LL37-coated ZPF-2 compound and method for detecting content and in-vitro release of LL37 in gel of LL37-coated ZPF-2 compound

Abstract

The invention relates to the field of analytical chemistry, in particular to a method for determining the content of LL37 in an LL37-coated ZPF-2 compound and a gel thereof by adopting a high performance liquid chromatography, carrying out an in-vitro release test on the LL37-coated ZPF-2 compound gel by adopting a vertical diffusion cell method, and then determining the release amount of the LL37 at each time point by adopting the high performance liquid chromatography, so as to obtain the in-vitro release rate. According to the content detection method disclosed by the invention, the content of LL37 in the LL37-coated ZPF-2 compound and the gel thereof is measured by adopting a high performance liquid chromatography through exploration of specific detection conditions and repeated test screening, and the method is simple to operate, high in specificity and high in sensitivity, has good precision and durability, and can be used for detecting the content of LL37 in the LL37-coated ZPF-2 compound and the gel thereof. Meanwhile, by changing different adding sequences of water and a citric acid-sodium citrate buffer solution, the problem of inaccurate detection result caused by different dissolution rates of LL37 and LL37 ZPF-2 compounds in the same solvent system is solved.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to the determination of the LL37 content in the LL37@ZPF-2 complex and its gel by high performance liquid chromatography (HPLC), the in vitro release test of the LL37@ZPF-2 complex gel by vertical diffusion cell method, and the release amount of LL37 at each time point by HPLC to obtain the in vitro release rate. Background Technology

[0002] Human Cathelicidin-like antimicrobial peptide LL37 (molecular formula: C 205 H 340 N 60 O 53 LL37 (CAS No.: 145947-66-7) is an antimicrobial peptide derived from an amphiphilic cathepsin, consisting of 37 amino acid residues and possessing angiogenic activity. Its amino acid sequence is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, and its molecular weight is approximately 4493. It exhibits strong chemotactic and immunomodulatory properties, broad-spectrum antimicrobial activity, and activities promoting wound repair and regeneration, making it highly valuable in the treatment of bacterial infections, wound healing, and chronic ulcers. Studies have shown that unlike the slightly acidic environment of normal skin, the wound microenvironment is alkaline. This alkaline environment leads to a decrease in the α-helix content of the antimicrobial peptide LL37, resulting in reduced solubility and antimicrobial properties. Therefore, the main challenge in applying the antimicrobial peptide LL37 is the decline in its activity and antimicrobial efficacy in the wound environment.

[0003] LL37@ZPF-2 is a complex formed by in-situ encapsulation of LL37 in MOF material ZPF-2. Studies have shown that LL37@ZPF-2 exhibits superior antibacterial efficacy, cell migration effect, and wound healing effect compared to LL37. Under specific conditions, the LL37@ZPF-2 complex can gradually release LL37. The gel matrix in its thermosensitive gel undergoes a phase transition to a semi-solid state on the skin surface, further slowing down the release rate of LL37. This inhibits the reduction of LL37 activity and antibacterial efficacy in the wound environment. The characteristic of LL37@ZPF-2 releasing the active ingredient LL37 under specific conditions makes it possible to determine the LL37 content in the LL37@ZPF-2 complex and its formulations.

[0004] While there are reports on the use of enzyme-linked immunosorbent assay (ELISA) kits to detect LL37 content in existing technologies, there are no reports on the use of high-performance liquid chromatography (HPLC) for LL37 content determination, especially for the detection of LL37 content in the LL37@ZPF-2 complex and its gel. Content is a key attribute for drug quality control and is crucial to ensuring drug efficacy; therefore, there is an urgent need for a rapid and accurate method to quantitatively determine the LL37 content in the LL37@ZPF-2 complex and its gel.

[0005] High-performance liquid chromatography (HPLC) is a technique used to separate, identify, and quantify components in mixtures. However, several technical challenges exist when determining the LL37 content in the LL37@ZPF-2 complex and its gel. These challenges include: inconsistent solubility of LL37, the LL37@ZPF-2 complex, and its gel, affecting analytical accuracy; the need to select a suitable chromatographic column for good separation; optimization of parameters such as mobile phase composition and ionic strength to achieve optimal separation; potential instability of samples during preparation and analysis, leading to decomposition or chemical changes; interference from other components in the sample; and finally, rigorous validation of the HPLC method, including linear range, limit of quantitation, precision, accuracy, and robustness, is required to ensure its reliability.

[0006] Regarding the in vitro release detection and evaluation of LL37@ZPF-2 complex gel, there are no reports in the existing technology on using the Franz diffusion cell method, i.e., the vertical diffusion cell method, for in vitro release testing of LL37@ZPF-2 complex gel, nor are there any reports on using high performance liquid chromatography to detect the LL37 content in the in vitro release receiving solution.

[0007] The literature "Preparation and Evaluation of Novel Topical Gel Preparations for Wound Healing in Diabetics" discloses a membrane dialysis method for studying the in vitro release characteristics of drugs in wound repair gels. This method involves encapsulating the drug-containing gel in a semi-permeable standard cellophane membrane, immersing it in a release medium, and allowing the drug to diffuse into the medium through the membrane pores. Samples are taken periodically to measure the cumulative drug release. However, this method requires at least 24 hours of testing, and the manual sampling and fluid replenishment limit measurement accuracy and result in poor reproducibility.

[0008] The literature "Assessment of the Efficacy of an LL-37-Encapsulated Keratin Hydrogel for the Treatment of Full-Thickness Wounds" describes a method where a pH 7.4 phosphate buffer is added to the top of a self-encapsulated LL-37 keratin hydrogel, and fresh pH 7.4 phosphate buffer is replaced at 1.5h, 3h, 6h, 12h, and 24h, maintaining this method for one week to assess the LL37 release rate. The released LL37 is quantified by enzyme-linked immunosorbent assay (ELISA). This method, which tests the release rate by placing the hydrogel in a buffer solution, differs significantly from the actual application method of applying the gel to the skin, resulting in poor in vitro-in vivo correlation of the release characteristics data.

[0009] Furthermore, the development of an in vitro release method for the LL37@ZPF-2 complex gel presents several challenges: First, it is crucial to ensure that the inert synthetic membrane does not adsorb the drug during the experiment, thus affecting drug release. Second, considering the temperature-sensitive nature of the LL37@ZPF-2 complex gel, which is liquid before use and transforms into a semi-solid gel upon skin contact, a suitable sample loading method must be selected. Third, it is essential to minimize the reverse osmosis effect of the receiving liquid on the gel during the experiment (i.e., the receiving liquid permeates the inert synthetic membrane in reverse, causing gel erosion), to avoid unreasonable burst release of the drug due to erosion of the gel by the release medium after reverse osmosis, thereby failing to achieve linear steady-state drug release. Fourth, it is necessary to balance detection efficiency and accuracy, enabling the drug to achieve linear steady-state release within a short time, and ensuring that the release amount can be accurately quantified. Fifth, the established in vitro release method needs to be validated through precision, reproducibility, linearity, and discriminant power. Similarly, the quantitative analysis method for LL37 in the corresponding sample solution also needs to undergo rigorous validation, including linear range, limit of quantitation, precision, accuracy, and robustness, to ensure the reliability of the method.

[0010] The above factors collectively influence the determination of the inert membrane material, inert membrane pore size, sample loading method, sample loading volume, receiving solution composition, sampling time, sampling time point selection, diffusion cell volume, and rotor stirring speed in the diffusion cell, as well as the determination of the chromatographic conditions used for quantitative analysis of LL37 in the in vitro release method. The final determined in vitro release method and quantitative analysis method can achieve the purpose of evaluating the in vitro release characteristics (i.e., in vitro release rate) of LL37 in LL37@ZPF-2 complex gel in a specific, accurate, rapid, and convenient manner. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting the LL37 content in the LL37@ZPF-2 complex and its gel. Based on the accurate quantification of LL37, this invention provides a rapid, accurate, and reliable test method for evaluating the in vitro release characteristics of LL37 in the temperature-sensitive gel of the LL37@ZPF-2 complex.

[0012] A method for determining the LL37 content in LL37@ZPF-2 complex and its gel, using high performance liquid chromatography, specifically includes the following steps:

[0013] S1. Preparation of reference solution: Accurately weigh an appropriate amount of LL37 reference standard, first dissolve it in water to prepare a solution with a concentration of 0.1~0.4 mg / ml, then dilute it with 0.1 mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution with a concentration of 0.05~0.2 mg / ml, shake well, and the solution is ready; S2. Chromatographic conditions: Octadecyl-bonded silica gel was used as the stationary phase; a 0.05%–0.2% trifluoroacetic acid aqueous solution was used as phase A, and acetonitrile or methanol was used as phase B; gradient elution was used, with the following elution program: 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B; a diode array detector was used, with a detection wavelength of 205 nm–230 nm; the column temperature was 25–45 °C; and the flow rate was 0.8–1.2 ml / min. S3. Determination method: Inject the reference solution and the test sample solution into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the test sample by peak area according to the external standard method; The amount of reference solution used is 10~40μl, and the amount of test sample detection solution used is 10~40μl; If the test sample is an LL37@ZPF-2 complex, the preparation steps for the LL37@ZPF-2 complex are as follows: accurately weigh an appropriate amount of the LL37@ZPF-2 complex, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution with a concentration of 0.8~2.0mg / ml, then dilute with water to prepare a solution with a concentration of 0.4~1.0mg / ml, shake well, and the solution is obtained. If the test sample is LL37@ZPF-2 complex gel, the preparation steps of LL37@ZPF-2 complex gel are as follows: accurately weigh an appropriate amount of LL37@ZPF-2 complex gel, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution with a concentration of 300~800mg / ml, then dilute with water to prepare a solution with a concentration of 150~400mg / ml, shake well, and the solution is obtained.

[0014] Furthermore, in step S1, the concentration of LL37 reference standard in the reference solution is 0.1 mg / ml.

[0015] Furthermore, in step S2, phase A is a 0.1% aqueous solution of trifluoroacetic acid, phase B is acetonitrile; the detection wavelength is 215 nm; the column temperature is 35 °C; and the flow rate is 1.0 ml / min.

[0016] Furthermore, in step S3, the volume of the reference solution is 20 μl, and the volume of the test sample detection solution is 20 μl; If the test sample is an LL37@ZPF-2 complex, the concentration of the LL37@ZPF-2 complex in the test sample solution is 0.64 mg / ml; If the test sample is an LL37@ZPF-2 complex gel, then the concentration of the LL37@ZPF-2 complex gel in the test sample solution is 300 mg / ml.

[0017] A method for detecting the in vitro release of LL37 in an LL37@ZPF-2 complex gel, comprising the following steps: An in vitro release test of the LL37@ZPF-2 complex gel is conducted using a vertical diffusion cell method, and the content of LL37 in the receiving solution is determined using the above method. S1. Conduct in vitro release tests on a transdermal diffusion apparatus. Use a mixed cellulose ester membrane as the filter membrane. The receiving solution is a pH 5.0 citrate-sodium citrate buffer solution. The diffusion cell temperature is 31℃~33℃, the rotation speed is 200~400rpm, the diffusion cell volume is 7~12ml, the sample loading volume is at least 1.0ml, the sampling volume does not exceed 1.5ml, the replenishment volume is the same as the sampling volume, and the sampling time does not exceed 6h. S2. Determination of LL37 content in the receiving solution: Octadecyl-bonded silica gel was used as the packing material; a 0.05%–0.2% trifluoroacetic acid aqueous solution was used as phase A, and acetonitrile or methanol was used as phase B; the gradient elution program was 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B; a diode array detector was used, with a detection wavelength of 205 nm–230 nm; the column temperature was 25–45 °C; the flow rate was 0.8–1.2 ml / min; 10–40 μl of the receiving solution sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the LL37 content in the receiving solution was calculated by peak area using the external standard method.

[0018] Furthermore, in step S1, the concentration of the receiving liquid is 0.05~0.1mol / L, and the sampling volume is 1.0~1.5ml.

[0019] Furthermore, in step S2, phase A is a 0.08%~0.12% trifluoroacetic acid aqueous solution, phase B is acetonitrile, the detection wavelength is 210nm~220nm, the column temperature is 30~40℃, and the flow rate is 0.9~1.1ml / min; Accurately measure 15-25 μl of the receiving liquid sample, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the receiving liquid by peak area using the external standard method.

[0020] Furthermore, in step S1, the pore size of the filter membrane is 0.45 μm; the receiving solution is 0.05 mol / L pH 5.0 citrate-sodium citrate buffer; the rotation speed is 200 rpm; the diffusion cell volume is 10 ml; the sample loading volume is 1.0 ml; the sampling volume is 1.0 ml; the replenishment volume is the same as the sampling volume; and the sampling time points are 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h.

[0021] Furthermore, in step S1, the rotation speed is 400 rpm; the diffusion cell volume is 7 ml; the sample loading volume is 1.5 ml; and the sampling volume is 1.5 ml.

[0022] Further, in step S2, phase A is a 0.1% trifluoroacetic acid aqueous solution; phase B is acetonitrile; the gradient elution program is 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector is used, the detection wavelength is 215 nm; the column temperature is 35℃; the flow rate is 1.0 ml / min; the determination method is as follows: accurately measure 20 μl of the receiving liquid sample, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the receiving liquid by peak area according to the external standard method.

[0023] Compared with the prior art, the present invention has significant advantages and beneficial effects, specifically reflected in the following aspects: (1) The content detection method of the present invention, through exploration of specific detection conditions and repeated experimentation and screening, uses high performance liquid chromatography to determine the content of LL37 in the LL37@ZPF-2 complex and its gel. The method is simple to operate, highly specific, highly sensitive, and has good precision and durability. At the same time, by changing the order of adding water and citrate-sodium citrate buffer, the problem of inaccurate detection results caused by the different dissolution rates of LL37 and LL37@ZPF-2 complex in the same solvent system is overcome.

[0024] (2) The in vitro release method of the present invention uses a standardized automatic transdermal diffusion instrument based on the vertical diffusion cell method, which has the advantages of high test accuracy and good reproducibility. It directly uses high performance liquid chromatography to measure the cumulative release of the drug at different sampling time points, and then obtains the in vitro release rate by establishing a linear release equation. The release data conforms to the Higuchi equation, which is particularly suitable for transdermal drug delivery systems, simulates the skin barrier, and is closer to the in vivo application scenario. It can effectively evaluate the in vitro release characteristics of LL37@ZPF-2 complex gels, including but not limited to thermosensitive gels, and evaluate their intra-batch and inter-batch quality differences. Its strong discriminative power can also be used as an evaluation tool for formulation optimization. Attached Figure Description

[0025] Figure 1 This is the chromatogram of the blank solvent in Example 1; Figure 2 The chromatogram of the LL37 reference solution in Example 1 is shown below. Figure 3 The chromatogram of the LL37@ZPF-2 complex test sample solution from batch 1 of Example 1 is shown below. Figure 4 The chromatogram of batch 240617 of the LL37@ZPF-2 complex gel test solution in Example 1 is shown below. Figure 5 This is the chromatogram of batch 240701 of the LL37@ZPF-2 complex gel released in vitro for 6 hours in Example 9; Figure 6 The in vitro release curve and linear release equation of batch 240701 LL37@ZPF-2 complex gel in Example 9 are shown. Figure 7 The linear equation obtained from the in vitro release assay of the LL37@ZPF-2 complex gel in Example 14 is shown. Detailed Implementation

[0026] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Furthermore, unless otherwise specified, the above embodiments and features described herein can be combined with each other.

[0027] The in vitro release method established in this invention employs a transdermal diffusion apparatus (automatic transdermal diffusion device). A drug-containing gel is placed on an inert synthetic membrane, and the drug diffuses through the membrane into the receiving solution. The cumulative amount of drug released through the membrane at sampling time points is measured. Then, a linear release equation is established to calculate the in vitro release rate (i.e., slope) and its coefficient of variation (CV), and the linear correlation coefficient r is evaluated. 2 If r 2If the coefficient of performance (CV) is ≥0.97 and the CV is ≤15%, then the in vitro release method meets the linearity requirement; otherwise, the method is unreliable. The drug-containing gel, isolated by an inert synthetic membrane, does not come into contact with the release medium. Compared to membrane dialysis, this method emphasizes release and transdermal performance, and its testing scenario is closer to the clinical application scenario of gel application to wounds, making it more conducive to the quality assessment of transdermal drug delivery systems.

[0028] The first aspect of this invention is to protect a method for detecting the LL37 content in the LL37@ZPF-2 complex and its gel, specifically comprising the following steps: (1) Preparation of reference solution: Accurately weigh an appropriate amount of LL37 reference standard, first dissolve it in water to prepare a solution containing 0.1~0.4 mg per ml, then dilute it with 0.1mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing 0.05~0.2 mg per ml, shake well, and the solution is obtained; The best embodiment of the preparation of the reference solution of the present invention is: first dissolve it in water to prepare a solution containing 0.2 mg per ml, then dilute it with 0.1mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing 0.1 mg per ml.

[0029] (2) Preparation of LL37@ZPF-2 complex test solution: Accurately weigh an appropriate amount of LL37@ZPF-2 complex, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution containing 0.8~2.0mg per ml, then dilute with water to prepare a solution containing 0.4~1.0mg per ml, shake well, and the solution is obtained; The best embodiment for preparing the LL37@ZPF-2 complex test solution of the present invention is: first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution containing 1.28mg per ml, then dilute with water to prepare a solution containing 0.64mg per ml.

[0030] (3) Preparation of LL37@ZPF-2 complex gel test solution: Accurately weigh an appropriate amount of LL37@ZPF-2 complex gel, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve and prepare a solution containing 300~800mg per ml, then dilute with water to prepare a solution containing 150~400mg per ml, shake well, and the solution is obtained; The best embodiment for preparing the LL37@ZPF-2 complex gel test solution of the present invention is: first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve and prepare a solution containing 600mg per ml, then dilute with water to prepare a solution containing 300mg per ml.

[0031] In the preparation process of the reference solution and the test solution of the present invention, water is added first and then citrate-sodium citrate buffer is added when preparing the reference solution, while citrate-sodium citrate buffer is added first and then water is added when preparing the test solution. At the same time, by changing the order of adding water and citrate-sodium citrate buffer, the problem of inaccurate detection results caused by the different dissolution rates of LL37 and LL37@ZPF-2 complex in the same solvent system is overcome.

[0032] (4) Chromatographic conditions: Octadecyl bonded silica gel was used as the packing material; a 0.05%~0.2% trifluoroacetic acid aqueous solution was used as phase A, and an organic solvent was used as phase B. Preferably, phase B was acetonitrile or methanol; gradient elution was performed, and the gradient elution program was: 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector was used, and the detection wavelength was 205nm~230nm; the column temperature was 25~45℃; and the flow rate was 0.8~1.2 ml / min. In this invention, as the optimal embodiment of chromatographic conditions, phase A is 0.1% trifluoroacetic acid solution, phase B is acetonitrile; the detection wavelength is 215 nm; the column temperature is 35 °C; and the flow rate is 1.0 ml / min.

[0033] (5) Determination method: Accurately measure 10~40 μl of reference solution, 10~40 μl of LL37@ZPF-2 complex test solution or 10~40 μl of LL37@ZPF-2 complex gel test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in LL37@ZPF-2 complex and LL37@ZPF-2 complex gel by peak area according to the external standard method.

[0034] The second aspect of this invention is to protect a method for detecting the in vitro release of LL37 in an LL37@ZPF-2 complex gel. This method employs a vertical diffusion cell method for the in vitro release test of the LL37@ZPF-2 complex gel and uses high-performance liquid chromatography (HPLC) to determine the LL37 content in the receiving solution. Specifically, it includes the following steps: (1) In vitro release test was carried out on a transdermal diffusion instrument. The filter membrane was a mixed cellulose ester membrane with a pore size of 0.45 μm. The receiving solution was a citrate-sodium citrate buffer solution with a concentration of 0.05~0.1 mol / L and a pH of 5.0. The diffusion cell temperature was 31~33℃, the rotation speed was 200~400 rpm, the diffusion cell volume was 7~12 ml, the sample loading volume was at least 1.0 ml, the sampling volume was no more than 1.5 ml, the replenishment volume was the same as the sampling volume, and the sampling time was no more than 6 h. (2) The content of LL37 in the receiving liquid taken at each sampling time point was determined by high performance liquid chromatography. The chromatographic conditions were as follows: octadecyl bonded silica gel was used as the packing material; 0.05%~0.2% trifluoroacetic acid aqueous solution was used as phase A, and acetonitrile or methanol was used as phase B; the gradient elution program was 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector was used, and the detection wavelength was 205nm~230nm; the column temperature was 25~45℃; the flow rate was 0.8~1.2ml / min; the determination method was as follows: 10~40μl of the receiving liquid sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the content of LL37 in the receiving liquid was calculated by peak area according to the external standard method.

[0035] As an embodiment of the present invention, a method for detecting the in vitro release of LL37 in an LL37@ZPF-2 complex gel specifically includes the following steps: (1) In vitro release test was carried out on a transdermal diffusion instrument. The filter membrane was a mixed cellulose ester membrane with a pore size of 0.45 μm. The receiving solution was a citrate-sodium citrate buffer solution with a concentration of 0.05~0.1 mol / L and a pH of 5.0. The diffusion cell temperature was 31~33℃, the rotation speed was 200~400 rpm, the diffusion cell volume was 7~12 ml, the sample loading volume was 1.0 ml, the sampling volume was 1.0~1.5 ml, the replenishment volume was the same as the sampling volume, and the sampling time did not exceed 6 hours. (2) The content of LL37 in the receiving liquid taken at each sampling time point was determined by high performance liquid chromatography. The chromatographic conditions were as follows: octadecyl bonded silica gel was used as the packing material; trifluoroacetic acid aqueous solution with a concentration of 0.08%~0.12% was used as phase A and acetonitrile was used as phase B; the gradient elution program was 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector was used with a detection wavelength of 210nm~220nm; the column temperature was 30~40℃; the flow rate was 0.9~1.1ml / min; the determination method was as follows: 15~25μl of the receiving liquid sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the content of LL37 in the receiving liquid was calculated by peak area according to the external standard method.

[0036] As an embodiment of the present invention, a method for detecting the in vitro release of LL37 in an LL37@ZPF-2 complex gel specifically includes the following steps: (1) In vitro release test was performed on a transdermal diffusion apparatus. The filter membrane was a mixed cellulose ester membrane with a pore size of 0.45 μm. The receiving solution was 0.05 mol / L pH 5.0 citrate-sodium citrate buffer. The diffusion cell temperature was 31~33℃, the rotation speed was 200 rpm, the diffusion cell volume was 10 ml, the sample loading volume was at least 1.0 ml, the sampling volume was 1.0 ml, the replenishment volume was the same as the sampling volume, and the sampling time points were 1h, 2h, 3h, 4h, 5h, and 6h. (2) The content of LL37 in the receiving liquid taken at each sampling time point was determined by high performance liquid chromatography. The chromatographic conditions were as follows: octadecyl bonded silica gel was used as the packing material; phase A was 0.1% trifluoroacetic acid aqueous solution; phase B was acetonitrile; the gradient elution program was 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector was used with a detection wavelength of 215 nm; the column temperature was 35℃; the flow rate was 1.0 ml / min; the determination method was as follows: 20 μl of the receiving liquid sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the content of LL37 in the receiving liquid was calculated by peak area according to the external standard method.

[0037] As an embodiment of the present invention, a method for detecting the in vitro release of LL37 in an LL37@ZPF-2 complex gel specifically includes the following steps: (1) In vitro release test was performed on a transdermal diffusion instrument. The filter membrane was a mixed cellulose ester membrane with a pore size of 0.45 μm. The receiving solution was 0.05 mol / L citrate-sodium citrate buffer solution with pH 5.0. The diffusion cell temperature was 31~33℃, the rotation speed was 400 rpm, the diffusion cell volume was 7 ml, the sample loading volume was at least 1.0 ml, the sampling volume was 1.5 ml, the replenishment volume was the same as the sampling volume, and the sampling time points were 1h, 2h, 3h, 4h, 5h, and 6h. (2) The content of LL37 in the receiving liquid taken at each sampling time point was determined by high performance liquid chromatography. The chromatographic conditions were as follows: octadecyl bonded silica gel was used as the packing material; phase A was 0.1% trifluoroacetic acid aqueous solution; phase B was acetonitrile; the gradient elution program was 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector was used with a detection wavelength of 215 nm; the column temperature was 35℃; the flow rate was 1.0 ml / min; the determination method was as follows: 20 μl of the receiving liquid sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the content of LL37 in the receiving liquid was calculated by peak area according to the external standard method.

[0038] In this invention, an Agilent 1260 high-performance liquid chromatograph was used for content determination; a Thermo Hypersil Gold column was used for LL37 content determination; an In vitro release test was conducted using a Retop RT800 automated sampling transdermal diffusion system; and an Agilent 1260 high-performance liquid chromatograph was used for LL37 content determination in the receiving liquid at each sampling time point.

[0039] <Example 1> In this embodiment, the LL37 reference standard was purchased from Wuhan Hanxiang Biotechnology Co., Ltd., batch number: 20231109.

[0040] Test samples: Three samples of LL37@ZPF-2 complex (self-made, numbered 1, 2 and 3 respectively). The preparation methods of the three samples of LL37@ZPF-2 complex were all in accordance with Example 1 in CN119818648.

[0041] Three portions of LL37@ZPF-2 complex gels (self-made, batch numbers 240617, 240701, and 24081402, respectively) were prepared according to Example 6 in CN119818648, wherein the mass ratio of LL@ZPF-2 complex to blank gel was approximately 1.8 mg / g.

[0042] The determination of LL37 content in the LL37@ZPF-2 complex and LL37@ZPF-2 complex gel specifically includes the following conditions: (1) Preparation of blank solvent: Accurately measure 5 ml of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer solution, place it in a 10 ml volumetric flask, dilute with water to the mark, and shake well. The chromatogram is shown below. Figure 1 As shown, by Figure 1 It can be seen that the blank solvent does not interfere with the detection.

[0043] (2) Preparation of the reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 50 ml volumetric flask, add about 25 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well. The chromatogram is shown below. Figure 2 As shown.

[0044] (3) Preparation of LL37@ZPF-2 complex test solution: Accurately weigh about 16 mg of LL37@ZPF-2 complex, place it in a 25 ml volumetric flask, add about 12.5 ml of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, sonicate to dissolve, dilute with water to the mark, and shake well.

[0045] (4) Preparation of the LL37@ZPF-2 complex gel test solution: Accurately weigh approximately 3 g of the LL37@ZPF-2 complex gel and place it in a 10 ml volumetric flask. Add approximately 5 ml of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer solution, sonicate to dissolve, dilute with water to the mark, and shake well. Obtain the chromatogram of the LL37@ZPF-2 complex test solution (number 1) as shown below. Figure 3 As shown.

[0046] (5) Chromatographic conditions: Octadecyl-bonded silica gel was used as the packing material; the column temperature was 35℃; mobile phase A was 0.1% trifluoroacetic acid aqueous solution, mobile phase B was acetonitrile, gradient elution was used, and the gradient elution program was: 0~10min, 40%~60%B; 10~11min, 60%~40%B; 11~21min, 40%B; a diode array detector was used, the detection wavelength was 215nm; the flow rate was 1.0ml / min. The chromatogram of the LL37@ZPF-2 complex gel test solution obtained from batch 240617 is shown below. Figure 4 As shown.

[0047] (6) Determination method: Accurately measure 20 μl of the above reference solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of LL37 in the LL37@ZPF-2 complex and its gel by peak area according to the external standard method. The specific detection results are shown in Table 1: Table 1

[0048] <Example 2> The determination of LL37 content in the LL37@ZPF-2 complex and its gel includes the following conditions: (1) Preparation of reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 100 ml volumetric flask, add about 50 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well.

[0049] (2) Preparation of LL37@ZPF-2 complex test solution: Accurately weigh about 20 mg of LL37@ZPF-2 complex, place it in a 50 ml volumetric flask, add about 25 ml of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, sonicate to dissolve, dilute with water to the mark, and shake well.

[0050] (3) Preparation of LL37@ZPF-2 complex gel test solution: Accurately weigh about 3g of LL37@ZPF-2 complex gel, place it in a 20ml volumetric flask, add about 10ml of 0.1mol / L pH5.0 citrate-sodium citrate buffer, sonicate to dissolve, dilute with water to the mark, and shake well.

[0051] (4)-(5) are consistent with (5) and (6) in Example 1.

[0052] The specific test results are shown in Table 2: Table 2

[0053] <Example 3> The determination of LL37 content in the LL37@ZPF-2 complex and its gel includes the following conditions: (1) Preparation of reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 25 ml volumetric flask, add about 12.5 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well.

[0054] (2) Preparation of LL37@ZPF-2 complex test solution: Accurately weigh about 25 mg of LL37@ZPF-2 complex, place it in a 25 ml volumetric flask, add about 25 ml of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, sonicate to dissolve, dilute with water to the mark, and shake well.

[0055] (3) Preparation of LL37@ZPF-2 complex gel test solution: Accurately weigh about 4g of LL37@ZPF-2 complex gel, place it in a 10ml volumetric flask, add about 5ml of 0.1mol / L pH5.0 citrate-sodium citrate buffer, sonicate to dissolve, dilute with water to the mark, and shake well.

[0056] (4)-(5) are consistent with (5) and (6) in Example 1.

[0057] The specific test results are shown in Table 3: Table 3

[0058] <Example 4> Validation of LL37 content determination method: Limit of quantitation The model of the high performance liquid chromatograph, the information on the reference standard, and the chromatographic conditions were all the same as in Example 1.

[0059] Accurately weigh approximately 20 mg of LL37 reference standard and place it in a 20 ml volumetric flask. Add water and sonicate to dissolve. Dilute with water to the mark and shake well. Accurately measure an appropriate amount and dilute with pH 5.0 citrate-sodium citrate buffer (0.1 mol / L) to prepare a solution containing approximately 0.001 mg per ml. Accurately measure 3 ml of this solution and place it in a 10 ml volumetric flask. Dilute with pH 5.0 citrate-sodium citrate buffer (0.1 mol / L) to the mark and shake well. This solution serves as the limit of quantitation solution.

[0060] Accurately measure 20 μl of the limit of quantitation solution and inject it into the liquid chromatograph. Repeat the injection six times and record the chromatograms. Calculate the signal-to-noise ratio (S / N) of the main peak to the blank baseline and the RSD (%) of the main peak area in the six injections.

[0061] The specific results are shown in Table 4: Table 4

[0062] As shown in Table 4, LL37 can be accurately quantified at a concentration of 0.0003 mg / ml.

[0063] <Example 5> Validation of LL37 content determination method: linear range The model of the high performance liquid chromatograph, the information on the reference standard, and the chromatographic conditions were all the same as in Example 1.

[0064] Preparation of linear solutions: Accurately weigh approximately 20 mg of LL37 reference standard, place it in a 20 ml volumetric flask, add water and sonicate to dissolve, dilute with water to the mark, shake well, accurately measure an appropriate amount, and dilute with pH 5.0 citrate-sodium citrate buffer (0.1 mol / L) to prepare solutions containing approximately 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg and 0.4 mg per ml, respectively, which are referred to as linear solutions 1 to 5.

[0065] Accurately measure 20 μl of each of the above linear solutions and inject them into the liquid chromatograph, recording the chromatograms. Perform linear regression with peak area (A) as the ordinate and concentration (C) as the abscissa, obtaining the regression equation: A = 10105C + 25.58, with a correlation coefficient r = 0.9998. Specific results are shown in Table 5. Table 5

[0066] As shown in Table 5, within the concentration range of 0.0003 mg / ml to 0.4176 mg / ml, the concentration of LL37 showed a good linear relationship with the peak area.

[0067] <Example 6> Validation of LL37 content determination method: recovery rate The model of the high performance liquid chromatograph, the information on the reference standard, and the chromatographic conditions were all the same as in Example 1.

[0068] Preparation of reference stock solution: Accurately weigh about 20 mg of LL37 reference standard, place it in a 20 ml volumetric flask, add water and sonicate to dissolve, dilute with water to the mark, and shake well to obtain the solution.

[0069] Preparation of reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer and shake well.

[0070] The preparation method of the test sample in this embodiment is as described in Example 6 of CN 119818648, that is, the composition and preparation method of the blank gel are the same as step (1) of Example 6. The preparation method of the LL37@ZPF-2 complex gel is as described in Example 6 of CN119818648, wherein the mass ratio of the LL37@ZPF-2 complex to the blank gel is approximately 1.8 mg / g, and the LL37 content in the LL37@ZPF-2 complex is 12.6%. Therefore, when the mass of LL37 in the LL37@ZPF-2 complex in the test sample solution of this embodiment is 1 mg, the mass of ZPF-2 material is 7 mg, the mass of the blank gel is 4.4 g, and so on.

[0071] Preparation of low-concentration test solution: Accurately measure 0.1 ml of the reference stock solution, 0.7 mg of ZPF-2 material and 0.44 g of blank gel into a 100 ml volumetric flask, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, shake well, and prepare 3 parallel aliquots (numbered 1 to 3) to obtain the test solution.

[0072] Preparation of medium-concentration test solution: Accurately measure 1 ml of reference stock solution, 7 mg of ZPF-2 material and 4.4 g of blank gel into a 100 ml volumetric flask, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, shake well, and prepare 3 parallel aliquots (numbered 4~6) to obtain the test solution.

[0073] Preparation of high-concentration test solution: Accurately measure 1.2 ml of reference stock solution, 8.4 mg of ZPF-2 material and 5.28 g of blank gel into a 100 ml volumetric flask, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, shake well, and prepare 3 parallel aliquots (numbered 7~9) to obtain the test solution.

[0074] Accurately measure 20 μl each of the above reference solution and test solution, inject them separately into the liquid chromatograph, record the chromatograms, calculate the content of LL37 in the test solution based on peak area using the external standard method, and compare it with the theoretical amount to calculate the recovery rate. Specific detection results are shown in Table 6: Table 6

[0075] As shown in Table 6, this method has high accuracy.

[0076] <Example 7> Validation of LL37 content determination method: robustness The model of the high-performance liquid chromatograph, the information on the test samples, and the test solution of the LL37@ZPF-2 complex gel were all the same as in Example 1.

[0077] The chromatographic conditions were adjusted, with the concentrations of phase A set at 0.08% and 0.12%, the initial proportions of phase B set at 38% and 42%, the column temperatures set at 30℃ and 40℃, the flow rates set at 0.8 ml / min and 1.2 ml / min, and the detection wavelengths set at 210 nm and 215 nm. The LL37@ZPF-2 complex gel sample solutions were analyzed, and the separation efficiency between the main peak and adjacent impurity peaks was used as the evaluation index to verify the robustness of the method. Specific results are shown in Table 7. Table 7

[0078] The results showed that, under the above chromatographic conditions, the main peak and adjacent impurity peaks were well separated.

[0079] <Example 8> Membrane inertness test The LL37 content was determined in the membrane inertness test using an Agilent 1260 high performance liquid chromatograph.

[0080] The information, chromatographic conditions, and assay method for LL37 reference standard are consistent with those in Example 1.

[0081] Test sample: LL37@ZPF-2 complex (self-made, number: 2).

[0082] Take an appropriate amount of LL37@ZPF-2 complex, dissolve and dilute it with 0.05mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing approximately 1 μg / ml of LL37. Prepare 6 parallel solutions, each 12 ml (consistent with the volume of the receiving liquid in the diffusion cell). Add a mixed cellulose ester membrane (0.45μm×25cm) to each of 3 of these solutions and soak them at 32±1℃ for 6 hours. Use the solution without membrane as a parallel control. At 0 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 0 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 6 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions with membrane, and take the average value as the 6 h drug concentration of the solution with membrane. Using the drug concentration of the unmembrane control at 6 h as the benchmark, the drug concentrations of the unmembrane control at 0 h and the membrane-coated solution at 6 h were compared with it. The results are shown in the table below.

[0083]

[0084] The results showed that the solution containing approximately 1 μg / ml LL37 obtained from the LL37@ZPF-2 complex was stable after 6 hours of storage at 32±1℃ in a receiving buffer of 0.05 mol / L pH 5.0 citrate-sodium citrate buffer. The average drug concentration of the solution containing the mixed cellulose ester membrane after 6 hours was 102.7% higher than that of the control solution without the membrane after 6 hours, indicating a recovery rate between 95% and 105%. This suggests that the mixed cellulose ester membrane is inert in 0.05 mol / L pH 5.0 citrate-sodium citrate buffer.

[0085] Test sample: LL37@ZPF-2 complex (self-made, number: 3).

[0086] Take an appropriate amount of LL37@ZPF-2 complex, dissolve and dilute it with 0.1mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing approximately 5 μg / ml of LL37. Prepare 6 parallel solutions, each 7 ml (consistent with the volume of the receiving liquid in the diffusion cell). Add a mixed cellulose ester membrane (0.45 μm × 25 cm) to each of 3 of these solutions and soak them at 32±1℃ for 6 h. Use the solution without membrane as a parallel control. At 0 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 0 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 6 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions with membrane, and take the average value as the 6 h drug concentration of the solution with membrane. Using the drug concentration of the unmembrane control at 6 h as the benchmark, the drug concentrations of the unmembrane control at 0 h and the membrane-coated solution at 6 h were compared with it. The results are shown in the table below.

[0087]

[0088] The results showed that the solution containing approximately 5 μg / ml LL37 obtained from the LL37@ZPF-2 complex was stable after 6 hours of storage at 32±1℃ in a receiving buffer of 0.1 mol / L pH 5.0 citrate-sodium citrate buffer. The average drug concentration of the solution containing the mixed cellulose ester membrane after 6 hours was 100.6% higher than that of the control solution without the membrane after 6 hours, indicating a recovery rate between 95% and 105%. This suggests that the mixed cellulose ester membrane is inert in 0.1 mol / L pH 5.0 citrate-sodium citrate buffer.

[0089] Test sample: LL37@ZPF-2 complex (self-made, number: 2).

[0090] Take an appropriate amount of LL37@ZPF-2 complex, dissolve and dilute it with 0.05mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing approximately 1 μg / ml of LL37. Prepare 6 parallel solutions, each 12 ml (consistent with the receiving liquid volume in the diffusion cell). Add a polyethersulfone membrane (0.45μm×25cm) to each of 3 of these solutions and soak them at 32±1℃ for 6 hours. Use the solution without membrane as a parallel control. At 0 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 0 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 6 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions with membrane, and take the average value as the 6 h drug concentration of the solution with membrane. Using the drug concentration of the unmembrane control at 6 h as the benchmark, the drug concentrations of the unmembrane control at 0 h and the membrane-coated solution at 6 h were compared with it. The results are shown in the table below.

[0091]

[0092] The results showed that the solution containing approximately 1 μg / ml of LL37 obtained from the LL37@ZPF-2 complex was stable after 6 hours of storage at 32±1℃ in a receiving buffer of 0.05 mol / L pH 5.0 citrate-sodium citrate buffer. The average drug concentration in the solution containing the polyethersulfone membrane after 6 hours was 91.6% of the average drug concentration in the control solution without the membrane after 6 hours, indicating that the polyethersulfone membrane adsorbs the drug in 0.05 mol / L pH 5.0 citrate-sodium citrate buffer and is therefore unsuitable as a membrane for in vitro release assays.

[0093] Test sample: LL37@ZPF-2 complex (self-made, number: 2).

[0094] Take an appropriate amount of LL37@ZPF-2 complex, dissolve and dilute it with 0.05mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution containing approximately 1 μg / ml of LL37. Prepare 6 parallel solutions, each 12 ml (consistent with the receiving liquid volume in the diffusion cell). Add a hydrophobic polyvinylidene fluoride membrane (0.45μm×25cm) to each of 3 of these solutions and soak them at 32±1℃ for 6 hours. Use the solution without membrane as a parallel control. At 0 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 0 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions without membrane, and take the average value as the 6 h drug concentration of the solution without membrane. At 6 h, take samples to measure the LL37 content in the 3 solutions with membrane, and take the average value as the 6 h drug concentration of the solution with membrane. Using the drug concentration of the unmembrane control at 6 h as the benchmark, the drug concentrations of the unmembrane control at 0 h and the membrane-coated solution at 6 h were compared with it. The results are shown in the table below.

[0095]

[0096] The results showed that the solution containing approximately 1 μg / ml of LL37 obtained from the LL37@ZPF-2 complex was stable after 6 hours of storage at 32±1℃ in a receiving buffer of 0.05 mol / L pH 5.0 citrate-sodium citrate buffer. The average drug concentration in the solution containing the hydrophobic polyvinylidene fluoride membrane after 6 hours was 93.2% of the average drug concentration in the control solution without the membrane after 6 hours. This indicates that the hydrophobic polyvinylidene fluoride membrane adsorbs the drug in 0.05 mol / L pH 5.0 citrate-sodium citrate buffer and is therefore unsuitable as a membrane for in vitro release assays.

[0097] <Example 9> In vitro release assay of LL37@ZPF-2 complex gel The information for the LL37 reference standard is consistent with that of Example 1.

[0098] Test sample: LL37@ZPF-2 complex gel (self-made, batch numbers: 24081402, 240701). The in vitro release test of the LL37@ZPF-2 complex thermosensitive gel was conducted according to the method shown in the table below.

[0099] The in vitro release test procedure is as follows. The equipment and operation method are only examples. The specific operation steps are adapted to the equipment used and are not intended to limit the in vitro release test equipment and operation method of this invention.

[0100] 1. Turn on the RT800 automatic sampling transdermal diffusion system, add approximately 80% of the specified receiving liquid volume to the diffusion cell, set the temperature to 32℃ and maintain the temperature.

[0101] 2. Immerse the mixed cellulose ester membrane in the receiving solution for 20-30 minutes. Remove the membrane, absorb the residual liquid with filter paper, fix it on the diffusion cell with a metering ring, add an appropriate amount of receiving solution to remove air, and then install the diffusion cell onto the diffuser.

[0102] 3. Enter the corresponding parameter values ​​on the equipment operation interface according to the in vitro release test method parameters in the table below, including diffusion cell volume, temperature, rotation speed, medium volume, sampling volume, replenishment volume, and sampling time.

[0103] 4. Use a pipette to add the specified amount of sample solution into the quantitative loop of the diffusion cell, and control the sample volume deviation within ±5%.

[0104] 5. After the sample loading is completed, the experiment will begin. Samples will be taken at 1h, 2h, 3h, 4h, 5h and 6h respectively, and the LL37 content will be determined by high performance liquid chromatography. The specific analytical method is as follows.

[0105] The in vitro release rate of the test samples was detected using the in vitro release test method shown in the table below (sample size n=6). During a 6-hour sampling period, the cumulative release amount of each diffusion cell was measured and calculated at sampling time points of 1h, 2h, 3h, 4h, 5h, and 6h. The x-axis was plotted as the square root of time (h), and the p-axis was plotted as the cumulative release amount per unit area (μg / cm²). 2 Plotting a standard curve with the x-axis as the y-axis, the slope of each linear release equation (n=6) is the in vitro release rate (IVRR), and the correlation coefficient r of the linear release equation is also obtained. 2 r 2 The value should not be less than 0.97. Calculate the CV value of IVRR (n=6), which should not be greater than 15% (e.g., Figure 5-6 (As shown).

[0106]

[0107] The analysis method is described as follows: A Thermo Hypersil Gold column was used at a column temperature of 35℃. Mobile phase A was 0.1% trifluoroacetic acid solution, and mobile phase B was acetonitrile. Gradient elution was performed with the following program: 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B. A diode array detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, and the injection volume was 20 μL.

[0108] Preparation of reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 50 ml volumetric flask, add about 25 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well.

[0109] Assay: Accurately measure 20 μl of the above reference solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of LL37 in the receiving liquid at each sampling time point by peak area using the external standard method.

[0110] The specific test results are as follows:

[0111] The in vitro release test of the above gel samples was repeated on different test days, and the test results are as follows:

[0112] The combined statistical analysis of the two tests showed that the inter-batch IVRR CV for 24081402 was 7.7% and that for 240701 was 6.3%, both less than 15%. 2 All values ​​were greater than 0.97, indicating that the intra-batch precision and inter-batch reproducibility determined by this method were good.

[0113] <Example 10> In vitro release assay of LL37@ZPF-2 complex gel The information for the LL37 reference standard is consistent with that of Example 1.

[0114] Test sample: LL37@ZPF-2 complex gel (self-made, batch numbers: 24081402, 240701).

[0115] The in vitro release test of the LL37@ZPF-2 complex thermosensitive gel was conducted according to the method in the table below.

[0116] The in vitro release rate of the test samples was detected using the in vitro release test method shown in the table below (sample size n=6). During a 6-hour sampling period, the cumulative release amount of each diffusion cell was measured and calculated at sampling time points of 1h, 2h, 3h, 4h, 5h, and 6h. The x-axis was plotted as the square root of time (h), and the p-axis was plotted as the cumulative release amount per unit area (μg / cm²). 2 Plotting a standard curve with the x-axis as the y-axis, the slope of each linear release equation (n=6) is the in vitro release rate (IVRR), and the correlation coefficient r of the linear release equation is also obtained. 2 r 2 The value should not be less than 0.97. Calculate the CV value of IVRR (n=6), which should not be greater than 15%.

[0117]

[0118] The analysis method is described as follows: A Thermo Hypersil Gold column was used at a column temperature of 35℃. Mobile phase A was 0.1% trifluoroacetic acid solution, and mobile phase B was acetonitrile. Gradient elution was performed with the following program: 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B. A diode array detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, and the injection volume was 20 μL.

[0119] Preparation of reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 50 ml volumetric flask, add about 25 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well.

[0120] Assay: Accurately measure 20 μl of the above reference solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of LL37 in the receiving liquid at each sampling time point by peak area using the external standard method.

[0121] The specific test results are as follows:

[0122] The in vitro release test of the above gel samples was repeated on different test days, and the test results are as follows:

[0123] The combined statistical analysis of the two tests showed that the inter-batch IVRR CV for 24081402 was 5.9% and that for 240701 was 4.7%, both less than 15%. 2 All values ​​were greater than 0.97, indicating that this method showed good intra-batch precision and inter-batch reproducibility in determining the self-made gel samples.

[0124] <Example 11> In vitro release assay of LL37@ZPF-2 complex gel The information for the LL37 reference standard is consistent with that of Example 1.

[0125] Test sample: LL37@ZPF-2 complex gel (self-made, batch numbers: 24081402, 240701).

[0126] The in vitro release test of the LL37@ZPF-2 complex thermosensitive gel was conducted according to the method in the table below.

[0127] The in vitro release rate of the test samples was detected using the in vitro release test method shown in the table below (sample size n=6). During a 6-hour sampling period, the cumulative release amount of each diffusion cell was measured and calculated at sampling time points of 1h, 2h, 3h, 4h, 5h, and 6h. The x-axis was plotted as the square root of time (h), and the p-axis was plotted as the cumulative release amount per unit area (μg / cm²). 2 Plotting a standard curve with the x-axis as the y-axis, the slope of each linear release equation (n=6) is the in vitro release rate (IVRR), and the correlation coefficient r of the linear release equation is also obtained. 2 r 2 The value should not be less than 0.97. Calculate the CV value of IVRR (n=6), which should not be greater than 15%.

[0128]

[0129] The analysis method is described as follows: A Thermo Hypersil Gold column was used at a column temperature of 35℃. Mobile phase A was 0.1% trifluoroacetic acid solution, and mobile phase B was acetonitrile. Gradient elution was performed with the following program: 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B. A diode array detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, and the injection volume was 20 μL.

[0130] Preparation of reference solution: Accurately weigh about 5 mg of LL37 reference standard, place it in a 50 ml volumetric flask, add about 25 ml of water, sonicate to dissolve, dilute to the mark with 0.1 mol / L pH 5.0 citrate-sodium citrate buffer, and shake well.

[0131] Assay: Accurately measure 20 μl of the above reference solution and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of LL37 in the receiving liquid at each sampling time point by peak area using the external standard method.

[0132] The specific test results are as follows:

[0133] The in vitro release test of the above gel samples was repeated on different test days, and the test results are as follows:

[0134] The combined statistical analysis of the two tests showed that the inter-batch IVRR CV for 24081402 was 5.5%, and the inter-batch CV for 240701 was 3.8%, both less than 15%. 2 All values ​​were greater than 0.97, indicating that this method showed good intra-batch precision and inter-batch reproducibility in determining the self-made gel samples.

[0135] <Example 12> In vitro release assay of LL37@ZPF-2 complex gel: selectivity The information, chromatographic conditions, and assay methods for LL37 reference standard are consistent with those in Example 9.

[0136] The method for preparing the LL37@ZPF-2 complex gel in this embodiment is consistent with Example 6 in CN119818648.

[0137] Test samples: LL37@ZPF-2 complex gel (self-made, batch numbers: 240701, 24081501, 24081502). Sample 240701, as a 100% specification sample, had an LL37 content of 0.226 mg / g. Sample 24081501, a 50% specification sample, was prepared using the same blank gel formulation, with an LL37 content of 0.112 mg / g. Sample 24081502, a 150% specification sample, had an LL37 content of 0.339 mg / g.

[0138] In vitro release tests were performed on 50% and 100% specification samples, and 100% and 150% specification samples, on the same day, using the same methods as in Example 9. (Based on USP40) <1724> The Mann-Whitney U test, used in Semisolid Drug Products—Performance Tests, is a statistical method for determining the 90% confidence interval of the IVRR ratio between two formulations. This study compared the IVRR results for 50%, 100%, and 150% strengths. If the 90% confidence interval for the IVRR ratio of the 50% and 100% strengths of the self-made samples was not within the range of 75%–133.33%, it indicated that their release rates were not equivalent. Similarly, if the 90% confidence interval for the IVRR ratio of the 100% and 150% strengths of the self-made samples was not within the range of 75%–133.33%, it also indicated that their release rates were not equivalent. The combined results of the inequitable IVRR results among the 50%, 100%, and 150% strengths of the self-made samples demonstrate a statistically significant difference in release rate, indicating that the IVRT method used for this product exhibits good selectivity.

[0139]

[0140] The results showed that the IVRR was not equivalent among the test samples of the three labeled specifications of 50%, 100%, and 150% (none of which were in the range of 75% to 133.33%), indicating that the IVRT method used in this product has good selectivity.

[0141] <Example 13> In vitro release assay of LL37@ZPF-2 complex gel: sensitivity Based on the IVRT test results under the optional item in Example 11, the IVRR values ​​of 50% labeled specification samples, 100% labeled specification samples, and 150% labeled specification samples were statistically analyzed. If the mean IVRR value of the 150% labeled specification samples is greater than that of the 100% labeled specification samples, and the IVRR value of the 100% labeled specification samples is greater than that of the 50% labeled specification samples, it indicates that the IVRT method of this product has good sensitivity.

[0142]

[0143] The results showed that the IVRR value was positively correlated with the change in drug content of the test sample of the LL37@ZPF-2 complex, indicating that the IVRT method used had good sensitivity.

[0144] <Example 14> In vitro release assay of LL37@ZPF-2 complex gel: specificity Based on the IVRT test results under the optional item in Example 11, a graph was plotted between the mean IVRR (Y) of the three labeled specifications (50%, 100%, and 150%) and the drug content (i.e., the LL37 content in the LL37@ZPF-2 gel sample) (X). Figure 7 If the correlation coefficient r of the obtained linear equation 2 It should conform to r 2 A requirement of ≥0.95 indicates that the IVRT method used in this product has good specificity.

[0145]

[0146] The results showed that the linear correlation coefficient r between the mean IVRR (Y) of the three labeled specifications (50%, 100%, and 150%) and the drug content (X) was the same. 2 The value is 0.9999, r 2 A value greater than 0.95 indicates that the IVRT method used has good specificity.

[0147] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the scope of protection of this invention.

Claims

1. A method for detecting the LL37 content in LL37@ZPF-2 complex and its gel, characterized in that, The determination is performed using high-performance liquid chromatography, specifically including the following steps: S1. Preparation of reference solution: Accurately weigh an appropriate amount of LL37 reference standard, first dissolve it in water to prepare a solution with a concentration of 0.1~0.4 mg / ml, then dilute it with 0.1 mol / L pH5.0 citrate-sodium citrate buffer to prepare a solution with a concentration of 0.05~0.2 mg / ml, shake well, and the solution is ready; S2. Chromatographic conditions: Octadecyl-bonded silica gel was used as the stationary phase; a 0.05%–0.2% trifluoroacetic acid aqueous solution was used as phase A, and acetonitrile or methanol was used as phase B; gradient elution was used, with the following elution program: 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B; a diode array detector was used, with a detection wavelength of 205 nm–230 nm; the column temperature was 25–45 °C; and the flow rate was 0.8–1.2 ml / min. S3. Determination method: Inject the reference solution and the test sample solution into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the test sample by peak area according to the external standard method; The amount of reference solution used is 10~40μl, and the amount of test sample detection solution used is 10~40μl; If the test sample is an LL37@ZPF-2 complex, the preparation steps for the LL37@ZPF-2 complex are as follows: accurately weigh an appropriate amount of the LL37@ZPF-2 complex, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution with a concentration of 0.8~2.0mg / ml, then dilute with water to prepare a solution with a concentration of 0.4~1.0mg / ml, shake well, and the solution is obtained. If the test sample is LL37@ZPF-2 complex gel, the preparation steps of LL37@ZPF-2 complex gel are as follows: accurately weigh an appropriate amount of LL37@ZPF-2 complex gel, first add 0.1mol / L pH5.0 citrate-sodium citrate buffer to dissolve it to prepare a solution with a concentration of 300~800mg / ml, then dilute with water to prepare a solution with a concentration of 150~400mg / ml, shake well, and the solution is obtained.

2. The method for detecting the LL37 content in the LL37@ZPF-2 complex and its gel according to claim 1, characterized in that, In step S1, the concentration of LL37 reference standard in the reference solution is 0.1 mg / ml.

3. The method for detecting the LL37 content in the LL37@ZPF-2 complex and its gel according to claim 1, characterized in that, In step S2, phase A is a 0.1% aqueous solution of trifluoroacetic acid, and phase B is acetonitrile; the detection wavelength is 215 nm; the column temperature is 35 °C; and the flow rate is 1.0 ml / min.

4. The method for detecting the LL37 content in the LL37@ZPF-2 complex and its gel according to claim 1, characterized in that, In step S3, the volume of the reference solution is 20 μl, and the volume of the test sample detection solution is 20 μl; If the test sample is an LL37@ZPF-2 complex, the concentration of the LL37@ZPF-2 complex in the test sample solution is 0.64 mg / ml; If the test sample is an LL37@ZPF-2 complex gel, then the concentration of the LL37@ZPF-2 complex gel in the test sample solution is 300 mg / ml.

5. A method for detecting the in vitro release of LL37 from an LL37@ZPF-2 complex gel, characterized in that, An in vitro release test of the LL37@ZPF-2 complex gel was conducted using a vertical diffusion cell method, and the content of LL37 in the receiving solution was determined using the method described in any one of claims 1-4. The test specifically included the following steps: S1. Conduct in vitro release tests on a transdermal diffusion apparatus. Use a mixed cellulose ester membrane as the filter membrane. The receiving solution is a pH 5.0 citrate-sodium citrate buffer solution. The diffusion cell temperature is 31℃~33℃, the rotation speed is 200~400rpm, the diffusion cell volume is 7~12ml, the sample loading volume is at least 1.0ml, the sampling volume does not exceed 1.5ml, the replenishment volume is the same as the sampling volume, and the sampling time does not exceed 6h. S2. Determination of LL37 content in the receiving solution: Octadecyl-bonded silica gel was used as the packing material; a 0.05%–0.2% trifluoroacetic acid aqueous solution was used as phase A, and acetonitrile or methanol was used as phase B; the gradient elution program was 0–10 min, 40%–60% B; 10–11 min, 60%–40% B; 11–21 min, 40% B; a diode array detector was used, with a detection wavelength of 205 nm–230 nm; the column temperature was 25–45 °C; the flow rate was 0.8–1.2 ml / min; 10–40 μl of the receiving solution sample was accurately measured and injected into the liquid chromatograph, the chromatogram was recorded, and the LL37 content in the receiving solution was calculated by peak area using the external standard method.

6. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 5, characterized in that, In step S1, the concentration of the receiving liquid is 0.05~0.1mol / L, and the sampling volume is 1.0~1.5ml.

7. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 5, characterized in that, In step S2, phase A is a 0.08%~0.12% trifluoroacetic acid aqueous solution, phase B is acetonitrile, the detection wavelength is 210nm~220nm, the column temperature is 30~40℃, and the flow rate is 0.9~1.1ml / min; Accurately measure 15-25 μl of the receiving liquid sample, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the receiving liquid by peak area using the external standard method.

8. The method for detecting the in vitro release of LL37 in the LL37@ZPF-2 complex gel according to claim 5, characterized in that, In step S1, the pore size of the filter membrane is 0.45 μm; the receiving solution is 0.05 mol / L pH 5.0 citrate-sodium citrate buffer; the rotation speed is 200 rpm; the diffusion cell volume is 10 ml; the sample loading volume is 1.0 ml; the sampling volume is 1.0 ml; the replenishment volume is the same as the sampling volume; and the sampling time points are 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h.

9. The method for detecting the in vitro release of LL37 in the LL37@ZPF-2 complex gel according to claim 8, characterized in that, In step S1, the rotation speed is 400 rpm; the diffusion cell volume is 7 ml; the sample loading volume is 1.5 ml; and the sampling volume is 1.5 ml.

10. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 8 or 9, characterized in that, In step S2, phase A is a 0.1% trifluoroacetic acid aqueous solution; phase B is acetonitrile; the gradient elution program is 0~10 min, 40%~60% B; 10~11 min, 60%~40% B; 11~21 min, 40% B; a diode array detector is used, the detection wavelength is 215 nm; the column temperature is 35℃; the flow rate is 1.0 ml / min; the determination method is as follows: accurately measure 20 μl of the receiving liquid sample, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of LL37 in the receiving liquid by peak area according to the external standard method.

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