CN111811999B - Method for measuring particle size of cross-linked dextran microspheres wrapped in cross-linked sodium hyaluronate gel - Google Patents
Method for measuring particle size of cross-linked dextran microspheres wrapped in cross-linked sodium hyaluronate gel Download PDFInfo
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- CN111811999B CN111811999B CN202010556731.XA CN202010556731A CN111811999B CN 111811999 B CN111811999 B CN 111811999B CN 202010556731 A CN202010556731 A CN 202010556731A CN 111811999 B CN111811999 B CN 111811999B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Abstract
The invention provides a method for measuring the particle size of a cross-linked dextran microsphere wrapped in a cross-linked sodium hyaluronate gel. According to the method, the crosslinked sodium hyaluronate gel is subjected to enzymolysis in a water bath at 37 ℃ by hyaluronidase, and after centrifugal washing, the particle size of the crosslinked dextran microspheres wrapped in the crosslinked sodium hyaluronate gel is measured by a laser particle size analyzer. The method is simple and quick to operate, small in human error, good in repeatability and accurate and reliable in result.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for measuring particle size of cross-linked dextran microspheres wrapped in cross-linked sodium hyaluronate gel.
Background
Dextran is a high molecular polysaccharide compound formed by dehydrating glucose units. The cross-linked dextran is a polymer compound, usually a white bead microsphere, having a cross-linked three-dimensional network structure formed by reacting purified linear dextran (pyran-type dextran) with epichlorohydrin and introducing glycerol side chains, and forming ether bonds between the dextran chains.
The cross-linked dextran microspheres have the effects of improving the gel stability, slowing down the gel release rate and the like, and have higher application value. Clinically, the composition can be used for accelerating wound healing, treating arthritis and the like. The cosmetic has effects in keeping moisture and resisting skin aging. As a filler, the particle size of the cross-linked dextran microspheres is a key index that enables the microspheres to smoothly pass through a needle, and not be phagocytized by phagocytes, nor excessively stimulate collagen proliferation.
However, no related detection technology for determining the particle size of the endoglucans coated in the crosslinked sodium hyaluronate gel is reported in the prior art, and the particle size of the endoglucans coated in the crosslinked sodium hyaluronate gel is difficult to be accurately determined by an instrument, so that an accurate and reliable value cannot be obtained.
Disclosure of Invention
The invention aims at solving the defects of the prior art and provides a method for measuring the particle size of cross-linked dextran microspheres coated in cross-linked sodium hyaluronate gel, which can be used for measuring the particle size of the cross-linked dextran microspheres coated in the cross-linked sodium hyaluronate gel by a laser particle sizer. For this purpose, the invention adopts the following technical scheme:
a method for determining the particle size of cross-linked dextran microspheres encapsulated in a cross-linked sodium hyaluronate gel, the method comprising the steps of:
step one: weighing a certain amount of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding a hyaluronidase solution, and performing enzymolysis in a constant-temperature water bath until the crosslinked hyaluronic acid nanogel is completely subjected to enzymolysis;
step two: washing the product after enzymolysis with purified water, and centrifuging to remove the upper layer solution to obtain a cross-linked dextran microsphere test solution;
step three: and (3) dripping a proper amount of cross-linked dextran microsphere test solution into a dispersion medium, balancing the test solution in the dispersion medium for a period of time, and measuring the particle size by a laser particle sizer.
On the basis of adopting the technical scheme, the invention can also adopt the following further technical schemes or use the further technical schemes in combination:
the water bath temperature was 37 ℃.
In the first step, the preparation method of the hyaluronidase solution comprises the following steps: and dissolving a proper amount of hyaluronidase into a hyaluronidase solution with a corresponding concentration by using 0.9% sodium chloride.
In the first step, the mass ratio of the crosslinked sodium hyaluronate gel to the hyaluronidase solution is 1:2, and the concentration of the hyaluronidase solution is inversely proportional to the complete enzymolysis time.
In the first step, when the concentration of the glass plum solution is 50U/mL, the time for complete gel enzymolysis is 4-8h.
And step two, washing the residual test solution from which the upper layer solution is removed by centrifugation with purified water, removing the upper layer solution by centrifugation, and repeating the steps for a plurality of times to obtain the residual test solution from which the upper layer solution is removed, wherein the residual test solution from which the upper layer solution is removed is the cross-linked dextran microsphere test solution.
In the third step, the balancing time is preferably 5-15min.
In the third step, the parameter setting of the laser particle analyzer is preferably 5-15% of shading degree, and the rotating speed of the pump is 1000-2000r/min.
The beneficial effects of the invention are as follows: a method for measuring the particle size of the cross-linked dextran microspheres coated in the cross-linked sodium hyaluronate gel by using a laser particle sizer is established. According to the method, the gel is subjected to enzymolysis by the hyaluronidase in a water bath at 37 ℃, and then the particle size of the cross-linked dextran microspheres wrapped in the cross-linked sodium hyaluronate gel is measured by a laser particle sizer. The method fills the technical blank of particle size detection of the crosslinked dextran microspheres coated in the crosslinked sodium hyaluronate gel, and has important significance.
Drawings
FIG. 1 is a graph of particle size determined for a gel sample of example 1 without pretreatment
FIG. 2 is a graph of particle size determined for the gel sample of example 2
FIG. 3 is a graph of particle size determined for the gel sample of example 3
FIG. 4 is a graph of particle size determined for the gel sample of example 4
FIG. 5 is a graph of particle size determined for the gel sample of example 5
FIG. 6 is a graph of particle size determined for the gel sample of example 6
Detailed Description
The invention will be further described with reference to examples in order to better explain the flow and aspects of the invention. The specific examples described herein are intended to be illustrative of the invention and are not intended to be limiting.
And (3) a step of: determination of particle size of sephadex microsphere coated in sodium Cross-linked hyaluronate gel
Example 1
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres in a container, adding dispersion medium purified water, balancing for 10min, and measuring the particle size by using a laser particle sizer, wherein the parameter setting range is that the shading degree is 5% -15%, and the pump rotating speed is 1500r/min.
Example 2
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding 8g of a 50U/mL hyaluronidase solution, and performing enzymolysis for 4 hours in a constant-temperature water bath at 37 ℃; washing the product after enzymolysis with purified water, centrifuging to remove the upper layer solution, and repeating the operation for 3 times to obtain the cross-linked dextran microsphere test solution; and (3) dripping a proper amount of cross-linked dextran microsphere test solution into dispersion medium purified water, balancing for 10min, and measuring the particle size by using a laser particle analyzer, wherein the parameter setting range is that the shading degree is 5-15%, and the pump rotating speed is 1500r/min.
Example 3
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding 8g of a 50U/mL hyaluronidase solution, and carrying out enzymolysis for 8 hours in a constant-temperature water bath at 37 ℃; washing the product after enzymolysis with purified water, centrifuging to remove the upper layer solution, and repeating the operation for 3 times to obtain the cross-linked dextran microsphere test solution; and (3) dripping a proper amount of cross-linked dextran microsphere test solution into dispersion medium purified water, balancing for 10min, and measuring the particle size by using a laser particle analyzer, wherein the parameter setting range is that the shading degree is 5-15%, and the pump rotating speed is 1500r/min.
Example 4
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding 8g of a 50U/mL hyaluronidase solution, and performing enzymolysis for 4 hours in a constant-temperature water bath at 37 ℃; washing the product after enzymolysis with purified water, centrifuging to remove the upper layer solution, and repeating the operation for 3 times to obtain the cross-linked dextran microsphere test solution; and (3) dripping a proper amount of cross-linked dextran microsphere test solution into dispersion medium purified water, balancing for 5min, and measuring the particle size by using a laser particle analyzer, wherein the parameter setting range is that the shading degree is 5-15%, and the pump rotating speed is 1500r/min.
Example 5
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding 8g of a 50U/mL hyaluronidase solution, and performing enzymolysis for 4 hours in a constant-temperature water bath at 37 ℃; washing the product after enzymolysis with purified water, centrifuging to remove the upper layer solution, and repeating the operation for 3 times to obtain the cross-linked dextran microsphere test solution; and (3) dripping a proper amount of cross-linked dextran microsphere test solution into dispersion medium purified water, balancing for 10min, and measuring the particle size by using a laser particle analyzer, wherein the parameter setting range is that the shading degree is 5-15%, and the pump rotating speed is 1000r/min.
Example 6
Weighing 4g of crosslinked hyaluronic acid nanogel containing crosslinked dextran microspheres, putting the crosslinked hyaluronic acid nanogel into a container, adding 8g of a 50U/mL hyaluronidase solution, and performing enzymolysis for 4 hours in a constant-temperature water bath at 37 ℃; washing the product after enzymolysis with purified water, centrifuging to remove the upper layer solution, and repeating the operation for 3 times to obtain the cross-linked dextran microsphere test solution; and (3) dripping a proper amount of cross-linked dextran microsphere test solution into dispersion medium purified water, balancing for 5min, and measuring the particle size by using a laser particle analyzer, wherein the parameter setting range is that the shading degree is 5-15%, and the pump rotating speed is 1000r/min.
And II: measurement result of particle size of Sephadex microsphere coated in Cross-linked sodium hyaluronate gel
Table 1: EXAMPLE 1 determination of particle size of gel sample without pretreatment
Conclusion: and the cross-linked dextran microspheres are wrapped by the gel without pretreatment operations such as enzymolysis and centrifugation, so that the measured particle size result is the mixed particle size of the gel and the cross-linked dextran microspheres, and the experimental result is inaccurate.
Table 2: measurement results under different conditions (D represents the diameter of the particle, D10 represents the diameter of the cumulative 10% point, D50 represents the diameter of the cumulative 50% point, and D90 represents the diameter of the cumulative 90% point)
Conclusion: using the same samples, the same person was conducted in accordance with each of examples 2 to 6 above, and the measurement results are shown in Table 2. The method has the advantages of small relative deviation of different measurement results, accurate results and wide application range.
Table 3: measurement results of the operations performed by different analysts according to example 2 (D represents the diameter of the particles, D10 represents the diameter of the cumulative 10% points, D50 represents the diameter of the cumulative 50% points, and D90 represents the diameter of the cumulative 90% points)
Conclusion: the same sample was measured 6 times by different analysts according to example 2 and the results are shown in table 3. The method has the advantages of small human error and good repeatability.
The foregoing is merely illustrative of the preferred embodiments of the present invention and the invention is not limited to the specific embodiments described above, but is capable of modification and variation without departing from the spirit and principles of the invention.
Claims (2)
1. A method for determining the particle size of cross-linked dextran microspheres encapsulated in a cross-linked sodium hyaluronate gel, the method comprising the steps of:
step one: weighing a certain amount of crosslinked sodium hyaluronate gel containing crosslinked dextran microspheres, placing the crosslinked sodium hyaluronate gel in a container, adding a hyaluronidase solution, and performing enzymolysis in a constant-temperature water bath until the crosslinked sodium hyaluronate gel is completely subjected to enzymolysis;
step two: washing the product after enzymolysis with purified water, and centrifuging to remove the upper layer solution to obtain a cross-linked dextran microsphere test solution;
step three: dripping a proper amount of cross-linked dextran microsphere test solution into a dispersion medium, balancing the test solution in the dispersion medium for a period of time, and measuring the particle size by a laser particle sizer;
in the first step, the mass ratio of the crosslinked sodium hyaluronate gel to the hyaluronidase solution is 1:2, and the concentration of the hyaluronidase solution is inversely proportional to the complete enzymolysis time; when the concentration of the glass plum solution is 50U/mL, the gel enzymolysis is completed for 4-8h; the water bath temperature is 37 ℃;
washing the residual test solution from which the upper layer solution is removed by centrifugation with purified water, removing the upper layer solution by centrifugation, and repeating the steps for a plurality of times to obtain the residual test solution from which the upper layer solution is removed, wherein the residual test solution is the cross-linked dextran microsphere test solution;
in the third step, the balancing time is 5-15min; the parameters of the laser particle size analyzer are set to 5-15% of shading degree, and the rotating speed of the pump is 1000-2000r/min.
2. A method for determining the particle size of cross-linked dextran microspheres entrapped in a cross-linked sodium hyaluronate gel according to claim 1, wherein: in the first step, the preparation method of the hyaluronidase solution comprises the following steps: and dissolving a proper amount of hyaluronidase into a hyaluronidase solution with a corresponding concentration by using 0.9% sodium chloride.
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| US3925018A (en) * | 1973-06-08 | 1975-12-09 | Technicon Instr | Method and apparatus for quantitative analysis utilizing particulate reagent material |
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| US3925018A (en) * | 1973-06-08 | 1975-12-09 | Technicon Instr | Method and apparatus for quantitative analysis utilizing particulate reagent material |
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| Temperature-Triggered enzyme immobilization and release based on cross-linked gelatin nanoparticles;Gab,ZH等;《web of science researherlD》;第7卷(第10期);第1-10页 * |
| 激光粒度分析仪测定交联透明质酸钠凝胶粒度方法的研究;朱莲等;《中国化工贸易》;第215页 * |
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