CN111700861B - Gelsolin inclusion and nanocrystallization method and suspension - Google Patents

Gelsolin inclusion and nanocrystallization method and suspension Download PDF

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CN111700861B
CN111700861B CN202010504265.0A CN202010504265A CN111700861B CN 111700861 B CN111700861 B CN 111700861B CN 202010504265 A CN202010504265 A CN 202010504265A CN 111700861 B CN111700861 B CN 111700861B
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gelsolin
cyclodextrin
inclusion
nanocrystallization
cyclodextrin inclusion
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李玮
范立嵩
郄淑燕
杭伟
袁双虎
雷晟暄
马志祥
孟祥伟
孙海航
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Shandong University
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Abstract

The invention belongs to the field of nanoparticle preparation in a biochemical technology, and particularly relates to a gelsolin inclusion and nanocrystallization method and suspension. The implementation scheme is as follows: according to the mass parts, the gelsolin and the hydroxypropyl-beta-cyclodextrin are mixed according to the proportion of 1: 10 into a mortar, adding a small amount of purified water, grinding for 4h, filtering, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound. And (3) nanocrystallizing the inclusion complex to obtain the nanosuspension of the gelsolin-cyclodextrin inclusion complex. The method does not use organic solvent and toxic cross-linking agent, and has the advantages of simple operation, good inclusion effect, good reproducibility, safety and reliability.

Description

Gelsolin inclusion and nanocrystallization method and suspension
Technical Field
The invention relates to a preparation method of nanoparticles in the field of biotechnology, in particular to a protein inclusion method.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The protein-coated material commonly used at present is chitosan. Chitosan is the only basic polysaccharide occurring in nature. Low toxicity, reproducibility, excellent biodegradability and biocompatibility. The molecular chain of the compound has abundant hydroxyl and amino, and chemical reaction is easy to carry out. However, chitosan is insoluble in water and common organic solvents, and soluble in dilute inorganic acids and certain organic acids. The drawback of chitosan being only soluble in acidic solutions limits its use. Since acidic media tend to interfere with the performance of sensitive drugs and are toxic to organisms. The protein medicine has high medicine effect, strong pertinence and low side effect, but the protein medicine is unstable in vivo and in vitro and is very sensitive, and the protein is easy to denature if the protein is included by an emulsion polymerization method, an automatic emulsion method and the like. Therefore, the method for preparing the protein-loaded nanoparticles with mild use conditions and simple operation is significant.
Cyclodextrins (often abbreviated as CDs) have good biocompatibility. The unique amphiphilic structure can enable cyclodextrin to be used as a 'host' and to use the inner cavity of the cyclodextrin to encapsulate various hydrophobic 'guest' compounds to form an inclusion complex, so that the solubilization of hydrophobic molecules is realized. And no covalent bond is generated, belonging to the category of typical supermolecular interaction. The inclusion process is thus also reversible, i.e. under suitable conditions the inclusion complex will dissociate and release the guest molecule. This property makes the cyclodextrin inclusion complex particularly suitable for use as a nano-drug carrier.
At present, substances encapsulated by cyclodextrin are mostly volatile oil, and the encapsulation of protein substances is not common yet. Compared with volatile oil, the protein substance has unstable property and is easy to inactivate, so that the inclusion of the protein substance needs innovation of a method.
Disclosure of Invention
In order to overcome the problems, the invention aims to provide a protein inclusion and nanocrystallization method. The method of the invention does not use organic solvent and toxic cross-linking agent, and has simple operation, good reproducibility, safety and reliability.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for cyclodextrin inclusion and nanocrystallization of a protein, comprising:
adopting cyclodextrin to carry out inclusion on gelsolin to form cyclodextrin/gelsolin inclusion compound;
dispersing the cyclodextrin/gelsolin inclusion compound in an aqueous solution containing a stabilizing agent to obtain a primary suspension;
shearing the primary suspension at a high speed to obtain a coarse suspension;
and carrying out micro-jet high-pressure homogenization on the coarse suspension to obtain the gelsolin-cyclodextrin inclusion compound nano suspension.
The research of the invention finds that: the cyclodextrin can be used as a 'host' by grinding, and the gelsolin as a 'guest' compound is encapsulated by the cyclodextrin through the inner cavity of the cyclodextrin, so that the solubilization effect on the gelsolin is realized, and no covalent bond is generated; at the same time, under appropriate conditions, the inclusion complex will dissociate and release gelsolin.
The invention also provides the gelsolin-cyclodextrin inclusion complex nanosuspension prepared by any one of the methods.
The gelsolin-cyclodextrin inclusion complex nano suspension has stable properties and good solubility, and can be used as a nano drug carrier.
The invention also provides application of the gelsolin-cyclodextrin inclusion complex nano-suspension in preparing medicaments for treating inflammatory diseases, tumors, burns and wounds.
The invention has the beneficial effects that:
(1) the method does not use organic solvent and toxic cross-linking agent, and has good inclusion effect, simple operation, good reproducibility, safety and reliability.
(2) The method is simple, low in cost, strong in practicability and easy to popularize.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows UV-verified spectra of example 1 of the present invention, wherein 1. gelsolin, 2.HP- β -CD, 3. simple mixture of gelsolin and HP- β -CD, and 4. clathrate of gelsolin and HP- β -CD.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention aims to provide a protein inclusion and nanocrystallization method. The method comprises the following steps:
(1) according to the mass parts, the gelsolin and the hydroxypropyl-beta-cyclodextrin are mixed according to the proportion of 1: 10 into a mortar, adding a small amount of purified water, grinding for 4h, filtering, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
(2) Dispersing the inclusion complex in an aqueous solution containing a stabilizer to obtain a primary suspension; the mass ratio of the inclusion complex to the stabilizer is 5: 1-8: 1;
(3) shearing the primary suspension prepared in the step (2) at a high speed to obtain a coarse suspension;
(4) homogenizing the coarse suspension prepared in the step (3) under high pressure by microjet to obtain a nano suspension of the inclusion complex;
the stabilizer is a surfactant, or the mass ratio of the surfactant to the polymer suspending agent is 4: 1-0.25: 1.
In the step (1), the type of the surfactant can be selected according to the routine in the field, and the surfactant is preferably one or more of lecithin, sodium dodecyl sulfate, tween surfactants, span surfactants, fatty glyceride and sodium deoxycholate. The Tween surfactant is preferably one or more of Tween-20, Tween-40, Tween-60 and Tween-80. The span surfactant is preferably one or more of span-20, span-40, span-60 and span-80. The type of the polymer suspending agent can be selected according to the routine in the field, and the invention preferably selects one or more of polyethylene glycol polymer suspending agent, polyvinylpyrrolidone and cellulose polymer suspending agent. The type of the cellulose-based polymer suspending agent can be selected according to the conventional methods in the field, such as sodium carboxymethyl cellulose and/or hydroxypropyl methyl cellulose; the type of the polyethylene glycol polymer suspending agent can be selected according to the routine in the field, and PEG6000 and/or PEG4000 are preferred in the invention. The polyvinylpyrrolidone is preferably polyvinylpyrrolidone-K30. When the stabilizer is a stabilizer with the mass ratio of the surfactant to the polymer suspending agent being 4: 1-0.25: 1, the stabilizer is preferably a stabilizer with the mass ratio of the surfactant to the polymer suspending agent being 2: 1-0.5: 1.
In the step (2), the mass ratio of the inclusion complex to the stabilizer is preferably 6:1 to 3:1 (e.g., 4: 1). So as to avoid the agglomeration and increase of the nano particles and improve the long-term stability of the nano suspension.
In step (2), the water is water conventionally used in the art, and is generally water for injection. The water for injection is generally distilled water or distilled water of deionized water, and is also called double distilled water.
In the step (3), in the field of preparation of a drug nanosuspension, the high-speed shearing is known by those skilled in the art, which generally means that a high-shear disperser is used for dispersing the primary suspension, and the agglomerated particles are subjected to primary deagglomeration so as to meet the requirement of the particle size range of a microfluid high-pressure homogenizer, wherein the requirement of the microfluid high-pressure homogenizer in the field is generally 5-30 μm (such as 20 μm); the rotating speed of the high-speed shearing in the field is 10000-30000 rpm generally, and 22000rpm is preferred in the application. The high-speed shearing time in the invention is preferably 10-30 minutes, and more preferably 15-20 minutes. The high-speed shearing temperature can be selected according to the routine in the field, and is generally 0-10 ℃. The high shear described in the present invention is preferably carried out in an ice-water bath.
In the step (4), the conditions of the high-pressure homogenization by the micro-jet flow can be selected according to the particle size of the inclusion complex in the prepared inclusion complex nanosuspension, and the conditions are preferably as follows: the pressure of the high-pressure homogenization of the microjet is 22000-35000 psi, and more preferably 30000-35000 psi; the circulation frequency of the microjet high-pressure homogenization is preferably 15-30 times, and more preferably 20-24 times; the particle diameter D50 of the inclusion particle of the inclusion nanoparticle suspension is preferably 400-700 nm (for example, 500 or 600 nm).
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The drug concentration in the following examples is the ratio of the mass (mg) of the inclusion complex to the volume (mL) of water for injection.
Example 1
1. Preparation of the inclusion complex
10g of HP-beta-CD is weighed, placed in a mortar, added with 20ml of distilled water with the temperature of 35 ℃ and ground for 15 minutes. Weighing 1g of gelsolin, completely dissolving in 10ml of distilled water, slowly dripping into a mortar by using a constant flow pump, mixing with HP-beta-CD, grinding while dripping, continuing grinding after dripping is finished, and controlling the total grinding time to be 4 h. And placing the mixture into a dialysis bag, filtering, transferring into a freeze dryer, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
2. Detection of inclusion characterization
The formation of inclusion was verified by uv spectroscopy. The ultraviolet spectrum of the gelsolin HP-beta-CD inclusion compound is different from that of a pure product of the gelsolin and a simple mixture of the two; the pure gelsolin product has an obvious absorption peak at 280nm, and HP-beta-CD hardly absorbs at the wavelength; the peak of the simple mixture of gelsolin and HP-beta-CD is 280nm, which proves that the gelsolin serving as the main body does not form an inclusion compound with the HP-beta-CD; the ultraviolet absorption curve of the inclusion complex is extremely similar to that of HP-beta-CD, and the ultraviolet absorption at 280nm is extremely weak and is almost not absorbed; further illustrating the gelsolin being encapsulated and the formation of the inclusion complex, as shown in FIG. 1.
3. Preparation of inclusion complex nanosuspension
(1) 2.4g of Tween-20 and 1.5g of PEG4000 were dispersed in 100mL of water for injection to obtain a dispersion medium containing a stabilizer.
(2) Dispersing 24g of gelsolin-cyclodextrin inclusion complex in the dispersing medium containing the stabilizing agent in the step (1) to obtain an initial suspension with the concentration of 240 mg/mL.
(3) Shearing the primary suspension obtained in the step (2) in an ice water bath at a high speed of 22000rpm for 20 minutes to obtain a crude suspension of the inclusion complex.
(4) And (4) carrying out micro-jet high-pressure homogenization on the coarse suspension obtained in the step (3), wherein the homogenization pressure is 30000psi, and the cycle times are 20 times, so as to obtain the final product, namely the gelsolin-cyclodextrin inclusion compound nano suspension.
Example 2
1. Preparation of the inclusion complex
HP-beta-CD 8g is weighed, placed in a mortar, added with 20ml of distilled water at 30 ℃ and ground for 15 minutes. Weighing 1g of gelsolin, completely dissolving in 10ml of distilled water, slowly dripping into a mortar by using a constant flow pump, mixing with HP-beta-CD, grinding while dripping, continuing grinding after dripping is finished, and controlling the total grinding time to be 4 h. And placing the mixture into a dialysis bag, filtering, transferring into a freeze dryer, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
2. Detection of inclusion characterization
The formation of inclusion was verified by uv spectroscopy. The ultraviolet spectrum of the gelsolin HP-beta-CD inclusion compound is different from that of a pure product of the gelsolin and a simple mixture of the two; the pure gelsolin product has an obvious absorption peak at 280nm, and HP-beta-CD hardly absorbs at the wavelength; the peak of the simple mixture of gelsolin and HP-beta-CD is 280nm, which proves that the gelsolin serving as the main body does not form an inclusion compound with the HP-beta-CD; the ultraviolet absorption curve of the inclusion complex is extremely similar to that of HP-beta-CD, and the ultraviolet absorption at 280nm is extremely weak and is almost not absorbed; further, it is described that gelsolin is encapsulated and an inclusion complex is formed.
3. Preparation of inclusion complex nanosuspension
(1) 0.75g of Tween-20 and 1.5g of PEG4000 were dispersed in 100mL of water for injection to obtain a dispersion medium containing a stabilizer.
(2) Dispersing 24g of gelsolin-cyclodextrin inclusion complex in the dispersing medium containing the stabilizing agent in the step (1) to obtain an initial suspension with the concentration of 240 mg/mL.
(3) And (3) shearing the primary suspension obtained in the step (2) in an ice water bath at a high speed of 10000rpm for 30 minutes to obtain a crude suspension of the inclusion complex.
(4) And (4) carrying out micro-jet high-pressure homogenization on the coarse suspension obtained in the step (3), wherein the homogenization pressure is 35000psi, and the cycle times are 15 times, so as to obtain the final product, namely the gelsolin-cyclodextrin inclusion compound nano suspension.
Example 3
1. Preparation of the inclusion complex
HP-beta-CD 9g is weighed, placed in a mortar, added with 20ml of distilled water at 40 ℃ and ground for 15 minutes. Weighing 1g of gelsolin, completely dissolving in 10ml of distilled water, slowly dripping into a mortar by using a constant flow pump, mixing with HP-beta-CD, grinding while dripping, continuing grinding after dripping is finished, and controlling the total grinding time to be 4 h. And placing the mixture into a dialysis bag, filtering, transferring into a freeze dryer, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
2. Detection of inclusion characterization
The formation of inclusion was verified by uv spectroscopy. The ultraviolet spectrum of the gelsolin HP-beta-CD inclusion compound is different from that of a pure product of the gelsolin and a simple mixture of the two; the pure gelsolin product has an obvious absorption peak at 280nm, and HP-beta-CD hardly absorbs at the wavelength; the peak of the simple mixture of gelsolin and HP-beta-CD is 280nm, which proves that the gelsolin serving as the main body does not form an inclusion compound with the HP-beta-CD; the ultraviolet absorption curve of the inclusion complex is extremely similar to that of HP-beta-CD, and the ultraviolet absorption at 280nm is extremely weak and is almost not absorbed; further, it is described that gelsolin is encapsulated and an inclusion complex is formed.
3. Preparation of inclusion complex nanosuspension
(1) 3.0g of Tween-20 and 1.5g of PEG4000 were dispersed in 100mL of water for injection to obtain a dispersion medium containing a stabilizer.
(2) Dispersing 24g of gelsolin-cyclodextrin inclusion complex in the dispersing medium containing the stabilizing agent in the step (1) to obtain an initial suspension with the concentration of 240 mg/mL.
(3) Shearing the primary suspension obtained in the step (2) in an ice water bath at a high speed of 30000rpm for 10 minutes to obtain a crude suspension of the inclusion complex.
(4) And (4) carrying out micro-jet high-pressure homogenization on the coarse suspension obtained in the step (3), wherein the homogenization pressure is 22000psi, and the cycle times are 30 times, so as to obtain the final product, namely the gelsolin-cyclodextrin inclusion compound nano suspension.
Example 4
1. Preparation of the inclusion complex
10g of HP-beta-CD is weighed, placed in a mortar, added with 20ml of distilled water with the temperature of 35 ℃ and ground for 15 minutes. Weighing 1g of gelsolin, completely dissolving in 10ml of distilled water, slowly dripping into a mortar by using a constant flow pump, mixing with HP-beta-CD, grinding while dripping, continuing grinding after dripping is finished, and controlling the total grinding time to be 4 h. And placing the mixture into a dialysis bag, filtering, transferring into a freeze dryer, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
2. Detection of inclusion characterization
The formation of inclusion was verified by uv spectroscopy. The ultraviolet spectrum of the gelsolin HP-beta-CD inclusion compound is different from that of a pure product of the gelsolin and a simple mixture of the two; the pure gelsolin product has an obvious absorption peak at 280nm, and HP-beta-CD hardly absorbs at the wavelength; the peak of the simple mixture of gelsolin and HP-beta-CD is 280nm, which proves that the gelsolin serving as the main body does not form an inclusion compound with the HP-beta-CD; the ultraviolet absorption curve of the inclusion complex is extremely similar to that of HP-beta-CD, and the ultraviolet absorption at 280nm is extremely weak and is almost not absorbed; further, it is described that gelsolin is encapsulated and an inclusion complex is formed.
3. Preparation of inclusion complex nanosuspension
(1) 2.4g of Tween-20 and 1.5g of PEG4000 were dispersed in 100mL of water for injection to obtain a dispersion medium containing a stabilizer.
(2) Dispersing 24g of gelsolin-cyclodextrin inclusion complex in the dispersing medium containing the stabilizing agent in the step (1) to obtain an initial suspension with the concentration of 240 mg/mL.
(3) Shearing the primary suspension obtained in the step (2) in an ice water bath at a high speed of 22000rpm for 15 minutes to obtain a crude suspension of the inclusion complex.
(4) And (4) carrying out micro-jet high-pressure homogenization on the coarse suspension obtained in the step (3), wherein the homogenization pressure is 28000psi, and the cycle times are 22 times, so as to obtain the final product, namely the gelsolin-cyclodextrin inclusion compound nano suspension.
Example 5
1. Preparation of the inclusion complex
10g of HP-beta-CD is weighed, placed in a mortar, added with 20ml of distilled water with the temperature of 30 ℃ and ground for 15 minutes. Weighing 1g of gelsolin, completely dissolving in 10ml of distilled water, slowly dripping into a mortar by using a constant flow pump, mixing with HP-beta-CD, grinding while dripping, continuing grinding after dripping is finished, and controlling the total grinding time to be 4 h. And placing the mixture into a dialysis bag, filtering, transferring into a freeze dryer, and freeze-drying to obtain the cyclodextrin/gelsolin inclusion compound.
2. Detection of inclusion characterization
The formation of inclusion was verified by uv spectroscopy. The ultraviolet spectrum of the gelsolin HP-beta-CD inclusion compound is different from that of a pure product of the gelsolin and a simple mixture of the two; the pure gelsolin product has an obvious absorption peak at 280nm, and HP-beta-CD hardly absorbs at the wavelength; the peak of the simple mixture of gelsolin and HP-beta-CD is 280nm, which proves that the gelsolin serving as the main body does not form an inclusion compound with the HP-beta-CD; the ultraviolet absorption curve of the inclusion complex is extremely similar to that of HP-beta-CD, and the ultraviolet absorption at 280nm is extremely weak and is almost not absorbed; further, it is described that gelsolin is encapsulated and an inclusion complex is formed.
3. Preparation of inclusion complex nanosuspension
(1) 1.5g of Tween-20 and 1.5g of PEG4000 were dispersed in 100mL of water for injection to obtain a dispersion medium containing a stabilizer.
(2) Dispersing 24g of gelsolin-cyclodextrin inclusion complex in the dispersing medium containing the stabilizing agent in the step (1) to obtain an initial suspension with the concentration of 240 mg/mL.
(3) Shearing the primary suspension obtained in the step (2) in an ice water bath at a high speed of 22000rpm for 18 minutes to obtain a crude suspension of the inclusion complex.
(4) And (4) carrying out micro-jet high-pressure homogenization on the coarse suspension obtained in the step (3), wherein the homogenization pressure is 30000psi, and the cycle times are 22 times, so as to obtain the final product, namely the gelsolin-cyclodextrin inclusion compound nano suspension.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (13)

1. A method for cyclodextrin inclusion and nanocrystallization of a protein, comprising:
adopting cyclodextrin to carry out inclusion on gelsolin to form cyclodextrin/gelsolin inclusion compound;
dispersing the cyclodextrin/gelsolin inclusion compound in an aqueous solution containing a stabilizing agent to obtain a primary suspension;
shearing the primary suspension at a high speed to obtain a coarse suspension;
and carrying out micro-jet high-pressure homogenization on the coarse suspension to obtain the gelsolin-cyclodextrin inclusion compound nano suspension.
2. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 1, wherein the mass ratio of cyclodextrin to gelsolin is 8-10: 1.
3. the method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 1, wherein the cyclodextrin is hydroxypropyl- β -cyclodextrin.
4. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 1, wherein the inclusion comprises the following specific steps: mixing cyclodextrin and gelsolin uniformly, adding water, grinding, filtering, and freeze drying.
5. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 4, wherein the milling time is 3 to 6 hours.
6. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 1, wherein the stabilizing agent is a surfactant, or a surfactant and a polymeric suspending agent.
7. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 6, wherein the surfactant is one or more of lecithin, sodium dodecyl sulfate, a tween surfactant, a span surfactant, a fatty acid glyceride, and sodium deoxycholate.
8. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 6, wherein the polymer suspending agent is one or more of a polyethylene glycol-based polymer suspending agent, polyvinylpyrrolidone, and a cellulose-based polymer suspending agent.
9. The method for protein-directed cyclodextrin inclusion and nanocrystallization of claim 1, wherein the mass ratio of the gelsolin-cyclodextrin inclusion compound to the stabilizer is 5:1 to 8: 1.
10. The method for cyclodextrin inclusion and nanocrystallization of a protein according to claim 1, wherein the high shear is applied at a rotation speed of 10000 to 30000rpm for 10 to 30 minutes at a temperature of 0 to 10 ℃;
the pressure of the microjet high-pressure homogenization is 22000-35000 psi, and the cycle times are 15-30 times; the particle size range of the micro-jet high-pressure homogenization is 5-30 mu m.
11. The method for protein-directed cyclodextrin inclusion and nanocrystallization of claim 10, wherein the number of cycles is 20 to 24.
12. A gelsolin-cyclodextrin inclusion complex nanosuspension prepared by the method of any one of claims 1 to 11.
13. Use of the gelsolin-cyclodextrin inclusion complex nanosuspension according to claim 12 in the manufacture of a medicament for the treatment of inflammatory diseases, anti-tumor, burn, and trauma.
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