Synthesis of acid-responsive amphiphilic liposomes and their application in tumor therapy

Abstract

The application relates to the field of triple-negative breast cancer treatment and relates to synthesis of an acid-responsive amphiphilic liposome and application of the acid-responsive amphiphilic liposome in tumor treatment. A chemotherapeutic drug, a phospholipid, cholesterol and a polyethylene glycolized lipid are dissolved in an organic solvent chloroform, vacuum rotary evaporation is carried out under certain temperature and rotation rate, a water phase is added for hydration, a polypeptide nanogold cluster solution is added for ultrasonic treatment, then crushing, molecular sieve screening, dialysis and ultrafiltration are carried out to obtain a pH-sensitive liposome; in the preparation process, the phospholipid spontaneously forms a kind of biological membrane-like phospholipid bilayer membrane vesicle in water, and the chemotherapeutic drug and the nanogold cluster are wrapped in the vesicle. The pH-sensitive liposome can target the triple-negative breast cancer tumor site through pH response, can improve the solubility of the chemotherapeutic drug, can realize high-efficiency treatment effect of the drug at a low dose, can obviously reduce the toxic side effect of the chemotherapeutic drug, and can realize effective treatment of the triple-negative breast cancer.

Description

Technical Field

[0001] This invention relates to a drug delivery system that can target tumors. It can be summarized as a method for synthesizing pH-sensitive liposomes that possess the ability to target tumor sites through pH response, while simultaneously encapsulating the polypeptide gold cluster GA and DTX, a commonly used chemotherapy drug for triple-negative breast cancer, to achieve a low-dose, high-efficiency therapeutic effect. Furthermore, in vitro and in vivo experiments have verified that this material can achieve targeted delivery of chemotherapy drugs to triple-negative breast cancer tumors, prolonging the drug release duration, achieving high-efficiency treatment with low drug dosage and low toxicity, and maximizing drug efficacy. Background Technology

[0002] Breast carcinoma (BC) is a malignant tumor that occurs in the glandular epithelial tissue of the breast, affecting 99% of women and 1% of men. According to the latest epidemiological statistics, breast cancer has become the leading cause of cancer-related deaths among women in my country, earning it the title of "the number one killer of women's health" in the medical community. The incidence rate of breast cancer in China is increasing by 2%-3% annually, and its mortality rate ranks fourth among female malignant tumors, seriously threatening women's lives and health. Breast cancer is often diagnosed through pathological examination. Triple-negative breast cancer (TNBC) has the worst prognosis among all breast cancers, characterized by early onset, high heterogeneity, poor differentiation, high invasiveness, and a high rate of metastasis and recurrence. Literature reports a 3-year recurrence rate of 19%, with a median survival of only 12 months after recurrence. Furthermore, due to the lack of common therapeutic targets such as ER, PR, and HER2, patients with triple-negative breast cancer often do not benefit much from traditional anti-HER2 therapy and endocrine therapy. Therefore, chemotherapy is the primary treatment method, but chemotherapy remission often has a short duration and is accompanied by strong side effects.

[0003] Currently, docetaxel (DTX) is a commonly used chemotherapy drug for treating triple-negative breast cancer. It is known to have low oral bioavailability due to its low water solubility, poor membrane permeability, and sensitivity to first-pass metabolism in the liver, and is currently administered intravenously. Its anticancer mechanism is similar to paclitaxel, but compared to paclitaxel, it has a higher intracellular concentration, a longer intracellular residence time, and stronger antitumor activity. Although its water solubility has improved somewhat compared to paclitaxel, it is still poorly soluble in water and still requires the use of the nonionic surfactant Tween 80 as a solubilizer, along with a 13% ethanol solution for dissolution. However, Tween 80 is hemolytic and highly viscous, leading to significant allergic reactions in most patients. This is mainly related to the formulation, and the main toxicities are neutropenia, peripheral neuropathy, and hypersensitivity reactions. Given these results, there is a stronger reason to use a carrier that is safer and better tolerable than Tween 80 for the re-delivery of taxane drugs. Furthermore, DTX affects cell division, so this drug is cytotoxic not only to cancer cells but also to hair follicles, bone marrow, and other germ cells, leading to a range of side effects, including hair loss. Simultaneously, DTX is a substrate of the drug efflux pump P-glycoprotein (P-gp), which reduces its concentration near cells and contributes to drug resistance. Therefore, to prevent excessive side effects and the development of drug resistance in cancer cells, the concentration of chemotherapy drugs must be strictly controlled and cannot be used continuously. Thus, there is an urgent need to develop new and effective chemotherapy strategies to improve overall treatment efficacy and patient prognosis.

[0004] In previous work, our research group had demonstrated in vitro and in vivo experiments that the peptide-gold cluster GA could act as a nanosensitizer, profoundly sensitizing triple-negative breast cancer cells to docetaxel, promoting endoplasmic reticulum stress in 4T1 cells induced by low concentrations of docetaxel, thereby enhancing cancer cell death and significantly improving the drug's anti-tumor activity. This provided a solid theoretical and practical foundation for our subsequent ideas. However, when we tried lower concentrations of DTX, we failed to achieve the desired therapeutic effect. We speculate that this may be due to the complexity of drug delivery to the tumor site and tumor heterogeneity, making it difficult for the drug to achieve its maximum efficacy.

[0005] In fact, the addition of GA only improves the therapeutic effect of chemotherapy drugs and reduces drug concentration, but does not solve the problem of improving drug toxicity. To address this issue, we need to start from two aspects: (1) improve and overcome the solubility problem of DTX; (2) improve the targeting of DTX to tumor sites. Based on this, we need to seek a biosafety-compatible nanoparticle formulation for targeted delivery of cytotoxic drugs. Among many drug carriers, pH-sensitive liposomes are the most commonly used nanocarriers in clinical practice for delivering cytotoxic drugs. Since their discovery in 1965, they have become one of the recognized preferred drug delivery carriers.

[0006] pH-sensitive liposomes are phospholipid bilayer membrane vesicles (approximately 4 nm thick) that spontaneously form in water, resembling biological membranes. Their structure is similar to that of human biological membranes, offering vast and flexible modification possibilities. Due to their amphiphilic nature, both hydrophilic and hydrophobic drugs can be loaded into liposomes. Liposomes are non-toxic, harmless, non-immunogenic, target tumor sites, highly selective, provide sustained release to prolong drug action, and are easily degraded in vivo. While stable at normal physiological pH levels, they become unstable in acidic conditions (pH 6.5-5.0) of pathological tissues (such as primary tumors, metastases, local ischemia, inflammation, and infection). This causes protonation of fatty acid carboxyl groups, forming a hexagonal structure that promotes fusion between the liposome membrane and cell membrane. The liposome is then stably internalized by the cell via endocytosis, effectively evading lysosomal isolation and degradation. This process allows the drug to accumulate at the tumor site, improving therapeutic efficacy while reducing side effects. Therefore, based on the designability and targeting of pH-sensitive liposomes, pH-sensitive liposomes that can target triple-negative breast cancer tumor sites can be designed. While retaining the property of GA peptide nanoparticles to promote DTX-induced cancer cell death, their tumor-targeting efficiency can be increased, and tumor activity can be inhibited more effectively.

[0007] A novel and effective treatment for triple-negative breast cancer involves improving the solubility of docetaxel while simultaneously killing tumor cells. This allows for specific targeting of the tumor, achieving a low-dose, high-efficiency therapeutic effect of chemotherapy while reducing side effects on other normal cells in the breast. Recent research data shows that docetaxel liposomes already exist, encapsulating docetaxel and significantly improving water solubility, eliminating the need for adding Tween 80. In vivo animal experiments have verified that this effectively improves therapeutic efficacy and significantly reduces toxic side effects. This provides data support for achieving a low-dose, high-efficiency therapeutic effect by simultaneously encapsulating docetaxel and peptide gold nanoparticle clusters in liposomes. This invention provides a novel, synthesized, pH-sensitive liposome for triple-negative breast cancer, simultaneously encapsulating peptide gold nanoparticles and the small-molecule chemotherapy drug DTX, named GA-DTX@Lipo. The GA peptide nanoparticles significantly promote DTX-induced cancer cell death in a synergistic effect, possessing the ability to target triple-negative breast cancer tumor sites, improving DTX solubility, and reducing damage to other normal germ cells and toxic side effects while enhancing therapeutic efficacy. pH-sensitive liposomes significantly enhance the antitumor killing effect of chemotherapy drugs by enabling targeted drug delivery and prolonging the drug's action time. They also significantly reduce drug concentration and toxic side effects, achieving a low-dose, high-efficiency therapeutic effect. This provides a new approach for the application of chemotherapy drugs in the treatment of triple-negative breast cancer and offers a new potential means to promote more effective and safer treatment of triple-negative breast cancer tumors in clinical practice. Summary of the Invention

[0008] This invention relates to the preparation and application of pH-sensitive liposomes simultaneously encapsulating a peptide-gold nanocluster (GA) and the chemotherapeutic drug docetaxel. This invention can be used to enhance the tumor-killing function of chemotherapeutic drugs. Furthermore, compared to peptide-gold nanoclusters and chemotherapeutic drugs alone, it can increase the targeting efficiency to lesion sites and does not produce toxic side effects on normal cells.

[0009] The technical solution adopted in this invention is as follows:

[0010] A method for preparing pH-sensitive liposomes targeting breast tumors, comprising the following steps:

[0011] Chemotherapy drugs, phospholipids, cholesterol, and PEGylated lipids were dissolved together in the organic solvent chloroform. The mixture was then subjected to vacuum rotary evaporation at a specific temperature and rotation rate. After hydration with an aqueous phase, a solution of peptide-gold nanoclusters was added and sonicated. The resulting solution was then broken up using an ultrasonic homogenizer, screened through a molecular sieve, and finally purified by dialysis and ultrafiltration to obtain pH-sensitive liposomes. During this preparation process, phospholipids spontaneously formed biomembrane-like phospholipid bilayer vesicles in water, encapsulating the chemotherapy drugs and gold nanoclusters within these vesicles.

[0012] The chemotherapy drugs mentioned include, but are not limited to, commonly used clinical chemotherapy drugs such as docetaxel (DTX), paclitaxel, and cisplatin. The peptide gold nanoclusters are glutathione gold nanoclusters (GA) with a "peptide shell" structure.

[0013] Meanwhile, the phospholipids, cholesterol, and polyethylene glycol-modified lipids used are DOPE, DPSG, and DSPE-mPEG2000, respectively.

[0014] DOPE, DPSG, and DSPE-mPEG2000 can be weighed in a molar ratio of 5-8:2-4:0.2-1 (preferably 7:3:0.6). The mass concentration of DPSG is 1 mL of chloroform for every 0.5 mg-1.5 mg (preferably 1 mg). The mass ratio of chemotherapy drugs to cholesterol is 0.1-10:1 (the specific amount can be adjusted as needed), and the mass ratio of polypeptide gold nanoclusters to cholesterol is 0.197-1.97:1 (the specific amount can be adjusted as needed).

[0015] Chemotherapy drugs, phospholipids, cholesterol, and polyethylene glycol-modified lipids were completely dissolved in chloroform and placed in a rotary evaporator for rotary evaporation. For example, the water bath temperature was set to 50°C and the rotation speed was 20 r / min until all organic solvents evaporated and a colorless and transparent film appeared on the bottle wall. Ultrapure water was added and hydration was continued for 1 hour. Finally, a GA solution of peptide nanogold clusters (1 mM, 3 mM, 4 mM, 6 mM, 8 mM, 10 mM, preferably 4 mM) was added and sonicated for 1 hour under ice bath conditions. The optimal solution was selected based on the actual synthesis.

[0016] The obtained solution was ultrasonically disrupted for 2 minutes, then screened through a 0.22 μm molecular sieve. Finally, pH-sensitive liposomes were obtained by purification methods such as dialysis and ultrafiltration. The hydrated particle size of the liposomes ranged from 30 to 170 nm. The hydrated particle size of the liposomes was generally around 68.1 nm.

[0017] The present invention utilizes pH-sensitive liposomes targeting breast tumors to specifically address pH responsiveness, which can be used to prepare tumor therapeutic drugs. In a triple-negative breast cancer tumor model, this GA-DTX@Lipo liposome enhances the tumor-killing effect of chemotherapeutic drugs while reducing toxic side effects.

[0018] The application of pH-sensitive liposomes that simultaneously encapsulate gold nanoclusters and small molecule drugs is used to prepare a targeted drug delivery system that enhances the therapeutic effect of chemotherapeutic drugs on tumors (preferably, the molar ratio of DOPE, DPSG, and DSPE-mPEG2000 is 7:3:0.6, the synthesis system is 1 mg of DPSG, then 2.6 mg of DOPE, 0.6 mg of DSPE-mPEG2000, a GA concentration of 4 mM (corresponding to a volume of 1 mL), and a DTX concentration of 5 mg / mL (added in a volume of 500 μL). After encapsulating the chemotherapeutic drugs docetaxel and gold nanoclusters, it has a targeted and synergistic therapeutic effect on breast tumors.

[0019] The application of pH-sensitive liposomes that simultaneously encapsulate gold nanoclusters and small molecule drugs aims to synergistically enhance the sensitizing effect of gold nanoclusters on the chemotherapy drug DTX in the treatment of triple-negative breast cancer, thereby improving drug targeting and therapeutic efficacy in triple-negative breast cancer.

[0020] The beneficial results of this invention are:

[0021] (1) The liposomes synthesized in this invention have good biocompatibility, are non-toxic and have diverse drug carrying capacity. The preparation steps are simple, the cost is low and the amount of reagents required is small.

[0022] (2) This invention can target tumor sites, has pH responsiveness, has the ability to release slowly and controllably, prolongs the duration of drug action, and solves the problem that current chemotherapy drugs lack effective targeted treatment methods for tumor sites.

[0023] (3) This invention improves the water solubility of docetaxel, has less toxic side effects on normal cells, and is highly safe.

[0024] (4) Compared with the existing synergistic therapy of chemotherapy drugs and peptide nanogold clusters, the present invention can also achieve targeted drug delivery, so that the drug accumulates in large quantities at the tumor site, and can effectively improve its toxic side effects on normal physiological tissues while improving the therapeutic effect.

[0025] (5) The present invention has better therapeutic effect in triple-negative breast cancer animal models. Compared with the injected chemotherapy drug DTX and nano gold cluster GA, it significantly reduces toxic side effects and makes the treatment safer and more effective. Attached Figure Description

[0026] Figure 1a The particle size distribution of Example 1 (blank liposomes) of the present invention at different system concentrations with the preferred molar ratio of DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6 is shown. The particle size of the 1 mg and 1.5 mg DPSG groups is about 58.8 nm, while the particle size of the 0.5 mg DPSG group is about 38 nm.

[0027] Figure 1b This is a particle size distribution chart of Example 1 of the present invention, showing the addition of different concentrations of GA to a system containing 1 mg of DPSG.

[0028] Figure 1c The particle size distribution is shown in Example 1 of the present invention. The particle size of the present invention is approximately 68.1 nm.

[0029] Figure 1d This is a statistical chart of particle size at different times in Embodiment 1 of the present invention;

[0030] Figure 1e This is a bar chart showing the encapsulation efficiency of Embodiment 1 of the present invention.

[0031] Figure 2a This is a particle size distribution chart of Example 1 of the present invention in buffer solutions with different pH values.

[0032] Figure 2b This invention relates to ICP-MS quantitative analysis of gold elements based on in vitro pH-responsiveness detection, as described in Example 1 of the present invention.

[0033] Figure 3a The particle size distribution is shown in Example 2 of the present invention. The particle size of the present invention is approximately 68.1 nm.

[0034] Figure 3b This is a particle size statistical diagram of Example 2 of the present invention at different times;

[0035] Figure 3c This is a bar chart showing the GA and DTX encapsulation rates in Example 2 of the present invention.

[0036] Figure 4a This is a particle size distribution chart of Example 2 of the present invention in buffer solutions with different pH values;

[0037] Figure 4b ICP-MS quantitative analysis of gold element in vitro based on pH response detection, Example 2 of this invention;

[0038] Figure 4c This is a DTX quantitative analysis by HPLC for in vitro pH-responsive detection in Example 2 of the present invention.

[0039] Figure 5a This is a line graph showing the tumor growth curve in an in vivo animal experiment according to Example 2 of the present invention;

[0040] Figure 5b This is a bar chart showing the tumor size in the animal experiments of Example 2 of the present invention;

[0041] Figure 5c This is a bar chart showing the tumor mass in an in vivo animal experiment according to Example 2 of the present invention.

[0042] Figure 5d This is a line graph showing the weight of mice throughout the entire in vivo animal experiment cycle of Example 2 of the present invention. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can understand and implement the present invention, and further recognize its advantages.

[0044] Unless otherwise defined in this specification, all technical terms herein are used according to their conventional definitions as commonly used and understood by one of ordinary skill in the art. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; and the reagents and materials described are commercially available unless otherwise specified.

[0045] This invention provides a novel method for preparing acid-responsive amphiphilic liposomes and their application in targeted therapy for nervous system tumors. Essentially, it involves the spontaneous formation of bilayer vesicles (i.e., phospholipid bilayers) from amphiphilic phospholipids and cholesterol in an aqueous solution, forming closed spherical vesicles. The addition of polyethylene glycol can enhance the long-circulation effect of the liposomes. Under certain ratios, temperatures, and gold cluster concentrations, hydrophilic polypeptide gold nanoclusters and lipophilic small molecule drug docetaxel are simultaneously encapsulated within the liposomes.

[0046] This invention provides a method for preparing pH-sensitive liposomes for targeted therapy of breast tumors to enhance the therapeutic effect of chemotherapy drugs, comprising:

[0047] DOPE, DPSG, and DSPE-mPEG2000 can be weighed in a molar ratio of 5-8:2-4:0.2-1 (preferably 7:3:0.6) to control the synthesis system. That is, if DPSG is weighed as 0.5mg-1.5mg (preferably 1mg), then DOPE should be weighed as 2.6mg and DSPE-mPEG2000 as 0.6mg. The small molecule chemotherapy drug DTX can be prepared by dissolving 1-10 mg (preferably 5 mg) in 1 mL of chloroform to prepare DTX solutions of different drug concentrations, preferably 5 mg / mL. Take 500 μL of the 5 mg / mL DTX solution, along with the weighed DOPE, DPSG, and DSPE-mPEG200, and dissolve them in 1 mL of chloroform. Sonicate for 1 min to ensure complete dissolution and mixing, then place the mixture in a round-bottom flask on a rotary evaporator. Set the water bath temperature to 50°C and the rotation speed to 20 r / min. Evaporate until the chloroform completely disappears, obtaining a colorless and transparent film. Then, add 1 mL of ultrapure water for 1 h of hydration, followed by 1 mL of GA (1-10 mM, preferably 4 mM). Sonicate for 1 h under ice bath conditions, then continue to disrupt the cell structure using a cell disruptor under ice bath conditions for 2 seconds on and 4 seconds off. Finally, filter using a 0.22 μm filter membrane. The liposome GA-DTX@Lipo is now synthesized.

[0048] Next, dialysis purification can be performed using a 100kDa dialysis bag to remove unencapsulated gold clusters and small molecule drugs. Finally, ultrafiltration (MWCO: 100kDa) is used at 5000 rpm for 30 min, followed by washing with PBS until no fluorescence is detected, yielding the purified liposomes. The DPSG mass was selected at (0.5 mg, 1 mg, 1.5 mg), with a 1 mg DPSG system being preferred. The concentration of added gold clusters was (1-10 mM), preferably 4 mM.

[0049] The liposomes prepared by the method of the present invention have a hydrated particle size of about 68.1 nm and an encapsulation efficiency of 10.4%.

[0050] The present invention provides a method for preparing pH-sensitive liposomes that simultaneously encapsulates both hydrophilic and hydrophobic drugs. This method employs a thin-film hydration process, utilizes inexpensive raw materials, and has a simple synthesis method. The resulting pH-sensitive liposomes exhibit high stability and are suitable for targeting breast tumors. This enhances the therapeutic effect of chemotherapy drugs, reduces toxic side effects, lowers drug concentration, and reduces the likelihood of drug resistance in cancer cells, making treatment safer and more effective.

[0051] The liposomes prepared in this invention can be designed and modified independently to meet the requirements of specific biomedical applications in terms of targeting, water solubility, drug encapsulation, size, and surface properties.

[0052] The pH-sensitive liposomes of the present invention have several advantages for the treatment of triple-negative breast cancer: (1) Due to their similarity to the cell membrane structure, they can protect drugs from chemical and biological degradation, prolong the drug's half-life, and improve the drug's stability and bioavailability; (2) They are amphiphilic and can simultaneously encapsulate hydrophilic and lipophilic drugs, improving drug solubility and exhibiting good biosafety, thus reducing adverse effects on the human body; (3) Compared with the combined treatment of chemotherapy drugs and nano-gold clusters, they can improve the drug's targeting and selectivity for triple-negative breast cancer and reduce toxic side effects on other normal cells.

[0053] The preparation method and application of the liposomes of the present invention will be further described below with reference to specific embodiments. In the examples, the phospholipids, cholesterol, and polyethylene glycol used are DOPE, DPSG, and DSPE-mPEG2000. The size of the synthesized liposomes was characterized using dynamic light scattering. The feasibility of this liposome for targeting triple-negative breast cancer and improving therapeutic efficacy was verified through a series of in vitro pH-responsive experiments and in vivo animal tumor model treatment experiments. Example 1: Optimization and Characterization of Liposome Preparation Conditions

[0054] 1) According to the preferred synthesis molar ratio, i.e. DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6, weigh 1 mg of DPSG, 2.6 mg of DOPE and 0.6 mg of DSPE-mPEG200 into 1.5 mL centrifuge tubes, add 1 mL of chloroform and use a vortex mixer to completely dissolve them.

[0055] 2) Transfer the mixture obtained in step (1) to a 10 mL round-bottom flask, place it on a rotary evaporator, set the water bath temperature to 50 °C, turn on the instrument, control the rotation speed to 20 r / min, adjust the position of the round-bottom flask so that the liquid inside can be completely placed in an environment of 50 °C, and rotary evaporate until the chloroform inside completely disappears and a colorless and transparent film is formed on the flask wall.

[0056] 3) Add 1 mL of ultrapure water to the round-bottom flask and hydrate for 1 h. Then turn off the rotary evaporator, add 1 mL of prepared GA (1-10 mM) of different concentrations, seal the mouth of the round-bottom flask with sealing film, sonicate for 1 h under ice bath conditions, take out the liquid in the round-bottom flask and place it in a 5 mL centrifuge tube.

[0057] 4) Continue to disrupt the cells using a cell sonicator under ice bath conditions for 2 minutes, turning it on for 2 seconds and off for 4 seconds. Finally, filter the mixture through a 0.22 μm filter membrane. The liposome synthesis is now complete.

[0058] Unencapsulated gold nanoclusters were removed by dialysis using a dialysis bag (MWCO: 100 kDa) and ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. Dynamic light scattering (DLS) analysis revealed that the particle size of liposomes in different systems was not entirely consistent. Blank liposomes were synthesized according to the optimized molar ratio of DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6. The liposome synthesis system was first optimized, such as... Figure 1a As shown, according to the order of DPSG at different masses of 0.5 mg, 1 mg, and 1.5 mg (while maintaining the ratio of DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6), the particle sizes were approximately 37.8 nm, 58.8 nm, and 58.8 nm, respectively. It was found that the particle size distributions of the 1 mg and 1.5 mg DPSG groups had a high overlap rate and similar sizes; therefore, the 1 mg DPSG system was selected. After determining the 1 mg DPSG system (i.e., 2.6 mg DOPE and 0.6 mg DSPE-mPEG200), we began to experiment with incorporating different concentrations of gold nanoclusters GA (1–10 mM) to synthesize liposomes GA@Lipo, and screened for the optimal concentration for incorporating gold nanoclusters, such as... Figure 1b As shown, the particle size distribution of GA remained stable and the particle size was suitable at concentrations of 4 mM, 6 mM, and 10 mM, all around 68.1 nm. 4 mM GA was chosen to conserve materials. Dynamic light scattering (DLS) was used to detect the particle size of the prepared 1 mg DPSG system containing 4 mM GA-encapsulated liposomes GA@Lipo. Figure 1c The particle size shown is approximately 68.1 nm. Next, liposomes containing 1 mg of DPSG and 4 mM of GA (GA@Lipo) were used. Dynamic light scattering (DLS) was used to measure the particle size at different times after being placed at 4°C to assess their stability. It was found that the particle size remained essentially unchanged after 24 h and 48 h. Figure 1d This demonstrated its stability. The encapsulation efficiency of the GA@Lipo liposome was then determined using ultracentrifugation at 200,000 r / min for 1 h. Free drug and liposomes were separated, and equal volumes of the supernatant from the centrifuged liposomes and the uncentrifuged liposomes were taken. The gold content was measured by ICP-MS, representing the content of free (unencapsulated) drug and the total drug content in the liposomes, respectively. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = 1 - (Unencapsulated drug in liposomes / Total drug in liposomes) × 100%. The final GA concentration of GA@Lipo was found to be 0.28 mM, and its encapsulation efficiency was calculated to be 10.4%.

[0059] Example 2: In vitro detection of pH responsiveness of GA@Lipo

[0060] To verify the pH responsiveness of liposomes in vitro, 50 μL of purified GA@Lipo liposomes obtained in Example 1, containing 1 mg of DPSG and 4 μmol of GA, were taken according to the preferred molar ratio (DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6), and added to 1 mL of buffer solutions with pH values ​​of 4.5, 6.5, and 7.4, respectively. The solutions were then placed in a 37°C oven for 24 h. The particle size change was first measured using a dynamic light scattering instrument. Figure 2a As shown, the particle size remained essentially unchanged under neutral conditions (pH 7.4), but increased or decreased in size under acidic conditions. This is likely due to the rupture and aggregation of liposomes, verifying its pH-responsiveness. Furthermore, measuring the Au content in the supernatant of the pH buffer solution at different time points using inductively coupled plasma mass spectrometry (ICP-MS) further confirms its pH-responsive release. Figure 2b As shown, under acidic conditions, the gold content exhibited a time-dependent effect, gradually increasing over time, demonstrating the controlled release capability of the liposomes. Meanwhile, under neutral conditions (pH 7.4), the Au content in the supernatant remained essentially unchanged, further validating the pH responsiveness of the liposome GA@Lipo.

[0061] Example 3: Synthesis and Characterization of GA-DTX@Lipo

[0062] For liposomes synthesized using a preferred molar ratio of DOPE:DPSG:DSPE-mPEG200 = 7:3:0.6, 1 mg of DPSG was selected and 4 μmol of GA was incorporated into the liposomes. Further attempts were made to incorporate the small molecule chemotherapy drug docetaxel (DTX). 5 mg of the optimized DTX (1–10 mg) was weighed and completely dissolved in 1 mL of chloroform. 500 μL of this solution was added to 1 mL of chloroform along with DOPE, DPSG, and DSPE-mPEG2000 (DOPE:DPSG:DSPE-mPEG200 molar ratio = 7:3:0.6). Subsequent experimental steps were performed consistent with the liposome synthesis method to obtain GA-DTX@Lipo. The particle size of the prepared GA-DTX@Lipo was determined using dynamic light scattering (DLS). Figure 3a The particle size shown is approximately 68.1 nm. Next, dynamic light scattering (DLS) was used to measure the particle size of the liposomes at different times to determine their stability. It was found that the particle size remained essentially unchanged after 24 h and 48 h. Figure 3bThe stability of the liposomes was demonstrated. The encapsulation efficiency of the liposomes was determined using ultracentrifugation. The centrifugation speed was set at 200,000 r / min for 1 h, separating the free drug and liposomes. Equal volumes of the supernatant from the centrifuged liposomes and the uncentrifuged liposomes were taken, and the Au content was measured by ICP-MS, representing the content of free (unencapsulated) drug and the total drug content in the liposomes, respectively. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = 1 - (Unencapsulated drug in liposomes / Total drug in liposomes) × 100%. The concentration of GA in GA-DTX@Lipo was found to be 0.23 mM, indicating a GA encapsulation efficiency of 11.5%. Furthermore, the concentration of DTX in the liposomes was detected using high-performance liquid chromatography (HPLC), finding to be 0.175 mg / mL, and the DTX encapsulation efficiency of GA-DTX@Lipo was calculated to be 14%.

[0063] Example 4: In vitro detection of pH responsiveness of GA-DTX@Lipo

[0064] To verify the pH responsiveness of liposomes in vitro, 50 μL of the purified liposome GA-DTX@Lipo obtained in Example 3 was added to 1 mL of buffer solutions with pH values ​​of 4.5, 6.5, and 7.4, respectively. The solutions were then placed in a 37°C oven for 24 h. The particle size change was first measured using a dynamic light scattering instrument. Figure 4a As shown, the particle size remained essentially unchanged under neutral conditions (pH 7.4), but increased or decreased in size under acidic conditions. This is likely due to liposome rupture and aggregation, verifying its pH responsiveness. Furthermore, measuring the Au content in the supernatant of the pH buffer solution at different time points using inductively coupled plasma mass spectrometry (ICP-MS) further confirms its pH-responsive release. Similar to the results from GA@Lipo, as... Figure 4b As shown, under acidic conditions, the gold content exhibited a time-dependent effect, gradually increasing over time, demonstrating the controlled-release capability of the liposomes. Meanwhile, at pH 7.4, the Au content in the supernatant remained essentially unchanged, further validating the pH-responsiveness of the liposome GA-DTX@Lipo.

[0065] Furthermore, the DTX content in buffer solutions at different pH values ​​at different time points was detected using a high-resolution liquid chromatography column, and the results were similar to those for Au content. Figure 4c As shown, the DTX content was found to be time-dependent, with the drug concentration gradually increasing over time, which also demonstrates the controlled-release capability of the liposomes. Furthermore, it was found that the DTX content was significantly higher under acidic conditions than under neutral conditions, further demonstrating the pH-responsiveness of the liposomes.

[0066] Example 5: The therapeutic effect of GA-DTX@Lipo on triple-negative breast cancer in an in vivo tumor model.

[0067] Based on the fact that GA enhances the killing effect of the chemotherapy drug DTX on triple-negative breast cancer cells, we further investigated the therapeutic effect of GA-DTX@Lipo liposomes on a triple-negative breast cancer tumor model. 6 4T1 cells were subcutaneously seeded into the mammary glands of mice and divided into three groups (Saline, GA+DTX, and GA-DTX@Lipo) for treatment. The GA+DTX group (with separate additions of GA and DTX) received GA every two days and DTX every four days. The GA-DTX@Lipo group (synthesized in Example 3) received DTX every four days via intraperitoneal injection. Tumor growth at the inoculation site was observed daily, and mouse weight was measured and tumor volume recorded using calipers. 肿瘤 (mm 3 = 0.5 x length x width 2 .

[0068] Figures 5a-5c The images show tumor growth curves, tumor volume bar charts, and tumor mass bar charts, respectively. It can be seen that the liposome GA-DTX@Lipo (200 μL per injection) has similar therapeutic effects to GA (10 mg / kg per injection) + DTX (15 mg / kg per injection). Both can reduce tumor growth volume and mass, and have anti-tumor activity. In triple-negative breast cancer tumor animal models, it can exert a tumor-killing effect at low drug concentrations.

[0069] Figure 5d The figures represent mouse body weight. It can be seen that compared with the DTX+GA injection group, the daily body weight of mice in the GA-DTX@Lipo group did not decrease significantly, indicating that the GA-DTX@Lipo of this invention has good biocompatibility, achieves targeted therapy, and can effectively improve the toxic side effects of drugs.

Claims

1. A method for preparing pH-sensitive liposomes targeting breast tumors, characterized in that, A method for preparing pH-sensitive liposomes capable of targeting breast tumors includes the following steps: Chemotherapy drugs, phospholipids, cholesterol, and PEGylated lipids were dissolved together in the organic solvent chloroform. The mixture was then subjected to vacuum rotary evaporation at a specific temperature and rotation rate. After hydration with an aqueous phase, a solution of peptide-gold nanoclusters was added and sonicated. The resulting solution was then broken up using an ultrasonic homogenizer, screened through a molecular sieve, and finally purified by dialysis and ultrafiltration to obtain pH-sensitive liposomes. During this preparation process, phospholipids spontaneously formed biomembrane-like phospholipid bilayer vesicles in water, encapsulating the chemotherapy drugs and gold nanoclusters within these vesicles.

2. A method for preparing pH-sensitive liposomes targeting breast tumors according to claim 1, characterized in that, The chemotherapy drugs mentioned include docetaxel (DTX), paclitaxel, and cisplatin, and the peptide gold nanoclusters are glutathione gold nanoclusters with a "peptide shell" structure.

3. A method for preparing pH-sensitive liposomes targeting breast tumors according to claim 1, characterized in that, Phospholipids, cholesterol, and PEGylated lipids are DOPE, DPSG, and DSPE-mPEG2000, respectively.

4. A method for preparing pH-sensitive liposomes targeting breast tumors according to claim 1, characterized in that, Phospholipids, cholesterol, and polyethylene glycol-modified lipids are weighed in a molar ratio of 5–8:2–4:0.2–1 (preferably 7:3:0.6), and the mass concentration of DPSG is 1 mL of chloroform for every 0.5 mg–1.5 mg (preferably 1 mg).

5. A method for preparing pH-sensitive liposomes targeting breast tumors according to claim 1, characterized in that, The mass ratio of chemotherapy drugs to cholesterol is 0.1–10:1, and the mass ratio of polypeptide gold nanoclusters to cholesterol is 0.197–1.97:

1.

6. A method for preparing pH-sensitive liposomes targeting breast tumors according to claim 1, characterized in that, Specifically, chemotherapy drugs, phospholipids, cholesterol, and PEGylated lipids were completely dissolved in chloroform and placed in a rotary evaporator for rotary evaporation until all organic solvents evaporated and a colorless, transparent film appeared on the flask wall. Ultrapure water was added for further hydration for 1 hour. Finally, a peptide nanoparticle gold cluster GA solution was added and sonicated in an ice bath for 1 hour. The resulting solution was then sonicated for 2 minutes, screened through a 0.22 μm molecular sieve, and finally purified by dialysis, ultrafiltration, and other methods to obtain pH-sensitive liposomes GA-DTX@Lipo.

7. pH-sensitive liposomes targeting breast tumors are prepared according to the method of any one of claims 1-6.

8. The application of pH-sensitive liposomes targeting breast tumors prepared by the method according to any one of claims 1-6, for the preparation of tumor therapeutic drugs.

9. The application according to claim 8, in the preparation of a drug for treating triple-negative breast cancer tumors.

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