CN112956473B - Artificial transparent belt and preparation method thereof - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
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Abstract
The invention provides an artificial transparent belt which is prepared from sodium alginate and polyornithine. The artificial transparent belt provided by the invention can store a small amount of sperms (including single sperms) in the cavity of the inner core, so that the loss of sperms is effectively avoided, the biocompatibility of the used materials is good, the activity of sperms is not influenced, and the problems of ethics, DNA or foreign heterologous protein pollution and the like are avoided.
Description
Technical Field
The invention belongs to the technical field of auxiliary reproduction, and particularly relates to an artificial transparent belt and a preparation method thereof.
Background
Infertility is a major disease that severely jeopardizes the reproductive health of humans, and the World Health Organization (WHO) predicts that infertility in the 21 st century will be the third world disease next to cancer and cardiovascular disease. WHO survey data showed that infertility incidence was about 10-15% of women of childbearing age worldwide, with men's factors accounting for about 40-50%. 70% of male infertility patients are caused by semen abnormalities and are mainly manifested by oligospermia, dysspermia, abnormal sperm or azoospermia. The incidence rates of oligospermia, azoospermia and oligospermia in men are statistically on an ascending year-by-year basis. The azoospermia accounts for 10% -15% of the infertility men, wherein 80% of the azoospermia is non-obstructive azoospermia, and no effective treatment means exists at present. In vitro fertilization by testis microscopy in combination with intracavitary single sperm injection (ICSI) techniques is the only hope that non-obstructive azoospermia patients will acquire genetic offspring.
The pregnancy rate of the clinical ICSI treatment cycle was only 50% and some patients required repeated ICSI treatments in order to obtain pregnancy. Since testis extraction is an invasive procedure, each extraction may cause testicle atrophy or further decrease of testicle function, thereby completely losing seminiferous function. In order to reduce the pain of repeated semen extraction of patients as much as possible and avoid complete loss of testicular seminiferous function, it is necessary to freeze-preserve trace motile sperm obtained by diagnostic puncture of azoospermia patients or remaining in the ICSI treatment cycle so as to resuscitate the sperm for ICSI treatment on the day of egg extraction of women, which greatly improves the flexibility and reliability of assisted fertility treatment of azoospermia patients. Meanwhile, the sperm motility of the patient with severe oligospermia is unstable, and the sperm of the patient with preventive freezing is also necessary, so that the period is canceled because no sperm is available in the day of egg taking of women is avoided. Therefore, the cryopreservation of trace sperm is a key technology for improving the success rate of pregnancy.
Cryopreservation of a small amount of sperm is a difficult problem in the assisted reproduction field, and no ideal cryopreservation method exists at present. The zona pellucida is a better method for freezing a small amount of sperms, and the zona pellucida is a layer of glycoprotein wrapped outside the oocytes and plays an important role in ovum generation, ovum protection and multi-sperm fertilization prevention. The zona pellucida contents were removed using micromanipulation techniques, and then small amounts of sperm were injected into the empty zona pellucida using an ICSI microinjection needle. In 1997, walmsley et al reported that the first infant successfully fertilized with empty zona pellucida frozen testis sperm was born, 3 of 5 cycles were pregnant, and 5 of them were twins. The freezing recovery rate of the sperm can be obviously improved by freezing the empty zona pellucida, the motility, the chromatin and the DNA integrity of the sperm are maintained, researches show that the freezing recovery rate of the frozen sperm of the zona pellucida is 88.3%, the fertilization rate of the ICSI motile sperm reaches 60%, the damage of the freezing of the empty zona pellucida to the sperm is lower, and the freezing of a small amount or single sperm can be well protected. In 2005, yellow lotus phoenix and Xu Chenming take a clear belt with evacuated cytoplasm as a cryopreservation carrier to freeze and store sperms, firstly, the testis sperms of biopsy are put into a closed clear belt, then, the addition and elution of a sperm cryoprotectant are carried out, and the sperms are closed and stored in the clear belt in the processes of cryopreservation and resuscitation, so that the loss of the sperms is avoided, and the sperm recovery rate is as high as 70 percent and is far higher than that of the conventional cryopreservation method. Although recovery rate of zona pellucida freezing is over 70%, there are problems such as that the method uses zona pellucida of mammal or human egg, material source is difficult (zona pellucida is difficult to obtain), ethical disputes exist (host DNA is at risk of being injected into human egg together with sperm in ICSI treatment), and there is also a risk of zoonotic infection, so that the method is difficult to be widely applied in assisted reproduction clinic.
In conclusion, the zona pellucida method is a better cryopreservation method for a small amount or a single sperm, and the cryopreservation operation of the sperm is simple and quick, but the method also has the problems of ethics, unavoidable pollution of DNA or foreign heterologous proteins and the like, and limits the clinical application of the zona pellucida method.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide an artificial transparent belt and a preparation method thereof, wherein the artificial transparent belt provided by the invention has good biocompatibility, does not influence sperm activity, and has no problems of ethics, DNA or foreign heterologous protein pollution and the like.
The invention provides an artificial transparent belt which is prepared from sodium alginate and polyornithine.
Preferably, the artificial transparent belt is a shell with a hollow structure, and the shell is a sodium alginate and polyornithine polyelectrolyte composite film;
the size of the artificial transparent belt is 80-200 mu m, preferably 100-150 mu m; the film thickness of the artificial transparent belt is 5 to 50. Mu.m, preferably 10 to 30. Mu.m.
Preferably, the polyornithine has a number average molecular weight of from 10kDa to 100kDa, preferably from 10kDa to 50kDa.
The invention also provides a preparation method of the artificial transparent belt, which comprises the following steps:
a) Mixing calcium alginate hydrogel microspheres with polyornithine solution, performing a film forming reaction, and forming a polyornithine polyelectrolyte complex film outside the microspheres to obtain coated hydrogel microspheres;
b) And mixing the coated hydrogel microspheres with sodium citrate solution, and performing liquefaction reaction to obtain the artificial transparent belt with the hollow structure.
Preferably, the calcium alginate hydrogel microspheres are prepared by an electrostatic liquid drop method or an emulsion internal gelation method.
Preferably, the raw materials for preparing the calcium alginate hydrogel microspheres are sodium alginate solution and calcium chloride solution, wherein the viscosity of the sodium alginate solution is 50-200 mPa.s, preferably 80-150 mPa.s; the mass concentration of the calcium chloride solution is 0.5% -2%, preferably 0.8% -1.5%.
Preferably, the polyornithine solution has a mass concentration of 0.01% to 3%, preferably a mass concentration of 0.05% to 2%.
Preferably, in step a), the temperature of the reaction is room temperature and the time of the reaction is 1 to 20 minutes, preferably 3 to 10 minutes.
Preferably, the sodium citrate concentration is 30 to 70mM, preferably 40 to 60mM.
Preferably, the liquefaction reaction time is 5-30 min, preferably 10-20 min; the temperature of the liquefaction reaction is room temperature.
Compared with the prior art, the invention provides an artificial transparent belt which is prepared from sodium alginate and polyornithine. The artificial transparent belt provided by the invention can store a small amount of sperms (including single sperms) in the cavity of the inner core, so that the loss of sperms is effectively avoided, the biocompatibility of the used materials is good, the activity of sperms is not influenced, and the problems of ethics, DNA or foreign heterologous protein pollution and the like are avoided.
Drawings
FIG. 1 is a microscopic photograph of the artificial transparent tape prepared in example 1;
FIG. 2 is an artificial zona pellucida loaded with sperm.
Detailed Description
The invention provides an artificial transparent belt which is prepared from sodium alginate and polyornithine.
In the invention, the artificial transparent belt is a shell with a hollow structure, and the shell is a sodium alginate and polyornithine polyelectrolyte composite film.
The size of the artificial transparent belt is 80-200 mu m, preferably 100-150 mu m; the film thickness of the artificial transparent belt is 5 to 50. Mu.m, preferably 10 to 30. Mu.m.
The molecular weight of the polyornithine is 10kDa to 100kDa, preferably 10kDa to 50kDa.
The invention also provides a preparation method of the artificial transparent belt, which comprises the following steps:
a) Mixing calcium alginate hydrogel microspheres with polyornithine solution, performing a film forming reaction, and forming a polyornithine polyelectrolyte complex film outside the microspheres to obtain coated hydrogel microspheres;
b) And mixing the coated hydrogel microspheres with sodium citrate solution, and performing liquefaction reaction to obtain the artificial transparent belt with the hollow structure.
The method for preparing the calcium alginate hydrogel microsphere is not particularly limited, and the method is known to those skilled in the art. Preferably, the invention adopts an electrostatic liquid drop method or an emulsifying internal gelation method to prepare the calcium alginate hydrogel microsphere.
The raw materials for preparing the calcium alginate hydrogel microspheres are sodium alginate solution and calcium chloride solution, wherein the viscosity of the sodium alginate solution is 50-200 mPa.s, preferably 80-150 mPa.s; the mass concentration of the calcium chloride solution is 0.5% -2%, preferably 0.8% -1.5%. The calcification time of the hydrogel microsphere is 10-30 min, preferably 15-25 min; microsphere preparation temperature is room temperature.
After the calcium alginate hydrogel microsphere is obtained, mixing the calcium alginate hydrogel microsphere with polyornithine solution, performing film forming reaction, and forming a polyornithine polyelectrolyte complex film outside the microsphere to obtain the coated hydrogel microsphere. Wherein, the molecular weight of the polyornithine is 10 kDa-100 kDa, preferably 10 kDa-50 kDa, the mass concentration of the solution is 0.01-3%, preferably 0.05-2%; the reaction time is 1-20 min, preferably 3-10 min; the reaction temperature was room temperature.
And mixing the coated hydrogel microspheres with sodium citrate solution, and performing liquefaction reaction to obtain the artificial transparent belt with the hollow structure. Wherein,,
the concentration of the sodium citrate solution is 30-70 mM, preferably 40-60 mM; liquefying time is 5-30 min, and optimizing time is 10-20 min; the reaction temperature was room temperature.
The artificial zona pellucida provided by the invention is used for cryopreserving a small amount of sperms with high sperm activity, and contains single sperms such as testis-piercing sperms, epididymal-piercing sperms, sperms of patients with severe oligospermia and the like.
The invention provides an artificial transparent belt for cryopreservation of a small amount of sperms and a preparation method thereof, and aims to provide an artificial transparent belt which has small volume, is provided with a closed space, is easy to operate and has high sperm activity and is suitable for cryopreservation of a small amount of sperms (including single sperms such as testis-piercing sperms, epididymal-piercing sperms, sperms of patients with severe oligospermia and asthenozoospermia and the like).
The technical scheme provided by the invention has the following technical advantages:
1. the artificial transparent belt provided by the invention has the advantages that the materials are nontoxic, the biocompatibility is good, the loaded sperms are not influenced, and the activity of the sperms is kept.
2. The artificial transparent belt provided by the invention has small volume, and can easily find and grasp sperms in micromanipulation, thereby being beneficial to maintaining the activity of sperms.
3. The artificial transparent belt provided by the invention has higher strength, unchanged form after freezing and keeping intact, effectively avoids the loss of sperms in the freezing and recovery processes, and has high sperm recovery rate.
4. The artificial transparent belt provided by the invention has a simple preparation process and is easy to amplify and realize industrial production.
In order to further understand the present invention, the artificial transparent belt and the preparation method thereof are described below with reference to examples, and the scope of the present invention is not limited by the following examples.
Example 1
The invention provides an artificial transparent belt for cryopreservation of a small amount of sperms and a preparation method thereof,
1. preparation of polyornithine artificial transparent belt
(1) Preparing a sodium alginate solution with the mass concentration of 1.2%, measuring the viscosity to be 90.3 mPa.s by a viscometer, dripping the sodium alginate solution into a calcium chloride solution with the mass concentration of 1.1% by adopting an electrostatic liquid drop method to form calcium alginate hydrogel microspheres, and removing the calcium chloride solution after calcification for 30 min;
(2) Adding calcium alginate hydrogel microspheres into polyornithine (number average molecular weight 22 kD) solution with the mass concentration of 0.1% to perform film forming reaction, reacting for 10min under stirring, forming polyelectrolyte complex film outside the microspheres, and washing with deionized water to remove unreacted polyornithine;
(3) And adding the cleaned coated microspheres into a sodium citrate solution with the mass concentration of 1.4% to liquefy for 5min, completely liquefying the internal gel to form a hollow structure, and cleaning the microspheres with deionized water to remove the sodium citrate solution, thereby obtaining the hollow artificial transparent belt.
2. Cryopreservation of sperm
During freezing, firstly, 1 drop of sperm culture solution (containing 30 sperms) is sucked and placed in a sperm culture dish to form a liquid drop (about 0.5 mu l), then 10ml of mineral oil is added to cover the liquid drop, 30 hollow microsphere carriers are sucked and placed in the liquid drop of the sperm culture solution, 1 movable sperm is caught by a microinjection needle and placed in 1 cryopreservation carrier, a cryopreservation tube placed in the 30 cryopreservation carriers is sucked by a suction tube, sperm cryopreservation liquid is added in the cryopreservation tube, liquid nitrogen steam with the height of 4cm above the liquid nitrogen surface is precooled and cooled for 3min (cooling speed is 20 ℃/min), and then the cryopreservation tube is put into liquid nitrogen for quick freezing and preservation.
3. Sperm resuscitation
During resuscitation, the freezing tube is taken out from the liquid nitrogen tank, the frozen stock solution containing the microsphere carrier is taken out from the freezing tube by a suction tube, the frozen stock solution is quickly added into a culture dish (5 ml of sperm culture solution containing 37 ℃), the sperm is searched in the frozen carrier, the sperm is grabbed by a microinjection needle and transferred into another culture dish containing sperm culture medium, and the sperm is used for subsequent sperm braking and ICSI of mature ovum, see fig. 2, and fig. 2 is an artificial transparent belt for loading the sperm.
Referring to FIG. 1, FIG. 1 is a microscopic image of the artificial transparent belt prepared in example 1, wherein the particle diameter of the microspheres is 130 μm and the film thickness is 15. Mu.m; the artificial zona pellucida after freezing is 100% in integrity, 100% in sperm recovery and 93.3% in sperm survival; the human egg zona pellucida has 100% of cryopreservation integrity, 100% of sperm recovery, 86.7% of sperm survival rate, and no significant difference from artificial zona pellucida.
Example 2
The invention provides an artificial transparent belt for cryopreservation of a small amount of sperms and a preparation method thereof,
1. preparation of polyornithine artificial transparent belt
(1) Preparing a sodium alginate solution with the mass concentration of 1.5%, measuring the viscosity to be 90.6 mPa.s by a viscometer, dripping the sodium alginate solution into a calcium chloride solution with the mass concentration of 1.1% by adopting an electrostatic liquid drop method to form calcium alginate hydrogel microspheres, and removing the calcium chloride solution after calcification for 30 min;
(2) Adding calcium alginate hydrogel microspheres into polyornithine (number average molecular weight 22 kD) solution with the mass concentration of 0.1% to perform film forming reaction, reacting for 8min under stirring, forming polyelectrolyte complex film outside the microspheres, and washing with deionized water to remove unreacted polyornithine;
(3) And adding the cleaned coated microspheres into a sodium citrate solution with the mass concentration of 1.4% to liquefy for 5min, completely liquefying the internal gel to form a hollow structure, and cleaning the microspheres with deionized water to remove the sodium citrate solution, thereby obtaining the hollow artificial transparent belt.
2. Cryopreservation of sperm
During freezing, firstly, 1 drop of sperm culture solution (containing 30 sperms) is sucked and placed in a sperm culture dish to form a liquid drop (about 0.5 mu l), then 10ml of mineral oil is added to cover the liquid drop, 30 hollow microsphere carriers are sucked and placed in the liquid drop of the sperm culture solution, 1 movable sperm is caught by a microinjection needle and placed in 1 cryopreservation carrier, a cryopreservation tube in which 10 cryopreservation carriers are placed is sucked by a suction tube, sperm cryopreservation liquid is added in the cryopreservation tube, and after liquid nitrogen steam with the height of 4cm above the liquid nitrogen surface is precooled and cooled for 3min (cooling speed is 20 ℃/min), the cryopreservation tube is put into liquid nitrogen for quick freezing and preservation.
(3) Sperm resuscitation
During resuscitation, the freezing tube is taken out from the liquid nitrogen tank, the frozen stock solution containing the microsphere carrier is taken out from the freezing tube by a suction tube, the frozen stock solution is quickly added into a culture dish (5 ml of sperm culture solution containing 37 ℃), sperms are searched in the frozen carrier, and the sperms are grabbed by a microinjection needle and transferred into another culture dish containing a sperm culture medium for subsequent sperm braking and ICSI of mature ova.
The particle diameter of the microsphere is 200 mu m, and the film thickness is 15 mu m; microsphere integrity rate after freezing is 90%, sperm recovery rate is 86.7%, and sperm survival rate is 82.4%.
Example 3
The invention provides an artificial transparent belt for cryopreservation of a small amount of sperms and a preparation method thereof,
1. preparation of polyornithine artificial transparent belt
(1) Preparing a sodium alginate solution with the mass concentration of 1.8%, measuring the viscosity to be 180.9 mPa.s by a viscometer, dripping the sodium alginate solution into a calcium chloride solution with the mass concentration of 1.1% by adopting an electrostatic liquid drop method to form calcium alginate hydrogel microspheres, and removing the calcium chloride solution after calcification for 30 min;
(2) Adding calcium alginate hydrogel microspheres into polyornithine (number average molecular weight 22 kD) solution with the mass concentration of 0.1% to perform film forming reaction, reacting for 8min under stirring to form microsphere shells, and washing the microspheres with deionized water to remove polyornithine solution;
(3) And adding the cleaned coated microspheres into a sodium citrate solution with the mass concentration of 1.4% to liquefy for 5min, completely liquefying the internal gel to form a hollow structure, and cleaning the microspheres with deionized water to remove the sodium citrate solution, thereby obtaining the hollow artificial transparent belt.
2. Cryopreservation of sperm
During freezing, firstly, 1 drop of sperm culture solution (containing 40 sperms) is sucked and placed in a sperm culture dish to form a liquid drop (about 0.5 mu l), then 10ml of mineral oil is added to cover the liquid drop, 20 hollow microsphere carriers are sucked and placed in the liquid drop of the sperm culture solution, 10 movable sperms are grabbed by a microinjection needle and placed in 1 cryopreservation carrier, a cryopreservation tube in which the 10 cryopreservation carriers are placed is sucked by a suction tube, sperm cryopreservation liquid is added into the cryopreservation tube, and after liquid nitrogen steam with the height of 4cm above the liquid nitrogen surface is precooled and cooled for 3min (cooling speed is 20 ℃/min), the cryopreservation tube is put into liquid nitrogen for quick freezing and preservation.
(3) Sperm resuscitation
During resuscitation, the freezing tube is taken out from the liquid nitrogen tank, the frozen stock solution containing the microsphere carrier is taken out from the freezing tube by a suction tube, the frozen stock solution is quickly added into a culture dish (5 ml of sperm culture solution containing 37 ℃), sperms are searched in the frozen carrier, and the sperms are grabbed by a microinjection needle and transferred into another culture dish containing a sperm culture medium for subsequent sperm braking and ICSI of mature ova.
Microsphere particle size 280 μm and film thickness 15 μm; microsphere integrity rate after freezing is 65%, sperm recovery rate is 62.5%, and sperm survival rate is 55%.
Example 4
The invention provides an artificial transparent belt for cryopreservation of a small amount of sperms and a preparation method thereof,
1. preparation of polyornithine artificial transparent belt
(1) Preparing a sodium alginate solution with the mass concentration of 1.5%, measuring the viscosity to be 90.6 mPa.s by a viscometer, dripping the sodium alginate solution into a calcium chloride solution with the mass concentration of 1.1% by adopting an electrostatic liquid drop method to form calcium alginate hydrogel microspheres, and removing the calcium chloride solution after calcification for 30 min;
(2) Adding calcium alginate hydrogel microspheres into polyornithine (number average molecular weight 63 kD) solution with the mass concentration of 0.1% to perform film forming reaction, reacting for 12min under stirring, forming polyelectrolyte complex film outside the microspheres, and washing with deionized water to remove unreacted polyornithine;
(3) And adding the cleaned coated microspheres into a sodium citrate solution with the mass concentration of 1.4% to liquefy for 5min, completely liquefying the internal gel to form a hollow structure, and cleaning the microspheres with deionized water to remove the sodium citrate solution, thereby obtaining the hollow artificial transparent belt.
2. Cryopreservation of sperm
During freezing, firstly, 1 drop of sperm culture solution (containing 30 sperms) is sucked and placed in a sperm culture dish to form a liquid drop (about 0.5 mu l), then 10ml of mineral oil is added to cover the liquid drop, 30 hollow microsphere carriers are sucked and placed in the liquid drop of the sperm culture solution, 1 movable sperm is caught by a microinjection needle and placed in 1 cryopreservation carrier, a cryopreservation tube in which 10 cryopreservation carriers are placed is sucked by a suction tube, sperm cryopreservation liquid is added in the cryopreservation tube, and after liquid nitrogen steam with the height of 4cm above the liquid nitrogen surface is precooled and cooled for 3min (cooling speed is 20 ℃/min), the cryopreservation tube is put into liquid nitrogen for quick freezing and preservation.
(3) Sperm resuscitation
During resuscitation, the freezing tube is taken out from the liquid nitrogen tank, the frozen stock solution containing the microsphere carrier is taken out from the freezing tube by a suction tube, the frozen stock solution is quickly added into a culture dish (5 ml of sperm culture solution containing 37 ℃), sperms are searched in the frozen carrier, and the sperms are grabbed by a microinjection needle and transferred into another culture dish containing a sperm culture medium for subsequent sperm braking and ICSI of mature ova.
The particle diameter of the microsphere is 200 mu m, and the film thickness is 10 mu m; microsphere integrity rate after freezing is 67.3%, sperm recovery rate is 66.4%, and sperm survival rate is 62.2%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. An artificial transparent belt is characterized by being prepared from sodium alginate and polyornithine; the artificial transparent belt is a shell with a hollow structure, and the shell is a sodium alginate and polyornithine polyelectrolyte composite film;
the size of the artificial transparent belt is 130 μm; the film thickness of the artificial transparent belt is 15 μm;
the polyornithine has a number average molecular weight of 22kDa;
the artificial transparent belt is prepared according to the following method:
mixing calcium alginate hydrogel microspheres with polyornithine solution with the mass concentration of 0.1%, and performing a film forming reaction to form polyornithine polyelectrolyte complex films outside the microspheres to obtain coated hydrogel microspheres;
and adding the coated hydrogel microspheres into a sodium citrate solution with the mass concentration of 1.4% for liquefying for 5min, completely liquefying the internal gel to form a hollow structure, and washing the microspheres with deionized water to remove the sodium citrate solution to obtain the artificial transparent belt.
2. A method of making an artificial transparent belt according to claim 1, comprising the steps of:
a) Mixing calcium alginate hydrogel microspheres with polyornithine solution with the mass concentration of 0.1%, and performing a film forming reaction to form polyornithine polyelectrolyte complex films outside the microspheres to obtain coated hydrogel microspheres;
b) And mixing the coated hydrogel microspheres with a sodium citrate solution with the mass concentration of 1.4%, and performing liquefaction reaction to obtain the artificial transparent belt with the hollow structure.
3. The method according to claim 2, wherein the calcium alginate hydrogel microspheres are prepared by an electrostatic droplet method or an emulsion internal gelation method.
4. The preparation method according to claim 3, wherein the raw materials for preparing the calcium alginate hydrogel microspheres are sodium alginate solution and calcium chloride solution, and the viscosity of the sodium alginate solution is 90.3 mpa.s; the mass concentration of the sodium alginate solution is 1.2%; the mass concentration of the calcium chloride solution is 1.1%.
5. The method according to claim 2, wherein in step a), the temperature of the reaction is room temperature and the time of the reaction is 10min.
6. The method of claim 2, wherein the liquefaction reaction is for 5 minutes; the temperature of the liquefaction reaction is room temperature.
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