Preparation method and application of albumin-loaded berberine hydrochloride nanocomplex

Abstract

The application belongs to the technical field of biological medicine, and relates to a preparation method and application of an albumin-loaded berberine hydrochloride nanocomposite, in particular to preparation of the albumin-loaded berberine hydrochloride nanocomposite and application of the albumin-loaded berberine hydrochloride nanocomposite in preparation of a drug for treating and / or preventing cancer. The albumin-loaded berberine hydrochloride nanocomposite comprises berberine hydrochloride and serum albumin, and the mass ratio of the serum albumin to the berberine hydrochloride is 1:0.1-0.4. The albumin-loaded berberine hydrochloride nanocomposite is prepared by a desolvation-chemical crosslinking method with the serum albumin as a carrier material, ethanol as a dehydrating agent and glutaraldehyde as a crosslinking agent. The albumin-loaded berberine hydrochloride nanocomposite has obvious antitumor activity and can be used for preparing an antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a preparation method and application of albumin-loaded berberine hydrochloride nanocomplexes, in particular to the preparation of albumin-loaded berberine hydrochloride nanocomplexes and the application thereof in the preparation of drugs for treating and / or preventing cancer. BACKGROUND

[0002] Berberine hydrochloride (BBM) is a bis-benzyl isoquinoline alkaloid extracted from the Berberidaceae family, which has the pharmacological effects of increasing white blood cell count, anti-inflammatory effect, anti-arrhythmia, anti-hyperglycemia, anti-tumor, immune regulation and prevention of myocardial ischemia-reperfusion injury. It has the advantages of rich source, low price, low toxicity and small side effects. At present, only berberine tablets are on the market, which are used for treating various leukopenia caused by smoking and preventing leukopenia after cancer radiotherapy and chemotherapy. In China, it has been used for postoperative leukocytosis, and the effect is good.

[0003] Recent studies have shown that berberine hydrochloride has inhibitory effect on various malignant tumor cells and has strong anticancer activity, including colorectal cancer (colon cancer + rectal cancer), lung cancer, breast cancer, hepatocellular carcinoma, bladder cancer, melanoma, pancreatic cancer, gastric cancer, head and neck squamous cell carcinoma, etc. Berberine hydrochloride mainly inhibits the activity, growth and invasion of cancer cells in a concentration-dependent manner, increases the apoptosis rate of cancer cells, induces cell cycle arrest at the G0 / G1 phase, and enhances the in vitro and in vivo anticancer activity of BBM by inhibiting different signaling pathways in various cancers, mainly affecting the JAK / STAT, CAMKII / c-Myc and TGF / SMAD signaling pathways, and it has less toxicity to normal cells. The potential advantages of high efficiency and low toxicity make it a research hotspot in the field of tumor treatment. However, due to its short half-life, low bioavailability and non-specific tissue distribution, the drug concentration at the tumor site is low after systemic administration, which may cause other toxic side effects, and its clinical application is limited. Therefore, it is of great significance to develop targeted preparations or controlled-release preparations of berberine drugs to achieve sustained delivery of therapeutic doses of berberine hydrochloride to the tumor site, improve its in vivo anti-tumor effect, and further combined with other anti-tumor drugs.

[0004] Albumin is an ideal drug delivery carrier. In 2005, the FDA first approved Abraxane, a paclitaxel albumin nanoparticle preparation, for the treatment of metastatic breast cancer. Albumin nanoparticles have both active targeting and passive targeting to tumor tissues. In active targeting, albumin nanoparticles interact with several specific receptors in cancer cells, including glycoprotein (Gp18, Gp30 and Gp60) receptors and cysteine-rich secretory protein receptors, both of which are highly expressed in cancer cells, resulting in the internalization of albumin nanoparticles in tumors Sleep D. Albumin and its application in drug delivery[J]. Expert Opinion On Drug Delivery, 2015, 12(5): 793-812. In passive targeting, albumin nanoparticles accumulate at tumor targets with enhanced permeability and retention effect (EPR effect). The binding of albumin to 60kDa glycoprotein on endothelial cells leads to the activation of caveolin-1 and the transport of intact albumin nanoparticles across the cell membrane Solanki R, Rostamabadi H, Patel S, et al. Anticancer nano-delivery systems based on bovine serum albumin nanoparticles: A critical review[J]. International Journal of Biological Macromolecules, 2021, 193(Pt A): 528-540.). In addition, albumin has many advantages as a delivery carrier, such as easy purification and control, natural non-toxicity, low immunogenicity, biocompatibility and biodegradability, high binding capacity for various drugs, good tolerance, and certain targeting without serious side effects, so it has become a rapidly developing drug delivery system for cancer treatment and has attracted widespread attention.

[0005] Currently, only berberine hydrochloride tablets are used in clinical practice, and there is no related report on albumin nanoparticles of berberine hydrochloride. Preparing berberine hydrochloride into albumin nanoparticles is expected to improve the bioavailability of the drug, increase the anti-tumor effect in vivo, and reduce the toxic side effects. SUMMARY

[0006] The purpose of the present application is to provide an albumin-loaded berberine hydrochloride nanoparticle (BBM-BSA-NPs) and a preparation method thereof, in order to solve the problems of poor bioavailability of berberine hydrochloride and difficulty in sustained action at tumor sites.

[0007] Based on the above purpose, the present application uses albumin as a carrier material, ethanol as a dehydrating agent, and glutaraldehyde as a cross-linking agent to prepare a berberine hydrochloride albumin nanoparticle complex (BBM-BSA-NPs).

[0008] Specifically, the present application uses the following technical solutions to achieve this:

[0009] An albumin-loaded berberine hydrochloride nanoparticle complex comprises berberine hydrochloride and serum albumin, and the mass ratio of serum albumin to berberine hydrochloride is 1:0.1-0.4.

[0010] The serum albumin is selected from bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin (OVA).

[0011] The albumin-loaded berberine hydrochloride nanoparticle complex is prepared by a desolvation-chemical cross-linking method.

[0012] Berberine hydrochloride and serum albumin are dissolved in deionized water to form a mixed solution. The pH is adjusted to 8.5-9.0, anhydrous ethanol is added and stirred until nanoparticles are formed, then glutaraldehyde is added and stirred for cross-linking. The ethanol is removed by rotary evaporation, and the berberine hydrochloride albumin nanoparticle complex is obtained by freeze-drying.

[0013] The specific preparation steps of the berberine hydrochloride albumin nanoparticle complex (BBM-BSA-NPs) are as follows:

[0014] (1) Precisely weigh the prescribed amount of berberine hydrochloride and serum albumin, dissolve them in deionized water, adjust the pH to 8.5-9.0, and obtain a berberine hydrochloride albumin aqueous solution as the water phase;

[0015] (2) Add anhydrous ethanol to the water phase of step (1) under stirring;

[0016] (3) Add a glutaraldehyde solution to the solution of step (2), stir and solidify, and remove the ethanol by rotary evaporation to obtain a BBM-BSA-NPs colloidal solution;

[0017] (4) Centrifuge, precipitate, and remove unreacted substances.

[0018] (4) Centrifuge, precipitate, and remove unreacted substances.

[0019] In step (1), the mass ratio of serum albumin to berberine hydrochloride is 1:0.1-0.4;

[0020] In step (1), the mass concentration of the albumin aqueous solution is 3-7 mg / mL, preferably 4-6 mg / mL;

[0021] In the step (1), the mass-volume concentration of berberine hydrochloride in the solution is 1-3 mg / mL, preferably 1-2 mg / mL;

[0022] In the step (2), the stirring mode is magnetic stirring or vortex mode, preferably magnetic stirring mode.

[0023] In the step (2), the ethanol dropwise speed is 1-6 mL / min, preferably 2-6 mL / min.

[0024] In the step (2), the volume ratio of water phase to anhydrous ethanol is 1:2-1:5, preferably 1:2-1:4.

[0025] In the step (3), the concentration of glutaraldehyde solution is 20%-25%, and the curing time is 2-24 hours, preferably 4-8 hours.

[0026] Further, in order to improve the stability of the BBM-BSA-NPs, a freeze-drying protective agent is added to the BBM-BSA-NPs to prepare a BBM-BSA-NPs freeze-dried powder.

[0027] The freeze-drying protective agent is one or two of lactose, mannitol and trehalose, preferably lactose+trehalose (1:1), mannitol+trehalose (1:1) and lactose+mannaol (1:1).

[0028] The amount of the freeze-drying protective agent is 2-7% (w / v), preferably 3-5% (w / v).

[0029] The albumin-loaded berberine hydrochloride nanocomposite provided by the application has a hydrodynamic diameter of 100-200 nm, preferably 140-180 nm, and a Zeta potential of -39 to -29 mV.

[0030] The application also provides the use of the albumin-loaded berberine hydrochloride nanocomposite or the freeze-dried powder thereof in the preparation of an antitumor drug.

[0031] The tumor is colorectal cancer, liver cancer, ovarian cancer, gastric cancer, bladder cancer, breast cancer, lung cancer.

[0032] The application also provides the use of the albumin-loaded berberine hydrochloride nanocomposite or the freeze-dried powder thereof in the preparation of a chemotherapeutic or immunotherapeutic drug sensitizer.

[0033] This invention employs a desolvation-chemical crosslinking method to prepare albumin-loaded berberine hydrochloride nanocomposites. Albumin is desolvated (or aggregated) using an organic solvent. Anhydrous ethanol is continuously added dropwise to an aqueous albumin solution under stirring until a pale blue opalescence appears, thus obtaining nanoparticles. During the addition of ethanol to the solution, the tertiary structure of albumin is gradually altered, its water solubility decreases, and the albumin undergoes phase separation, forming an increasingly hydrophobic substance that encapsulates the hydrophobic drug within its hydrophobic structure. This substance tends to form small-sized desolvated albumin aggregates (agglomerate layers). Albumin particles that have not formed a sufficiently stable morphology can be redissolved after dispersion in water. Glutaraldehyde hardens the aggregate layer through crosslinking, where the amino portion of the lysine residue and the arginine portion of the albumin guanidine side chain solidify through a condensation reaction with the aldehyde group of glutaraldehyde, resulting in stable nanoparticles with a particle size of 100-200 nm.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1) Retain the antitumor efficacy of berberine hydrochloride while overcoming its limitations of rapid metabolism and small molecular weight; increase the volume and mass of the formulation to improve the sustained-release properties of the drug.

[0036] 2) Using serum albumin as the backbone material, it has good human compatibility, is safe and non-toxic, is biodegradable, and can increase the concentration of circulating drugs in the blood.

[0037] 3) Albumin was used as a carrier to prepare berberine hydrochloride albumin nanoparticles, which exerted a synergistic anti-tumor effect on the tumor site through the EPR effect.

[0038] 4) Albumin is easily absorbed by the lymphatic system, which can effectively prevent lymphatic metastasis and liver metastasis.

[0039] 5) Albumin itself has targeting properties, which can reduce the toxic side effects on non-target organs throughout the body. Attached image description:

[0040] Figure 1 Electron micrograph of bovine serum albumin nanoparticles (BBM-BSA-NPs) containing berberine hydrochloride;

[0041] A: Electron microscope scale bar 200 nm; B: Electron microscope scale bar 500 nm; C: Electron microscope scale bar 1.0 μM;

[0042] Figure 2 The in vitro drug release curves for BBM-BSA-NPs and BBM are shown. Data are expressed as mean ± standard deviation.

[0043] Figure 3 Survival rate and IC50 of CT26 cell line treated with BBM-BSA-NPs and BBM. 50 value.

[0044] A, B, C, D, E, and F represent cell viability and IC50 values ​​at 24h, 48h, and 72h after drug administration, respectively. 50 Value; Mean±SEM; n=3, *P<0.05; **P<0.01, ***P<0.001.

[0045] Figure 4 Survival rate and IC50 of HT29 cell line treated with BBM-BSA-NPs and BBM. 50 value.

[0046] A, B, and C represent the values ​​at 24 hours, 48 ​​hours, and 72 hours after drug administration, respectively; D represents the IC50 value at 24 hours after drug administration. 50 Value; Mean±SEM; n=3, *P<0.05; **P<0.01, ***P<0.001.

[0047] Figure 5 The effects of bovine serum albumin groups Blank-BSA-NPs-1 and Blank-BSA-NPs-2 on the survival of CT26 and HT29 cells.

[0048] A: CT26-24h, B: CT26-48h, C: CT26-72hHT29, D: HT29-24h, E: HT29-48h, F: HT29-72h; Data are expressed as Mean±SEM; n=3, *P<0.05.

[0049] Figure 6 The 24-hour survival rate and IC50 of AGS, CAOV3, and HepG2 cells treated with BBM-BSA-NPS and BBM were compared. 50 value.

[0050] A, D: AGS, administered for 24 hours; B, E: CAOV3, administered for 24 hours; C, F: HepG2, administered for 24 hours; Data are expressed as mean ± SEM; n = 3, *P < 0.05; **P < 0.01, ***P < 0.001.

[0051] Figure 7 Survival rates of HEK-293 cells treated with BBM-BSA-NPs and BBM.

[0052] A: 24h, B: 48h, C: 72h; Mean±SEM; n=3, *P<0.05.

[0053] Figure 8 Morphological changes in CT26 cells after 24 h of treatment with BBM-BSA-NPs (10X).

[0054] A: Normal cells; B: Cell morphology after administration at a concentration of 16 μM; C: Cell morphology after administration at a concentration of 32 μM; D: Cell morphology after administration at a concentration of 56 μM; n = 3.

[0055] Figure 9 The apoptosis of CT26 cells was observed in different concentrations of BBM-BSA-NPs.

[0056] Data are expressed as Mean±SEM; n=3, *P<0.05; **P<0.01.

[0057] Figure 10 The apoptosis of CT26 cells was investigated at concentrations of 16 μM, 32 μM, and 56 μM for BBM and BBM-BSA-NPs.

[0058] Data are expressed as Mean±SEM; n=3, *P<0.05; **P<0.01.

[0059] Figure 11 Tumor images of COC CT26 tumor-bearing mice treated with different drug groups.

[0060] A: On day 14, mice in each group were sacrificed, tumors were removed, and tumor sizes were measured in different drug administration groups; B: Typical tumor-bearing mice in each group on day 14.

[0061] Figure 12 Changes in tumor volume in mice on day 14 after administration of saline, Blank-BSA-NPs, BBM-BSA-NPs, and BBM. Data are expressed as mean ± standard deviation. n = 10, *P < 0.05; ****P < 0.0001.

[0062] Figure 13 The mean tumor weight of tumor-bearing mice on day 14 after treatment with saline, Blank-BSA-NPs, BBM-BSA-NPs, and BBM.

[0063] Data are expressed as Mean±SEM; n=10, *P<0.05; **P<0.01, ****P<0.0001.

[0064] Figure 14 The study measured the changes in body weight of tumor-bearing mice on day 14 after treatment with saline, Blank-BSA-NPs, BBM-BSA-NPs, and BBM. Data are expressed as mean ± SEM; n = 10.

[0065] Figure 15 The effects of BBM and BBM-BSA-NPs on the viability of human tumor cells.

[0066] A: Bladder cancer T24 B: Breast cancer MCF-7 C: Liver cancer HepG2 D: Lung cancer H129.

[0067] Among them, BBM-BSA-NPs is a nanocomposite based on albumin and berberine hydrochloride, where BBM is berberine hydrochloride, BSA is bovine serum albumin, NPs are nanoparticles, and Control is the saline control group. Detailed implementation method:

[0068] The present invention will now be described with reference to embodiments. However, the present invention is not limited to the embodiments described below.

[0069] The present invention relates to serum albumin nanoparticles loaded with berberine hydrochloride, which are composed of berberine hydrochloride and bovine serum albumin (BSA). Deionized water, anhydrous ethanol, sodium hydroxide, and glutaraldehyde were all commercially available in the preparation process.

[0070] In this invention, the stirring method, the order of adding BSA drug solution and ethanol, the ethanol dropping rate, the concentration of BSA solution, the dosage (concentration of BBM), the pH value, the volume ratio of ethanol to water phase, the amount of glutaraldehyde used, and the curing time all have different degrees of influence on the nanocomposite.

[0071] The order in which BSA drug solution and ethanol are added dropwise significantly affects the particle size, encapsulation efficiency, and drug loading of BBM-BSA-NPs. Compared to reverse addition, adding BSA drug solution dropwise to ethanol results in smaller particle sizes, but the latter leads to higher encapsulation efficiency and drug loading, more uniform particle size distribution, and a better polydispersity index. This is because when ethanol is added dropwise to the BSA drug solution, the instantaneous ethanol concentration at the solution interface is higher, leading to faster desolvation of albumin and larger albumin aggregates. During this process, the drug is encapsulated within the hydrophobic structure of albumin along with the desolvation. This drug loading reaction continues until the ethanol addition is complete, resulting in more drug being encapsulated within the hydrophobic structure of albumin and a higher encapsulation efficiency. Therefore, this invention employs the sequential addition of BSA drug solution to ethanol for preparation.

[0072] pH value has a significant impact on nanoparticle size. At pH 7.00, the solution quickly turns white and turbid during the addition of ethanol, with large albumin aggregates forming flocculent precipitates at the bottom. As the pH increases, the flocculents disappear, the solution gradually becomes clear, and the particle size gradually decreases. At pH 9, after the addition of ethanol, the solution is transparent, exhibiting a pale blue opalescence, and the particle size is small. However, when pH > 9, the albumin nanosphere formation rate and yield decrease, requiring more organic solvent ethanol to obtain the same nanoparticle yield. pH value controls the aggregation of BSA molecules during the desolventization process, providing both electrostatic and hydrophobic interactions. When pH is above 7, the high electrostatic repulsion condition of BSA molecules (isoelectric point pI of approximately 4.9) limits the coagulation of protein-protein interactions, thus allowing the formation of smaller BSA nanoparticles. The preferred pH value in this invention is 8.5-9.0.

[0073] This invention investigated the effects of stirring methods on the particle size, encapsulation efficiency, and drug loading of nanoparticles. The results showed that the particle size of drug-loaded nanoparticles prepared by vortex stirring was superior to that prepared by magnetic stirring, but the encapsulation efficiency of vortex stirring fluctuated significantly and was lower than that of nanoparticles prepared by magnetic stirring. Furthermore, the artificially fixed vortex preparation method is difficult to control, and its stability is hard to guarantee. Considering particle size, encapsulation efficiency, drug loading, and process operability, magnetic stirring was chosen for nanoparticle preparation.

[0074] The ethanol dropping rate has a significant impact on the nanoparticle size. As the ethanol dropping rate increases, the particle size decreases sharply, while the polydispersity index and encapsulation efficiency decrease slightly. Considering the particle size, encapsulation efficiency, and drug loading, the ethanol dropping rate was determined to be 1-6 mL / min, preferably 2-6 mL / min.

[0075] BSA concentration affects particle size, encapsulation efficiency, and drug loading. The average particle size of nanoparticles increases with increasing BSA concentration, while the drug loading gradually decreases and the encapsulation efficiency gradually increases. However, with further increases in BSA concentration, the encapsulation efficiency decreases. Therefore, a BSA concentration of 3-7 mg / mL was determined, preferably 4-6 mg / mL.

[0076] BBM concentration affects particle size, encapsulation efficiency, and drug loading. As drug concentration increases, both encapsulation efficiency and particle size of the nanoparticles increase; however, when drug concentrations are 2.0 and 3.0 mg / mL, the nanoparticle size and encapsulation efficiency are similar. This invention preferably uses a BBM concentration of 1-3 mg / mL, more preferably 1-2 mg / mL.

[0077] The volume ratio of ethanol to BSA (aqueous phase) has a significant impact on particle size. As the volume of ethanol increases, the nanoparticle size increases sharply. However, the encapsulation efficiency and drug loading show a trend of first increasing and then decreasing. The encapsulation efficiency and drug loading of the nanoparticles reach their highest values ​​when the volume ratio is 1:3, and then begin to decrease. Therefore, the volume ratio of BSA solution to ethanol is 1:2-1:5, preferably 1:2-1:4.

[0078] The addition of glutaraldehyde primarily affects the internal cross-linking morphology and in vitro release behavior of nanoparticles. The principle is that the amino groups on the lysine residues and the guanidino side chains on the arginine residues of albumin undergo a condensation reaction with the aldehyde groups of glutaraldehyde, solidifying to form a network-like framework. With increasing glutaraldehyde dosage, the internal cross-linking of the nanoparticles deepens, the porosity further decreases, and the overall nanoparticle structure becomes more compact, leading to a smaller particle size, reduced drug loading, and slower drug release. As the glutaraldehyde dosage increases, the nanoparticle size gradually decreases, but the encapsulation efficiency and drug loading gradually decrease. When the glutaraldehyde concentration is 25%, the dosage is 5-25 μL, preferably 5 μL.

[0079] Increasing the curing time from 2 hours to 4 hours resulted in an increase in both the average nanoparticle size and encapsulation efficiency, with 4 hours being the optimal time. Subsequently, with further extension of the curing time, the nanoparticle size increased slowly, while the drug loading and encapsulation efficiency remained relatively stable. Therefore, the curing time is 2-24 hours, preferably 4-8 hours.

[0080] Example 1: Preparation of BBM-BSA-NPs

[0081] Accurately weigh BSA and BBM, add them to 2 mL of deionized water, and sonicate until dissolved. Adjust the pH of the BSA drug solution with 0.10 mol / L sodium hydroxide. Under constant temperature and magnetic stirring at 37°C, add appropriate amounts of anhydrous ethanol dropwise to the aqueous phase at different dropping rates. Finally, add a certain amount of 25% glutaraldehyde solution, continue stirring to solidify, and remove the organic solvent by rotary evaporation at 40°C to obtain the BBM-BSA-NPs colloidal solution. Centrifuge at 12000 rpm for 45 min in a high-speed refrigerated centrifuge, collect the precipitate, redisperse it in an appropriate amount of water, and repeat the operation twice to remove unreacted BSA, residual glutaraldehyde, anhydrous ethanol, and free drug, thus obtaining BBM-BSA-NPs.

[0082] In this invention, the aqueous phase and ethanol are mixed in the order of adding BSA drug solution dropwise to ethanol, and BBM-BSA-NPs are prepared by magnetic stirring (Schemes 1-7). The results are shown in Table 1:

[0083] Table 1 Effect of different preparation conditions on the properties of BBM-BSA-NPs

[0084]

[0085] The results showed that when the pH was 8.5-9.0, the BSA concentration was 4-6 mg / mL, the BBM concentration was 1-3 mg / mL, the ethanol dropping rate was 1-6 mL / min, the volume ratio of aqueous phase to anhydrous ethanol was 1:2-1:4, the curing agent concentration was 25%, the dosage was 5-25 μL, and the curing time was 4-24 hours, the particle size of the nanoparticles was less than 200 nm, the PDI was less than 0.2, the encapsulation efficiency was greater than 50%, and the drug loading was greater than 15%.

[0086] When the pH is 9.0, the BSA concentration is 4-6 mg / mL, the BBM concentration is 1-2 mg / mL, the ethanol dropping rate is 2-6 mL / min, the volume ratio of aqueous phase to anhydrous ethanol is 1:2-1:4, the curing agent concentration is 25%, the dosage is 5 μL, and the curing time is 4-24 hours, the particle size of the nanoparticles is less than 200 nm, the PDI is less than 0.2, the encapsulation efficiency is greater than 85%, and the drug loading is greater than 25%.

[0087] Example 2: Screening of lyophilization protectants in the preparation of BBM-BSA-NPs lyophilized powder

[0088] 2 mL of BBM-BSA-NPs (Example 1, Scheme 2) was placed in a vial, and 3% (w / v) of lactose, mannitol, trehalose, lactose + trehalose (1:1), mannitol + trehalose (1:1), and lactose + mannitol (1:1) were added as lyophilization protectants until completely dissolved. The solution was pre-frozen at -20°C for 2 hours, then frozen at -80°C for 12 hours, and finally dried in a vacuum freeze dryer for 12 hours to obtain BBM-BSA-NPs lyophilized powder.

[0089] After taking out the lyophilized powder sample, compare and record its appearance characteristics, then add 1 mL of deionized water to re-dissolve it, observe its resolubility and solution state, and screen nano-lyophilization protectants.

[0090] The types of freeze-drying protectants were initially screened based on the uniformity of appearance, color, and redispersibility of the freeze-dried powder. The best appearance was characterized by a smooth, even surface, free from lumps, collapse, and wrinkles; the best color was characterized by uniformity, absence of patches, and a fine, even texture; the best redispersibility was characterized by the ability of the freeze-dried powder to quickly recover to a clear, homogeneous solution free of visible fine particles after being added to the original volume of deionized water and shaken, with a shorter reconstitution time being better.

[0091] Freeze-dried powders were prepared by adding 3% (w / v) of the freeze-drying protectants lactose, mannitol, trehalose, lactose + trehalose (1:1), mannitol + trehalose (1:1), and lactose + mannitol (1:1). The appearance of each group of freeze-dried powders is shown in Table 2. Table 2 shows that the appearance of lactose and mannitol alone is better, and the resolubility is better. When using the combined protectants lactose + trehalose and lactose + mannitol in a 1:1 ratio, the freeze-dried powders have a neat appearance, uniform color, and better resolubility and redispersibility than those with a single protectant, as shown in Table 3. Therefore, the lactose + trehalose combination and the lactose + mannitol combination were selected as freeze-drying protectants.

[0092] Table 2 Appearance results of different lyophilization protectants (n=3)

[0093]

[0094] Table 3 Appearance results using different lyophilization protectants (n=3)

[0095]

[0096] Example 3: Screening of the Dosage of Lyophilizing Protectant in the Preparation of BBM-BSA-NPs Lyophilized Powder

[0097] BBM-BSA-NPs (Example 1, Scheme 2) were selected, and lactose + trehalose (1:1) was used as the freeze-drying protectant. The freeze-drying effect of different amounts of lactose + trehalose (1:1) was further compared, and the results are shown in Table 4. Table 4 shows that when the amount of lactose + trehalose (1:1) was 2-7% (w / v), the particle size was less than 250 nm and the PDI was less than 0.3. When the amount was 3-5%, the particle size was less than 200 nm and the PDI was less than 0.2. The freeze-dried powder showed good resolubility and redispersibility; therefore, 3-5% lactose + trehalose (1:1) was selected as the freeze-drying protectant.

[0098] Table 4. Particle size, PDI, and Zeta potential measurements of lyophilized lactose + trehalose (1:1) powders after reconstitution (n=3)

[0099]

[0100] Example 4: Preparation of BBM-BSA-NPs lyophilized powder

[0101] 2 mL of BBM-BSA-NPs (Example 1, Scheme 2) was placed in a vial, and 3% (w / v) lactose + trehalose (1:1) was added as a lyophilization protectant until it was completely dissolved. The solution was pre-frozen at -20°C for 2 hours, then frozen at -80°C for 12 hours, and finally dried in a vacuum freeze dryer for 12 hours to obtain BBM-BSA-NPs lyophilized powder.

[0102] The transmission electron microscopy results of BBM-BSA-NPs are as follows: Figure 1 ,Depend on Figure 1 It can be seen that BBM-BSA-NPs are regular spherical with smooth surfaces and uniform particle size distribution, proving that the BBM-BSA-NPs formulation was successfully prepared.

[0103] The particle size and zeta potential of the BBM-BSA-NPs lyophilized nanoparticle powder were determined after reconstitution with deionized water. The average particle size obtained was 147.60±3.33 nm, the average PDI was 0.02±0.01, and the average zeta potential was -29.69±0.58 mV.

[0104] Example 5: In vitro release of BBM-BSA-NPs nanocomposite.

[0105] Accurately weigh 6 mg of BBM active pharmaceutical ingredient into a 10 mL volumetric flask, and dilute to volume with PBS buffer at pH 5.8 to obtain a sample solution concentration of 0.6 mg / mL. Take three 2 mL aliquots of the solution and place them into pretreated dialysis bags. Clamp the ends of the dialysis bags tightly and place them into stoppered bottles containing 100 mL of release medium.

[0106] Accurately weigh three 35mg portions (containing 6mg BBM) of BBM-BSA-NPs lyophilized powder (Example 4), add 2mL of pH 5.8 PBS release medium to disperse and dissolve, then pack them into a pretreated dialysis bag, clamp both ends of the dialysis bag with clamps, and place them into a stoppered bottle containing 100mL of PBS release medium.

[0107] In vitro release assays were performed according to Method I of the 2020 edition of the Chinese Pharmacopoeia. The solution was shaken at 37°C and 100 rpm for 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, and 72 h, with simultaneous replenishment of the same release medium at the same temperature and volume. A suitable amount of the solution was filtered through a 0.45 μM filter, and the filtrate was analyzed by HPLC to determine the BBM content. The cumulative release percentage (Q) at each time point was calculated. In vitro release curves were plotted with release time (t) on the x-axis and cumulative release percentage (Q) on the y-axis to compare the release of BBM raw material and BBM-BSA-NPs. Results are shown below. Figure 2 .

[0108] Depend on Figure 2It can be seen that BBM active pharmaceutical ingredient (API) exhibits a burst release at 0.5 hours, with a cumulative release of 59.05±2.28%; at 2 hours, the cumulative release is 87.47±4.22%; and at 5 hours, the cumulative release reaches 96.27±1.44%, essentially achieving complete release. This indicates that BBM has a relatively fast release rate and can almost achieve complete release. BBM-BSA-NPs, on the other hand, exhibit a burst release at 0.5 hours, with a cumulative release of 21.96±4.44%; at 3 hours, a relatively large cumulative release of 41.07±4.47% is reached. Subsequently, the drug release becomes slower, with cumulative releases of 48.44±0.32%, 49.01±0.57%, and 49.91±0.42% at 24 hours, 48 ​​hours, and 72 hours, respectively, indicating that BBM-BSA-NPs possess certain sustained-release characteristics.

[0109] Example 6: Antitumor activity of berberine hydrochloride albumin nanocomposite

[0110] Cell lines: CT26, HEK 293, HT29, AGS, CAOV3 and HepG2 cells were all obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0111] Cell culture:

[0112] Mouse COC cells CT26 were cultured in RMPI-1640 medium containing 10% FBS and 1% penicillin antibiotics at 37°C in an incubator containing 5% CO2. When the cells reached 70-90% confluence, they were passaged using trypsin containing EDTA. The entire cell culture process was conducted under sterile conditions, and all reagents and equipment used were sterilized by high-temperature sterilization or alcohol disinfection.

[0113] The culture methods and procedures for human COC cells HT29, human renal epithelial cells 293, human gastric adenocarcinoma cells AGS, human ovarian cancer cells CAOV3, and human liver cancer cells HepG2 were the same as those for CT26 cells. However, the culture medium for HT29 was changed to 5A medium containing 10% FBS and 1% penicillin-dextrose antibody, and the culture media for the other cells were changed to DMEM containing 10% FBS and 1% penicillin-dextrose antibody.

[0114] In vitro cytotoxicity assay

[0115] Take CT26 mouse COC cells in logarithmic growth phase from an incubator, add 500 μL of trypsin solution containing EDTA, and incubate at 37°C for 5 min. Add 1 mL of RPMI-1640 medium to terminate digestion, collect the cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 1 mL of RPMI-1640 serum medium, repeatedly pipette to prepare a single-cell suspension, observe under a microscope, calculate the cell concentration using a counting chamber, add an appropriate amount of RPMI-1640 medium, and dilute to a cell suspension of 50,000 cells / mL. Seed the cell suspension into the first to second-to-last rows of nine 96-well plates, and add 100 μL of PBS to the last row of wells. After 24 hours, once the cells were observed to be adhering and growing normally, the culture medium was removed using a pipette, and different concentrations (2, 4, 8, 16, 32, 56 μM) of BBM and BBM-BSA-NPs solutions were added to replace the medium. Six parallel wells were set up for each concentration group in each 96-well plate. The experiment was divided into three groups, with three 96-well plates per group. After culturing each group of 96-well plates in an incubator for 24 h, 48 h, and 72 h, the effect of BBM and BBM-BSA-NPs on cell viability was determined using the CCK8 assay. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was determined by fluorescence intensity, and the IC50 was calculated. 50 The values ​​were then analyzed statistically.

[0116] Based on the BBM administration concentration range, corresponding concentrations of Blank-BSA-NPs-1 (3.5, 7, 14, 28, 56, and 98 μg / mL) and doubled concentrations of Blank-BSA-NPs-2 (7, 14, 56, 196, and 343 μg / mL) were established to investigate the effect of Blank-BSA-NPs treatment on the viability of CT26 and HT29 cells after 24 h, 48 h, and 72 h. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was determined by fluorescence intensity and IC50 was calculated. 50 value.

[0117] Cell viability is calculated using the following formula: Cell viability = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%

[0118] The effects of BBM and BBM-BSA-NPs on the survival rate of human COC cells HT29 were determined using the same method, but the culture medium was replaced with 5A medium.

[0119] The inhibitory effects of BBM and BBM-BSA-NPs on human renal epithelial cells 293, human gastric adenocarcinoma cells AGS, human ovarian cancer cells CAOV3, and human liver cancer cells HepG2 were determined using the same method.

[0120] In vitro cytotoxicity test results

[0121] Figure 3 Survival rate and corresponding IC50 of CT26 cells after drug administration of BBM and BBM-BSA-NPs 50 The results showed that after different treatment times, BBM and BBM-BSA-NPs at different concentrations significantly inhibited the survival rate of CT26 cells. Furthermore, the inhibitory effect gradually increased with increasing drug concentration and treatment time, exhibiting a concentration- and time-dependent relationship. Figure 3 (AC). At 24h, 48h, and 72h, the cell inhibition rate of BBM-BSA-NPs was higher than that of the BBM group (significant differences were observed at drug concentrations of 8, 16, and 32 μM, P < 0.05). The results also showed that after 24h, 48h, and 72h of drug treatment, the difference in cell inhibition between BBM-BSA-NPs and BBM became more significant at drug concentrations of 16 and 32 μM (P < 0.05). This is because in the BBM-BSA-NPs nanostructure, BBM and BSA are more tightly bound, exhibiting a certain sustained-release effect. With prolonged treatment time, more BBM is released from the bound nanostructure, gradually enhancing the cell inhibition effect, whereas free BBM does not have this mechanism.

[0122] BBM-BSA-NPs were used to treat CT26 cells at 24, 48, and 72 hours of IC50. 50 The concentrations were 9.82 μM, 9.321 μM, and 8.949 μM, respectively, all lower than the IC50 values ​​after 24, 48, and 72 hours of BBM treatment. 50 The values ​​were 16.71 μM, 14.88 μM, and 12.59 μM (which were 1.70, 1, 60, and 1.41 times higher than those of the BBM-BSA-NPs group, respectively). Figure 3 (DF). The above results indicate that, compared with BBM, BBM-BSA-NPs can significantly enhance the inhibitory effect of BBM on COC CT26 cells.

[0123] Figure 4 The results showed that BBM and BBM-BSA-NPs exhibited similar pharmacodynamic trends in HT29 cells as they did in CT26 cells. BBM and BBM-BSA-NPs also showed significant inhibitory effects on HT29 cells, with these effects being concentration- and time-dependent. Figure 4(AC). Meanwhile, it was found that HT29 cells were more sensitive to the drug than CT26 cells. At 48 h, under the treatment of a very low concentration of 2 μM BBM-BSA-NPs, the survival rate of HT29 cells was 40.12% ± 3.81%, below 50%, while the cell survival rate of the BBM group was 75.84% ± 6.25% (P < 0.05). At 72 h, the cell survival rates of the BBM-BSA-NPs and BBM groups were 12.50% ± 0.65% and 15.83% ± 1.97%, respectively (P < 0.05). Since the cell survival rate was already below 50% at low concentrations at 48 h and 72 h, only the IC50 of the BBM-BSA-NPs and BBM groups at 24 h was compared. 50 Value, see Figure 4 IC of Group D, BBM 50 The value is 1.36 times that of the BBM-BSA-NPs group.

[0124] Figure 5 The effects of bovine serum albumin-based Blank-BSA-NPs-1 and Blank-BSA-NPs-2 on the survival rate of CT26 and HT29 cells were investigated. In nanodelivery systems, the non-toxicity, biocompatibility, and safety of the carrier are crucial. Therefore, the effects of Blank-BSA-NPs (Blank-BSA-NPs-1 and Blank-BSA-NPs-2) at various concentration ranges and doubled concentrations on cell survival were examined. At 24 h, 48 h, and 72 h after administration of Blank-BSA-NPs-1 and Blank-BSA-NPs-2, respectively, the survival rates of CT26 and HT29 cells were approximately 100%, and there was no significant difference between the two different concentrations of Blank-BSA-NPs (P>0.05). Within the administered concentration range and at doubled concentrations, Blank-BSA-NPs had almost no effect on the survival rate of CT26 and HT29 cells. The above results indicate that the carrier material BSA has no cytotoxic effect on CT26 and HT29 cells, and exhibits good biocompatibility and safety. Figure 5 AF).

[0125] Figure 6 The 24-hour survival rate and IC50 of AGS, CAOV3, and HepG2 cells treated with BBM-BSA-NPS and BBM were compared. 50 The values ​​were [value missing]. Both BBM and BBM-BSA-NPs significantly inhibited AGS, CAOV3, and HepG2. After 24 hours of administration, BBM and BBM-BSA-NPs showed similar inhibitory effects on AGS cell viability; the IC50 value of the BBM-BSA-NPs group was [value missing]. 50 The value was 43.42 μM, slightly lower than the IC50 of the BBM group. 50The value was 44.33 μM, with no statistically significant difference. Figure 6 (A and D). In CAOV3 cells, at high concentrations, BBM showed stronger cytotoxicity than BBM-BSA-NPs, with a statistically significant difference (P<0.05). This may be due to the sustained-release effect of BBM-BSA-NPs, which takes longer to reach a sufficient effective concentration. However, the IC50 of the BBM-BSA-NPs group was significantly lower. 50 The value was 40.80 μM, lower than the 43.05 μM in the BBM group. Figure 6 (B and E). At various drug concentrations, the cell viability of the BBM-BSA-NPs group in HepG2 cells was significantly lower than that of the BBM-treated group (P<0.05), with a lower IC50 at 24 h. 50 The value was 40.27 μM, lower than the IC50 value of the BBM group. 50 Value 44.47 μM ( Figure 6 C and F).

[0126] The above experimental results show that BBM-BSA-NPs have a certain targeting selectivity for in vitro cytotoxicity against different types of cancer cells, with stronger selectivity for liver cancer than for gastric adenocarcinoma and ovarian cancer, and better in vitro anti-tumor effect than BBM.

[0127] Example 7: Toxicity of BBM and BBM-BSA-NPs on HEK-293 Cells

[0128] Drug nephrotoxicity is an important aspect of drug safety evaluation and toxicology research. HEK-293 cells, or human embryonic kidney cells, derived from human kidneys, are commonly used to study in vitro cytotoxicity. This invention uses HEK-293 cells to conduct a preliminary evaluation of the nephrotoxicity of the drugs BBM-BSA-NPs and BBM. Figure 7AC). After 24 hours of treatment with BBM-BSA-NPs and BBM, the survival rate of HEK-293 cells remained around 100%, with no significant change. With prolonged administration, after 48 hours, the cell survival rate in the low-concentration group showed almost no change. However, at a higher concentration of 32 μM, the cell survival rates of both the BBM-BSA-NPs and BBM groups began to decrease, with survival rates of 87.54 ± 3.59% and 86.46 ± 0.15%, respectively. At the maximum concentration of 56 μM, the cell survival rates of both the BBM-BSA-NPs and BBM groups... Cell viability further decreased, to 72.84±4.68% and 68.37±5.74%, respectively. After 72 hours of administration, cell viability remained unchanged in the low-concentration group. At a concentration of 32 μM, cell viability in the BBM-BSA-NPs and BBM groups showed little change, at 88.75±8.44% and 80.87±3.76%, respectively, with no statistically significant difference. At the maximum concentration of 56 μM, cell viability in the BBM-BSA-NPs and BBM groups decreased, to 63.55±9.22% and 62.43±6.20%, respectively. The results indicate that BBM-BSA-NPs and BBM exhibit some selectivity for cells. After 24 hours of administration, the drugs did not affect the viability of HEK-293 cells and showed no nephrotoxicity. With prolonged administration, low concentrations of 2, 4, 8, and 16 μM showed no toxicity to HEK-293 cells, but the high concentration of 56 μM showed some toxicity.

[0129] Example 8: Observation of the effect of BBM-BSA-NPs on CT26 cell morphology using an inverted microscope

[0130] The effect of BBM-BSA-NPs on the morphology of CT26 cells was observed using an inverted microscope. The results are as follows: Figure 8 In the normal control group, cells grew normally, with smooth cell membranes, clear edges, good growth status, and high density. 24 hours after drug administration, the cells treated with 16 μM BBM-BSA-NPs became rounder, with brighter edges, and dead cells appeared. The number of adherent cells decreased, intercellular spaces widened, and density decreased. With increasing drug concentration, the number of rounder, brighter cells increased, their morphology became more irregular, adherent cells further decreased, floating dead cells increased, and intercellular spaces widened further. At the highest drug concentration of 56 μM, the cell morphology became completely irregular, most cells died, and a small number of adherent cells remained round and bright.

[0131] Example 9 Apoptosis Experiment

[0132] Concentration-dependent experiment of BBM-BSA-NPs in CT26 cells

[0133] The apoptosis induced by BBM-BSA-NPs in CT26 cells was analyzed using Annexin V-FITC / PI double staining. After treating CT26 cells with BBM-BSA-NPs at concentrations of 0, 16, 32, and 56 μM for 24 hours, the percentages of total apoptotic cells (early and late apoptotic cells) in each concentration group were 12.94±9.53%, 20.97±8.39%, 41.61±12.96%, and 52.80±8.43%, respectively; the percentages of early apoptotic cells were 0.83±0.32%, 5.51±2.98%, 10.27±8.07%, and 12.20±3.07%, respectively; and the percentages of late apoptotic cells were 12.11±9.21%, 15.47±6.58%, 31.33±6.02%, and 40.60±11.19%, respectively. Figure 9 The results showed that with increasing drug concentration, the proportion of apoptotic CT26 cells gradually increased, and the total number of apoptotic cells in the highest concentration group was significantly higher than that in the control group and the low concentration group (P<0.05), indicating that BBM-BSA-NPs have a significant apoptosis-inducing effect on CT26 cells in a concentration-dependent manner.

[0134] Example 10: Comparison of the effects of BBM and BBM-BSA-NPs on apoptosis in CT26 cells.

[0135] The apoptosis of CT26 cells was compared using Annexin V-FITC / PI double staining at three drug concentrations: low (16 μM), medium (32 μM), and high (56 μM). At medium and high concentrations, the percentage of apoptosis in the BBM-BSA-NPs and BBM groups was significantly higher than that in the control group (P < 0.05). At low concentrations, although the percentage of apoptosis in the BBM group was higher than that in the control group, the difference was not significant (P > 0.05), while the percentage in the BBM-BSA-NPs group was significantly higher than that in the control group. Figure 10 (P<0.05). Notably, at all three drug concentrations, the percentage of apoptosis in the BBM-BSA-NPs group was significantly higher than that in the BBM group (P<0.05). This indicates that BBM-BSA-NPs has a stronger effect on inducing apoptosis in CT26 cells than BBM, and albumin nanodelivery of BBM can significantly enhance the anticancer effect of BBM.

[0136] Examples 6-10 demonstrate that BBM-BSA-NPs not only exhibit good toxicity against COC cells CT26 and HT29, but also against other cancer cells, including human gastric adenocarcinoma cells AGS, human ovarian cancer cells CAOV3, and human liver cancer cells HepG2. BBM-BSA-NPs strongly inhibited the proliferation of COC cells in vitro and promoted cancer cell apoptosis, showing significantly better in vitro antitumor activity than BBM. Furthermore, it showed no or slight toxicity inhibition against normal human kidney cells HEK 293, and the carrier material BSA was non-toxic to both CT26 and HT29 cells. This demonstrates that BBM-BSA-NPs have good biocompatibility and safety, exhibiting the advantages of high efficiency and low toxicity.

[0137] Example 11: In vivo antitumor activity of BBM-BSA-NPs

[0138] Experimental animals: 5-week-old Balb / c mice (SPF grade, female), weighing 20±2g, purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0139] Feeding conditions: maintain a temperature of 20-22℃, a humidity of 55%, and a day-night cycle of 12 hours.

[0140] Cell line: Mouse COC CT26 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0141] Experimental methods:

[0142] Frozen CT26 cells were rapidly thawed in a 37°C water bath, centrifuged, and the supernatant was discarded. RMPI-1640 culture medium containing 10% FBS and 1% penicillin was added, and the cells were cultured in a 37°C incubator containing 5% CO2. The entire cell culture process was conducted under sterile conditions; all reagents and equipment used were sterilized by high-temperature sterilization or alcohol disinfection. When the cells reached 70-90% confluence, they were passaged using trypsin containing EDTA until the cell number reached the required quantity for subsequent animal experiments. All cells were then collected, diluted with PBS, and cultured at a density of 1×10^6 cells / mL. 7 Cell suspensions of 1 cell per mL were prepared and stored in an ice box.

[0143] CT26 COC cells were subcutaneously injected into the back of mice, with each mouse receiving 0.1 mL of cell suspension. Tumor growth was observed daily, and the length and width of the tumors were measured and recorded to calculate the tumor volume. On the second day after inoculation, small granular protrusions appeared at the inoculation site, continuing to grow and forming a distinct tumor mass, indicating successful model establishment. Tumor volume = 0.5 × maximum diameter × minimum diameter 2 When the tumor grows to 100mm 3Around [time], begin drug administration. This is because the tumor volume determined in the preliminary experiment was 200 mm². 3 Around day 12, however, some mice in the control group developed excessively large and ulcerated tumors. Therefore, for ethical reasons, this experiment opted to begin administering the drug when the tumor volume was relatively small, specifically setting the tumor volume at 100 mm². 3 about.

[0144] Tumor-bearing mice were randomly divided into four groups (n=10): Control group (saline), Blank-BSA-NPs, BBM-BSA-NPs, and BBM group (30 mg / kg). Tumors were classified into four groups based on their mean tumor volume (100 mm²). 3 Mice were administered the drugs via tail vein injection on days 1, 3, 5, 7, and 9, every other day. Before each daily administration, mice were weighed, and changes in tumor volume were recorded; tumor volume was calculated using the formula. Two weeks later, mice were euthanized by cervical dislocation, and the tumor mass was removed and weighed. Mouse body weight, changes in tumor volume, and final tumor weight were used as indicators to evaluate the anticancer effects and toxicity of BBM-BSA-NPs and BBM in mice.

[0145] Data were statistically analyzed using GraphPad Prism 9 software, and results are expressed as mean ± standard error (Mean ± SEM). Statistical differences were compared using ANOVA and t-tests, with p < 0.05 considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0146] Experimental results:

[0147] The CT26 tumor-bearing mouse model was used to evaluate the in vivo antitumor effects and efficacy of Control (saline), Blank-BSA-NPs, BBM-BSA-NPs, and BBM. Tumor images of each group of mice taken at the end of the experiment are shown below. Figure 11 (Tail clipping represents tumor regression, and tumor volume is recorded as 0). As shown in the figure, tumor regression occurred in 2 mice in the BBM-BSA-NPs group and 1 mouse in the BBM group. Furthermore, the tumor volume in the BBM-BSA-NPs group was significantly smaller than that in the BBM group, indicating that BBM-BSA-NPs has a stronger anti-tumor effect than BBM. Compared with the control group and the Blank-BSA-NPs group, tumor growth in mice in both the BBM-BSA-NPs and BBM groups was inhibited to varying degrees, and the tumor volume was significantly smaller than that in the control group and the Blank-BSA-NPs group. This suggests that BBM itself has anti-tumor activity against COC, and BBM-BSA-NPs exhibits even stronger anti-tumor activity.

[0148] The tumor volume of mice in each group was measured and calculated before each administration. Finally, all data were statistically analyzed, and the results are as follows: Figure 12 Tumors grew rapidly in both the control group and the Blank-BSA-NPs group, with almost no difference in tumor volume. At day 14, the mean tumor volume was 894.60 ± 69.56 mm. 3 and 931.56±58.16mm 3 The value was significantly larger than that of the BBM-BSA-NPs group (279.19 ± 46.75 mm). 3 The BBM group's 456.75±54.64mm 3 Both the BBM-BSA-NPs group and the BBM group showed significant differences compared to the control group (*P<0.0001), while the tumor volume in the BBM-BSA-NPs group was significantly smaller than that in the BBM group (*P<0.05). These results indicate that the antitumor activity of BBM-BSA-NPs against COC is significantly stronger than that of BBM.

[0149] Drug administration was discontinued on day 10. Mouse weight and tumor volume were continuously observed and recorded. On day 14, after measuring weight and tumor volume, the mice were euthanized, and the tumor fragments were dissected and weighed. Statistical analysis of the tumor fragment weight data was performed. Results are shown below. Figure 13 As shown in the figure, among the four groups, the BBM-BSA-NPs group had the lowest average tumor mass weight, at 0.13±0.04g, significantly lower than the control group and the Blank-BSA-NPs group (P<0.0001), which were 0.58±0.06g and 0.52±0.05g, respectively. There was no significant difference in average tumor mass weight between the control group and the Blank-BSA-NPs group. The average tumor mass weight in the BBM group was 0.29±0.04g, lower than the control group (P<0.01) but higher than the BBM-BSA-NPs group (P<0.05). It can be observed that the tumor mass weight gradually decreased in the treatment groups, and the differences among the control group, BBM group, and BBM-BSA-NPs group were all statistically significant.

[0150] Therefore, based on the combined data of tumor volume growth and final tumor weight in mice, it can be demonstrated that, compared with other groups, the BBM-BSA-NPs group mice had the slowest tumor volume growth and the strongest in vivo anti-tumor activity, and the BSA nanocarrier can significantly enhance the anti-cancer activity of BBM against COC cells.

[0151] The body weight of mice in each group was measured and recorded before each administration. All data were then statistically analyzed, and the results are as follows: Figure 14Before drug administration, the body weights of mice in the control group, Blank-BSA-NPs group, BBM-BSA-NPs group, and BBM group were 22.33±0.23g, 22.16±0.30g, 22.63±0.17g, and 22.78±0.30g, respectively. On day 14 after drug administration, the body weights of each group were 20.43±0.24g, 20.52±0.30g, 21.41±0.28g, and 21.04±0.36g, respectively. This slight decrease in body weight may be due to the tumor continuously absorbing nutrients from the mice as the tumor grows. Compared with the control group and Blank-BSA-NPs group, the BBM-BSA-NPs and BBM groups showed the least weight loss, with the BBM-BSA-NPs group showing a slightly smaller weight change than the BBM group. It is speculated that the drug began to exert its therapeutic effect after administration, gradually reducing the tumor's growth rate and decreasing its consumption of the mouse's own nutrients, thereby alleviating the tumor's impact on the mouse's weight. This suggests that BBM-BSA-NPs have an improved effect on the mouse's physical function.

[0152] In vivo antitumor experiments in mice showed that BBM-BSA-NPs significantly inhibited tumor growth, with a significantly smaller tumor volume and lighter tumor mass compared to the control group. Furthermore, the BBM-BSA-NPs group exhibited a stronger inhibitory effect on tumors compared to the BBM group. It is speculated that the encapsulation of BBM with BSA prolongs the half-life and duration of action of BBM in vivo, allowing for sustained release of BBM from the nanocarrier. Additionally, BSA itself possesses both active and passive targeting mechanisms, enabling the encapsulated BBM to better target the tumor site, increasing the drug accumulation at the tumor site and thus achieving a more effective anticancer effect.

[0153] The effects of BBM and BBM-BSA-NPs on the viability of human tumor cells were investigated using the CCK-8 assay. Results are shown in […]. Figure 15 The results showed that BBM and BBM-BSA-NPs had good antitumor activity against human liver cancer, human bladder cancer, human breast cancer cells and human lung cancer cells.

Claims

1. The application of albumin-loaded berberine hydrochloride nanocomposite in the preparation of drugs for treating colorectal cancer, characterized in that, The albumin-loaded berberine hydrochloride nanocomposite comprises berberine hydrochloride and serum albumin, with a mass ratio of serum albumin to berberine hydrochloride of 1:0.1-0.

4. It is prepared via a solvent-desolvent-chemical crosslinking method using serum albumin as the carrier material, ethanol as the dehydrating agent, and glutaraldehyde as the crosslinking agent. The preparation method includes the following steps: (1) Accurately weigh the prescribed amount of berberine hydrochloride and serum albumin, dissolve them in deionized water, adjust the pH to 8.5-9.0, and obtain an aqueous solution of berberine hydrochloride and albumin as the aqueous phase; (2) Add anhydrous ethanol to the aqueous phase of step (1) under magnetic stirring; (3) Add glutaraldehyde solution to the solution in step (2), stir to solidify, and remove ethanol by rotary evaporation to obtain albumin-loaded berberine hydrochloride nanocomposite colloidal solution; (4) Centrifuge, precipitate, and remove unreacted substances to obtain the final product; In step (1), the mass-volume concentration of albumin aqueous solution is 4-6 mg / mL, and the mass-volume concentration of berberine hydrochloride in the solution is 1-3 mg / mL. In step (2), magnetic stirring is used and the ethanol dropping rate is 1-6 mL / min; in step (2), the volume ratio of aqueous phase to anhydrous ethanol is 1:2-1:

4.

2. The application according to claim 1, characterized in that, In step (3), the concentration of glutaraldehyde solution is 20%-25%, the volume is 5-25ul, and the curing time is 2-24 hours.

3. The application according to claim 2, characterized in that, The curing time is 4-8 hours.

4. The application according to claim 1, characterized in that, In step (1), the mass-volume concentration of berberine hydrochloride in the solution is 1-2 mg / mL.

5. The application according to claim 1, characterized in that, In step (2), the ethanol is added at a rate of 2-6 mL / min.

6. The application according to claim 1, characterized in that, The albumin-loaded berberine hydrochloride nanocomposite was prepared into a lyophilized powder by adding a lyophilization protectant, which was one or two of lactose, mannitol, and trehalose.

7. The application according to claim 6, characterized in that, The freeze-drying protectant is a combination of lactose and trehalose; the amount of freeze-drying protectant used is 2-7% w / v.

8. The application according to claim 7, characterized in that, The amount of the freeze-drying protectant is 3-5% w / v.

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