Topical formulations of arsenic trioxide for the treatment of superficial tumors and methods of making the same

Abstract

This invention discloses a topical drug delivery formulation loaded with arsenic trioxide for the treatment of superficial tumors, belonging to the field of pharmaceutical formulation and preparation technology. This invention encapsulates arsenic trioxide using phospholipids, cholesterol, etc., as liposome materials to obtain arsenic trioxide-containing liposomes. These liposomes are then combined with a thermosensitive hydrogel. Utilizing the in-situ formation and long-term controlled release characteristics of the thermosensitive in-situ gel, a phase transition occurs rapidly after injection, constructing an intratumoral arsenic trioxide drug reservoir, achieving localized long-term sustained release of arsenic trioxide. Furthermore, the arsenic trioxide-containing liposomes are introduced into soluble microneedles. Leveraging the minimally invasive and painless advantages of microneedles, a precise delivery channel for arsenic trioxide is established directly to the tumor, allowing for patient self-administration. This maximizes the therapeutic effect of arsenic trioxide at the tumor site, overcoming the limitations of traditional applications and providing an innovative and feasible formulation approach for the local treatment of superficial tumors.

Description

Technical Field

[0001] This invention relates to a topical administration formulation loaded with arsenic trioxide for the treatment of superficial tumors, belonging to the field of pharmaceutical preparations and their preparation technology. Background Technology

[0002] Arsenic trioxide is the main active ingredient in the traditional Chinese medicine arsenic, and its presence is recorded in ancient medical texts such as the *Compendium of Materia Medica*. In the 1970s, Professor Zhang Tingdong of Harbin Medical University was the first to discover the significant therapeutic effect of arsenic trioxide in treating acute promyelocytic leukemia. In recent years, numerous studies both domestically and internationally have further confirmed that arsenic trioxide also has a significant inhibitory effect on various solid tumors, including liver cancer, breast cancer, and skin cancer. Research indicates that arsenic trioxide can inhibit tumor cell proliferation, migration, and invasion, and induce programmed cell death.

[0003] Currently, the clinically used arsenic trioxide is mainly in the form of intravenous injection. However, due to its narrow therapeutic window, difficulty in controlling the dosage, and poor targeting, it often causes systemic toxicity, especially cardiotoxicity. Moreover, the intravenous arsenic trioxide currently used in clinical practice still cannot achieve precise local treatment of superficial tumors. Among them, for superficial tumors, the Chinese Clinical Application Guidelines for Tumor Hyperthermia (2017.V1.1) define them as: (1) Skin cancers in all parts of the body, including squamous cell carcinoma, adenocarcinoma, and melanoma. (2) Metastatic cancers in all superficial lymph nodes of the body, such as the neck, supraclavicular region, axilla, and groin. (3) Malignant tumors of superficial organs and limbs: ① Primary tumors of the head and neck that are relatively superficial, such as lip cancer, gingival cancer, buccal mucosal cancer, and cancers of the face, scalp, and auricle; ② Vulvar cancer and anal cancer; ③ Cancers of the limbs, such as soft tissue sarcoma and osteosarcoma; ④ Advanced breast cancer. (4) Tumors that have recurred or metastasized in the chest and abdominal walls.

[0004] To overcome this limitation, a metal ion gradient method has been used to encapsulate arsenic trioxide into liposomes. This strategy significantly improves the stability of the drug within the liposomes and effectively reduces premature leakage during delivery. However, it still cannot achieve precise local treatment of superficial tumors.

[0005] Therefore, it is essential to provide a new topical formulation of arsenic trioxide to advance its application in the treatment of superficial tumors in a safer, more efficient, and more user-friendly direction. Summary of the Invention

[0006] The present invention aims to provide a topical administration formulation loaded with arsenic trioxide for the treatment of superficial tumors.

[0007] The technical solution of the present invention:

[0008] One objective of this invention is to provide a topical drug delivery formulation loaded with arsenic trioxide for the treatment of superficial tumors, specifically comprising a thermosensitive hydrogel containing arsenic trioxide liposomes and soluble microneedles containing arsenic trioxide liposomes.

[0009] Further specifying, the thermosensitive hydrogel containing arsenic trioxide liposomes is prepared by arsenic trioxide liposomes, gel matrix, pH adjuster and purified water through swelling method, ion-induced gelation, blending composite method and photocrosslinking method.

[0010] Furthermore, the molar percentages of arsenic trioxide liposomes, gel matrix, pH adjuster, and purified water are 5%~40%, 6%~35%, 0.1%~2%, and 50%~70%, respectively.

[0011] Further specifying, the soluble microneedles containing arsenic trioxide liposomes are prepared by arsenic trioxide liposomes, microneedle matrix and purified water through molding method, 3D printing method and micro-injection molding method.

[0012] Furthermore, the molar percentages of arsenic trioxide liposomes, microneedle matrix, and purified water are 5%~30%, 10%~30%, and 40%~85%, respectively.

[0013] Furthermore, arsenic trioxide liposomes are prepared from arsenic trioxide, phospholipids, and cholesterol via thin-film dispersion, injection, ion gradient, reverse-phase evaporation, freeze-thaw, and extrusion methods.

[0014] To further specify, the phospholipid is one or a mixture of egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and polyethylene glycol-modified phospholipids.

[0015] Furthermore, the molar percentages of arsenic trioxide, phospholipids, and cholesterol in arsenic trioxide liposomes are 1%~40%, 1%~95%, and 1%~45%, respectively.

[0016] Furthermore, the molar percentages of arsenic trioxide, phospholipids, and cholesterol in arsenic trioxide liposomes are 1%~25%, 50%~70%, and 10%~25%, respectively.

[0017] Furthermore, the gel matrix is ​​one or more of the following: Pluronic acid, chitosan, gelatin, methylcellulose, and hydroxypropyl methylcellulose.

[0018] Furthermore, the molar percentages of arsenic trioxide liposomes, gel matrix, pH adjuster, and purified water are 5%~25%, 15%~35%, 0.5%~1%, and 50%~65%, respectively.

[0019] Furthermore, the microneedle matrix is ​​one or more of polyvinylpyrrolidone, hyaluronic acid, polyvinyl alcohol, and chitosan.

[0020] Furthermore, the molar percentages of arsenic trioxide liposomes, microneedle matrix, and purified water are 10%~20%, 20%~30%, and 50%~70%, respectively.

[0021] Beneficial effects:

[0022] This invention utilizes the in-situ formation and long-term controlled release characteristics of thermosensitive in-situ gels. After injection, a rapid phase transition occurs, constructing an intratumoral arsenic trioxide drug reservoir, achieving localized, long-term sustained release of arsenic trioxide. Furthermore, arsenic trioxide-containing liposomes are introduced into soluble microneedles. Leveraging the minimally invasive and painless advantages of microneedles, a precise delivery channel for arsenic trioxide is established directly to the tumor, allowing for patient self-administration. Simultaneously, both the thermosensitive in-situ gel and the soluble microneedles maximize the therapeutic effect of arsenic trioxide at the tumor site, overcoming the limitations of traditional applications and providing an innovative and feasible formulation approach for the local treatment of superficial tumors. Compared with existing technologies, it also has the following advantages:

[0023] (1) The present invention uses phospholipids, cholesterol and other liposomes as liposome materials. These liposomes can encapsulate arsenic trioxide, improve the stability of arsenic trioxide and promote its absorption, thereby improving the bioavailability of the drug.

[0024] (2) This invention uses chitosan, F127, and F68 as gel matrix materials to obtain an in-situ hydrogel matrix with thermosensitive properties. After injection, it undergoes a rapid phase transition, which can increase the local concentration of the drug and increase the amount and retention time of arsenic trioxide in the diseased tissue. Moreover, the gel has good biodegradability and can be absorbed and completely degraded by tissues under the skin, resulting in high biosafety.

[0025] (3) The present invention uses PVP and other materials as microneedle matrix materials, which can form a microneedle array with suitable mechanical strength, effectively piercing the stratum corneum of the skin, delivering the encapsulated arsenic trioxide liposomes into the tumor, reducing the side effects related to systemic exposure, and enhancing the therapeutic effect on superficial tumors. Attached Figure Description

[0026] Figure 1 Thermosensitive conversion temperature curves of the in-situ gels prepared in Example 1 and Comparative Examples 1-6;

[0027] Figure 2 This is a photograph of the arsenic trioxide liposomes prepared in Example 1.

[0028] Figure 3 The particle size distribution diagram of the arsenic trioxide liposomes prepared in Example 1 is shown.

[0029] Figure 4 Zeta potential diagram of arsenic trioxide liposomes prepared in Example 1;

[0030] Figure 5 Transmission electron microscopy image of arsenic trioxide liposomes prepared in Example 1;

[0031] Figure 6 Thermosensitive experiment of arsenic trioxide liposome in situ gel prepared in Example 1;

[0032] Figure 7 Needle penetration test of the arsenic trioxide liposome in situ gel prepared in Example 1;

[0033] Figure 8 This is a diagram illustrating the in vivo antitumor effect of the arsenic trioxide liposome in situ gel prepared in Example 1.

[0034] Figure 9 This is a photograph of the arsenic trioxide liposome microneedles prepared in Example 2.

[0035] Figure 10 Microscopic image of the arsenic trioxide liposome microneedles prepared in Example 2;

[0036] Figure 11 Skin puncture test of arsenic trioxide liposome microneedles prepared in Example 2;

[0037] Figure 12 The image shows the in vivo antitumor effect of the arsenic trioxide liposome microneedles prepared in Example 2. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0040] Example 1

[0041] Step 1: Preparation of arsenic trioxide liposomes:

[0042] Using soybean lecithin to cholesterol at a mass ratio of 6:1 (30 mg soybean lecithin) as the carrier material, the liposomes were dissolved in chloroform and then vacuum-dried at 30 °C for 90 min to form a lipid film. 2 mL of 300 mM nickel acetate solution was added, and the mixture was hydrated at 55 °C for 60 min and sonicated at 240 W for 10 min to obtain monolayer liposomes. A nickel ion concentration gradient was established by replacing the nickel salt in the aqueous phase with saturated sodium chloride through ultrafiltration centrifugation (8000 rpm, 30 min, 3 cycles). Subsequently, 1 mL of 2 mg / mL arsenic trioxide solution and 1 mL of deionized water were added, and the mixture was stirred and incubated at 60 °C and 400 rpm for 90 min. The nickel ion gradient drove trivalent arsenic into the liposome cavity, forming an insoluble Ni(AsO2)2 complex with nickel ions. Finally, free drug was removed by ultrafiltration centrifugation to obtain arsenic trioxide liposomes.

[0043] Step 2: Preparation of a thermosensitive hydrogel containing arsenic trioxide liposomes:

[0044] Take 0.04 g of chitosan and place it in a 5 mL volumetric flask. Add an appropriate amount of 0.5% acetic acid solution and stir magnetically until completely dissolved. After adjusting the volume, take 1.618 g of F127 and F68 powders in a mass ratio of 18:3 and add them to the above chitosan solution to adjust the volume to 5 mL. Swell in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. Take 4.636 mL of the blank gel and slowly add 0.364 mL of the arsenic trioxide liposomes prepared in step one. Stir slowly and evenly under low temperature conditions to fully load the liposomes into the gel, thus obtaining a thermosensitive hydrogel containing arsenic trioxide liposomes.

[0045] Example 2

[0046] Step 1: Preparation of arsenic trioxide liposomes:

[0047] Using soybean lecithin to cholesterol at a mass ratio of 6:1 (30 mg soybean lecithin) as the carrier material, the liposomes were dissolved in chloroform and then vacuum-dried at 30 °C for 90 min to form a lipid film. 2 mL of 300 mM nickel acetate solution was added, and the mixture was hydrated at 55 °C for 60 min and sonicated at 240 W for 10 min to obtain monolayer liposomes. A nickel ion concentration gradient was established by replacing the nickel salt in the aqueous phase with saturated sodium chloride through ultrafiltration centrifugation (8000 rpm, 30 min, 3 cycles). Subsequently, 1 mL of 2 mg / mL arsenic trioxide solution and 1 mL of deionized water were added, and the mixture was stirred and incubated at 60 °C and 400 rpm for 90 min. The nickel ion gradient drove trivalent arsenic into the liposome cavity, forming an insoluble Ni(AsO2)2 complex with nickel ions. Finally, free drug was removed by ultrafiltration centrifugation to obtain arsenic trioxide liposomes.

[0048] Step 2: Soluble microneedles containing arsenic trioxide liposomes:

[0049] Take 0.011 mL of the arsenic trioxide liposomes prepared in step one and mix them evenly with 4.5 mg of polyvinylpyrrolidone K90 gel matrix to prepare a drug-containing polyvinylpyrrolidone K90 solution. Inject this solution into a PDMS microneedle negative mold and centrifuge at 3000 rpm for 30 min to fill the micropores at the needle tip. After removal, carefully scrape off the excess matrix on the surface of the mold with a scraper and collect it. Then, add blank PVP solution on top of the mold as a support substrate and centrifuge again at 5000 rpm for 30 min to ensure tight adhesion to the needle tip layer. After thorough drying at room temperature, carefully demold to obtain soluble microneedles containing arsenic trioxide liposomes.

[0050] Example 3

[0051] The difference between this embodiment and Example 1 is as follows: In step one, a lipid film is formed by dissolving a carrier material of soybean lecithin and cholesterol at a mass ratio of 8:1 (30 mg of soybean lecithin) in chloroform and then vacuum drying at 30°C for 60 min. 2 mL of a 300 mM zinc acetate solution is added, and the mixture is hydrated at 55°C for 90 min and sonicated at 160 W for 10 min to obtain monolayer liposomes. A zinc ion concentration gradient is established by replacing the zinc salt in the external aqueous phase with saturated sodium chloride through ultrafiltration centrifugation (8000 rpm, 30 min, 3 cycles). Then, 1 mL of a 2 mg / mL arsenic trioxide solution and 1 mL of deionized water are added, and the mixture is stirred and incubated at 60°C and 400 rpm for 60 min. The zinc ion gradient drives trivalent arsenic into the liposome cavity, forming an insoluble Zn(AsO2)2 complex with zinc ions. Finally, free drug is removed by ultrafiltration centrifugation to obtain arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 1.

[0052] Example 4

[0053] The difference between this embodiment and Example 1 is as follows: In step one, a lipid film is formed by dissolving soybean lecithin and cholesterol in a mass ratio of 10:1 (30 mg soybean lecithin) in chloroform and then vacuum drying at 30°C for 30 min. 2 mL of a 300 mM manganese acetate solution is added, and the mixture is hydrated at 55°C for 120 min and sonicated at 80 W for 10 min to obtain monolayer liposomes. A manganese ion concentration gradient is established by replacing the manganese salt in the aqueous phase with saturated sodium chloride through ultrafiltration centrifugation (8000 rpm, 30 min, 3 cycles). Then, 1 mL of a 2 mg / mL arsenic trioxide solution and 1 mL of deionized water are added, and the mixture is stirred and incubated at 60°C and 400 rpm for 30 min. The manganese ion gradient drives trivalent arsenic into the liposome cavity, forming an insoluble Mn(AsO2)2 complex with manganese ions. Finally, free drug is removed by ultrafiltration centrifugation to obtain arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 1.

[0054] Example 5

[0055] The difference between this embodiment and Example 1 is as follows: In step two, 0.025g of chitosan is placed in a 5mL beaker, and an appropriate amount of 0.5% acetic acid solution is added. After magnetic stirring until completely dissolved and the volume is adjusted, 1.618g of F127 and F68 powders in a mass ratio of 18:5 are added to the above chitosan solution and the volume is adjusted to 5mL. The solution is then swollen in a refrigerator at 4℃ for 36h until completely dissolved to obtain a blank gel. 4.636mL of the blank gel is taken, and 0.364mL of arsenic trioxide liposomes prepared in Example 1 are slowly added. The solution is slowly stirred evenly under low temperature conditions to fully load the liposomes into the gel, thus obtaining a thermosensitive hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 1.

[0056] Example 6

[0057] The difference between this embodiment and Example 1 is as follows: In step two, 0.01g of chitosan is placed in a 5mL beaker, and an appropriate amount of 0.5% acetic acid solution is added. After magnetic stirring until completely dissolved and the volume is adjusted, 1.618g of F127 and F68 powders in a mass ratio of 18:7 are added to the above chitosan solution and the volume is adjusted to 5mL. The solution is then swollen in a 4℃ refrigerator for 24 hours until completely dissolved to obtain a blank gel. 4.636mL of the blank gel is taken, and 0.364mL of arsenic trioxide liposomes prepared in Example 1 are slowly added. The solution is slowly stirred evenly under low temperature conditions to ensure that the liposomes are fully loaded in the gel, thus obtaining a thermosensitive hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 1.

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 16:3 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0060] The thermosensitive ability of the hydrogel containing arsenic trioxide liposomes prepared in this comparative example was determined. The experimental results showed that the thermosensitive conversion temperature of the hydrogel was 50℃.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 16:5 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0063] Comparative Example 3

[0064] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 16:7 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a thermosensitive hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0065] Comparative Example 4

[0066] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 20:3 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0067] Comparative Example 5

[0068] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 20:5 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0069] Comparative Example 6

[0070] The difference between this comparative example and Example 1 is as follows: In step two, 0.04 g of chitosan was placed in a 5 mL beaker, and an appropriate amount of 0.5% acetic acid solution was added. After magnetic stirring until completely dissolved and the volume was adjusted, 1.618 g of F127 and F68 powders in a mass ratio of 20:7 were added to the above chitosan solution and the volume was adjusted to 5 mL. The solution was then swollen in a refrigerator at 4 °C for 48 h until completely dissolved to obtain a blank gel. 4.636 mL of the blank gel was taken, and 0.364 mL of arsenic trioxide liposomes prepared in Example 1 were slowly added. The solution was slowly stirred evenly under low temperature conditions to ensure that the liposomes were fully loaded into the gel, thus obtaining a thermosensitive hydrogel containing arsenic trioxide liposomes. The remaining process steps and parameter settings were the same as in Example 1.

[0071] The thermosensitive properties of the hydrogels containing arsenic trioxide liposomes prepared in Example 1 and Comparative Examples 1-6 were measured, and the results are as follows: Figure 1 As shown. In Example 1, when the mass ratio of F127:F68 is 18:3, the prepared system has a suitable thermosensitive conversion temperature, and can undergo gel phase transition under conditions close to human physiological temperature, meeting the requirements for human applications.

[0072] Example 7

[0073] The difference between this embodiment and Example 2 is as follows: In step two, 0.011 mL of the arsenic trioxide liposomes prepared in Example 1 is mixed evenly with 3.8 mg of polyvinylpyrrolidone K90 gel matrix to prepare a drug-containing polyvinylpyrrolidone K90 solution. This solution is injected into a PDMS microneedle negative mold and centrifuged at 4000 rpm for 20 min to fill the micropores at the needle tip. After removal, excess matrix on the surface of the mold is carefully scraped off with a scraper and recycled. Then, blank PVP solution is added on top of the mold as a support substrate, and centrifuged again at 4000 rpm for 20 min to ensure tight adhesion to the needle tip layer. After thorough drying at room temperature, the mold is carefully demolded to obtain soluble microneedles containing arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 2.

[0074] Example 8

[0075] The difference between this embodiment and Example 2 is as follows: In step two, 0.012 mL of the arsenic trioxide liposomes prepared in Example 1 is mixed evenly with 3 mg of polyvinylpyrrolidone K90 gel matrix to prepare a drug-containing polyvinylpyrrolidone K90 solution. This solution is injected into a PDMS microneedle negative mold and centrifuged at 5000 rpm for 10 min to fill the micropores at the needle tip. After removal, excess matrix on the surface of the mold is carefully scraped off with a scraper and recycled. Then, blank PVP solution is added on top of the mold as a support substrate, and centrifuged again at 5000 rpm for 10 min to ensure tight adhesion to the needle tip layer. After thorough drying at room temperature, the mold is carefully demolded to obtain soluble microneedles containing arsenic trioxide liposomes. The remaining process steps and parameter settings are the same as in Example 2.

[0076] Example of effect 1

[0077] The physicochemical properties of the thermosensitive hydrogel containing arsenic trioxide liposomes prepared in Example 1 were characterized, and are described in detail below:

[0078] (1) Examination of the morphology and particle size potential of the prepared arsenic trioxide liposomes

[0079] First, the prepared liposome solution was visually inspected to check for homogeneity and the presence of any precipitate or particles at the bottom. Second, at room temperature, 10 μL of arsenic trioxide liposomes were diluted 100-fold in purified water, and the particle size and potential of the arsenic trioxide liposomes were measured using a nanoparticle size and Zeta potential analyzer. Finally, the morphology of the lipid nanoparticles was observed using a transmission electron microscope (TEM). The diluted liposome solution was dropped onto a 300-mesh copper grid, negatively stained, allowed to air dry, and then its morphological characteristics were observed using TEM.

[0080] Experimental results are as follows Figure 2 The prepared liposome solution was a pale blue, homogeneous, and transparent liquid with no precipitate or particles at the bottom; the particle size of the prepared liposomes was 94.76 ± 1.63 nm (e.g., ...). Figure 3 As shown), the potential is 1.70 ± 0.51 mV (as shown). Figure 4 As shown in the image, the liposomes have a uniform particle size distribution, meeting the requirements for nano-formulations, and their potential is beneficial for maintaining their stable state. From TEM images (such as...) Figure 5 As shown in the figure, the prepared arsenic trioxide liposomes are clearly spherical with a relatively complete morphology and clear outline. Their particle size is consistent with the results measured by the particle size analyzer.

[0081] (2) Characterization of the encapsulation efficiency and drug loading of the prepared arsenic trioxide liposomes

[0082] Arsenic concentration was determined using an atomic fluorescence spectrometer under the following conditions: lamp current 70 mA, negative voltage 280 V, carrier solution 5% HCl, reducing agent 0.5% NaOH + 2.0% KHB4. Standard solutions with concentrations of 0, 1, 2, 4, 8, and 10 μg / L were prepared to form a standard curve. Sample preparation: 1 mL of freshly prepared liposome solution was placed in an ultrafiltration centrifuge tube and centrifuged at 8000 rpm for 30 min. The supernatant was diluted with deionized water. 1.0 mL of the sample solution was transferred to a 10.0 mL colorimetric tube, 0.5 mL of 10% thiourea solution and 0.5 mL of concentrated hydrochloric acid were added, and the solution was brought to volume with deionized water. The mixture was stirred and allowed to stand for 0.5 h. The content of unencapsulated arsenic trioxide in the supernatant was calculated based on the standard curve.

[0083] The formulas for calculating drug encapsulation efficiency (EE) and drug-loading content (LC) are as follows:

[0084] EE (%) = ×100% (1)

[0085] LC (%) = ×100% (2)

[0086] In the formula, M is the dosage of arsenic trioxide (mg), V is the volume of the centrifuged filtrate (mL), c is the concentration of arsenic trioxide in the filtrate (mg / mL), and Mn is the total mass of the lipid nanoparticles (mg).

[0087] The arsenic trioxide EE in the liposomes prepared in Example 1, calculated according to the formula, was 51.65±1.46%, and the LC was 2.97±0.08%.

[0088] (3) Determination of the thermosensitive ability of thermosensitive hydrogels containing arsenic trioxide liposomes

[0089] At room temperature, 2 mL of arsenic trioxide liposome hydrogel was placed in a vial, which was then placed in a heat-collecting magnetic stirrer. The temperature was raised to 37°C, and the changes in the hydrogel were observed. After a period of time, the temperature was lowered to 25°C, and the phenomena were observed and recorded.

[0090] like Figure 6 As shown, when the temperature is raised to 37°C and the vial is inverted, the solution stops flowing, indicating that the hydrogel successfully transitions from a sol to a gel state under these conditions. When the temperature is further reduced to room temperature, it can transition from a gel to a sol state, demonstrating that the thermosensitive state transition of the gel is reversible.

[0091] (4) Determination of the needle-passing ability of thermosensitive hydrogels containing arsenic trioxide liposomes

[0092] At room temperature, a thermosensitive hydrogel containing arsenic trioxide liposomes in sol state was placed into a 1 mL syringe and injected at a constant speed at room temperature. The result was measured to determine whether the hydrogel solution could pass through a long needle. Figure 7 As shown, arsenic trioxide-containing liposomes can pass through the syringe needle in a 1 mL syringe. The arsenic trioxide-containing liposomes that have passed through the needle are then placed in water at 37°C, and gelation is observed to be completed within approximately 1 minute. These results demonstrate that the thermosensitive properties of arsenic trioxide-containing liposomes prevent needle clogging during injection and allow for rapid formation of a structurally intact gel with sufficient support in vivo.

[0093] (5) In vivo antitumor pharmacodynamic study of thermosensitive hydrogels containing arsenic trioxide liposomes

[0094] SPF-grade C57 / BL6 mice (6-8 weeks old, male) were subcutaneously injected with 0.1 mL of B16 cell suspension (containing 1×10⁻⁶ cells) into their backs. 6 A melanoma solid tumor xenograft model was established using [number] cells. On day 5 post-inoculation, the tumor volume was increased to approximately 40 mm. 3 Mice bearing tumors were randomly divided into three groups (n=3): a control group, a free arsenic trioxide group (arsenic trioxide solution), and an arsenic trioxide liposome thermosensitive gel group (preparation group from Example 1). Each group received an intratumoral injection of 100 μL of 0.9% NaCl solution, 100 μL of arsenic trioxide solution, or 100 μL of the arsenic trioxide-containing liposome thermosensitive hydrogel prepared in Example 2 (all containing 0.5 mg / kg of arsenic trioxide). During treatment, the long axis (L) and short axis (W) of the transplanted tumor were measured every other day using electronic calipers. The tumor volume was calculated using the formula: Tumor volume = (L × W) / (L × W) 2 The tumor volume was calculated by dividing the tumor size by 2, and a tumor growth curve was plotted accordingly to dynamically evaluate the tumor-suppressing effect of each treatment group. Simultaneously, the mouse weight was measured and recorded periodically, and a weight change curve was plotted to preliminarily evaluate the in vivo toxicity of the treatment regimen. After treatment, all mice were euthanized, and the tumor masses were carefully dissected and photographed against a uniform background to obtain tumor images through direct comparison. Subsequently, the weight of each tumor mass was measured using a precision electronic balance, and a tumor weight statistical chart was plotted. Further quantitative analysis was used to verify the inhibitory effect of each group on tumor growth.

[0095] The results are as follows Figure 8As shown in Figure A, the tumor growth curves of subcutaneous xenografts in mice in each group are as follows: Compared with the control group, the free arsenic trioxide group showed a certain tumor growth inhibition effect; while the thermosensitive hydrogel containing arsenic trioxide liposomes prepared in Example 1 had a significantly smaller tumor volume at each time point than the control group and the free arsenic trioxide group, indicating that the preparation has better anti-tumor activity at the same dosage. Figure B shows the weight change curves of mice in each group. Throughout the observation period, the weight of mice in each group remained stable, and no obvious abnormalities were observed in activity, feeding, or fur condition, suggesting that the thermosensitive hydrogel containing arsenic trioxide liposomes has good in vivo tolerance. Figure C shows the tumor tissue after treatment. It can be seen that the tumor volume of the control group was the largest, followed by the free arsenic trioxide group, while the tumor volume of the arsenic trioxide liposome thermosensitive gel group was the smallest, and the tumor inhibition effect was the most significant. Figure D shows the tumor weight statistics for each group. Statistical analysis showed that the average tumor weight in the arsenic trioxide liposome thermosensitive gel group was significantly lower than that in the control group and the free arsenic trioxide group (**P<0.01), further confirming the potent inhibitory effect of this preparation on tumor growth.

[0096] Example 2

[0097] The physicochemical properties of the soluble microneedles containing arsenic trioxide liposomes prepared in Example 2 were characterized as follows:

[0098] (1) Morphological observation

[0099] The prepared microneedles were placed on the fingertip, and their tip morphology and distribution were observed with the naked eye. For example... Figure 9 As shown, the needles are arranged in a 10×10 pattern, neatly arranged, and the needles are intact.

[0100] (2) Observation under an optical microscope

[0101] Using a standard optical microscope, place the microneedle patch on a glass slide with the array side facing upwards. Observe the overall distribution using a 4x objective lens, and switch to a 10x objective lens for detailed examination of the local needle tips. Figure 10 As shown, the overall array structure of the microneedles is well-organized, with the microneedles having a length of approximately 840 μm, a diameter of 430 μm, and a tip-to-tip distance of 600 μm.

[0102] (3) Skin puncture test (methylene blue staining method)

[0103] Take 1cm from the back of an isolated mouse 2 For skin penetration, vertically press the microneedle patch into the skin, maintain pressure for 1-2 minutes, gently remove the microneedles, immediately apply 0.4% methylene blue solution to cover the insertion area, let stand for 5 minutes, gently rinse the surface with physiological saline, and observe and photograph the blue micropore array under a microscope. Analyze the number, diameter, and uniformity of the micropores to assess the penetration success rate and needle tip strength.

[0104] Test results are as follows Figure 11 The results showed that after removing the microneedles and staining, a clear and regular array of blue micropores could be observed on the skin surface. The micropores were round, uniform in diameter, and evenly distributed, closely matching the design structure of the microneedle array. The micropore formation rate (i.e., penetration success rate) was extremely high, indicating that the microneedles possess excellent mechanical strength and can effectively and completely penetrate the skin's stratum corneum barrier to form drug delivery channels, confirming their good skin penetration ability and structural reliability.

[0105] (4) Drug loading determination

[0106] The tip portion of the microneedle patch was precisely scraped off with a blade (to avoid substrate interference). The PVP matrix was dissolved in 0.5 mL of chloroform to disrupt the liposome structure and release arsenic trioxide. The patch was sonicated for 10 min to ensure complete drug dissolution. The patch was then centrifuged (12000 rpm, 3 min) to remove insoluble impurities. The supernatant was collected, and the arsenic concentration was measured and calculated using an atomic fluorescence spectrophotometer.

[0107] The soluble microneedles containing arsenic trioxide liposomes prepared in Example 2 were determined to have an average arsenic trioxide content of 4.471 ± 0.095 μg / tablet, which can achieve effective drug loading at the needle tip and meet the dosage accuracy requirements of transdermal drug delivery formulations.

[0108] (5) In vivo antitumor pharmacodynamic study of soluble microneedles containing arsenic trioxide liposomes

[0109] SPF-grade C57 / BL6 mice (6-8 weeks old, male) were subcutaneously injected with 0.1 mL of B16 cell suspension (containing 1×10⁻⁶ cells) into their backs. 6 A melanoma solid tumor xenograft model was established using [number] cells. On day 5 post-inoculation, the tumor volume was increased to approximately 40 mm. 3 Mice bearing tumors were randomly divided into three groups (n=3): a control group, a free arsenic trioxide group (arsenic trioxide solution), and an arsenic trioxide liposome microneedle group (preparation group from Example 2). The control group received intratumoral injections of 100 μL of arsenic trioxide, 0.9% NaCl solution, an intratumoral injection of an equal volume of arsenic trioxide solution (arsenic trioxide content 0.5 mg / kg), and local percutaneous drug administration using soluble microneedles containing arsenic trioxide liposomes prepared in Example 2 (arsenic trioxide content 0.22 mg / kg). During treatment, the long diameter (L) and short diameter (W) of the transplanted tumor were measured every other day using electronic calipers. The tumor volume was calculated using the formula: Tumor volume = (L × W) / (L × W) / W. 2The tumor volume was calculated by dividing the tumor size by 2, and a tumor growth curve was plotted accordingly to dynamically evaluate the tumor-suppressing effect of each treatment group. Simultaneously, the mouse weight was measured and recorded periodically, and a weight change curve was plotted to preliminarily evaluate the in vivo toxicity of the treatment regimen. After treatment, all mice were euthanized, and the tumor masses were carefully dissected and photographed against a uniform background to obtain tumor images through direct comparison. Subsequently, the weight of each tumor mass was measured using a precision electronic balance, and a tumor weight statistical chart was plotted. Further quantitative analysis was used to verify the inhibitory effect of each group on tumor growth.

[0110] Test results are as follows Figure 12 As shown in Figure A, the tumor growth curves of subcutaneous xenografts in mice in each group are as follows: Compared with the control group, the free arsenic trioxide group showed a certain inhibitory effect on tumor growth, but the effect was limited; while the soluble microneedles containing arsenic trioxide liposomes prepared in Example 2 had significantly smaller tumor volumes than the control group and the free arsenic trioxide group throughout the entire treatment period, indicating that the microneedle preparation can achieve sustained and effective anti-tumor activity through local delivery. Figure B shows the weight change curves of mice in each group. During the observation period, the weight of mice in each group remained stable, and no obvious abnormalities were observed in activity, feeding, or fur condition, indicating that the percutaneous administration route of arsenic trioxide liposome microneedles was safe and had no significant systemic toxicity. Figure C shows the tumor tissue that was peeled off after treatment. It can be seen that the tumor volume of the control group was the largest, followed by the free arsenic trioxide group, while the tumor volume of the arsenic trioxide liposome microneedle group was the smallest, and the tumor inhibition effect was the most significant. Figure D shows the tumor weight statistics for each group. Statistical analysis showed that the average tumor weight in the arsenic trioxide liposome microneedle group was significantly lower than that in the control group and the free arsenic trioxide group (***P<0.001, **P<0.01, *P<0.05), further confirming the potent inhibitory effect of this preparation on tumor growth.

[0111] In summary, this invention successfully prepared arsenic trioxide liposomes, a thermosensitive hydrogel containing arsenic trioxide liposomes, and soluble microneedles containing arsenic trioxide liposomes for local drug delivery. Preparation process studies showed that the obtained liposomes had regular morphology, uniform particle size, and good encapsulation efficiency; the thermosensitive liposome gel underwent a sol-gel phase transition under physiological conditions, and the liposome microneedles exhibited a neat array and suitable mechanical strength, all demonstrating excellent formulation properties. In vivo pharmacodynamic evaluation results showed that in a melanoma-bearing mouse model, the thermosensitive hydrogel containing arsenic trioxide liposomes and the soluble microneedles containing arsenic trioxide liposomes prepared in this invention showed significantly smaller tumor volumes at all time points compared to the free arsenic trioxide group, indicating a more prominent tumor-suppressing effect. Photographs of the dissected tumor tissue and tumor weight statistics after treatment further confirmed that both formulations effectively inhibited tumor growth, with significantly better efficacy than the free drug group. Regarding safety evaluation, the weight of mice in each group remained stable throughout the observation period, and no significant abnormalities were observed in activity, food intake, or fur condition, indicating that both formulations had good in vivo tolerability. The above results indicate that the present invention achieves enhanced efficacy and reduced toxicity in superficial tumors by improving drug delivery efficiency and local retention, and has potential clinical application value.

[0112] In addition, the arsenic trioxide liposome in situ gel of the present invention can also be administered by topical application, and is suitable for the treatment of superficial tumors.

[0113] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A topical arsenic trioxide-loaded formulation for treating superficial tumors, characterized in that, This includes thermosensitive hydrogels containing arsenic trioxide liposomes and soluble microneedles containing arsenic trioxide liposomes.

2. The topical administration formulation according to claim 1, characterized in that, Thermosensitive hydrogel containing arsenic trioxide liposomes was prepared by arsenic trioxide liposomes, gel matrix, pH adjuster and purified water through swelling method, ion-induced gelation, blending and composite method and photocrosslinking method. The molar percentages of the arsenic trioxide liposomes, gel matrix, pH adjuster, and purified water are 5%~40%, 6%~35%, 0.1%~2%, and 50%~70%, respectively.

3. The topical administration formulation according to claim 1, characterized in that, Soluble microneedles containing arsenic trioxide liposomes were prepared by arsenic trioxide liposomes, microneedle matrix and purified water through molding method, 3D printing method and micro-injection molding method. The molar percentages of the arsenic trioxide liposomes, microneedle matrix, and purified water are 5%~30%, 10%~30%, and 40%~85%, respectively.

4. The topical administration formulation according to claim 2 or 3, characterized in that, Arsenic trioxide liposomes were prepared from arsenic trioxide, phospholipids and cholesterol via thin-film dispersion, injection, ion gradient, reverse-phase evaporation, freeze-thaw, and extrusion methods. The phospholipid is one or a mixture of egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and polyethylene glycol-modified phospholipids; The molar percentages of arsenic trioxide, phospholipids, and cholesterol in the arsenic trioxide liposomes are 1%~40%, 1%~95%, and 1%~45%, respectively.

5. The topical administration formulation according to claim 4, characterized in that, The molar percentages of arsenic trioxide, phospholipids, and cholesterol in arsenic trioxide liposomes are 1%–25%, 50%–70%, and 10%–25%, respectively.

6. The topical administration formulation according to claim 2, characterized in that, The gel matrix is ​​one or more of the following: Pluronic acid, chitosan, gelatin, methylcellulose, and hydroxypropyl methylcellulose.

7. The topical administration formulation according to claim 2, characterized in that, The molar percentages of arsenic trioxide liposomes, gel matrix, pH adjuster, and purified water are 5%~25%, 15%~35%, 0.5%~1%, and 50%~65%, respectively.

8. The topical administration formulation according to claim 3, characterized in that, The microneedle matrix is ​​one or more of polyvinylpyrrolidone, hyaluronic acid, polyvinyl alcohol, and chitosan.

9. The topical administration formulation according to claim 3, characterized in that, The molar percentages of arsenic trioxide liposomes, microneedle matrix, and purified water are 10%~20%, 20%~30%, and 50%~70%, respectively.

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