Biomembrane vesicles for targeted delivery of bortezomib and methods of making and using the same

Abstract

This invention discloses a targeted delivery biomembrane vesicle for bortezomib, its preparation method, and its applications, specifically relating to the field of biomedical technology. This invention utilizes the reversible covalent chemistry between bortezomib and the glycocalyx on the surface of the biomembrane vesicle to construct a tumor microenvironment-responsive biomembrane vesicle-protected bortezomib nanoprodrug for targeted intratumoral delivery. This vesicle retains the biomimetic homologous targeting properties of the cell membrane, thereby achieving efficient tumor tissue enrichment and specifically triggering the cleavage of bortezomib ester bonds in the acidic tumor microenvironment, thus precisely releasing bortezomib and achieving specific killing of tumor cells. This not only improves drug utilization but also reduces damage to normal tissues. Based on this "turning enemy into friend" strategy, the development of this invention will provide a theoretical basis for developing new, highly effective, and low-toxicity cancer chemotherapy regimens.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a biomembrane vesicle for targeted delivery of bortezomib, its preparation method, and its application. Background Technology

[0002] Bortezomib (BTZ), a highly effective proteasome inhibitor, is a first-line treatment for malignant tumors such as multiple myeloma. However, BTZ lacks tumor targeting specificity, and its boric acid active group readily interacts with endogenous substances through non-specific recognition, often leading to severe toxic side effects such as dose-limiting peripheral neuropathy and thrombocytopenia, significantly restricting its clinical application. Therefore, developing novel BTZ nanoprodrugs with targeting capabilities is one effective way to overcome the current limitations in the clinical application of BTZ.

[0003] In recent years, biomembrane vesicles have attracted much attention due to their excellent biointerface properties. Unlike traditional nanocarriers, they effectively circumvent complex chemical synthesis and modification processes. Surface proteins of biomembrane vesicles not only enhance tumor targeting but also endow them with immune evasion capabilities, prolonging their blood circulation time. Notably, like cell membranes, biomembrane vesicles are composed of a phospholipid bilayer and also contain functional proteins, lipids, and, as reported in recent years, RNA. Numerous studies have shown that these biomolecules exist in glycosylated forms and can act as anchoring sites for borate-containing substances through reversible borate ester bonds. Existing methods remain only at the theoretical level, lacking complete and reproducible technical approaches; therefore, this invention is proposed. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to provide a BTZ nanoprodrug that is simple to prepare, highly targeted, biosafe, and capable of precise drug release in response to the tumor microenvironment, and its application in anti-tumor therapy.

[0005] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a targeted delivery of BTZ biomembrane vesicles, which uses tumor cell membrane (CCM) vesicles as carriers and forms BTZ covalently linked to the surface of CCM vesicles via borate ester bonds; the borate ester bonds are generated by the reaction of the boric acid groups of BTZ with the cis-dihydroxy structures on the sugar calyx of the CCM vesicle surface.

[0006] Preferably, the compounds containing a cis-dihydroxy structure on the sugar calyx of the CCM vesicle surface include, but are not limited to, one or more of sialic acid, galactose, and mannose.

[0007] Preferably, the tumor includes, but is not limited to, one or a combination of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignant tumors.

[0008] More preferably, the tumor is cancer.

[0009] More preferably, the cancer is cervical cancer (HeLa).

[0010] More preferably, the particle size of the biomembrane vesicles is 100-200 nm.

[0011] Uniform biomembrane vesicles were prepared with a particle size controlled between 100 and 200 nm. The core advantage of this size is that it facilitates the delivery of vesicles to the tumor site through high permeability and retention effects, thereby promoting tumor targeting and efficient uptake of the biomembrane vesicles.

[0012] More preferably, the zeta potential of the biomembrane vesicles is -32 to -32.5 mV, and more preferably -32.4 mV.

[0013] Notably, the borate group of BTZ can reversibly covalently bind with compounds containing the cis-dihydroxy characteristic structure. This borate affinity interaction exhibits pH-responsive characteristics: when the ambient pH reaches or exceeds the pKa of borate, borate forms a tetrahedral borate anion (sp³ hybridization), which then reacts with compounds containing the cis-dihydroxy characteristic structure to form five- or six-membered cyclic esters. Conversely, when the ambient pH is significantly lower than the pKa of borate, borate reverts to a planar triangular configuration (sp² hybridization), leading to the dissociation of the borate-cis-dihydroxy complex.

[0014] BTZ can undergo pH-dependent, reversible covalent binding with glycocalyxes on cell membranes. Under physiological conditions (pH 7.35–7.45), BTZ reacts with the glycocalyxes of cell membrane vesicles, covalently anchoring BTZ to the vesicle surface via borate ester bonds. Unlike non-covalent interactions, this binding mode exhibits better stability and reduces drug leakage during blood transport. Conversely, when this nanoprodrug is taken up by cells and transported to lysosomes, the borate ester bonds break in response to the weakly acidic environment within the lysosome, leading to rapid release of the BTZ drug. This property provides an ideal chemoswitch for constructing stimulus-responsive drug delivery systems.

[0015] This invention also provides a method for preparing the above-mentioned targeted BTZ delivery biomembrane vesicles, comprising the following steps: (1) Extraction of tumor cell membrane: After culturing tumor cells and collecting the cells, extract tumor cell membrane fragments.

[0016] (2) Formation of BTZ-loaded biomembrane vesicles (BTZ@CCM): BTZ and tumor cell membrane fragments obtained in step (1) were co-incubated under weakly alkaline conditions, washed, and extruded to obtain BTZ@CCM.

[0017] Preferably, in step (2), the mass ratio of BTZ to tumor cell membrane fragments is (1-1.5):(1-1.5), and more preferably 1:1.

[0018] The 1:1 mass ratio of BTZ to tumor cell membrane fragments aims to balance drug loading rate and therapeutic dose requirements: on the one hand, it avoids the waste of free BTZ drug caused by a decrease in loading rate due to excessive BTZ; on the other hand, it prevents the preparation of BTZ@CCM nanoprodrugs from being limited due to insufficient BTZ, which would then require an increased dosage.

[0019] Preferably, in step (2), the weakly alkaline condition is pH 7.35~7.45, and more preferably pH 7.4.

[0020] The weakly alkaline condition is the optimal pH selected based on the physiological environment in the body.

[0021] Preferably, in step (2), the incubation time is 2-3 hours, and more preferably 2 hours.

[0022] Preferably, in step (2), the washing method is to wash with PBS to remove unbound BTZ.

[0023] Preferably, in step (2), the extrusion method is through a liposome extruder, and the number of extrusions is 10-15 times, more preferably 11 times.

[0024] The present invention also provides a drug combination comprising the above-described biomembrane vesicles for targeted delivery of BTZ.

[0025] Preferably, it may also contain a pharmaceutically or immunologically acceptable carrier or excipient.

[0026] The present invention also provides the application of the above-mentioned targeted delivery of BTZ biomembrane vesicles or drug combinations in the preparation of drugs for treating malignant tumors.

[0027] Preferably, the tumor includes, but is not limited to, one or a combination of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignant tumors.

[0028] More preferably, the tumor is cancer.

[0029] More preferably, the cancer is cervical cancer.

[0030] The beneficial effects of this invention are as follows: 1. The biomembrane vesicles for targeted delivery of BTZ proposed in this invention possess excellent targeting and immune evasion properties. Based on the homologous targeting properties of biomembrane vesicles, BTZ nanoprodrugs can actively recognize and accumulate in homologous tumor tissues, overcoming the shortcomings of BTZ itself in lacking targeting; at the same time, natural biomembrane vesicles have "self" marking, which can effectively evade the recognition and clearance of the immune system and prolong their circulation time in vivo.

[0031] 2. The drug combination proposed in this invention enables intelligent drug release. BTZ can form pH-responsive borate ester bonds with the glycocalyx on the surface of biomembrane vesicles, acting as a "chemical switch." When the BTZ nanoprodrug enters tumor cells via endocytosis and is transported to acidic lysosomes, the borate ester bonds break in the acidic environment, achieving precise and controllable release of BTZ within tumor cells, greatly improving drug utilization and reducing damage to normal tissues.

[0032] 3. The biomembrane vesicles for targeted BTZ delivery proposed in this invention are simple to prepare and have high biocompatibility. Employing a "top-down" strategy, no complex chemical synthesis and modification steps are required. A multifunctional integrated delivery system can be constructed through simple co-incubation and physical extrusion, resulting in a simple process with good reproducibility. Furthermore, the delivery system is composed of bio-derived materials and exhibits excellent biocompatibility.

[0033] 4. This invention constructs a tumor microenvironment-responsive targeted BTZ delivery biomembrane vesicle, which achieves effective killing of tumor cells and effectively reduces toxic side effects through specific recognition and precise release of BTZ.

[0034] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the principle of BTZ@CCM, which responds to the tumor microenvironment, for BTZ-targeted intratumoral delivery in Embodiment 1 of the present invention. Figure 2 This is a transmission electron microscope image of BTZ@CCM in Embodiment 1 of the present invention; Figure 3 This is a hydration particle size diagram of BTZ@CCM in Example 1 of the present invention; Figure 4 This is a Zeta potential diagram of CCM and BTZ@CCM in Embodiment 1 of the present invention; Figure 5 This is an SDS-PAGE gel electrophoresis image of CCM, BTZ, and BTZ@CCM in Example 1 of this invention; Figure 6 The fluorescence spectra of Alizarin Red S and BTZ in Example 1 of this invention are shown. Figure 7 Mass spectrometry analysis of the products generated by the reaction of BTZ with (A) sialic acid, (B) galactose and (C) mannose in Example 1 of this invention; Figure 8 This is a graph showing the change in hydrated particle size of BTZ@CCM in PBS and DMEM cell culture medium over 5 days in Example 1 of this invention. Figure 9 The UV-Vis absorption spectra of BTZ@CCM at different time points in phosphate buffer at pH 5.0, pH 6.5, and pH 7.4 in Example 2 of this invention are shown. Figure 10 This is a laser confocal image of HeLa cells and HK-2 cells after incubation with DiD-labeled liposomes and BTZ@CCM for 3 hours, respectively, in Example 3 of this invention; Figure 11 The flow cytometry analysis results of DiD-labeled BTZ@CCM and liposomes after incubation with HeLa cells for different times (0, 3, 6 and 9 hours) in Example 3 of this invention are shown. Figure 12 This is a laser confocal image of DiD-labeled liposomes and BTZ@CCM cells co-incubated with RAW264.7 cells for 3 hours in Example 3 of this invention. Figure 13 This is a graph showing the cell viability of HeLa cells after incubation with different doses of CCM, BTZ, and BTZ@CCM for 48 hours in Example 4 of the present invention. Figure 14 This is a staining image of HeLa cells after 48 hours of treatment with moderate doses of CCM, BTZ, and BTZ@CCM in Example 4 of the present invention (green: live cells, red: dead cells). Figure 15 This is an analysis of the expression levels of apoptosis-related proteins after treatment with moderate doses of CCM, BTZ, and BTZ@CCM in Example 4 of the present invention. Figure 16 Biochemical and routine blood analysis of blood from healthy nude mice 30 days after tail vein administration of different doses of BTZ@CCM in Example 5 of this invention; PBS was used as a negative control. Figure 17 The images show H&E staining of vital organs (heart, liver, spleen, lung, and kidney) in healthy nude mice 30 days after tail vein administration of different doses of BTZ@CCM in Example 5 of this invention. PBS is used as a negative control. Figure 18 These are in vivo fluorescence imaging images and quantitative analysis images of tumor-bearing mice in each group in Example 6 of the present invention; Figure 19 This is a graph showing the change in tumor-bearing mouse volume over time in each group in Example 7 of the present invention; Figure 20 These are optical photographs of isolated tumors from tumor-bearing mice treated for 15 days in each group in Example 7 of this invention. The data in the attached figures are expressed as mean ± standard deviation. The Student's test was used to analyze the significance of the differences. P < 0.001 was considered highly significant, denoted as ***; 0.001 ≤ P < 0.01 was considered highly significant, denoted as **; and 0.01 < P ≤ 0.05 was considered significant, denoted as *. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0037] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0038] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0039] Terms and definitions: As used herein, the term “tumor” refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative lesion,” and “tumor” are not mutually exclusive in this text.

[0040] As used herein, “tumor cell” means any tumor cell present in a tumor or its sample. Tumor cells can be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or methods described herein.

[0041] Example 1: Preparation of BTZ@CCM Culture human cervical cancer HeLa cells and mouse cervical cancer U14 cells (both purchased from Shanghai Fuheng Biotechnology Co., Ltd.) to approximately 5 × 10⁶ cells / year. 3Cells were collected in quantities of tens of thousands, washed with PBS, scraped, and centrifuged to collect the cell pellet. Following the procedures of the cell membrane protein and cytoplasmic protein extraction kit (purchased from Beyotime), cells were processed with a membrane protein extraction reagent containing 1% PMSF, dispersed, and incubated on ice for 15 minutes. The mixture was then subjected to three freeze-thaw cycles at 37°C and liquid nitrogen. The nuclei and debris were removed by centrifugation at 700 g, and the supernatant was collected by centrifugation at 12000 g to collect the precipitate, which was the HeLa cell membrane fragment. 20 μL of BTZ in DMSO solution was mixed with the cell membrane fragment in PBS (pH 7.4, 980 μL), with a BTZ to cell membrane fragment ratio of 1:1. The mixture was reacted with shaking at room temperature for 2 hours. After the reaction, unbound BTZ was removed by centrifugation and washing. The resulting precipitate was resuspended in PBS and repeatedly extruded 11 times using a liposome extruder (200 nm pore size) to obtain the BTZ@CCM nanoprodrug.

[0042] like Figure 1 The diagram shows the preparation process of BTZ@CCM and its intratumoral targeted delivery strategy. BTZ@CCM achieves intratumoral targeted delivery, reduces the toxic side effects of BTZ, and achieves specific killing of tumor cells.

[0043] like Figure 2 The transmission electron microscope image of BTZ@CCM shown below indicates that BTZ@CCM consists of spherical or near-spherical vesicles with good dispersibility and uniform particle size.

[0044] like Figure 3 The dynamic light scattering measurement of BTZ@CCM shows that its hydrated particle size is approximately 149 nm.

[0045] like Figure 4 The diagram shows the zeta potential of CCM and BTZ@CCM. The potential of CCM is approximately -32.8 mV, while the potential of BTZ@CCM formed after binding with BTZ is approximately -32.3 mV, indicating that the formation of borate ester bonds does not change the surface charge properties of CCM.

[0046] like Figure 5 The image shown is an SDS-PAGE gel electrophoresis image of CCM and BTZ@CCM. The protein bands of BTZ@CCM and CCM are in the same position, confirming that the preparation process basically preserves the proteins on the cell membrane.

[0047] like Figure 6The image shows further evaluation of BTZ@CCM using the Alizarin Red S (ARS) indicator method. ARS specifically binds to free boric acid groups to produce fluorescence. Three groups of samples were taken: 1) ARS + BTZ; 2) the supernatant after the reaction of ARS + BTZ with the cell membrane; 3) ARS + PBS. Their fluorescence spectra were detected. The results showed that compared with the blank group 3) ARS + PBS, group 1 (ARS + BTZ) showed a significant fluorescence enhancement peak, proving the presence of free boric acid groups. The fluorescence intensity of group 2 (supernatant) was significantly lower than that of group 1, similar to the blank group, indicating that the initially added BTZ had reacted and bound with the CCM calyx, consuming the free boric acid groups, thus indirectly proving the successful formation of the borate ester bond between BTZ and the CCM calyx.

[0048] like Figure 7 The image shows the mass spectrometry analysis of the products from the reactions of BTZ with sialic acid, galactose, and mannose, respectively. The product peaks appear at 656.2758, 527.2289, and 527.2292 (m / z, [M]), respectively. - At the location indicated, a borate ester bond was formed.

[0049] like Figure 8 The figure shows the changes in hydrated particle size of BTZ@CCM dispersed in PBS and DMEM media over 5 days. The results show that the hydrated particle size of BTZ@CCM did not change significantly, indicating that BTZ@CCM has good stability in in vitro physiological solutions.

[0050] Example 2: pH-responsive drug release test of materials: The prepared BTZ@CCM was placed in phosphate buffer solutions of different pH values ​​(5.0, 6.5, 7.4) and shaken at 37°C. Samples were taken at predetermined time points, centrifuged, and the absorbance of the supernatant was measured at 269 nm using a UV-Vis spectrophotometer to calculate the cumulative release rate of BTZ.

[0051] like Figure 9 As shown, BTZ release was slow over 24 hours at pH 7.4. BTZ release accelerated significantly at pH 6.5 and pH 5.0, exhibiting a clear pH dependence, demonstrating that the delivery system possesses good acid-triggered drug release performance.

[0052] Example 3: In vitro experiments on the targeting and immune escape functions of BTZ@CCM: Using HK-2 cells as a control, we evaluated the homologous targeting ability of BTZ@CCM on HeLa cells.

[0053] First, HeLa and HK-2 cells were seeded separately in confocal dishes and cultured for 24 hours. Then, BTZ@CCM cells labeled with the fluorescent dye DiD and liposomes were co-incubated with HeLa and HK-2 cells for 3 hours, respectively. The cells were washed three times with PBS, stained with DAPI for 10 minutes in the dark, and the intracellular fluorescence signal was observed using a laser confocal microscope. Figure 10 As shown, the red fluorescence signal of the BTZ@CCM group was significantly higher than that of other groups in HeLa tumor cells, indicating that BTZ@CCM has good tumor targeting performance.

[0054] HeLa cells were seeded in confocal dishes and cultured for 24 hours. Then, BTZ@CCM labeled with the fluorescent dye DiD and liposomes were incubated with HeLa cells for different times (3, 6, and 9 hours). After enzymatic digestion, centrifugation, and redispersement with PBS, the fluorescence intensity of BTZ@CCM and liposomes in HeLa cells was quantitatively analyzed by flow cytometry. Figure 11 As shown, with prolonged incubation time, the fluorescence intensity of BTZ@CCM in HeLa cells was significantly higher than that of liposomes, indicating that the homologous targeting of BTZ@CCM promotes the uptake of tumor cells.

[0055] RAW264.7 cells were seeded in confocal dishes and cultured for 24 hours. Then, DiD-labeled BTZ@CCM and liposomes were co-incubated with RAW264.7 macrophages for 3 hours. Cells were washed three times with PBS, stained with DAPI for 10 minutes, and intracellular fluorescence signals were observed using a laser confocal microscope. Figure 12 As shown, RAW264.7 cells treated with liposomes exhibited strong fluorescence signals, while RAW264.7 cells treated with BTZ@CCM showed weak fluorescence signals, indicating that BTZ@CCM has good immune evasion capabilities.

[0056] Example 4: In vitro cytotoxicity assay of BTZ@CCM: First, the effect of different formulations on cell viability was detected using a CCK-8 assay kit. Specifically, HeLa cells were seeded in 96-well plates with 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours. Different doses of CCM, BTZ, and BTZ@CCM were added to each well and incubated for 48 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. The absorbance of each well was measured at 450 nm using a multi-mode microplate reader, and the relative viability of cells in each treatment group was calculated.

[0057] The results are as follows Figure 13As shown, blank CCM exhibited no significant cytotoxicity within the tested concentration range and demonstrated good biocompatibility. Cell viability in both the BTZ and BTZ@CCM treatment groups showed a concentration-dependent decrease, indicating that BTZ@CCM possesses good tumor-killing activity, primarily derived from BTZ-mediated cell killing.

[0058] Furthermore, we further investigated the effect of different formulations on cell viability using a live / dead cell staining assay. HeLa cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. CCM, BTZ, and BTZ@CCM were added to each well, followed by incubation for 48 hours. Cells were washed with PBS, and 4 μM calcein and 4 μM propidium iodide were added to each well. Cells were then incubated at 37°C in the dark for 10 minutes. Cell viability was then observed under an inverted fluorescence microscope. Green signals indicate live cells, and red signals indicate dead cells.

[0059] The results are as follows Figure 14 As shown, compared with the PBS blank control group and the CCM group, the BTZ group and the BTZ@CCM group exhibited stronger red fluorescence signals, indicating that they have a stronger ability to promote tumor cell apoptosis. This is consistent with the above CCK-8 results.

[0060] The study then investigated the changes in the expression levels of apoptosis-related proteins after treatment with different drugs, including Bcl-2, Bax, and Cleaved Caspase-3. like Figure 15 As shown, the BTZ@CCM treatment group significantly downregulated the expression of the apoptosis protein Bcl-2 and upregulated the expression of the pro-apoptotic proteins Bax and Cleaved Caspase-3, elucidating the molecular mechanism by which it induces apoptosis.

[0061] Example 5: Biosafety of BTZ@CCM: Normal female BALB / c mice were administered different doses of BTZ@CCM (0.3, 0.6, 1.2 mg / kg) via intravenous injection. -1 (3 animals per group). After 30 days of drug administration, blood samples were collected from each group for blood chemistry and routine analysis. In addition, major organs were collected for hematoxylin and eosin staining.

[0062] like Figure 16As shown, after 30 days of intravenous administration of different doses of BTZ@CCM, compared with the PBS group, there were no statistically significant differences in liver and kidney function (liver function indicators: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase; kidney function indicators: creatinine, uric acid, blood urea nitrogen) and routine blood indicators in mice, indicating that BTZ@CCM has no obvious acute toxicity. Meanwhile, H&E staining results of important organs (heart, liver, spleen, lungs, and kidneys) showed no obvious pathological abnormalities (e.g., ...) in the tissue sections. Figure 17 (As shown).

[0063] Example 6: In vivo tumor-targeting experiment of BTZ@CCM: A cervical cancer ectopic xenograft model was constructed by subcutaneously inoculating murine cervical cancer U14 cells into the axilla of normal female BALB / c nude mice. The specific experimental method was as follows: After the U14 cells entered the logarithmic growth phase, the cells were collected, centrifuged, and diluted to a suitable concentration with PBS. Finally, 100 μL of the diluted U14 cell suspension was injected into the left axilla of each mouse, at a dose of 1 × 102. 6 Each cell. The implanted tumor-bearing mice were randomly divided into 3 groups (tumor volume 150 mm²). 3 Near-infrared fluorescent dye ICG-labeled CCM or ICG-labeled liposomes were injected into mice via the tail vein, with an equal dose of PBS injected as a control group. Near-infrared fluorescence images at different time points (0, 2, 4, 6, 8, 12, and 24 hours) were acquired using an AniView100 in vivo imaging system, and fluorescence quantification was performed.

[0064] The results are as follows Figure 19 As shown, after administration, the fluorescence signal of CCM at the tumor site continuously increased, reaching a peak intensity at 4 hours. In contrast, nanoliposomes showed a weaker signal at the mouse tumor site. These results indicate that CCM can effectively accumulate at the tumor site and prolong its retention time. Furthermore, corresponding real-time fluorescence quantification also demonstrated that the enrichment degree of CCM at the tumor site was higher than that of the liposome group.

[0065] Example 7: In vivo experiment on the anti-tumor effect of BTZ@CCM: U14 tumor-bearing mice prepared according to the method described in Example 6 were randomly divided into 4 groups and injected with equal doses of CCM, BTZ, and BTZ@CCM via tail vein. The PBS group served as a control. Tumor growth and body weight changes in the tumor-bearing mice were monitored every 3 days. The formula for calculating tumor volume is: V = (L × W) 2 ) / 2, where L and W represent the length and width of the tumor.

[0066] like Figure 19As shown, compared to other groups, the BTZ@CCM group exhibited the most significant tumor inhibition rate, indicating its superior anti-tumor effect. Furthermore, optical photographs of ex vivo tumors from each group 15 days after treatment also confirmed the above experimental results (e.g., Figure 20 (As shown).

[0067] In summary, this invention successfully constructs a pH-responsive biomembrane vesicle-protected BTZ nanoprodrug. This system integrates homology targeting, immune escape, and tumor microenvironment-triggered drug release, demonstrating excellent tumor targeting, enhanced antitumor efficacy, and good biosafety in both in vitro and preliminary in vivo experiments. It provides a novel and promising solution for overcoming the bottlenecks in the clinical application of BTZ.

[0068] Example 8: The difference between this embodiment and Example 1 is that BTZ and CCM vesicles were incubated at pH 7.35 for 2 hours. The mass ratio of BTZ to tumor cell membrane fragments was 1.5:1. Everything else was the same as in Example 1.

[0069] Comparative Example 1: BTZ and CCM vesicles were incubated for 2 hours at pH < 7. Incubation at excessively low pH increases the Lewis acidity of boric acid, causing it to exist more as an uncharged boric acid form. Simultaneously, the protonation degree of the hydroxyl groups in cis-diol also increases. These two factors together significantly decrease the equilibrium constant for ester bond formation between the two, resulting in a substantial reduction or even complete failure of borate ester formation efficiency. This reduces drug loading and the killing efficacy against tumor cells.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomembrane vesicle for targeted delivery of bortezomib, characterized in that, It uses tumor cell membrane vesicles as carriers and is formed by covalently linking bortezomib to the surface of tumor cell membrane vesicles through borate ester bonds; the borate ester bonds are generated by the reaction of the boric acid group of bortezomib with the cis-dihydroxy structure on the sugar calyx of the tumor cell membrane vesicle surface.

2. The biomembrane vesicle according to claim 1, characterized in that, The compound containing a cis-dihydroxy structure on the glycocalyx of the tumor cell membrane vesicle is one or more of sialic acid, galactose, and mannose; the tumor is one or a combination of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignant tumor.

3. The biomembrane vesicle according to claim 1, characterized in that, The biomembrane vesicles have a particle size of 100-200 nm and a zeta potential of -32 to -32.5 mV.

4. The method for preparing biomembrane vesicles according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Extraction of tumor cell membrane: After culturing tumor cells and collecting the cells, extract tumor cell membrane fragments; (2) Formation of bortezomib-loaded biomembrane vesicles: Bortezomib and tumor cell membrane fragments obtained in step (1) were co-incubated under weakly alkaline conditions and washed; Bortezomib-loaded biomembrane vesicles were obtained by extrusion.

5. The preparation method according to claim 4, characterized in that, In step (2), the mass ratio of bortezomib to tumor cell membrane fragments is (1-1.5):(1-1.5).

6. The preparation method according to claim 4, characterized in that, In step (2), the weakly alkaline conditions are pH 7.35~7.45; the incubation time is 2-3 hours.

7. The preparation method according to claim 4, characterized in that, In step (2), the washing method is to wash with PBS to remove unbound bortezomib; the extrusion method is to extrude through a liposome extruder, and the number of extrusions is 10-15 times.

8. A drug combination, characterized in that, It comprises the biomembrane vesicles as described in any one of claims 1-3 or the biomembrane vesicles prepared by the preparation method as described in any one of claims 4-7.

9. The use of the biomembrane vesicles according to any one of claims 1-3, or the biomembrane vesicles prepared by the preparation method according to any one of claims 4-7, or the drug combination according to claim 8, in the preparation of a drug for treating malignant tumors.

10. The application according to claim 9, characterized in that, The tumor is one or a combination of carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignant tumor.

Images