Erythromycin nano-liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses erythromycin nano-liposome as well as a preparation method and application thereof. Vitamin E acetate and resveratrol are adopted to construct a dual stable system, a high-pressure homogenization method is adopted to prepare the erythromycin nano-liposome, and the erythromycin nano-liposome has the advantages of high transdermal efficiency, excellent bioavailability, strong stability, high encapsulation efficiency, accurate targeting, medication safety and the like. On the basis, the invention further provides the erythromycin in-situ gel which is good in clinical efficacy, safe and comfortable in medication and good in stability and biocompatibility, meanwhile, the appearance smoothness after wound healing can be improved, and the dual requirements of patients for wound healing efficiency and healing quality can be better met. The invention can be applied to the field of external preparations.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and specifically discloses an erythromycin nanoliposome, its preparation method, and its application. Background Technology

[0002] In the clinical treatment of skin infections, topical preparations are a common treatment for mild infections caused by minor skin injuries, small-area burns, or ulcers. These preparations can act directly on the lesion site and have significant advantages such as high local drug concentration, definite efficacy, effective avoidance of the first-pass effect of the gastrointestinal tract and liver, and ease of use, and are widely used in clinical practice.

[0003] Erythromycin, a typical macrolide antibiotic, has a well-defined antibacterial mechanism: it specifically binds to the 50S subunit of bacterial ribosomes, inhibiting peptidyl transferase activity, thereby interfering with ribosome translocation, hindering peptide chain elongation, and ultimately inhibiting bacterial protein biosynthesis, thus achieving its antibacterial effect. Based on this mechanism, erythromycin ointment has become a commonly used topical skin preparation in clinical practice, widely applied to the treatment of mild to moderate bacterial skin and soft tissue infections, small-area burns, and infected ulcers.

[0004] However, traditional ointments and creams for topical application of erythromycin have several shortcomings: firstly, the large particle size of the active ingredient leads to low transdermal absorption and poor bioavailability; secondly, insufficient drug stability makes it prone to burst release, resulting in significant fluctuations in blood drug concentration, which not only affects the stability of therapeutic effects but may also increase the risk of local adverse reactions. These deficiencies limit the clinical application of traditional topical erythromycin preparations. Therefore, developing a topical erythromycin preparation with high transdermal absorption efficiency, high bioavailability, and good stability has significant clinical value and application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an erythromycin nanoliposome, its preparation method, and its applications. This invention provides an erythromycin nanoliposome with high transdermal efficiency, high encapsulation efficiency, excellent bioavailability, strong stability, precise targeting, and safe administration. Using erythromycin nanoliposomes and polysaccharide-grafted N-isopropylacrylamide as raw materials, a further erythromycin in-situ gel with advantages such as high transdermal efficiency, precise targeting, long-lasting efficacy, safe and comfortable administration, and multifunctional synergistic repair is also provided.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an erythromycin nanoliposome comprising 5 g / L-10 g / L erythromycin, 10 g / L-100 g / L lipid raw material, 20 g / L-50 g / L emulsifier, 1 g / L-5 g / L vitamin E acetate and 0.1 g / L-5 g / L resveratrol.

[0007] The erythromycin nanoliposomes provided by this invention have advantages such as high transdermal efficiency, excellent bioavailability, strong stability, high encapsulation efficiency, precise targeting, and safe administration. This invention utilizes liposome nanocompositing technology to form erythromycin into nanoliposome structures with small particle size and large specific surface area. This structure not only effectively overcomes the skin's stratum corneum barrier, promoting rapid drug penetration into deep skin tissues and significantly improving drug permeability, but also overcomes the shortcomings of traditional formulations, such as large active ingredient particle size, poor transdermal absorption, and difficulty in reaching deep skin layers to exert its effect. Furthermore, it enables slow drug release, effectively avoiding the sudden release phenomenon common in traditional formulations and significantly reducing fluctuations in blood drug concentration.

[0008] This invention employs a dual-stabilization system constructed from vitamin E acetate and resveratrol. Vitamin E acetate acts as a stabilizer, significantly enhancing the antioxidant capacity of nanoliposomes, reducing oxidative degradation during storage or use, and improving drug encapsulation efficiency to ensure stable loading of erythromycin within the liposomes. Resveratrol further strengthens the stabilizing effect, synergistically working with vitamin E acetate to substantially improve the overall stability of the formulation, extend shelf life, and ensure uniform formulation quality. Furthermore, resveratrol possesses targeted delivery capabilities, interacting with specific receptors or signaling molecules at inflamed lesions to guide nanoliposomes to actively aggregate at the site of inflammation, achieving targeted drug delivery. This characteristic not only further increases drug concentration at the lesion site, enhancing therapeutic specificity, but also reduces drug distribution in normal tissues, minimizing potential irritation to healthy skin, thus optimizing medication safety while improving efficacy.

[0009] Preferably, the lipid raw material includes at least one of glyceryl monostearate, glyceryl caprylate, lecithin, cholesterol, or polyethylene glycol phospholipid.

[0010] Preferably, the emulsifier comprises at least one of poloxamer 188 or Tween-80. Secondly, the present invention provides a method for preparing the above-mentioned erythromycin nanoliposomes, the method comprising the following steps: S1. melting the lipid raw material and mixing it with erythromycin to obtain an oil phase; The emulsifier, vitamin E acetate, resveratrol and water were mixed to obtain an aqueous phase; S2. The oil phase and the aqueous phase are mixed to obtain an initial dispersion system; S3. Erythromycin nanoliposomes were prepared by high-pressure homogenization of the initial dispersion system.

[0011] This invention uses a high-pressure homogenization method to prepare erythromycin nanoliposomes. This preparation method has core advantages such as strong process adaptability, controllable product quality, and outstanding industrialization potential. It can also form a synergistic effect with the formulation design of nanoliposomes, further ensuring product performance.

[0012] The reasons for choosing high-pressure homogenization to prepare erythromycin nanoliposomes in this invention are as follows: First, high-pressure homogenization, through the synergistic effect of high-pressure shearing, impaction, and cavitation, can fully break down and uniformly disperse lipid particles in the initial oil-water dispersion system, resulting in erythromycin nanoliposomes with a narrow particle size distribution and uniform particle morphology. Second, the strong mechanical forces during high-pressure homogenization promote the full fusion of erythromycin and lipid raw materials, allowing the drug to be more tightly encapsulated in the liposome bilayer or inner aqueous phase structure, significantly improving the drug encapsulation efficiency. Simultaneously, this process reduces liposome aggregation and drug leakage during preparation, and, combined with the dual stabilizing effect of vitamin E acetate and resveratrol, further enhances the drug-carrying stability of the nanoliposomes. Third, the mild preparation environment of high-pressure homogenization protects the antioxidant activity of vitamin E acetate and the structural stability of resveratrol, preventing functional loss; while the uniform liposome structure provides a stable carrier basis for the specific binding of resveratrol to receptors at inflammatory sites, ensuring efficient targeted aggregation.

[0013] In summary, the preparation method of erythromycin nanoliposomes provided by this invention is simple, efficient and controllable, and can accurately ensure the uniformity of particle size, high encapsulation efficiency and stability of erythromycin nanoliposomes, and has the potential for industrial application.

[0014] Preferably, in the high-pressure homogenization method, the pressure is 60000kPa-80000kPa, and the number of cycles is 3-6.

[0015] Thirdly, the present invention provides the application of the erythromycin nanoliposomes provided in the first aspect in the preparation of erythromycin in situ gel.

[0016] Fourthly, the present invention provides an erythromycin in situ gel comprising the erythromycin nanoliposomes described in the first aspect, and at least one substance selected from temperature-sensitive matrix materials, thickeners, humectants, transdermal penetration enhancers, epidermal growth factors, or preservatives.

[0017] The erythromycin in situ gel provided by this invention not only has the advantages of high transdermal efficiency, good targeting, long-lasting efficacy, safe and comfortable medication, and excellent degradation performance, but also can improve the appearance and smoothness of wound healing and shorten the wound healing time, thus better meeting the dual needs of patients for wound healing efficiency and healing quality.

[0018] Erythromycin in situ gel not only possesses the inherent advantages of in situ gels, such as flexible administration, adaptability to complex wounds, comfortable user experience, outstanding drug control and retention capabilities, improvement of wound dryness, and repair of skin barrier function, but also combines the advantages of erythromycin nanoliposomes, including effective penetration of the stratum corneum barrier, increased drug permeability, slow drug release, and strong targeting. Furthermore, the in situ gel and the active pharmaceutical ingredient, erythromycin nanoliposomes, form a synergistic system: on the one hand, the in situ gel network structure can further regulate the drug release rate of erythromycin nanoliposomes, and combined with the sustained-release properties of the nanoliposomes themselves, achieve dual controlled release, effectively avoiding sudden increases in local concentration caused by burst drug release and prolonging the duration of efficacy; on the other hand, the gel carrier can protect the structural integrity of the nanoliposomes, reducing their damage during application and gel formation, ensuring the targeted guidance function of resveratrol, achieving dual precise drug delivery through gel carrier locking and nanoliposome targeting, reducing drug distribution in normal skin tissue, and further improving drug safety and targeted efficacy.

[0019] Preferably, the temperature-sensitive matrix material is a polysaccharide-grafted N-isopropylacrylamide.

[0020] Thermosensitive in-situ gel formulations can avoid gastrointestinal reactions caused by oral medications. The medication is applied to the skin in liquid form and rapidly undergoes a phase transition to form a gel. This gel adheres to the administration site, not only prolonging the drug's retention time but also achieving long-lasting and sustained release, greatly improving patient compliance.

[0021] More preferably, the polysaccharide-grafted N-isopropylacrylamide includes at least one of Bletilla striata polysaccharide-grafted N-isopropylacrylamide, Tremella fuciformis polysaccharide-grafted N-isopropylacrylamide, Astragalus membranaceus polysaccharide-grafted N-isopropylacrylamide, or Glycyrrhiza uralensis polysaccharide-grafted N-isopropylacrylamide.

[0022] The grafting rate of the polysaccharide grafted with N-isopropylacrylamide was 260%-340%.

[0023] This invention constructs a temperature-sensitive matrix material by grafting polysaccharide with N-isopropylacrylamide (NIPAAm). This material has advantages such as biodegradability, excellent biocompatibility, and high safety for clinical application, providing a reliable guarantee for the biosafety and clinical applicability of in-situ gel formulations.

[0024] For example, the preparation method of polysaccharide grafted N-isopropylacrylamide in this invention includes the following steps: dissolving Bletilla striata polysaccharide, Tremella fuciformis polysaccharide, Astragalus membranaceus polysaccharide or Glycyrrhiza uralensis polysaccharide in 3% sulfuric acid solution to prepare a polysaccharide solution with a concentration of 0.1%-3% w / v; Dissolve N-isopropylacrylamide in water to prepare a NIPAAm solution with a final concentration of 1.2%-6% w / v; The above polysaccharide solution and NIPAAm solution were mixed evenly and stirred under a nitrogen atmosphere for 0.5 h to obtain a polysaccharide-NIPAAm mixture. A 0.1%-0.6% w / v ammonium persulfate solution was placed in a dropping funnel and added dropwise to the above polysaccharide-NIPAAM mixture. The polymerization reaction was carried out at 60°C under a nitrogen atmosphere for 5-7 hours. The reaction product was then placed in a dialysis bag and dialyzed for 3 days. After freeze-drying for 48 hours, polysaccharide-grafted NIPAAM was obtained.

[0025] In the polysaccharide-NIPAAm mixture, the mass ratio of polysaccharide to NIPAAm is 1:2-1:10; In the polymerization reaction, the mass ratio of polysaccharide to ammonium persulfate is 3:1 to 3:3; The molecular weight cutoff of the dialysis bag is 10,000 Da to 14,000 Da. For example, the present invention selects a dialysis bag with a molecular weight cutoff of 14,000 Da.

[0026] Preferably, the thickener includes at least one of polyethylene glycol-400, povidone K30, sodium alginate, chitosan quaternary ammonium salt, or hydroxybutyl chitosan.

[0027] Preferably, the moisturizer includes at least one of glycerin, squalane, hyaluronic acid, sorbitol, butylene glycol, or polyethylene glycol.

[0028] Preferably, the epidermal growth factor includes at least one of recombinant human epidermal growth factor or recombinant bovine basic fibroblast growth factor.

[0029] Studies have shown that the combined use of erythromycin and recombinant human epidermal growth factor can promote wound healing, shorten scab removal time, facilitate skin recovery and smoothness, and improve the color and appearance of lesions.

[0030] Preferably, the transdermal penetration enhancer includes at least one of propylene glycol or water-soluble laurocapram.

[0031] Preferably, the preservative includes at least one of benzoic acid, benzalkonium bromide, potassium sorbate, or parabens.

[0032] More preferably, the erythromycin in situ gel comprises the following components at the following concentrations: 134 g / L erythromycin nanoliposome powder, 70 g / L-90 g / L temperature-sensitive matrix material, 3 g / L-70 g / L thickener, 50 g / L-100 g / L humectant, 0.008 g / L-0.01 g / L epidermal growth factor, 30 g / L-60 g / L transdermal penetration enhancer, and 0.6 g / L-2 g / L preservative. The solvent is phosphate buffer. The erythromycin nanoliposome powder is lyophilized erythromycin nanoliposome powder.

[0033] Compared with traditional erythromycin cream, the erythromycin in situ gel provided by this invention not only has high transdermal efficiency, long-lasting efficacy, safe and comfortable use, and good stability, but also can shorten healing time, improve clinical efficacy, and improve the appearance and smoothness of the wound after healing, thus better meeting patients' dual needs for wound healing efficiency and healing quality.

[0034] Compared with erythromycin in situ gel using poloxamer as the matrix material, the erythromycin in situ gel provided by the present invention uses polysaccharide-grafted N-isopropylacrylamide grafted product as a temperature-sensitive gel matrix, which has superior degradation performance and biocompatibility. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The diagram shows the state of erythromycin in situ gel I at different temperatures in Example 3 of the present invention; wherein, Figure 1 a) is the state at 25℃. Figure 1 b) is the state at 34℃; Figure 2 Examples 5 illustrate the wound conditions of different groups after 10 days of treatment according to the present invention; wherein, Figure 2 a) is the positive control group; Figure 2 b) is the treatment group; Figure 3 This is a comparison chart of the degradation rates of erythromycin in situ gel I and erythromycin in situ gel III in Example 6 of the present invention; Figure 4 This is a comparison chart of cell survival rates in different groups in Example 6 of the present invention. Detailed Implementation

[0037] 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.

[0038] To better illustrate the embodiments provided by the present invention, further examples are given below.

[0039] In this invention, Bletilla striata polysaccharide, Tremella fuciformis polysaccharide, Astragalus membranaceus polysaccharide, and Glycyrrhiza uralensis polysaccharide were all purchased from Shanxi Baichuan Biotechnology Co., Ltd.

[0040] Example 1 This embodiment provides an erythromycin nanoliposome and its preparation method, wherein the raw materials for preparing the erythromycin nanoliposome include: 5g of erythromycin, 25g of lipid raw material, 12g of emulsifier, 2.5g of vitamin E acetate and 2g of resveratrol; The lipid raw materials include 19.7g of soybean lecithin and 5.3g of cholesterol; The emulsifier is poloxamer 188.

[0041] The preparation method of erythromycin nanoliposomes includes the following steps: S1. Take the above lipid raw material, heat and melt it, add erythromycin, mix well, melt, and obtain the oil phase; The emulsifier, vitamin E acetate and resveratrol were mixed evenly, and deionized water was added to make up to 500 mL. The mixture was ultrasonically dispersed at a frequency of 20 kHz until completely dissolved, and then placed in a constant temperature water bath at 37 ℃ to obtain the aqueous phase. S2. Under the stirring of a constant temperature magnetic stirrer, slowly add the oil phase to the aqueous phase, continue stirring, stop heating, and obtain the initial dispersion system; S3. The initial dispersion system is placed in a high-pressure homogenizer, the pressure is set to 70000 kPa, and the number of cycles is 3 to obtain erythromycin nanoliposomes I.

[0042] Example 2 This embodiment provides an erythromycin nanoliposome and its preparation method, wherein the raw materials for preparing the erythromycin nanoliposome include: 4g of erythromycin, 40g of lipid raw material, 12.5g of emulsifier, 2g of vitamin E acetate and 2.5g of resveratrol; The lipid raw materials include 36g of soybean lecithin and 4g of cholesterol; The emulsifier is Tween-80.

[0043] The preparation method of erythromycin nanoliposomes includes the following steps: S1. Take the above lipid raw material, heat and melt it, add erythromycin, mix well, melt, and obtain the oil phase; The emulsifier, vitamin E acetate and resveratrol were mixed evenly, and deionized water was added to make up to 500 mL. The mixture was ultrasonically dispersed at a frequency of 20 kHz until completely dissolved, and then placed in a constant temperature water bath at 37 ℃ to obtain the aqueous phase. S2. Under the stirring of a constant temperature magnetic stirrer, slowly add the oil phase to the aqueous phase, continue stirring, stop heating, and obtain the initial dispersion system; S3. The initial dispersion system is placed in a high-pressure homogenizer, the pressure is set to 60000 kPa, and the number of cycles is 4 to obtain erythromycin nanoliposomes II.

[0044] Example 3 This embodiment provides an erythromycin nanoliposome and its preparation method, wherein the raw materials for preparing the erythromycin nanoliposome include: 2.5g of erythromycin, 30g of lipid raw material, 15g of emulsifier, 1.5g of vitamin E acetate and 1.5g of resveratrol; The lipid raw materials include 12g of caprylic / capric glyceride and 18g of glyceryl monostearate; The emulsifier is Tween-80.

[0045] The preparation method of erythromycin nanoliposomes includes the following steps: S1. Take the above lipid raw material, heat and melt it, add erythromycin, mix well, melt, and obtain the oil phase; The emulsifier, vitamin E acetate and resveratrol were mixed evenly, and deionized water was added to make up to 500 mL. The mixture was ultrasonically dispersed at a frequency of 20 kHz until completely dissolved, and then placed in a constant temperature water bath at 37 ℃ to obtain the aqueous phase. S2. Under the stirring of a constant temperature magnetic stirrer, slowly add the oil phase to the aqueous phase, continue stirring, stop heating, and obtain the initial dispersion system; S3. The initial dispersion system is placed in a high-pressure homogenizer, the pressure is set to 70000 kPa, and the number of cycles is 3 to obtain erythromycin nanoliposomes III.

[0046] Example 4 This embodiment provides an erythromycin in situ gel and its preparation method, wherein the erythromycin in situ gel includes the following components at the following concentrations: 134 g / L erythromycin nanoliposome powder, 70 g / L temperature-sensitive matrix material, 5 g / L thickener, 60 g / L humectant, 0.1 g / L epidermal growth factor, 30 g / L transdermal penetration enhancer, 2 g / L preservative, and the balance being phosphate buffer solution with a pH of 6.4.

[0047] The temperature-sensitive matrix material is N-isopropylacrylamide grafted onto Bletilla striata polysaccharide with a grafting rate of 280%; the thickener is sodium alginate; the moisturizer is glycerin; the transdermal penetration enhancer is propylene glycol; the epidermal growth factor is recombinant human epidermal growth factor; and the preservative is potassium sorbate.

[0048] The method for preparing the Bletilla striata polysaccharide grafted with N-isopropylacrylamide is as follows: dissolve 3g of Bletilla striata polysaccharide in 3% sulfuric acid solution to prepare a polysaccharide solution with a concentration of 3% w / v. Dissolve 30g of N-isopropylacrylamide in water to prepare a NIPAAm solution with a final concentration of 5% w / v; The above polysaccharide solution and NIPAAm solution were mixed evenly and stirred under a nitrogen atmosphere for 0.5 h to obtain a polysaccharide-NIPAAm mixture. A 0.3% w / v ammonium persulfate solution was placed in a dropping funnel and added dropwise to the above polysaccharide-NIPAAM mixture. After polymerization at 60°C under a nitrogen atmosphere for 6 hours, the reaction product was placed in a dialysis bag and dialyzed for 3 days. The product was then freeze-dried for 48 hours to obtain polysaccharide-grafted NIPAAM.

[0049] In the polymerization reaction, the mass ratio of polysaccharide to ammonium persulfate is 3:2; The dialysis bag has a molecular cutoff of 14000 Da.

[0050] The preparation method of erythromycin in situ gel provided in this embodiment includes the following steps: freeze-drying the erythromycin nanoliposome I prepared in Example 1 to obtain erythromycin nanoliposome powder; This embodiment uses the preparation of 100mL of erythromycin in situ gel as an example. 13.4g of erythromycin nanoliposome powder was added to 70mL of phosphate buffer with pH 6.4 and sonicated for 20min to fully disperse the erythromycin nanoliposome powder. The thermosensitive gel matrix, thickener, moisturizer, transdermal penetration enhancer and preservative were added according to the formula and mixed evenly. The volume was adjusted to 100mL with the above-mentioned phosphate buffer and stirred thoroughly with a magnetic stirrer at a low temperature of 3-8℃ for 60min. Then it was placed in an environment of 4℃ for 24h to swell, resulting in a clear, bubble-free and clumpy erythromycin in situ gel I.

[0051] Example 5 This embodiment provides an erythromycin in situ gel and its preparation method, wherein the erythromycin in situ gel comprises the following components at the following concentrations: 140 g / L erythromycin nanoliposome powder, 80 g / L temperature-sensitive matrix material, 20 g / L thickener, 60 g / L humectant, 0.1 g / L epidermal growth factor, 50 g / L transdermal penetration enhancer, 1.5 g / L preservative, and the balance being phosphate buffer solution with a pH of 6.4.

[0052] The temperature-sensitive matrix material is tremella polysaccharide grafted with N-isopropylacrylamide, with a grafting rate of 310%; the thickener is povidone K30; the moisturizer is squalane; the transdermal penetration enhancer is water-soluble laurocapram; the epidermal growth factor is recombinant bovine basic fibroblast growth factor; and the preservative is potassium sorbate.

[0053] The method for preparing the grafted N-isopropylacrylamide onto the Tremella polysaccharide is as follows: dissolve 3g of Tremella polysaccharide in a 3% sulfuric acid solution to prepare a polysaccharide solution with a concentration of 3% w / v. Dissolve 20g of N-isopropylacrylamide in water to prepare a NIPAAm solution with a final concentration of 4% w / v; The above polysaccharide solution and NIPAAm solution were mixed evenly and stirred under a nitrogen atmosphere for 0.5 h to obtain a polysaccharide-NIPAAm mixture. A 0.4% w / v ammonium persulfate solution was placed in a dropping funnel and added dropwise to the above polysaccharide-NIPAAM mixture. After polymerization at 60°C under a nitrogen atmosphere for 6 hours, the reaction product was placed in a dialysis bag and dialyzed for 3 days. The product was then freeze-dried for 48 hours to obtain polysaccharide-grafted NIPAAM.

[0054] In the polymerization reaction, the mass ratio of polysaccharide to ammonium persulfate is 3:3; The dialysis bag has a molecular cutoff of 14000 Da.

[0055] The preparation method of erythromycin in situ gel provided in this embodiment is basically the same as that provided in Example 4, except that: (1) the concentration of each raw material in the erythromycin in situ gel is as described above; (2) the erythromycin nanoliposomes II prepared in Example 2 are freeze-dried to obtain erythromycin nanoliposome powder, which is used for the preparation of erythromycin in situ gel in this embodiment. The erythromycin in situ gel finally obtained is denoted as erythromycin in situ gel II.

[0056] Example 6 This embodiment provides an erythromycin in situ gel and its preparation method, wherein the erythromycin in situ gel comprises the following components at the following concentrations: 145 g / L erythromycin nanoliposome powder, 90 g / L temperature-sensitive matrix material, 10 g / L thickener, 8 g / L humectant, 0.1 g / L epidermal growth factor, 55 g / L transdermal penetration enhancer, 1 g / L preservative, and the balance being phosphate buffer solution with a pH of 6.4.

[0057] The temperature-sensitive matrix material is astragalus polysaccharide grafted with N-isopropylacrylamide, with a grafting rate of 265%; the thickener is polyethylene glycol-400; the moisturizer is hyaluronic acid; the transdermal penetration enhancer is water-soluble laurocapram; the epidermal growth factor is recombinant bovine basic fibroblast growth factor; and the preservative is parabens.

[0058] The method for preparing the tremella polysaccharide grafted with N-isopropylacrylamide is as follows: dissolve 3g of astragalus polysaccharide in a 3% sulfuric acid solution to prepare a polysaccharide solution with a concentration of 3% w / v. Dissolve 30g of N-isopropylacrylamide in water to prepare a NIPAAm solution with a final concentration of 5% w / v; The above polysaccharide solution and NIPAAm solution were mixed evenly and stirred under a nitrogen atmosphere for 0.5 h to obtain a polysaccharide-NIPAAm mixture. A 0.5% w / v ammonium persulfate solution was placed in a dropping funnel and added dropwise to the above polysaccharide-NIPAAM mixture. After polymerization at 60°C under a nitrogen atmosphere for 6 hours, the reaction product was placed in a dialysis bag and dialyzed for 3 days. The product was then freeze-dried for 48 hours to obtain polysaccharide-grafted NIPAAM.

[0059] In the polymerization reaction, the mass ratio of polysaccharide to ammonium persulfate is 3:2; The dialysis bag has a molecular cutoff of 14000 Da.

[0060] The preparation method of erythromycin in situ gel provided in this embodiment is basically the same as that provided in Example 4, except that: (1) the concentration of each raw material in the erythromycin in situ gel is as described above; (2) the erythromycin nanoliposomes III prepared in Example 3 is freeze-dried to obtain erythromycin nanoliposome powder, which is used for the preparation of erythromycin in situ gel in this embodiment. The erythromycin in situ gel finally obtained is denoted as erythromycin in situ gel III.

[0061] Example 7 This embodiment provides an erythromycin in situ gel and its preparation method, wherein the erythromycin in situ gel comprises the following components at the following concentrations: 137 g / L erythromycin nanoliposome powder, 70 g / L temperature-sensitive matrix material, 8 g / L thickener, 70 g / L humectant, 0.08 g / L epidermal growth factor, 40 g / L transdermal penetration enhancer, 2 g / L preservative, and the balance being phosphate buffer solution with a pH of 6.4.

[0062] The temperature-sensitive matrix material is licorice polysaccharide grafted with N-isopropylacrylamide, with a grafting rate of 340%; the thickener is hydroxybutyl chitosan; the humectant is butylene glycol; the transdermal penetration enhancer is propylene glycol; the epidermal growth factor is recombinant human epidermal growth factor; and the preservative is potassium sorbate.

[0063] The method for preparing the licorice polysaccharide grafted with N-isopropylacrylamide is as follows: dissolve 3g of astragalus polysaccharide in a 3% sulfuric acid solution to prepare a polysaccharide solution with a concentration of 3% w / v. Dissolve 25g of N-isopropylacrylamide in water to prepare a NIPAAm solution with a final concentration of 3% w / v; The above polysaccharide solution and NIPAAm solution were mixed evenly and stirred under a nitrogen atmosphere for 0.5 h to obtain a polysaccharide-NIPAAm mixture. A 0.4% w / v ammonium persulfate solution was placed in a dropping funnel and added dropwise to the above polysaccharide-NIPAAM mixture. After polymerization at 60°C under a nitrogen atmosphere for 6 hours, the reaction product was placed in a dialysis bag and dialyzed for 3 days. The product was then freeze-dried for 48 hours to obtain polysaccharide-grafted NIPAAM.

[0064] In the polymerization reaction, the mass ratio of polysaccharide to ammonium persulfate is 3:1; The dialysis bag has a molecular cutoff of 14000 Da.

[0065] The preparation method of erythromycin in situ gel provided in this embodiment is basically the same as that provided in Example 4, except that: (1) the concentration of each raw material in the erythromycin in situ gel is as described above; (2) the erythromycin nanoliposome I prepared in Example 1 is freeze-dried to obtain erythromycin nanoliposome powder, which is used for the preparation of erythromycin in situ gel in this embodiment. The erythromycin in situ gel finally obtained is referred to as erythromycin in situ gel IV.

[0066] Comparative Example 1 This comparative example provides an erythromycin nanoliposome, wherein the raw materials used to prepare the erythromycin nanoliposome are basically the same as those in Example 1, except that vitamin E acetate is not added. The erythromycin nanoliposome was prepared according to the method described in Example 1, and is designated as Erythromycin Nanoliposome Pair I.

[0067] Comparative Example 2 This comparative example provides an erythromycin in situ gel, the components and concentrations of which are basically the same as those in Example 4, except that the erythromycin nanoliposome powder is replaced with an equal amount of erythromycin as described in Example 4. The drug loading of the erythromycin nanoliposomes used in Example 4 was determined to be 7.5%. The erythromycin in situ gel prepared according to the method described in Example 4 is designated as Erythromycin In Situ Gel Pair I.

[0068] Comparative Example 3 This comparative example provides an erythromycin in situ gel, the components and concentrations of which are basically the same as those in Example 4, except that epidermal growth factor is not added. The erythromycin in situ gel was prepared according to the method described in Example 4, and is designated as Erythromycin In Situ Gel Comparison II.

[0069] Comparative Example 4 This comparative example provides an erythromycin in situ gel, the components and concentrations of which are basically the same as in Example 5, except that the temperature-sensitive matrix material is replaced with a poloxamer series temperature-sensitive material. Specifically, the 80 g / L temperature-sensitive matrix material in Example 5 is replaced with 230 g / L poloxamer 407 and 50 g / L poloxamer 188, and the phosphate buffer ratio is reduced accordingly. The erythromycin in situ gel was prepared according to the method described in Example 5, and is designated as Erythromycin In Situ Gel Pair III.

[0070] Example 1 The erythromycin nanoliposomes prepared in Examples 1-3 were freeze-dried to obtain the corresponding erythromycin nanoliposome powders. The encapsulation efficiency of different erythromycin nanoliposomes was determined by dialysis in this invention.

[0071] The supernatant was filtered and the erythromycin content was determined by HPLC. This invention uses a Shimadzu 2030 HPLC system for determination. The chromatographic method is as follows: mobile phase 0.2 mol / L ammonium acetate solution: acetonitrile (70:30); detection wavelength 210 nm; flow rate 1 mL / min; column temperature 30℃. The encapsulation efficiency was calculated according to Formula 1. Each sample was measured in triplicate, and the results are shown in Table 1.

[0072] Formula 1 Table 1

[0073] Erythromycin nanoliposomes were diluted with an appropriate amount of phosphate buffer (pH 6.4) to prepare an aqueous dispersion system. The particle size and zeta potential of the nanoparticles were determined by wet laser particle size analyzer. Each sample was measured in triplicate, and the results are shown in Table 2.

[0074] Table 2

[0075] As can be seen from the above data, the encapsulation efficiency of the erythromycin nanoliposomes provided by the present invention is greater than 80%, the average particle size is less than 200 nm, and the distribution is uniform and stable.

[0076] Example 2 The leakage rate of erythromycin nanoliposomes prepared in Examples 1-3 and Comparative Example 1 was determined in this invention. The specific determination method is as follows: Erythromycin nanoliposomes were placed at three different temperatures (4℃, 25℃) for 30 days. Specifically, 10 mg of erythromycin nanoliposomes prepared in different examples or comparative examples were placed in dialysis bags, and the bags were placed in 50 mL beakers. Each beaker was soaked in PBS buffer (pH 6.4). Samples were taken on days 0, 10, 20, and 30 to observe the appearance. 1 mL of the soaking solution was filtered and analyzed by high-performance liquid chromatography (HPLC) using the same method as in Example 1. The leakage rate was calculated according to Formula 2.

[0077] Formula 2 In the formula: EE 初始 Encapsulation efficiency of erythromycin nanoliposomes as determined on day 0; EE 特定贮藏时间 The encapsulation efficiency of erythromycin nanoliposomes was determined after storage for a specific time; the leakage rate of different erythromycin nanoliposomes is shown in Table 3.

[0078] Table 3

[0079] As shown in Table 3, compared with Comparative Example 1, the erythromycin nanoliposomes prepared in this invention have a significantly lower leakage rate during storage, thus improving storage stability.

[0080] Example 3 This invention investigated the thermosensitivity of the erythromycin in situ gels prepared in Examples 4-7. Details are as follows: According to the "inversion method," 4 mL of different erythromycin in situ gel solutions at room temperature were taken into vials and placed in a constant temperature water bath. The water temperature was adjusted and slowly increased from 25°C. The fluidity of the gel solution was observed by inverting the vial for every 1°C increase. The center temperature of the in situ gel in the vial was recorded using a probe thermometer. The temperature at which the in situ gel just became completely non-flowable was taken as the gelation temperature of the gel solution. Figure 1 The diagram shows the state of erythromycin in situ gel I at different temperatures. Figure 1 a) is the state at 25℃. Figure 1 b) is the state at 34℃.

[0081] Depend on Figure 1 It can be seen that at room temperature (25℃), erythromycin in situ gel I is liquid; at 34℃, erythromycin in situ gel I is solid.

[0082] The gelation temperature determination results of different erythromycin in situ gels are shown in Table 4.

[0083] Table 4

[0084] As shown in Table 4, the erythromycin in situ gel provided by this invention has good temperature sensitivity, and the excipients do not have an adverse effect on the temperature sensitivity of the material, which can meet the application requirements of clinical scenarios.

[0085] Example of effect 4 This invention compares the differences in erythromycin permeation in transdermal tests between erythromycin liposome in situ gel I, erythromycin in situ gel I, and commercially available erythromycin ointment (1% erythromycin content). Details are as follows: Using the Franz diffusion cell as a transdermal experimental setup, detached skin or skin substitutes were fixed in the diffusion cell, the drug was placed in the supply cell, a magnetic stir bar was placed in the receiving cell, and phosphate buffer (pH 6.4) was added. The concentration of erythromycin in the receiving cell was measured at intervals to study the permeation effect of erythromycin and to calculate the cumulative permeation amount of the drug through the skin.

[0086] This invention uses isolated skin from male mice. The mouse skin is made into a square with sides of approximately 2 cm. The surface liquid of the mouse skin is absorbed with filter paper. The stratum corneum is placed face down on the supply pool, covering the receiving pool, and then the release pool is covered. Finally, it is secured with clips. The instrument is set to a constant temperature water bath of 36℃ and a rotation speed of 200 rpm. The device is placed in the transdermal test apparatus. After equilibration for 30 minutes, the receiving solution is replaced, and air bubbles are removed. After the receiving solution reaches a constant temperature, 200 μL of erythromycin nanoliposome in situ gel and 0.2 g of commercially available erythromycin ointment are placed on one side of the stratum corneum of the mouse skin. Samples of 1 mL of each batch are taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. The samples are centrifuged at 10000 rpm for 30 min, and the supernatant is filtered and the erythromycin content is determined by HPLC. The results of the erythromycin permeation of different samples at 12 h are shown in Table 5.

[0087] Table 5

[0088] As shown in Table 5, compared with erythromycin ointment and erythromycin in situ gel I, the erythromycin in situ gel I prepared in this invention has a significantly increased permeability and improves the burst release phenomenon of the drug.

[0089] Example 5 This invention compares the effects of commercially available erythromycin ointment (1% erythromycin content), erythromycin in situ gel I (1% erythromycin content), and erythromycin in situ gel II (1% erythromycin content) on wound healing ability. The specific details are as follows: Sixty rats with minor burns were selected: the treatment group consisted of 15 females and 15 males, with wound areas ranging from 3 to 8 cm². 2 The average length was (6.32±0.5) cm. 2 Superficial second-degree burns. The positive control group consisted of 15 females and 15 males, with wound areas ranging from 4 to 8 cm². 2 The average length was (7.04±0.5) cm. 2 Superficial second-degree burns.

[0090] The control group of scalded rats were treated with erythromycin ointment, applied to the wound in appropriate amounts twice daily until healing. The treatment group of scalded rats were treated with erythromycin in situ gel I, applied to the wound in appropriate amounts twice daily until healing.

[0091] Clinical efficacy evaluation criteria. (1) Evaluation criteria for marked efficacy and effective efficacy. Marked efficacy: The wound heals and scabs fall off within 10 days, the skin returns to a smooth state, the color of the lesion is close to that of normal skin, and the appearance is aesthetically pleasing; Effective: The wound is basically healed within 10 days but the scabs have not fallen off, and the color of the affected area has faded; Ineffective: The above conditions do not improve or worsen within 10 days. (2) Comparison of wound healing. The wound healing time, scab formation, and scab fall-off time are statistically analyzed, and the smoothness of the wound after healing is observed.

[0092] Statistical analysis showed that in the positive control group: 11 cases showed significant improvement (36.7%), 15 cases showed improvement (50%), and 4 cases showed no improvement (13.3%). In the treatment group: 20 cases showed significant improvement (66.7%), 8 cases showed improvement (26.6%), and 2 cases showed no improvement (6.7%). The experimental results indicate that the efficacy of erythromycin in situ gel I provided by this invention is significantly higher than that of the positive control group, demonstrating better clinical efficacy.

[0093] Table 6 shows the statistics on healing time, scab formation, and scab shedding time. The wound condition of different groups at 10 days after treatment is also shown in the table. Figure 2 As shown. Among them, Figure 2 a) is the positive control group; Figure 2 b) is the treatment group.

[0094] Table 6

[0095] The data in Table 6 show that the erythromycin in situ gel I provided by the present invention can shorten the healing time, improve the clinical efficacy, and improve the appearance and smoothness of the wound after healing, thus better meeting the patients' dual needs for wound healing efficiency and healing quality.

[0096] Example 6 This invention compares the differences between erythromycin in situ gel I and erythromycin in situ gel III in terms of degradation performance and safety, as detailed below: 1. Degradation capacity test The degradation of gel samples in phosphate-buffered saline (PBS) solution was assessed. Degradation was characterized by monitoring the weight change of the in-situ gel during immersion. 30 mg of erythromycin in-situ gel I and erythromycin in-situ gel III samples were placed separately into 6-well plates containing PBS buffer. The plates were sealed and placed in a constant-temperature shaker at 37°C and 150 rpm. The gel samples were removed at preset time points, surface moisture was wiped off, and the samples were lyophilized and weighed. All experiments were performed in triplicate. The degradation capacity of in-situ gel samples prepared with different matrices was calculated.

[0097] The comparison of the degradation rates of erythromycin in situ gel I and erythromycin in situ gel III is shown in the figure below. Figure 3 As shown.

[0098] Depend on Figure 3 It is evident that the erythromycin in situ gel I, using Bletilla striata polysaccharide grafted with N-isopropylacrylamide as a temperature-sensitive matrix material, provided by this invention, can be completely degraded within 25 hours, while the erythromycin in situ gel III, using poloxamer series as a matrix material, still has a high residual rate of 71% after 48 hours. These results demonstrate that the erythromycin in situ gel provided by this invention possesses excellent biodegradability.

[0099] 2. Cytotoxicity test 0.1 g of erythromycin in situ gel I and erythromycin in situ gel III samples were immersed in 10 mL of NIH-3T3 cell culture medium and incubated at 37°C for 12 h. The residual gel was then filtered off using a filter membrane to obtain the gel extract. NIH-3T3 cells were then seeded into 96-well plates at a density of 4000 cells / well and incubated overnight. Subsequently, 100 μL of the gel extract was added to each well. 100 μL of PBS buffer was added to each well as a control. Cells after adding the gel extract or PBS buffer were then incubated at 37°C in a 5% CO2 incubator for 48 h. After the incubation period, cell viability was assessed using a CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in 5% CO2 for 2 h. Cell viability was then detected using an enzymatic method.

[0100] The comparison of cell survival rates in different groups is shown in the figure below. Figure 4As shown.

[0101] Depend on Figure 4 It can be seen that, compared with erythromycin in situ gel III, the erythromycin in situ gel I provided by the present invention has better biocompatibility and better safety.

[0102] In summary, compared with erythromycin in situ gel using poloxamer as the matrix material, the erythromycin in situ gel provided by the present invention has superior degradation performance and biocompatibility.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An erythromycin nanoliposome, characterized in that, It includes: erythromycin 5g / L-10g / L, lipid raw material 10g / L-100g / L, emulsifier 20g / L-50g / L, vitamin E acetate 1g / L-5g / L and resveratrol 0.1g / L-5g / L.

2. The erythromycin nanoliposomes as described in claim 1, characterized in that, The lipid raw material includes at least one of glyceryl monostearate, glyceryl caprylate, lecithin, cholesterol, or polyethylene glycol phospholipids; and / or The emulsifier includes at least one of poloxamer 188 or Tween-80.

3. The method for preparing erythromycin nanoliposomes according to claim 1 or 2, characterized in that, The preparation method includes the following steps: S1. Melt the lipid raw material and mix it with erythromycin to obtain an oil phase; The emulsifier, vitamin E acetate, resveratrol and water were mixed to obtain an aqueous phase; S2. The oil phase and the aqueous phase are mixed to obtain an initial dispersion system; S3. Erythromycin nanoliposomes were prepared by high-pressure homogenization of the initial dispersion system.

4. The method for preparing erythromycin nanoliposomes as described in claim 3, characterized in that, In the high-pressure homogenization method, the pressure is 60000kPa-80000kPa, and the number of cycles is 3-6.

5. The use of the erythromycin nanoliposomes according to claim 1 or 2 in the preparation of erythromycin in situ gel.

6. An erythromycin in situ gel, characterized in that, It includes the erythromycin nanoliposomes as described in claim 1 or 2, and at least one substance selected from temperature-sensitive matrix materials, thickeners, humectants, transdermal penetration enhancers, epidermal growth factors, or preservatives.

7. The erythromycin in situ gel as described in claim 6, characterized in that, The temperature-sensitive matrix material is a polysaccharide-grafted N-isopropylacrylamide.

8. The erythromycin in situ gel as described in claim 7, characterized in that, The polysaccharide-grafted N-isopropylacrylamide includes at least one of the following: Bletilla striata polysaccharide-grafted N-isopropylacrylamide, Tremella fuciformis polysaccharide-grafted N-isopropylacrylamide, Astragalus membranaceus polysaccharide-grafted N-isopropylacrylamide, or Glycyrrhiza uralensis polysaccharide-grafted N-isopropylacrylamide; and / or The grafting rate of the polysaccharide grafted with N-isopropylacrylamide was 260%-340%.

9. The erythromycin in situ gel as described in claim 6, characterized in that, The thickener includes at least one of polyethylene glycol-400, povidone K30, sodium alginate, chitosan quaternary ammonium salt, or hydroxybutyl chitosan; and / or The moisturizer includes at least one of glycerin, squalane, hyaluronic acid, sorbitol, butylene glycol, or polyethylene glycol; and / or The epidermal growth factor includes at least one of recombinant human epidermal growth factor or recombinant bovine basic fibroblast growth factor; and / or The transdermal penetration enhancer includes at least one of propylene glycol or water-soluble laurocapram; and / or The preservative includes at least one of benzoic acid, benzalkonium bromide, potassium sorbate, or parabens.

10. The erythromycin in situ gel according to any one of claims 6-9, characterized in that, The erythromycin in situ gel comprises the following components at the following concentrations: erythromycin nanoliposome powder 108g / L-160g / L, temperature-sensitive matrix material 70g / L-90g / L, thickener 3g / L-70g / L, humectant 50g / L-100g / L, epidermal growth factor 0.008g / L-0.01g / L, transdermal penetration enhancer 30g / L-60g / L, and preservative 0.6g / L-2g / L; The erythromycin nanoliposome powder is lyophilized erythromycin nanoliposome powder.

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