Hyaluronic acid and sodium alginate double-layer modified liposome microsphere and preparation method thereof

Abstract

The invention belongs to the technical field of liposome preparation, and relates to hyaluronic acid and sodium alginate double-layer modified liposome microspheres and a preparation method thereof.The preparation method comprises the steps that firstly, lecithin, ginsenoside Rg3 and tripterine are weighed according to a certain proportion and added into ethyl alcohol, ultrasonic treatment is conducted to obtain an ethyl alcohol mixed solution, then a hydration solution is obtained through a thin film hydration method, and then the liposome microspheres are prepared. The preparation method comprises the following steps: firstly, carrying out ultrasonic treatment on a hydrated solution, then carrying out ice-bath treatment on the hydrated solution, carrying out ultrasonic crushing and filtering to obtain a liposome solution, firstly modifying hyaluronic acid on the surface of the liposome, and then coating sodium alginate to form a double-layer liposome microsphere with targeting transportation and pH response, and the microsphere can respond and release a drug in a weak acid environment in an oral administration manner, so that the drug delivery efficiency is improved. The stability and the targeting property of the medicine are effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome preparation, and relates to a hyaluronic acid and sodium alginate double-layer modified liposome microsphere and a preparation method thereof. Background Art

[0002] Colon cancer is a common malignancy with high morbidity and mortality. With changing lifestyles and an aging population, the risk of colon cancer is increasing year by year. Traditional treatments such as surgery, chemotherapy, and radiotherapy, while able to prolong patient survival to a certain extent, are often accompanied by significant side effects and a high risk of recurrence. Therefore, the development of novel, highly effective, and low-toxic treatments for colon cancer is crucial.

[0003] Despite progress in current colorectal cancer treatments, limitations remain, such as inefficient drug delivery, significant side effects, and poor patient tolerance. Traditional chemotherapy drugs often lack targeting, leading to uneven drug distribution throughout the body, which not only compromises therapeutic efficacy but also increases side effects for patients. Furthermore, some colorectal cancer patients develop resistance to chemotherapy drugs, significantly compromising treatment effectiveness.

[0004] While liposomes have shown great potential as drug carriers in tumor therapy, their application in colorectal cancer treatment still faces certain limitations. On the one hand, conventional liposomes lack targeting, making it difficult to accurately deliver drugs to tumor sites, resulting in uneven drug distribution in the body and reduced therapeutic efficacy. On the other hand, the complexity of the tumor microenvironment, such as acidic pH and high osmotic pressure, can affect liposome stability and drug release behavior, thereby limiting its therapeutic efficacy. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a liposome microsphere modified with a double layer of hyaluronic acid and sodium alginate and a preparation method thereof. The present invention replaces the cholesterol in traditional liposomes by using ginsenoside Rg3, and combines the first layer of hyaluronic acid modification and the second layer of sodium alginate wrapping. Through the specific binding of hyaluronic acid and sodium alginate, the responsive release and targeted transport of liposome microspheres in a specific pH environment are achieved, while improving the stability and biocompatibility of liposome microspheres, enhancing their targeting to target cells and drug release efficiency, and providing a new solution for the delivery of poorly soluble drugs such as tripterygium wilfordii.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weighing lecithin, ginsenoside Rg3 and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, and then rotary evaporating the ethanol mixture to dryness to obtain a lipid film; The lipid film is mixed with a phosphate buffered saline solution and then hydrated to obtain a hydrated solution, which is then ultrasonically broken in an ice bath to obtain a liposome solution to be filtered; Filtering the liposome solution to be filtered through a filter membrane to obtain a liposome solution, mixing the liposome solution with a hyaluronic acid solution, incubating with stirring, and allowing to stand to obtain a hyaluronic acid-modified liposome solution; The hyaluronic acid modified liposome solution and the sodium alginate solution are mixed and stirred, and then added dropwise into the calcium chloride solution to obtain liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate.

[0007] Hyaluronic acid in this invention is a natural polysaccharide with excellent biocompatibility and degradability. It can bind to various cell surface receptors (such as CD44) to achieve targeted delivery. Sodium alginate is an anionic polysaccharide with excellent film-forming properties and stability, which can further enhance the stability and bioavailability of the microspheres. In addition, replacing cholesterol in traditional liposomes with ginsenoside Rg3 not only reduces cholesterol-related side effects such as pulmonary hypertension, but also imparts enhanced bioactivity and pharmacological effects to the liposome microspheres. In a preferred embodiment of the present invention, the ratio of ammonium molybdate powder to azeotropic solution is 550 g: 2 L to 3 L.

[0008] In a preferred embodiment of the present invention, the mass ratio of lecithin, ginsenoside Rg3 and tripterygium wilfordii is 100:30:1-8, and the mass fraction of ethanol is 100%.

[0009] In a preferred embodiment of the present invention, the evaporation temperature of the ethanol mixture to dryness is 48°C to 50°C, the evaporation time is 0.75h to 1h, the hydration reaction temperature is 55°C to 60°C, and the hydration reaction time is 1h to 1.5h.

[0010] In a preferred embodiment of the present invention, the ratio of the lipid film to the phosphate buffered saline solution in the hydration reaction is 1:1 to 1.2.

[0011] In a preferred embodiment of the present invention, the ice bath ultrasonic conditions are: ultrasonic power of 270W to 290W, ultrasonic temperature of 25°C to 28°C, intermittent ultrasonication, ultrasonication on for 3s to 5s and off for 5s, and ultrasonication time of 10min to 25min.

[0012] In a preferred embodiment of the present invention, the pore size of the filter membrane in the membrane filtration is 0.22 μm, the volume ratio of the liposome solution to the hyaluronic acid solution is 1:1-2, the mass fraction of hyaluronic acid is 1%, the stirring incubation time is 2.5h-3h, the standing time is 0.5h-1h, and the rotation speed is 300r / min-500r / min.

[0013] In a preferred embodiment of the present invention, the volume ratio of the hyaluronic acid-modified liposome solution to sodium alginate is 1:1-4, and the concentration of the sodium alginate solution is 0.1M.

[0014] In a preferred embodiment of the present invention, the sodium alginate solution is prepared by weighing 2 g of sodium alginate powder and adding it to 100 ml of ultrapure water. The solution is then placed in a rotor and transferred to a magnetic stirrer for heating and stirring until the ammonium alginate is completely dissolved.

[0015] In a preferred embodiment of the present invention, the calcium chloride solution is prepared by weighing 5.55 g of calcium chloride powder and dissolving it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution.

[0016] Another object of the present invention is to provide a hyaluronic acid and sodium alginate double-layer modified liposome microsphere prepared by any of the preparation methods described above, wherein the hyaluronic acid and sodium alginate double-layer modified liposome microsphere has targeted transport and pH response functions.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 10. The present invention weighs lecithin, ginsenoside Rg3 and tripterygium wilfordii, adds them to ethanol, and performs ultrasonic treatment to obtain an ethanol mixture, the ethanol mixture is rotary evaporated to dryness to obtain a lipid film, the lipid film is mixed with a phosphate buffered saline solution and then subjected to a hydration reaction to obtain a hydrated liquid, the hydrated liquid is ultrasonically crushed in an ice bath to obtain a liposome solution to be filtered, the liposome solution to be filtered is filtered through a filter membrane to obtain a liposome solution, the liposome solution is mixed with a hyaluronic acid solution, incubated with stirring, and allowed to stand to obtain a hyaluronic acid-modified liposome solution, the hyaluronic acid-modified liposome solution is mixed and stirred with a sodium alginate solution, and then added dropwise to a calcium chloride solution to obtain liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate, which can achieve precise delivery of tripterygium wilfordii at the lesion site, reduce the distribution of the drug in normal tissues, and thus reduce the systemic toxicity and side effects of the drug. The liposome microspheres of the present invention are not only suitable for the delivery of tripterine, but can also be used for the delivery of other drugs with similar physicochemical properties and pharmacological activities. In addition, the preparation method can be adjusted and optimized according to specific needs to meet the delivery requirements of different drugs.

[0018] 11. This invention uses hyaluronic acid as the first layer of the modified material. Hyaluronic acid can specifically bind to receptors (such as CD44) on the surfaces of specific cells (such as cancer cells), thereby achieving active targeted drug delivery, increasing drug concentration at the lesion site, and reducing systemic side effects. The modified liposomes, coated with sodium alginate as the outer layer, are pH-responsive. In acidic environments (such as the tumor microenvironment), sodium alginate undergoes a gelation transition, thereby regulating the drug release rate. This pH-responsive release mechanism facilitates sustained, controlled drug release at the lesion site, enhancing therapeutic efficacy. Using ginsenoside Rg3 instead of traditional cholesterol as the liposome membrane material significantly improves liposome stability. Ginsenoside Rg3 has a steroid structure similar to cholesterol, but possesses greater bioactivity and compatibility, helping to maintain liposome structural integrity and prolong drug circulation in vivo. Therefore, liposome microspheres modified with a hyaluronic acid and sodium alginate bilayer can enhance drug targeting, pH-responsive release, improve liposome stability, and reduce drug toxicity. They can also be used to deliver other drugs with similar physicochemical properties and pharmacological activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a flow chart of the preparation process of the sodium alginate and hyaluronic acid double-modified liposomes of the present invention.

[0020] Figure 2 This is a low-speed electron microscopy image of the sodium alginate and hyaluronic acid dual-modified liposomes prepared in Example 1 of the present invention.

[0021] Figure 3 This is an electron micrograph of the medium-speed group of liposomes doubly modified with sodium alginate and hyaluronic acid prepared in Example 2 of the present invention.

[0022] Figure 4 This is a high-speed electron microscope image of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 3 of the present invention.

[0023] Figure 5 This is a high-speed electron microscope image of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 4 of the present invention.

[0024] Figure 6 This is a high-speed electron microscope image of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 5 of the present invention.

[0025] Figure 7 This is a high-speed electron microscope image of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 8 of the present invention.

[0026] Figure 8 This is a particle size analysis diagram of the low-speed group of liposomes dual-modified with sodium alginate and hyaluronic acid prepared in Example 1 of the present invention.

[0027] Figure 9 This is a particle size analysis diagram of the medium-speed group of liposomes double-modified with sodium alginate and hyaluronic acid prepared in Example 2 of the present invention.

[0028] Figure 10 This is a particle size analysis diagram of the high-speed group of liposomes dual-modified with sodium alginate and hyaluronic acid prepared in Example 3 of the present invention.

[0029] Figure 11 This is a particle size analysis diagram of the unmodified liposome prepared in Comparative Example 1 of the present invention.

[0030] Figure 12 This is a physical picture of the sodium alginate and hyaluronic acid double-modified liposome microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The following is a detailed description of the technical solutions in the embodiments of the present invention, using preferred embodiments and accompanying drawings in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] It should be noted that all professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0033] A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate, such as Figure 1 As shown, the following steps are included: Weighing lecithin, ginsenoside Rg3 and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, and then rotary evaporating the ethanol mixture to dryness to obtain a lipid film; The lipid film is mixed with a phosphate buffered saline solution and then hydrated to obtain a hydrated solution, which is then ultrasonically broken in an ice bath to obtain a liposome solution to be filtered; filtering the liposome solution to be filtered through a filter membrane to obtain a liposome solution, mixing the liposome solution with a hyaluronic acid solution, incubating with stirring, and allowing to stand to obtain a hyaluronic acid-modified liposome solution; The hyaluronic acid modified liposome solution and the sodium alginate solution are mixed and stirred, and then added dropwise into the calcium chloride solution to obtain liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate.

[0034] Example 1 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Lecithin, ginsenoside Rg3, and celastrol in a mass ratio of 100:30:1 were weighed and added to ethanol, and ultrasonically treated to obtain an ethanol mixture. The ethanol mixture was then placed on a rotary evaporator and evaporated to dryness under vacuum conditions at 50°C to remove the ethanol and obtain a lipid film. The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonicated at a power of 270 W and a temperature of 25°C, with intermittent ultrasonication (3 s on, 5 s off) for 25 min to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 300 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0035] Example 2 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weighing 100:30:1 lecithin, ginsenoside Rg3, and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, then placing the mixture on a rotary evaporator and rotary evaporating it to dryness at 50°C under vacuum conditions to remove the ethanol and obtain a lipid film; The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonicated at a power of 270 W and a temperature of 25°C, with intermittent ultrasonication (3 s on, 5 s off) for 25 min to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 400 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0036] Example 3 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weighing 100:30:1 lecithin, ginsenoside Rg3, and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, then placing the mixture on a rotary evaporator and rotary evaporating it to dryness at 50°C under vacuum conditions to remove the ethanol and obtain a lipid film; The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonicated at a power of 270 W and a temperature of 25°C, with intermittent ultrasonication (3 s on, 5 s off) for 25 min to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0037] Example 4 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weighing 100:30:1 lecithin, ginsenoside Rg3, and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, then placing the mixture on a rotary evaporator and rotary evaporating it to dryness at 50°C under vacuum conditions to remove the ethanol and obtain a lipid film; The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonic power was set to 270 W, ultrasonic temperature was set to 25°C, and ultrasonication was performed intermittently, with ultrasonication for 5 seconds on and 5 seconds off, and ultrasonication was performed for 10 minutes to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0038] Example 5 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weigh 200:60:3 of lecithin, ginsenoside Rg3, and triptolide into ethanol, and perform ultrasonic treatment to obtain an ethanol mixture. The ethanol mixture is then placed on a rotary evaporator and evaporated to dryness under vacuum conditions at 50°C to remove the ethanol and obtain a lipid film. The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonic power was set to 270 W, ultrasonic temperature was set to 25°C, and ultrasonication was performed intermittently, with ultrasonication for 5 seconds on and 5 seconds off, and ultrasonication was performed for 10 minutes to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0039] Example 6 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weigh 50:15:1 lecithin, ginsenoside Rg3, and triptolide in ethanol, and perform ultrasonic treatment to obtain an ethanol mixture. The ethanol mixture is then placed on a rotary evaporator and evaporated to dryness under vacuum conditions at 50°C to remove the ethanol and obtain a lipid film. The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonic power was set to 270 W, ultrasonic temperature was set to 25°C, and ultrasonication was performed intermittently, with ultrasonication for 5 seconds on and 5 seconds off, and ultrasonication was performed for 10 minutes to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0040] Example 7 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weigh 50:15:3 lecithin, ginsenoside Rg3, and triptolide into ethanol, and perform ultrasonic treatment to obtain an ethanol mixture. The ethanol mixture is then placed on a rotary evaporator and evaporated to dryness under vacuum conditions at 50°C to remove the ethanol and obtain a lipid film. The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonic power was set to 270 W, ultrasonic temperature was set to 25°C, and ultrasonication was performed intermittently, with ultrasonication for 5 seconds on and 5 seconds off, and ultrasonication was performed for 10 minutes to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0041] Example 8 A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate comprises the following steps: Weigh 50:15:4 lecithin, ginsenoside Rg3, and triptolide into ethanol, and perform ultrasonic treatment to obtain an ethanol mixture. The ethanol mixture is then placed on a rotary evaporator and evaporated to dryness under vacuum conditions at 50°C to remove the ethanol and obtain a lipid film. The lipid film was then mixed with 10 mL of phosphate buffered saline at 55°C for hydration to obtain a hydrated solution. The hydrated solution was placed in an ice bath and ultrasonic power was set to 270 W, ultrasonic temperature was set to 25°C, and ultrasonication was performed intermittently, with ultrasonication for 5 seconds on and 5 seconds off, and ultrasonication was performed for 10 minutes to obtain a liposome solution to be filtered. The liposome solution to be filtered was filtered through a 0.22 μm filter membrane to obtain a liposome solution, which was mixed with a hyaluronic acid solution at a volume ratio of 1:1, stirred at 500 rpm for 3 h, and allowed to stand for 1 h to obtain a hyaluronic acid-modified liposome solution; Weigh 2 g of sodium alginate powder and add it to 100 mL of ultrapure water. Place the rotor and transfer it to a magnetic stirrer while heating and stirring until the sodium alginate is completely dissolved to obtain a sodium alginate solution. Weigh 5.55 g of calcium chloride powder and dissolve it in 500 ml of deionized water to prepare a 0.1 M calcium chloride solution. Mix the hyaluronic acid-modified liposome solution with the sodium alginate solution in a volume ratio of 1:4, stir for 0.5 h, and then add the mixture dropwise to the 0.1 M calcium chloride solution through a 26G syringe needle to obtain double-layer liposome microspheres.

[0042] Comparative Example 1 A method for preparing liposomes loaded with tripterygium wilfordii comprises the following steps: Weigh 100:30:1 lecithin, ginsenoside Rg3 and tripterygium wilfordii into ethanol and perform ultrasonic treatment to obtain an ethanol mixture; place the ethanol mixture on a rotary evaporator and evaporate it to dryness under vacuum conditions at 50°C to remove the ethanol to obtain a lipid film; at 55°C, mix the lipid film with 10 ml of PBS solution and perform a hydration reaction for 1 hour to obtain a hydrated solution, put the hydrated solution in an ice bath, set the ultrasonic power to 270 W, the ultrasonic temperature to 25°C, perform intermittent ultrasonication, ultrasonicate for 3 seconds and stop for 5 seconds, and ultrasonically crush for 25 minutes to obtain a liposome solution to be filtered; filter the liposome solution to be filtered through a 0.22 μm filter membrane to obtain liposomes loaded with tripterygium wilfordii.

[0043] Comparative Example 2 A method for preparing liposomes loaded with tripterygium wilfordii comprises the following steps: Weigh 100:30:1 lecithin, ginsenoside Rg3 and tripterygium wilfordii into ethanol and perform ultrasonic treatment to obtain an ethanol mixture; place the ethanol mixture on a rotary evaporator and evaporate it to dryness under vacuum conditions at 50°C to remove the ethanol to obtain a lipid film; at 55°C, mix the lipid film with 10 ml of PBS solution and perform a hydration reaction for 1 hour to obtain a hydrated solution, put the hydrated solution in an ice bath, set the ultrasonic power to 270 W, the ultrasonic temperature to 25°C, perform intermittent ultrasonication, ultrasonicate for 5 seconds and stop for 5 seconds, and ultrasonically crush for 10 minutes to obtain a liposome solution to be filtered; filter the liposome solution to be filtered through a 0.22 μm filter membrane to obtain liposomes loaded with tripterygium wilfordii.

[0044] Result Analysis The average particle size difference between the low-speed liposomes doubly modified with sodium alginate and hyaluronic acid and the unmodified liposomes is 27.19 nm; the average particle size difference between the medium-speed liposomes doubly modified with sodium alginate and hyaluronic acid and the unmodified liposomes is 15.03 nm; the average particle size difference between the high-speed liposomes doubly modified with sodium alginate and hyaluronic acid and the unmodified liposomes is 26.07 nm. It can be concluded that the particle size difference between the low-speed and high-speed grades is large, and the hyaluronic acid wraps the liposomes more completely. The particle size difference between the medium-speed grades is smaller, and the hyaluronic acid wrapping may be uneven, which also needs to be observed in combination with electron microscope images.

[0045] Figures 2 to 4 The electron microscopic images of the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 1 to 3 at low, medium and high speeds show that: although the hyaluronic acid-modified liposomes in the low-speed group of Example 1 have the largest difference in particle size, their morphology is irregular. Figure 5The electron microscope image of the high-speed liposomes of sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 4 shows that the liposome structure of the high-speed group with the same drug dosage is complete, the packaging is uniform, the morphology is round and smooth, and it has a clear double-layer membrane structure, which proves that low speed affects the integrity of the liposome structure. The medium-speed group of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 2 has the smallest particle size difference and is not suitable as the final speed. The high-speed group of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Example 3 has a round and full morphology, hyaluronic acid is evenly wrapped on the surface of the liposomes, and has a clear double-layer membrane structure, which proves that 500r / min should be used as the final speed.

[0046] Figure 5 、 Figure 6 、 Figure 7 Electron micrographs of high-speed liposomes double-modified with sodium alginate and hyaluronic acid prepared in Example 4, Example 5, and Example 8, respectively. Except for the amount of tripterygium wilfordii added, the other parameters are the same. The amount of tripterygium wilfordii added in Example 4 is 1 mg, the amount of tripterygium wilfordii added in Example 5 is 1.5 mg, and the amount of tripterygium wilfordii added in Example 8 is 8 mg. Figures 5-7 Available, Figure 5 That is, the liposomes in the electron microscopy image of Example 4 have the most regular morphology, the most rounded and smooth surface, and the most obvious double-layer membrane structure. Therefore, 1 mg was selected as the final dosage.

[0047] Table 1 is a summary table of the average particle size differences between the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 1 to 8 and the celastrol-loaded liposomes prepared in Comparative Examples 1 and 2. The particle size data of the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 4 to 8 and Examples 1 to 3 can be concluded that after changing the ultrasonic crushing parameters to 5s on and 5s off, and the crushing time to 10min, the particle size of the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 4 to 8 is significantly smaller than that in Examples 1 to 3, proving that changing the ultrasonic crushing parameters optimizes the liposome structure, which is conducive to the preparation of liposomes with smaller and better particle sizes. By comparing the particle size difference data of the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 4 to 8 and the celastrol-loaded liposomes prepared in Comparative Example 2, it can be concluded that the maximum particle size difference between Example 4 and Comparative Example 1 is 22.93 nm, and the particle size differences of the sodium alginate and hyaluronic acid doubly modified liposomes prepared in Examples 5 to 8 and the celastrol-loaded liposomes prepared in Comparative Example 1 are 8.74 nm, 16.28 nm, 9.29 nm, and 6.87 nm, respectively. At the same rotation speed, a celastrol dosage of 1 mg should be selected as the final dosage.

[0048] Figures 8-11The particle size analysis diagrams of the sodium alginate and hyaluronic acid double-modified liposomes prepared in Examples 1 to 3 and Comparative Example 1 are shown. Figures 8-11 It can be seen that the liposome particles prepared by the present invention have uniform size distribution and are of uniform size, which is beneficial to the consistency and repeatability of subsequent drug delivery.

[0049] Figure 12 This is a physical picture of the sodium alginate and hyaluronic acid double-modified liposome microspheres prepared in Example 1. It can be seen from the figure that the prepared microspheres are uniform in size, round in shape, and complete in structure.

[0050] Table 1 is a summary of the average particle size differences between the sodium alginate and hyaluronic acid dual-modified liposomes prepared in Examples 1 to 8 of the present invention and the liposomes loaded with tripterygium wilfordii prepared in Comparative Examples 1 and 2. It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0051] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate, characterized in that: The following steps are involved: Weighing lecithin, ginsenoside Rg3 and triptolide into ethanol, and ultrasonically treating the mixture to obtain an ethanol mixture, and then rotary evaporating the ethanol mixture to dryness to obtain a lipid film; The lipid film is mixed with a phosphate buffered saline solution and then hydrated to obtain a hydrated solution, which is then ultrasonically broken in an ice bath to obtain a liposome solution to be filtered; filtering the liposome solution to be filtered through a filter membrane to obtain a liposome solution, mixing the liposome solution with a hyaluronic acid solution, incubating with stirring, and allowing to stand to obtain a hyaluronic acid-modified liposome solution; The hyaluronic acid modified liposome solution and the sodium alginate solution are mixed and stirred, and then added dropwise into the calcium chloride solution to obtain liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate.

2. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The mass ratio of the lecithin, ginsenoside Rg3 and tripterygium wilfordii is 100:30:1-8.

3. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The evaporation temperature of the ethanol mixture to dryness is 48° C. to 50° C., the evaporation time is 0.75 h to 1 h, the hydration reaction temperature is 55° C. to 60° C., and the hydration reaction time is 1 h to 1.5 h.

4. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The ratio of the lipid film to the phosphate buffered saline solution in the hydration reaction is 131 mg:10 ml.

5. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The ice bath ultrasonic conditions are as follows: ultrasonic power of 270W to 290W, ultrasonic temperature of 25°C to 28°C, intermittent ultrasonication, ultrasonication on for 3s to 5s and off for 5s, and ultrasonication time of 10min to 25min.

6. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The pore size of the filter membrane in the membrane filtration is 0.22 μm, the volume ratio of the liposome solution to the hyaluronic acid solution is 1:1-2, the mass fraction of hyaluronic acid is 1%, the stirring incubation time is 2.5h-3h, the standing time is 0.5h-1h, and the rotation speed is 300r / min-500r / min.

7. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The volume ratio of the hyaluronic acid-modified liposome solution to the sodium alginate solution is 1:1-4, and the concentration of the sodium alginate solution is 0.1M.

8. The method for preparing liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 1, characterized in that: The concentration of the calcium chloride solution is 0.1M.

9. A liposome microsphere modified with a double layer of hyaluronic acid and sodium alginate prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate have targeted transport and pH response functions.

10. Use of the liposome microspheres modified with a double layer of hyaluronic acid and sodium alginate according to claim 9 in preparing a drug for treating colon cancer.

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