pipette. Add 10mL of basal culture medium and mix well. Centrifuge at 1000rpm / min for 5min, discard the supernatant, and resuspend the cells in fresh complete culture medium. Gently pipette to mix well, and transfer the cell suspension to a sterile culture flask. Incubate overnight at 37°C in a 5% CO2 incubator. Change the culture medium after 24 hours. Observe the cell status under a microscope. When the cells have grown to cover 70% of the culture flask area, discard the supernatant and wash 2-3 times with PBS to remove loosely adhered or dead cells. Add 1mL of trypsin per 25T flask and digest in a 37°C incubator until the cytoplasm shrinks and the cells no longer clump together. Quickly add 3mL of complete culture medium to stop the digestion. Transfer the liquid from the culture flask to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add fresh complete culture medium to resuspend the cells, gently pipette to mix thoroughly, transfer the cell suspension to a sterile culture flask, and incubate at 37°C with 5% CO2, changing the culture medium as needed.

[0053] Take appropriate amounts of DTX, Tween 80, and 13% ethanol solution (v / v) and prepare a 20 mg / mL Taxotere stock solution according to the instructions. Dilute with the corresponding basal culture medium to obtain a 1 mg / mL injection solution. Further dilute to obtain 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µg / mL DTX injection solutions as controls.

[0054] Take 1 mg / mL of the prepared mPR-DTX polymer micelle solution and add the corresponding basic culture medium to obtain DTX injection solutions of 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µg / mL.

[0055] Take the mPR polymer solution and dilute it with the corresponding basal culture medium to obtain two concentration gradient injection solutions of 0.15, 0.3, 1.5, 3, 15, 30, 150, 300 µg / mL and 50, 100, 200, 400, 600, 800, 1000, 1200 µg / mL.

[0056] Experimental conclusions: For 4T1 and MCF-7 breast cancer cells, the growth inhibitory effects of paclitaxel, blank carrier, and mPR-DTX polymer micelles all increased with increasing drug concentration. Furthermore, after 24 and 48 hours of drug administration, the IC50 of mPR-DTX polymer micelles on MCF-7 and 4T1 cells was significantly higher. 50 The values ​​were all significantly lower than those in the Taxostat group. Specifically, when the administration time was 48 hours, the IC50 values ​​of mPR-DTX polymer micelles on 4T1 cells were significantly lower. 50The value was 0.35±0.14 μg / mL, indicating that Taxotere's IC50 against 4T1 cells was... 50 The value was 4.89±2.11 μg / mL, and there was a significant difference between the two (p<0.01), indicating that the mPR-DTX polymer micelles had a stronger inhibitory effect on tumor cell growth. The blank carrier also had a certain inhibitory effect on the growth of MCF-7 and 4T1, and the inhibitory effect of the blank carrier on tumor cell growth became more obvious with the extension of time. It is speculated that this is because the blank carrier contains a retinoic acid block. Retinoic acid is a pharmacologically active molecule with certain anti-tumor activity, and therefore shows a certain killing effect on 4T1 and MCF-7.

[0057] For fibroblast 3T3 cells, the IC50 of the blank vector was evaluated after 24 hours. 50 The value was 376.4 μg / mL, and the IC50 value after 48 hours was... 50 The value was 366.2 μg / mL, IC50 50 A higher value indicates that the blank vector has higher safety. Example 4

[0058] In vivo toxicity studies in mice Male Balb / c mice, weighing (20 ± 2) g, were randomly divided into 11 groups of 6 mice each. The mice were administered the drug via tail vein injection on days 0, 4, and 9, with each mouse receiving 0.2 mL of the drug. The control group received an equal volume of physiological saline. Dosage is shown in Table 5. From the date of administration, the mice were observed for toxicity symptoms, and their weight changes and survival were recorded daily for 14 consecutive days. The maximum tolerated dose (MTD) of the mice to mPR polymer, mPR-DTX polymer micelles, and Taxotere injection was calculated.

[0059] The indicators for observing animal responses after drug administration include the following aspects: Central nervous system and neuromuscular system: abnormal posture, restlessness, lethargy, spasms, convulsions, paralysis, motor incoordination, hypersensitivity or sluggishness in response to external stimuli; respiratory system: dyspnea, deep breathing, tachypnea, wasp waist; skin and hair: congestion, cyanosis, ruffled and dirty coat; eyes: ptosis, exophthalmos, conjunctival congestion, corneal opacity; digestive system: diarrhea, anorexia.

[0060] The maximum tolerated dose should result in a weight loss in animals not exceeding 20% ​​of that in negative control animals.

[0061] Calculate the weight loss rate using the following formula: .

[0062] Table 1. In vivo toxicity test in mice

[0063] Experimental Conclusions: Analysis of survival rate and weight change rate: On day 10 after administration, the weight of mice in the mPR blank vector group showed an increasing trend with no deaths. The maximum administered dose was 800 mg / kg, indicating good safety of the blank vector. One mouse died on day 12 in the mPR-DTX-PM-30 mg / kg group and one mouse died on day 9 in the Taxotere-30 mg / kg group; no deaths were observed in the other groups. Except for the saline group, the weight of mice in the other groups showed a continuous decreasing trend after administration. The weight loss ranged from 15-20% on day 7 in the 30 mg / kg group, from 10-15% in the 20 mg / kg group, and less than 10% in the 10 mg / kg and 5 mg / kg groups. After the third administration, the weight loss rate in the mPR-DTX-PM-10 mg / kg and Taxotere-5 mg / kg groups was around 15%, while the weight loss in the other groups exceeded 20%. With prolonged recovery time, the mPR-DTX polymer micelle group showed a significantly better trend in weight recovery than the Taxodia group.

[0064] Analysis of animal reactions after drug administration: After administration of the blank carrier at 800 mg / kg, mice experienced slight abdominal bloating, possibly caused by retinoic acid irritating the gastric mucosa. This returned to normal within half an hour. Mice in the 400 mg / kg and 200 mg / kg groups showed no abnormalities, indicating that the blank carrier had good safety. On day 11 after administration, mice in the mPR-DTX-PM-30 mg / kg and Taxotere-30 mg / kg groups experienced limb paralysis, even deformities, and facial edema. This is related to the peripheral nerve damage caused by docetaxel: docetaxel can cause numbness in the hands and feet, and peripheral nerve damage. This side effect is the most persistent and easily leads to fluid retention. In the groups with doses less than or equal to 20 mg / kg, mice had healthy limbs and no edema or deformities.

[0065] The maximum tolerated dose obtained from the above experimental results is: 20 mg / kg for the mPR-DTX polymer micelle group and 10 mg / kg for the Taxodia group. Example 5

[0066] In vivo pharmacokinetic studies in rats With a drug loading ratio of 1:2.4 and ethanol as the film-forming solvent, a micelle solution was prepared by rotary evaporation at 35°C and hydration with 5 mL of ultrapure water. The mPR-DTX polymer micelle solution was then used. Experimental animals: Female SD rats, weighing (180 ± 20) g, purchased from Nanjing Qinglongshan Breeding Farm, animal qualification certificate number: 20230206Aazz0100000405.

[0067] Blood sample processing method: Take 90 μL of plasma sample, add 10 μL of 200 ng / mL PTX internal standard solution, vortex mix, add 1 mL of acetonitrile solution, vortex for 2 min, centrifuge at 4℃ and 9200 rpm for 10 min, take 200 μL of supernatant, evaporate to dryness with nitrogen; add 200 μL of acetonitrile to reconstitute, centrifuge at 4℃ and 9200 rpm for 10 min, take 150 μL of supernatant in a liquid chromatography vial, inject for analysis, plot the average blood drug concentration-time curve, and calculate the pharmacokinetic parameters.

[0068] Twelve SD rats (200±20g) were randomly divided into two groups (n=6): the first group received mPR-DTX polymer micelles, and the second group received Taxoldi. Both groups received the drug via tail vein administration at a dose of 10mg / kg. Rats were fasted for 12 hours prior to the experiment, but water was allowed throughout the experiment. Following tail vein administration from each SD rat, 0.5mL of blood was collected from the orbital sinus at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h. The blood samples were placed in heparin sodium centrifuge tubes, immediately mixed, centrifuged at 4000rpm for 10min, and the supernatant was collected and stored at -20℃ for later use.

[0069] After the plasma samples were processed as described above, LC-MS was performed to determine the peak areas of DTX and internal standard PTX. The DTX content in the plasma samples at each time point was calculated using the internal standard method. Pharmacokinetic parameters were calculated, and an average plasma drug concentration-time curve was plotted.

[0070] Table 2. Pharmacokinetic parameters

[0071] The pharmacokinetic parameters of mPR-DTX polymer micelles and Taxotere are shown in the table above. The results indicate that, compared with the Taxotere group, the overall DTX exposure of the mPR-DTX polymer micelle group was slightly lower. The peak concentration and time to peak concentration of the Taxotere group were slightly higher than those of the mPR-DTX polymer micelle group, but there was no significant difference between the two. The time to peak concentration (Tmax) was 0.40±0.12h and 0.35±0.12h, respectively, which were both relatively short, and the blood drug concentration decreased rapidly, indicating that both drugs can be rapidly distributed to various tissue receptors and have a rapid onset of action. In addition, the clearance rate (CI) of the mPR-DTX polymer micelle group was higher than that of the Taxotere group, and there was a significant difference between the two. This may be because the mPR-DTX polymer micelles are in thermodynamic and kinetic equilibrium in vitro. Once injected into the blood, the micelles are immediately diluted and mixed with blood cells, plasma proteins and other components. Under the action of blood shear force and plasma proteins, they rapidly dissociate, and the polymer micelles tend to separate into monomers, resulting in the rapid release of DTX. After being metabolized by the liver metabolic enzyme CYP3A4, it is finally excreted from the body through bile. Example 6

[0072] Pharmacodynamic experiments With a drug loading ratio of 1:2.4 and ethanol as the film-forming solvent, micelle solutions were prepared by rotary evaporation at 35°C and hydration with 5 mL of ultrapure water. The mPR-DTX polymer micelle solution was then used.

[0073] Experimental animals: Female Balb / C mice, weighing (18±2) g, purchased from Nanjing Qinglongshan Breeding Farm, animal qualification certificate number: 20230206Abzz0100000124. Normal Balb / C mice were inoculated with 4T1 cells to establish a mouse tumor 4T1 model. The tumor volume was approximately 80-150 mm. 3 Twenty-four tumor-bearing mice were randomly divided into four groups: mPR-DTX polymer micelle group (5, 10 mg / kg), Taxotere group (10 mg / kg), and saline group, with six mice in each group. mPR-DTX polymer micelles and Taxotere were diluted to appropriate concentrations with 0.9% sodium chloride injection and administered via tail vein injection on days 0, 4, 9, and 16, respectively. The day of the first administration was designated as day 0. Specific dosage details are shown in Table 1.

[0074] Table 3. Pharmacodynamic experimental group settings, dosage and method

[0075] After drug administration, the tumor diameter was measured every two days, and the tumor volume and mouse weight were calculated and recorded. Tumor growth curves, tumor growth inhibition rate curves, and body weight change curves were plotted with time as the x-axis. At the end of the drug administration cycle, the mice were weighed, euthanized by cervical dislocation, and the tumors were removed and weighed. These tumors, along with the removed heart, liver, spleen, lungs, and kidneys, were then fixed in 4% paraformaldehyde. The heart, liver, spleen, lungs, and kidneys were stained with H&E to examine their morphology.

[0076] Table 4. Results of animal pharmacodynamic experiments

[0077] Experimental results: The tumor growth inhibition rate was 73.4% in the mPR-DTX-PM group (5 mg / kg), 81.3% in the 10 mg / kg group, and 60.7% in the Taxotere-10 mg / kg group. These results indicate that the mPR-DTX polymer micelle group showed significantly better tumor growth inhibition than the Taxotere group. The medium-dose mPR-DTX polymer micelle group (5 mg / kg) showed better tumor growth inhibition than the high-dose Taxotere group (10 mg / kg), possibly due to the polymer carrier exerting its anti-tumor effect. Example 7

[0078] In vivo distribution study of mPR-DTX polymer micelles in a mouse 4T1 tumor model 4T1 cells were cultured in 1640 medium containing 10% FBS (with 100 U / mL penicillin and streptomycin antibiotics) at 37°C and 5% CO2. Cells in the logarithmic growth phase were selected, trypsinized, centrifuged, counted, and the cell density was adjusted to 1×10⁻⁶. 7 Cells / mL: After disinfecting the skin of the second pair of mammary glands of each Balb / C mouse with alcohol, 100 μL of cell suspension was subcutaneously injected. The mice were then fed for another 2 weeks, and tumors with a volume of approximately 80-150 mm were selected. 3 Tumor-bearing mice were used in subsequent experiments.

[0079] Take 200 mg of mPR polymer and 1 mg of Dir (1.0 mL of 1 mg / mL Dir ethanol solution), dissolve in an appropriate amount of ethanol, remove the ethanol by rotary evaporation at 37℃, and dilute with physiological saline to 0.1 mg / mL to obtain mPR-Dir injection; take 1 mg of Dir (1.0 mL of 1 mg / mL Dir ethanol solution), dilute with physiological saline to 0.1 mg / mL to obtain free Dir injection.

[0080] Twelve tumor-bearing mice were randomly divided into two groups. The in vivo distribution of mPR-DTX polymer micelles was studied using a Dir fluorescent probe to simulate the hydrophobic chemotherapy drug DTX. Both the free Dir group and the mPR-Dir injection group were administered via tail vein injection; dosage details are shown in Table 4. Following administration, mice were anesthetized with isoflurane at 1, 2, 4, 6, 8, 12, and 24 hours, and fluorescence imaging was performed using a small animal in vivo imaging system. Three mice were sacrificed at 24 hours, and various organs (heart, liver, spleen, lung, kidney, and tumor) were harvested and placed under in vivo imaging to detect the fluorescence signal intensity of each tissue.

[0081] Table 5. In vivo distribution of dosage

[0082] Experimental conclusions: In the mPR-Dir-PM group, the fluorescence intensity at the tumor site gradually increased 4 hours after administration; at 8 hours, it showed a relatively concentrated distribution at the tumor site; and at 24 hours, the fluorescent dye was still mainly concentrated at the tumor site, possibly because the EPR effect increased the retention time of micelles at the tumor site. In the free Dir group, the fluorescent dye gradually distributed throughout the body 2 hours after administration; with the extension of time, the fluorescence intensity in the liver and kidneys increased, indicating that Dir was gradually metabolized and excreted in the body.

[0083] 24-hour ex vivo organ fluorescence imaging showed that the mPR-Dir-PM group exhibited the strongest fluorescence at the tumor site, indicating that the polymer micelles have a strong targeting effect on the tumor site. The drug can accumulate more easily in the tumor tissue, thereby increasing the local drug concentration and enhancing the therapeutic effect.

Claims

1. A docetaxel micelle nanomedicine, characterized in that, Made from docetaxel and polymer mPR; The structural formula of the polymer mPR is as follows: ，n=1000。 2. The docetaxel micelle nanomedicine according to claim 1, characterized in that, The mass ratio of docetaxel to polymer mPR is 1:2-1:

3.

3. The method for preparing docetaxel micelle nanomedicine according to claim 1 or 2, characterized in that, Polymer micelles were prepared by mixing and dispersing docetaxel and polymer mPR into a drug film and then hydrating it.

4. The preparation method according to claim 3, characterized in that, Docetaxel and polymer mPR were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a drug film. A hydration medium was added to prepare polymer micelles.

5. The preparation method according to claim 4, characterized in that, The organic solvent is selected from acetonitrile, methanol, ethanol, and chloroform, with ethanol being preferred.

6. The preparation method according to claim 4, characterized in that, The mass ratio of docetaxel to polymer mPR is 1:3, 1:2.5, 1:2.4, 1:2.2 or 1:2, preferably 1:2.

4.

7. The preparation method according to claim 4, characterized in that, The temperature of the rotary evaporation is 40°C, 37°C, 35°C or 33°C, preferably 35°C.

8. The preparation method according to claim 4, characterized in that, The hydration medium is selected from ultrapure water, physiological saline, and PBS buffer, with physiological saline being preferred.

9. The use of the docetaxel micelle nanomedicine according to claim 1 or 2 in the preparation of tumor therapeutic drugs.

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