Apatinib-immune synergistic nano-drug based on bacterium source vesicles as well as preparation method and application of apatinib-immune synergistic nano-drug

Abstract

The invention discloses an apatinib-immune synergistic nano-drug based on bacterial source vesicles as well as a preparation method and application of the apatinib-immune synergistic nano-drug. The nano-drug is Apatinib-MVs, and the nano-drug takes a membrane vesicle secreted by parabacteroides coumarini as a carrier to entrap an anti-angiogenesis drug apatinib; in-vitro anti-tumor experiment results show that the nano-drug can exert an efficient anti-tumor effect through a dual synergistic mechanism of inhibiting tumor angiogenesis and activating anti-tumor immune response at the same time.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine technology, specifically relating to an apatinib-immunosynergistic nanomedicine based on bacterial vesicles, and also to the preparation method of the nanomedicine and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Treatment strategies include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Apatinib, an anti-angiogenic drug, is a highly selective vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor that can inhibit tumor growth and metastasis by blocking tumor angiogenesis. However, apatinib has problems such as easy development of drug resistance and toxic side effects.

[0003] Nanomedicine delivery systems provide an effective means to improve drug targeting and reduce toxic side effects. Bacterial-derived membrane vesicles (MVs) are a novel type of bio-based nanocarrier with advantages such as good biocompatibility, ease of modification, natural targeting of tumors, and activation of immune responses. Parabacteroides goldsteinii, as a gut probiotic, has membrane vesicles derived from it that are highly safe and possess unique immunomodulatory properties, making it an ideal drug delivery platform.

[0004] Currently, there are no reports of using *Pseudomonas aeruginosa* membrane vesicles as a carrier to encapsulate apatinib for synergistic treatment of breast cancer. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an apatinib-immunosynergistic nanomedicine (Apatinib-MVs) based on bacterial vesicles. This nanomedicine can exert a dual effect of anti-angiogenesis and immune activation, achieving highly efficient synergistic treatment of breast cancer.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned nanomedicine.

[0007] Another object of the present invention is to provide the application of the above-mentioned nanomedicines.

[0008] An apatinib-immunosynergistic nanomedicine based on bacterial vesicles, wherein the nanomedicine uses outer membrane vesicles secreted by *Pseudomonas guillier* as a carrier, and the vesicles encapsulate the anti-angiogenic drug apatinib.

[0009] Preferably, the mass ratio of apatinib to outer membrane vesicles in the nanomedicine is 2:1.

[0010] Preferably, the apatinib nanomedicine has an encapsulation efficiency of 68.5% in the outer membrane vesicles and a drug loading of 9.2%.

[0011] A method for preparing an apatinib-immunosynergistic nanomedicine based on bacterial vesicles includes the following steps: First, *Pseudomonas aeruginosa* is cultured under anaerobic conditions for 48–72 hours, the culture supernatant is collected, and outer membrane vesicles are obtained by ultracentrifugation purification; then, apatinib is incubated with the outer membrane vesicles at 37°C in the dark for 12 hours, and unencapsulated free drug is removed by dialysis to obtain the nanomedicine Apatinib-MVs.

[0012] Application of a bacterial vesicle-based apatinib-immunosynergistic nanomedicine in the preparation of drugs for treating breast cancer.

[0013] Preferably, the nanomedicine can exert a highly effective anti-tumor effect through a dual synergistic mechanism of inhibiting tumor angiogenesis and activating anti-tumor immune response.

[0014] Preferably, the nanomedicine includes a pharmaceutically acceptable excipient, which includes one or more of a diluent, stabilizer, buffer, preservative, or carrier.

[0015] The apatinib-immunosynergistic nanomedicine based on bacterial vesicles disclosed in this application can achieve the following beneficial effects: (1) Intelligent targeted delivery: The outer membrane vesicles of *Pseudomonas guillierii* have natural tumor targeting properties and certain immune adjuvant effects, which can improve the accumulation of drugs in tumor sites; (2) Simple preparation and high biosafety: The preparation method is mild and the steps are clear; the outer membrane vesicles derived from probiotics are used as carriers, which have good biocompatibility and low potential toxicity.

[0016] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0018] Figure 1 is a flow chart of the nanomedicine preparation process provided in the embodiments of this application; Figure 2 is a diagram showing the physicochemical characterization results of the nanomedicine provided in the embodiments of this application; Figure 3 is an in vitro antitumor effect diagram of the nanomedicine provided in the embodiments of this application; Figure 4 is a diagram illustrating the in vitro antitumor mechanism of the nanomedicine provided in the embodiments of this application; Figure 5 is a flowchart of the in vivo antitumor experimental design of nanomedicines provided in the embodiments of this application; Figure 6 shows the effect of the nanomedicine provided in the embodiments of this application on tumor-infiltrating CD8. + Flow cytometry results showing the effect of T cell activation. Detailed Implementation

[0019] To make the inventive purpose, technical solution and beneficial technical effects of this application clearer, the following detailed description of this application is provided in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0020] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included in the range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form an unspecified range.

[0021] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0022] The above-described invention content is not intended to describe every disclosed implementation or every method of implementation in this application. Instead, the following description provides more specific examples of exemplary implementation methods. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. In each instance, the examples are listed only as representative groups and should not be construed as exhaustive.

[0023] Example 1: Preparation of Nanomedicines This embodiment details the preparation process of the nanomedicine; see [link to documentation]. Figure 1 It mainly includes the following two core steps: 1.1 Extraction and purification of *Pseudomonas guilloché* membrane vesicles Extraction of bacterial membrane vesicles: *Pseudomonas goutii* was inoculated into an anaerobic medium and cultured at 37°C under anaerobic conditions until the late logarithmic growth phase; the bacterial suspension was collected and centrifuged at 4°C and 8000×g for 15 minutes to remove the bacterial cells and collect the supernatant; the supernatant was then filtered sequentially through 0.45μm and 0.22μm microporous membranes to completely remove residual bacteria.

[0024] Purification of bacterial membrane vesicles: The filtrate was ultracentrifuged at 150,000×g for 2 hours at 4°C. The supernatant was discarded, and the precipitate was the crude bacterial membrane vesicle. The precipitate was resuspended in pre-cooled sterile phosphate buffer (PBS, pH 7.4) and further purified by sucrose density gradient centrifugation. The vesicle components were collected, dialyzed, and high-purity bacterial membrane vesicles (MVs) were obtained and stored at -80°C for later use.

[0025] 1.2 Apatinib encapsulation The purified MVs were mixed with apatinib hydrochloride powder at a mass ratio of 20:1 (based on the protein content of the MVs) in PBS; the mixture was incubated at 37°C in the dark with shaking for 12 hours to effectively encapsulate apatinib into the MVs; subsequently, the mixture was repeatedly centrifuged and washed using an ultrafiltration centrifuge (molecular weight cutoff 100 kDa) to completely remove any unencapsulated free drug, thus obtaining the nanomedicine Apatinib-MVs.

[0026] Example 2: Physicochemical Characterization of Nanomedicines See Figure 2 The physicochemical properties of the Apatinib-MVs prepared in Example 1 were analyzed.

[0027] 2.1 Experimental Methods The physicochemical characterization of Apatinib-MVs was performed using ultraviolet-visible spectrophotometry: First, a concentration-absorbance standard curve (R0) at 260 nm was plotted using apatinib standards. 2 = 0.9999); Subsequently, the prepared Apatinib-MVs were subjected to 100 kDa ultrafiltration centrifugation to separate the free drug, and the absorbance of the filtrate or the lysed drug-loaded vesicle solution was measured. The drug content was calculated by substituting the absorbance into the standard curve, and then the encapsulation efficiency and drug loading were obtained.

[0028] 2.2 Experimental Results UV absorption characteristics: See Figure 2 B. Apatinib has a characteristic absorption peak at 260 nm. After drug loading, the absorbance of MVs at this wavelength is significantly increased, indicating successful drug loading; Standard curve and drug loading parameters: see [link to standard curve]. Figure 2 A, Based on the apatinib concentration-absorbance standard curve (R 2 = 0.9999), and calculations show that when the mass ratio of MVs to drug is 2:1, the encapsulation efficiency reaches 68.5% and the drug loading is 9.2%. See [reference needed]. Figure 2 CD; In vitro release behavior: see Figure 2E, under pH 6.5 (simulating tumor microenvironment) conditions, the cumulative drug release rate reached 85% within 72 hours, which was significantly higher than that of the pH 7.4 group, indicating that it has microenvironment-responsive sustained-release characteristics; the above results confirm that Apatinib-MVs have good drug loading capacity and controllable release performance.

[0029] Example 3: Evaluation of the in vitro antitumor effect of nanomedicines See Figure 3 The human triple-negative breast cancer cell line MDA-MB-231 (purchased from the American Type Culture Collection, ATCC) was used. ® CRM-HTB-26 ™ In vitro functional experiments were conducted.

[0030] 3.1 Experimental Procedure Cell proliferation experiment: Cells were inoculated at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 12-well plates. After adhesion, Apatinib-MVs (containing apatinib at a final concentration of 5 μM) were added, and an untreated control group was set up. After incubation for 24 hours, cells were labeled with CFSE fluorescent dye and cultured for another 48 hours. Cells were then collected and their proliferation number was detected by flow cytometry.

[0031] Transwell invasion assay: Matrigel was diluted and coated onto the upper chamber of a Transwell chamber, with 3 × 10⁶ cells per well. 5 MDA-MB-231 cells resuspended in serum-free medium were added to the lower chamber with complete medium containing 10% FBS and Apatinib-MVs. After 48 hours of culture, the cells were fixed, stained with crystal violet, and the number of cells that had permeated the membrane was counted.

[0032] Scratch assay: When the cells in the 6-well plate reached 80% confluence, scratches were created using a 200 μL pipette tip. After washing with PBS, fresh culture medium containing Apatinib-MVs was added. Photos were taken at 0 h and 24 h, and the scratch healing rate was calculated using ImageJ software.

[0033] 3.2 Experimental Results See Figure 3 A. CFSE flow cytometry results showed that the cell proliferation rate was significantly reduced in the Apatinib-MVs treatment group, indicating that it can effectively inhibit the proliferation of breast cancer cells; see also Figure 3 B. Transwell invasion assay showed that the number of transmembrane cells in the Apatinib-MVs group was significantly lower than that in the control group, indicating that it can effectively inhibit the invasive ability of breast cancer cells; see also Figure 3C. Scratch assay showed that the scratch healing rate of the Apatinib-MVs group was significantly lower than that of the control group after 24 hours, suggesting that it significantly inhibited cell migration. In summary, Apatinib-MVs can significantly inhibit the proliferation, migration and invasion of breast cancer cells in vitro.

[0034] Example 4: The in vitro antitumor mechanism of nanomedicines 4.1 Experimental Methods To investigate the antitumor mechanism of Apatinib-MVs, the following in vitro experimental methods were used: HUVECs tube formation assay: Matrigel (300 μg / mL) was spread on 96-well plates and incubated at 37°C for 1 hour to form a gel; then 5 × 10⁶ cells were inoculated. 4 Human umbilical vein endothelial cells (HUVECs) per well were added, along with Apatinib-MVs (containing apatinib at a final concentration of 5 μM), while the control group was given an equal volume of PBS. After 6 hours of culture, three fields of view were randomly selected under an inverted microscope for photographing, and the total length of the tubules was quantitatively analyzed using ImageJ software.

[0035] Apoptosis detection: MDA-MB-231 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 12-well plates and treated with Apatinib-MVs for 48 hours after adhesion. Cells were collected, double-stained with Annexin V-PE and 7-AAD, and the proportion of early and late apoptotic cells was detected by flow cytometry.

[0036] Western Blot protein expression analysis: After treating MDA-MB-231 cells for 48 hours, total protein was extracted, and the concentration was determined. SDS-PAGE electrophoresis and membrane transfer were then performed. The cells were incubated overnight at 4°C with primary antibodies against Vimentin, β-catenin, Bcl-2, and Cleaved Caspase-3, respectively, followed by incubation at room temperature for 2 hours with the corresponding secondary antibodies. Chemiluminescence was used for imaging, and semi-quantitative analysis was performed with β-actin as an internal control.

[0037] 4.2 Experimental Results See Figure 4 A. HUVECs tubule formation assays showed that Apatinib-MVs significantly inhibited the formation of angioid structures, reducing the total tubule length by approximately 70% compared to the control group; see also Figure 4 B. Annexin V / 7-AAD double staining flow cytometry results showed that the total proportion of early and late apoptotic cells was significantly increased in the Apatinib-MVs treatment group; see also Figure 4C. Western blot results showed that after treatment with apatinib-MVs, the expression of EMT-related proteins Vimentin and β-catenin was significantly downregulated, the expression of VEGFA protein was significantly downregulated, and the level of the anti-apoptotic protein Bcl-2 was significantly reduced. These results indicate that apatinib-MVs can exert anti-breast cancer effects through multiple mechanisms, including inhibiting angiogenesis, inducing tumor cell apoptosis, and reversing epithelial-mesenchymal transition (EMT).

[0038] Example 5: In vivo antitumor effects of nanomedicines 5.1 Experimental Methods Twenty female Balbc mice aged 4-5 weeks (SPF grade) were randomly divided into four groups (n=5): ① PBS group; ② Apatinib group; ③ MVs group; ④ Apatinib-MVs group, see [link to relevant documentation]. Figure 5 Immunization pretreatment was performed by subcutaneous injection of the corresponding preparation at designated time points; on day 7 after the last immunization, MDA-MB-231 cells (5 × 10⁶) were inoculated into the mammary fat pads of mice. 5 Mice were euthanized 21 days after inoculation, and the tumors were removed.

[0039] Tumor tissue was used to prepare a suspension of tumor-infiltrating lymphocytes (TILs), and 1×10⁻⁶ TILs were collected. 6 Cells were stained sequentially for surface markers (CD8) and intracellular cytokines (IFN-γ, TNF-α). Samples were then analyzed using flow cytometry and FlowJo software.

[0040] 5.2 Experimental Results See Figure 6 Compared with the PBS control group, the apatinib monotherapy group, and the MVs treatment group, the apatinib-MVs group showed a higher concentration of CD8+ in tumor tissues of mice. + The proportion of T cells and the production of IFN-γ (CD8) + IFN-γ + ) and TNF-α (CD8) + TNF-α + Activated CD8 + The proportions of T cell subsets were significantly increased.

[0041] These results demonstrate that Apatinib-MVs nanomedicines can effectively recruit and activate CD8+ in the tumor microenvironment. +T lymphocytes are stimulated to secrete key anti-tumor cytokines IFN-γ and TNF-α, thereby significantly enhancing the body's antigen-specific cellular immune response. This immune activation effect, combined with the anti-angiogenic effect of apatinib, constitutes the synergistic therapeutic mechanism of this nanomedicine for breast cancer.

[0042] The above embodiments demonstrate that the present invention has successfully constructed apatinib-MVs, an apatinib-immunosynergistic nanodrug based on *Pseudomonas guillierii* membrane vesicles. The nanodrug has a well-defined preparation process, high drug loading efficiency and sustained-release properties, and can exert a highly effective anti-tumor effect through a synergistic mechanism of "anti-angiogenesis-immunoactivation," providing a strong basis for the development of novel combination therapy strategies for breast cancer.

Claims

1. An apatinib-immunosynergistic nanomedicine based on bacterial vesicles, characterized in that, The nanomedicine uses outer membrane vesicles secreted by *Pseudomonas guillier* as a carrier, and the vesicles encapsulate the anti-angiogenic drug apatinib.

2. The nanomedicine according to claim 1, characterized in that, The mass ratio of apatinib to outer membrane vesicles is 2:

1.

3. The nanomedicine according to claim 1, characterized in that, The apatinib had an encapsulation efficiency of 68.5% and a drug loading of 9.2%.

4. A method for preparing a nanomedicine as described in any one of claims 1-3, characterized in that, Includes the following steps: First, *Pseudomonas guillierii* was cultured under anaerobic conditions for 48–72 hours, and the culture supernatant was collected and purified by ultracentrifugation to obtain outer membrane vesicles. Then, apatinib was incubated with the outer membrane vesicles at 37°C in the dark for 12 hours, and the unencapsulated free drug was removed by dialysis to obtain the nanomedicine Apatinib-MVs.

5. The use of a nanomedicine as described in any one of claims 1-3 in the preparation of a treatment for breast cancer.

6. The application according to claim 5, characterized in that, The nanomedicine can exert a highly effective anti-tumor effect through a dual synergistic mechanism of inhibiting tumor angiogenesis and activating anti-tumor immune response.

7. The application according to claim 5, characterized in that, The nanomedicine includes pharmaceutically acceptable excipients, which include one or more of diluents, stabilizers, buffers, preservatives, or carriers.

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