CN112089826A - Slow-release medicine containing active biological factor and preparation method thereof - Google Patents

Slow-release medicine containing active biological factor and preparation method thereof Download PDF

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CN112089826A
CN112089826A CN201910528009.2A CN201910528009A CN112089826A CN 112089826 A CN112089826 A CN 112089826A CN 201910528009 A CN201910528009 A CN 201910528009A CN 112089826 A CN112089826 A CN 112089826A
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chitosan
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starch
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李校堃
林丽
张宏宇
冯治国
肖健
龚方华
杨丽珠
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Hangzhou Center For Biomedical Research And Innovation
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract

The invention aims to provide a slow-release medicine containing active biological factors and a preparation method thereof. The sustained-release medicament is prepared by an emulsion crosslinking method, the preparation method is simple, the obtained sustained-release medicament can greatly reduce the burst release rate of the active biological factor, can obtain longer sustained-release capability, has good medicament-loading rate and encapsulation rate, is suitable for clinical popularization of the active biological factor, and has great application value.

Description

Slow-release medicine containing active biological factor and preparation method thereof
Technical Field
The invention belongs to the field of active biological factors, and particularly relates to a slow-release medicament containing the active biological factors and a preparation method thereof.
Background
Fibroblast growth factor is an important cytokine, which has various physiological functions and plays an important role in life activities. The fibroblast growth factor family is currently known to comprise 23 members, with 30-80% homology between family members. Research on fibroblast growth factors has been focused on FGF-1(a-FGF, acidic fibroblast growth factor), FGF-2(b-FGF, basic fibroblast growth factor), FGF-13, FGF-21, and the like.
FGF-2, namely basic fibroblast growth factor, is originally separated from bovine pituitary and brain tissue extracts and can promote division and proliferation of 2T3 cells, and subsequent researches show that FGF-2 is widely distributed in vivo, can be separated from pituitary, brain, hypothalamus, retina, adrenal gland, thymus, corpus luteum, kidney, heart, liver and placenta and can be expressed in various tumor tissues. The human FGF-2 gene is a single copy gene, is positioned on the short arm of chromosome 4, has a total length of more than 40kb, has non-coding sequences which play a role in regulating gene transcription at the 5 'end and the 3' end of the gene, has an mRNA with the length of about 6802bp, and generates low Mr FGF-2 and 4 high Mr FGF-2 due to different translation initiation sites.
FGF-2 is a multifunctional cytokine, which can promote mitosis of cells, induce angiogenesis, promote repair of damaged vascular endothelial cells, promote repair of soft tissue injury, promote repair of cartilage and bone tissue injury, promote repair and regeneration of nerve tissue, and the like. In conclusion, the fibroblast growth factor, especially the basic fibroblast growth factor, has great clinical value and can be used for treating or assisting in treating various clinical diseases.
The in vivo half-life of exogenous active biomolecules such as fibroblast growth factors is short, for example, the in vivo half-life of FGF-2 is only 3-10min, and the effect of systemic or local administration cannot meet the clinical application requirement. In order to meet clinical requirements, the research of the slow release carrier of the active biological factor becomes a hot point of research. At present, various sustained-release carriers can be used for drug sustained release, and natural biodegradable high polymer materials and artificially synthesized biodegradable high polymer materials are common, wherein the research of chitosan is concerned.
The chitosan is the only naturally-occurring alkaline polysaccharide, is a safe, nontoxic and biodegradable natural polymer, has biocompatibility, and has the effects of resisting bacteria, stopping bleeding, inhibiting cancer cell metastasis and the like. The chitosan slow release carrier can be prepared into various forms, such as nano particles, microspheres, tablets, membranes, gels and the like, and the microspheres are the slow release forms of medicines which are actually applied at present. The chitosan microsphere is a microspherical entity formed by dissolving or dispersing a drug in a carrier, and the particle size ranges from 1 to 250 mu m. However, when a sustained release carrier such as chitosan is used for preparing sustained release microspheres, the controlled release effect on bioactive molecules is not ideal enough, and the drug release rate is fast. Therefore, there is a need for further improvement of the slow release microsphere carrier of chitosan to further improve its controlled release effect and/or reduce its burst rate.
The invention takes FGF-2 as an example, and researches the slow release capability of a slow release microsphere carrier prepared from chitosan and starch on active biological factors.
Disclosure of Invention
In order to solve the defects of high burst release rate and unsatisfactory sustained release effect of the chitosan sustained release microsphere carrier in the prior art, the invention prepares the sustained release microsphere carrier by combining chitosan and starch, thereby greatly reducing the burst release rate of the sustained release microsphere carrier and having good sustained release effect.
Specifically, the invention provides a slow-release medicine containing an active biological factor, which comprises a slow-release carrier, the active biological factor and/or a stabilizer.
Further, the slow release carrier is selected from starch, chitosan or a mixture thereof, preferably, the slow release carrier is prepared from chitosan and starch.
Further, the active biological molecule is fibroblast growth factor, nerve growth factor, epidermal growth factor, insulin, preferably fibroblast growth factor, more preferably FGF-2.
Further, the stabilizer is one or more of albumin, polyethylene glycol, cyclodextrin and dextran.
Further, the sustained-release medicine further comprises a preservative selected from sodium benzoate, ethyl p-hydroxybenzoate and methyl p-hydroxybenzoate.
The invention also provides a preparation method of the slow-release medicine containing the active biological factors, which comprises the following steps:
dissolving starch and/or chitosan in acetic acid solution, dissolving an active biological factor in 2-6mmol/L HCl solution, mixing the two solutions to obtain mixed solution, slowly adding the mixed solution into octanol solution containing tween-80, slowly stirring for 2h, adding 80-150g/L sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1-2h, precipitating, repeatedly rinsing with isopropanol and double distilled water to prepare a microsphere carrier containing the active biological factor, and further preparing the microsphere carrier into a sustained-release medicament.
Further, the molecular weight of the chitosan is 60-200kDa, and the deacetylation degree is more than 90%; the starch is soluble starch; preferably, the chitosan has a molecular weight of about 100 kDa.
Further, the dosage of the starch is 50-150mg, and the dosage of the chitosan is 50-300 mg; preferably, the amount of starch is 50mg and the amount of chitosan is 150 mg.
Furthermore, the dosage of the active biological factor is 5-20 mug, preferably 10 mug.
Further, 50mg of starch and 150mg of chitosan (100kDa) are dissolved in 9mL of 20mL/L acetic acid solution, 10 mu g of active biological factor is dissolved in 1mL of 4mmol/L HCl solution, the 2 solutions are mixed to obtain a mixed solution, the mixed solution is slowly added into 90mL of octanol solution containing 5mL/L of Tween-80, a magnetic stirrer is used for stirring for 2h, 100g/L sodium tripolyphosphate aqueous solution is added for crosslinking, the stirring is continued for 1h, and the precipitate is repeatedly rinsed by isopropanol and double distilled water to prepare the active biological factor chitosan microsphere.
Advantageous effects
The slow-release medicine has lower burst release rate and good slow-release effect, can maintain the activity of the active biological factors, and is suitable for clinical popularization; can effectively reduce the times of medication of patients and obtain better treatment effect.
The sustained-release microsphere carrier is prepared by an emulsification crosslinking method, and the preparation method is simple and convenient for industrial application.
Drawings
FIG. 1: scanning electron microscope images of FGF-2 sustained release microspheres prepared from chitosan and starch.
FIG. 2: in vitro release profile of FGF-2 sustained release microspheres.
FIG. 3: cell proliferation activity of FGF-2 sustained release microspheres.
Detailed Description
The present invention is described in more detail below to facilitate an understanding of the present invention.
It should be understood that the terms or words used in the specification and claims should not be construed as having meanings defined in dictionaries, but should be interpreted as having meanings that are consistent with their meanings in the context of the present invention on the basis of the following principles: the concept of terms may be defined appropriately by the inventors for the best explanation of the invention.
The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to the routine experimental procedures in the art or according to the conditions recommended by the manufacturers.
The molecular weight range of the chitosan in the invention is 60-200kDa, and the deacetylation degree is more than 90%; the starch is soluble starch.
Example 1: preparation of chitosan slow-release microsphere
Generally, the chitosan microsphere carrier is prepared by a spray drying method and an emulsion crosslinking method in the field, and the characteristics of the protein/polypeptide, high temperature resistance, easy inactivation and denaturation and the like of the used active biological factor are comprehensively considered, so the emulsion crosslinking method is selected to prepare the chitosan microsphere carrier in the embodiment.
The existing research shows that the slow release microsphere carrier prepared by chitosan alone has the defects of easy burst release of active substances and insufficient encapsulation efficiency, so that in an early pre-screening experiment, the chitosan is mixed with microsphere carrier materials such as starch, dextran, sodium hyaluronate, albumin, collagen, sodium alginate and the like, and sodium tripolyphosphate is used as a cross-linking agent for cross-linking to prepare microspheres, and the result shows that a regular spherical carrier can be obtained only when the chitosan is mixed with the starch, and the encapsulation efficiency and the burst release efficiency are further improved. Therefore, the following experiments detail the preparation of chitosan and starch microsphere carriers.
For active biological factors, FGF-2 is specifically selected to prepare a slow-release microsphere carrier, and the steps are as follows:
dissolving a certain amount of starch and chitosan (100kDa) in 9mL of 20mL/L acetic acid solution, dissolving 5 mu g of FGF-2 in 1mL of 4mmol/L HCl solution, mixing the 2 solutions to obtain a mixed solution, slowly adding the mixed solution into 90mL of octanol solution containing 5mL/L of Tween-80, stirring for 2h by using a magnetic stirrer, adding 100g/L sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1h, precipitating, repeatedly rinsing with isopropanol and double distilled water, and preparing the FGF-2 chitosan microspheres.
On the basis of pre-experiments, other parameters are unchanged, and the influences of various parameters on the shape, the particle size and the yield of the microspheres are detected by adjusting the dosage (50mg, 100mg, 150mg and 300mg) of chitosan and the dosage (50mg, 100mg and 150mg) of starch through single-factor and orthogonal experiments. Yield is the mass of microspheres obtained divided by the mass of chitosan and starch added.
The results are shown in Table 1:
Figure BDA0002098809670000051
from the above results, it can be seen that when 150mg of chitosan and 50mg of starch are used to prepare a sustained release microsphere carrier, regular and uniform microspheres having a spherical shape with a particle size of about 80 μm can be obtained, and a higher microsphere yield can be obtained. Thus, in the microsphere drug loading experiments described below, a slow release microsphere carrier prepared with 150mg chitosan and 50mg starch was selected.
And (3) determining the drug loading rate of the FGF-2 sustained release microspheres:
slowly adding 20mg of sustained-release microsphere carriers prepared by FGF-2 with different concentrations into 4ml of 2% acetic acid solution, fully stirring until the microspheres are completely dissolved, measuring the drug loading and encapsulation efficiency of the FGF-2 microspheres by using an FGF-2 kit (Shanghai Jianglai Biotech Co., Ltd.), respectively adding a diluted standard solution and a sample solution diluted to different concentrations into a prepared pore plate according to the kit specification, reserving a control pore, adding no sample solution, placing the pore plate into a centrifugal device after carrying out heat preservation in a thermostat, centrifuging and drying the liquid in the pore plate, respectively adding an FGF-2 antibody reagent into the pores of samples with different concentrations, placing the pore plate into the thermostat again for cultivation, repeatedly washing the pore plate and carrying out constant-temperature cultivation, finally adding a stop solution to finish reaction, and then measuring the absorbance value of the pore plate at the wavelength of 450nm by using an enzyme reader, and calculating the concentration of the sample in each hole according to the absorbance curve, and finally calculating the drug loading rate and the encapsulation efficiency of the FGF-2-loaded sustained-release microspheres.
The drug loading rate is the mass of the drug in the solution divided by the mass of the microspheres in the solution; the encapsulation efficiency is the drug loading multiplied by the total mass of the microspheres divided by the total mass of the FGF-2 added.
The results of adjusting the amount of FGF-2 used to determine its effect on drug loading and encapsulation efficiency are shown in Table 2:
FGF-2(μg) drug loading (%) Encapsulation efficiency (%)
5 16.3±1.1 77.8±2.9
10 22.3±1.8 93.4±3.1
20 14.7±3.1 70.4±4.8
40 11.8±2.8 65.7±2.4
From the above results, it can be seen that when 10 μ g of FGF-2 is used to prepare the sustained release microsphere carrier, the best drug loading and encapsulation efficiency can be obtained, which is beneficial to the effective utilization of active biomolecules, and the production cost can be significantly reduced and a better clinical treatment effect can be obtained.
Further compares the drug loading, encapsulation efficiency and in vitro release data of FGF-2 sustained release microsphere carrier (150mg chitosan and 50mg starch) and FGF-sustained release microsphere carrier (150mg chitosan).
Taking 150mg of chitosan (100kDa) and 50mg of starch, or independently dissolving 150mg of chitosan (kDa) in 9mL of 20mL/L acetic acid solution respectively, dissolving 10 mu g of FGF-2 in 1mL of 4mmol/LHCl solution, mixing the 2 solutions for a certain amount to obtain a mixed solution, slowly adding the mixed solution into 90mL of octanol solution containing 5mL/L of Tween-80, stirring for 2h by using a magnetic stirrer, adding 100g/L of sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1h, precipitating, repeatedly rinsing by using isopropanol and double distilled water to respectively prepare FGF-2 slow-release microsphere carriers, repeating for 5 times, and respectively measuring the drug loading amount, the encapsulation efficiency and the in-vitro release data.
Drug loading and encapsulation efficiency measurements were as previously described.
The detection of the in vitro FGF-2 sustained release microsphere release comprises the following steps:
placing a certain mass of microsphere carrier in a sterile test tube, adding 10-containing solution into the test tube7And (3) putting the test tube into a PBS (pH 7.4) solution of U/L lysozyme, placing the test tube into a constant-temperature shaking box at 37 ℃, continuously shaking and uniformly mixing until the slow-release microspheres are completely dissolved, centrifuging the mixed solution at different time points, taking supernate at different time points as a sample solution, then adding the PBS solution again, continuously shaking and uniformly mixing, measuring the sample solution by using an FGF-2 kit, and measuring the drug loading amount and the encapsulation rate in the steps. Repeating the operation for 3 times, averaging the absorbance values of the samples at different time points, calculating the amount of FGF-2 in the samples according to the existing curve, and drawing an FGF-2 sustained-release carrier in-vitro release curve according to the measured amount of FGF-2.
The results show that:
the drug loading rate of the FGF-2 sustained-release microsphere carrier prepared from chitosan and starch is 21.9 +/-1.5%, and the encapsulation rate is 93.5 +/-2.7%; the drug loading rate of the FGF-2 sustained-release microsphere carrier prepared from chitosan alone is 16.9 +/-2.0%, and the encapsulation rate is 85.3 +/-2.7%, namely the sustained-release microsphere carrier of the active biomolecules prepared from chitosan and starch can obtain better drug loading rate and encapsulation rate.
In vitro release data show that compared with an FGF-2 sustained-release microsphere carrier prepared from chitosan alone, the release rate of the FGF-2 sustained-release microsphere carrier prepared from chitosan and starch at the initial release stage is remarkably reduced, the release rates of the chitosan and the starch at 6h are respectively 26.6 +/-1.7% and 15.3 +/-0.9%, the release rates at 10 days are respectively 89.5 +/-2.3% and 98.2 +/-3.0%, and specific release curves are shown in figure 2.
Example 2: FGF-2 slow-release microsphere carrier cell proliferation activity determination method
Since the active FGF-2 has cell proliferation activity, the activity of the microsphere carrier is tested by the proliferation capacity of the FGF-2 sustained-release microsphere carrier on Balb/c3T3 cells.
The method comprises the following specific steps:
balb/c3T3 cells in logarithmic growth phase are diluted to 5-6 x 10 by 1640 culture medium containing 10% calf serum 4100. mu.l of the suspension of (4) was inoculated into a 96-well plateNo cells were added to the limbal wells, only medium was added as a cell-free blank, 37 ℃ with 5% CO2After culturing for 8 hours in an incubator to allow the cells to adhere to the walls, the cells in the 96-well plate were washed twice with 1640 medium containing 0.5% calf serum, and cultured for 48 hours by adding 100. mu.l of 1640 medium containing 0.5% calf serum, and the cells were allowed to stand still by serum starvation. During the period, the culture medium is replaced by 1640 culture medium containing 0.5% calf serum once, the culture solution is discarded, the DMEM culture medium (10% calf serum) containing 40ng/ml FGF-2 and the DMEM culture medium (10% calf serum) containing 40ng/ml FGF-2 sustained-release microsphere carrier are respectively added once on the beginning day of the experiment, the DMEM culture medium (10% calf serum) is used as a blank control, the temperature is 37 ℃, and the temperature is 5% CO2Culturing in an incubator, collecting cells on days 1, 2, 3, 5, 7 and 9 of culture, and sequentially detecting absorbance of each hole by using a microplate reader, wherein the detection wavelength is 450 nm.
As shown in fig. 3, it can be seen from fig. 3 that after 1 day of culture, the FGF-2 group and the FGF-2 sustained release microsphere carrier group have no significant difference (P > 0.05) with respect to the blank control group, when 2 days of culture, the FGF-2 group has a significant difference (P < 0.05) with respect to the FGF-2 sustained release microsphere carrier group and the blank control group, when 3, 5, 7, and 9 days of culture, the FGF-2 group and the FGF-2 sustained release microsphere carrier group have a significant difference (P < 0.05) with respect to the blank control group, and after 3 days of culture, the cell proliferation activity of the FGF-2 sustained release microsphere carrier group is significantly greater than that of the FGF-2 group because the FGF-2 sustained release microsphere carrier can sustain FGF-2 for a longer period of time and maintain the activity thereof for a longer period.
Example 3: stability of FGF-2 sustained release microsphere carrier
The purpose of this example is to study the effect of FGF-2 sustained release microsphere carrier on the stability of FGF-2, and the experiment is divided into the following three groups: standing at 4 deg.C, room temperature, and 40 deg.C, measuring biological activity of the sample at 5, 10, 20, and 30 days, and comparing the measurement results.
The preparation method of the FGF-2 sustained-release microsphere carrier comprises the following steps of taking 150mg of chitosan (100kDa) and 50mg of starch (experimental group) or separately dissolving 150mg of chitosan (kDa) (control group) in 9mL of 20mL/L acetic acid solution respectively, dissolving 10 mu g of FGF-2 in 1mL of 4mmol/LHCl solution, mixing 2 solutions for a certain amount to obtain a mixed solution, slowly adding the mixed solution into 90mL of 5mL/L Tween-80-containing octanol solution, stirring for 2 hours by a magnetic stirrer, adding 100g/L of sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1 hour, precipitating, repeatedly rinsing with isopropanol and double distilled water, and respectively preparing the FGF-2 sustained-release microsphere carrier.
Referring to example 2, the method for testing the biological activity of the FGF-2 sustained release microsphere specifically comprises the following steps:
balb/c3T3 cells in logarithmic growth phase are diluted to 5-6 x 10 by 1640 culture medium containing 10% calf serum4The suspension of (4) was inoculated into a 96-well plate in an amount of 100. mu.l per well, and only the medium was added as a cell-free blank at 37 ℃ in marginal wells without adding cells and with 5% CO2After culturing for 8 hours in an incubator to allow the cells to adhere to the walls, the cells in the 96-well plate were washed twice with 1640 medium containing 0.5% calf serum, and cultured for 48 hours by adding 100. mu.l of 1640 medium containing 0.5% calf serum, and the cells were allowed to stand still by serum starvation. During the period, the culture medium is replaced by 1640 culture medium containing 0.5% calf serum once, the culture solution is discarded, the DMEM culture medium (10% calf serum) containing 40ng/ml FGF-2 (standard group) and the DMEM culture medium (10% calf serum) containing 40ng/ml FGF-2 sustained release microsphere carrier (experimental group or control group) are respectively added once on the day of the beginning of the experiment, the DMEM culture medium (10% calf serum) is used as a blank control, the temperature is 37 ℃, and the temperature is 5% CO2Culturing in an incubator, collecting cells respectively on the 7 th day of culture, and sequentially detecting the absorbance of each hole by using an enzyme-labeling instrument, wherein the detection wavelength is 450 nm.
The activity of the experimental group or the control group is marked by the proportion of the standard group, namely the proportion of the activity of the experimental group or the control group relative to the activity of the standard group is respectively tested after the experimental group or the control group is stored at different temperatures for different time.
The stability and activity experiments were repeated three times.
The stability test results are shown in table 3.
Table 3: stability of FGF-2 sustained-release microspheres
Figure BDA0002098809670000091
From the above results, it is presumed that, when a sustained release microsphere carrier is prepared using FGF-2 prepared from chitosan and starch, the stability of FGF-2 can be greatly enhanced in comparison to a sustained release microsphere carrier of FGF-2 prepared from chitosan alone, whether at 4 ℃, room temperature, or 40 ℃, because the sustained release microsphere carrier prepared from FGF-2 prepared from chitosan and starch has a better encapsulation efficiency and a lower burst release rate, thereby being able to encapsulate FGF-2 in the microsphere carrier to protect the stable existence of its activity.
According to the above results, FGF-2 sustained-release microsphere carriers prepared from chitosan and starch can be preserved at room temperature in consideration of cost and convenience.
Without limitation, the sustained-release microspheres of the present invention are suitable for use with a variety of active biological factors.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (10)

1. A sustained release medicament comprising an active biological factor, characterized in that: the slow release drug comprises a slow release carrier, active biological factors and/or a stabilizing agent.
2. The sustained-release drug according to claim 1, characterized in that: the slow release carrier is selected from starch, chitosan or a mixture thereof, and is preferably prepared from chitosan and starch.
3. The sustained-release drug according to claim 1, characterized in that: the active biological molecules are fibroblast growth factors, nerve growth factors, epidermal growth factors and insulin, preferably the fibroblast growth factors, and more preferably FGF-2.
4. The sustained-release drug according to claim 1, characterized in that: the stabilizer is one or more of albumin, polyethylene glycol, cyclodextrin and dextran.
5. The sustained-release drug according to claim 1, characterized in that: the slow release medicine further comprises a preservative selected from sodium benzoate, ethyl p-hydroxybenzoate and methyl p-hydroxybenzoate.
6. A process for the preparation of a sustained release medicament as claimed in any one of claims 1 to 5, wherein: the method comprises the following steps:
dissolving starch and/or chitosan in acetic acid solution, dissolving an active biological factor in 2-6mmol/L HCl solution, mixing the two solutions to obtain mixed solution, slowly adding the mixed solution into octanol solution containing tween-80, slowly stirring for 2h, adding 80-150g/L sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1-2h, precipitating, repeatedly rinsing with isopropanol and double distilled water to prepare a microsphere carrier containing the active biological factor, and further preparing the microsphere carrier into a sustained-release medicament.
7. The method of claim 6, wherein: the molecular weight of the chitosan is 60-200kDa, and the deacetylation degree is more than 90%; the starch is soluble starch; preferably, the chitosan has a molecular weight of about 100 kDa.
8. The method of claim 6, wherein: the dosage of the starch is 50-150mg, and the dosage of the chitosan is 50-300 mg; preferably, the amount of starch is 50mg and the amount of chitosan is 150 mg.
9. The method of claim 6, wherein: the dosage of the active biological factor is 5-20 mug, preferably 10 mug.
10. The method of claim 6, wherein: dissolving 50mg of starch and 150mg of chitosan (100kDa) in 9mL of 20mL/L acetic acid solution, dissolving 10 mu g of active biological factor in 1mL of 4mmol/LHCl solution, mixing the 2 solutions to obtain a mixed solution, slowly adding the mixed solution into 90mL of octanol solution containing 5mL/L of Tween-80, stirring for 2h by using a magnetic stirrer, adding 100g/L sodium tripolyphosphate aqueous solution for crosslinking, continuously stirring for 1h, precipitating, repeatedly rinsing by using isopropanol and double distilled water, and preparing the active biological factor chitosan microsphere.
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