Bortezomib nano composition and preparation method thereof

Abstract

The invention relates to the technical field of medicine preparation, and provides a bortezomib nano composition, and the bortezomib nano composition comprises the following components: bortezomib, albumin and a medium. The bortezomib nano composition can be delivered in a targeted manner, is low in toxicity and stable, and provides a new strategy for treatment of multiple myeloma.

Description

Technical Field

[0001] This application relates to the field of drug preparation technology, and in particular to a bortezomib nanocomposition and its preparation method. Background Technology

[0002] Multiple myeloma is a malignant tumor originating from plasma cells in the bone marrow. It is characterized by abnormal clonal proliferation of plasma cells and the secretion of large amounts of M protein, leading to symptoms such as bone pain, anemia, and renal insufficiency. Current treatments include traditional chemotherapy, immunomodulatory drugs, and hematopoietic stem cell transplantation, but all have significant limitations.

[0003] Traditional chemotherapy: Although it can relieve symptoms, it is easy to develop drug resistance, and middle-aged and elderly patients have poor tolerance to it, making it difficult to cure the disease.

[0004] Immunomodulatory drugs: adverse reactions such as hematologic toxicity exist.

[0005] Hematopoietic stem cell transplantation: autologous transplantation has a high risk of relapse, while allogeneic transplantation carries serious complications such as transplant-related mortality and graft-versus-host disease.

[0006] Therefore, there is an urgent need to research a drug composition that has targeted delivery capabilities, low toxicity, and stability, to provide a new strategy for the treatment of multiple myeloma. Summary of the Invention

[0007] In view of this, this application aims to provide a bortezomib nanocomposition that can provide a targeted delivery, low-toxicity and stable multiple myeloma treatment drug.

[0008] To achieve the above objectives, the technical solution of this application is implemented as follows: A bortezomib nanocomposition, wherein the components of the bortezomib nanocomposition include bortezomib, albumin, and a medium.

[0009] Furthermore, the albumin is either bovine serum albumin or human serum albumin.

[0010] Furthermore, the medium can be any one of water, sodium chloride solution, or glucose solution.

[0011] Furthermore, the mass ratio of bortezomib to albumin is between 0.05 and 0.20.

[0012] Compared with the prior art, this application has the following advantages: The bortezomib nanocomposition of this application comprises albumin, a medium, and bortezomib. The bortezomib nanocomposition prepared by the above components does not require the addition of surfactants or stabilizers. It relies on the self-crosslinking of albumin to form a stable nanostructure, avoiding the use of toxic solvents and reducing toxicity. At the same time, the nanostructure formed by the self-crosslinking of albumin in this application enables the composition to have the function of targeted delivery and improves the encapsulation efficiency of bortezomib drug and the stability of the nanocomposition.

[0013] This application also proposes a method for preparing bortezomib nanocomposites to improve the encapsulation efficiency and stability of the drug.

[0014] To achieve the above objectives, the technical solution of this application is implemented as follows: A method for preparing a bortezomib nanocomposition, the method comprising: Disperse albumin in a medium and stir to prepare an aqueous solution; Bortezomib was dispersed in anhydrous ethanol and sonicated to prepare an organic phase solution. The organic phase solution is added to the aqueous phase solution to prepare a primary emulsion system; The colostrum system is placed under a preset homogenization pressure and homogenized a preset number of times. During each homogenization interval, it needs to be cooled to a preset temperature to form a stable nanoparticle system. The nanoparticle system was subjected to ultrafiltration and centrifugation, and the precipitate was collected to obtain the bortezomib nanocomposition.

[0015] Furthermore, the step of dispersing albumin in a medium, stirring, and preparing an aqueous solution includes: Bovine serum albumin was dispersed in a medium and placed in a water bath at a preset temperature. The mixture was stirred at 800 rpm to 1400 rpm to prepare an aqueous solution.

[0016] Furthermore, the step of adding the organic phase solution to the aqueous phase solution to prepare the primary emulsion system includes: The organic phase solution was added to the aqueous phase solution in small amounts multiple times while continuously stirring at a speed of 800 rpm to 1400 rpm to prepare the primary emulsion system.

[0017] Furthermore, the preset homogenization pressure is between 500 Bar and 800 Bar.

[0018] Furthermore, the preset number of homogenization cycles is between 5 and 20.

[0019] Furthermore, the preset temperature is between 2℃ and 8℃.

[0020] Compared with the prior art, this application has the following advantages: The method for preparing the bortezomib nanocomposition described in this application promotes drug binding by breaking non-covalent bonds of albumin with ethanol, and induces oxidative cross-linking of albumin thiol groups by high shear force generated by high-pressure homogenization. This method avoids the use of toxic solvents and improves the encapsulation efficiency and stability of the bortezomib nanocomposition.

[0021] Meanwhile, the bortezomib nanocomposition prepared by the method of this application can deliver drugs in a targeted manner, resulting in a drug enrichment at the tumor site that is significantly higher than that of free drugs, improving cellular uptake efficiency, and also having sustained-release properties that can prolong the effective concentration maintenance time. In addition, it is safe and can reduce hepatotoxicity. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the preparation process of the bortezomib nanocomposite described in the embodiments of this application; Figure 2 To verify the release curve of BNPs at pH 6.8 in Example 1; Figure 3 To verify the release curve of BNPs at pH 7.4 in Example 1; Figure 4 To verify the preliminary storage stability of BNPs in Example 1; Figure 5 To verify the inhibitory effect of bortezomib nanocomposite and free bortezomib on the proliferation of RPMI 8226 cells in Example 2 after 24 hours; Figure 6 To verify the inhibitory effect of bortezomib nanocomposite and free bortezomib on the proliferation of RPMI 8226 cells after 48 hours in Example 2; Figure 7 To verify the apoptosis effect of RPMI 8226 cells in Example 2; Figure 8 To verify the flow cytometry-detected cell uptake graph in Example 2; Figure 9 To verify the evaluation of tumor growth inhibition effect in Example 3 (a: image of tumor tissue at the end of the experiment; b: tumor volume change curves of mice in different treatment groups; c: average tumor volume of mice in each group at the end of treatment); Figure 10 To verify the effect of different treatment methods on mouse body weight in Example 3; Figure 11To verify the effects of different treatment methods on liver and kidney function in tumor-bearing mice in Example 3 (a and b are liver function-related indicators, and c and d are kidney function-related indicators). Figure 12 The figure shows the effects of different drug administration groups on the major tissues and organs of mice with multiple myeloma in Example 3. Detailed Implementation

[0023] To make the technical solution and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0025] For items where specific conditions are not specified in this application, conventional conditions or conditions recommended by the manufacturer of the equipment used shall apply. For items where the manufacturer of the reagents or instruments used is not specified, conventional products that can be purchased commercially shall be used. As for the technical means or processes involved, if specific conditions are not specified, they shall be carried out in accordance with the existing methods in the relevant field.

[0026] The present application will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] An embodiment of the first aspect of this application provides a bortezomib nanocomposition capable of providing a targeted delivery, low-toxicity, and stable multiple myeloma treatment drug.

[0028] In view of this, in order to overcome the shortcomings of the prior art, the bortezomib nanocomposition of this embodiment is designed to include bortezomib, albumin and mediator.

[0029] Specifically, bortezomib, one of the components mentioned above, is an important anti-tumor drug, belonging to the proteasome inhibitor class. It is widely used in the treatment of hematologic malignancies. It works by inhibiting proteasome activity within tumor cells, preventing tumor cells from breaking down specific proteins, leading to the accumulation of these proteins within the cells and ultimately inducing tumor cell apoptosis. Simultaneously, it can also affect the tumor microenvironment, inhibiting tumor cell growth and spread. Bortezomib is clinically used to treat multiple myeloma and mantle cell lymphoma.

[0030] Albumin, as a natural protein, has been widely used in the field of nanoparticles due to its excellent biocompatibility, biodegradability, low immunogenicity, and unique structural and functional properties. In antitumor therapy, albumin can encapsulate hydrophobic antitumor drugs within albumin nanoparticles, solving the problems of low drug solubility and high toxicity. Albumin-based nanoparticles offer advantages such as high biocompatibility, targeting potential, strong drug loading capacity, and ease of modification. In the drug composition of this embodiment, the preferred carrier for the drug delivery system is either bovine serum albumin or human serum albumin. Furthermore, the molecular weight of the albumin selected in this embodiment is preferably between 40 kDa and 100 kDa.

[0031] The medium is used as a solvent in the nanocomposition of this embodiment, preferably, for example, any one of water, sodium chloride solution or glucose solution.

[0032] Among these, ultrapure water is preferred, referring to water that has undergone deep purification treatment and is virtually free of any impurities (including ions, organic matter, microorganisms, particulate matter, etc.), with a resistivity typically reaching 18.2 MΩ・cm (25℃). The application of ultrapure water is irreplaceable in the research, preparation, and quality control of nanoparticle formulations, as its high purity directly affects the stability, uniformity, and biosafety of these formulations.

[0033] The sodium chloride solution is preferably a 0.9% sodium chloride solution, and the glucose solution is preferably a 5% glucose solution.

[0034] In this embodiment, the mass ratio of bortezomib to albumin in the bortezomib nanocomposition is preferably, for example, between 0.05 and 0.20.

[0035] Based on the above description of each component, the bortezomib nanocomposition of this embodiment is prepared using albumin, a medium, and bortezomib without the addition of surfactants or stabilizers. It relies on the self-crosslinking of albumin to form a stable nanostructure, avoiding the use of toxic solvents and reducing toxicity. At the same time, the nanoparticles formed by albumin enable the composition to have the function of targeted delivery, and improve the encapsulation efficiency of bortezomib drug and the stability of the nanocomposition.

[0036] The second aspect of this application provides a method for preparing a bortezomib nanocomposition to avoid the use of toxic solvents and improve the encapsulation efficiency and stability of the drug.

[0037] In existing technologies, the main methods for preparing albumin nanoparticles include solvent removal and Nab™ technology. Solvent removal induces aggregation by disrupting the albumin structure with organic solvents (such as ethanol), but suffers from poor particle stability, the risk of organic solvent residue, and low encapsulation efficiency. Nab™ technology utilizes high-pressure homogenization combined with a Class II toxic solvent (such as chloroform) to promote albumin cross-linking into a shell. While this improves stability, solvent toxicity limits its clinical application, and it is only suitable for hydrophobic drugs with high binding rates.

[0038] In view of this, in order to overcome the shortcomings of the prior art, the preparation method of the bortezomib nanocomposition in this embodiment, as an exemplary implementation, is combined with... Figure 1 As shown, the preparation method may include, for example, the following steps.

[0039] Step S1: Disperse albumin in a medium and stir to prepare an aqueous solution.

[0040] In step S1 above, albumin is dispersed in a medium and stirred to form an aqueous solution. As a preferred embodiment, this may include, for example, dispersing albumin in a medium, placing it in a water bath at a preset temperature, and stirring at a speed of 800 rpm to 1400 rpm to form an aqueous solution.

[0041] Among them, the preset temperature can preferably be 75°C. Heating the aqueous solution to 75°C can optimize the spatial structure of albumin and expose more drug binding sites.

[0042] Step S2: Dissolve bortezomib in anhydrous ethanol and sonicate to prepare an organic phase solution.

[0043] The use of ultrasound-assisted dissolution in step S2 above is beneficial for bortezomib to be uniformly dispersed in the organic phase solution.

[0044] Step S3: Add the organic phase solution to the aqueous phase solution to prepare the colostrum system.

[0045] In step S3 above, the organic phase solution is added to the aqueous phase solution to prepare a primary emulsion system. As a preferred embodiment, this may include, for example, adding the organic phase solution to the aqueous phase solution in small amounts multiple times while continuously stirring at a speed of 800 rpm to 1400 rpm to obtain the primary emulsion system.

[0046] Step S4: Place the colostrum system under a preset homogenization pressure and cycle it for a preset number of times. During each homogenization interval, it needs to be cooled to a preset temperature to form a stable nanoparticle system.

[0047] In step S4 above, the preset homogenization pressure is preferably between 500 Bar and 800 Bar, the preset number of homogenization cycles is preferably between 5 and 20, and the preset temperature is preferably between 2°C and 8°C. Appropriate homogenization pressure and cycles can balance the encapsulation efficiency and optimized particle size of the nanocomposite.

[0048] Step S5: Ultrafiltration and centrifugation of the nanoparticle system, collection of the precipitate, and preparation of bortezomib nanocomposition.

[0049] In step S5 above, ultrafiltration centrifugation is used to separate unencapsulated free bortezomib.

[0050] Based on the above description, the preparation method of the bortezomib nanocomposition in this embodiment combines solvent removal and high-pressure homogenization technology. Ethanol is used to break the non-covalent bonds of albumin to promote drug binding, and the high shear force generated by high-pressure homogenization induces the oxidative cross-linking of albumin thiol groups. This avoids the use of toxic solvents and significantly improves the encapsulation efficiency and nanoparticle stability of the composition.

[0051] It is worth noting that, regarding the bortezomib nanocomposition and its preparation method in this embodiment, based on the above preferred embodiments, the following preparation examples can be referred to in specific implementation.

[0052] Example This example demonstrates the preparation of the aforementioned bortezomib nanocomposition, and the specific preparation steps are as follows: Step S1: Weigh 25 mg BSA and dissolve it in 7.5 mL of ultrapure water. Place the solution in a 75℃ constant temperature water bath with a magnetic stirrer (1200 rpm) and stir to form a clear solution, thus obtaining an aqueous solution.

[0053] Step S2: Weigh 5 mg of BTZ and dissolve it in 5 mL of ethanol. Sonicate the solution until it is clear and transparent to obtain an organic phase solution.

[0054] Step S3: While continuously stirring (1200 rpm), add the organic phase to the aqueous phase in small amounts several times to form a milky white promulgation system.

[0055] Step S4: Transfer the colostrum to a high-pressure microfluidic nano-homogenizer, set the homogenization pressure to 700 Bar, cycle the homogenization 10 times, and cool it to 2-8℃ at intervals. During the homogenization process, the high shear force induces the oxidation of thiol groups between BSA molecules to form disulfide bonds, which crosslink to form a stable nanoparticle system.

[0056] Step S5: Unencapsulated free BTZ is separated by ultrafiltration centrifugation, and the nanoparticle precipitate is collected to obtain bortezomib nanocomposite.

[0057] Verification Example 1 Example 1 of this verification demonstrates that bortezomib nanocomposition has higher encapsulation efficiency and stability through characterization, and the specific methods are as follows.

[0058] Experimental methods 1. Solvent-free thermosetting method and Nab were used respectively. TM Bortezomib albumin nanoparticles were prepared by a method, and then compared with the encapsulation efficiency and stability of the bortezomib nanocomposite prepared in this example.

[0059] 1.1 Solvent-free thermosetting method First, ethanol was slowly added dropwise to the albumin solution under heating conditions, with continuous stirring to disrupt the non-covalent bonds and secondary structures of albumin, promoting protein separation and the formation of nanoparticles. As more organic solvent was added, the solution gradually became turbid, indicating that albumin had precipitated. Unreacted organic solvent and other impurities were removed by ultrafiltration and centrifugation to obtain an albumin nanoparticle suspension.

[0060] 1.2 Nab TM Law Nab TM The technical process is as follows: hydrophobic drugs are dissolved in class II organic solvents such as chloroform, mixed with albumin aqueous solution, and formed into coarse droplets by stirring or shearing. The particle size is controlled by high-pressure homogenization. After removing the organic solvent, the cavitation effect of bubbles caused by high shear force during homogenization is used to oxidize the thiol groups of albumin or break the disulfide bonds by superoxide ions, promoting albumin cross-linking to form a nanoparticle system to encapsulate the drug.

[0061] 1.3 Results In a comparison of different preparation methods, the preparation method in this example demonstrates advantages. As shown in Table 1, the encapsulation efficiency of the binding method reached 61.31±6.36%, significantly higher than that of the solvent removal method and Nab. TM The method showed that it had a better encapsulation effect on the drug. In addition, the nanoparticles prepared by the combined method had a particle size of 126.16±25.18 nm, which was significantly smaller than that of the other two methods, and its PDI was 0.157±0.056, indicating that the bortezomib nanocomposition prepared in this example had a more uniform system.

[0062] Table 1 Screening of preparation methods

[0063] 2. Drug release test By studying the release behavior of nano-formulations under different pH conditions, BNPs were found to possess significant sustained-release properties. Figure 2 and 3As shown, under both pH 6.8 and pH 7.4 conditions, the cumulative release rate of BNPs increased slowly over time, exhibiting a more gradual and sustained release rate compared to Free BTZ. This indicates that the release behavior of BNPs is not significantly affected by pH, consistently maintaining a slow drug release. This sustained-release effect is significant for drug therapy, not only allowing drugs to maintain effective concentrations in the body for extended periods, reducing dosing frequency and improving patient compliance, but also mitigating the toxic side effects caused by drug concentration fluctuations. This provides strong experimental evidence and theoretical support for the application of BNPs in pharmaceutical formulations.

[0064] 3. Preliminary stability assessment from Figure 4 Based on the data and appearance of BNPs, under room temperature conditions, BNPs maintained a clear and uniform appearance without any instability such as turbidity or precipitation. Regarding particle size, although there were some variations, the values ​​remained within a similar range without a significant increase or decrease; and the PDI remained within a reasonable range over 15 days. These data indicate that BNPs exhibited good stability during the 15-day observation period, with both appearance and particle size performance being quite ideal.

[0065] Verification Example 2 This validation example 2 systematically evaluated the effects of bortezomib nanocomposition on multiple myeloma cells through in vitro cell experiments, aiming to reveal its mechanism of action in antitumor therapy. First, in vitro cytotoxicity experiments were conducted to examine the selective toxicity of bortezomib nanocomposition to multiple myeloma cells, assessing its specific killing effect on tumor cells. Second, in vitro apoptosis experiments were conducted to study the apoptosis effect of bortezomib nanocomposition on multiple myeloma cells. Finally, in vitro cell uptake experiments were conducted to explore the uptake characteristics of bortezomib nanocomposition. Specific validation methods are as follows.

[0066] Materials and Methods 1. Instruments and reagents 1.1 Instruments

[0067] 1.2 Medicines and Reagents

[0068] 2. Experimental Methods 2.1 Cell Culture Human multiple myeloma cells (RPMI 8226) were purchased from Wuhan Pronosai Biotechnology Co., Ltd., and cultured using RMPI8226 cell-specific medium, with the medium changed every 1-2 days.

[0069] 2.2 Cytotoxicity test The CCK-8 assay was used to examine the cytotoxicity of different formulation groups. RPMI 8226 cells in the logarithmic growth phase were collected, counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 Cells were seeded at 100 μL per well in 96-well plates. Subsequently, different concentrations of free bortezomib (Free BTZ), blank nanoparticles (Blank NPs), and BNPs solutions were added, along with a control group. Each well contained 100 μL of BNPs, with six replicates per group. A blank control group (without cells) was also included, with 200 μL per well and six replicates. The cells were incubated at 37 °C and 5% CO2 for 24 and 48 h, respectively. Then, 20 μL of CCK-8 reagent was added to each well, and incubation continued for 2 to 6 h. The absorbance (optical density, OD value) of each well was measured using a microplate reader at an excitation wavelength of 450 nm. The cell proliferation inhibition rate was calculated based on the OD value using the following formula:

[0070] 2.3 Detection of tumor cell apoptosis rate Annexin V and Propidium iodide (PI) double staining is commonly used to detect apoptosis. RPMI-8226 cells in the logarithmic growth phase were selected, counted, and their concentration adjusted to 1×10⁻⁶. 6 Cells / mL. In a six-well plate, 1 mL of cell suspension was seeded in each well, with three replicates. The IC50 was calculated based on the CCK-8 assay results. 50 Free-BTZ and BNPs drug solutions were prepared. A blank group and a blank albumin nanoparticle group were set up as control groups, with 1 mL of the corresponding solution added to each well. The six-well plates were incubated in a cell culture incubator for 12 h. After incubation, cells from each group were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in pre-chilled PBS and washed twice. Then, the cells were resuspended in 100 μL of 1×Binding Buffer, centrifuged again at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of 1×Binding Buffer. 100 μL of cell suspension from each group was transferred to a labeled flow cytometry tube, 5 μL of Annexin V-FITC was added, and the mixture was thoroughly mixed. The reaction was carried out at room temperature in the dark for 15 min. Afterward, 5 μL of PI was added, the mixture was mixed again, and the reaction was carried out at room temperature in the dark for another 5 min. Finally, the mixture was diluted with 500 μL of PBS and detected by flow cytometry (FCM) within 1 h.

[0071] 2.4 Cell uptake experiment Inoculate 1×10⁻⁶ cells per well in a six-well plate. 6 RPMI-8226 cells were incubated with free coumarin-6 and albumin nanoparticles loaded with free coumarin-6, respectively, at concentrations of 0.1 and 0.2 μg / mL. The cells were incubated for 4 h. After incubation, the cells were washed three times with PBS and then resuspended in 0.5 mL of PBS. Intracellular fluorescence intensity was detected by FCM, with the FITC channel selected as the excitation channel.

[0072] 3 Results 3.1 Cytotoxicity test The CCK-8 assay results showed that the BNPs drug system had a significant inhibitory effect on tumor cell proliferation. Free BTZ and BNPs inhibited the proliferation of RPMI8226 cells by an IC50 value within 24 h. 50 The values ​​were 56.78 ng / mL and 43.31 ng / mL, respectively. According to... Figure 5 and 6 The results showed that the blank control group and the blank albumin nanoparticle group had weak inhibitory effects on tumor cell proliferation, exhibiting low inhibitory efficacy. However, both Free-BTZ and BNPs showed dose-dependent proliferation inhibition at the same treatment time; and compared with the blank control group and the blank albumin nanoparticle group, they both had stronger proliferation inhibition effects. Notably, the BNPs drug system showed stronger inhibitory effects on tumor cell proliferation than Free-BTZ at both 24 h and 48 h, demonstrating higher inhibitory efficacy.

[0073] 3.2 In vitro effect on promoting tumor cell apoptosis Annexin V binds to phosphatidylserine (PS) residues exposed on the outer side of the cell membrane, while PS residues are located on the inner side of the membrane in normal cells and evert during apoptosis. FITC-labeled Annexin V can detect early apoptotic cells (green fluorescence). PI is a DNA dye that cannot penetrate intact cell membranes and can only enter cells with damaged membranes. Therefore, PI staining (red fluorescence) can distinguish between late apoptotic and necrotic cells. A single positivity for Annexin V-FITC indicates early apoptosis, while a double positivity indicates late apoptotic or necrotic cells. Figure 7 Flow cytometry results showed that, under the same concentration and time conditions, the BNPs group had a stronger pro-apoptotic effect on tumor cells than the BTZ group.

[0074] 3.3 Cellular uptake The cellular uptake of MPC-11 was examined using flow cytometry, and the results are as follows: Figure 8As shown, the uptake level of albumin nanoparticles by cells was significantly higher than that of free coumarin-6, indicating that this formulation can be more effectively taken up by cells and enter the cell interior compared to the free drug. Furthermore, the uptake of nanoparticles by cells showed a trend of increasing with increasing drug concentration, and correspondingly, the fluorescence intensity of cellular uptake also gradually increased.

[0075] Verification Example 3 This validation example 3 comprehensively evaluates its efficacy and safety characteristics in animal models through in vivo experiments, and clarifies its mechanism of action and effects in vivo. The specific validation methods are as follows.

[0076] 1. Materials and Methods 1.1 Instruments

[0077] 1.2 Medicines and Reagents

[0078] 2. Experimental Methods 2.1 Animal husbandry Female BCLB / c mice aged 7-8 weeks and weighing between 16 and 20 g were housed in a specific pathogen-free (SPF) animal laboratory at an experimental animal center.

[0079] 2.2 Establishment of a tumor-bearing mouse model The mouse plasmacytoma cell line (MPC-11) was purchased from Haixing Biotechnology Co., Ltd., and cultured using MPC-11 cell-specific medium with daily medium changes.

[0080] Mice were given an environmental acclimatization period of approximately one week. During this period, the mice maintained stable weight and showed no significant adverse reactions, allowing subsequent experiments to proceed. MPC-11 cells in the logarithmic growth phase were collected and counted. The cells were washed twice with PBS, then resuspended in pre-cooled PBS and thoroughly mixed to achieve a cell density of 6 × 10⁶ cells / mL. 6 A cell suspension of 0.1 mL / mL was administered subcutaneously to the right axilla of each mouse after preparation. Tumor growth was closely monitored daily after inoculation.

[0081] 2.3 Tumor volume measurement The longest and shortest diameters of the tumor were measured using vernier calipers, and the tumor volume was calculated using the following formula:

[0082] V: Tumor volume; a: Longest diameter of the tumor; b: Shortest diameter of the tumor. 2.4 Antitumor therapeutic effects of BNPs Once the mouse tumors reached approximately 100 mm³, the mice were randomly divided into five groups of six each. The five groups were: a saline control group (Control), blank albumin nanoparticles (Blank NPs), free bortezomib (Free BTZ), commercially available injectable bortezomib (BTZ), and bortezomib albumin nanoparticles (BNPs). Clear labeling of each group facilitated subsequent experimental procedures and data recording.

[0083] The BTZ dosage was 1 mg / kg, administered via tail vein injection, once every two days, with each injection consisting of 0.2 mL. During treatment, changes in mouse body weight and tumor volume were closely monitored, along with continuous observation of the diet, mental state, and fecal characteristics of mice in different groups.

[0084] 2.5 In vivo safety studies of BNPs drug system After a 14-day treatment cycle, the mice were anesthetized, and then venous blood was collected from them. Blood cell analyzers were used to perform routine blood tests and liver and kidney function tests to assess the systemic toxicity of the mice during the treatment period.

[0085] The safety of the formulation was assessed by histopathological analysis of major organs in mice using the hematoxylin-eosin staining (H&E) assay. Mice were sacrificed after the last administration, and heart, liver, spleen, lung, and kidney tissues were collected. After fixation in 4% paraformaldehyde, the tissues were embedded in paraffin, sectioned, stained with hematoxylin-eosin, and observed and photographed using an optical microscope to analyze the presence of obvious lesions in different tissues.

[0086] 3 Results 3.1 In vivo antitumor effects of BNPs During treatment, changes in tumor volume in mice were observed and recorded every other day, such as... Figure 9 As shown, MM tumors grow slowly in the early stages and rapidly in the later stages. BNPs exhibit a significant advantage in inhibiting tumor growth. Compared with Control, Blank NPs, Free BTZ, and commercially available BTZ, the tumor volume growth in the BNPs treatment group was slower. Especially during days 6-14, the tumor volume in the BNPs group was significantly lower than in other treatment groups. This indicates that BNPs have a good effect in inhibiting tumor growth.

[0087] 3.2 In vivo safety studies of BNPs drug system 3.2.1 Weight Changes Monitor the weight changes of mice and plot the weight change curve, such as... Figure 10 As shown, no significant decrease in mouse weight was observed in the formulation group compared to the control group, indicating that the formulation has low systemic toxicity.

[0088] 3.2.2 Blood biochemistry analysis like Figure 11 As shown, BTZ itself carries a risk of liver and kidney damage. In mice, aspartate aminotransferase (AST) and blood urea nitrogen (UREA) were significantly abnormal after drug administration, reflecting disorders of liver metabolism and kidney excretion. [7] Meanwhile, MM disease itself can cause imbalances in key liver and kidney function indicators in mice, leading to elevated transaminase and blood urinary nitrogen levels. BNPs can alleviate liver and kidney damage caused by BTZ to some extent.

[0089] 3.3 H&E pathological staining H&E staining results are as follows Figure 12 As shown, in the BNPs group, cardiomyocytes were neatly arranged with no signs of damage; liver cells were morphologically intact and portal areas were normal; spleen nodules were clearly defined and cells were evenly distributed; alveolar structures in the lungs were intact without inflammation or other pathological changes; and glomeruli and tubules in the kidneys were clearly defined with normal interstitium. Notably, the Free BTZ group and the commercially available injectable bortezomib BTZ group showed slight changes in cell morphology in the liver and kidneys of mice, suggesting that this type of drug may potentially cause damage to these organs. In conclusion, BNPs demonstrated good safety after in vivo administration, laying the foundation for its further research and application.

[0090] The results of Verification 1 show that the bortezomib nanocomposition prepared in this example has sustained-release properties and higher encapsulation stability.

[0091] Example 2, apoptosis analysis, validated the antitumor activity of BNPs. At the same concentration and treatment time, the BNPs group induced significantly stronger apoptosis than the BTZ group. Cell uptake experiments showed that RPMI 8226 cells uptake significantly more BNPs than free coumarin-6, and the fluorescence intensity increased with increasing drug concentration, reflecting the superior cellular uptake efficiency of BNPs. In conclusion, compared to free drugs, BNPs not only improve drug delivery efficiency but also enhance antitumor activity due to the structural advantages of nanocarriers, demonstrating stronger therapeutic potential and providing important experimental evidence for the development of targeted antitumor drugs.

[0092] The results of this validation example 3 demonstrate that BNPs can effectively inhibit tumor growth in MM tumors and exhibit good safety. This provides experimental evidence for BNPs as a potential anti-tumor therapy and holds promise for providing a new strategy for the treatment of multiple myeloma.

[0093] The above descriptions are merely some embodiments of this application and are not intended to limit this application. The technical features or structures in the foregoing different embodiments can be arbitrarily combined to form other specific technical solutions as needed. For those skilled in the art, this application can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of the claims of this application.

Claims

1. A bortezomib nano-composition, characterized in that: The components of the bortezomib nano-composition include bortezomib, albumin and medium. 2.The bortezomib nano-composition of claim 1, characterized in that: The albumin is any one of bovine serum albumin or human serum albumin. 3.The bortezomib nano-composition of claim 1, characterized in that: The medium is any one of water, sodium chloride solution or glucose solution. 4.The bortezomib nano-composition of claim 1, characterized in that: The mass ratio of bortezomib to albumin is between 0.05 and 0.

20.

5. A method of preparing a bortezomib nanocomposition, characterized in that, The preparation method comprises: dispersing albumin in medium, stirring to prepare an aqueous phase solution; dispersing bortezomib in anhydrous ethanol, ultrasonic to prepare an organic phase solution; adding the organic phase solution to the aqueous phase solution to prepare a primary emulsion system; placing the primary emulsion system under a preset homogenization pressure, cycling a preset number of times of homogenization, cooling to a preset temperature during each homogenization interval to form a stable nanoparticle system; ultrafiltration centrifugation of the nanoparticle system, collecting the precipitate to prepare the bortezomib nano-composition.

6. The method of claim 5, wherein the bortezomib nano-composition is prepared by, The dispersing albumin in medium, stirring to prepare an aqueous phase solution comprises: dispersing albumin in medium, placing in a water bath at a preset temperature, stirring at a speed of 800 rpm-1400 rpm to prepare an aqueous phase solution.

7. The method of claim 5, wherein the bortezomib nano-composition is prepared by, The adding the organic phase solution to the aqueous phase solution to prepare a primary emulsion system comprises: adding the organic phase solution to the aqueous phase solution in small amounts and multiple times under continuous stirring at a speed of 800 rpm-1400 rpm to prepare the primary emulsion system. 8.The preparation method of the bortezomib nano-composition of claim 5, characterized in that: The preset homogenization pressure is between 500 Bar and 800 Bar. 9.The preparation method of the bortezomib nano-composition of claim 5, characterized in that: The preset number of times of homogenization is between 5 and 20. 10.The preparation method of the bortezomib nano-composition of claim 5, characterized in that: The preset temperature is between 2℃ and 8℃.

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