Composition for preventing and / or treating hand-foot syndrome and synergistically resisting breast cancer as well as preparation method and application of composition

Abstract

The invention relates to the technical field of medicines for treating hand-foot syndrome and breast cancer, in particular to a composition for preventing and / or treating hand-foot syndrome (HFS) and synergistically resisting breast cancer as well as a preparation method and application of the composition. The composition comprises CuB-DOX (at) Lip, the CuB is cucurbitacine B, the DOX is doxorubicin, and the Lip is liposome. The mass ratio of CuB to DOX is 1: 5. The preparation method comprises the following steps: 1, preparing CuB-loaded lipidosome by using a film dispersion method; and 2, preparing the liposome CuB-DOX-coated Lip co-loaded with the two drugs by using an ammonium sulfate gradient method. In-vitro experiments prove that the combined application of the two medicines has the effect of treating the breast cancer. In-vivo experiments show that the compound has an obvious targeting effect. Meanwhile, the tumor growth inhibition effect of the CuB-DOX-coated Lip is obviously higher than that of a free drug group and a DOX-coated Lip group. Compared with a DOX-coated Lip group, IL-6 in a mouse in the CuB-DOX-coated Lip group is remarkably reduced, and IL-10 in the mouse in the CuB-DOX-coated Lip group is remarkably increased, which indicates that the CuB-DOX-coated Lip effectively inhibits inflammatory response in the mouse, so that the effect of preventing HFS is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating hand-foot syndrome and breast cancer, and in particular to a composition capable of preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, as well as a preparation method and application thereof. Background Art

[0002] Currently, the treatments for breast cancer include surgical resection, radiotherapy, chemotherapy, hormone regulation therapy, targeted therapy and traditional Chinese medicine treatment. When treating breast cancer, we should adopt the principle of both precise and comprehensive treatment. According to the actual condition of the patient, multiple treatment methods should be used together, and local treatment and systemic treatment should be adopted at the same time to improve the efficacy and improve the patient's quality of life.

[0003] Doxrubicin (DOX), also known as adriamycin, has a broad anti-tumor spectrum and is suitable for breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, etc. It is a representative anti-cancer drug of the classic anthracycline anti-tumor drugs.

[0004] In view of the serious cardiotoxicity of doxorubicin injection, Sequus Pharmaceuticals in the United States developed doxorubicin liposomes (drug name: Doxil, DOX@Lip) in 1995. It is the first anti-tumor nanodrug approved by the FDA and has obtained clinical indications for breast cancer, ovarian cancer, myeloma, and AIDS-KS. DOX@Lip reduces cardiac toxicity while achieving targeted therapeutic effects. The core of this method is to encapsulate DOX into liposomes containing methoxypolyethylene glycol (MPEG). This step is called polyethylene glycolation (PEGylation). It can prevent the liposomes from being phagocytosed by the mononuclear phagocyte system (MPS), increase their residence time in the bloodstream, and slow the accumulation of DOX in cardiac tissue, ultimately helping to reduce cardiac side effects.

[0005] Although DOX@Lip significantly reduces its cardiotoxicity, there are still some unavoidable side effects in practical applications, such as bone marrow suppression, hand-foot syndrome, and allergic reactions.

[0006] Hand-foot syndrome (HFS), also known as palmar-toe sensory erythema syndrome, is a common adverse reaction to anticancer drugs, characterized by decreased sensation or severe pain in the hands and feet. It is a skin toxicity reaction that typically occurs in the hands and feet, primarily during chemotherapy or targeted therapy for cancer patients.

[0007] The main symptoms of HFS include hypoesthesia, numbness, tingling, paresthesia, and pain. Initial symptoms typically include itching of the hands and feet, followed by redness, swelling, and pain on the tips of the fingers and toes. As the condition worsens, it may cause rashes, dryness, and cracking of the skin on the hands and feet. Rarely, it may lead to cut-like skin lesions. Common symptoms include blisters, extravasation, and even ulcers, which increase the risk of reinfection. Individuals with HFS may be afraid to move due to the intense pain in their hands and feet, and more severe cases may affect their ability to perform daily activities. There have been reports of patients developing HFS during their fifth cycle of adjuvant chemotherapy. These patients developed numerous red patches and skin lesions on various areas of the body, such as between the fingers, on the soles of the feet, around the navel, under the arms, and on the tongue. The largest lesion was approximately 20 square centimeters, covering over 15% of the body surface and continuing to expand. These lesions were also extremely painful, and some foot wounds even leaked tissue fluid. It is reported that the incidence of HFS caused by DOX@Lip can reach 58% to 84%.

[0008] Cucurbitacin B (CuB) is a tetracyclic triterpenoid compound derived from the Cucurbitaceae family. It exhibits anti-inflammatory, anti-tumor, hypoglycemic, and hepatoprotective properties. CuB possesses broad and comprehensive anti-tumor properties, primarily through its ability to regulate multiple signaling pathways, including JAK / STAT3, MARK, PI3K / Akt, Notch, Wnt, CIP2A / PP2A, integrin HER2, Hippo-YAP, VEGF / FAK / MMP-9, and others. Furthermore, CuB significantly reduces the growth rate of breast cancer MCF-7 cells, induces their apoptosis, and initiates autophagy, ultimately inhibiting tumor growth.

[0009] Liposomes, as drug carriers, are closed vesicles composed of phospholipids dispersed in water, forming a lipid bilayer with an aqueous interior. Patients treated with DOX@Lip demonstrated favorable therapeutic responses, further demonstrating its anti-tumor activity and relatively low adverse reactions. Both DOX@Lip and paclitaxel liposomes are currently marketed drugs and demonstrate excellent anti-tumor activity.

[0010] Although DOX@Lip can effectively reduce the cardiotoxicity of DOX, the adverse reaction HFS will occur during its clinical application, which limits the clinical application of DOX@Lip. Summary of the Invention

[0011] To comprehensively address these issues, this study co-encapsulated CuB and DOX in liposomes (CuB-DOX@Lip). The anti-inflammatory effects of CuB were exploited to prevent HFS, a side effect of DOX@Lip. CuB and DOX also demonstrated a synergistic anti-breast cancer effect. The formulation of CuB-DOX@Lip, prepared at varying CuB dosages, was selected based on its HFS prevention efficacy, encapsulation efficiency, and drug loading. The HFS prevention efficacy and synergistic anti-breast cancer potential were evaluated in both in vitro and in vivo experiments. This strategy, which reduces the adverse effects of DOX@Lip while increasing its efficacy, holds significant research value and practical implications.

[0012] In order to achieve the above objectives, the first aspect of the present invention provides a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, which is composed of CuB-DOX@Lip, wherein CuB is cucurbitacin B, DOX is doxorubicin, and Lip is liposome.

[0013] Preferably, the mass ratio of CuB to DOX is 1:5.

[0014] The second aspect of the present invention provides a compound having the effects of preventing hand-foot syndrome and synergistically combating breast cancer, and the use of the compound in preparing a drug for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer.

[0015] The third aspect of the present invention provides a method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, comprising:

[0016] Step 1: Prepare CuB-loaded liposomes using a thin film dispersion method;

[0017] Step 2: The ammonium sulfate gradient method was used to prepare the two-drug co-loaded liposomes CuB-DOX@Lip.

[0018] Preferably, step 1 comprises:

[0019] Step 1.1: Mix HSPC, cholesterol, DSPE-PEG2000 and appropriate amount of CuB in an eggplant-shaped flask;

[0020] Step 1.2: Add dichloromethane and anhydrous ethanol to completely dissolve the mixture;

[0021] Step 1.3: A thin lipid film is formed by rotary evaporation under reduced pressure at room temperature.

[0022] Preferably, step 2 includes:

[0023] Step 2.1: Hydrate the lipid film with ammonium sulfate, sonicate with a probe, and filter through a filter membrane;

[0024] Step 2.2: The product filtered in step 2.1 was dialyzed against a glucose solution to remove free ammonium sulfate and obtain CuB liposomes CuB@Lip.

[0025] Step 2.3: Dissolve DOX in glucose solution and mix with CuB@Lip, incubate and filter the membrane to obtain the two-drug co-loaded liposomes CuB-DOX@Lip.

[0026] Preferably, the volume ratio of dichloromethane to anhydrous ethanol is 1:2.

[0027] Preferably, the hydration conditions in step 2.1 are 60° C. and 30 minutes.

[0028] Preferably, the incubation condition in step 2.2 is 60° C. for 45 minutes.

[0029] Preferably, the mass ratio of CuB to DOX in step 2 is 1:5.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. This invention co-encapsulates CuB and DOX in liposomes for the first time, leveraging the anti-inflammatory effect of CuB to prevent HFS, an adverse reaction caused by the clinical use of DOX@Lip. The synergistic effect of the two drugs inhibits breast cancer, providing a new approach and strategy for addressing HFS, an adverse reaction of DOX@Lip, while also improving the drug's efficacy.

[0032] 2. This invention uses the hand-foot syndrome score and the two inflammatory factors IL-6 and IL-10 as indicators for evaluating HFS. The optimal formulation selected based on encapsulation efficiency, drug loading, and HFS prevention efficacy is: HSPC 47.5mg, cholesterol 15.95mg, DSPE-PEG2000 15.95mg, CuB 2.5mg, ammonium sulfate 100mg, DOX 10mg, dichloromethane 1mL, anhydrous ethanol 2mL, and purified water 5mL.

[0033] 3. The CuB-DOX@Lip prepared according to the optimized formulation achieved average encapsulation efficiency and drug loading of 76.03±0.4% and 2.39±0.5% for CuB, and 95.12±0.1% and 11.98±0.3% for DOX, respectively. The average particle size was 133.20±0.60 nm, the zeta potential was -41.10±1.01 mV, and the PDI was 0.195±0.600. Transmission electron microscopy revealed spherical bilayers with good dispersion. The CuB-DOX@Lip exhibited excellent stability over 7 days of storage, consistent with the serum stability trend of DOX@Lip. The cumulative release of both free CuB and DOX reached over 70% at 8 h, while the cumulative release of CuB and DOX from the CuB-DOX@Lip did not exceed 30% during the same period, demonstrating the excellent sustained-release effect of the CuB-DOX@Lip.

[0034] 4. In vitro cell uptake experiments suggest that CuB can enhance DOX uptake in 4T1 and MCF-7 cells. The inhibitory effects of FreeCuB, Free DOX, and CuB-DOX@Lip on cells were concentration-dependent. Due to the slow release of the drug from the liposomes, Free CuB+DOX exhibited the best cell growth inhibition, but CuB-DOX@Lip exhibited a stronger inhibitory effect than DOX@Lip. Cell wound healing, cell migration and invasion, and apoptosis assays also showed similar trends in both cell types.

[0035] 5. In vitro experiments demonstrated that the combination of the two drugs had a synergistic effect against breast cancer. In vivo experimental results showed that the fluorescence intensity of DiR@Lip in tumor-bearing mice was stronger than that of free DiR (Free DiR) as observed by small animal live imaging, indicating a significant targeting effect. Furthermore, the inhibitory effect of CuB-DOX@Lip on tumor growth was significantly higher than that of the free drug group and the DOX@Lip group. In vivo safety evaluation results showed that the body weight changes in the CuB-DOX@Lip group were not significantly different from those in the other groups. H&E tissue section results showed no significant toxicity in the heart, liver, spleen, lungs, or kidneys, indicating that CuB-DOX@Lip has a targeted anti-tumor effect. Elisa results showed that compared with the DOX@Lip group, IL-6 in the CuB-DOX@Lip group was significantly downregulated, while IL-10 was significantly upregulated, indicating that CuB-DOX@Lip effectively inhibited the inflammatory response in mice, thereby preventing HFS. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0037] In the attached figure:

[0038] Figure 1 HFS of CuB-DOX@Lip with different CuB dosages (n=5);

[0039] Figure 2 Foot skin sections of rats in the control group and HFS group;

[0040] Figure 3 Expression of IL-6 and IL-10 in rats of each group (n=5), *p<0.05, **p<0.01, ***p<0.001;

[0041] Figure 4 Appearance and morphology of CuB-DOX@Lip. (A) Appearance of the preparation; (B) Transmission electron microscopy image of CuB-DOX@Lip (×220,000);

[0042] Figure 5 Particle size and potential of CuB-DOX@Lip. (A) Particle size distribution of CuB-DOX@Lip; (B) Potential distribution of CuB-DOX@Lip;

[0043] Figure 6 Stability of CuB-DOX@Lip (n=3);

[0044] Figure 7 In vitro release of CuB, DOX, and CuB-DOX@Lip (n=3);

[0045] Figure 8 DOX uptake by 4T1 cells (×200);

[0046] Figure 9 DOX uptake by MCF-7 cells (×200);

[0047] Figure 10 DOX uptake by 4T1 and MCF-7 cells. (A), (B) Quantitative analysis of DOX uptake by 4T1 cells; (C), (D) Quantitative analysis of DOX uptake by MCF-7 cells (n=3), *p<0.05, **p<0.01;

[0048] Figure 11 Effects of blank liposomes on the survival rates of 4T1 and MCF-7 cells (n=3);

[0049] Figure 12 Inhibitory rate of CuB and DOX on 4T1 cells (n=3);

[0050] Figure 13Inhibitory rate of CuB and DOX on MCF-7 cells (n=3);

[0051] Figure 14 Inhibitory rate of CuB+DOX on 4T1 and MCF-7 cells (n=3);

[0052] Figure 15 The inhibitory index of the combined application of CuB and DOX on 4T1 and MCF-7 cells;

[0053] Figure 16 Inhibitory effect of CuB-DOX@Lip on 4T1 and MCF-7 cells. (A) Inhibitory effect of CuB-DOX@Lip on 4T1 cells; (B) Inhibitory effect of CuB-DOX@Lip on MCF-7 cells (n=3), *p<0.05, **p<0.01, ***p<0.001;

[0054] Figure 17 The control effect of different drug groups on apoptosis of 4T1 and MCF-7 cells and the statistical results of apoptosis rate. (A) The control effect on apoptosis of 4T1 cells and the statistical results of apoptosis rate; (B) The control effect on apoptosis of MCF-7 cells and the statistical results of apoptosis rate (n=3), **p<0.01, ***p<0.001;

[0055] Figure 18 (A), (B) Effects of different drug groups on wound healing of 4T1 cells and quantitative analysis; (C), (D) Effects of different preparation groups on wound healing of MCF-7 cells and quantitative analysis (n=3), *p<0.05, **p<0.01;

[0056] Figure 19 (A) Effects of different drug groups on 4T1 cell migration and invasion; (B) Effects of different preparations on 4T1 cell migration rate; (C) Effects of different preparations on 4T1 cell invasion rate (n=3), ***p<0.001;

[0057] Figure 20 (A) Effects of different drug groups on the migration and invasion of MCF-7 cells; (B) Effects of different preparations on the migration rate of MCF-7 cells; (C) Effects of different preparations on the invasion rate of MCF-7 cells (n=3), *p<0.05, **p<0.01;

[0058] Figure 21DiR was used as a fluorescent probe to investigate the in vivo targeting effect of CuB-DOX@Lip. (A) Fluorescence distribution in mice at various time points; (B) Fluorescence distribution in isolated tumors and major organs; (C) Quantitative analysis of fluorescence in mice at various time points; (D) Quantitative analysis of fluorescence in mice in supine position at 48 hours; (E) Quantitative analysis of fluorescence in isolated tumors (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001.

[0059] Figure 22 Changes in tumor volume in Balb / c mice treated with saline, Free DOX, CuB+DOX, DOX@Lip, and CuB-DOX@Lip (n=5), ***p<0.001;

[0060] Figure 23 (A) Visual view of isolated tumor; (B) In vitro tumor weight; (C) Tumor inhibition rate of each drug group (n=5), *p<0.05, **p<0.01;

[0061] Figure 24 H&E staining of ex vivo tumors;

[0062] Figure 25 Body weight changes of mice in each group during the administration period (n=5);

[0063] Figure 26 H&E staining of the main organs of mice in each group;

[0064] Figure 27 CuB-DOX@Lip preventive effect on HFS (n=5), *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0065] The following combination Figure 1-Figure 27 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0066] Experimental Materials

[0067] The cell lines used in this experiment were the mouse breast cancer cell line 4T1 and the human breast cancer cell line MCF-7, both obtained from the Cell Bank of the Chinese Academy of Sciences. The animals used in this experiment were female SD rats and female Balb / c mice, obtained from Heilongjiang Yuheng Veterinary Technology Service Co., Ltd. The animal experiments were reviewed and approved by the Qiqihar Medical College Laboratory Animal Ethics Committee, confirming that the research content and experimental design conformed to international ethical standards for the use of laboratory animals and all regulations established by the Qiqihar Medical College Laboratory Animal Ethics Committee.

[0068] Main reagents

[0069]

[0070]

[0071] Example 1:

[0072] A composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer is provided. The composition comprises CuB-DOX@Lip, wherein CuB represents cucurbitacin B, DOX represents doxorubicin, and Lip represents liposomes. The mass ratio of CuB to DOX is 1:5.

[0073] Example 2:

[0074] A composition capable of preventing hand-foot syndrome and synergistically combating breast cancer is provided, and the composition is used for preparing a medicament for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer.

[0075] Example 3:

[0076] A method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, comprising:

[0077] Step 1: Prepare CuB-loaded liposomes using a thin film dispersion method;

[0078] Step 1.1: Mix 47.5 mg of HSPC, 15.95 mg of cholesterol, 15.95 mg of DSPE-PEG2000, and an appropriate amount of CuB in a 500 ml eggplant-shaped bottle;

[0079] Step 1.2: Add 1 ml of dichloromethane and 2 ml of anhydrous ethanol at 60°C to completely dissolve the mixture;

[0080] Step 1.3: A thin lipid film is formed by rotary evaporation under reduced pressure at room temperature.

[0081] Step 2: The ammonium sulfate gradient method was used to prepare the two-drug co-loaded liposomes CuB-DOX@Lip.

[0082] Step 2.1: The thin lipid film was hydrated with 5 mL of ammonium sulfate (200 mM) at 60°C for 30 minutes, then sonicated with a probe for 10 minutes and filtered through 0.45 μm and 0.22 μm filters in sequence.

[0083] Step 2.2: The product filtered in step 2.1 was dialyzed against 1000 ml of 5% glucose solution for 3 hours (the buffer was changed every hour) to remove free ammonium sulfate and obtain CuB liposomes CuB@Lip.

[0084] Step 2.3: Dissolve 10 mg of DOX in 500 μl of 5% glucose solution and mix with CuB@Lip. Incubate at 60°C for 45 minutes and filter through a 0.22 μm filter to obtain the two-drug co-loaded liposomes CuB-DOX@Lip.

[0085] Preferably, in step 2, the mass ratio of CuB to DOX is 1:5.

[0086] 1. Determination of the optimal prescription:

[0087] CuB-DOX@Lip with CuB dosage of 1 mg, 2.5 mg, and 5 mg was prepared, and the encapsulation efficiency and drug loading capacity of CuB-DOX@Lip prepared at each dosage were investigated. The results are shown in Table 1.

[0088] Table 1 Encapsulation efficiency and drug loading of CuB and DOX at different CuB dosages

[0089]

[0090] The pharmacodynamics of the three preparations were investigated to observe their effectiveness in preventing HFS. 25 female SD rats aged 6-8 weeks, weighing approximately 200 g, were housed in a well-ventilated environment with a temperature of 21±1.5°C, a humidity of 55±15%, and a light / dark cycle of 12 hours. The rats had free access to food and water. The rats were randomly divided into 5 groups, with 5 rats in each group. The 5 groups were the positive control group, the negative control group, and the CuB-DOX@Lip groups with a dosage of 1 mg, 2.5 mg, and 5 mg. The positive control group, the 1 mg, 2.5 mg, and 5 mg CuB-DOX@Lip groups were all administered 10 mg / kg DOX via the tail vein 4 times, with an interval of 3 days between doses. The negative control group did not receive any treatment.

[0091] The concentration of DOX@Lip was 2 mg / ml and was diluted to 10 mg / kg with 5% glucose solution before administration. CuB-DOX@Lip with CuB dosages of 1 mg, 2.5 mg, and 5 mg were also diluted with 5% glucose solution to a DOX concentration of 10 mg / kg. After 4 administrations, a visual inspection was performed: the hands and feet of the rats in each group were observed. If redness, swelling, or sloughing occurred, it was considered grade 3 HFS, and photos were taken. The results are shown in Figure 1 , Table 2. According to Figure 1 It can be seen intuitively from Table 2 that encapsulating CuB in DOX@Lip has a good effect in preventing HFS.

[0092] Table 2 The incidence of claw syndrome in rats in each group

[0093]

[0094] The skin of the rat's foot was removed for pathological sectioning and H&E staining. Figure 2 As shown. It can be observed that the epidermis of the rat foot skin is thinning, the basal layer cells are swollen, vacuolar degeneration, the stratum corneum is separated and some tissues are hyperkeratotic, the dermis is loose and edematous, and inflammatory cells are infiltrated, which are symptoms of inflammation. Blood was collected from the abdominal aorta of each group of rats, and serum was obtained after standing. The enzyme-linked immunosorbent assay (ELISA) method was used to determine the content of IL-6 and IL-10 in rat serum. Figure 3 As shown, it can be observed that the pro-inflammatory factor IL-6 in the serum of rats in the DOX@Lip group was significantly increased compared with the blank control group, while the anti-inflammatory factor IL-10 content was significantly reduced, and the rats also showed inflammatory symptoms. The levels of inflammatory factors IL-6 and IL-10 in the serum of rats in each group were measured. Compared with the DOX@Lip group, the 1mg, 2.5mg, and 5mg dosages of CuB-DOX@Lip all improved the inflammation in the rats, but there was no significant difference in the two inflammatory factors in the 1mg group compared with the DOX@Lip group. Although the 5mg dosage group had a better effect in inhibiting the inflammatory response than the 2.5mg dosage group, there was no significant difference in the two inflammatory factors between the two groups.

[0095] Since the HFS prevention efficacy of CuB at 2.5 mg and 5 mg was similar, and there was no significant difference in the improvement of serum inflammatory cytokine levels, a 2.5 mg CuB dose was selected based on the HFS prevention efficacy, the encapsulation efficiency of CuB and DOX, and the drug loading. Therefore, the optimal formulation of CuB-DOX@Lip was: 47.5 mg HSPC, 15.95 mg cholesterol, 15.95 mg DSPE-PEG2000, 2.5 mg CuB, 100 mg ammonium sulfate, 10 mg DOX, 1 mL dichloromethane, 2 mL anhydrous ethanol, and 5 mL purified water.

[0096] 2. Characterization of CuB-DOX@Lip

[0097] 2.1 Appearance and morphology of CuB-DOX@Lip

[0098] Since DOX is red, CuB-DOX@Lip is a clear solution with red opalescence. Figure 4 A. Under transmission electron microscopy, it can be observed that CuB-DOX@Lip presents a round bilayer liposome structure with uniform distribution. Figure 4 Middle B.

[0099] 2.2 Particle size and potential of CuB-DOX@Lip

[0100] The particle size of CuB-DOX@Lip was 133.20±0.60nm and PDI was 0.195±0.600 as measured by a particle size analyzer. Figure 5 A. The Zeta potential of the CuB-DOX@Lip preparation was measured by a particle size analyzer: -41.10±1.01mV, see Figure 5 Middle B.

[0101] 2.3 Drug loading and encapsulation efficiency of CuB-DOX@Lip

[0102] Calculated by high-performance liquid chromatography, in CuB-DOX@Lip, the encapsulation efficiency of CuB was 76.03±0.4%, and the drug loading was 2.39±0.5%; the encapsulation efficiency of DOX was 95.12±0.1%, and the drug loading was 11.98±0.3%.

[0103] 2.4 Storage and serum stability of CuB-DOX@Lip

[0104] The particle sizes of CuB-DOX@Lip and DOX@Lip were measured at the same time within 7 days. Figure 6 The particle size of CuB-DOX@Lip remained almost unchanged when stored at 4°C in PBS for 7 days, while the change trend was the same as that of DOX@Lip at 37°C in FBS, which proved that encapsulating CuB in DOX@Lip did not affect its stability.

[0105] 2.5 Release of CuB-DOX@Lip

[0106] like Figure 7 The cumulative release of free CuB and DOX reached over 70% at 8 hours, while the cumulative release of CuB and DOX from CuB-DOX@Lip did not exceed 30% during the same period. At 72 hours, the cumulative release of CuB and DOX from CuB-DOX@Lip exceeded 60%, indicating that the CuB-DOX@Lip formulation can continuously release drugs and avoid sudden drug release, achieving a sustained release effect.

[0107] Example 4:

[0108] The invention discloses a composition CuB-DOX@Lip for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, and its application in vitro in combating breast cancer.

[0109] Unless otherwise specified, the cells used in in vitro experiments were 4T1 and MCF-7 cells, and the experimental groups were FreeDOX group, Free CuB+DOX group, DOX@Lip group, and CuB-DOX@Lip group.

[0110] 1. Cellular Uptake Experiment

[0111] 1.1 Qualitative cellular uptake experiments

[0112] like Figure 8 、 Figure 9 As shown, the blue part in the picture is the cell nucleus stained blue with Hoechst33258 dye, and the red part is DOX, which represents the cell's uptake of DOX. The results show that, whether in 4T1 cells or MCF-7 cells, the Free CuB+DOX group has the strongest fluorescence intensity, followed by the Free DOX group. The DOX@Lip and CuB-DOX@Lip groups have a sustained release effect on the drug due to the encapsulation of liposomes, so the fluorescence intensity generated is less than that of the free drug. It is worth noting that the CuB-DOX@Lip group has a stronger fluorescence intensity than the DOX@Lip group, and the same situation also occurs in the free drug group. Therefore, we speculate that CuB may enable cells to take up more DOX.

[0113] 1.2 Quantitative cell uptake experiments

[0114] In order to better understand the cellular uptake and verify our hypothesis, we conducted a quantitative analysis of cellular uptake. Figure 10 In both 4T1 and MCF-7 cells, the CuB+DOX group was positioned at the far right, indicating the strongest uptake capacity. This result is consistent with the qualitative uptake analysis. The quantified data histogram shows that the cellular uptake capacity of CuB+DOX in the free drug group was statistically significant higher than that of the DOX alone group. Furthermore, the cellular uptake capacity of CuB-DOX@Lip in the liposome drug group was statistically significant higher than that of DOX@Lip. This further confirms our hypothesis that CuB can enhance cellular DOX uptake.

[0115] 2 Cytotoxicity assay

[0116] First, the effect of blank liposomes on the survival rate of mouse breast cancer 4T1 cells and human breast cancer MCF-7 cells was investigated. After the two cells were treated with blank liposomes at concentrations of 0.5, 1, 5, 10, 50, 100, and 500 μg / ml, the survival rates of 4T1 and MCF-7 cells were all above 80%. Therefore, it can be shown that blank liposomes have little toxicity to 4T1 and MCF-7 cells. Figure 11 .

[0117] like Figure 12 、 Figure 13 The concentrations of CuB and DOX were set at 0.02, 0.06, 0.1, 0.14, 0.18, and 0.2 μg / mL, respectively, and the concentrations of DOX were set at 0.1, 0.3, 0.5, 0.7, 0.9, and 1 μg / mL, respectively. Blank complete medium was used as the control group and the cells were treated with 4T1 and MCF-7 cells. The results showed that both CuB and DOX had a certain inhibitory effect on the growth of 4T1 and MCF-7 cells in a dose-dependent manner.

[0118] The calculation of encapsulation efficiency and drug loading showed that the concentration ratio of CuB to DOX in the prepared CuB-DOX@Lip was 1:5. To study the combined effect of the two drugs at a ratio of 1:5, CuB+DOX solutions with total concentrations of 0.3, 0.5, 0.7, 0.9, and 1 μg / ml were prepared (where CuB concentration: DOX concentration = 1:5). Figure 14 As shown in the figure, it can be observed that with the increase of concentration, the cell inhibition rate of CuB+DOX on 4T1 and MCF-7 cells also increases continuously, indicating that the inhibitory effect of the two drugs on 4T1 and MCF-7 cells is also dose-dependent when used in combination.

[0119] Calculated IC50 values ​​for CuB against 4T1 and MCF-7 cells were 0.19 μg / ml and 0.21 μg / ml, respectively, while those for DOX were 0.28 μg / ml and 0.35 μg / ml, respectively. Combined administration yielded IC50 values ​​of 0.16 μg / mL and 0.11 μg / mL for 4T1 and MCF-7 cells, respectively, significantly lower than the IC50 values ​​for either drug alone. These IC50 values ​​demonstrate that the inhibitory activity of CuB combined with DOX is stronger than that of either DOX or CuB alone, demonstrating a synergistic effect. This may be due to CuB promoting increased DOX uptake in 4T1 and MCF-7 cells, thereby reducing drug efflux.

[0120] In order to verify the effect of the CuB:DOX ratio of 1:5 in the CuB-DOX@Lip system, the experimental data were input into the CompuSyn software, and the software system was used to analyze whether there was a synergistic effect when the CuB and DOX ratio was 1:5. Figure 15 ,It can be seen from the Fa-CI curve that the CI value is less than 1 when the two drugs are ,used together. Therefore, it can be ,said that a synergistic effect can be exhibited when the ratio of CuB and DOX is ,1:5.

[0121] The concentrations of CuB-DOX@Lip and DOX@Lip solutions were 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 μg / ml, respectively. Free CuB+DOX and DOX groups with the same concentrations were then set up to investigate the cytotoxicity of the preparations. The results are shown in the table. Figure 16. Compared with the Free CuB+DOX group and the DOX group at the same dose, the combined use of the two drugs can increase the cell inhibition rate of 4T1 and MCF-7 cells, proving that combined administration can increase the toxicity to 4T1 and MCF-7 cells. Due to the encapsulation of liposomes, CuB and DOX are slowly released, so the cell inhibition rates of the CuB-DOX@Lip and DOX@Lip groups are lower than those of the corresponding free drugs. However, it is not difficult to see that at the same dose, whether it is the free drug group or the liposome drug group, the cell inhibition rate after the combined use of CuB is higher than that of a single drug. This is because CuB and DOX have a synergistic effect at a ratio of 1:5, so the cell inhibition effect is better than that of the single drug group at the same concentration.

[0122] 3. Cell apoptosis experiment

[0123] Flow cytometry was used to analyze the apoptosis of 4T1 and MCF-7 cells 24 hours after administration. Figure 17 . According to the quantitative results of the total apoptosis rate. It can be observed that the apoptosis-inducing effect of the combination of CuB and DOX is better than that of DOX alone, whether in the free drug group or the liposome drug group, and the difference is statistically significant. The drug in the liposome group is encapsulated in the liposomes, resulting in sustained release of the drug, while the free drug directly contacts the cells. Therefore, the apoptosis-inducing effect of the liposome group is not as good as that of the free drug. This result further proves that CuB and DOX have a synergistic effect, which is consistent with the results of the cytotoxicity experiment.

[0124] 4. Cell scratch assay

[0125] like Figure 18 As shown, in 4T1 and MCF-7 breast cancer cells, the wound healing rate of 4T1 cells in the negative control group was approximately 45.12% after 24 hours. The Free CuB+DOX group exhibited the highest metastasis inhibition effect, with a healing rate of only 9.28% after 24 hours. The wound healing rates of the FreeDOX, CuB-DOX@Lip, and DOX@Lip groups were 18.74%, 25.67%, and 32.85% after 24 hours, respectively. The wound healing rate of MCF-7 cells in the negative control group was approximately 49.11% after 24 hours. Similarly, the Free CuB+DOX group exhibited the highest metastasis inhibition effect, with a healing rate of only 8.27% after 24 hours. The wound healing rates of the Free DOX, CuB-DOX@Lip, and DOX@Lip groups were 12.25%, 28.37%, and 34.41% after 24 hours, respectively. In both cell lines, the combination of CuB and DOX exhibited a stronger metastasis inhibition effect than DOX alone, regardless of whether it was a free drug or a liposomal drug.

[0126] 5. Cell migration and invasion assay

[0127] Figure 19、 Figure 20 The Free CuB+DOX group showed the strongest inhibitory effect on the migration of 4T1 and MCF-7 cells. The migration rates of 4T1 cells treated with FreeCuB+DOX were approximately 11.15%, and those of MCF-7 cells were approximately 9.89%. The migration rates of 4T1 cells treated with CuB-DOX@Lip were approximately 39.74%, and those of MCF-7 cells were approximately 32.58%. In both cell lines, the combined treatment group exhibited significantly stronger inhibition of cell migration than DOX alone, with statistically significant differences. The invasion rates of 4T1 and MCF-7 cells treated with FreeCuB+DOX were 10.25% and 8.32%, respectively. These results demonstrate that the combined use of CuB and DOX can significantly inhibit the migration and invasion of 4T1 and MCF-7 cells.

[0128] Example 5:

[0129] Disclosed is an application of a composition CuB-DOX@Lip for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer in vivo.

[0130] A 4T1 breast cancer tumor-bearing mouse model was constructed and used for experimental research when the tumor volume reached 100 mm3.

[0131] 1 Evaluation of tumor targeting and distribution in mice

[0132] DiR@Lip was prepared using the same method as CuB@Lip, using DiR as a probe. Six mice were randomly divided into two groups of three. Each group received a 100 μL injection of free DiR or DiR@Lip via the tail vein at a DiR dose of 500 μg / kg. The in vivo distribution of the liposomes was observed and photographed using an IVIS Lumina III in vivo imaging system at 0, 2, 4, 6, 8, 10, 12, 24, and 48 hours after injection. Figure 21 Figure A shows the fluorescence distribution of each DiR preparation at different time points. Fluorescence signals were detected at the tumor site in both the Free DiR group and the DiR@Lip group at 0 h, but the fluorescence intensity of the DiR@Lip group was significantly stronger than that of the Free DiR group. To better compare the fluorescence intensity of the Free DiR group and the DiR@Lip group at the tumor site, the mice were photographed in a supine position at 48 h. The fluorescence intensity at each time point was quantitatively analyzed, as shown in Figure 5. Figure 21 In Figure C, it can be observed that the fluorescence intensity of the tumor site in both the Free DiR group and the DiR@Lip group increased with time, and the fluorescence intensity of the tumor site in the DiR@Lip group was stronger than that in the Free DiR group at each time point. Figure 21Middle D shows the quantitative analysis of the fluorescence intensity of the tumor site in the supine position of mice at 48 hours. The fluorescence intensity of the DiR@Lip group was also stronger than that of the Free DiR group, and the difference was statistically significant.

[0133] After 48 hours, the mice in both groups were killed to obtain tumors and major organs, and the distribution of fluorescence in the isolated tumors and tissues was observed. Figure 21 In Figure B, the DiR fluorescence signals in the Free DiR and DiR@Lip groups were mainly concentrated in the tumor, liver, and spleen. The results for isolated tumors and organs were consistent with the in vivo imaging results in mice. Figure 21 Figure E shows the fluorescence intensity in an ex vivo tumor. The DiR@Lip group showed stronger fluorescence than the Free DiR group, with a statistically significant difference. These results demonstrate that liposome encapsulation can effectively target the drug through the EPR effect, allowing it to be preferentially retained and accumulated at the tumor site.

[0134] 2 In vivo anti-tumor efficacy and safety evaluation

[0135] Twenty-five mice were randomly divided into five groups, each containing five mice. The treatment groups included Free DOX, Free CuB+DOX, DOX@Lip, and CuB-DOX@Lip, with a normal saline group serving as the control group. Administration was via tail vein injection every two days, with 200 μl administered each time at a dose of 5 mg / kg (calculated based on DOX concentration). Tumor volume and body weight were measured every other day. The mice were sacrificed on the second day after four doses, and the tumors were removed, photographed, and weighed. The hearts, livers, spleens, lungs, and kidneys were also removed. Tumors, hearts, livers, spleens, lungs, and kidneys in each group were fixed in 4% paraformaldehyde solution. After paraffin embedding and sectioning, H&E staining was performed, and pathological sections were prepared. H&E-stained sections of the heart, liver, spleen, lungs, and kidneys were observed microscopically and photographed. The in vivo toxicity of Free DOX, Free CuB+DOX, DOX@Lip, and CuB-DOX@Lip was evaluated based on organ damage and changes in mouse body weight. The necrosis of tumors in each group of mice was observed by H&E staining pathological sections of tumor tissues to evaluate the in vivo anti-breast cancer effect of CuB-DOX@Lip.

[0136] 2.1 Determination of tumor growth volume inhibition ability

[0137] Tumor growth volume Figure 22The results showed that Free DOX, Free CuB+DOX, DOX@Lip, and CuB-DOX@Lip inhibited tumor growth in Balb / c mice with breast cancer. While the tumor volume in the saline group increased significantly, the Free CuB+DOX group still showed stronger tumor inhibition than Free DOX alone. Notably, the CuB-DOX@Lip group showed the strongest tumor growth inhibition, even stronger than Free CuB+DOX, and the difference was statistically significant. This is because the drug is encapsulated in liposomes, which give the drug long circulation and passive targeting, resulting in better tumor growth inhibition.

[0138] 2.2 Determination of in vitro tumor weight and tumor inhibition rate

[0139] In vitro tumor weight Figure 23 Figure B shows that compared to the saline, Free DOX, Free CuB+DOX, and DOX@Lip groups, the CuB-DOX@Lip group had the smallest tumor weight. Due to the passive targeting and long circulation of liposomes, CuB-DOX@Lip allows for greater drug accumulation in tumors, thus demonstrating the best tumor inhibitory effect. Figure 23 The in vitro tumor image in Figure A shows that Free DOX, Free CuB+DOX, and DOX@Lip all have anti-tumor effects compared to the saline group, but the CuB-DOX@Lip group has the strongest inhibitory effect on tumor growth. The tumor inhibition rate of each group is calculated as follows: Figure 23 As shown in Figure C, the CuB-DOX@Lip group had the highest tumor inhibition rate, further indicating that CuB-DOX@Lip has a stronger effect in inhibiting tumor growth.

[0140] 2.3 Tumor-killing effect of CuB-DOX@Lip

[0141] The killing effect of CuB-DOX@Lip on tumors is as follows Figure 24 H&E staining of the ex vivo tumor after pathological sectioning showed that the tumor in the CuB-DOX@Lip group was the most damaged, which further proved that CuB-DOX@Lip has the strongest tumor-killing effect.

[0142] 3 Safety evaluation

[0143] 3.1 Changes in mouse weight

[0144] The body weight of mice was measured every other day after administration, and the systemic toxicity of CuB-DOX@Lip on Balb / c tumor-bearing mice was investigated based on the changes in body weight. Figure 25As shown in the data, there was no significant change in the body weight of mice in the normal saline, Free DOX, Free CuB+DOX, DOX@Lip, and CuB-DOX@Lip groups during the administration period, indicating that CuB-DOX@Lip had less toxicity to tumor-bearing mice.

[0145] 3.2 Toxicity in major organs of tumor-bearing mice

[0146] Toxicity to major organs of tumor-bearing mice Figure 26 H&E staining showed that Free DOX, Free CuB+DOX, DOX@Lip, and CuB-DOX@Lip did not cause significant damage to the major organs of tumor-bearing mice. These results indicate that CuB-DOX@Lip has good safety.

[0147] Example 6:

[0148] Evaluation of the effect of CuB-DOX@Lip, a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, on preventing hand-foot syndrome

[0149] Mouse paws are small and their skin is thin, making it difficult to observe the development of HFS. Previous studies have confirmed that the development of HFS is accompanied by an inflammatory response, and the regulatory effects of CuB-DOX@Lip on IL-6 and IL-10 have also been verified in the SD rat HFS model. Therefore, the effectiveness of CuB-DOX@Lip in preventing HFS can be assessed by monitoring the expression of inflammatory factors. When mice were sacrificed in 5.2, blood was collected from each group of mice via eyeballs. After blood collection, the blood was placed in a test tube rack and allowed to stand for 2 hours. Serum was then centrifuged at 4°C and 3000 rpm for 15 minutes to obtain serum. ELISA was used to measure the levels of IL-6 and IL-10 in the serum of each group of mice. Figure 27 The results showed that compared with the DOX-free group, the Free CuB+DOX group showed a significant decrease in inflammation levels in mice, which is attributed to the anti-inflammatory effects of CuB. The DOX@Lip group showed the highest inflammation levels, which may be the cause of HFS. Compared with the DOX@Lip group, the CuB-DOX@Lip group showed a significant decrease in IL-6 levels and a significant increase in IL-10 levels. This result is consistent with the previous results of the preventive effect of CuB-DOX@Lip on HFS in SD rats, indicating that CuB-DOX@Lip effectively reduces inflammation levels in breast cancer tumor-bearing mice, thereby preventing the occurrence of HFS.

[0150] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, characterized in that: The composition is CuB-DOX@Lip, where CuB is cucurbitacin B, DOX is doxorubicin, and Lip is liposome.

2. A composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 1, characterized in that: The mass ratio of CuB and DOX was 1:

5.

3. A composition for preventing hand-foot syndrome and synergistically combating breast cancer, characterized in that: Use for preparing medicine for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer.

4. A method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer, characterized in that: include: Step 1: Prepare CuB-loaded liposomes using a thin film dispersion method; Step 2: The ammonium sulfate gradient method was used to prepare the two-drug co-loaded liposomes CuB-DOX@Lip.

5. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 4, characterized in that: Step 1 includes: Step 1.1: Mix HSPC, cholesterol, DSPE-PEG2000, and CuB in an eggplant-shaped flask; Step 1.2: Add dichloromethane and anhydrous ethanol to completely dissolve the mixture; Step 1.3: A thin lipid film is formed by rotary evaporation under reduced pressure at room temperature.

6. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 5, characterized in that: Step 2 includes: Step 2.1: Hydrate the lipid film with ammonium sulfate, sonicate with a probe, and filter through a filter membrane; Step 2.2: The product filtered in step 2.1 was dialyzed against a glucose solution to remove free ammonium sulfate and obtain CuB liposomes CuB@Lip. Step 2.3: Dissolve DOX in glucose solution and mix with CuB@Lip, incubate and filter the membrane to obtain the two-drug co-loaded liposomes CuB-DOX@Lip.

7. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 6, characterized in that: The volume ratio of dichloromethane to anhydrous ethanol is 1:

2.

8. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 7, characterized in that: The hydration conditions in step 2.1 are 60°C and 30 minutes.

9. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 8, characterized in that: The incubation condition in step 2.2 is 60°C for 45 minutes.

10. The method for preparing a composition for preventing and / or treating hand-foot syndrome and synergistically combating breast cancer according to claim 9, characterized in that: In step 2, the mass ratio of CuB to DOX was 1:5.

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