A degradable 177 Lu-plga radioactive microspheres, preparation method and application thereof

Abstract

The application discloses a degradable 177 Lu-PLGA radioactive microspheres, a preparation method and application thereof, and relate to the technical field of medicines.The method comprises the following steps: preparing a primary oil-in-water emulsion by using a gelatin aqueous solution and a dichloromethane solution of polylactic acid-glycolic acid copolymer; collecting double emulsion droplets based on a microfluidic double emulsion system by taking the primary oil-in-water emulsion as an intermediate phase and taking a polyvinyl alcohol aqueous solution as an external water phase; obtaining dry mesoporous polylactic acid-glycolic acid copolymer microspheres by solidifying the double emulsion droplets, purifying the double emulsion droplets after removing the gelatin, and freeze-drying; and obtaining degradable 177 Lu radioactive nuclide-labeled dry mesoporous polylactic acid-glycolic acid copolymer microspheres. 177 Lu-PLGA radioactive microspheres.The application overcomes the problems of in-vivo leakage evaluation difficulty of existing radioactive microspheres and in-vivo lifelong retention of glass microspheres and resin microspheres, thereby being beneficial to secondary embolization treatment after tumor recurrence.

Description

Technical Field

[0001] This invention relates to a biodegradable 177 Lu-PLGA radioactive microspheres, their preparation methods, and applications belong to the field of medical technology. Background Technology

[0002] Hepatocellular carcinoma (HCC) is characterized by delayed detection, difficulty in diagnosis and treatment, rapid disease progression, and poor prognosis.

[0003] Currently, treatment methods for liver cancer encompass a variety of approaches, including surgical resection, radiofrequency ablation, liver transplantation, chemotherapy, external beam radiotherapy, and in vivo radionuclide therapy. Among these, TARE (Transarterial Radioembolization) is an innovative treatment method that precisely delivers radionuclide-loaded microspheres to the tumor's blood supply arteries via the arterial system, achieving high-intensity radiotherapy to the tumor area while minimizing damage to surrounding healthy tissues. These microspheres not only effectively embolize tumor vessels, cutting off their blood supply, but also directly kill tumor cells through radiation. Compared to traditional radiotherapy, TARE significantly reduces the radiation dose received by normal tissues, exhibiting better therapeutic effects and lower systemic toxicity, and has become a key advancement in the treatment of advanced liver cancer.

[0004] To date, three types of radioactive microspheres have been successfully developed for clinical use: 90Y-labeled glass microspheres (TheraSpheres), 90Y-labeled resin microspheres (SIR-Spheres), and 166Ho-labeled polylactic acid (PLLA) microspheres (QuiremSpheres). However, these microspheres all have certain limitations. For example, the production of 90Y glass microspheres and 166Ho polylactic acid microspheres relies on neutron activation technology. 90Y resin microspheres have relatively weak mechanical strength and may rupture or deform during injection, which can affect their distribution within the liver and thus the therapeutic effect.

[0005] Currently, the vast majority of carrier materials for radioactive microspheres used clinically are non-biodegradable. This presents certain limitations, including the possibility of microspheres permanently remaining in blood vessels, potentially causing chronic inflammation, vascular fibrosis, and foreign body reactions, increasing the potential risk of tissue damage, and affecting subsequent treatment for tumor recurrence. The materials of some non-biodegradable microspheres may also affect subsequent imaging examinations, interfering with the assessment of disease progression. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biodegradable... 177Lu-PLGA radioactive microspheres, their preparation methods, and applications overcome the difficulties in assessing in vivo leakage of existing radioactive microspheres and the problem of lifelong retention of glass microspheres and resin microspheres in vivo, thus facilitating secondary embolization therapy after tumor recurrence.

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0008] In a first aspect, the present invention provides a biodegradable 177 The preparation method of Lu-PLGA radioactive microspheres includes:

[0009] A primary oil-in-water emulsion was prepared using a gelatin aqueous solution and a dichloromethane solution of polylactic acid-glycolic acid copolymer.

[0010] Using a primary oil-in-water emulsion as the internal phase and a polyvinyl alcohol aqueous solution as the external phase, droplets of the dual emulsion system were collected based on a microfluidic dual emulsion system.

[0011] After solidifying the dual emulsion droplets, removing the gelatin, purifying, and freeze-drying, dried mesoporous polylactic acid-glycolic acid copolymer microspheres were obtained.

[0012] use 177 Lu radionuclide-labeled dried mesoporous polylactic acid-glycolic acid copolymer microspheres were obtained to obtain biodegradable... 177 Lu-PLGA radioactive microspheres.

[0013] Furthermore, the preparation of the primary oil-in-water emulsion using a gelatin aqueous solution and a dichloromethane solution of polylactic acid-glycolic acid copolymer includes:

[0014] A mixed solution was obtained by mixing an aqueous solution of gelatin and a dichloromethane solution of polylactic acid-glycolic acid copolymer;

[0015] The mixed solution was treated using ultrasonic emulsification technology, and after standing to remove air bubbles, a primary oil-in-water emulsion was obtained.

[0016] Furthermore, the gelatin aqueous solution is in the range of 5-10 wt%;

[0017] And / or, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 1~3:1;

[0018] And / or, the molecular weight range of the polylactic acid-glycolic acid copolymer is 10~100kDa;

[0019] And / or, the volume ratio of the gelatin aqueous solution and the dichloromethane solution of the polylactic acid-glycolic acid copolymer is 1:1~4.

[0020] Furthermore, the microfluidic dual emulsion system employs a glass capillary or a polydimethylsiloxane microfluidic chip;

[0021] And / or, the range of the polyvinyl alcohol aqueous solution is 0.5~2wt%;

[0022] And / or, the flow rate of the intermediate phase is in the range of 1~2µL / min, and the flow rate of the outer phase is in the range of 3~6µL / min.

[0023] Furthermore, the process of curing the dual emulsion droplets, removing gelatin, purifying, and lyophilizing to obtain dried mesoporous polylactic acid-glycolic acid copolymer microspheres comprises:

[0024] By allowing the double emulsion droplets to stand, dichloromethane diffuses and evaporates, forming a porous polylactic acid-glycolic acid copolymer microsphere framework.

[0025] The porous polylactic acid-glycolic acid copolymer microsphere framework was transferred to a buffer containing collagenase for incubation to enzymatically remove gelatin and form mesoporous microspheres.

[0026] After washing, pre-freezing, and vacuum freeze-drying, mesoporous polylactic acid-glycolic acid copolymer microspheres were obtained.

[0027] Furthermore, the settling time ranges from 12 to 24 hours;

[0028] And / or, the concentration of collagenase in the collagenase-containing buffer solution is in the range of 0.1~0.2 mg / mL;

[0029] And / or, the incubation conditions include incubation at 32-37°C for 2-4 hours;

[0030] And / or, the pre-freezing conditions include pre-freezing for 12 to 24 hours within the range of -80 to -60°C;

[0031] And / or, the conditions for vacuum freeze drying include vacuum freeze drying for 24 to 48 hours at a temperature ≤ -60°C and a vacuum degree < 0.1 mbar.

[0032] Furthermore, the aforementioned utilization 177 Lu radionuclide-labeled dried mesoporous polylactic acid-glycolic acid copolymer microspheres were obtained to obtain biodegradable... 177 Lu-PLGA radioactive microspheres include:

[0033] A dispersion was obtained by uniformly dispersing dried mesoporous polylactic acid-glycolic acid copolymer microspheres in ultrapure water.

[0034] Add to dispersion 177 After the first incubation stage with LuCl3, potassium phosphate buffer solution was added and the second incubation stage was continued. After centrifugation, the supernatant was discarded to obtain labeled microspheres.

[0035] The labeled microspheres were washed to obtain biodegradable products. 177 Lu-PLGA radioactive microspheres.

[0036] Furthermore, the concentration range of the dried mesoporous polylactic acid-glycolic acid copolymer microspheres and ultrapure water in the dispersion is 1 mg: 40~500 µL;

[0037] And / or, the 177 The radioactivity of LuCl3 ranges from 0.5 to 2 mCi;

[0038] And / or, the conditions for the first incubation stage include oscillation at 35-40°C for 1-3 minutes, and the conditions for the second incubation stage include oscillation at 35-40°C for 2-5 minutes;

[0039] And / or, the potassium phosphate buffer solution has a pH range of 12-13, a concentration of 0.1-0.5M, and a volume ratio of 1:1-5 with the dispersion.

[0040] And / or, the centrifugation includes centrifugation at 3000~6000 rpm for 2~5 min;

[0041] And / or, the washing is performed using a phosphate-buffered saline solution with a pH range of 7.2 to 7.4.

[0042] Secondly, the present invention also provides a biodegradable 177 Lu-PLGA radioactive microspheres, via biodegradable as described in any of the first aspects 177 Lu-PLGA radioactive microspheres were prepared using a specific method.

[0043] Thirdly, the present invention also provides a biodegradable 177 Applications of Lu-PLGA radioactive microspheres include the treatment of hepatocellular carcinoma, with methods including delivery via the arterial system. 177 Lu-PLGA radioactive microspheres are delivered to the tumor's blood supply arteries to achieve tumor vascular embolization, cutting off its blood supply, and destroying tumor cells through radiation.

[0044] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0045] This invention uses polylactic acid-glycolic acid copolymer (PLGA) material and 177 Lu constructed a novel biodegradable polymer radioactive microsphere with good radiostability. 177Lu-PLGA, using SPECT / CT imaging technology for in vitro monitoring of microspheres, overcomes the difficulties in assessing in vivo leakage of 90Y microspheres and the problem of lifelong retention of glass and resin microspheres in vivo, thus facilitating secondary embolization therapy after tumor recurrence. Furthermore, this method can achieve optimal integration of radiotherapy and embolization therapy, ensuring the gradual degradation of microspheres after radionuclide decay to a safe level, providing a solid experimental foundation for clinical translation.

[0046] The radioactive microsphere-loaded nuclide of the present invention 177 Lu, 177 Lu emits beta rays with low energy, with a maximum energy of 0.497 MeV and a penetration depth between 0.2 and 2 mm. This low penetration depth makes... 177 The radioactivity of Lu microspheres is more concentrated on the local tumor, thereby greatly reducing damage to the surrounding healthy tissues; 177 Lu has a longer half-life of 6.7 days, making it easier to transport and use in clinical applications; 177 Lu can also emit low doses of gamma rays, which allows for observation via single-photon emission computed tomography (SPECT). 177 The localization of Lu microspheres offers possibilities. This feature greatly facilitates doctors' visual assessment of microsphere distribution, treatment efficacy, and post-treatment monitoring;

[0047] The carrier material of this invention is PLGA. As a biodegradable high molecular weight organic compound, the degradation rate of PLGA can be precisely controlled by adjusting the synthesis conditions. In the human body, the final metabolic products of PLGA are water and carbon dioxide. PLGA material has the advantages of excellent biocompatibility, biodegradability, non-toxicity, non-immunogenicity and suitable mechanical strength. Attached Figure Description

[0048] Figure 1 This is a SEM schematic diagram of the mesoporous PLGA microspheres of Embodiment 1 of the present invention. 177 A schematic diagram of the size distribution of Lu-PLGA radioactive microspheres, where a is a SEM image of mesoporous PLGA microspheres under 400x and 10000x magnification, and b is... 177 Schematic diagram of the size distribution of Lu-PLGA radioactive microspheres;

[0049] Figure 2 Example 1 of the present invention: mesoporous PLGA microspheres under different pH ranges of potassium phosphate buffer solutions. 177 Labeling efficiency of Lu and the effect of potassium phosphate buffer solution on mesoporous PLGA microspheres at pH 11 177 A schematic diagram of Lu labeling efficiency, where 'a' represents the labeling efficiency of mesoporous PLGA microspheres under different pH ranges of potassium phosphate buffer solutions.177 A schematic diagram of Lu labeling efficiency (n=3); b represents mesoporous PLGA microspheres under potassium phosphate buffer solution conditions at pH=11. 177 A diagram illustrating the labeling efficiency of Lu over time (n=3);

[0050] Figure 3 In Embodiment 2 of the present invention 177 A schematic diagram comparing the radiostability of Lu-PLGA radioactive microspheres in 1X PBS and 10% fetal bovine serum;

[0051] Figure 4 In Embodiment 2 of the present invention 177 Schematic diagram of Fourier transform infrared spectroscopy analysis of Lu-PLGA radioactive microspheres;

[0052] Figure 5 In Embodiment 2 of the present invention 177 Schematic diagram of EDS mapping of Lu-PLGA radioactive microspheres;

[0053] Figure 6 Intratumoral injection in Example 2 of the present invention 177 Lu-PLGA radioactive microspheres (100 μCi) and 177 SPECT / CT illustration of H22 tumor-bearing mice in LuCl3 solution (100 μCi);

[0054] Figure 7 In Example 2 of the present invention, a gamma counter was used to measure intratumoral injection. 177 Lu-PLGA radioactive microspheres (100 μCi) and 177 A schematic diagram of radiometric contrast of organs and tissues extracted after SPECT-CT imaging study of H22 tumor-bearing mice in LuCl3 solution (100 μCi).

[0055] Figure 8 5mg in Example 2 of the present invention 177 Degradation and weight change of Lu-PLGA radioactive microspheres under an optical microscope over 100 days, where a is 5 mg. 177 A schematic diagram of the degradation of Lu-PLGA radioactive microspheres under an optical microscope over 100 days, where b represents 5 mg. 177 A schematic diagram of the weight change of Lu-PLGA radioactive microspheres over 100 days (n=3).

[0056] Figure 9 In Embodiment 3 of the present invention 177 A schematic diagram of in vitro cell experiments using Lu-PLGA radioactive microspheres, where a represents different concentrations. 177A schematic diagram illustrating the relative viability of H22 and HepG2 cells incubated with Lu-PLGA radioactive microspheres (n=5), where b represents the radioactivity in the range of 0–50 μCi. 177 Schematic diagram of the relative viability of H22 and HepG2 cells after 24 hours of incubation with Lu-PLGA radioactive microspheres (n=5).

[0057] Figure 10 In Example 3 of the present invention, mesoporous PLGA microspheres (0.5 mg / mL) and 177 Schematic diagram of immunofluorescence confocal imaging of HepG2 cells incubated with Lu-PLGA radioactive microspheres (0.5 mg / mL, 200 μCi / mL);

[0058] Figure 11 In Example 3 of the present invention, PBS, ultrapure water, and 177 A schematic diagram showing the results of treating mouse blood with Lu-PLGA radioactive microspheres (0.5 mg / mL) and calculating the hemolysis rate using an ELISA reader (n=3). Detailed Implementation

[0059] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0060] Example 1

[0061] This embodiment provides a 177 The preparation method of Lu-PLGA radioactive microspheres includes the following steps:

[0062] A 7.5 wt% aqueous gelatin solution (as the inner aqueous phase) was mixed with a 2 wt% dichloromethane (DCM) solution of polylactic acid-glycolic acid copolymer (PLGA, with a molar ratio of lactic acid to glycolic acid of 1:1 and a molecular weight range of 40 kDa) (as the oil phase) at a volume ratio of 1:2.

[0063] Subsequently, a W / O primary oil-in-water emulsion was prepared by ultrasonic emulsification (ultrasonic treatment at 30W power for 30 seconds), and allowed to stand until any potential air bubbles were completely eliminated in preparation for subsequent microfluidic operations.

[0064] Using glass capillaries or polydimethylsiloxane (PDMS) microfluidic chips, combined with a precision injection pump, a W / O / W dual emulsion system is precisely constructed through a flow focusing mechanism. In this embodiment, a microfluidic chip is used, specifically:

[0065] A W / O primary oil-in-water emulsion was used as the intermediate phase, and a 1 wt% aqueous solution of polyvinyl alcohol (PVA) was used as the external aqueous phase. Both phases were injected into a microfluidic chip at precisely controlled flow rates. To achieve the preparation of uniform droplets with an average diameter of approximately 50 μm, the flow rate of the intermediate phase was 1 µL / min, and the flow rate of the external aqueous phase was 3 µL / min. The W / O / W dual emulsion droplets were collected in a PVA solution in an ice bath.

[0066] It is important to note that the dual emulsion droplets must exhibit a high degree of size uniformity. In this embodiment, the inner aqueous phase of the W / O / W dual emulsion droplets is a gelatin aqueous solution, the middle oil phase is a PLGA-DCM solution, and the outer aqueous phase is a PVA aqueous solution.

[0067] The two emulsion droplets are left to stand overnight at 4°C. This process allows the organic solvent DCM to slowly diffuse into the external aqueous phase and evaporate. The DCM is removed by stirring with a glass rod for 1 hour, thereby forming a porous PLGA microsphere framework, which is the cured microsphere.

[0068] Subsequently, the solidified microspheres were transferred to a buffer solution containing collagenase (0.2 mg / mL collagenase solution, incubated at 37°C for 2 hours) to enzymatically remove the gelatin template, thereby forming a mesoporous structure. After enzymatic hydrolysis, the mesoporous structure was washed three times with ultrapure water (Milli-Q purified water, resistivity 18.2 MΩ·cm) to thoroughly remove residual DCM, PVA, and gelatin degradation products.

[0069] The washed microspheres were pre-frozen at -80℃ for 24 hours, and then subjected to vacuum freeze drying (24 hours, temperature ≤−60℃, vacuum degree <0.1 mbar) to finally obtain dry and stable mesoporous PLGA microspheres.

[0070] Weigh 5 mg of dried mesoporous PLGA microspheres and disperse them in 500 μL of ultrapure water (Milli-Q pure water, resistivity 18.2 MΩ·cm) in a 1.5 mL centrifuge tube. Sonicate or shake gently until the microspheres are evenly dispersed.

[0071] Then, add 1 μL of 177 In a LuCl3 (1 mCi) solution, incubate at 37°C with shaking for 2 minutes. Then, add 100 μL of K3PO4 solution (pH 13), and continue shaking for 3 minutes under the same conditions. Centrifuge to obtain the final product. 177 Lu-PLGA radioactive microspheres.

[0072] Mesoporous PLGA microspheres were observed using scanning electron microscopy (SEM). 177 The surface and internal morphology of Lu-PLGA radioactive microspheres were analyzed to confirm the presence and distribution of mesoporous structures, such as... Figure 1As shown in Figure a, the morphological characteristics of PLGA microspheres at magnifications of 400 and 10000 are displayed. The PLGA microspheres are spherical and possess a certain degree of porosity. The PLGA matrix contains a highly porous and interconnected irregular network of pores, resembling a honeycomb or sponge-like structure. The size distribution is as follows... Figure 1 As shown in b, 177 The size of the Lu-PLGA radioactive microspheres is 57.78 ± 3.563 μm, which effectively demonstrates that their size is suitable for hepatic artery embolization in the treatment of hepatocellular carcinoma.

[0073] Comparative Example 1

[0074] because 177 Lu 3+ Precipitation reactions are generally pH-dependent, so K3PO4 and KOH are used to adjust the pH of the reaction medium. Therefore, in Comparative Example 1, all other steps of Example 1 are kept unchanged, only the pH of the K3PO4 solution is changed, and the reaction is tested. 177 The labeling efficiency of Lu, such as Figure 2 As shown in a, from Figure 2 As can be seen from a, the radiolabeling efficiency increases sharply with the increase of pH value, reaching a plateau of about 98.77% ± 0.24% at pH 11.

[0075] Comparative Example 2

[0076] For research and 177 The effect of reaction time of LuCl3 solution on radiolabeling efficiency: This comparative example keeps other steps unchanged from Example 1, only changing the pH value of K3PO4 solution to 11, and continuously changing... 177 The reaction time of LuCl3 solution, the results are as follows Figure 2 As shown in b, the radiolabeling efficiency can reach over 98% within 5 minutes. However, there is no significant change in the radiolabeling efficiency when the reaction time is increased. This indicates that there is no linear relationship between the radiolabeling efficiency and the reaction time.

[0077] Next, regarding 177 The radiostability of Lu-PLGA radioactive microspheres in 1X PBS and 10% fetal bovine serum was tested, with 1 mg (100 μCi) of the microspheres. 177 Lu-PLGA radioactive microspheres were placed in 1 mL of PBS and 10% FBS, respectively. The solutions were centrifuged multiple times over 120 hours (4000 rpm, 3 minutes). After separating the supernatant, the radioactivity of the microspheres was measured using an activity meter, and the radiostability was calculated. The supernatant was then returned to the centrifuge tube. Results are as follows: Figure 3 As shown, the test results are 177The radiostability of Lu-PLGA radioactive microspheres in 1X PBS and 10% fetal bovine serum was above 90% within 120 hours, indicating that... 177 Lu-PLGA radioactive microspheres can be used for further in vivo and in vitro experiments.

[0078] Then, for different radioactive... 177 Fourier transform infrared (FTIR) spectroscopy and EDS mapping analysis were performed on Lu-PLGA radioactive microspheres. The Fourier transform infrared spectra are as follows: Figure 4 As shown, 3660cm -1 The peak at 2985 cm⁻¹ represents the stretching vibration of OH. -1 The peak at 1740 cm⁻¹ represents the stretching vibration of CH, originating from the methyl group in PLGA. -1 The peak at 1445 cm⁻¹ represents the stretching vibration of C=O. -1 and 1386cm -1 The two peaks at 1272 cm⁻¹ represent the asymmetric and symmetric bending vibrations of CH, respectively. -1 ~1072cm -1 The peaks within the range are all stretching vibrations of CO, 860 cm⁻¹. -1 ~695cm -1 Peaks within this range are generally due to out-of-plane bending vibrations of cyclic structures at CH, but PLGA does not contain cyclic structures, which may be vibrational modes of conjugated structures in the polymer. EDS mapping analysis results are as follows... Figure 5 As shown, 177 Lu-PLGA radioactive microspheres 177 EDS mapping of Lu and its decayed Hf confirmed the nuclide loading in the microspheres.

[0079] Example 2

[0080] For evaluation 177 The SPECT-CT imaging capability of Lu-PLGA radioactive microspheres was utilized to construct an H22 subcutaneous tumor mouse model, using 2×10 6 H22 cells were suspended in 50 μL PBS and subcutaneously injected into the right ventral region of each mouse. After successful tumor bearing, intratumoral injection was performed. 177 Lu-PLGA radioactive microspheres (100 μCi) and 177 LuCl3 solution (100 μCi) was used for SPECT scans at different time points after injection, such as... Figure 6 As shown, 177 The signal from Lu-PLGA radioactive microspheres was still visible on the seventh day after injection, while 177 SPECT / CT images of the LuCl3 solution group show that, over time, 177Lu showed significant spread from the tumor site in mice to the whole body. More importantly, no radioactivity was found in other organs and tissues, indicating that the radioactive microspheres were very stable in vivo, providing a prerequisite for subsequent therapeutic experiments.

[0081] In order to accurately track 177 The distribution of Lu was determined using a gamma counter, which was used to measure the radioactivity of organs and tissues extracted after imaging studies. The results are as follows: Figure 7 As shown, this confirms 177 Lu-PLGA radioactive microspheres release almost no radiation. 177 Lu ions, because if delivered in free form, can be detected in other organs and tissues besides tumors, such as bones. 177 The radioactivity of Lu.

[0082] Finally, for 177 The degradation ability of Lu-PLGA radioactive microspheres was evaluated by using 5 mg of... 177 Lu-PLGA radioactive microspheres were placed in 10% FBS, and their morphology was observed under a light microscope every 20 days. Figure 8 As shown in 'a', it can be seen that 177 Lu-PLGA radioactive microspheres underwent significant degradation and gradual rupture after approximately two months. At each time point, the supernatant was removed, and the microspheres were lyophilized and weighed. The overall mass of the microspheres showed a gradual decrease, reaching 1.677 ± 0.066 mg at 100 days. Figure 8 As shown in b, based on existing research, nuclides decay through four half-lives ( 177 The safety level can be achieved within approximately one month, thus effectively demonstrating that radiotherapy can achieve optimal integration with embolization therapy.

[0083] Example 3

[0084] This embodiment evaluates the biosafety and efficacy of mesoporous PLGA microspheres using the Cell Counting Kit-8 (CCK8) cell assay. 177 The cytotoxicity of Lu-PLGA radioactive microspheres is demonstrated through the following procedures:

[0085] Different concentrations of mesoporous PLGA microspheres (0 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL) were incubated with human hepatocellular carcinoma cells (HepG2) and mouse hepatocellular carcinoma cells (H22) for 24 hours, respectively. The results are as follows: Figure 9 As shown in a, even at the highest concentration of 1000 μg / mL, mesoporous PLGA microspheres did not significantly inhibit cell viability.

[0086] Subsequently, different radioactive concentrations were... 177 Lu-PLGA radioactive microspheres (0 μCi / mL, 1.56 μCi / mL, 3.125 μCi / mL, 6.25 μCi / mL, 12.5 μCi / mL, 25 μCi / mL, 50 μCi / mL) were incubated with human hepatocellular carcinoma cells (HepG2) and mouse hepatocellular carcinoma cells (H22) for 24 hours, respectively. The results are as follows: Figure 9 As shown in b, 177 Lu-PLGA microspheres induced half-maximal cell death at 50 μCi / mL, demonstrating that the radionuclide... 177 Lu is responsible for cell damage.

[0087] Next, the results were assessed by γ-H2AX staining. 177 DNA double-strand breaks induced by Lu-PLGA radioactive microspheres were observed in HepG2 cells at 5 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per confocal microsphere. After 24 hours of culture, they were seeded separately with 1 mL of PBS, mesoporous PLGA microspheres (1 mg / mL), and [other solutions]. 177 Cells were treated with Lu-PLGA (1 mg / mL, 100 mCi / mL) radioactive microspheres for 12 hours. After treatment, cells were stained with γ-H2AX antibody and DAPI (4',6-diamidindo-2-phenylindole), and then observed and imaged using a confocal microscope. The confocal images are shown below. Figure 10 As shown, 177 Lu-labeled microspheres can cause extensive DNA damage in tumor cells.

[0088] In addition, a hemolysis experiment was conducted to verify whether mesoporous PLGA microspheres would cause hemolysis. Specifically, blood was collected from mouse eyeballs and treated with 5 mg of mesoporous PLGA microspheres, 1X PBS (pH 7.2-7.4), and ultrapure water, respectively, and then left to stand for half an hour. The samples were centrifuged, and the hemolysis was observed. The hemoglobin absorbance was measured using an ELISA reader, and the hemolysis rate was calculated. The results are shown below. Figure 11 As shown, the hemolysis rate of mesoporous PLGA microspheres is less than 0.5%, which effectively demonstrates their good biocompatibility.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A degradable 177 A method for preparing Lu-PLGA radioactive microspheres, characterized in that, The application relates to a method for preparing mesoporous polylactic acid-glycolic acid copolymer microspheres. The method comprises the following steps: preparing a primary oil-in-water emulsion by using a gelatin aqueous solution and a dichloromethane solution of polylactic acid-glycolic acid copolymer; collecting double emulsion droplets based on a microfluidic double emulsion system by taking the primary oil-in-water emulsion as an intermediate phase and taking a polyvinyl alcohol aqueous solution as an external water phase; purifying the double emulsion droplets after solidification and removal of gelatin, and obtaining dry mesoporous polylactic acid-glycolic acid copolymer microspheres after freeze-drying, which comprises the following steps: standing the double emulsion droplets to diffuse and volatilize dichloromethane, so as to form a porous polylactic acid-glycolic acid copolymer microsphere skeleton; transferring the porous polylactic acid-glycolic acid copolymer microsphere skeleton to a buffer solution containing collagenase for incubation to remove gelatin by enzymolysis, so as to form a mesoporous structure microsphere; washing the mesoporous structure microsphere, pre-freezing, and vacuum freeze-drying to obtain dry mesoporous polylactic acid-glycolic acid copolymer microspheres, wherein: the standing time ranges from 12 to 24 hours; and / or, the concentration of collagenase in the buffer solution containing collagenase ranges from 0.1 to 0.2 mg / mL; and / or, the incubation condition parameters include incubation at 32-37 DEG C for 2-4 hours; and / or, the pre-freezing condition parameters include pre-freezing at -80 to -60 DEG C for 12-24 hours; Utilizing 177 Lu radionuclide-labeled dried mesoporous polylactic-co-glycolic acid microspheres to obtain degradable 177 Lu-PLGA radiomicropsheres, comprising: and / or, the vacuum freeze-drying condition parameters include vacuum freeze-drying at a temperature of less than or equal to -60 DEG C and a vacuum degree of less than 0.1 mbar for 24-48 hours; To the dispersion was added 177 LuCl3, after the first incubation stage, a second incubation stage was continued after the addition of a potassium phosphate buffer solution, and the supernatant was discarded after centrifugation to obtain the labeled microspheres; The labeled microspheres are washed to obtain degradable 177 Lu-PLGA radioactive microspheres, wherein: uniformly dispersing the dry mesoporous polylactic acid-glycolic acid copolymer microspheres in ultrapure water to obtain a dispersion liquid; and / or, the 177 The radioactivity of LuCl3ranges from 0.5 to 2 mCi. the concentration of the dispersion liquid ranges from 1 mg:40 to 500 muL; and / or, the condition parameters of the first incubation stage include oscillation at 35-40 DEG C for 1-3 min, and the condition parameters of the second incubation stage include oscillation at 35-40 DEG C for 2-5 min; and / or, the pH of the potassium phosphate buffer solution ranges from 12 to 13, the concentration is 0.1-0.5 M, and the volume ratio of the dispersion liquid to the potassium phosphate buffer solution is 1:1-5; and / or, the centrifugation includes centrifugation at 3000-6000 rpm for 2-5 min; 2. The degradable 177 A method for preparing Lu-PLGA radioactive microspheres, characterized in that, and / or, the washing adopts phosphate buffered saline with a pH ranging from 7.2 to 7.

4. The method for preparing the primary oil-in-water emulsion by using the gelatin aqueous solution and the dichloromethane solution of polylactic acid-glycolic acid copolymer comprises the following steps: mixing the gelatin aqueous solution and the dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a mixed solution; 3. The degradable 177 A method for preparing Lu-PLGA radioactive microspheres, characterized in that, processing the mixed solution by using ultrasonic emulsification technology, and obtaining the primary oil-in-water emulsion after standing and removing air bubbles. The gelatin aqueous solution ranges from 5 to 10 wt%; and / or, the molar ratio of lactic acid and glycolic acid in the polylactic acid-glycolic acid copolymer ranges from 1 to 3:1; and / or, the molecular weight of the polylactic acid-glycolic acid copolymer ranges from 10 to 100 kDa; 4. The degradable 177 A method for preparing Lu-PLGA radioactive microspheres, characterized in that, and / or, the volume ratio of the gelatin aqueous solution to the dichloromethane solution of polylactic acid-glycolic acid copolymer ranges from 1:1 to 4. The microfluidic double emulsion system adopts a glass capillary or a polydimethylsiloxane microfluidic chip; and / or, the polyvinyl alcohol aqueous solution ranges from 0.5 to 2 wt%. And / or, the flow rate of the intermediate phase ranges from 1 to 2 µL / min and the flow rate of the external aqueous phase ranges from 3 to 6 µL / min.

5. A degradable 177 Lu-PLGA radioactive microspheres characterized in that, The degradable 177 The Lu-PLGA radioactive microspheres were prepared by the method.

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