CN1268325C - Ternary composite microsphere formulation and its preparation method - Google Patents

Abstract

The present invention provides a triple composite microsphere preparation and its preparation method. The main components of the preparation are model drugs, calcium alginate, chitosan, and glycolide-lactide copolymer. The preparation method is to use W/O emulsification , isopropanol washing to prepare small-sized calcium alginate microcapsules, and further wrap the calcium alginate microcapsules with chitosan to form a denser structure of alginic acid-chitosan double-layer microcapsules, and then use emulsification-solvent The alginic acid-chitosan double-layer microcapsules were encapsulated in the glycolide-lactide copolymer by the evaporation method to form a three-layer composite microsphere. The composite microspheres provided by the invention can make protein and polypeptide drugs well protected in the environment of hydrophilic sodium alginate-chitosan microcapsules and significantly reduce the phenomenon of sudden release and incomplete release, which can reach The drug is released for a long time, and the release mode of the drug can be adjusted by changing the composition of PLGA. The preparation form of the three-layer composite microsphere preparation of the present invention includes injection dosage form and oral dosage form.

Description

Triple composite microsphere preparation and preparation method thereof

Technical field

The invention belongs to the field of pharmacy, and relates to a biodegradable microcapsule and microsphere preparation and a preparation method thereof, in particular to a calcium alginate-chitosan-glycolide-lactide copolymer triple composite microsphere preparation and a preparation method thereof. This new compound preparation is of great significance to solving the bottleneck problem of protein vaccine administration.

technical background

Vaccination is the most effective public health measure against infectious diseases other than the provision of purified water. It’s just that some vaccines such as hepatitis B vaccine and tetanus vaccine often need to be inoculated 2-3 times to achieve the expected immune effect. Therefore, complete immunization upon first vaccination is one of the main goals of the World Health Organization's vaccine development programme. The development of a single-dose vaccine that provides full immunity with a single vaccination is an ideal solution. The key is to choose suitable biodegradable polymer materials and establish a delivery system for long-term pulse release of antigens. Vaccine carriers are commonly used polyester, polyamino acid, polyanhydride and so on. Copolymer of glycolide and lactide (PLGA) is commonly used in polyesters ^[1] , and PLGA microspheres have an adjuvant effect for vaccine administration. At present, the biggest resistance encountered in the development of PLGA microspheres as a controlled-release delivery system for water-soluble protein and polypeptide vaccines is the stability of such drugs and the sudden and incomplete release of drugs from the PLGA matrix. The hydrophobicity of PLGA makes the encapsulation effect of hydrophilic protein drugs poor.

First of all, there are very few types of organic solvents that can dissolve PLGA, and dichloromethane is the most commonly used. Repeated sonication or stirring of protein drugs and organic solvents leads to protein aggregation and impaired activity. Second, the acidic components generated along with the degradation of polymers are also an important reason for protein inactivation. Existing many method reports are used to avoid the activity loss of albumen: (1) when preparing, directly disperse in organic solvent with albumen lyophilized powder, to increase the stability of albumen ^[2] ; (2) make W/O earlier Emulsion, the oil in the primary emulsion can separate the protein drug and the organic phase of the polymer; (3) using a new encapsulation technology, using mild organic solvents such as acetic acid, benzyl alcohol, glycerol triacetate and N, N'-dimethylethyl Amide dissolves PLGA to minimize protein denaturation ^[3] ; (4) Add protein protective agents, such as polyhydroxy substances such as trehalose, cyclodextrin, polyethylene glycol (PEG), and surfactants such as Tween 20 and bovine serum albumin. They either adsorb on the two-phase interface before the protein, reduce the contact between the encapsulated protein and the organic solvent, or rely on polyhydroxyl to surround the protein to fix the natural conformation of the protein ^[4] ; (5) eliminate the acidity generated during the degradation of PLGA The adverse effects of substances create a microenvironment with a relatively stable pH value for the antigen. Stabilizers that can be added include gelatin, gum arabic, acetylated chitin, sodium glutamate, etc. Alkaline substances such as magnesium hydroxide, magnesium carbonate, and zinc carbonate can also be directly applied, and even protein drugs can be directly dissolved in phosphate buffer. agent to maintain the pH during the degradation of PLGA. Investigation on the stability of bovine serum albumin (BSA) encapsulated in PLGA microspheres with 3% Mg(OH) ₂ and 10% sucrose: 81% of the protein aggregated when the microspheres were incubated at 37°C for 4 weeks without additives , 50% after adding sugar; less than 7% after adding alkali ^[5] . (6) Encapsulating protein drugs in agarose hydrogel particles, and further dispersing them in PLGA microspheres. This composite microsphere has a high encapsulation efficiency, fast hydration, and more importantly, the agarose hydrogel has a stabilizing effect on the protein, but it fails to change the burst release of the protein drug ^[6] .

In addition, the preparation of water-soluble protein PLGA microspheres often adopts the W/O/W double emulsion method, which can often form porous microspheres, and vacuum drying makes the porosity larger ^[7] . There are also a series of methods used to reduce drug burst release: such as reducing the volume ratio of the inner aqueous phase; adding buffer salts to the outer aqueous phase; changing the composition of the organic phase or adding glycerin to the organic phase; changing the operation method such as replacing it with reduced pressure solvent evaporation Atmospheric pressure solvent evaporation method, ultrasonic emulsification instead of high-speed stirring, etc. ^[8] . However, the problems of sudden release of water-soluble proteins and polypeptide drugs from PLGA microspheres and incomplete release due to protein aggregation and other reasons have not been well resolved.

Natural or synthetic biodegradable materials are increasingly used in pharmaceutical formulations. Compared with non-biodegradable materials, it has more advantages: such as more convenient drug administration, good compliance, a certain drug release rate in the later stage of application, and even pulse drug administration may occur with changes in degraded materials. However, no matter whether it is a hydrophilic material or a hydrophobic material, the drug delivery system used alone for protein drugs is not ideal. Hydrophilic materials have good biocompatibility, but often have a faster drug release rate. On the other hand, hydrophobic materials have a slow-release effect, but they are incompatible with water-soluble proteins and polypeptide drugs, which will cause the stretching of the natural protein structure in the folded state, resulting in a change in activity, and the encapsulation efficiency is also very low. Difficult to improve.

Contents of the invention

The present invention provides a new compound microsphere preparation and its preparation method, that is: triple compound microsphere preparation and its preparation method, the purpose is to provide water-soluble polypeptide, protein vaccines with a stable hydrophilic microenvironment, thus good Protect the protein drug from the loss of activity caused by the organic solvent and the pH drop caused by the biodegradation of PLGA, and at the same time improve the drug encapsulation rate, greatly reduce the sudden release of the drug, and pass through the outermost layer of PLGA itself The adjustment of the ratio of polylactic acid (PLA) to polyglycolic acid (PGA) makes it possible for the final controlled release of the drug. This new type of compound preparation is of great significance to solve the bottleneck problem of protein vaccine administration.

The triple compound microsphere preparation provided by the present invention and its preparation method are as follows: dissolving the medicine in sodium alginate solution as water phase, adopting W/O emulsification and washing with isopropanol to prepare small-diameter calcium alginate microcapsules, and Chitosan further wraps calcium alginate microcapsules to form alginic acid-chitosan double-layer microcapsules with a denser structure, and then emulsification-solvent evaporation method is used to wrap alginic acid-chitosan double-layer microcapsules into BG Three-layer composite microspheres are formed in the ester-lactide copolymer.

The main components of the microsphere preparation of the present invention are: model drug, calcium alginate, chitosan, glycolide-lactide copolymer, emulsifier, and medicinal dressing.

Preparation method of the present invention mainly realizes by following scheme:

(1) Preparation of calcium alginate microcapsules: Dissolve the model drug in 0.5% to 2% (w/v) sodium alginate solution as the water phase, and take the first emulsifier separately, and mix it with 4% to 6% (w/v) v) The concentration is dissolved in isooctane as the oil phase, the volume ratio of the two phases is 1:2, the rotation speed of the high-speed emulsion is 5000-20000rpm, and the second emulsifier is added dropwise after the two-phase high-speed emulsion for 1-5 minutes, and then the emulsion is evenly mixed After 1 to 5 minutes, add 1% to 10% (w/v) cross-linking agent drop by drop and still mix for 1 to 5 minutes, add isopropanol and finally mix for 1 to 3 minutes at the same speed. Calcium alginate microcapsules were prepared after centrifugation.

(2) Preparation of calcium alginate-chitosan double-layer microcapsules: incubate calcium alginate microcapsules with 0.1% to 2.0% chitosan solution at pH 4 to 6 for 10 to 40 minutes, collect the microcapsules by centrifugation and use the shell Washing with polysaccharide solution and/or water, freeze-drying to obtain calcium alginate-chitosan double-layer microcapsules with uniform particle size and round shape.

(3) Preparation of three-layer composite microspheres: disperse the above-mentioned double-layer microspheres into 3.5-4.0 g of dichloromethane at a concentration of 1% to 1.5% (w/w), and use a probe-type ultrasonic instrument of 50-200w, 10-40 Sonicate for 1 second, then weigh 1 g of glycolide-lactide copolymer (PLGA), racemic PLGA (DL-PLGA) and left-handed PLA (L-PLA) (according to the ratio of 40:54:6) and add it to the above suspension vortex to dissolve as the oil phase; prepare another 5-10ml of 0.1%-0.5% sodium carboxymethylcellulose solution as the water phase, add the oil phase to the water phase, stir at 1200-2000rpm for 3-5 minutes , subsequently, it is added dropwise to 50-200ml, in 0.5%-1% polyvinyl alcohol (PVA) solution, and the emulsion is homogenized under 500-600rpm for 1-2 minutes, then the speed is increased to 800rpm-2000rpm and stirring is continued for 1-3 minutes, The dichloromethane was removed by rotary evaporation under reduced pressure, the composite microspheres were collected by centrifugation, washed with distilled water and freeze-dried.

(4) Calcium alginate-chitosan microcapsules can also be dispersed in the acetonitrile solution of glycolide-lactide copolymer (PLGA) with a concentration of ≤1.2% (w/v) (microspheres: PLGA=1: 4～1:10), probe type ultrasonic instrument 50～200w, after 5～30 seconds of ultrasonication, it is used as the suspension water phase, and Span 80 is dissolved in peanut oil at 4%～8% (w/v) as the oil phase. The oil phase is slowly dripped into the above suspension under stirring at 500-600rpm, and then, increase the speed to 800-2000rpm and continue to stir for 1-2 minutes, reduce acetonitrile by rotary evaporation under reduced pressure, collect the composite microspheres by centrifugation, and wash with petroleum ether Evaporate or vacuum dry at 37°C.

The model drugs of the present invention mainly include polypeptide and protein drugs.

The content of the sodium alginate solution is 0.5% to 2%.

The amount of the second emulsifier is 15%-49% of the amount of the first emulsifier, the first emulsifier is preferably Span 80, and the second emulsifier is preferably Tween 80.

The PLGA used in the preparation of the three-layer composite microsphere has a ratio of PLA to PGA of 50-100:50-0.

The particle size of the calcium alginate-chitosan microcapsule is 1-5 μm. The particle size of the composite microspheres is 20 μm to 50 μm.

The preparation form of the three-layer composite microsphere preparation of the present invention includes injection dosage form and oral dosage form. Advantages and positive effects of the present invention

(1) The invention first adopts mild modified emulsification and ion cross-linking methods to wrap water-soluble polypeptides and protein vaccines into calcium alginate-chitosan double-layer microcapsules, on the one hand to provide a stable hydrogel microenvironment , so as to well protect the protein drugs from or less affected by the activity of organic solvents. On the other hand, the pH drop caused by the biodegradation of PLGA can also take away part of the H ⁺ with the dissolution of chitosan, thus Reduce the loss of activity due to the acidity of degradation products, and reduce the incomplete release of proteins.

(2) Most protein drugs have high solubility in water. When preparing sodium alginate microcapsules by emulsification and cross-linking technology, the encapsulation rate of microcapsules prepared by water washing method is 4% to 18%, which is far lower than that of alcohol washing method. 80% to 100%. At the same time, the amount of microcapsules obtained by the water washing method is about 50% of that of the alcohol washing method. Therefore, the present invention further prepares calcium alginate microcapsules wrapped with chitosan by alcohol washing method, so that the first step of composite microcapsule preparation is established on a higher starting point. The obtained alginic acid microcapsules are further incubated in chitosan solution, encapsulated by electrostatic interaction and then encapsulated by hydrophobic PLGA material for the third time, and the encapsulation efficiency can be close to 100%, which not only makes water-soluble protein drugs The encapsulation efficiency in the hydrophobic PLGA microspheres is greatly improved, and the leakage and rapid release of the water-soluble drugs by the alginic acid microcapsules can be blocked, so that the drug release mode can be better controlled.

(3) The composite microspheres can significantly reduce the sudden release of the drug, from about 50% of the drug released within 24 hours from the simple PLGA microspheres to about 10% of the composite microspheres, and the ratio of PLA to PGA is adjusted to control the drug through the outermost PLGA final release mode.

Description of drawings

Figure 1 is a scanning electron micrograph of the triple composite microspheres of calcium alginate-chitosan-glycolide-lactide copolymer.

Figure 2 is a comparison of the yield, encapsulation efficiency and drug loading ratio of the calcium alginate microspheres prepared by the water washing method and the alcohol washing method.

Figure 3 is a release comparison of alginic acid-chitosan microspheres prepared after incubation with different concentrations of chitosan.

Figure 4 is a comparison of the in vitro release of alginic acid-chitosan microspheres prepared under different pH conditions.

Fig. 5 is a scanning electron microscope image of the particle size and morphology of alginic acid-chitosan microspheres.

Figure 6 is the in vitro cumulative release diagram of BSA in simple PLGA microspheres and triple composite microspheres.

Detailed ways

Example 1 A preparation method of composite microspheres of the present invention

(1) Preparation of calcium alginate microcapsules: using BSA as a model drug, dissolve BSA with a small amount of distilled water and mix well with 1% sodium alginate solution. The first emulsifier is Span 80, which is soluble in isooctane 5% (w/v), the volume ratio of the two phases is 1:2, the high-speed milking speed is 12000rpm, and the time is 3 minutes, then add the second emulsifier, namely Tween 80, and the concentration is 30% (w/w) , the dosage is 45% of Span 80, and then milk for 3 minutes, add cross-linking agent calcium chloride solution, the concentration is 8% (w/v), and then milk for 3 minutes, add isopropanol to separate out the extraction of microcapsules The time is 2 minutes. Calcium alginate microcapsules were prepared after centrifugation.

(2) Preparation of calcium alginate-chitosan microspheres: the calcium alginate microcapsules prepared above were incubated with 1.0% pH4 chitosan solution for 10-40 minutes, the microcapsules were collected by centrifugation, and then the same chitosan Wash once with sugar solution, wash twice with water (or wash with water all three times), and freeze-dry to obtain calcium alginate-chitosan microcapsules with a size of <5 μm, uniform particle size, and round shape.

(3) Preparation of three-layer composite microspheres: disperse the above-mentioned microcapsules into 3.5-4.0 g of dichloromethane at a concentration of 1.2% (w/w), use a probe-type ultrasonic instrument at 200w for 40 seconds, and then weigh PLGA, DL - 1 g of PLGA and L-PLA (40:54:6 ratio) was added to the above suspension, and vortexed to dissolve as the oil phase; another 8 ml of 0.2% sodium carboxymethylcellulose solution was prepared as the water phase. Add the oil phase to the water phase, stir at 1800rpm for 5 minutes, then add it dropwise to 150ml, 0.5% PVA solution, mix at 600rpm for 2 minutes, increase the speed to 1200rpm and continue stirring for 3 minutes, and rotate under reduced pressure Evaporate to remove dichloromethane, collect the composite microspheres by centrifugation, wash with distilled water, and then freeze-dry. The particle size of the composite microspheres obtained is 35 μm, and the particle size of the microspheres is shown in Figure 1. The composite microsphere prepared by the method of the invention can obviously reduce the burst release phenomenon, and can adjust the release mode of the drug by changing the composition ratio of the PLGA in the outer layer.

In the present invention, water-soluble protein or polypeptide vaccines are firstly dissolved in a certain concentration of sodium alginate solution to form an initial protection, and calcium alginate microcapsules are prepared by emulsification-ion cross-linking method ^[9] , and the alcohol washing method improves drug packaging. Encapsulation efficiency, and incubate in chitosan solution to form double-layer composite microcapsules, so that the structure of calcium alginate microcapsules is more compact, reducing the burst release of encapsulated drugs, and providing drugs with double hydrophilic polymers protection, creating a suitable hydrophilic microenvironment. Moreover, due to the rapid initial degradation rate of PLGA, the acidic components produced are conducive to the dissolution of chitosan and the first release of the drug, and it does not affect the first release of the drug under the premise of reducing or eliminating the drug burst release, and can even Take away most of the H ⁺ produced by PLGA degradation. The problem of protein stability and burst release can be solved. In addition, the encapsulation efficiency of alginic acid-chitosan microcapsules on the model protein BSA can be as high as 97%, and the second PLGA encapsulation can also be greater than 90%, so the drug encapsulation efficiency can be greatly improved. Since the sodium alginate microcapsules are further coated with chitosan, the leakage and rapid release of the water-soluble drug by the alginate microcapsules can also be blocked, reducing the burst release of the drug, thereby better controlling the release mode of the drug. The second encapsulation also uses the W/O emulsification method, one of which refers to the PLGA phase diagram and polymer alloy theory in the literature ^[10] . At the same time, the different release modes of the drug can be controlled by changing the ratio of PLA to PGA in PLGA, ranging from 50:50 to 100:0 or mixing different ratios of PLGA microspheres. The preparation of this kind of composite microspheres not only protects proteins and polypeptide drugs with alginic acid gel hydrophilic microenvironment as the first layer of protection, but the addition of chitosan greatly reduces the sudden release of drugs and further stabilizes the protein. , the outermost layer of PLGA, through the adjustment of the ratio of PLA and PGA, the final controlled release of the drug is possible. Therefore, it is of great significance to solve the bottleneck problem of protein vaccine preparations.

Embodiment 2 The second preparation method of composite microspheres of the present invention

The rotational speed of the two-phase high-speed emulsion in the first step is 5000rpm; the particle size of the double-layer composite microcapsules obtained in the second step is <8 μm; the particle size of the microspheres obtained in the third step is <50 μm; the rest of the steps are the same as in Example 1.

Example 3 The third preparation method of composite microspheres of the present invention

In the first step, the rotation speed of the two-phase high-speed emulsion is 20000rpm, the particle size of the double-layer composite microcapsules obtained in the second step is <2 μm, and the particle size of the microspheres obtained in the third step is <30 μm, and the rest of the steps are the same as in Example 1.

Embodiment 4 The 4th preparation method of the composite microsphere of the present invention

The crosslinking agent is zinc chloride solution, the particle size of the double-layer composite microcapsules obtained in the second step is <5 μm, the particle size of the microspheres obtained in the third step is <40 μm, and the rest of the steps are the same as in Example 1.

Example 5 The fifth preparation method of composite microspheres of the present invention

The cross-linking agent is barium chloride solution, the particle diameter of the double-layer composite microcapsules obtained in the second step is <5 μm, the particle diameter of the microspheres obtained in the third step is <40 μm, and the rest of the steps are the same as in Example 1.

Example 6 The sixth preparation method of composite microspheres of the present invention

The steps are basically the same as in Example 1, except that 1% pH6 chitosan solution is used to incubate to obtain composite microcapsules with the same particle size and shape.

Example 7 The seventh preparation method of the composite microspheres of the present invention

The steps are basically the same as in Example 1, but the third step uses 0.1% sodium carboxymethyl cellulose solution to obtain composite microcapsules with a particle size of <30 μm.

Example 8 The eighth preparation method of the composite microspheres of the present invention

The steps are basically the same as in Example 1, but the third step uses 0.5% sodium carboxymethylcellulose solution to obtain the composite microcapsules with a particle size of <50 μm.

Example 9 The ninth preparation method of the composite microspheres of the present invention

The steps are basically the same as in Example 1, but in the third step, the rotational speed is increased to 2000 rpm to obtain a composite microcapsule with a particle size of less than 30 μm.

Example 10 The ninth preparation method of composite microspheres of the present invention

The model drug is superoxide dismutase (SOD). The rest of the preparation method is the same as in Example 1, and the particle size, shape and release characteristics of the prepared composite microspheres are the same as those of BSA in Example 1.

Example 11 The eleventh preparation method of the composite microspheres of the present invention

The first step and the second step adopt the same method as in Example 1. The third step method is:

Disperse calcium alginate-chitosan microcapsules at a concentration of 0.86% (w/v) in an acetonitrile solution of poly(lactide glycolide) (PLGA) (microcapsules: PLGA = 1:6), and probe ultrasound Instrument 200w, 20 seconds of ultrasonication as the suspension water phase, Span 80 dissolved in peanut oil at 6.67% (w/v) as the oil phase. The oil phase was slowly dripped into the above suspension under stirring at 600rpm, then, the rotation speed was increased to 1200rpm and stirring was continued for 2 minutes, the acetonitrile was removed by rotary evaporation under reduced pressure, and the composite microspheres were collected by centrifugation. After washing with petroleum ether, evaporate to dry at 37°C or dry in vacuum. The particle size of the prepared composite microspheres is 30.1 μm. See Figure 1 for the particle size of the microspheres.

The method of the present invention utilizes the mild cross-linking method of the hydrophilic biodegradable material sodium alginate and calcium ions to provide water-soluble polypeptides and protein vaccines with a stable hydrophilic microenvironment, and then uses another hydrophilic biodegradable After incubation with the degraded polymer chitosan solution, the double-layer alginic acid-chitosan microcapsule structure produced by the electrostatic interaction of anion and cation is more dense, so that the protein drug is further protected from or less affected by organic solvents and pH generated with PLGA biodegradation The loss of activity caused by the decrease, so as to protect the protein drug well, improve the drug encapsulation rate at the same time, greatly reduce the burst release of the drug, and further disperse the above-mentioned double-layer microcapsules in PLGA microspheres to make triple composite microspheres, and Finally, sustained-release preparations are obtained, and the release of the drug is controlled within the required time by adjusting the ratio of polylactic acid (PLA) to polyglycolic acid (PGA) in the outermost layer of PLGA itself.

Example 12 Comparison of yield, encapsulation efficiency and drug loading rate of calcium alginate microcapsules prepared by water washing method and alcohol washing method

Due to the high solubility of BSA in water, the encapsulation rate of calcium alginate microcapsules prepared by water washing method is 18.83%±0.11%, which is far lower than that of alcohol washing method. 99.95% ± 0.06%. At the same time, the amount of microcapsules obtained by the water washing method is 49.43% of that of the alcohol washing method, and the results are shown in Figure 2.

Example 13 Comparison of the release of alginic acid-chitosan microcapsules prepared in physiological saline after incubation with different concentrations of chitosan

The release of protein is gradually inhibited as the concentration of chitosan encapsulating calcium alginate microspheres increases. , the 48h protein release rate in normal saline was 99.50% ± 0.01%, 93.20% ± 0.42%, 44.19% ± 0.11%, and if it continued to increase to 1.0%, there was no greater delaying effect, which was 54.00 ± 0.85%. The results can be found in Attached Figure 3.

Example 14 Comparison of in vitro release of alginic acid-chitosan microcapsules

The alginic acid-chitosan microcapsules prepared by incubating 1% alginic acid microcapsules with 1% chitosan solutions of pH 4 and pH 6 respectively, compared with the in vitro release, showed that 1%, chitosan at pH 4 The solution has a stronger blocking effect on protein release than the pH 6 solution. The former 48h protein release rate was 20.35±1.07%, the latter was 55.96%±3.00%. The release of protein in the double-layer composite microcapsules can be delayed from 48h to more than 7d in physiological saline, and the results are shown in Figure 4.

Example 15 Particle size and morphology of composite microcapsules Scanning electron microscope

The alginic acid microcapsules prepared by washing with alcohol are coated with a chitosan solution with a pH of 4 and a concentration in the range of 0.2% to 0.5%, and the size is uniform, spherical, smooth, and dispersed in normal saline or phosphate buffer. Good performance, the average particle size is 1-2 μm, and the encapsulation rate is greater than 85%. The scanning electron microscope results are shown in Figure 5, and the volume average particle size is 1.15±0.21 μm.

Example 16 The in vitro cumulative release comparison of BSA in simple PLGA microspheres and triple composite microspheres

Precisely weigh 30 mg of simple PLGA microspheres or 150 mg of triple composite microspheres into an eppendorf tube, add 3.0 ml of normal saline as the release medium, vortex to disperse, and set a constant temperature oscillator at 37°C to perform the release test at 700 rpm. Centrifuge the sample solution at 1, 2000rpm (PLGA microspheres) or 4000rpm (triple compound microspheres) for 15 minutes at a certain interval, take 1.0ml supernatant and measure the content of released protein with a micro BCA kit, and use 1.0ml fresh medium for precipitation Make up, continue to oscillate after dispersing. To calculate the cumulative drug release, take the percentage of the cumulative drug release at each experimental point and the total amount of protein contained in the microspheres as the ordinate, and each experimental sampling point as the abscissa to make a release curve. The composite microspheres prepared by the method of the present invention can significantly reduce the burst release phenomenon, and can adjust the release mode of the drug by changing the composition ratio of the outer layer of PLGA. For comparison with the in vitro release of the PLGA microspheres of BSA, see Figure 6.

Some references involved in the present invention:

1. O′Hagan DT, Singh M, Gupta RK. Poly(1actide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines. Advanced Drug Delivery Reviews 1998; 32: 225-246

2. Diwan M, Park TG. Pegylation enhances protein stability stability during gencapsulation in PLGA microspheres. J Controlled Release2001; 73: 233-244

3. Kinam P.A new microencapsulation process for protein drugs. New ProjectX (2000)

4. He Ying, Liu Lin, Wei Shuli, etc. Study on protein stability in tetanus toxoid polylactic acid microspheres. Chinese Journal of Pharmaceutical Sciences 2001; 36(6): 391-394

5. Kang J, Schwendeman SP. Comparison of the effects of Mg(OH) ₂ and sucrose on the stability of bovine serum albumin encapsulated in injectable poly(d, l-lactide-co-glycolide) implants. Biomaterials2002; 23(1): 239 -245

6.Wang N, Wu XS.A novel approach to stabilization of protein drugsin poly(lactic-co-glycolic acid)microspheres using agarosehydrogel.International J Pharm1998;166:1-14

7. Li Yan, Sun Dianjia, Bi Dianzhou. Research progress on the preparation of polylactic acid and poly(lactic-co-glycolic acid) microspheres and the factors affecting their in vitro release. Chinese Modern Applied Pharmacy 2002; 19(4): 281-284

8. Zhao Feng, Gao Yongliang. Research progress of polylactic acid microspheres prepared by emulsification dispersion method. Chinese Journal of New Drugs 2002: 11(2): 123-126

9. Lemoine D, Wauters F, Bouchend’homme S, et al., Preparation and characterization of alginate microspheres containing a model antigen. Int J Pharm1998; 176: 9-19

10. Matsumoto A, Matsukawa Y, Suzuki T, et al. The polymer-alloys method as a new preparation method of biodegradable microspheres: principle and application to cisplatin-loaded microspheres. J Controlled Release 997; 48(1): 19-27

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Without further elaboration, it is believed that those skilled in the art can use the content disclosed above to the maximum extent, and these equivalent forms also fall within the scope defined by the appended claims of the present application. Furthermore, the foregoing preferred specific embodiments should be understood as illustrative only and not limiting the scope of the invention in any way.

Claims (6)

1. the preparation method of triple complex microspheres, its feature comprises: it is water that water soluble polypeptide, protein medicaments are dissolved in the sodium alginate soln, adopt W/O emulsifying, washed with isopropyl alcohol to prepare the small particle diameter sodium alginate microcapsule, and further wrap up sodium alginate microcapsule with chitosan, form the more fine and close alginic acid-chitosan double-layer microcapsule of structure, adopt emulsifying-solvent evaporation method that alginic acid-chitosan double-layer microcapsule is wrapped up again and form three layers of complex microsphere in the Vicryl Rapide.

2. the preparation method of triple complex microspheres according to claim 1, its feature also comprises: the consisting of of preparation: water soluble polypeptide, protein medicaments 3%, calcium alginate 10%, chitosan 2%, Vicryl Rapide 85%.

3. the preparation method of triple complex microspheres according to claim 1, its feature comprises: realize by following scheme:

(1) sodium alginate microcapsule preparation: with water soluble polypeptide, protein medicaments is dissolved in and is water in 0.5%～2% (w/v) sodium alginate soln, other gets first emulsifying agent, be dissolved in isobutyltrimethylmethane. as oil phase by 4%～6% (w/v) concentration, biphase volume ratio is 1: 2, the even rotating speed of breast is 5000～20000rpm at a high speed, even second emulsifying agent that drips after 1～5 minute of biphase high speed breast, breast is even 1～5 minute again, dropwise added behind 1%～10% (w/v) cross-linking agent still breast even 1～5 minute, add isopropyl alcohol last breast under same rotational speed and spare 1～3 minute, make sodium alginate microcapsule after centrifugal, wherein the second emulsifying agent consumption is 15%～49% of the first emulsifying agent consumption, first emulsifying agent is selected sorbester p17 for use, and second emulsifying agent is selected Tween 80 for use;

(2) calcium alginate-chitosan double-layer microcapsule preparation: with 0.1%～2.0%, the chitosan solution of pH 4～6 hatched sodium alginate microcapsule 10～40 minutes, centrifugal, clean with chitosan solution and/or water after collecting microcapsule, lyophilization obtain particle diameter evenly, the calcium alginate-chitosan double-layer microcapsule of form rounding;

(3) three layers of complex microsphere preparation: above-mentioned double-layer microcapsule is dispersed in 3.5～4.0g dichloromethane with 1%～1.5% (w/w) concentration, probe type ultrasonic instrument 50～200w, 10～40 seconds ultrasonic, take by weighing Vicryl Rapide in 40: 54: 6 ratios again, racemization PLGA and left-handed PLA 1g altogether add in the above-mentioned suspension, vortex makes dissolving as oil phase, other prepares 0.1%～0.5% carboxymethylcellulose sodium solution, 5～10ml as water, oil phase is added to water, 1200～2000rpm stirred stimulating milk secretion even 3～5 minutes, subsequently, it is dropwise added 50～200ml, in 0.5%～1% poly-vinyl alcohol solution, even rotating speed to the 800～2000rpm that improves after 1～2 minute of 500～600rpm stimulating milk secretion continues to stir 1～3 minute, the decompression rotary evaporation, remove dichloromethane, centrifugal collection complex microsphere, the distilled water wash postlyophilization promptly gets triple complex microspheres.

4. the preparation method of triple complex microspheres according to claim 3, its feature comprises: in the step (3) by microcapsule: PLGA=1: with≤1.2% (w/v) concentration be dispersed to calcium alginate-chitosan double-layer microcapsule in the acetonitrile solution of Vicryl Rapide at 4～1: 10, probe type ultrasonic instrument 50～200w, ultrasonic back was as the suspendible water in 5～30 seconds, sorbester p17 is dissolved in Oleum Arachidis hypogaeae semen as oil phase by 4%～8% (w/v), oil phase slowly splashes into above-mentioned suspension under 500～600rpm stirs, subsequently, improve rotating speed to 800～2000rpm and continue and stirred 1～2 minute, the decompression rotary evaporation, remove acetonitrile, centrifugal collection complex microsphere, 37 ℃ volatilize or vacuum drying promptly gets triple complex microspheres after the petroleum ether.

5. the preparation method of triple complex microspheres according to claim 3, its feature comprises: the used Vicryl Rapide of step (3), its polylactic acid is 50～100: 50～0 with the ratio of poly-hydroxyl glycolic.

6. the preparation method of triple complex microspheres according to claim 3, its feature comprises: the particle diameter of calcium alginate-chitosan microcapsules is 1～5 μ m, triple complex microsphere particle diameters are 20 μ m～50 μ m.

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