A co-loaded vismodegib and bms-1 biomimetic nanodelivery system, and a preparation method and application thereof

Abstract

The application discloses a kind of co-loading Vismodegib and BMS-1 biomimetic nano delivery system and preparation method and application thereof, belong to the field of biological medicine technology.Vismodegib and BMS-1 are made into nanomicelle core in the present application, then red blood cell vesicle is used to make co-loading Vismodegib and BMS-1 biomimetic nano delivery system.It is verified by test that the biomimetic nano delivery system prepared in the present application inhibits the growth of mouse orthotopic tumor and the formation of lung / liver tumor metastasis, and the effect is significantly better than Vismodegib or BMS-1 alone, Vismodegib and BMS-1 are used in combination and the nano delivery carrier of single-loading Vismodegib or BMS-1.The present application provides a new idea for the clinical treatment of triple-negative breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by its negative estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 (HGF-2) status. It is characterized by its aggressiveness, high postoperative recurrence rate, and poor prognosis. Traditional endocrine therapy and targeted therapy are ineffective against TNBC, but the rise of immunotherapy has brought hope to TNBC patients. Immune checkpoint inhibitors targeting programmed death protein-1 (PD-1) or programmed death-ligand 1 (PD-L1) have provided clinical benefit to some PD-L1-positive patients as monotherapy or in combination with chemotherapy. However, the high heterogeneity of TNBC and its dysfunctional tumor immune microenvironment lead to poor treatment response or drug resistance in most patients. Studies show that a key feature of TNBC is the presence of dense fibrotic stroma, often accompanied by overactive cancer-associated fibroblasts (CAFs) and excessive extracellular matrix (ECM) deposition. Dense interstitium can increase solid stress, raise interstitial fluid pressure, and compress tumor blood vessels, thereby establishing a series of pathological barriers that block the transport and infiltration of circulating therapeutic drugs or effector T cells into the tumor parenchyma.

[0003] Previous studies have confirmed that tumor cells secrete Hedgehog ligands such as Shh to regulate fibroblasts in the tumor microenvironment, promoting their activation into cancer cells (CAFs). The pro-fibrotic matrix secreted by CAFs forms a dense and robust physical barrier around the tumor nest. Furthermore, overactivated Hedgehog signaling pathways can also promote the polarization of tumor-recruited macrophages towards the pro-tumor M2 phenotype. Therefore, effectively inhibiting the Hedgehog signaling pathway may improve the efficacy of TNBC immunotherapy by suppressing CAF activation and increasing the M1 / M2 infiltration ratio of tumor-associated macrophages. Preclinical studies have shown that the Hedgehog signaling pathway inhibitor vismodegib reduces the degree of extracellular matrix fibrosis by inhibiting CAF activation, reducing compression of tumor vessels, increasing therapeutic drug perfusion, and reversing CD8+. + T-cell immune rejection of tumors increases the number of cytotoxic immune cells within the tumor, restoring the therapeutic response to immune checkpoint blockade therapy. Summary of the Invention

[0004] The purpose of this invention is to provide a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1, its preparation method, and its application, in order to solve the problems existing in the prior art. The biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​prepared by this invention can be used in the treatment of triple-negative breast cancer.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1, comprising the following steps:

[0007] Add Vismodegib solution and BMS-1 ​​solution to mPEG 5K -PAE 10K The organic phase was obtained by vortexing in the solution and then added to the aqueous phase PBS. After stirring, sonication, centrifugation and filtration, the nanomicelle cores V / B@NM were obtained.

[0008] A biomimetic nanomedicine solution was prepared by co-extruding V / B@NM with erythrocyte vesicles using liposomes. After centrifugation, a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​was obtained.

[0009] Optionally, the erythrocyte vesicles are prepared by the following steps:

[0010] Red blood cells are added to PBS and swell. After centrifugation, a white precipitate is obtained, which is the red blood cell membrane.

[0011] After being treated with ultrasound, the red blood cell membrane is extruded using a liposome extruder to obtain red blood cell vesicles.

[0012] Preferably, the Vismodegib solution is obtained by dissolving Vismodegib in dimethyl sulfoxide, the BMS-1 ​​solution is obtained by dissolving BMS-1 ​​in tetrahydrofuran, and the mPEG... 5K -PAE 10K Solution by mPEG 5K -PAE 10K Obtained by dissolving in tetrahydrofuran;

[0013] Vismodegib, BMS-1 ​​and mPEG 5K -PAE 10K The mass ratio is 0.5:0.5:10;

[0014] The volume ratio of the organic phase to the aqueous phase is 1:2.

[0015] Preferably, the stirring conditions are 400 rpm and 50°C for 4 hours;

[0016] The ultrasound conditions were: power 450W, ultrasound duration 2 seconds, interval 2 seconds, total time 10 minutes.

[0017] Preferably, the mass ratio of V / B@NM to erythrocyte vesicles is 1:1.

[0018] The present invention also provides a biomimetic nanodelivery system for co-loaded Vismodegib and BMS-1 ​​prepared according to the preparation method described above.

[0019] The present invention also provides the application of the aforementioned biomimetic nanodelivery system in the preparation of drugs that inhibit the growth and metastasis of triple-negative breast cancer.

[0020] The present invention also provides the application of the aforementioned biomimetic nanodelivery system in the preparation of drugs that enhance the immunotherapy effect of triple-negative breast cancer.

[0021] The present invention also provides a medicament for treating triple-negative breast cancer, the medicament comprising the aforementioned biomimetic nanodelivery system.

[0022] Optionally, the drug may also contain pharmaceutically acceptable excipients.

[0023] The present invention discloses the following technical effects:

[0024] Biomimetic nanodelivery systems based on cell membrane modification utilize in vivo biological membranes to biomimeticly modify functionalized nanocarriers, effectively combining the "self-sustaining" properties of natural biological membranes with the advantages of "artificial" nanocarriers. This can significantly improve the biocompatibility of nanomedicines, reduce immunogenicity, prolong blood circulation time, while simultaneously enhancing tumor targeting and the effectiveness of immunotherapy, and mitigating off-target toxicity. Based on this, this invention utilizes a pH-responsive block copolymer carrier to encapsulate the Hedgehog signaling pathway inhibitor Vismodegib and the PD-1 / PD-L1 interaction inhibitor BMS-1 ​​into a nanomicelle core. A red blood cell membrane coating is then applied to the surface of the nanomicelle core to create a co-loaded Vismodegib and BMS-1 ​​biomimetic nanodelivery system, designed to improve the efficacy of immunotherapy for triple-negative breast cancer.

[0025] Experimental verification showed that the biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​prepared in this invention inhibited the growth of in situ tumors and the formation of lung / liver tumor metastases in mice, with significantly better effects than Vismodegib or BMS-1 ​​alone, Vismodegib and BMS-1 ​​in combination, and nanodelivery carriers carrying Vismodegib or BMS-1 ​​alone. This inhibitory effect is related to Vismodegib's inhibition of CAF activation, remodeling of the tumor extracellular matrix (such as reduced collagen fiber formation and decreased Collagen I expression), promotion of M2 tumor-associated macrophage polarization to M1, and synergistic reprogramming of the tumor immunosuppressive microenvironment and enhancement of CD8 by the PD-1 / PD-L1 inhibitor BMS-1. + T-cell infiltration is closely related to the immune response. This invention provides a new approach for the clinical treatment of triple-negative breast cancer. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Characterization of the biomimetic nanodelivery system; A: Morphology of V / B@NM; B: Morphology of RV / B@NM; C: Particle size of V / B@NM and RV / B@NM; D: Expression of erythrocyte membrane protein CD47 on RV / B@NM; E: pH-responsive drug release of Vismodegib and BMS-1;

[0028] Figure 2 The distribution of RV / B@NM in various organs and tumor tissues of tumor-bearing mice over time; A: Distribution of various organs and tumor tissues in tumor-bearing mice within 24 hours after injection in each group; B: Relative fluorescence intensity of Figure A; C: Accumulation of various organs and tumor tissues in each group 24 hours after injection in each group; D: Relative fluorescence intensity of Figure C.

[0029] Figure 3 The effects of RV / B@NM on the growth of orthotopic tumors in tumor-bearing mice were investigated. A: Effects of each group on the survival time of tumor-bearing mice; B: Effect of the biomimetic nanodelivery system on the volume of orthotopic tumors in tumor-bearing mice; C: Tumor inhibition rate of the biomimetic nanodelivery system; D: Pathological analysis of orthotopic tumors in tumor-bearing mice using the biomimetic nanodelivery system; E: Immunohistochemical analysis of Ki-67 orthotopic tumors in tumor-bearing mice using the biomimetic nanodelivery system.

[0030] Figure 4The effects of RV / B@NM on lung / liver metastases in tumor-bearing mice; A: Representative photographs of the effects of each group on lung metastases in tumor-bearing mice and statistical graphs of the effects on lung metastatic tumor nodules in mice, where red circles represent metastatic tumor nodules; B: Pathological analysis of lung metastatic tumor lesions in tumor-bearing mice in each group; C: Pathological analysis of liver metastatic tumor lesions in tumor-bearing mice in each group.

[0031] Figure 5 The effect of RV / B@NM on collagen production in tumor tissue;

[0032] Figure 6 RV / B@NM targets CAF markers α-SMA, extracellular matrix protein Collagen I, and CD8 in tumor tissue. + The effect of T cell marker CD8α expression;

[0033] Figure 7 For RV / B@NM to target CD8 in tumor tissue + The influence of T cell marker expression;

[0034] Figure 8 The effect of RV / B@NM on the expression of markers of M2 tumor-associated macrophages (M2-TAMs) and M1 tumor-associated macrophages (M1-TAMs) in tumor tissues;

[0035] Figure 9 Safety assessment of RV / B@NM; A: Pathological analysis of the heart, spleen and kidney of tumor-bearing mice by the biomimetic nanodelivery system; B: Effects on liver and kidney function in tumor-bearing mice; C: Effects on blood routine tests in tumor-bearing mice. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Example 1

[0042] 1. Experimental materials

[0043] 1.1 Experimental Cells

[0044] The mouse triple-negative breast cancer cell line 4T1 was purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. 4T1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at a sterile environment of 37°C in a 5% CO2 incubator.

[0045] 1.2 Laboratory Animals

[0046] One hundred BALB / c female mice aged 5–6 weeks and weighing between 16 ± 2.0 g were purchased from Beijing Vital River Laboratory Animal Co., Ltd., and all animals were housed in an SPF-grade barrier enclosure at the Animal Center of Ningxia Medical University.

[0047] 1.3 Main Experimental Reagents and Chemicals

[0048] Vismodegib (L10258B007) was purchased from Adooq Bioscience; BMS-1 ​​(RNO232051) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; mPEG 5K -PAE 10K(R-650051-15K) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; Cy7 (R-CY2007) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; CD8 (584230), CD86 (560582), CD3 (553061), CD206 (571024), and F4 / 80 (565410) were all purchased from Youningwei Biotechnology Co., Ltd.

[0049] 2. Experimental Methods

[0050] 2.1 Preparation, optimization, characterization, and in vitro drug release of the Vismodegib and BMS-1 ​​co-loaded biomimetic nanodelivery system

[0051] 2.1.1 Preparation of Vismodegib and BMS-1 ​​co-loaded biomimetic nanodelivery system (RV / B@NM)

[0052] 2.1.1.1 Preparation of V / B@NM

[0053] V / B@NM was prepared by a self-assembly-solvent evaporation method.

[0054] ① Weigh 0.1 mg Vismodegib and add it to 50 μL of dimethyl sulfoxide (DMSO), and add 0.1 mg BMS-1 ​​to 50 μL of tetrahydrofuran (THF). The mass ratio of the two drugs is 1:1.

[0055] ②2mg mPEG 5K -PAE 10K Add to 100 μL THF, mix thoroughly with a pipette until completely dissolved, then add the Vismodegib solution and BMS-1 ​​solution to mPEG. 5000 -PAE 10000 After vortexing the solution, add it dropwise over 1 minute to 1 mL of PBS (pH 7.4) (organic phase: aqueous phase = 1:5).

[0056] ③ After stirring with a magnetic stirrer at 400 rpm and 50℃ for 4 hours, use a probe-type ultrasonic instrument to perform ultrasonication in an ice bath at a power of 450W, for 2 seconds at a time, with a 2-second interval, for a total of 10 minutes.

[0057] ④ Use a low-temperature high-speed centrifuge (4000 rpm, 10 min) to remove free mPEG. 5K -PAE 10K Polymers, BMS-1, and Vismodegib.

[0058] ⑤ Then filter with a 0.22μm filter membrane to obtain V / B@NM solution.

[0059] 2.1.1.2 Preparation of Red Blood Cell Vesicles

[0060] ① After anesthetizing BALB / c mice with sodium pentobarbital, 0.5 mL of blood was added to a 10 mL centrifuge tube containing 200 μL of heparin sodium. 1×PBS buffer was added to about 5 mL, and the mixture was repeatedly aspirated with a dropper. The mixture was centrifuged for 5 min (4℃, 3500 rpm) to remove the supernatant plasma and white blood cells. After pre-cooling with 1×PBS, the mice were washed twice to obtain red blood cells.

[0061] ② Add 0.25×PBS (10 times the volume), incubate at 4℃ for 2 hours to allow full expansion in hypotonic solution, centrifuge for 10 minutes (4℃, 12000 rpm), discard the supernatant, and centrifuge twice more with 1×PBS (centrifugation conditions: 4℃, 12000 rpm). The resulting white precipitate is the pure red blood cell membrane. Dissolve the white precipitate in 2 mL of 1×PBS solution.

[0062] ③ Finally, the red blood cell membrane was treated with 200W ultrasound in a 4℃ water bath for 10 minutes, and then the cell membrane was passed through 50nm and 20nm polycarbonate films in sequence using a liposome extruder, with each pore diameter being 9 times, to obtain red blood cell vesicles.

[0063] 2.1.1.3 Preparation of RV / B@NM

[0064] ① The total red blood cell vesicles and nano-drugs were co-extruded and vibrated under ultrasound for 3 minutes to ensure uniformity.

[0065] ② Using a liposome extruder, the solution was passed sequentially through 200nm and 100nm porous polycarbonate films 11 times each to obtain a biomimetic nanomedicine solution.

[0066] ③ Introduce the biomimetic nanomedicine solution into an ultrafiltration tube, centrifuge for 15 min (1500 rpm) to remove free nanoparticles, and obtain RV / B@NM. Store at 4℃ for later use.

[0067] 2.1.2 Optimization of the preparation process of nanomicelle cores co-loaded with Vismodegib and BMS-1 ​​(V / B@NM)

[0068] Based on the experimental methods described above, the optimal preparation process for V / B@NM was determined by studying the effects of ultrasonic intensity, organic-to-aqueous phase volume ratio, ultrasonic emulsification time, carrier-to-drug mass ratio, and stirring temperature on the obtained V / B@NM.

[0069] 2.1.2.1 Selection of Ultrasonic Intensity

[0070] When preparing nanosystems, ultrasonic probes are needed for dispersion; however, excessively strong ultrasound can cause breakage and uneven dispersion of the nanosystems. When the ultrasonic intensity is too low, the nanoparticle size increases. Experimental results are shown in Table 1. When the ultrasonic power used is 450W, the nanomedicine has a smaller particle size.

[0071] Table 1. Effect of ultrasonic intensity on biomimetic nanomedicines

[0072]

[0073] 2.1.2.2 Selection of the volume ratio of organic phase to aqueous phase

[0074] The size of the nanosystem was controlled by adjusting the organic / aqueous phase volume ratio (1:2, 1:4, 1:6, 1:8, 1:10). Table 2 shows that the ratio of organic to water has a significant impact on the size of the nanosystem. A suitable water phase ratio facilitates the diffusion of the organic phase into the aqueous phase and reduces its migration, thus avoiding particle size increase due to insufficient space. Analysis of the experimental results determined the optimal organic-to-aqueous phase ratio to be 1:2.

[0075] Table 2. Effect of organic phase / aqueous phase volume ratio on biomimetic nanomedicines

[0076]

[0077] 2.1.2.3 Effect of ultrasonic emulsification time on V / B@NM

[0078] Ultrasonic emulsification is a crucial step in the preparation of nanomedicines. However, if the ultrasonic treatment time is too short, the organic and aqueous phases cannot complete the emulsion process, resulting in significant size variations in the prepared nanosystems. Conversely, excessive ultrasonic treatment time can easily cause the nanosystems to break down. Therefore, the appropriate ultrasonic treatment time is a key factor determining the quality of the nanosystem. Under magnetic field stirring, an ultrasonic treatment time of 10 minutes yielded the smallest drug-loaded nanoparticle size, with a particle size index (PDI) < 0.2.

[0079] Table 3. Effect of ultrasound time on biomimetic nanomedicines

[0080]

[0081] 2.1.2.4 Effect of carrier to drug mass ratio on V / B@NM

[0082] Based on the above, this invention aims to study the effect of changing the mass ratio of carrier to drug on the particle size of nanomedicines. However, when the drug mass is too large, it is difficult to encapsulate and form nanoparticles, and a large amount of sedimentation occurs, leading to drug loss and decreased drug loading efficiency. At the same time, the size of the obtained nanoparticles is too large. Therefore, a carrier mass: drug mass = 10:1 was selected as the optimal mixing ratio, as shown in Table 4.

[0083] Table 4. Effect of carrier-to-drug mass ratio on biomimetic nanomedicines

[0084]

[0085] 2.1.2.5 Effect of stirring temperature on V / B@NM

[0086] Higher temperatures lead to more intense Brownian motion and collisions between molecules, making it less likely for them to aggregate. However, there is a temperature threshold. After screening experimental conditions, the smallest nanoparticle size was obtained when the stirring temperature was 50℃.

[0087] Table 5 Effect of stirring temperature on the preparation of biomimetic nanosystems

[0088]

[0089]

[0090] Based on the above results, an optimal method for preparing nanomicelle cores (V / B@NM) co-loaded with Vismodegib and BMS-1 ​​was established:

[0091] V / B@NM was prepared using a self-assembly-solvent evaporation method. 0.1 mg Vismodegib was added to 200 μL of dimethyl sulfoxide (DMSO), and 0.1 mg BMS-1 ​​was added to 200 μL of tetrahydrofuran (THF), with a drug-to-drug mass ratio of 1:1. 2 mg of mPEG was also added. 5K -PAE 10K Add to 100 μL THF, mix thoroughly with a pipette until completely dissolved, and measure the carrier mass (2 mg mPEG). 5K -PAE 10K The drug-to-mass ratio (0.1 mg Vismodegib + 0.1 mg BMS-1) is 10:1. The Vismodegib and BMS-1 ​​solutions are added to the mPEG solution. 5K -PAE 10KAfter vortexing to mix, the solution was added dropwise over 1 minute to 1 mL of PBS (pH 7.4), with an organic phase ratio of 1:2 (200 μL DMSO, 300 μL THF) to aqueous phase (1 mL PBS). The mixture was stirred with a magnetic stirrer at 400 rpm and 50°C for 4 hours, followed by sonication in an ice bath using a probe-type sonicator at 450 W, with 2-second intervals for a total of 10 minutes. The supernatant free mPEG was removed by low-temperature high-speed centrifugation (4000 rpm, 10 minutes). 5K -PAE 10K Polymers, BMS-1, and Vismodegib were then used. The nanomicelle cores V / B@NM were obtained by filtration through a 0.22 μm sterile filter membrane.

[0092] Based on this, the influence of different total red blood cell vesicle counts to nanomedicine mass ratios on the biomimetic nanosystem was investigated. Finally, the preparation process of the biomimetic nanosystem was obtained. The results of investigating the influence of different total red blood cell vesicle counts to nanomedicine mass ratios on the biomimetic nanosystem are shown in Table 6 below. It can be seen that when the mass ratio is 1:1, the prepared RV / B@NM particles have the best particle size and dispersion index.

[0093] Table 6 Total Erythrocyte Vesicle Count: The Influence of Nanodrug Quality on Biomimetic Nanodrugs

[0094]

[0095] Therefore, based on the optimized formulation in Table 6, RV / B@NM was prepared using a co-extrusion method with a ratio of total erythrocyte vesicles to nanomicelle core mass of 1:1. Specifically, a certain number of erythrocyte vesicles were taken, V / B@NM was added, and the mixture was vibrated under ultrasonication for 3 min to ensure homogenization. Using a liposome extruder, the mixture was sequentially passed through 200 nm and 100 nm porous polycarbonate films, 11 times each, to obtain a biomimetic nanomedicine solution. This solution was then introduced into an ultrafiltration tube and centrifuged for 15 min (1500 rpm) to remove free nanoparticles, thus obtaining the erythrocyte membrane-modified biomimetic nanodelivery system RV / B@NM, which was stored at 4℃ for later use.

[0096] Simultaneously, the Vismodegib-loaded biomimetic nanodelivery system RV@NM was prepared using the same method as RV / B@NM, except that 0.2 mg of Vismodegib was added to 400 μL of dimethyl sulfoxide (DMSO). 2 mg of mPEG was also added. 5K -PAE 10K Add to 100 μL THF, mix thoroughly with a pipette until completely dissolved, and measure the carrier mass (2 mg mPEG). 5K -PAE 10KThe ratio of Vismodegib solution to drug mass (0.2 mg Vismodegib) is 10:1. Add the Vismodegib solution to mPEG. 5K -PAE 10K in solution.

[0097] Preparation of the single-loaded BMS-1 ​​biomimetic nanodelivery system RB@NM: The preparation method is the same as that of RV / B@NM, except that 0.2 mg of BMS-1 ​​was added to 400 μL of tetrahydrofuran (THF). 2 mg of mPEG was also added. 5K -PAE 10K Add to 100 μL THF, mix thoroughly with a pipette until completely dissolved, and measure the carrier mass (2 mg mPEG). 5K -PAE 10K The ratio of drug mass (0.2 mg BMS-1) to mPEG is 10:1. Add the BMS-1 ​​solution to the mPEG. 5K -PAE 10K in solution.

[0098] 2.1.3 Characterization of drug-loaded biomimetic nanosystems

[0099] The physicochemical properties of the biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1, prepared according to the optimal method, were investigated. This invention mainly evaluates the physicochemical properties of the nanosystem from four aspects: particle size distribution, zeta potential, drug loading and encapsulation efficiency, and surface morphology of the nanosystem.

[0100] (1) Particle size and distribution of nanosystems, and determination of zeta potential

[0101] 1 mL of biomimetic nanomedicine solution (V / B@NM and RV / B@NM, three replicates) was taken and the particle size, zeta potential and dispersion index (PDI) of the biomimetic nanomedicine were determined by dynamic light scattering at room temperature.

[0102] (2) Determination of encapsulation efficiency and drug loading of Vismodegib and BMS-1

[0103] Take the precipitate after centrifugation, add 10 times the amount of methanol, mix well, precipitate the polymer, filter the sample through a 0.22μm organic microporous membrane, and take the filtrate as the free drug. Use HPLC to determine the drug content and encapsulation rate.

[0104] Conditions for HPLC determination of Vismodegib content:

[0105] Column: Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm);

[0106] Mobile phase: 0.1% CH3OH (mobile phase A), H2O (mobile phase B), 0.1% CH3CN (mobile phase C), the proportions of which are shown in Table 7;

[0107] Flow rate: 1.0 mL / min; column temperature: 25℃; detection wavelength: 236 nm; injection volume: 10 μL.

[0108] Table 7 Conditions for determining Vismodegib content with different mobile phase ratios

[0109]

[0110] Conditions for HPLC determination of BMS-1 ​​content:

[0111] Column: Agilent ZORBAX Eclipse Plus C18 (4.6×250mm, 5μm);

[0112] Mobile phase: CH3CN (mobile phase A), H2O (mobile phase B), the proportions of which are shown in Table 8;

[0113] Flow rate: 1.0 mL / min; column temperature: 25℃; detection wavelength: 280 nm; injection volume: 10 μL.

[0114] Table 8. Conditions for determining BMS-1 ​​content under different mobile phase ratios

[0115]

[0116] Formulas for calculating encapsulation efficiency and drug loading:

[0117] Drug loading = Mass of BMS-1 ​​or Vismodegib in the nanomedicine / Mass of the nanomedicine × 100%;

[0118] Encapsulation efficiency = (mass of BMS-1 ​​or Vismodegib in the nanomedicine / dosage of BMS-1 ​​or Vismodegib) × 100%.

[0119] (3) Characterization of RV / B@NM membrane proteins

[0120] The content of erythrocyte membrane proteins in RV / B@NM was measured by Western blot, with CD47 selected as the erythrocyte membrane marker protein.

[0121] 2.1.4 In vitro drug release performance of pH-responsive drug-loaded nanosystems

[0122] The release rate of biomimetic nanomedicines at pH 7.4, pH 6.5, and pH 5.0 was evaluated using in vitro dialysis. Details are as follows:

[0123] ① Place three batches of fresh biomimetic nanomedicine suspensions, tighten both ends of the dialysis bag (8000D, 24mm wide), and put the sample into an Erlenmeyer flask containing 50mL of dissolving solution. Stir at 37℃ and 1200rpm.

[0124] ② At predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48h), collect 1mL of the release solution, add an equal volume of fresh dissolution solution, and filter through a 0.22μm filter membrane.

[0125] ③ The drug release rate of biomimetic nanomedicines was analyzed by high performance liquid chromatography, and the cumulative release curves of Vismodegib and BMS-1 ​​were calculated.

[0126] 2.2 Small animal live imaging

[0127] A mouse triple-negative breast cancer tumor model was established by inoculating 4T1 triple-negative breast cancer cells into the mammary fat pads of BALB / c mice. Mice with successful modeling were randomly divided into three groups (n=8): the Cy7 group (injected via tail vein with free Cy7 dye solution), the V / B@NM (Cy7) group (injected via tail vein with Cy7-modified V / B@NM), and the RV / B@NM (Cy7) group (injected via tail vein with Cy7-modified RV / B@NM). The average tumor volume was approximately 500 mm². 3 In vivo targeted distribution experiments were conducted. Mice were anesthetized with 0.3% sodium pentobarbital via intraperitoneal injection (0.10 mL / 10 g). Imaging was performed at 2, 6, 12, and 24 hours using an IVIS spectral imager with excitation / emission wavelengths of 750 / 773 nm. Subsequently, the major organs (spleen, liver, heart, lung, and kidney) and tumor tissues of the mice were removed for ex vivo imaging, and the results were analyzed using imaging system analysis software.

[0128] 2.3 Pharmacodynamic evaluation of the Vismodegib and BMS-1 ​​co-loaded biomimetic nanodelivery system

[0129] A mouse triple-negative breast cancer tumor model was established by inoculating 4T1 triple-negative breast cancer cells into the mammary fat pads of BALB / c mice. The average tumor volume of the tumor-bearing mice reached 100 mm. 3At approximately 10:00 AM, the mice were randomly divided into 7 groups (n=10) and treated according to the following administration regimens: (1) Model group: After modeling, the mice were injected with the same volume of physiological saline as the experimental group via tail vein for 3 days; (2) Vismodegib group: Vismodegib was injected via tail vein at 360 μg / kg / 3 days; (3) BMS-1 ​​group: BMS-1 ​​was injected via tail vein at 345 μg / kg / 3 days; (4) Vismodegib combined with BMS-1 ​​group: Vismodegib (208 μg / kg / 3 days) and BMS-1 ​​were injected via tail vein. 1 (204 μg / kg / 3d); (5) RV@NM group: RV@NM was administered via tail vein injection, 5 mg / kg / 3d (Vismodegib dosage was 360 μg, with a drug loading of 7.2%); (6) RB@NM group: RB@NM was administered via tail vein injection, 5 mg / kg / 3d (BMS-1 ​​dosage was 345 μg, with a drug loading of 6.9%); (7) RV / B@NM group: RV / B@NM was administered via tail vein injection, 5 mg / kg / 3d (Vismodegib dosage was 208 μg, BMS-1 ​​dosage was 204 μg). Normal mice served as the blank control group.

[0130] During the experiment, the mortality of mice in each group was recorded to monitor the impact of each group on the survival of tumor-bearing mice.

[0131] The length 'a' and longitudinal length 'b' of the tumor were measured using the vernier caliper method, and the tumor volume V = a × b was calculated. 2 / 2, and plotted tumor growth curves; the day after treatment, mice were euthanized by cervical dislocation, and tumors were dissected to measure tumor weight and calculate tumor inhibition rate. A portion of tumor tissue was also taken to prepare a single-cell suspension, and flow cytometry was used to analyze the CD8+ of tumors in each group of mice. + The expression of T cell markers CD3 and CD8; M2-TAMs markers F4 / 80 and CD206; M1-TAMs markers F4 / 80 and CD86; fixation of some tumor tissues for pathological histological staining and immunohistochemistry; and excising tissues from the heart, lungs, liver, spleen, and kidneys for pathological analysis.

[0132] 2.4 Multicolor immunofluorescence analysis

[0133] The tyramide signal amplification (TSA) technique was used to achieve fluorescence imaging of multiple target proteins. In short, after antigen retrieval, tumor tissue sections were first incubated with 3% H2O2 at room temperature. Then, the blocked tissue sections were incubated with α-SMA primary antibody, followed by incubation with the corresponding horseradish peroxidase-labeled goat anti-rabbit IgG. After washing, the sections were incubated with freshly prepared TSA dye working solution (diluted 1:100 with signal amplification solution). Sections after the first round of staining were placed in antigen retrieval solution for a second and third round of retrieval, and incubated with Collagen I and CD8α primary antibodies and their corresponding secondary antibodies, respectively. Finally, the cell nuclei were counterstained with DAPI and the sections were mounted. Imaging was recorded using a fluorescence scanning imaging system.

[0134] 2.5 Liver and kidney function and blood routine tests in tumor-bearing mice

[0135] After treatment, blood was collected from the orbital cavity of the mice, allowed to stand for 1–2 hours, centrifuged (3000 rpm, 10 min), and the supernatant pale yellow transparent liquid was aspirated with a pipette to obtain mouse serum. The serum was processed according to the kit instructions, and changes in alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen levels were measured in each group of mice.

[0136] Separately, mouse blood was collected, and a reagent kit was used to detect changes in blood routine indicators in each group of mice, including white blood cells, lymphocytes, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red blood cell distribution width.

[0137] 3 Results and Analysis

[0138] 3.1 Characterization of RV / B@NM

[0139] V / B@NM was successfully prepared using a self-assembly-solvent evaporation method. RV / B@NM was then prepared by co-extruding erythrocyte vesicles with V / B@NM using a liposome extruder. The morphology and particle size of the nanosystems were analyzed using transmission electron microscopy and a laser particle size analyzer. The results showed that both V / B@NM and RV / B@NM were spherical with uniform shapes. The particle sizes of V / B@NM and RV / B@NM were 149.2±3.0 nm and 184.6±1.4 nm, respectively. The latter, due to its outer layer of erythrocyte membrane, had a particle size approximately 30 nm larger than the former. Figure 1 Furthermore, the PDI of RV / B@NM was measured to be 0.17±0.04, indicating a relatively uniform particle size distribution. The Zeta potential was -22.71±1.87mV (Table 10), and the expression of erythrocyte membrane protein CD47 in RV / B@NM was detected by Western blot. Figure 1 (D). The above results fully demonstrate that the erythrocyte membrane was successfully coated on the V / B@NM surface.

[0140] Table 10 Particle size, Zeta potential, and PDI of R-V / B@NM

[0141]

[0142] The encapsulation efficiency and drug loading of Vismodegib and BMS-1 ​​in RV / B@NM were determined by high performance liquid chromatography (HPLC). The results are shown in Table 11. The encapsulation efficiency of Vismodegib was 72.48±1.23%, and the drug loading was 4.16±0.05%. The encapsulation efficiency of BMS-1 ​​was 70.51±0.89%, and the drug loading was 4.07±0.03%.

[0143] Table 11 Encapsulation efficiency and drug loading of Vismodegib and BMS-1 ​​in R-V / B@NM

[0144]

[0145] Furthermore, the pH-responsive drug release of RV / B@NM was determined using high-performance liquid chromatography. For example... Figure 1 As shown in Figure E, the drug release of Vismodegib and BMS-1 ​​in the biomimetic nanodelivery system gradually increased as the pH decreased from 7.4 to 5.0. At pH 5.0, the maximum drug release after 50 hours reached 81.62% and 91.05%, respectively, indicating that the Vismodegib and BMS-1 ​​co-loaded biomimetic nanodelivery system possesses acid-responsive drug release characteristics, which is beneficial for the controlled release of drugs in the acidic tumor microenvironment in vivo.

[0146] Distribution of 3.3RV / B@NM tumor-bearing mice in vivo

[0147] Tumor-bearing mice were administered Cy7-labeled nanomedicine via the tail vein, and drug accumulation in the mice was measured at 2, 6, 12, and 24 hours using a small animal in vivo imaging system. Figure 2 According to AB, compared with the V / B@NM(Cy7) group, the fluorescence intensity of various organs in mice in the RV / B@NM(Cy7) group was significantly enhanced, and the fluorescence accumulation intensity at the tumor site was more obvious over time; subsequently, the fluorescence intensity of Cy7 was detected in isolated tumors and major organs (heart, lung, liver, spleen, and kidney). Figure 2 As observed in the CD, compared with the V / B@NM group, the RV / B@NM group showed stronger tumor fluorescence, while the fluorescence intensity of other major organs was relatively weaker, indicating that the erythrocyte membrane-modified biomimetic nanodrug RV / B@NM can effectively accumulate in the tumor site of tumor-bearing mice.

[0148] 3.4RV / B@NM inhibits in situ tumor growth and lung / liver metastasis in tumor-bearing mice.

[0149] To preliminarily evaluate the efficacy of RV / B@NM, the effect of RV / B@NM on the survival of tumor-bearing mice was first monitored. The results are as follows: Figure 3 As shown in Figure A, all mice in the model group, Vismodegib group, BMS-1 ​​group, Vismodegib combined with BMS-1 ​​group, RV@NM group, and RB@NM group died on days 40, 43, 47, 48, 46, and 47, respectively, with a survival rate of 0%. However, the survival rate of mice in the RV / B@NM group was still 40% on day 51, indicating that RV / B@NM can effectively prolong the survival time of tumor-bearing mice.

[0150] Based on the established advantage of biomimetic nanodelivery systems over free drugs in antitumor efficacy, this invention further evaluates the inhibitory effect of biomimetic nanodelivery systems on tumor growth in tumor-bearing mice. For example... Figure 3 As shown in Figure B, the tumor volume of the model group mice increased exponentially. After administration of RV@NM or RB@NM, the tumor growth rate of the tumor-bearing mice slowed down. However, after administration of RV / B@NM, the tumor volume growth rate was significantly reduced, and tumor growth even stopped in the later stage of treatment. Figure 3 The tumor inhibition rate results of RV / B@NM further verified that the RV / B@NM treatment group could significantly inhibit the growth of in situ tumors in mice. Compared with the tumor inhibition rates of RV@NM and RB@NM (36.4% ± 3.6% and 43.8% ± 2.2%, respectively), the inhibition rate of RV / B@NM reached 71.4% ± 4.3%, indicating that RV / B@NM significantly improved the inhibitory effect on tumor growth. Pathological analysis of mouse tumors using H&E staining technology showed that the tumor cells in the model group had large nuclei with significant mitosis and dense chromatin staining; while after treatment with RV@NM, RB@NM, and RV / B@NM, the tumor cells showed varying degrees of morphological changes such as shrinkage, vacuolation, and nuclear chromatin condensation, especially the RV / B@NM group showed the most significant changes. Figure 3 In addition, immunohistochemical detection of Ki-67 expression showed that the RV / B@NM group mice had the weakest tumor nuclear proliferation ability, indicating that it significantly inhibited tumor cell division and proliferation. Figure 3 (E).

[0151] Furthermore, the number of metastatic tumor nodules on the surface of the excised lung tissue was counted. The results showed that the number of metastatic tumor nodules in the lung tissue of the model group mice was approximately 17.0 ± 2.0. After treatment with RV@NM, RB@NM, and RV / B@NM, the number of metastatic tumor nodules in the lung tissue of mice decreased to 7.0 ± 2.0, 5.0 ± 1.0, and 2.0 ± 1.0, respectively. Figure 4In section A, H&E staining was used to perform pathological evaluation of mouse lung and liver tissues. Figure 4 The results from the BC study showed that the model group mice had a large number and area of ​​tumor metastatic lesions in the lung and liver tissues. Treatment with RV@NM, RB@NM, and RV / B@NM reduced the number and size of tumor lesions, with the RV / B@NM treatment group showing the most significant inhibitory effect on metastasis. These results indicate that RV / B@NM can significantly inhibit the spontaneous metastasis of tumors from their original location to distant organs (liver / lung) in tumor-bearing mice.

[0152] 3.5RV / B@NM remodels the extracellular matrix of mouse tumor cells and increases CD8. + T-cell intratumoral infiltration

[0153] The fibrogenic matrix secreted by CAFs forms a tight physical barrier around the cancer nest, hindering CD8... + T cell infiltration into the tumor parenchyma leads to tumor immune rejection. First, the effect of RV / B@NM on collagen fiber content in the extracellular matrix of mouse tumor cells was assessed using Sirius red and Masson's staining. Figure 5 As shown, the higher collagen fiber content in the tumors of the model group indicates a higher degree of extracellular matrix fibrosis, while the collagen fiber content in mouse tumors significantly decreased after RV / B@NM treatment. Further analysis of activated CAFs markers α-SMA, extracellular matrix proteins Collagen I and CD8 in tumor tissues of mice in each group of mice... + Multicolor immunofluorescence analysis of the T cell marker CD8α showed that α-SMA and Collagen I were highly expressed in the model group, while CD8α was lowly expressed, indicating that the matrix components secreted by activated CAFs weakened CD8α expression. + T cell infiltration into tumors, while the expression of α-SMA and Collagen I decreased and the expression of CD8α increased in mouse tumors after RV / B@NM treatment. Figure 6 Further flow cytometry analysis was used to analyze CD8+ in mouse tumor tissue. + T infiltration analysis also confirmed that RV / B@NM significantly increased intratumoral CD8. + The infiltration ratio of T ( Figure 7 This result indicates that RV / B@NM can effectively inhibit the activation of CAFs, reduce Collagen I content, and increase CD8+. + T-cell intratumoral infiltration.

[0154] 3.6RV / B@NM promotes the polarization of M2 tumor-associated macrophages to M1 tumor-associated macrophages.

[0155] The tumor immunosuppressive microenvironment is a major reason for the poor response to immunotherapy in triple-negative breast cancer, and M2 tumor-associated macrophages (TAMs) are important immunosuppressive cells in the tumor microenvironment. Flow cytometry analysis of mouse tumor tissues showed that, compared with the model group, RV / B@NM significantly reduced intratumoral infiltration of M2-TAMs (approximately 12.2%); and compared with RV@NM or RB@NM monotherapy groups, the combination therapy of Vismodegib and BMS-1 ​​based on RV / B@NM also significantly reduced the proportion of M2-TAMs infiltration. Conversely, RV / B@NM treatment significantly promoted intratumoral infiltration of M1-TAMs (approximately 15.7% higher than the model group). Figure 8 The above results indicate that RV / B@NM treatment significantly increases the number of pro-tumor immune-related cells, including CD8, in tumor-bearing mice. + Vismodegib significantly reduced intratumoral infiltration of T cells and M1-TAMs, while significantly reducing the recruitment of immunosuppressive-associated cells (M2-TAMs) to the tumor, suggesting that the combination of Vismodegib and BMS-1 ​​can synergistically enhance CD8+. + T-cell infiltration and immune response enhance the efficacy of immunotherapy for triple-negative breast cancer.

[0156] 3.7 Biosafety evaluation of RV / B@NM

[0157] In vivo drug delivery safety is a prerequisite for the clinical translation of nanomedicines. Pathological analysis of major tissues in tumor-bearing mice, including the heart, spleen, and kidneys, showed that after multiple intravenous administrations of RV / B@NM, no significant pathological damage was observed in the major organs of the mice. Figure 9 (A) Further evaluation was conducted on changes in serum liver function indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and renal function indicators, creatinine (CRE) and blood urea nitrogen (BUN), in each group of mice. Figure 9 The results of the B-cell assay showed that, compared with the model group, there were no significant changes in liver and kidney function indicators in any of the treatment groups; the results of routine blood tests in mice showed that the levels of white blood cells (WBC), lymphocytes (LYMPH), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW) were all unchanged from those in the model group. Figure 9 (C). The above results show that RV / B@NM has good biocompatibility.

[0158] Based on the above experimental results, the biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​prepared in this invention inhibited the growth of in situ tumors in mice and the formation of lung / liver tumor metastases. The effect was significantly superior to Vismodegib or BMS-1 ​​alone, Vismodegib or BMS-1 ​​in combination, and nanodelivery carriers carrying Vismodegib or BMS-1 ​​alone. This inhibitory effect is related to Vismodegib's inhibition of CAF activation, remodeling of the tumor extracellular matrix (such as reduced collagen fiber formation and decreased Collagen I expression), and promotion of M2-type tumor-associated macrophage polarization to M1 type. Furthermore, the synergistic effect of Vismodegib in combination with the PD-1 / PD-L1 inhibitor BMS-1 ​​reprogrammed the tumor immunosuppressive microenvironment, enhancing CD8+. + T-cell infiltration is closely related to the immune response. This invention provides a new approach for the clinical treatment of triple-negative breast cancer.

[0159] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1, characterized in that, Includes the following steps: The Vismodegib solution and the BMS-1 solution are added to mPEG 5K -PAE 10K The organic phase is vortexed and mixed, and then added to the aqueous PBS phase, and then stirred, ultrasonically treated, centrifuged, and filtered to obtain the nanomicelle core V / B@NM; the ultrasonic treatment is performed at a power of 450 W, for 2 s, with an interval of 2 s, and a total time of 10 min. A biomimetic nanomedicine solution was prepared by co-extruding V / B@NM with erythrocyte vesicles using liposomes. After centrifugation, a biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​was obtained. The red blood cell vesicles are prepared by the following steps: red blood cells are added to PBS to swell, and after centrifugation, a white precipitate is obtained as the red blood cell membrane; the red blood cell membrane is treated with ultrasound and then extruded using a liposome extruder to obtain red blood cell vesicles; The Vismodegib solution was obtained by dissolving Vismodegib in dimethyl sulfoxide, and the BMS-1 ​​solution was obtained by dissolving BMS-1 ​​in tetrahydrofuran. The mPEG... 5K -PAE 10K Solution by mPEG 5K -PAE 10K The Vismodegib, BMS-1, and mPEG were obtained by dissolving in tetrahydrofuran. 5K -PAE 10K The mass ratio is 0.5:0.5:10; the volume ratio of the organic phase to the aqueous phase is 1:2; The stirring conditions were 400 rpm and 50°C for 4 hours. The mass ratio of V / B@NM to erythrocyte vesicles is 1:

1.

2. The biomimetic nanodelivery system co-loaded with Vismodegib and BMS-1 ​​prepared by the preparation method described in claim 1.

3. The application of the biomimetic nanodelivery system according to claim 2 in the preparation of drugs that inhibit the growth and metastasis of triple-negative breast cancer.

4. The application of the biomimetic nanodelivery system as described in claim 2 in the preparation of a drug that enhances the immunotherapy effect of triple-negative breast cancer.

5. A drug for treating triple-negative breast cancer, characterized in that, The drug comprises the biomimetic nanodelivery system of claim 2.

6. The drug as described in claim 5, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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