Atorvastatin calcium albumin nanoparticle and preparation and application thereof

Abstract

The application discloses atorvastatin calcium albumin nanoparticles and a preparation method and application thereof. The atorvastatin calcium albumin nanoparticles are composed of atorvastatin calcium and serum albumin, and the mass ratio of atorvastatin calcium to serum albumin is 1:3-1:10. The preparation method comprises the following steps: weighing atorvastatin calcium, fully dissolving the atorvastatin calcium in anhydrous ethanol to obtain an organic phase; weighing serum albumin, fully dissolving the serum albumin in deionized water, adjusting the pH of the solution by sodium hydroxide to obtain an aqueous phase; under stirring, dropping the organic phase into the aqueous phase, adding glutaraldehyde solution, continuously stirring and crosslinking, after the crosslinking is completed, removing the anhydrous ethanol by rotary evaporation, centrifuging the obtained solution by using a high-speed refrigerated centrifuge, and taking the lower precipitate as the atorvastatin calcium albumin nanoparticles. The atorvastatin calcium albumin nanoparticles prepared in the application can prolong the in-vivo circulation time of atorvastatin calcium, improve the bioavailability, and especially improve the antitumor activity of atorvastatin calcium.

Description

Atorvastatin calcialbumin nanoparticles and their preparation and application Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to an atorvastatin calcium albumin nanoparticle, its preparation method, and its application. Background Technology

[0002] Atorvastatin calcium (AtvCa) is a white or off-white powder, slightly soluble in water and acetonitrile, slightly soluble in ethanol, and readily soluble in methanol.

[0003] AtvCa is a commonly used statin drug in clinical practice. It can lower triglyceride and low-density lipoprotein cholesterol levels and increase high-density lipoprotein cholesterol levels. Clinically, it is mainly used to treat hypercholesterolemia, atherosclerosis, and coronary heart disease.

[0004] Colorectal cancer (COC) is a cancer formed by the abnormal growth of colon cells. Due to its high pathological similarity to rectal cancer (RC), it is collectively referred to as colorectal cancer (CRC). According to global cancer statistics in 2020, CRC has become the third leading cause of cancer death worldwide, second only to breast cancer and lung cancer in incidence. In 2020, there were approximately 1.932 million new cases of CRC globally, and approximately 935,000 deaths, accounting for a staggering 48.4%. Currently, the first-line treatment regimen is chemotherapy combined with anti-epidermal growth factor receptor (EGFR) targeted therapy, or chemotherapy combined with anti-angiogenic inhibitors. Major treatment drugs include fluorouracil, leucovorin, oxaliplatin, and irinotecan. These chemotherapy drugs have a powerful tumor-killing effect, but while killing cancer cells, they also have significant toxic side effects on normal tissue cells, especially blood cells and lymphocytes that play an important role in the body's immune defense. This greatly affects the patient's intraoperative adaptability and poses a significant risk of postoperative infection.

[0005] However, AtvCa suffers from poor water solubility, low bioavailability, short plasma half-life, first-pass effect in the liver, toxic side effects, and a relatively high effective antitumor dose, which limits its further clinical application in antitumor therapy. These limitations can be overcome by combining the drug with an appropriate delivery carrier.

[0006] Bovine serum albumin (BSA), a natural protein, possesses high biocompatibility and is a high-performance drug delivery carrier. Studies have reported that BSA can actively target the GP60 receptor on the surface of tumor cells. As a natural protein, BSA can simultaneously achieve both active and passive targeting of tumor tissues. Its unique protein structure allows for the loading of AtvCa, mitigating its poor water solubility and reducing its unfavorable factors in formulation, thereby enhancing antitumor efficacy.

[0007] Sodium oxamate (SO) is a lactate dehydrogenase (LDH) inhibitor. LDH is a glycolytic enzyme that mediates the bidirectional conversion between pyruvate and lactate and is one of the important metabolic enzymes regulating energy metabolism in tumor cells. Under hypoxic conditions, tumor cells exhibit reduced mitochondrial oxidative phosphorylation, leading to the production of more lactate under LDH catalysis. Even when oxygen is sufficient to support mitochondrial oxidative phosphorylation, tumor cells tend to engage in glycolysis. With ample nutrient supply, excess energy is excreted as lactate, promoting rapid cell division. Therefore, inhibiting lactate production from glycolysis is an effective strategy for suppressing cancer cell development. Furthermore, drug-resistant tumor patients often exhibit dysfunction of the mismatch repair (MMR) system. Studies have shown that MMR dysfunction weakens the ability to detect DNA damage, allowing tumor cells to generate more mutations, increase adaptability, and develop resistance to anticancer drugs. SO, while inhibiting glycolysis, can upregulate the expression of the MLH1 gene in the MMR system, enhancing the antitumor effect of drugs and improving tumor resistance to medications.

[0008] There are currently no reports on preparing AtvCa into nanoparticles or administering AtvCa in combination with sodium oxalate for anti-tumor, especially anti-CRC. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention prepares BSA nanoparticles (AtvCa-BSA-NPs) loaded with AtvCa, aiming to prolong the in vivo circulation time of AtvCa and improve its bioavailability and anti-COC activity.

[0010] The technical solution adopted in this invention is:

[0011] An atorvastatin calcium albumin nanoparticle is composed of atorvastatin calcium and serum albumin, wherein the mass ratio of atorvastatin calcium to serum albumin is 1:3-1:10, preferably 1:4-1:5.

[0012] The serum albumin mentioned is one of bovine serum albumin, human serum albumin, or ovalbumin.

[0013] The present invention also provides a method for preparing the atorvastatin calcialbumin nanoparticles, comprising the following steps:

[0014] 1) Weigh AtvCa, add an appropriate amount of anhydrous ethanol to dissolve it completely, and obtain the organic phase;

[0015] 2) Weigh serum albumin, dissolve it thoroughly in deionized water, adjust the pH of the solution with sodium hydroxide to obtain the aqueous phase;

[0016] 3) While stirring, add the organic phase dropwise to the aqueous phase, add glutaraldehyde solution, and continue stirring for cross-linking. After cross-linking is completed, remove anhydrous ethanol by rotary evaporation. Centrifuge the resulting solution using a high-speed refrigerated centrifuge and take the lower precipitate as atorvastatin calcium albumin nanoparticles.

[0017] In step 2), the pH is 7.5-9.0, preferably 8.0-9.0;

[0018] In step 2), the concentration of serum albumin is 5-20 mg / mL; preferably 10-15 mg / mL.

[0019] In step 3), the volume ratio of the aqueous phase to the organic phase is 1:2 to 1:4.

[0020] In step 3), the mass ratio of AtvCa in the aqueous phase to serum albumin in the organic phase is 10:1-3:1.

[0021] In step 3), the concentration of the glutaraldehyde solution is 20%-25%.

[0022] In step 3), the crosslinking time is 2-24 hours, preferably 4-24 hours.

[0023] Furthermore, the preparation method includes the following steps:

[0024] 1) Weigh 8 mg of atorvastatin calcium (AtvCa), add 12 mL of anhydrous ethanol and dissolve thoroughly to obtain the organic phase;

[0025] 2) Weigh 40 mg of bovine serum albumin (BSA), add 4 mL of deionized water to dissolve it completely, and adjust the pH of the solution to 8.5 with sodium hydroxide to obtain the aqueous phase;

[0026] 3) After stirring the aqueous phase for 10 min, add the organic phase dropwise to the aqueous phase while stirring, add 10 μL of glutaraldehyde solution, and continue stirring for crosslinking for 12 h. After crosslinking is completed, remove anhydrous ethanol by rotary evaporation at 45 °C. Centrifuge the resulting solution at 4 °C and 12000 rpm for 60 min using a high-speed refrigerated centrifuge. Take the lower precipitate as atorvastatin calcium albumin nanoparticles AtvCa-BSA-NPs.

[0027] A lyophilized powder of atorvastatin caloprotein nanoparticles is prepared by the following steps: atorvastatin caloprotein nanoparticles (AtvCa-BSA-NPs) are prepared into a solution; an AtvCa-BSA-NPs solution is taken, and a lyophilization protectant is added to make the concentration of the lyophilization protectant 2-5% by mass-volume percentage; the mixture is stirred until it is evenly dispersed; the resulting product is placed in a -80℃ freezer and freeze-dried under vacuum to obtain the AtvCa-BSA-NPs lyophilized powder.

[0028] Furthermore, the freeze-drying protectant is one or a combination of two or more of mannitol, trehalose, or glucose.

[0029] This invention also provides the application of atorvastatin calpain nanoparticles or lyophilized powder thereof in the preparation of antitumor drugs.

[0030] Furthermore, the tumor in question is colorectal cancer.

[0031] The beneficial effects of this invention are:

[0032] This invention successfully prepared AtvCa-BSA-NPs with sustained-release properties using a solvent removal method. In vitro experiments showed that AtvCa-BSA-NPs inhibited the proliferation of mouse COC CT26 cells and various human COC cells, and had the ability to inhibit migration and induce apoptosis in mouse COC CT26 cells. Moreover, the inhibitory ability on proliferation and migration and the ability to induce apoptosis were stronger than those of AtvCa, making it a safe and effective AtvCa nano-formulation. In vivo tumor-bearing mouse experiments showed that AtvCa-BSA-NPs significantly inhibited tumor growth compared to AtvCa. Furthermore, the combination of AtvCa-BSA-NPs with sodium oxalate further enhanced the in vivo anti-COC effect of AtvCa-BSA-NPs, providing a new research idea for the further anti-COC application of AtvCa nanocarriers. Attached Figure Description

[0033] Figure 1 is an appearance diagram of the lyophilized powder prepared in Example 2 using different mixed protective agents.

[0034] Figure 2 shows the particle size distribution (A) and potential distribution (B) of AtvCa-BSA-NPs prepared in Example 3.

[0035] Figure 3 is a transmission electron microscope image of AtvCa-BSA-NPs prepared in Example 3.

[0036] Figure 4 shows the in vitro release curves of AtvCa and AtvCa-BSA-NPs;

[0037] The data are represented by Mean±SD, n=3, ****P<0.0001.

[0038] Figure 5 shows the effects of AtvCa and AtvCa-BSA-NPs on CT26 cells;

[0039] Among them, AB: the effects of AtvCa, AtvCa-BSA-NPs and Blank-BSA-NPs on CT26 cell viability and corresponding IC50 after 24 hours of drug administration. 50 CD: Effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on CT26 cell viability and corresponding IC50 values ​​48 h after drug administration. 50 E: Effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on CT26 cell viability 72 h after drug administration. Data are expressed as Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0040] Figure 6 shows the effects of AtvCa and AtvCa-BSA-NPs on HCT116 cells;

[0041] In this study, A represents the effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on HCT116 cell viability 24 hours after drug administration; BC represents the effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on HCT116 cell viability and corresponding IC50 values ​​48 hours after drug administration. 50 DE: Effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on HCT116 cell viability and corresponding IC50 values ​​72 h after drug administration. 50 Data are expressed as Mean±SD, n=3, *P<0.05, **P<0.01, ***P<0.001.

[0042] Figure 7 shows the effects of AtvCa, AtvCa-BSA-NPs on HT29, SW480 and SW620;

[0043] Among them, A: the effect of AtvCa, AtvCa-BSA-NPs and Blank-BSA-NPs on the viability of HT29 cells 24 h after drug administration; BC: the effect of AtvCa, AtvCa-BSA-NPs and Blank-BSA-NPs on the viability of SW480 cells 24 h after drug administration and the corresponding IC50 values. 50 DE: Effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on SW620 cell viability and corresponding IC50 values ​​24 h after drug administration. 50Data are expressed as Mean±SD, n=3, *P<0.05, **P<0.01, ***P<0.001.

[0044] Figure 8 shows the effects of AtvCa and AtvCa-BSA-NPs on HEK293 cells;

[0045] Among them, AC: the effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on HEK293 cell viability after 24h (A), 48h (B), and 72h (C) of drug administration. Data are expressed as Mean ± SD, n = 3, *P < 0.05.

[0046] Figure 9 shows the effects of AtvCa and AtvCa-BSA-NPs on NIH3T3 cells;

[0047] Among them, AC: the effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on NIH3T3 cell viability after 24h (A), 48h (B), and 72h (C) of drug administration. Data are expressed as Mean ± SD, n = 3, **P < 0.01.

[0048] Figure 10 shows the 24-hour scratch healing of CT26 by AtvCa and AtvCa-BSA-NPs;

[0049] In this diagram, A represents the cell scratch morphology observed under an inverted microscope, and B shows the relationship between scratch healing rate and concentration. Data are expressed as Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0050] Figure 11 shows the uptake of AtvCa-BSA-NPs by CT26 cells at 2h and 12h;

[0051] In this image, blue fluorescence represents DAPI-labeled cell nuclei, green fluorescence represents Cou-6-labeled AtvCa-BSA-NPs, and Merge represents the image after overlaying the DAPI and Cou-6 channels. Scale bar: 100 μm.

[0052] Figure 12 shows the apoptosis of CT26 cells under different concentrations of AtvCa and AtvCa-BSA-NPs;

[0053] Wherein, AG: A represents the control group, and BD represents AtvCa administration concentrations of 10 μM (B) and 20 μM, respectively.

[0054] (C) Flow cytometry plots of apoptosis at 48 h with 40 μM (D). EG represents flow cytometry plots of apoptosis at 48 h with AtvCa-BSA-NPs concentrations of 10 μM (E), 20 μM (F), and 40 μM (G); HJ: Percentage of total apoptotic cells at 48 h with AtvCa-BSA-NPs concentrations of 10 μM (H), 20 μM (I), and 40 μM (J). Data are expressed as Mean ± SD, n = 3, *P < 0.05, **P < 0.01.

[0055] Figure 13 shows the tumor growth curves of tumor-bearing mice after different treatment groups;

[0056] Data are expressed as Mean±SD, n=8, *P<0.05, ***P<0.001, ****P<0.0001.

[0057] Figure 14 shows the final tumor results in tumor-bearing mice after different treatment groups;

[0058] In the figure, A is an in vitro visual image of the tumor mass in each group, and B is a statistical chart of the final tumor mass weight in each group. Data are expressed as Mean ± SD, n = 8, *P < 0.05, **P < 0.01.

[0059] Figure 15 shows the changes in body weight of mice in each group;

[0060] The data is represented by Mean±SD, and n=8.

[0061] Figure 16 shows the H&E staining of major tissues from CT26 tumor-bearing mice;

[0062] Magnification: ×400, Scale bar: 50μM.

[0063] Figure 17 shows the serum ALT, AST, UREA, and CREA levels in each group of mice;

[0064] Where A represents the ALT level, B represents the AST level, C represents the UREA level, and D represents the CREA level. Data are expressed as Mean ± SD, and n = 3. Detailed Implementation

[0065] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0066] BSA, as a novel drug carrier, exhibits good encapsulation effects for hydrophobic drugs with minimal toxicity. BSA nanoparticles can be prepared using various methods, including solvent removal, emulsification, spraying, self-assembly, pH / thermally sensitive gelation, and Nab... TM Technologies, etc. The solvent removal method, based on introducing hydrophobic drugs with organic solvents, has the advantages of being simple and readily available, producing small nanoparticles, but requires the use of suitable cross-linking agents. The emulsification method first requires preparing a water-in-oil (W / O) emulsion under conditions such as stirring, ultrasonication, and high-pressure homogenization, followed by solidification and removal of the organic solvent. However, the resulting nanoparticles are not stable enough, often requiring the addition of emulsifiers, which affects the protein's own activity. In recent years, research has used a water-in-oil (W / O / W) double emulsification method to obtain more stable albumin nanoparticles. Nab TM The first technique is an improvement on the emulsification method, applicable to many hydrophobic drugs. High-pressure homogenization of the oil-water mixture causes the free thiol groups of BSA to crosslink into disulfide bonds, creating cavities that encapsulate the drug inside. This crosslinking method is simple to operate and does not introduce organic solvents, but it requires a high protein binding rate. Preliminary experiments showed that this method was not effective for encapsulating AtvCa. The self-assembly method involves the spontaneous formation of nanoparticles from a BSA solution using a reducing agent when the concentration exceeds the critical micelle concentration and the critical co-solution temperature. Its advantage is the small particle size, but this method requires a reducing agent, and residual reducing agent may cause biotoxicity. The pH / thermally sensitive gel method requires precise control of pH and temperature. Based on the above analysis, this invention determines the use of a solvent-free method to prepare AtvCa-BSA-NPs. This method is suitable for laboratory preparation environments, is controllable, and produces nanoparticles with small sizes.

[0067] The particle size and zeta potential of AtvCa-BSA-NPs were analyzed using a laser particle size analyzer.

[0068] The microstructure of AtvCa-BSA-NPs was observed using transmission electron microscopy.

[0069] The encapsulation efficiency (EE) and drug loading (LC) of AtvCa-BSA-NPs are calculated according to the following formulas (1) and (2):

[0070] EE=(m0-m1) / m0×100%………………(1)

[0071] LC=(m0-m1) / (m0+m BSA )×100%…………(2)

[0072] Where m0 is the total AtvCa dosage (mg), m1 is the amount of unencapsulated AtvCa (mg), and m BSA The amount of BSA added (in mg).

[0073] Example 1: Preparation of Atorvastatin Caloprotein Nanoparticles

[0074] An atorvastatin calpain nanoparticle is prepared by the following steps.

[0075] 1) Weigh out atorvastatin calcium AtvCa, dissolve it completely in anhydrous ethanol to obtain the organic phase;

[0076] 2) Weigh bovine serum albumin (BSA), add 4 mL of deionized water to dissolve it completely, and adjust the pH of the solution to 7.5–9.0 with sodium hydroxide to obtain the aqueous phase;

[0077] 3) After pre-stirring the aqueous phase for 10 min, add the organic phase dropwise to the aqueous phase while stirring, add 5-20 μL of glutaraldehyde solution, and continue stirring for crosslinking for 2-24 h. After crosslinking is completed, remove anhydrous ethanol by rotary evaporation at 45 °C. Centrifuge the resulting solution at 12,000 rpm for 60 min at 4 °C using a high-speed refrigerated centrifuge. Take the lower precipitate as atorvastatin calcium albumin nanoparticles AtvCa-BSA-NPs.

[0078] (I) Investigation of solution pH value

[0079] Following the above preparation method, with atorvastatin calcium AtvCa 8mg, ethanol volume 10mL, BSA dosage 40mg, crosslinking agent dosage 10μL, and crosslinking time 12h kept constant, the effects of BSA solution pH values ​​of 7.5, 8.0, 8.5, and 9.0 on particle size, PDI, encapsulation efficiency, and drug loading were investigated. The results are shown in Table 1.

[0080] Table 1. Effects of solution pH on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0081]

[0082] The isoelectric point of BSA is 4.9. When the pH is higher than 7, the electrostatic repulsion between BSA molecules increases, weakening BSA aggregation and making it easier to form stable nanoparticles. As shown in Table 1, the pH value in the range of 7.5-9.0 has a significant impact on the nanoparticle size and encapsulation efficiency. At a pH of 7.5, white flocculent matter begins to appear in the sample at the initial stage of stirring, and the flocculent matter gradually aggregates to form a precipitate. As the pH value increases to 8.0, 8.5, and 9.0, no precipitate appears in the solution, and the particle size gradually decreases. At a pH value of 8.5, the encapsulation efficiency and drug loading reach their maximum. Therefore, considering both particle size and encapsulation efficiency, the pH value of the solution in this invention is 8.0-9.0, preferably 8.5.

[0083] (II) Investigation of BSA Dosage

[0084] Following the above preparation method, with atorvastatin calcium (AtvCa) 8 mg, ethanol volume 12 mL, solution pH 9.0, crosslinking agent volume 10 μL, and crosslinking time 12 h constant, the changes in particle size, PDI, encapsulation efficiency, and drug loading were investigated when 20, 40, 60, and 80 mg of BSA (BSA concentration of 5-20 mg / mL) were added to 4 mL of deionized water. The results are shown in Table 2.

[0085] Table 2 shows the effect of BSA dosage on particle size, PDI, encapsulation efficiency, and drug loading (n=3).

[0086]

[0087] As shown in Table 2, with a fixed BSA solution volume of 4 mL, the particle size gradually increased with increasing BSA dosage, the encapsulation efficiency initially increased and then decreased, and the drug loading gradually decreased. When the BSA dosage was 20 mg, the encapsulation efficiency was the lowest, and precipitation began to appear in the sample after storage at 4℃ for 1 h. This may be due to insufficient BSA, causing unencapsulated drug to precipitate. When the BSA dosage was 60 mg, the encapsulation efficiency was slightly higher than that of nanoparticles with 40 mg BSA, but the particle size was much larger. Therefore, considering particle size, encapsulation efficiency, and drug loading, the preferred BSA dosage in this invention is 40 mg, at which point the mass ratio of AtvCa to BSA is 1:5, and the BSA concentration is 10 mg / mL.

[0088] (III) Investigation of Ethanol Usage

[0089] Following the above preparation method, with atorvastatin calcium AtvCa 8mg, BSA dosage 40mg, volume 4mL, solution pH 9.0, crosslinking agent dosage 10μL, and crosslinking time 12h fixed, the effects of ethanol dosage of 8, 10, 12, and 16mL on particle size, PDI, encapsulation efficiency, and drug loading were investigated. The results are shown in Table 3.

[0090] Table 3. Effects of ethanol dosage on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0091]

[0092]

[0093] As shown in Table 3, with the increase of ethanol volume, the particle size and encapsulation efficiency gradually decrease. PDI reaches its maximum when the ethanol volume is 10 mL, and the overall drug loading does not change significantly. When the ethanol volume is increased to 12 mL and 16 mL, the particle size reaches its minimum, but the decreasing trend slows down. Since the encapsulation efficiency gradually decreases with increasing ethanol volume, and the amount of organic reagent should be as small as possible, considering the effects of particle size and encapsulation efficiency, the preferred method in this invention is to use 12 mL of ethanol, at which point the volume ratio of the aqueous phase to the organic phase is 1:3.

[0094] (iv) Investigation of drug dosage ratio

[0095] Following the above preparation method, with the ethanol volume fixed at 10 mL, BSA dosage at 40 mg, solution pH at 9.0, crosslinking agent dosage at 10 μL, and crosslinking time at 10 h, the effects of AtvCa mass ratios of 4 mg, 5 mg, 8 mg, 10 mg, and 13.3 mg (i.e., BSA to AtvCa mass ratios of 10:1, 8:1, 5:1, 4:1, and 3:1) on particle size, PDI, encapsulation efficiency, and drug loading were investigated. The results are shown in Table 4.

[0096] Table 4. Effects of drug dosage ratio on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0097]

[0098] As shown in Table 4, the particle size and drug loading gradually increase with the increase of AtvCa dosage. The encapsulation efficiency does not show a clear trend, but reaches its maximum at a dosage ratio of 4:1, followed by 5:1. The particle size is relatively smallest at dosage ratios of 10:1, 8:1, and 5:1, and the PDI is lowest at a dosage ratio of 5:1. Considering the effects of particle size, PDI, encapsulation efficiency, and drug loading, the preferred dosage ratio in this invention is 5:1.

[0099] (V) Investigation on the Dosage of Crosslinking Agent

[0100] Glutaraldehyde is a cross-linking agent with high molecular flexibility. It can connect with the free amino groups of two BSA molecules to form two "arms" linked by Schiff bases, creating a stable and spatially flexible network cross-linked structure. Cross-linking BSA with glutaraldehyde can improve the encapsulation of drugs by nanoparticles. Following the above preparation method, with atorvastatin calcium (AtvCa) 8 mg, ethanol volume 12 mL, BSA dosage 40 mg, solution pH 8.0, and cross-linking time 12 h constant, the effects of adding glutaraldehyde (5, 10, 15, and 20 μL) on particle size, PDI, encapsulation efficiency, and drug loading were investigated. The results are shown in Table 5.

[0101] Table 5. Effects of crosslinking agent dosage on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0102]

[0103] As shown in Table 5, the particle size of the nanoparticles gradually decreased with increasing glutaraldehyde dosage. This is likely due to the formation of a more compact skeletal structure from the cross-linking of more glutaraldehyde. The encapsulation efficiency showed a trend of first increasing and then decreasing, reaching its maximum at a glutaraldehyde dosage of 10 μL. This is likely because less cross-linking agent resulted in less formation of the BSA network skeleton, while more cross-linking agent significantly reduced the nanoparticle size, compressing the space for encapsulating AtvCa. Considering the biotoxicity of glutaraldehyde, as well as the effects of particle size and encapsulation efficiency, the preferred cross-linking agent dosage in this invention is 10 μL.

[0104] (vi) Crosslinking time investigation

[0105] Following the above preparation method, with atorvastatin calcium AtvCa 8mg, ethanol volume 12mL, BSA dosage 40mg, solution pH 9.0 and crosslinking agent dosage 10μL fixed, the effects of glutaraldehyde crosslinking time of 2, 4, 8, 12 and 24h on particle size, PDI, encapsulation efficiency and drug loading were investigated. The results are shown in Table 6.

[0106] Table 6. Effects of crosslinking time on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0107]

[0108] As shown in Table 6, with the increase of glutaraldehyde crosslinking time, the nanoparticle size gradually decreases, the PDI first decreases and then increases, and the encapsulation efficiency reaches its maximum at 12 h. Considering the biotoxicity of glutaraldehyde, as well as the influence of particle size and encapsulation efficiency, the preferred crosslinking time in this invention is 12 h.

[0109] Example 2: Preparation of Atorvastatin Calcium Albumin Nanoparticle Lyophilized Powder

[0110] A lyophilized powder of atorvastatin calcium albumin nanoparticles, the preparation method of which includes the following steps:

[0111] 1) Weigh 8 mg of atorvastatin calcium (AtvCa), add 12 mL of anhydrous ethanol and dissolve thoroughly to obtain the organic phase;

[0112] 2) Weigh 40 mg of bovine serum albumin (BSA), add 4 mL of deionized water to dissolve it completely, and adjust the pH of the solution to 8.5 with sodium hydroxide to obtain the aqueous phase;

[0113] 3) After stirring the aqueous phase for 10 min, add the organic phase dropwise to the aqueous phase while stirring, add 10 μL of glutaraldehyde solution, and continue stirring for crosslinking for 12 h. After crosslinking is completed, remove anhydrous ethanol by rotary evaporation at 45 °C. Centrifuge the resulting solution at 4 °C and 12000 rpm for 60 min using a high-speed refrigerated centrifuge. Take the lower precipitate as atorvastatin calcium albumin nanoparticles AtvCa-BSA-NPs.

[0114] 4) Atorvastatin calpain nanoparticles AtvCa-BSA-NPs were reconstituted to obtain an AtvCa-BSA-NPs solution. A lyophilization protectant was added to make the concentration of the lyophilization protectant 3% by mass volume. The mixture was stirred until it was evenly dispersed. The resulting product was placed in a -80℃ freezer and freeze-dried under vacuum to obtain AtvCa-BSA-NPs lyophilized powder.

[0115] (a) Selection of type of freeze-drying protectant

[0116] Using the above preparation method, trehalose, mannitol, and glucose were used as freeze-drying protectants. The properties of the resulting freeze-dried powder are shown in Table 7.

[0117] Table 7. Effects of different types of lyophilization protectants on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0118]

[0119] As shown in Table 7, the mannitol group yielded the largest particle size, while the glucose group produced the smallest lyophilized powder. Furthermore, although the mannitol group had a larger particle size, it exhibited a smooth appearance, uniform texture, and no collapse or clumping. The lyophilized powders from the trehalose and glucose groups both showed varying degrees of collapse. Considering both particle size and appearance, and requiring a lyophilized powder that balances both appearance and particle size encapsulation efficiency, this invention preferably uses a mixture of mannitol and glucose as the lyophilization protectant.

[0120] (II) Selection of the ratio of mixed lyophilization protectants

[0121] According to the above preparation method, mannitol and glucose were used as freeze-drying protectants. The total concentration of the mixed freeze-drying protectant was 3% (w / v). The properties and appearance of freeze-dried powders obtained by using mannitol and glucose with different mixing ratios of 3:7, 5:5 and 7:3 as freeze-drying protectants are shown in Table 8 and Figure 1.

[0122] Table 8. Effects of different types of lyophilization protectants on particle size, PDI, encapsulation efficiency, and drug loading (n=3)

[0123]

[0124] As shown in Table 8, the smaller the proportion of mannitol, the smaller the particle size of the reconstituted lyophilized powder, reaching its minimum at a mixing ratio of 3:7. Figure 1 shows that as the proportion of mannitol in the lyophilization protectant increases, the collapse of the lyophilized powder gradually decreases, with a smooth and uniform appearance at mannitol to glucose mixing ratios of 5:5 and 7:3. Considering both particle size and appearance, this invention preferably uses a 3% (w / v) mannitol-glucose mixed lyophilization protectant with a mannitol-to-glucose mass ratio of 5:5-3:7.

[0125] Example 3: Application of Atorvastatin Calcopenin Nanoparticles in the Treatment of Colon Cancer (I) Preparation of Atorvastatin Calcopenin Nanoparticles

[0126] 1) Weigh 8 mg of atorvastatin calcium (AtvCa), add 12 mL of anhydrous ethanol and dissolve thoroughly to obtain the organic phase;

[0127] 2) Weigh 40 mg of bovine serum albumin (BSA), add 4 mL of deionized water to dissolve it completely, and adjust the pH of the solution to 8.5 with sodium hydroxide to obtain the aqueous phase;

[0128] 3) After stirring the aqueous phase for 10 min, the organic phase was added dropwise to the aqueous phase while stirring, followed by 10 μL of glutaraldehyde solution. The mixture was stirred and crosslinked for 12 h. After the crosslinking was completed, anhydrous ethanol was removed by rotary evaporation at 45 °C. The resulting solution was centrifuged at 12,000 rpm for 60 min at 4 °C using a high-speed refrigerated centrifuge. The lower precipitate was taken as atorvastatin calcium albumin nanoparticles (AtvCa-BSA-NPs).

[0129] (II) Characterization

[0130] 1. Particle size and potential measurement

[0131] The particle size and zeta potential of the prepared AtvCa-BSA-NPs are shown in Figure 2. The particle size of AtvCa-BSA-NPs is 174.4±3.59 nm, with uniform particle size distribution and good peak shape; the zeta potential is -27.55±0.26 mV, with good electronegativity, indicating that the AtvCa-BSA-NPs system is stable.

[0132] 2. Morphological observation

[0133] Figure 3 shows the results of transmission electron microscopy observation of AtvCa-BSA-NPs. The AtvCa-BSA-NPs are clearly spherical with distinct edges and uniform diameter, indicating successful preparation. A small amount of particle aggregation is observed, which may be due to the high sample concentration.

[0134] 3. In vitro release study of AtvCa-BSA-NPs

[0135] Figure 4 shows the in vitro release results of AtvCa and AtvCa-BSA-NPs. AtvCa-BSA-NPs exhibited rapid release from 0 to 4 hours, followed by a slower release rate, reaching a maximum cumulative release of 78.21 ± 1.50% at 24 hours. AtvCa showed a burst release from 0 to 18 hours, with a cumulative release of 94.77 ± 3.45% at 12 hours, 96.14 ± 2.04% at 18 hours, and complete release by 24 hours. Comparison of the release curves of AtvCa and AtvCa-BSA-NPs indicates that AtvCa-BSA-NPs slowed the release rate of AtvCa, demonstrating its good sustained-release properties.

[0136] (III) In vitro anti-colon cancer effect of AtvCa-BSA-NPs

[0137] Experimental cells: Mouse COC cells CT26, mouse fibroblasts NIH3T3, human embryonic kidney cells HEK293, and human COC cells HCT116, HT29, SW480, and SW620 were all obtained from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0138] Cell culture: CT26, HCT116, HEK 293, HT29, NIH3T3, SW480, and SW620 cells were cultured in a constant temperature incubator at 37°C with 5% CO2. All complete culture media used contained 1% antibiotics and 10% fetal bovine serum. CT26 cells were cultured in 1640 medium, HEK 293 and NIH3T3 cells in DMEM medium, SW480 and SW620 cells in L-15 medium, and HT29 and HCT116 cells in 5A medium.

[0139] Cell passage: Collect cells in the logarithmic growth phase, discard the original culture medium, wash three times with an appropriate amount of PBS to remove metabolic waste and dead cells, add an appropriate amount of trypsin solution containing EDTA, and incubate in a CO2 incubator for digestion. After observing under a microscope that most cells float and move like sand grains, add an appropriate amount of complete culture medium to stop digestion and carefully pipette. Place the cell suspension in a centrifuge and centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with an appropriate amount of culture medium, transfer to three culture dishes, and incubate in a CO2 incubator.

[0140] Cell plating: After diluting the cells as needed, count them under a microscope to obtain a cell suspension of the target concentration. For example, in a 96-well plate, the cell suspension is diluted to a density of 5 × 10⁻⁶ cells / well. 3 Seeds were placed in 96-well plates and transferred to an incubator until the cells were fully adhered to the plate.

[0141] 1. Cytotoxicity test

[0142] 1.1) Cytotoxic effects of AtvCa-BSA-NPs and AtvCa on CT26

[0143] The cytotoxicity of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs against CT26 cells was determined using the CCK-8 assay. CT26 cell plates were prepared and treated with different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs for 24 h, 48 h, and 72 h. The control group was treated with fresh 1640 medium containing the same concentration of DMSO. The AtvCa-BSA-NPs and AtvCa groups were each tested in triplicate. At the time points, the old medium was discarded, and 100 μL of fresh medium containing 10% CCK-8 reagent was added to each well. The plates were incubated for an appropriate time and then placed in a microplate reader to measure absorbance at 450 nm. The cell viability and overall IC50 were calculated under different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs. 50 The result is shown in Figure 5.

[0144] Cell growth survival rate is calculated using the following formula:

[0145] CV = (A2 - A0) / (A1 - A0) × 100%

[0146] Where V represents cell growth and survival rate, A0 represents absorbance of the blank group, A1 represents absorbance of the control group, and A2 represents absorbance of the drug-treated group.

[0147] As shown in Figure 5, the cytotoxic effects of AtvCa, AtvCa-BSA-NPs, and Blank-BSA-NPs on CT26 cells at 24h, 48h, and 72h were detected using a CCK-8 assay. The results showed that Blank-BSA-NPs had no significant toxicity to CT26 cells. AtvCa and AtvCa-BSA-NPs at different concentrations exhibited varying degrees of inhibition on CT26 cell proliferation (A, C, and E in Figure 5). Overall, the inhibitory effect increased with increasing drug concentration and time. Furthermore, AtvCa-BSA-NPs showed a higher inhibitory effect on cell survival than AtvCa, which was significant at many concentrations. For example, at drug concentrations of 40μM and 60μM, AtvCa-BSA-NPs showed superior inhibition of CT26 cells at 24h and 48h compared to AtvCa (P<0.05), indicating that the sustained-release structure of AtvCa-BSA-NPs prolongs the action time of the encapsulated AtvCa, resulting in a more sustained inhibitory effect on cells. After 72 hours of drug treatment, a concentration of 20 μM caused significant cell killing, while at a concentration of 40 μM, the cell survival rate was only 1.66 ± 0.51%.50 It can quantitatively evaluate the antitumor activity of AtvCa-BSA-NPs, IC50 50 The lower the value, the stronger the anticancer activity. At 24h and 48h after drug administration, AtvCa showed an IC50 value for CT26 cells. 50 The IC50 values ​​of AtvCa-BSA-NPs on CT26 cells were 55.34 μM and 23.92 μM, respectively. 50 The IC50 values ​​of AtvCa-BSA-NPs were 33.48 μM and 19.49 μM (B and D in Figure 5), respectively. 50 Compared to the active pharmaceutical ingredient AtvCa, both concentrations were reduced. At 48h and 72h after administration, high concentrations of AtvCa and AtvCa-BSA-NPs had already killed the vast majority of cells, so there was no significant difference. At 72h after administration, even lower concentrations caused a significant decrease in cell viability, therefore IC50 was not considered. 50 The above results indicate that AtvCa-BSA-NPs have stronger cytotoxicity against CT26 and can significantly enhance the growth inhibition of CT26 by AtvCa.

[0148] 1.2) Cytotoxic effects of AtvCa-BSA-NPs and AtvCa on HCT116

[0149] The cytotoxic effects of different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs on HCT116 cells after 24 h, 48 h, and 72 h of treatment were investigated using the CCK-8 assay (method as described in 1.1). The results are shown in Figure 6.

[0150] As shown in Figure 6, AtvCa and AtvCa-BSA-NPs exhibited certain cytotoxicity against HCT116 cells at 24h, 48h, and 72h, while Blank-BSA-NPs showed no significant inhibitory effect on HCT116 (A, B, and D in Figure 6). At 24h of administration, AtvCa and AtvCa-BSA-NPs showed no inhibitory effect on HCT116 cells, and cell viability exceeded 90% at all administered concentrations. At 48h of administration, high-concentration groups began to show cytotoxicity, and AtvCa-BSA-NPs showed superior inhibition compared to AtvCa at concentrations of 60μM and 80μM. At 72h of administration, both higher concentrations of AtvCa and AtvCa-BSA-NPs exhibited cytotoxicity, with AtvCa-BSA-NPs showing stronger cytotoxicity at 100μM (P<0.05). At 24h and 48h of administration, the IC50 of AtvCa on HCT116 cells... 50 The IC50 values ​​of AtvCa-BSA-NPs on CT26 cells were 88.65 μM and 37.95 μM, respectively. 50The concentrations were 62.22 μM and 36.58 μM (C and E in Figure 6), respectively. The results indicate that AtvCa-BSA-NPs exhibited stronger HCT116 cytotoxicity than AtvCa, but lower sensitivity than CT26.

[0151] 1.3) Cytotoxic effects of AtvCa-BSA-NPs and AtvCa on HT29, SW480, and SW620 cells

[0152] The cytotoxic effects of different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs on HT29, SW480, and SW620 cells after 24 h were investigated using the CCK-8 assay (method as described in 1.1). The results are shown in Figure 7.

[0153] As shown in Figure 7, at 24 h, AtvCa and AtvCa-BSA-NPs all exhibited varying degrees of inhibitory effects on HT29, SW480, and SW620, while Blank-BSA-NPs had no significant effect (A, B, and D in Figure 7). After 24 h of drug administration, AtvCa-BSA-NPs, compared to AtvCa, showed a statistically significant difference in inhibitory effect on HT29 at a concentration of 60 μM (P < 0.05), and the cell viability at 100 μM was 52.41 ± 2.22%. 24 h after administration, AtvCa and AtvCa-BSA-NPs also exhibited certain toxic effects on SW480 and SW620. At 100 μM, AtvCa-BSA-NPs showed stronger inhibition of SW480 than AtvCa (P<0.05), while AtvCa-BSA-NPs showed stronger inhibition of SW620 than AtvCa at 60 μM (P<0.05). However, when the administered concentration was increased to 80 μM and 100 μM, the difference in efficacy between AtvCa and AtvCa-BSA-NPs was not significant (P>0.05). The 24 h IC50 of AtvCa-BSA-NPs against SW480 and SW620 was not specified. 50 The values ​​are 55.57 μM and 50.18 μM, respectively, which are smaller than the IC values ​​of AtvCa for SW480 and SW620. 50 The values ​​of 76.34 μM and 62.13 μM (C and E in Figure 7) indicate that AtvCa-BSA-NPs have a stronger inhibitory effect on SW480 and SW620 than AtvCa, and the effect on SW620 is better.

[0154] 1.4) Cytotoxic effects of AtvCa-BSA-NPs and AtvCa on HEK293 cells

[0155] The cytotoxic effects of different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs on HEK293 cells after 24 h, 48 h, and 72 h were investigated using the CCK-8 assay to verify their safety (method as in 1.1). The results are shown in Figure 8.

[0156] As shown in Figure 8, 24 h after administration, AtvCa and AtvCa-BSA-NPs had no significant inhibitory effect on HEK293 cells (Figure 8A). After 48 h of treatment with AtvCa and AtvCa-BSA-NPs at concentrations of 10-40 μM, the cell viability of HEK293 cells was above 90%. However, the cell viability of the higher concentration groups began to decrease. At the maximum administered concentration of 100 μM, the cell viability of the AtvCa and AtvCa-BSA-NPs groups decreased to 71.55±4.72% and 68.33±4.07%, respectively, with no statistically significant difference (P>0.05). At an administered concentration of 10 μM, AtvCa and AtvCa-BSA-NPs showed a significant difference in cell viability, but this had no practical impact on the overall results (Figure 8B). 72 h after administration, there was no significant difference in the effects of different concentrations of AtvCa and AtvCa-BSA-NPs on cell viability. At the maximum administered concentration of 100 μM, the cell viability of the AtvCa and AtvCa-BSA-NPs groups was the lowest, at 70.31 ± 1.94% and 65.70 ± 3.30%, respectively (Figure 8C). Blank-BSA-NPs showed no significant toxicity to HEK293 cells. The experimental results indicate that AtvCa and AtvCa-BSA-NPs had no significant effect on HEK-293 cell viability at concentrations ranging from 10 to 100 μM, but exhibited slight toxicity at high concentrations or prolonged treatment, suggesting that AtvCa-BSA-NPs inhibited the aforementioned tumor cells without causing significant toxicity to renal cells.

[0157] 1.5) Cytotoxic effects of AtvCa-BSA-NPs and AtvCa on NIH3T3

[0158] Evaluating whether the drug affects the proliferation of NIH3T3 cells is fundamental to subsequent in vivo experiments and a necessary step to ensure in vivo safety. The cytotoxic effects of different concentrations of AtvCa-BSA-NPs, AtvCa, and Blank-BSA-NPs on NIH3T3 cells at 24h, 48h, and 72h were investigated using the CCK-8 assay (method as described in 1.1). The results are shown in Figure 9.

[0159] As shown in Figure 9, Blank-BSA-NPs showed no significant toxicity to NIH3T3, while AtvCa and AtvCa-BSA-NPs exhibited relatively weak toxicity to NIH3T3. However, the toxicity increased with increasing concentration. After 24 hours of administration, the cell viability rates of AtvCa and AtvCa-BSA-NPs at the maximum concentration of 100 μM were 81.36 ± 3.22% and 78.94 ± 3.06%, respectively (Figure 9A), with no statistically significant difference (P > 0.05). With increasing administration time, the cytotoxicity of the low-concentration group showed no significant change, while the cytotoxicity of the high-concentration group increased slightly. After 48 hours of administration, the cell viability of AtvCa and AtvCa-BSA-NPs at a concentration of 100 μM was 71.97 ± 3.33% and 64.19 ± 6.75%, respectively (Figure 9B). After 72 hours of administration, the cell viability of AtvCa and AtvCa-BSA-NPs at a concentration of 100 μM was 49.68 ± 2.61% and 48.42 ± 4.37%, respectively.

[0160] (Figure 9, C). The experimental results showed that AtvCa and AtvCa-BSA-NPs at different concentrations had low cytotoxicity to NIH3T3 cells after 24 h and 48 h of administration, but exhibited slight toxicity under prolonged and high-concentration treatment.

[0161] 2. Cell migration assay

[0162] Tumor cell migration and metastasis are closely related. A scratch assay was used to observe the horizontal migration of CT26 cells treated with different concentrations of AtvCa-BSA-NPs and AtvCa for 24 hours. CT26 cells were treated with 6 × 10⁻⁶... 5 Cells were seeded per well in a 6-well plate. Using a sterile 200 μL pipette tip, the cells were vertically scratched along the center line of the well, controlling the pressure to maintain equal scratch width. Exfoliated cells were washed away with PBS. Different concentrations of AtvCa-BSA-NPs and AtvCa were added to each well as a medium change. One scratch field of view was selected from each well and labeled. Since the concentration of the drugs in the cell scratch assay should not have a cytotoxic effect on cells, the drug concentrations were determined to be 0, 5, 10, and 20 μM based on the cytotoxicity results of AtvCa-BSA-NPs and AtvCa on CT26 cells. Each AtvCa-BSA-NPs and AtvCa concentration group was tested in triplicate, and images were taken using an inverted microscope at time point 0 h. After recording and photographing, the 6-well plate was placed in a cell culture incubator. After 24 h, it was removed and photographed again using an inverted microscope. The results are shown in Figure 10. The experimental results were quantitatively analyzed using ImageJ to obtain the migration area. Cell migration rate (MR) was calculated using the following formula:

[0163] MR=(S0-S 24) / S0×100%

[0164] Where S0 is the scratch area at 0h, S 24 Scratch area over 24 hours

[0165] Figure 10 shows the wound healing degree of AtvCa and AtvCa-BSA-NPs at different time points when the drug concentration was 5 μM. There was no significant difference in the scratch healing degree of CT26 cells within 24 h compared to the control group, and there was also no significant difference between AtvCa and AtvCa-BSA-NPs (A and B in Figure 10). As the drug concentration of AtvCa and AtvCa-BSA-NPs increased to 10 and 20 μM, the scratch healing rate showed a decreasing trend. When the drug concentration reached 10 μM, the scratch healing rates of AtvCa and AtvCa-BSA-NPs within 24 h were 37.98±4.20% and 26.65±3.75%, respectively. At this concentration, the AtvCa-BSA-NPs group showed a significantly stronger inhibitory effect on cell migration compared to the control group (P<0.01). When the drug concentration reached 20 μM, the scratch healing rates of AtvCa and AtvCa-BSA-NPs within 24 h were 34.20±4.87% and 21.14±2.60%, respectively. At this concentration, the AtvCa-BSA-NPs group showed a significant difference compared with the control group (P<0.001). Furthermore, at drug concentrations of 10 μM and 20 μM, the scratch healing rate of the AtvCa-BSA-NPs group was consistently lower than that of the AtvCa group (P<0.05). The experimental results indicate that AtvCa and AtvCa-BSA-NPs can limit the migration ability of CT26 in a concentration-dependent manner. Moreover, the encapsulation of AtvCa by AtvCa-BSA-NPs more effectively inhibited the horizontal migration of CT26 and weakened its horizontal diffusion and transfer ability.

[0166] 3. Cell uptake assay

[0167] Preparation of Cou-6 labeled AtvCa-BSA-NPs: 8 mg AtvCa and 2.0 mg coumarin 6 (Cou 6) powder were accurately weighed and dissolved in ethanol. The mixture was sonicated to dissolve completely as the organic phase, thus obtaining coumarin 6 labeled AtvCa-BSA-NPs.

[0168] 3.1) Investigation on the uptake of AtvCa-BSA-NPs by CT26 cells

[0169] Qualitative analysis was performed using laser confocal microscopy to observe the uptake of AtvCa-BSA-NPs by CT26 cells. CT26 cells in the logarithmic growth phase were digested and counted at a concentration of 1.2 × 10⁻⁶. 5Cells / well were diluted into confocal cell culture dishes and placed in a cell culture incubator. After adhesion, AtvCa-BSA-NPs labeled with Cou-6 were added, diluted with culture medium to a Cou-6 concentration of 0.03 μg / mL, and cultured for 2 h and 12 h respectively. After culture, the culture medium was discarded, and the cells at the bottom of the dish were slowly washed three times with PBS. 200 μL of 4% paraformaldehyde fixative was added to each dish and fixed at room temperature for 20 min. After three more slow washes with PBS, an appropriate amount of DAPI was added to each dish to ensure that all cells were completely submerged in DAPI, and incubated at room temperature for 5 min. The supernatant was then discarded, and the cells were washed three times with PBS, leaving 100 μL of PBS in each dish. The entire process was carried out under dark conditions, with three replicates at each time point. The results were captured and recorded using a laser confocal microscope. The results are shown in Figure 11.

[0170] As shown in Figure 11, in order to preliminarily investigate the uptake of AtvCa-BSA-NPs by CT26 cells, AtvCa-BSA-NPs were fluorescently labeled in this invention. The results showed that the cell nuclei at different time points all showed obvious blue fluorescence, indicating that the cells were in good condition, while the cytoplasm showed green fluorescence. The fluorescence intensity increased with the increase of drug administration time, indicating that AtvCa-BSA-NPs can be taken up by CT26 cells, and the cumulative uptake increases with the extension of time.

[0171] 4. Apoptosis assay

[0172] 4.1) Comparison of the effects of AtvCa and AtvCa-BSA-NPs on apoptosis in CT26 cells

[0173] Phosphatidylserine (PS) is primarily expressed on the inner side of the cell membrane in healthy cells. In the early stages of apoptosis, PS can translocate from the inner side of the cell membrane to the surface, becoming exposed to the extracellular environment. Annexin-V is a phospholipid-binding protein that specifically binds to PS. Labeling Annexin-V with Alexa Fluor 488 as a fluorescent probe allows for the detection of early apoptosis using flow cytometry or fluorescence microscopy. Propidine iodide (PI) is a nucleic acid dye that cannot penetrate the cell membrane of normal cells. However, due to the damaged cell membranes of late-stage apoptotic cells and dead cells, PI can pass through the cell membrane and enter the nucleus. By simultaneously using Annexin-V, Alexa-Fluor 488, and PI in flow cytometry, cells in the early and late stages of apoptosis, as well as dead cells, can be distinguished.

[0174] CT26 cells were divided into 5×10 5Cells were seeded at a concentration of 10, 20, and 40 μM AtvCa and AtvCa-BSA-NPs per well in 6-well plates and incubated until uniform adhesion. In the experimental groups, the culture medium was changed to contain 10, 20, and 40 μM AtvCa and AtvCa-BSA-NPs, respectively. The control group received the same drug-free culture medium. Cells were incubated for 48 h. After incubation, the culture medium was discarded, and the cells were carefully washed once with PBS. 200 μL of EDTA-free trypsin was added to each well to digest the cells, and the plates were incubated at room temperature. To avoid over-digestion by trypsin which could damage the cell membrane and affect apoptosis results, digestion was stopped when cells could be easily detached by gentle pipetting. Cells were collected by centrifugation and washed three times with PBS. Cells were resuspended in 200 μL of Binding Buffer. For each sample, 10 μL of Annexin-VAlexa-Fluor 488 and 5 μL of PI were added under light-protected conditions and mixed thoroughly. Staining was performed for 10 min in the dark, followed by the addition of 200 μL of Binding Buffer. Apoptosis was then detected using flow cytometry. In the flow cytometry four-quadrant plot, the lower left quadrant represents live cells; the upper left quadrant represents cell debris and dead cells; the upper right quadrant represents necrotic cells and late-stage apoptotic cells; and the lower right quadrant represents early-stage apoptotic cells. The total apoptosis rate for each group was calculated. The results are shown in Figure 12.

[0175] The apoptosis results of CT26 cells after 48 h of Annexin-VAlexa-Fluor 488 and PI double staining are shown in Figure 12. At drug concentrations of 20 μM and 40 μM, the total apoptosis percentage of AtvCa and AtvCa-BSA-NPs groups was significantly higher than that of the control group (P<0.05), and the apoptosis percentage increased with increasing drug concentration. At 20 μM and 40 μM, the total apoptosis percentages of AtvCa and AtvCa-BSA-NPs reached 24.7±6.4% and 40.7±7.4%, and 32.1±5.1% and 49.8±8.3%, respectively. At drug concentrations of 10 μM, 20 μM, and 40 μM, the total apoptosis percentage of the AtvCa-BSA-NPs group was significantly higher than that of the AtvCa group (P<0.05). The results showed that the encapsulation of AtvCa by AtvCa-BSA-NPs significantly promoted the induction of apoptosis in CT26, exhibiting a stronger anti-cancer effect.

[0176] In summary, in vitro experiments demonstrated that AtvCa-BSA-NPs exhibited good cytotoxicity against COC cells CT26, HCT116, HT29, SW480, and SW620, showing significantly greater cytotoxicity compared to AtvCa. It also showed weak cytotoxicity against human kidney cells HEK 293 and mouse fibroblasts NIH3T3. AtvCa-BSA-NPs inhibited the 24-hour migration of CT26 cells at concentrations of 10 μM and 20 μM, with the inhibitory effect positively correlated with the administered concentration. AtvCa-BSA-NPs could be taken up by CT26 cells and accumulate intracellularly. Apoptosis experiments showed that inducing apoptosis may be the mechanism by which AtvCa-BSA-NPs exert their effects; the percentage of apoptosis in CT26 cells was significantly higher at both 20 μM and 40 μM concentrations than with AtvCa, indicating a more significant pro-apoptotic effect.

[0177] (iv) In vivo anti-colon cancer effect of atorvastatin calcium albumin nanoparticles

[0178] Establishment of a tumor-bearing mouse model: Each mouse was subcutaneously injected with 100 μL of CT26 cell suspension into the lower right back. Tumor growth was observed every two days. The longer and shorter diameters of the tumor were measured and recorded using calipers. Tumor volume (V) was calculated using the following formula. The tumor was considered complete when it reached approximately 100 mm². 3 Start administering medication at that time.

[0179] V = A × B 2 / 2

[0180] Where A is the longer diameter of the tumor (in mm) and B is the shorter diameter of the tumor (in mm).

[0181] 1. Evaluation of antitumor activity in tumor-bearing mice

[0182] Tumor-bearing mice were randomly divided into 6 groups of 8 mice each, based on tumor volume: (1) Control (saline); (2) Blank-BSA-NPs; (3) AtvCa; (4) AtvCa-BSA-NPs; (5) Sodium oxalate SO; and (6) AtvCa-BSA-NPs+SO. The concentrations of AtvCa and AtvCa-BSA-NPs were 20 mg / kg, and the concentration of SO was 100 mg / kg. Tumors were treated with a tumor volume of 100 mm². 3 Dosing was initiated every other day, with SO administered intratumorally as a solution, and the rest via tail vein injection. When the maximum tumor volume approached 2000 mm² (as per animal ethics guidelines), administration was initiated. 3Mice were euthanized, their organs and tissues were separated, tumors were dissected, washed with physiological saline, and weighed. The antitumor effects of AtvCa and AtvCa-BSA-NPs, as well as the effect of SO on the antitumor effect of AtvCa-BSA-NPs, were evaluated using tumor volume and final tumor weight as indicators. The results are shown in Figure 13.

[0183] As shown in Figure 13, in the Blank-BSA-NPs group, some mice on day 12 of drug administration showed tumor volumes approaching the ethically recommended maximum tumor volume of 2000 mm². 3 Therefore, the experiment was terminated on day 12 of drug administration. The tumor volume in the AtvCa group on day 12 was 1434.41 ± 538.90 mm. 3 Compared to the saline control group (1557.73±321.88 mm), 3 The mean value of the Blank-BSA-NPs group was 1320.73 ± 584.52 mm. 3 There was no statistically significant difference in tumor volume between the two groups (P>0.05), indicating that 20 mg / kg AtvCa raw material had no significant antitumor effect. This may be because AtvCa is partially decomposed in the blood after intravenous injection and is rapidly metabolized, resulting in less accumulation at the tumor site. On day 12, the tumor volume in the AtvCa-BSA-NPs group was 782.57±364.44 mm. 3 The tumor volume was lower than that of the saline control group (P<0.001) and also lower than that of the Blank-BSA-NPs group (P<0.05). Furthermore, the tumor volume was smaller than that of the AtvCa group (P<0.05), indicating that AtvCa-BSA-NPs exhibited superior antitumor activity against CT26 compared to AtvCa. Notably, the tumor volume in the AtvCa-BSA-NPs+SO group on day 12 was only 413.24±290.43 mm. 3 Compared to the AtvCa-BSA-NPs group, the tumor volume was further reduced (P<0.05), and there was also a statistically significant difference compared to the SO group (P<0.05), while the tumor volume in the SO group was 1213.43±754.02 mm. 3 There was no statistically significant difference compared with the saline control group (P>0.05), indicating that the antitumor effect of 100 mg / kg SO alone was not significant, but the combination with AtvCa-BSA-NPs could further enhance the antitumor effect of AtvCa-BSA-NPs.

[0184] The mice and tumor tissues of each group were photographed and recorded, and the tumor weight was statistically analyzed. The results are shown in Figure 14. The tumors in the AtvCa-BSA-NPs group were smaller than those in the AtvCa group. The tumors in the AtvCa-BSA-NPs+SO group were smaller than those in the AtvCa-BSA-NPs group. There was no significant difference between the SO group and the saline control group (Figure 14A). The mean tumor mass weight in the AtvCa-BSA-NPs group was 0.61±0.36g, which was lower than that in the control group (1.18±0.44g, P<0.05) and the Blank-BSA-NPs group (1.26±0.66g, P<0.05), and also lower than that in the AtvCa group (1.24±0.74g, P<0.05). The mean tumor mass weight in the SO group was 1.32±0.67g, which was not statistically different from that in the saline control group and the Blank-BSA-NPs group. However, the mean tumor mass weight in the AtvCa-BSA-NPs+SO group was 0.30±0.21g, which was lower than that in the SO group (P<0.01) and the AtvCa-BSA-NPs group (P<0.05) (Figure 14B).

[0185] Based on the combined results of tumor tissue imaging and final tumor weight, it was demonstrated that the AtvCa-BSA-NPs group exhibited slower tumor growth and smaller final tumor volume compared to AtvCa, significantly enhancing the antitumor activity of AtvCa. Furthermore, the combined use of AtvCa-BSA-NPs and SO further enhanced the antitumor activity of AtvCa-BSA-NPs.

[0186] 2. Evaluation of the toxic side effects of the delivery system

[0187] 2.1) Mouse body weight

[0188] The health status of the mice was observed before each administration, and the weight of all mice was measured and recorded. A mouse weight-day curve was plotted. The results are shown in Figure 15.

[0189] As shown in Figure 15, the body weight of mice in each group was measured and recorded before each administration. Over time, all experimental groups showed similar trends in body weight change: from the first administration, the mice's body weight increased slowly, then remained stable, and continued to increase slowly after day 8. This indicates that no abnormal decrease or fluctuation in body weight occurred in any group, and the overall changes were not significantly different, demonstrating the safety of the drugs and administration methods.

[0190] 2.2) H&E staining of mouse tissue

[0191] Hematoxylin and eosin (H&E) staining was used to stain the heart, liver, spleen, lung, kidney, and tumor tissues of mice in each experimental group to evaluate changes in tissue morphology. First, paraffin sections were prepared. Mouse heart, liver, spleen, lung, and kidney sections were fixed in 4% paraformaldehyde fixative. The fixed tissues were rinsed three times with running water, dehydrated with different concentrations of alcohol, and then embedded in paraffin. After the paraffin cooled, the sections were cut into uniform thickness sections using a tissue slicer. The sections were then dewaxed with xylene and ethanol, rinsed twice with double-distilled water, and stained with hematoxylin dye. Staining was terminated by rinsing with running water to remove the dye, followed by differentiation in 1% hydrochloric acid ethanol for a few seconds, rinsing with running water to regain blue color, dehydration in a gradient of alcohols, and complete immersion in xylene for 20 minutes. Finally, neutral resin was added for mounting. The stained tissue sections were observed and photographed under a microscope. The results are shown in Figure 16.

[0192] The H&E staining results are shown in Figure 16. After the treatment of each group, no obvious inflammatory damage or lesions were found in the heart, liver, spleen, lungs, and kidneys of the mice in each experimental group. This indicates that the drugs and the administration methods of intravenous injection and intratumoral injection have no toxic side effects on the organs and tissues of mice, proving that AtvCa-BSA-NPs and AtvCa-BSA-NPs combined with SO have a more effective anti-tumor effect.

[0193] 2.3) Liver and kidney indicators in mice

[0194] Hepatic and nephrotoxicity in mice was further evaluated by analyzing serum biochemical indicators. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important indicators of liver function; ALT and AST levels in the blood increase when hepatocellular is damaged. Creatinine (CREA) and urea (UREA) are important indicators of kidney function. When kidney function is impaired, the concentration of CREA and UREA excreted through renal filtration in the urine increases.

[0195] Mouse blood was collected and allowed to stand at room temperature for 15 minutes. The blood was then centrifuged at 2000 rpm for 10 minutes. The supernatant was collected and centrifuged again under the same conditions to remove residual blood cells, yielding clear, colorless serum. The levels of ALT, AST, UREA, and CREA in the serum were detected using appropriate kits. The results are shown in Figure 17.

[0196] Figure 17 shows the results of ALT, AST, UREA, and CREA index detection in mouse liver and kidneys. All ALT, UREA, and CREA indices in each group were within the normal reference range. Furthermore, there were no significant differences between the Blank-BSA-NPs group, AtvCa group, AtvCa-BSA-NPs group, SO group, and AtvCa-BSA-NPs+SO group and the control group. The AST index in the Blank-BSA-NPs group slightly exceeded the normal reference range, presumably due to sampling error. The AST indices in the other groups were normal. The AST indices in the AtvCa group, AtvCa-BSA-NPs group, SO group, and AtvCa-BSA-NPs+SO group were all within the normal reference range and showed no significant differences compared to the control group. These results demonstrate that AtvCa-BSA-NPs and AtvCa-BSA-NPs+SO have no significant toxic side effects on mouse liver and kidneys.

[0197] This invention established a CT26 tumor-bearing mouse model and used tumor volume change trends and tumor weight as indicators to preliminarily evaluate the in vivo tumor-suppressive effects of AtvCa, AtvCa-BSA-NPs, and the combination of AtvCa-BSA-NPs and SO. AtvCa-BSA-NPs showed a significantly increased anti-tumor effect compared to AtvCa. The combined use of AtvCa-BSA-NPs and SO further enhanced tumor inhibition. The safety of tail vein injection of AtvCa-BSA-NPs and intratumoral injection of SO was demonstrated through mouse body weight change trends, liver and kidney indicators, and H&E staining. This invention proves that using BSA as an AtvCa delivery carrier can prolong the in vivo action time of the drug and achieve a stronger tumor therapeutic effect in COC, while SO can further enhance the therapeutic effect, providing ideas and a research basis for the further clinical application of AtvCa.

Claims

1. The application of atorvastatin calcium albumin nanoparticles in the preparation of drugs for treating colorectal cancer, characterized in that, Composed of atorvastatin calcium and serum albumin, with a mass ratio of atorvastatin calcium to serum albumin of 1:4-1:5, the atorvastatin calcium-albumin nanoparticles are prepared by the following method: 1) Weigh atorvastatin calcium, add an appropriate amount of anhydrous ethanol and dissolve it completely to obtain an organic phase; 2) Weigh serum albumin, add deionized water and dissolve it completely, adjust the pH of the solution with sodium hydroxide to obtain an aqueous phase; 3) Under stirring, add the organic phase dropwise to the aqueous phase, add glutaraldehyde solution, and continue stirring for crosslinking. After crosslinking, remove the anhydrous ethanol by rotary evaporation, and centrifuge the resulting solution using a high-speed refrigerated centrifuge. The lower precipitate is taken as the atorvastatin calcium-albumin nanoparticles. In step 2), the pH is 8.5; the concentration of serum albumin is 10 mg / mL; in step 3), the volume ratio of the aqueous phase to the organic phase is 1:2-1:4; in step 3), the concentration of the glutaraldehyde solution is 20%-25%, the amount used is 10 µL, and the crosslinking time is 8-12 hours.

2. The application according to claim 1, characterized in that, The serum albumin mentioned is one of bovine serum albumin, human serum albumin, or ovalbumin.

3. The application according to claim 1, characterized in that, The atorvastatin calcium mentioned above can be replaced by atorvastatin or other salts, wherein the salts are potassium salts, sodium salts, magnesium salts, ammonium salts, iron salts, zinc salts, selenium salts, molybdenum salts, chromium salts, cobalt salts, and manganese salts of atorvastatin.

4. The application according to any one of claims 1-3, characterized in that, The atorvastatin calcium albumin nanoparticles were added with a lyophilization protectant and then freeze-dried under vacuum to prepare atorvastatin calcium albumin nanoparticle lyophilized powder.

5. The application according to claim 4, characterized in that, The freeze-drying protectant is one or a combination of two or more of mannitol, trehalose, or glucose.

6. The application according to claim 5, characterized in that, The freeze-drying protectant is a combination of mannitol and glucose in a mass ratio of 3:7 to 7:

3.

7. The application according to claim 1, characterized in that, The atorvastatin calcium albumin nanoparticles are used in combination with sodium oxalate.

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