An arsenical liposome gel drug delivery system, and a preparation method and application thereof

Abstract

The present application relates to an arsenical liposome gel drug delivery system and its preparation method and application, and belongs to the technical field of biological medicine. In order to solve the problems of unsafe carrier, single function, drug resistance and insufficient microenvironment response of ATO in the local treatment of TBC cancer wound, the present application provides an arsenical liposome gel drug delivery system, which comprises 20(S)-protopanaxadiol-based intelligent gel and folate-modified ATO / cGAMP co-loaded liposomes dispersed therein, and constructs an injectable in-situ gel reservoir composite pH / GSH double-response folate-targeted liposome double-layer drug delivery architecture. The intratumoral injection method is used for intervention treatment, forming a multiple synergistic treatment effect of metabolic sensitization preconditioning + targeted drug release killing + immune microenvironment remodeling. The outer gel pretreatment and the inner liposome precise drug release timing coordination, function complementation, completely break through the limitation of single drug treatment, realize the integrated synergistic treatment of antitumor and wound repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an arsenic liposome gel drug delivery system, its preparation method and application. Background Technology

[0002] Skin ulceration and other cancerous wounds caused by triple-negative breast cancer (TNBC) are serious complications of advanced-stage tumors. These wounds possess a unique and complex pathological microenvironment, primarily characterized by a weakly acidic environment with a pH of 5.5–6.5, high glutathione levels (2–10 times higher than normal tissue), and chronic inflammation accompanied by an immunosuppressive state characterized by massive infiltration of M2 macrophages and regulatory T cells. Current mainstream clinical treatments include wound debridement, routine dressing changes, and local chemotherapy. However, these approaches generally suffer from limitations such as insufficient drug penetration depth, short local retention time, and inability to precisely respond to drug release, failing to adapt to the specific microenvironment of cancerous wounds and resulting in limited clinical efficacy.

[0003] Arsenic trioxide (ATO) is a classic antitumor drug used clinically to treat acute promyelocytic leukemia (TNBC). Recent studies have confirmed that it can effectively inhibit TNBC progression by inducing tumor immunogenic cell death, showing promising potential in the treatment of TNBC-related cancerous lesions. However, existing local delivery systems for ATO primarily use albumin or metal ions such as iron and manganese as carriers to achieve stable drug encapsulation and microenvironment-responsive release. Exogenous albumin easily induces immune rejection, and metal ions are prone to metabolic accumulation toxicity and trigger persistent local inflammation. These pose safety risks for cancerous lesion treatment scenarios requiring long-term, repeated administration, hindering clinical translation.

[0004] Furthermore, arsenic trioxide struggles to overcome metabolic and immune resistance in TNBC (tumor nephrotic syndrome) lesions, limiting overall treatment efficacy. Attempts to combine arsenic trioxide with metabolic inhibitors and immune agonists often involve simple physical mixing of drugs, lacking precise synergistic design and failing to simultaneously and effectively address tumor resistance phenotypes. This results in weak synergistic effects and poor system stability. Moreover, existing arsenic trioxide delivery formulations are functionally limited, only exhibiting tumor cell killing effects and failing to effectively regulate wound inflammation, inhibit abnormal angiogenesis, or promote wound tissue repair. Simultaneously, the sustained cytotoxicity of these formulations can damage normal wound repair cells and delay wound healing, making it difficult to simultaneously address the dual needs of anti-tumor therapy and wound repair.

[0005] Currently, most liposomes and hydrogel formulations used in cancerous wounds are designed for single-environment response. Even the few formulations with dual-response capabilities are difficult to synergistically respond to the unique weak acid and high glutathione dual microenvironment signals of TNBC wounds, and cannot simultaneously meet the therapeutic needs of deep targeted drug penetration and long-term local retention.

[0006] In summary, current treatment of TNBC cancerous lesions faces multiple technical challenges, including a mismatch between carrier safety and therapeutic functionality, a conflict between antitumor effects and wound repair, difficulty in simultaneously addressing metabolic and immune drug resistance, and a challenge in balancing drug penetration and long-term retention. There is an urgent need for a metal-free, exogenous protein-free, and locally intelligent arsenic trioxide drug delivery system that can synergistically solve these multiple problems and achieve multi-mechanism synergistic effects. Summary of the Invention

[0007] To address the problems of unsafe carriers, limited functionality, drug resistance, and insufficient microenvironment response in the local treatment of TNBC cancerous lesions with existing ATO, this invention provides an arsenic liposome gel drug delivery system, its preparation method, and its application.

[0008] The technical solution of the present invention:

[0009] An arsenic-based liposome gel delivery system comprises a 20(S)-protopanaxadiol-based smart gel and folic acid-modified ATO / cGAMP co-loaded liposomes dispersed therein, wherein the mass ratio of the folic acid-modified ATO / cGAMP co-loaded liposomes to the 20(S)-protopanaxadiol-based smart gel is 1.5~2.5:100.

[0010] The 20(S)-protopanaxadiol-based smart gel contains the following components in parts by weight: 60-140 parts of 20(S)-protopanaxadiol, 60-140 parts of dioleoylphosphatidylglycerol, 2.5-10 parts of quercetin, and 0.5-2.5 parts of cGAMP;

[0011] When preparing the folic acid-modified ATO / cGAMP co-loaded liposomes, the feed contains the following components in parts by weight: 2-5 parts ATO, 1-4 parts cGAMP, and 80-98 parts lipid membrane material; the lipid membrane material is composed of hydrogenated soybean lecithin, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in a mass ratio of 70-80:5-15:10-20.

[0012] Furthermore, the mass ratio of the folic acid-modified ATO / cGAMP co-loaded liposomes to the 20(S)-protopanaxadiol-based smart gel is 2:100;

[0013] The 20(S)-protopanaxadiol-based smart gel contains the following components in parts by weight: 100 parts 20(S)-protopanaxadiol, 100 parts dioleoylphosphatidylglycerol, 5 parts quercetin and 1.25 parts cGAMP;

[0014] When preparing the folic acid-modified ATO / cGAMP co-loaded liposomes, the feed contains the following components in parts by weight: 4 parts ATO, 2 parts cGAMP and 94 parts lipid membrane material; the lipid membrane material is composed of hydrogenated soybean phospholipids, cholesterol hemisuccinate and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in a mass ratio of 75:10:15.

[0015] Furthermore, the particle size of the folic acid-modified ATO / cGAMP co-loaded liposomes is 100~150 nm.

[0016] A method for preparing an arsenic-based liposome gel drug delivery system includes the following steps:

[0017] Step 1: Preparation of 20(S)-Protopanaxadiol-based smart gel:

[0018] 20(S)-Protopanaxadiol and dioleoylphosphatidylglycerol were dissolved in an organic solvent, rotary evaporated to form a film, and then vacuum dried. A hydration medium containing quercetin, cGAMP and calcium chloride was added, and the mixture was ultrasonically treated and cooled to obtain 20(S)-Protopanaxadiol-based smart gel.

[0019] Step 2: Preparation of folic acid-modified ATO / cGAMP co-loaded liposomes:

[0020] Hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine were dissolved in an organic solvent, rotary evaporated to form a membrane, added a buffer containing ATO and cGAMP for hydration, ultrasonically dispersed, and extruded through a polycarbonate membrane to obtain folic acid-modified ATO / cGAMP co-loaded liposomes.

[0021] Step 3: Assemble the arsenic liposome gel drug delivery system:

[0022] The folic acid-modified ATO / cGAMP co-loaded liposomes obtained in step two are added to the 20(S)-protopanaxadiol-based smart gel obtained in step one, and stirred and dispersed to obtain the arsenic liposome gel drug delivery system.

[0023] Furthermore, the organic solvents mentioned in steps one and two are both mixed solvents of chloroform and methanol, with a volume ratio of chloroform to methanol of 2:1.

[0024] Furthermore, the rotary evaporation temperature in step one is 42~48℃, the vacuum drying time is 4~5h, the ultrasonic temperature is 42~48℃, the ultrasonic power is 200~300W, the ultrasonic time is 6~10min, and the concentration of calcium chloride in the hydration medium is 0.5~2mM.

[0025] Furthermore, in step two, the rotary evaporation temperature is 35~45℃, the ultrasonic dispersion is carried out at an ultrasonic power of 200~300W for 5~10 minutes, and the pore sizes of the polycarbonate membranes through which the extrusion passes are 400nm, 200nm and 100nm respectively, with each extrusion performed 3~5 times.

[0026] Furthermore, in step three, the stirring speed is 150~250 rpm, the time is 30~60 min, and the temperature is 4℃.

[0027] Application of an arsenic-based liposome gel drug delivery system in the preparation of a drug for treating cancerous lesions in triple-negative breast cancer.

[0028] Furthermore, the drug is an intratumoral injection preparation.

[0029] The beneficial effects of this invention are:

[0030] The arsenic-based liposome gel drug delivery system provided by this invention constructs an injectable in-situ gel reservoir combined with a pH / GSH dual-responsive folic acid-targeting liposome bilayer drug delivery architecture. Utilizing a layered carrier, it achieves spatial partitioning and temporal stepwise release of different active drugs. Addressing the shortcomings of existing cancer wound treatments, which are limited to topical application, insufficient drug penetration depth, and short local retention time, this invention breaks through traditional wound treatment approaches by innovatively applying this composite drug delivery formulation to intratumoral injection. Through in-situ gel formation, a long-lasting local drug reservoir is constructed, inhibiting the proliferation of solid tumors at the source and blocking tumor tissue invasion of the skin, establishing a new treatment logic of "treating the tumor is healing the wound."

[0031] Following intratumoral injection, the 20(S)-protopanaxadiol-based smart gel initially and continuously releases the metabolic sensitizer quercetin and the immune activator cGAMP. Western blot analysis showed a significant downregulation of GLUT1 expression, achieving "pre-feeding" metabolic sensitization. Simultaneously, cGAMP pre-activates the local STING pathway. Subsequently, folic acid-modified liposomes respond to the weakly acidic and high-GSH microenvironment within the tumor, explosively releasing the chemotherapeutic agents ATO and cGAMP. In vitro release showed a release rate of less than 20% under normal physiological conditions at 48 hours, reaching 55-65% in the intratumoral microenvironment, achieving "post-detonation" precise killing and immune hyperactivation. Multiplex immunofluorescence assays showed CD4+. + and CD8 + The significant increase in T cell infiltration and the decrease in the proportion of Tregs demonstrate that the immunosuppressive microenvironment was effectively reversed.

[0032] This system achieves a multi-faceted synergistic therapeutic effect through "metabolic sensitization and pre-regulation + targeted drug delivery and killing + immune microenvironment remodeling." The outer gel pretreatment and the inner liposome precise drug delivery are coordinated in timing and complement each other, completely breaking through the limitations of single-drug therapy and achieving integrated synergistic treatment for anti-tumor and wound repair. In vivo efficacy results in animals confirmed that after intratumoral intervention with this preparation, the skin ulceration symptoms at the primary tumor site in mice were significantly improved; pathological observation of wound tissue sections showed a significantly accelerated re-epithelialization process and a significant increase in epidermal tissue thickness.

[0033] This invention uses pharmaceutical-grade 20(S)-protopanaxadiol as the gel matrix framework, completely independent of protein and metal excipients, thus avoiding the immunogenicity and cumulative toxic side effects caused by carriers at the raw material level. In vivo organ safety and pathological testing results showed that no inflammatory infiltration, cell necrosis, or other pathological damage was observed in mouse heart and kidney tissue sections after complete administration, confirming the excellent safety of the formulation for systemic administration.

[0034] The arsenic liposome gel drug delivery system of this invention has a simple and easy-to-control preparation process and a low threshold for industrial scale-up. It has extremely high translational and application value in the field of clinical local targeted therapy for triple-negative breast cancer with cancerous lesions. Attached Figure Description

[0035] Figure 1 This is a photograph of the actual appearance of the AsLIP@PPD-Gel precursor fluid in Example 4;

[0036] Figure 2 The image shows a SEM image of AsLIP@PPD-Gel after gelation under physiological conditions in Example 4. The icon bar is 200 nm.

[0037] Figure 3 The images shown are: A) is an in-situ gelation observation of the AsLIP@PPD-Gel precursor liquid droplet added to a glass slide at room temperature; B) is an in-situ gelation observation of the AsLIP@PPD-Gel precursor liquid squeezed out by a medical syringe needle.

[0038] Figure 4 This is a hydration particle size distribution curve of AsLIP liposomes in Example 4;

[0039] Figure 5 The image shows the Zeta potential test spectrum of AsLIP liposomes in Example 4;

[0040] Figure 6 This is a comparison of the in vitro cumulative release curves of arsenic drugs from the AsLIP@PPD-Gel solid gel under different pH and GSH conditions in Example 4.

[0041] Figure 7 The image shows the XPS binding energy spectrum of arsenic in the AsLIP sample from Example 4.

[0042] Figure 8 The following are rheological property characterization diagrams of AsLIP@PPD-Gel in Example 4: A is the strain scan spectrum, B is the temperature scan spectrum, C is the frequency scan spectrum, and D is the multi-cycle strain thixotropic recovery curve.

[0043] Figure 9 The images show macroscopic characterization of tumor growth in two groups of TNBC-bearing mice in Example 5. A is an ex vivo photograph of the tumor, and B is a curve showing the change in tumor volume during the treatment period.

[0044] Figure 10 The images show the immunohistochemical characterization of tumor tissues from two groups of TNBC-bearing mice in Example 5. A is the immunohistochemical micrograph of Caspase-3 and Ki67 in the tumor tissue, B is the comparison of the average optical density of Caspase-3 immunohistochemistry, and C is the comparison of the average optical density of Ki67 immunohistochemistry.

[0045] Figure 11 These are characterization images of cancerous ulceration repair in two groups of TNBC-bearing mice in Example 5. A is a macroscopic photograph of the ulceration on the body surface of the tumor-bearing mice, and B is a pathological micrograph of the skin tissue at the ulceration site stained with HE.

[0046] Figure 12 The images show the characterization of glucose metabolism, STING pathway, and pyroptosis protein expression in two groups of TNBC tumor-bearing mice in Example 5. A is the Western blot electrophoresis pattern of GLUT1, STING, p-STING, and GSDME-N proteins; B is the comparison of the relative expression levels of GLUT1 protein; C is the comparison of the relative expression levels of phosphorylated p-STING protein; and D is the comparison of the relative expression levels of GSDME-N protein.

[0047] Figure 13 This is a characterization diagram of the T lymphocyte immune microenvironment in two groups of TNBC-bearing mice in Example 5. A represents CD4+ in tumor tissue. + CD8 + FOXP3 + And multiplex immunofluorescence staining overlay image, B represents CD4. + CD8 + FOXP3 + Comparison chart of the percentage of positive immune cells;

[0048] Figure 14 The images show HE staining images of the heart and kidney of two groups of TNBC tumor-bearing mice in Example 5. A is a panoramic and magnified microscopic image of HE staining of the heart tissue, and B is a panoramic and magnified microscopic image of HE staining of the kidney tissue. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0050] Student's t-test was used to analyze differences between groups. All experiments were performed in triplicate, and data are expressed as mean ± SD. This indicates that the difference is statistically significant. This indicates that the difference is statistically significant. This indicates that the difference is statistically significant.

[0051] Example 1

[0052] This embodiment provides a method for preparing an arsenic liposome gel delivery system (AsLIP@PPD-Gel).

[0053] The raw materials and their sources used in this embodiment are as follows:

[0054] Arsenic trioxide injection was purchased from Harbin Medical University Pharmaceutical Co., Ltd. (Harbin, China), with a specification of 1 mg / mL. 20(S)-Protopanaxadiol (20(S)-PPD), dioleoylphosphatidylglycerol (DOPG), hydrogenated soybean lecithin (HSPC), cholesterol hemisuccinate (CHEMS), and folic acid-PEG-DSPE were all pharmaceutical grade reagents. Quercetin, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), and other organic solvents were all analytical grade.

[0055] The preparation method of the arsenic liposome gel drug delivery system in this embodiment is as follows:

[0056] Step 1: Preparation of 20(S)-Protopanaxadiol-based Smart Gel PPD-Gel:

[0057] Weigh 100 mg of 20(S)-protopanaxadiol and 100 mg of dioleoylphosphatidylglycerol, dissolve them in 5 mL of chloroform-methanol mixed solvent (chloroform to methanol volume ratio 2:1), place in a round-bottom flask, and remove the organic solvent by rotary evaporation at 45 °C to form a uniform film. Continue vacuum drying for 4 h. Add 5 mL of physiological saline containing quercetin, cGAMP, and CaCl2. The concentration of quercetin in the physiological saline is 1 mg / mL, the concentration of cGAMP is 0.25 mg / mL, and the concentration of CaCl2 is 1 mM. Sonicate in a water bath at 45 °C for 300 W for 8 min, cool to room temperature, and a light yellow paste-like gel is formed, which is the 20(S)-protopanaxadiol-based smart gel PPD-Gel.

[0058] Step 2: Prepare folic acid-modified ATO / cGAMP co-loaded liposomes (AsLIP) using a thin-film dispersion-extrusion method:

[0059] The membrane material, weighed according to the mass ratio of hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine (75:10:15), with a total lipid content of 94 mg, was dissolved in 10 mL of a chloroform-methanol mixed solvent (chloroform to methanol volume ratio 2:1). The solution was placed in a round-bottom flask and evaporated in a 40°C water bath using rotary evaporation. The membrane was then vacuum-dried overnight. Hydration was achieved by adding 5 mL of pH 7.4 phosphate buffer containing 4 mg ATO and 2 mg cGAMP. After ultrasonic dispersion at 300 W for 5 min, the membrane was sequentially extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes three times each, yielding folic acid-modified ATO / cGAMP co-loaded liposomes (AsLIP) with an average particle size of approximately 120 nm.

[0060] The encapsulation efficiency of arsenic trioxide was determined to be 78% by inductively coupled plasma mass spectrometry (ICP-MS), and the encapsulation efficiency of cGAMP was determined to be 55% by high performance liquid chromatography (HPLC).

[0061] Step 3: Assemble the arsenic liposome gel delivery system (AsLIP@PPD-Gel):

[0062] Take 1 mL of suspension containing 20 mg of folic acid-modified liposomes obtained in step 2, and slowly add it to 1 g of 20(S)-protopanaxadiol-based smart gel prepared in step 1 at 4 °C. Stir at 200 rpm for 45 min to uniformly disperse the liposomes in the gel matrix, thus obtaining the arsenic liposome gel delivery system (AsLIP@PPD-Gel).

[0063] Example 2

[0064] This embodiment provides a method for preparing an arsenic liposome gel drug delivery system.

[0065] The raw materials and sources used in this embodiment are the same as in Example 1. The preparation method of the arsenic liposome gel drug delivery system is as follows:

[0066] Step 1: Preparation of 20(S)-Protopanaxadiol-based smart gel:

[0067] Weigh 60 mg of 20(S)-protopanaxadiol and 140 mg of dioleoylphosphatidylglycerol, dissolve them in 5 mL of chloroform-methanol mixed solvent (chloroform to methanol volume ratio 2:1), place in a round-bottom flask, and remove the organic solvent by rotary evaporation at 42 °C to form a uniform film. Continue vacuum drying for 4 h. Add 5 mL of physiological saline containing quercetin, cGAMP, and CaCl2. The concentration of quercetin in the physiological saline is 0.5 mg / mL, the concentration of cGAMP is 0.1 mg / mL, and the concentration of CaCl2 is 0.5 mM. Sonicate in a water bath at 42 °C for 300 W for 10 min, cool to room temperature, and a brownish-yellow paste-like gel is formed, which is the 20(S)-protopanaxadiol-based smart gel.

[0068] Step 2: Prepare folic acid-modified ATO / cGAMP co-loaded liposomes using a thin-film dispersion-extrusion method:

[0069] The membrane material, weighed at a mass ratio of hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine of 70:15:10, with a total lipid content of 97 mg, was dissolved in 10 mL of a chloroform-methanol mixed solvent (chloroform to methanol volume ratio of 2:1). The solution was placed in a round-bottom flask and evaporated in a water bath at 45°C to form a membrane. The membrane was then vacuum-dried overnight. Hydration was achieved by adding 5 mL of pH 7.4 phosphate buffer containing 2 mg ATO and 1 mg cGAMP. After ultrasonic dispersion at 300 W for 10 min, the membrane was sequentially extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes three times each, yielding folic acid-modified ATO / cGAMP co-loaded liposomes (AsLIP) with a particle size of approximately 120 nm.

[0070] The encapsulation efficiency of arsenic trioxide was determined to be 72% by inductively coupled plasma mass spectrometry (ICP-MS), and the encapsulation efficiency of cGAMP was determined to be 48% by high performance liquid chromatography (HPLC).

[0071] Step 3: Assemble the arsenic liposome gel drug delivery system:

[0072] Take 1 mL of suspension containing 15 mg of folic acid-modified liposomes obtained in step two, and slowly add it to 1 g of 20(S)-protopanaxadiol-based smart gel prepared in step one at 4 °C. Stir at 150 rpm for 60 min to uniformly disperse the liposomes in the gel matrix, thus obtaining the arsenic liposome gel drug delivery system.

[0073] Example 3

[0074] This embodiment provides a method for preparing an arsenic liposome gel drug delivery system.

[0075] The raw materials and sources used in this embodiment are the same as in Example 1. The preparation method of the arsenic liposome gel drug delivery system is as follows:

[0076] Step 1: Preparation of 20(S)-Protopanaxadiol-based smart gel:

[0077] Weigh 140 mg of 20(S)-protopanaxadiol and 60 mg of dioleoylphosphatidylglycerol, dissolve them in 5 mL of chloroform-methanol mixed solvent (chloroform to methanol volume ratio 2:1), place in a round-bottom flask, and remove the organic solvent by rotary evaporation at 48 °C to form a uniform film. Continue vacuum drying for 4 h. Add 5 mL of physiological saline containing quercetin, cGAMP, and CaCl2. The concentration of quercetin in the physiological saline is 2 mg / mL, the concentration of cGAMP is 0.5 mg / mL, and the concentration of CaCl2 is 2 mM. Sonicate in a water bath at 48 °C for 300 W for 6 min, cool to room temperature, and a brownish-yellow paste-like gel is formed, which is the 20(S)-protopanaxadiol-based smart gel.

[0078] Step 2: Prepare folic acid-modified ATO / cGAMP co-loaded liposomes using a thin-film dispersion-extrusion method:

[0079] The membrane material, weighed at a mass ratio of hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine of 75:15:20, with a total lipid content of 92 mg, was dissolved in 10 mL of a chloroform-methanol mixed solvent (chloroform to methanol volume ratio of 2:1). The solution was placed in a round-bottom flask and evaporated in a water bath at 45°C to form a membrane. The membrane was then vacuum-dried overnight. Hydration was achieved by adding 5 mL of pH 7.4 phosphate buffer containing 5 mg ATO and 3 mg cGAMP. After ultrasonic dispersion at 300 W for 5 min, the membrane was sequentially extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes three times each to obtain folic acid-modified ATO / cGAMP co-loaded liposomes with a particle size of approximately 120 nm.

[0080] The encapsulation efficiency of arsenic trioxide was determined to be 82% by inductively coupled plasma mass spectrometry (ICP-MS), and the encapsulation efficiency of cGAMP was determined to be 60% by high performance liquid chromatography (HPLC).

[0081] Step 3: Assemble the arsenic liposome gel drug delivery system:

[0082] Take 1 mL of suspension containing 25 mg of folic acid-modified liposomes obtained in step two, and slowly add it to 1 g of 20(S)-protopanaxadiol-based smart gel prepared in step one at 4 °C. Stir at 250 rpm for 30 min to uniformly disperse the liposomes in the gel matrix, thus obtaining the arsenic liposome gel drug delivery system.

[0083] Example 4

[0084] This embodiment uses the arsenic liposome gel delivery system AsLIP@PPD-Gel prepared in Example 1 as the research object to carry out macro / micro morphology of the formulation, injectability and in vitro gelation properties, low temperature stability observation, physicochemical property characterization and rheological property investigation.

[0085] I. Macroscopic and Microscopic Morphological Observation

[0086] Using AsLIP@PPD-Gel prepared in Example 1 as the precursor solution, such as Figure 1 As shown, the AsLIP@PPD-Gel precursor solution is a uniform pale yellow solution with a stable and homogeneous texture. There is no obvious precipitation, layering, flocculation, or visible insoluble particles, indicating that the gel precursor has good dispersibility and excellent homogeneous stability after preparation.

[0087] After the AsLIP@PPD-Gel precursor solution was induced to form an in situ gel in a 37°C aqueous physiological simulation environment, it was observed under a scanning electron microscope. Figure 2 As shown, dispersed spherical AsLIP liposome nanoparticles can be clearly observed in the gel matrix. The liposomes have regular morphology and are well dispersed in the gel substrate without obvious aggregation or agglomeration. This indicates that the gel matrix can effectively immobilize the loaded lipid nanocarriers, preventing the nanoparticles from agglomerating and settling, and providing structural protection for in-situ long-term drug delivery.

[0088] II. Observation of Injectable and In Vitro Gel Forming Properties

[0089] The in vitro injectability of AsLIP@PPD-Gel precursor solution was tested using a standard 1mL sterile medical syringe. Figure 3 As shown, the AsLIP@PPD-Gel precursor solution has good fluidity and injectability. It can be smoothly injected with a conventional syringe without significant resistance. The solution can be quickly solidified into a gel mass when dropped onto a glass slide at room temperature.

[0090] III. Low Temperature Stability Observation

[0091] The AsLIP@PPD-Gel precursor solution was stored at 4°C for 7 days for low-temperature stability study. The results showed that the overall appearance of the precursor solution remained consistent with the initial state, and no layering, precipitation or gel collapse occurred, indicating that its storage stability was good.

[0092] The above characterization data demonstrate that AsLIP@PPD-Gel meets the physicochemical properties standards, possesses excellent injectability and storage and in vivo gelation stability, and complies with the formulation usage conditions for intratumoral in situ injection.

[0093] IV. Physicochemical property characterization

[0094] (I) Particle size distribution determination of folic acid-modified ATO / cGAMP co-loaded liposomes AsLIP

[0095] The folic acid-modified ATO / cGAMP co-loaded liposomes AsLIP prepared in Example 1 were used to detect the hydrated particle size of the sample using a dynamic light scattering particle size analyzer. The sample was appropriately diluted with deionized water before testing. The scattered light intensity data were collected on the instrument at room temperature, and the instrument automatically output the average particle size and polydispersity index (PDI).

[0096] Particle size distribution results are as follows Figure 4 As shown, the average hydrated particle size of AsLIP liposomes is 136.7 nm, PDI=0.138, and the particle size distribution curve shows a single sharp peak without any impurities. This indicates that the prepared liposomes have uniform particle size, low aggregation, and excellent dispersion stability, making them suitable for subsequent embedding into 20(S)-protopanaxadiol-based smart gels.

[0097] It should be noted that the AsLIP liposomes freshly extruded had a particle size of approximately 120 nm, which swelled to 136.7 nm after hydration equilibrium. Both of these sizes are within the acceptable range for liposome particle size.

[0098] (II) Zeta potential assay of folic acid-modified ATO / cGAMP co-loaded liposomes AsLIP

[0099] The apparent Zeta potential of the AsLIP liposome samples diluted as described above was measured using a potentiometer, and the average value was taken from three parallel tests.

[0100] like Figure 5 As shown, the zeta potential of liposomes is -25.8mV, and the particle surface carries a significant negative charge. The electrostatic repulsion effectively inhibits particle flocculation and sedimentation, further confirming the storage and dispersion stability of the liposome colloidal system.

[0101] (III) In vitro dual-response drug release assay of AsLIP@PPD-Gel solid gel

[0102] In vitro drug release assays were conducted using dynamic dialysis: 1 g of AsLIP@PPD-Gel prepared in Example 1 was accurately weighed, placed into a dialysis bag with a molecular weight cutoff of 8000~14000 Da, and sealed. Six release media were prepared: three blank buffer solutions at pH 5.4, pH 6.5, and pH 7.4, and a buffer system with 10 mM GSH added at the corresponding pH. The sealed dialysis bag was immersed in each release medium, and the shaker was set to a constant temperature of 32±0.5℃ and a rotation speed of 100 rpm. At preset time points, 0.5 mL of the released solution was collected, and the same volume and temperature of fresh isothermal medium were simultaneously added. The concentration of arsenic (ATO) in the medium was quantitatively detected using ICP-MS, and the cumulative percentage of arsenic drug release was calculated.

[0103] The results are as follows Figure 6 As shown, in the three blank media (pH 5.4, 6.5, and 7.4) without GSH, the overall drug release was slow, with a cumulative release rate of less than 20% after 48 hours. The dense three-dimensional network of PPD gel can effectively bind the internal liposomes, achieving long-term sustained release in the physiological environment. After adding 10mM GSH under the same pH conditions, the drug release rate of each group was significantly improved. The cumulative drug release rates of the pH 5.4+GSH, pH 6.5+GSH, and pH 7.4+GSH groups were approximately 60%, 55%, and 48% after 48 hours, respectively. This confirms that AsLIP@PPD-Gel solid gel has significant pH-GSH dual reduction response drug release characteristics, which can trigger rapid drug release in the high GSH and weakly acidic microenvironment of tumors and achieve sustained release and retention in the normal physiological environment, thus meeting the needs of local in-situ drug delivery in tumors.

[0104] (iv) XPS elemental characterization of folic acid-modified ATO / cGAMP co-loaded liposomes AsLIP

[0105] The AsLIP liposome powder prepared in Example 1 was freeze-dried, and the characteristic binding energy peak of arsenic in the sample was collected by X-ray photoelectron spectroscopy in the range of 33-54 eV, which verified that the active arsenic component was successfully encapsulated inside the liposome.

[0106] The results are as follows Figure 7 As shown in the figure, the characteristic binding energy peak of arsenic appears at ~44eV, proving that the trivalent arsenic drug was successfully encapsulated inside the liposome, and the drug loading process achieved effective loading of the active component.

[0107] V. Rheological Performance Evaluation

[0108] The AsLIP@PPD-Gel room temperature liquid precursor liquid prepared in Example 1 was uniformly filled between the 20mm parallel plate and the substrate of the rheometer, with the gap between the upper and lower plates controlled at 300μm. The overflow liquid from the edge of the clamping plate was scraped off, and the test was started after the internal stress of the filling was eliminated by letting it stand. Four rheological tests were carried out in sequence, and the elastic modulus G' and viscous modulus G” were continuously collected in real time.

[0109] ①Strain scanning: The instrument continuously and linearly increases the oscillating strain from low to high, with the oscillating strain range being 0.1% to 200%;

[0110] ②Temperature scan: Under small constant strain conditions, the temperature is uniformly increased from 25℃ to 40℃ to simulate the process of changing from room temperature storage environment to human physiological temperature environment;

[0111] ③ Frequency scanning: With constant temperature and small strain, the oscillation shear frequency is gradually changed from a low frequency of 1 rad / s to a high frequency of 200 rad / s;

[0112] ④Thixotropic cycle test: 0.1% low strain for 60s → 50% high strain for 60s → switch back to 0.1% low strain for 60s, repeat 3 complete cycles to simulate the process of high-intensity shearing of the needle during injection and the self-healing process of the internal environment after the external force is removed after the injection is completed.

[0113] Figure 8 Figure A shows the strain scan results. In the low strain range of strain <10%, G' is always > G", and the gel exhibits solid elastic characteristics. When the strain is >10%, G' drops rapidly and falls below G" after exceeding the critical value. The gel structure collapses and thins under external force, meeting the injectable conditions for syringe extrusion flow.

[0114] Figure B shows the temperature scan results. In the physiological temperature range of 25~40℃, G' is consistently higher than G", and the modulus decreases slowly and slightly. At the human body temperature of 37℃, it still maintains a stable gel state. After being injected into the body, it can quickly solidify in situ and is not easily diluted and lost by body fluids.

[0115] Figure C shows the frequency scan results. The full scan interval G' > G', and there is no crossover of the modulus, indicating that the three-dimensional network structure of the gel is stable and not subject to external oscillation. It is not easy for the structure to collapse or for the drug to be leaked during storage at room temperature and pipeline transportation.

[0116] Figure D shows the thixotropic cycling results. High strain damages the gel, causing a sharp drop in modulus. After switching to low strain, G' recovers rapidly. Repeating this process three times can efficiently restore the initial modulus. The gel exhibits excellent thixotropy and self-healing capabilities, and structural damage caused by injection can be repaired and reshaped in vivo.

[0117] In summary, this gel exhibits shear thinning for easy injection, stabilizes at physiological temperatures within the body, and self-repairs after breakage, thus meeting the rheological requirements for in-situ intratumoral drug delivery.

[0118] Example 5

[0119] To systematically evaluate the in vivo tumor-suppressing effect, cancerous wound repair ability, anti-tumor molecular mechanism, tumor immune microenvironment regulation effect, and in vivo biosafety of the arsenic liposome gel delivery system of this invention via in situ injection on triple-negative breast cancer (TNBC), this embodiment uses the AsLIP@PPD-Gel precursor fluid prepared in Example 1 as the test drug and 4T1 orthotopic TNBC-bearing BALB / c mice as the animal model. In vivo tumor growth inhibition test, histopathological examination of ulcerated wound tissue, Western blotting verification of tumor protein expression, multiplex immunofluorescence analysis of tumor-infiltrating lymphocytes, and HE detection of safety in major organs such as the heart and kidneys are carried out sequentially.

[0120] I. In vivo tumor growth inhibition test

[0121] Eight-week-old SPF-grade female BALB / c mice, weighing 16–20 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. The mice were housed in an SPF-grade specific pathogen-free (SPF) environment with free access to food and water. All animal experiments in this study were conducted strictly in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Approval No.: YS085).

[0122] The method for establishing the mouse mammary gland orthotopic TNBC animal model is as follows:

[0123] Eight-week-old female BALB / c mice were weighed and then subcutaneously injected with 5×10⁻⁶ mg / L of the fat pad in the fourth pair of mammary glands. 5 Four 4T1 triple-negative breast cancer cells were observed. Tumor growth was monitored every other day, and the long diameter and the short diameter perpendicular to it were measured using calipers. Tumor volume was calculated using the formula V = long diameter × short diameter. 2 / 2 Calculation.

[0124] When the in situ tumor grows to approximately 100 mm 3 At that time, the model mice were randomly divided into a TNBC control group and an AsLIP@PPD-Gel administration group, with 6 mice in each group. The TNBC control group was injected intratumorally with an equal volume of sterile PBS solution, while the AsLIP@PPD-Gel administration group was injected intratumorally with the AsLIP@PPD-Gel precursor solution prepared in Example 1. The administration volume was 30 μL per mouse, and the administration frequency was once every 7 days, for a total of 2 administrations. The long and short diameters of the tumor were measured at regular intervals throughout the administration, and the tumor volume was calculated.

[0125] Mice were euthanized after the treatment cycle ended. The tumor tissue was completely dissected, photographed, weighed, fixed, embedded, and prepared into paraffin sections. Immunohistochemical staining was performed for the apoptosis marker Caspase-3 and the proliferation marker Ki67. Microscopic photographs were taken and the average optical density value of positive expression was quantitatively calculated.

[0126] The results of the comparison of tumor tissue samples and volumes between the two groups of mice are as follows: Figure 9 As shown, compared with the TNBC control group, the ex vivo tumor volume of the AsLIP@PPD-Gel treatment group was significantly smaller, and the tumor proliferation rate was significantly inhibited throughout the treatment cycle.

[0127] Immunohistochemical staining results as follows Figure 10 As shown, the Caspase-3 positive expression level was significantly upregulated in the AsLIP@PPD-Gel administration group. Ki67 tumor proliferation protein expression was significantly downregulated. This study confirms that AsLIP@PPD-Gel can achieve in vivo anti-triple-negative breast cancer effects by inducing tumor cell apoptosis and inhibiting malignant tumor proliferation.

[0128] II. Pathological examination of cancerous wound tissue

[0129] Before the final dissection of the animals treated with the drug, the appearance of the ulcerated tumor in situ in mice was photographed. Full-thickness skin tissue from the edge of the ulcer was taken, fixed in 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, and sectioned to obtain paraffin sections with a thickness of 4 μm. After hematoxylin-eosin (HE) standardized staining, the pathological morphological changes of the epidermis, dermis, and skin appendages were observed under an optical microscope.

[0130] The results are as follows Figure 11 As shown, in the TNBC control group, the skin at the tumor site had large-area ulceration and epidermal defects, while in the AsLIP@PPD-Gel treatment group, the area of ​​skin ulceration was significantly reduced.

[0131] HE pathological sections confirmed that in the TNBC control group, the entire epidermis was missing, a large number of skin appendages such as hair follicles and sebaceous glands atrophied and disappeared, and the dermis was abnormally fibrotic and thickened; in the AsLIP@PPD-Gel treatment group, the wound showed significant re-epithelialization, with intact and thickened epidermal structure and regular dermal layer arrangement, indicating that AsLIP@PPD-Gel can effectively repair cancerous skin ulceration secondary to triple-negative breast cancer.

[0132] III. Western Blot Validation of Tumor Protein Expression

[0133] Fresh tumor tissues were collected from mice in each group. Total protein was extracted from the tissues using RIPA lysis buffer, and protein concentration was determined using a BCA kit (Beyotime, China). Protein samples were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk powder at room temperature for 2 h, incubated with primary antibody at 4°C overnight, and incubated with secondary antibody at room temperature for 1 h. Protein bands were collected by ECL luminescent reagent, and the relative expression levels of each target protein were semi-quantitatively calculated using ImageJ software.

[0134] The results are as follows Figure 12 As shown, compared with the TNBC control group, the expression of glucose transporter GLUT1 in tumor tissue was significantly downregulated in the AsLIP@PPD-Gel treatment group. This study demonstrates that AsLIP@PPD-Gel exerts its antitumor effect in vivo by blocking glucose uptake and inhibiting tumor glycolysis, thereby enhancing metabolism. The phosphorylation level of STING protein (p-STING) is significantly increased, activating the cGAS-STING innate immune pathway. Simultaneously, the expression of the downstream pyroptosis activation marker protein GSDME-N is significantly upregulated, indicating that the pyroptosis pathway is effectively activated. This experiment confirms that AsLIP@PPD-Gel exerts its antitumor effect in vivo through a triple pathway of "metabolic inhibition + STING immune activation + induction of tumor cell pyroptosis."

[0135] IV. Multiplex Immunofluorescence Analysis of Tumor-Infiltrating Lymphocytes

[0136] Fresh tumor tissue was embedded in OCT to prepare frozen sections. Immunofluorescence staining with CD4, CD8, and FOXP3 markers was performed, and cell nuclei were counterstained with DAPI. The sections were mounted with anti-fluorescence quenching mounting medium, and fluorescence images were acquired using a laser confocal microscope. CD4 counts were then quantitatively analyzed. + Support T, CD8 + Cytotoxic T, FOXP3 + Percentage of regulatory T (Treg) positive cells.

[0137] The results are as follows Figure 13 As shown, CD4+ in tumor lesions after AsLIP@PPD-Gel administration + CD8 + The number of functional anti-tumor T cells infiltrating was significantly increased, and immunosuppressive FOXP3 cells were also observed. + The significant decrease in the proportion of Treg cells (***p<0.001) indicates that AsLIP@PPD-Gel reverses the tumor immunosuppressive microenvironment by activating the STING pathway, thereby generating a potent anti-tumor immune response in situ within the tumor.

[0138] V. Safety and pathological HE testing of major organs such as the heart and kidneys

[0139] After the treatment cycle, the heart and kidney organs of mice in each group were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with standardized HE, and the pathological damage of the organ tissues was observed under an optical microscope.

[0140] The results are as follows Figure 14 As shown, in the TNBC control group and the AsLIP@PPD-Gel administration group, the heart and kidney tissue cells were arranged densely and regularly, without pathological damage such as inflammatory cell infiltration, cell necrosis, tissue edema, and vacuolar degeneration. The organ structures were intact, which proves that the systemic toxicity of AsLIP@PPD-Gel administered locally within the tumor is extremely low and its in vivo biosafety is excellent.

[0141] The in vivo series of test results in this embodiment confirm that the AsLIP@PPD-Gel prepared in Example 1 can effectively inhibit the proliferation of triple-negative breast cancer in situ and repair cancerous ulceration. It exerts its efficacy through multiple mechanisms, including glucose metabolism blockade, STING pathway activation, induction of tumor pyroptosis, and remodeling of anti-tumor immunity, and has no obvious organ toxicity, thus meeting the application requirements of intratumoral in situ injection of anti-tumor agents.

Claims

1. An arsenic-based liposome gel drug delivery system, characterized in that, The invention comprises a 20(S)-protopanaxadiol-based smart gel and folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes dispersed therein, wherein the mass ratio of the folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes to the 20(S)-protopanaxadiol-based smart gel is 1.5~2.5:

100. The 20(S)-protopanaxadiol-based smart gel contains the following components in parts by weight: 60-140 parts of 20(S)-protopanaxadiol, 60-140 parts of dioleoylphosphatidylglycerol, 2.5-10 parts of quercetin, and 0.5-2.5 parts of cGAMP; When preparing the folic acid-modified arsenic trioxide / cGAMP co-supported liposomes, the feed contains the following components in parts by weight: 2-5 parts arsenic trioxide, 1-4 parts cGAMP, and 80-98 parts lipid membrane material; the lipid membrane material is composed of hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in a mass ratio of 70-80:5-15:10-20. The preparation method of the 20(S)-protopanaxadiol-based smart gel is as follows: 20(S)-protopanaxadiol and dioleoylphosphatidylglycerol are dissolved in an organic solvent, rotary evaporated to form a film, and then vacuum dried. Then, a hydration medium containing quercetin, cGAMP and calcium chloride is added, ultrasonically treated, and cooled to obtain the 20(S)-protopanaxadiol-based smart gel.

2. The arsenic liposome gel delivery system according to claim 1, characterized in that, The mass ratio of the folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes to the 20(S)-protopanaxadiol-based smart gel is 2:

100. The 20(S)-protopanaxadiol-based smart gel contains the following components in parts by weight: 100 parts 20(S)-protopanaxadiol, 100 parts dioleoylphosphatidylglycerol, 5 parts quercetin and 1.25 parts cGAMP; When preparing the folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes, the feed contains the following components in parts by weight: 4 parts arsenic trioxide, 2 parts cGAMP and 94 parts lipid membrane material; the lipid membrane material is composed of hydrogenated soybean lecithin, cholesterol hemisuccinate and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in a mass ratio of 75:10:

15.

3. The arsenic liposome gel delivery system according to claim 1 or 2, characterized in that, The folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes have a particle size of 100~150 nm.

4. A method for preparing an arsenic-based liposome gel drug delivery system as described in any one of claims 1-3, characterized in that, The steps are as follows: Step 1: Preparation of 20(S)-Protopanaxadiol-based smart gel: 20(S)-Protopanaxadiol and dioleoylphosphatidylglycerol were dissolved in an organic solvent, rotary evaporated to form a film, and then vacuum dried. A hydration medium containing quercetin, cGAMP and calcium chloride was added, and the mixture was ultrasonically treated and cooled to obtain 20(S)-Protopanaxadiol-based smart gel. Step 2: Preparation of folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes: Hydrogenated soybean phospholipids, cholesterol hemisuccinate, and folic acid-polyethylene glycol-distearate phosphatidylethanolamine were dissolved in an organic solvent, rotary evaporated to form a film, added a buffer containing arsenic trioxide and cGAMP for hydration, ultrasonically dispersed, and extruded through a polycarbonate membrane to obtain folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes. Step 3: Assemble the arsenic liposome gel drug delivery system: The folic acid-modified arsenic trioxide / cGAMP co-loaded liposomes obtained in step two are added to the 20(S)-protopanaxadiol-based smart gel obtained in step one, and stirred and dispersed to obtain the arsenic liposome gel drug delivery system.

5. The preparation method of the arsenic liposome gel drug delivery system according to claim 4, characterized in that, The organic solvents mentioned in steps one and two are both mixed solvents of chloroform and methanol, with a volume ratio of chloroform to methanol of 2:

1.

6. The method for preparing the arsenic liposome gel drug delivery system according to claim 4 or 5, characterized in that, The temperature of the rotary evaporation in step one is 42~48℃, the vacuum drying time is 4~5h, the temperature of the ultrasound is 42~48℃, the ultrasound power is 200~300W, the ultrasound time is 6~10min, and the concentration of calcium chloride in the hydration medium is 0.5~2mM.

7. The preparation method of the arsenic liposome gel drug delivery system according to claim 6, characterized in that, The rotary evaporation temperature in step two is 35~45℃, the ultrasonic dispersion is carried out at an ultrasonic power of 200~300W for 5~10 minutes, and the pore sizes of the polycarbonate membranes through which the extrusion is carried out are 400nm, 200nm and 100nm respectively, with each extrusion performed 3~5 times.

8. The preparation method of the arsenic liposome gel drug delivery system according to claim 7, characterized in that, The stirring speed in step three is 150~250 rpm, the time is 30~60 min, and the temperature is 4℃.

9. The use of an arsenic-based liposome gel delivery system as described in any one of claims 1-3 in the preparation of a medicament for treating cancerous lesions in triple-negative breast cancer.

10. The application according to claim 9, characterized in that, The drug is an intratumoral injection preparation.

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