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CN-121994959-A - LL37@ZPF-2 compound and method for detecting LL37 content and in-vitro release in gel thereof

CN121994959ACN 121994959 ACN121994959 ACN 121994959ACN-121994959-A

Abstract

The invention relates to the field of analytical chemistry, in particular to a method for measuring the content of LL37 in an LL37@ZPF-2 compound and gel thereof by adopting a high performance liquid chromatography, a method for carrying out in-vitro release test of the gel of the LL37@ZPF-2 compound by adopting a vertical diffusion cell method, and then measuring the release amount of the LL37 at each time point by adopting the high performance liquid chromatography, thereby obtaining the in-vitro release rate. The content detection method of the invention adopts the high performance liquid chromatography to measure the content of LL37 in the LL37@ZPF-2 compound and the gel thereof by searching and repeatedly testing and screening specific detection conditions, has simple operation, strong specificity, high sensitivity and good precision and durability, and simultaneously solves the problem of inaccurate detection results caused by different dissolution rates of the LL37 and the LL37@ZPF-2 compound in the same solvent system by changing the addition sequence of water and citric acid-sodium citrate buffer solution.

Inventors

  • WANG JINHU
  • LI HONG
  • ZHANG HUI
  • ZHANG WEN
  • ZHAO AILI
  • WANG YINGCHAO
  • REN YUMIN
  • QU GUANGMIN
  • LI DAWEI
  • ZHANG YUN
  • FU BINGYUE

Assignees

  • 山东省药学科学院

Dates

Publication Date
20260508
Application Date
20260210

Claims (10)

  1. 1. The method for detecting the LL37 content in the LL37@ZPF-2 complex and the gel thereof is characterized by comprising the following steps of: s1, preparing a reference substance solution, namely precisely weighing a proper amount of LL37 reference substance, adding water to dissolve the LL37 reference substance to prepare a solution with the concentration of 0.1-0.4 mg/ml, diluting the solution with 0.1mol/L of citric acid-sodium citrate buffer solution with the pH of 5.0 to prepare a solution with the concentration of 0.05-0.2 mg/ml, and shaking the solution uniformly to obtain the reference substance solution; s2, chromatographic conditions comprise using octadecyl bonded silica gel as a filler, using a trifluoroacetic acid aqueous solution with the concentration of 0.05% -0.2% as a phase A and acetonitrile or methanol as a phase B, performing gradient elution, wherein the gradient elution program comprises 0% -10 min,40% -60% B, 10% -11 min,60% -40% B, 11% -21 min and 40% B, adopting a diode array detector to detect the wavelength of 205 nm-230 nm, the column temperature of 25-45 ℃ and the flow rate of 0.8-1.2 ml/min; S3, the determination method comprises the steps of respectively injecting a reference substance solution and a sample detection liquid to be tested into a liquid chromatograph, recording a chromatogram, and calculating the content of LL37 in the sample to be tested according to an external standard method by using a peak area; Wherein the dosage of the reference substance solution is 10-40 mu l, and the dosage of the sample detection solution is 10-40 mu l; If the sample is LL37@ZPF-2 compound, the preparation method of the LL37@ZPF-2 compound comprises precisely weighing appropriate amount of the LL37@ZPF-2 compound, adding 0.1mol/L citric acid-sodium citrate buffer solution with pH of 5.0 to prepare a solution with concentration of 0.8-2.0 mg/ml, diluting with water to prepare a solution with concentration of 0.4-1.0 mg/ml, and shaking uniformly to obtain the sample; If the sample is LL37@ZPF-2 composite gel, the preparation method of the LL37@ZPF-2 composite gel comprises the steps of precisely weighing a proper amount of the LL37@ZPF-2 composite gel, adding 0.1mol/L of pH5.0 citric acid-sodium citrate buffer solution to dissolve to prepare a solution with the concentration of 300-800 mg/ml, diluting with water to prepare a solution with the concentration of 150-400 mg/ml, and shaking uniformly to obtain the sample.
  2. 2. The method for detecting the LL37 content in the LL37@ZPF-2 complex and the gel thereof according to claim 1, wherein in the step S1, the concentration of the LL37 reference substance in the reference substance solution is 0.1mg/ml.
  3. 3. The method for detecting the LL37 content in the LL37@ZPF-2 complex and a gel thereof according to claim 1, wherein in the step S2, the A phase is 0.1% aqueous trifluoroacetic acid solution, the B phase is acetonitrile, the detection wavelength is 215nm, the column temperature is 35 ℃, and the flow rate is 1.0ml/min.
  4. 4. The method for detecting the LL37 content in the LL37@ZPF-2 complex and gel thereof according to claim 1, wherein in the step S3, the amount of the control solution is 20. Mu.l, and the amount of the sample detection solution is 20. Mu.l; If the sample is LL37@ZPF-2 complex, the concentration of the LL37@ZPF-2 complex in the sample detection solution is 0.64mg/ml; If the sample to be tested is LL37@ZPF-2 complex gel, the concentration of the LL37@ZPF-2 complex gel in the sample to be tested is 300 mg/ml.
  5. 5. An in vitro release method for detecting LL37 in LL37@ZPF-2 composite gel, which is characterized in that a vertical diffusion cell method is adopted to carry out an in vitro release test of the LL37@ZPF-2 composite gel, and the method of any one of claims 1-4 is adopted to measure the content of LL37 in a receiving solution, and specifically comprises the following steps: S1, performing an in-vitro release test on a transdermal diffusion instrument, wherein a filter membrane adopts a mixed cellulose ester membrane, a receiving solution is citric acid-sodium citrate buffer solution with pH of 5.0, the temperature of a diffusion cell is 31-33 ℃, the rotating speed is 200-400 rpm, the volume of the diffusion cell is 7-12 ml, the sample loading amount is at least 1.0ml, the sample volume is not more than 1.5ml, the fluid replacement volume is the same as the sample volume, and the sampling time is not more than 6 hours; S2, detecting the content of LL37 in the receiving liquid, namely taking octadecyl bonded silica gel as a filling agent, taking an aqueous solution of trifluoroacetic acid with the concentration of 0.05% -0.2% as a phase A and acetonitrile or methanol as a phase B, carrying out gradient elution for 0-10 min,40% -60% of B, 10-11 min,60% -40% of B, 11-21 min and 40% of B, adopting a diode array detector to detect the content of LL37 in the receiving liquid with the wavelength of 205-230 nm, the column temperature of 25-45 ℃ and the flow rate of 0.8-1.2 ml/min, precisely measuring 10-40 mu l of the receiving liquid sample, injecting the receiving liquid sample into a liquid chromatograph, recording a chromatogram, and calculating the content of LL37 in the receiving liquid according to an external standard method by using the peak area.
  6. 6. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 5, wherein in step S1, the concentration of said receiving solution is 0.05-0.1 mol/L and the sampling volume is 1.0-1.5 ml.
  7. 7. The method for detecting the in-vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 5, wherein in step S2, the A phase is 0.08% -0.12% trifluoroacetic acid aqueous solution, the B phase is acetonitrile, the detection wavelength is 210-220 nm, the column temperature is 30-40 ℃, and the flow rate is 0.9-1.1 ml/min; And precisely measuring 15-25 mu l of the receiving liquid sample, injecting the receiving liquid sample into a liquid chromatograph, recording a chromatogram, and calculating the content of LL37 in the receiving liquid according to an external standard method by using a peak area.
  8. 8. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 5, wherein in the step S1, the pore diameter of the filter membrane is 0.45 μm, the receiving solution is 0.05mol/L of citric acid-sodium citrate buffer solution with pH of 5.0, the rotating speed is 200rpm, the volume of the diffusion cell is 10ml, the loading amount is 1.0ml, the sampling volume is 1.0ml, the fluid replacement volume is the same as the sampling volume, and the sampling time points are 1h, 2h, 3h, 4h, 5h and 6h.
  9. 9. The method for detecting the in vitro release of LL37 in LL37@ZPF-2 complex gel according to claim 8, wherein in step S1, the rotation speed is 400rpm, the diffusion cell volume is 7ml, the loading amount is 1.5ml, and the sampling volume is 1.5ml.
  10. 10. The method for detecting the in-vitro release of LL37 in the LL37@ZPF-2 complex gel according to claim 8 or 9, wherein in the step S2, the A phase is 0.1% trifluoroacetic acid aqueous solution, the B phase is acetonitrile, the gradient elution program is 0-10 min,40% -60% B, 10-11 min,60% -40% B, 11-21 min and 40% B, a diode array detector is adopted, the detection wavelength is 215nm, the column temperature is 35 ℃, the flow rate is 1.0ml/min, the determination method is that 20 μl of a receiving liquid sample is precisely measured, the liquid chromatograph is injected, the chromatogram is recorded, and the content of LL37 in the receiving liquid is calculated according to an external standard method by peak area.

Description

LL37@ZPF-2 compound and method for detecting LL37 content and in-vitro release in gel thereof Technical Field The invention relates to the field of analytical chemistry, in particular to a method for measuring the content of LL37 in an LL37@ZPF-2 compound and gel thereof by adopting a high performance liquid chromatography, a method for carrying out in-vitro release test of the gel of the LL37@ZPF-2 compound by adopting a vertical diffusion cell method, and then measuring the release amount of the LL37 at each time point by adopting the high performance liquid chromatography, thereby obtaining the in-vitro release rate. Background The humanized Cathelicidin antibacterial peptide LL37 (molecular formula: C 205H340N60O53, CAS number: 145947-66-7) has angiogenic activity, is an antibacterial peptide derived from amphiphilic cathepsin consisting of 37 amino acid residues, has the amino acid sequence of LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES and the molecular weight of about 4493, has strong chemotaxis and immunoregulation characteristics, has wide antibacterial activity and activity of promoting wound repair and regeneration, and has high application value in the fields of bacterial infection, wound healing, chronic ulcer and other disease treatment. Studies have shown that unlike normal skin, which exhibits slightly acidic properties, the wound microenvironment pH is alkaline, which results in a decrease in the alpha helix content of antibacterial peptide LL37, and a decrease in the dissolution activity and antibacterial properties. Thus, the major challenge faced in the application of the antimicrobial peptide LL37 is its reduced activity and antimicrobial efficacy in the wound environment. LL37@ZPF-2 is a compound formed by in-situ encapsulation of LL37 with MOFs material ZPF-2. Research has shown that LL37@ZPF-2 has superior antimicrobial efficacy, cell migration effect and wound healing effect compared to LL 37. The LL37@ZPF-2 compound can gradually release the LL37 under specific conditions, gel matrix in the temperature-sensitive gel agent can change phase into a semi-solid state on the surface of skin, so that the release rate of the LL37 is further slowed down, and the activity reduction and the antibacterial effect reduction of the LL37 in a wound environment are both inhibited to a certain extent. The property of LL37@ZPF-2 to release the active ingredient LL37 under specific conditions makes possible the measurement of the LL37 content in the LL37@ZPF-2 complex and its formulations. In the prior art, the detection of the content of LL37 by using an enzyme-linked immunosorbent assay (ELISA) kit is reported, but no related report of the content of LL37 by using a high performance liquid chromatography is yet available, and particularly, the detection method for the content of LL37 in the LL37@ZPF-2 complex and a gel thereof still belongs to the blank. The content is a key attribute of medicine quality control and is important to ensure the effectiveness of medicines, so a rapid and accurate detection method is urgently needed for quantitatively measuring the content of LL37 in the LL37@ZPF-2 compound and the gel thereof. High performance liquid chromatography is a technique for separating, identifying and quantifying individual components in a mixture, but there are a number of technical difficulties in determining the LL37 content in the ll37@zpf-2 complex and its gels. These difficulties include inconsistent solubility of LL37, ll37@zpf-2 complex and its gel, affecting analytical accuracy, selection of appropriate chromatographic columns to achieve good separation, optimization of parameters such as mobile phase composition, ionic strength, etc. to obtain optimal separation, possible instability of samples during preparation and analysis, easy decomposition or chemical changes, possible interference of other components in the samples on detection, and finally, strict verification of high performance liquid chromatography methods including linear range, quantitative limits, precision, accuracy, durability, etc. to ensure reliability of the methods. In the in vitro release detection and evaluation of LL37@ZPF-2 composite gel, no related report exists in the prior art that the in vitro release detection of the LL37@ZPF-2 composite gel is carried out by adopting a Franz diffusion cell method, namely a vertical diffusion cell method, and no report exists in the prior art that the LL37 content in an in vitro release receiving solution is detected by adopting a high performance liquid chromatography. In literature Preparation and Evaluation of Novel Topical Gel Preparations for Wound HEALING IN Diabetics, in-vitro release characteristic research of medicines in wound repair gel is disclosed by adopting a membrane dialysis method, namely, a semipermeable membrane standard cellophane membrane is adopted to encapsulate medicine-containing gel, the gel is immersed into a release medium, the medicines are diffus