Preparation method and application of high-efficiency polypeptide drug-loaded microspheres

Abstract

The invention discloses a preparation method and application of efficient polypeptide drug-loaded microspheres. The method comprises the following steps: 1) dissolving a degradable polymer material of which the tail end is carboxyl in an organic solvent to form an oil phase; 2) adding an internal water phase pore-foaming agent and homogenizing to form primary emulsion; 3) adding the primary emulsion into the outer water phase, and homogenizing to obtain W1 / O / W2 type multiple emulsion; 4) removing the organic solvent from the multiple emulsion in the curing process, and drying to obtain blank porous microspheres with certain porosity and pore size distribution; (5) selecting a proper incubation medium, so that the blank porous microspheres can expose more available terminal carboxyl groups, and co-incubating the medicine and the microspheres in the medium to realize'healing 'of medicine loading and pore channels; and 6) washing and freeze-drying to obtain the drug-loaded microspheres. According to the method, the technological process is controlled in the preparation process of the blank porous microspheres and the self-healing drug loading process, the carboxyl density of the blank porous microspheres is improved, the constructed blank particle carrier has more carboxyl sites to be combined with polypeptide, and high-encapsulation-efficiency drug loading is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to a preparation method and application of high-efficiency polypeptide drug-loaded microspheres. Background Art

[0002] Peptides are a class of drugs with unique pharmacological mechanisms and physicochemical properties. They are highly selective for their targets and have been widely used. Currently, more than 100 peptide drugs have been approved for marketing, but due to their short elimination half-life, they often require frequent injections.

[0003] Injectable sustained-release microspheres are tiny spherical entities formed based on polyester polymers, usually with a particle size between 10 and 100 μm. They slowly release drugs through the degradation of polymers. Clinically, they have the advantages of reducing the number of dosing times, smoothing blood drug concentrations, improving drug safety, and preventing passive drug delivery and loss. Traditional methods for preparing long-acting sustained-release microspheres include solvent evaporation, coacervation, spray drying, melt extrusion, etc. However, traditional methods have many problems: for example: ① The raw materials are expensive, and most of them have high water solubility. In traditional preparation methods, the drugs are very easy to diffuse into the external aqueous phase, resulting in a low encapsulation rate of microspheres; ② Microspheres loaded with peptides usually cannot be sterilized, so expensive aseptic processing and many unit operations are required, which is not conducive to large-scale production operations; ③ In traditional methods, as the organic solvent evaporates, the drug-containing emulsion droplets gradually solidify into spheres, and the internal and external aqueous phases and the organic solvent evaporation channels form pores inside the microspheres, so it will cause a very serious drug burst release phenomenon in the early stage of release.

[0004] Porous microspheres are micron-sized particles with an internal pore structure that can provide a large surface area and internal voids, and can effectively load and protect drug molecules. The binding mechanism between polypeptides and polymers is usually a tight bond formed through electrostatic interactions and hydrogen bonds. Then, by utilizing the binding mechanism of the two, if more terminal carboxyl groups are introduced into blank porous microspheres, the binding sites between polypeptides and polymer microspheres can be increased, thereby effectively improving the loading capacity and stability of the microspheres. Existing methods such as chemical modification and polymer modification can introduce more carboxyl groups into porous microspheres, but they are often complicated to operate, costly, and not conducive to product quality control. Therefore, it is necessary to develop a simple, efficient and controllable method for regulating carboxyl density.

[0005] CN111568878B discloses a method for preparing polypeptide drug microspheres based on submerged airflow spraying technology, wherein the method comprises dissolving a polypeptide water-soluble drug in water to obtain an inner aqueous phase drug solution, dissolving a carrier material in an organic solvent to obtain an oil phase polymer solution; then adding the oil phase to the inner aqueous phase drug solution, ultrasonically crushing or shearing and dispersing to form colostrum; then transporting the colostrum to the liquid inlet of the airflow nozzle at the bottom of the liquid drying tank, and simultaneously opening the gas inlet of the airflow nozzle to atomize the colostrum, and spraying the colostrum from the bottom to the top into the aqueous solution containing a surfactant in the liquid drying tank through the nozzle of the airflow nozzle, spraying the colostrum while stirring to form a double emulsion, and removing the organic solvent to obtain polypeptide drug microspheres. Although this method produces drug-loaded microspheres with high encapsulation efficiency and stable release, it has the disadvantages of complex production process, high difficulty and high cost, and many problems in large-scale production.

[0006] CN107537038A discloses a method for improving the encapsulation yield of a water-soluble drug microsphere drug-carrying system, and the method provides a method for improving the encapsulation yield of a water-soluble drug microsphere drug-carrying system: in the existing double emulsion method (W 1 / O / W 2 ) is added to the inner aqueous phase drug-containing solution of the emulsion with a mass fraction of 15% to 30%. Although this invention improves the encapsulation rate of the water-soluble drug microsphere drug delivery system, the preparation is complicated and the addition of gelatin brings many difficulties to subsequent testing and clinical approval.

[0007] Therefore, how to prepare microspheres loaded with polypeptide drugs with high encapsulation efficiency is an urgent problem to be solved. Summary of the invention

[0008] In order to solve the above technical problems and the shortcomings of the existing technical problems, the present invention provides a method for preparing microspheres with high efficiency of loaded polypeptide drugs. Based on the mechanism of action of the carboxyl end of the polymer and the polypeptide drug binding, the present invention develops a method for regulating the carboxyl density of blank porous microspheres, and uses it as a pharmaceutical excipient, co-incubates the blank porous microspheres with the drug solution under conditions that are conducive to the protonation of the polypeptide drug, and realizes a drug loading process with a high encapsulation rate after the polymer "self-heals". The method is simple to operate and makes large-scale industrialization possible.

[0009] The technical solution adopted by the present invention is:

[0010] The present invention provides a method for regulating the carboxyl density of blank porous microspheres to achieve efficient microspheres loaded with polypeptide drugs. The present invention first prepares blank porous microspheres with a certain porosity and pore size distribution and a high carboxyl density, and then uses polymer materials and certain polypeptide drugs to undergo stronger ionization in a suitable incubation medium, so that more terminal carboxyl groups are combined with the drug, and after ionization, the two are tightly combined with opposite charges, and finally the drug loading is completed by "self-healing". The method improves the encapsulation rate of the drug under mild conditions, is simple to operate, and is conducive to industrial production.

[0011] The design principle of the present invention is divided into the following three parts:

[0012] 1. Principle of increasing the carboxyl density of blank porous microspheres

[0013] First, in terms of polymer selection, we found that the carboxyl content has a key influence on the encapsulation rate, so when selecting the raw materials for microspheres, we should avoid using ester-terminated polymers and choose carboxyl-terminated polymers as the raw materials for microsphere preparation. This is a prerequisite for ensuring a high encapsulation rate. In addition, the larger the surface area of ​​the blank microspheres, the greater the possibility that the carboxyl end of the polymer is exposed on the surface, and therefore the number of exposed carboxyl ends will be greater. A larger surface area means that more surface area can be exposed to the incubation environment, so the terminal groups in the polymer chain (such as carboxyl groups) have more opportunities to be exposed on the surface. And the porous microspheres prepared with a certain porosity and pore size distribution usually have the most suitable specific surface area for drug loading. These pores provide a certain surface area, which can increase the number of carboxyl ends exposed on the surface.

[0014] 2. The principle of tight binding between peptide drugs and acid-terminated polymers

[0015] A polypeptide is formed by condensation of different amino acid molecules in the form of peptide bonds, and a peptide composed of three or more amino acid molecules is called a polypeptide. In the present invention, using common HEPES buffer, under neutral HEPES buffer, the polypeptide is positively charged after protonation, and is tightly bound to a high molecular polymer with a free carboxylic acid group. Taking leuprolide acetate as an example, its pKa is 9.82±0.15, then it is positively charged in a neutral environment, and the positively charged polypeptide will be mutually bound to a high molecular polymer with a carboxylic acid terminal. The titration experiment of isothermal titration calorimetry (ITC) determined that the mechanism of action of the combination of the two is: first, the combination of the two is a spontaneous binding reaction with entropy and enthalpy, and secondly, its interaction mechanism is mainly electrostatic interaction, and there is also an interaction force of hydrogen bonding and hydrophobic interaction.

[0016] 3. Principle of Self-Healing of Porous Microspheres

[0017] First, self-healing is a unique property of polymers, that is, the process of self-repairing damaged structures (such as cracks and pores) through the spontaneous rearrangement of polymer chains; secondly, the self-healing mechanism is to use the passive healing of polymer chains above the glass transition temperature (Tg) of the polymer to close the pores, thereby achieving a healing effect. Based on the unique self-healing properties of polymers, the present invention self-heals the microsphere pores under the conditions of suitable temperature and incubation medium, and the present invention finds that HEPES buffer can cause more violent rearrangement of acid-terminated polymers compared to other buffers, increase the activity of polymer chains, and is more conducive to the self-healing of microspheres. And the present invention also finds the most suitable porosity range and pore size distribution of blank porous microspheres, which can not only increase the number of terminal carboxyl groups, but also do not affect the self-healing degree of microspheres, thereby achieving efficient drug loading.

[0018] In a first aspect, the present invention provides a method for preparing blank porous microspheres with high carboxyl density.

[0019] The method for preparing blank porous microspheres with high carboxyl density provided by the present invention comprises the following steps:

[0020] 1) dissolving a degradable acid-terminated polymer material having a high carboxylic acid content in an organic solvent to form an oil phase;

[0021] 2) adding the internal water phase porogen into the oil phase in step 1) and homogenizing to form colostrum;

[0022] 3) adding the colostrum described in step 2) to the external aqueous phase and homogenizing to obtain W 1 / O / W 2 Type emulsion;

[0023] 4) W described in step 3) 1 / O / W 2 The type of multiple emulsion is cured, the organic solvent is removed during the curing process, and blank porous microspheres are obtained after washing and drying.

[0024] In step 1) of the above method, the organic solvent contains at least one organic solvent having a solubility in water of less than 10%; preferably, the organic solvent includes any one or a combination of at least two of dichloromethane, chloroform, dichloroethane, ethyl acetate, butyl acetate and acetone, more preferably dichloromethane.

[0025] In step 1) of the above method, the degradable acid-terminated polymer material with a high carboxylic acid content has the following characteristics: 1) an acid-terminated degradable polymer material, 2) a polymer material with a suitable molecular weight and viscosity, and 3) a large number of terminal carboxyl groups; further, the polymer material includes at least one of the following: acid-terminated polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), polyglutamic acid (PGA), polylactic acid-caprolactone copolymer (PLCL); further, the molecular weight of the polymer material is 6 to 100 kD.

[0026] In step 1) of the above method, in the oil phase, every 2 mL of organic solvent contains 200 to 400 mg of polymer material.

[0027] In step 2) of the above method, the porogen may include one or more of ammonium carbonate, sodium bicarbonate, sodium carbonate, sodium bicarbonate, sodium alginate, sodium chloride, zinc chloride, sucrose or dextran.

[0028] In step 2) of the above method, the porogen is added in the form of an aqueous solution, and its concentration may be 0.5-0.8% (w / v), specifically 0.5% (w / v), 0.6% (w / v), 0.8% (w / v).

[0029] In step 2) of the above method, the volume ratio of the inner water phase to the oil phase is 1:2 to 1:5, specifically 1:4.

[0030] In step 3) of the above method, the external aqueous phase may be a 0.5-2% (w / v) polyvinyl alcohol solution, preferably a 1% (w / v) polyvinyl alcohol solution. The molecular weight of the polyvinyl alcohol may be 82,000-124,000, specifically 85,000.

[0031] In step 3) of the above method, the volume ratio of the colostrum (oil phase) to the external water phase may be 1:10 to 1:15, specifically 1:12.

[0032] In step 4) of the above method, the emulsion is transferred to distilled water for solidification, and the solidification time may be 4-6 hours.

[0033] In step 4) of the above method, the washing is done with distilled water until no foam is produced.

[0034] In step 4) of the above method, the drying is vacuum drying or freeze drying.

[0035] Since the free carboxyl density of blank porous microspheres with different pores formed during the formation of blank microspheres is also different, the homogenization time in the preparation of colostrum also needs to be controlled to form blank porous microspheres with high carboxyl density.

[0036] In step 2) of the above method, the homogenization speed during the preparation of colostrum is 8,000-20,000 rpm; the homogenization time is 1-4 min, preferably 10,000-12,000 rpm for homogenization for 1.5-2 min.

[0037] In step 3) of the above method, W is prepared 1 / O / W 2 The homogenization speed during the emulsion is 4,000-10,000 rpm; the homogenization time is 0.5-5 min, preferably 5,000-8,000 rpm for homogenization for 1-2 min.

[0038] The blank porous microspheres with high carboxyl density prepared by the above method also belong to the protection scope of the present invention.

[0039] The blank porous microspheres with high carboxyl density have a certain porosity and pore size distribution. The porosity of the microspheres ranges from 78.62 to 81.89%, and it has been verified that the pore sizes of the pores inside the blank microspheres should be mostly distributed in the range of 0.010 to 5.324 μm, and there are no extremely fine pores less than 0.010 μm.

[0040] In a second aspect, the present invention provides a method for preparing microspheres that efficiently carry polypeptide drugs.

[0041] The method for preparing microspheres with high efficiency and carrying polypeptide drugs provided by the present invention comprises the following steps:

[0042] a) dissolving the polypeptide drug and the blank porous microspheres with high carboxyl density in a suitable incubation medium, and incubating them for a period of time under mild conditions to achieve "healing" of the drug loading and the pores;

[0043] b) Centrifuging the system in step a) to remove the supernatant, and then washing and drying the obtained microspheres to obtain drug-loaded microspheres.

[0044] In step a) of the above method, since the principle of drug loading is that the hydrophilic polypeptide is protonated under neutral conditions and tightly combined with the ionized polymer, the polypeptide drug used has the following characteristics: it is a hydrophilic substance, pKa≥7, and can be protonated in the incubation medium.

[0045] Furthermore, the hydrophilic polypeptide drug may include but is not limited to: leuprolide acetate, octreotide acetate, triptorelin, exenatide, lanreotide, pasireotide acid, etc.

[0046] Furthermore, the hydrophilic polypeptide drug is added in the form of a solution, and the drug concentration is 5 mg / mL to 50 mg / mL, specifically 20 mg / mL.

[0047] Furthermore, in the step a), the mass ratio of the polypeptide drug to the blank porous microspheres with a high carboxyl density is 1:3 to 1:20, preferably 1:8 to 1:15, specifically 1:9.

[0048] Since the drug loading process occurs under the premise of protonation of the drug and the terminal carboxyl group of the polymer, the incubation medium should also play a role in promoting the protonation of the drug and the polymer.

[0049] The incubation medium in step a) of the above method is HEPES buffer, with a concentration of 0.05-0.5M (preferably 0.05-0.1M), a pH of 5.8-9.8, an incubation temperature of 35°C-45°C (preferably 37°C-42°C), and an incubation time of 10-24h; more preferably, HEPES buffer, with a concentration of 0.1M, a pH of 7.4, and incubation at 42°C for 12h. It has been verified that HEPES buffer can make the protonation degree of the combination of the two higher, and has a higher encapsulation efficiency.

[0050] The microspheres containing high-efficiency polypeptide drugs prepared by the above method also fall within the protection scope of the present invention.

[0051] The present invention starts from the mechanism of action of the carboxyl terminal of the polymer and the polypeptide drug combining with each other. On the one hand, in the process of preparing blank porous microspheres, the porosity and pore size distribution of the microspheres are controlled within a certain range to form more available terminal carboxyl groups, so as to combine more polypeptide drugs and improve the encapsulation rate; on the other hand, in the process of drug loading, the incubation conditions are controlled to the conditions most suitable for the protonation of the carboxyl groups at the terminal of the microspheres, so that the two can be combined to improve the encapsulation rate. The method provided by the present invention can prepare a large number of blank microspheres with high carboxyl terminals and use them as a raw material for industrial production, and then mix and incubate with certain hydrophilic polypeptides under a mild environment, and then wash and freeze-dry to produce drug-loaded microspheres with high encapsulation rate and good release behavior in a simple way.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) Improving microsphere encapsulation efficiency: The present invention can provide more binding sites (carboxyl end) inside the microsphere by controlling the porosity and pore size distribution, thereby effectively improving the encapsulation efficiency. This allows more polypeptides to bind to the microspheres, improving the encapsulation efficiency of the drug.

[0054] (2) Easy operation and simplified production process: The present invention can increase the stability of polypeptide drugs and is easy to operate. When loading drugs, the present invention can achieve efficient drug loading by mixing polypeptide drugs with polymer particles under mild conditions. The present invention does not require complex unit operations and also increases the stability of polypeptide drugs.

[0055] (3) Enhance the stability of microspheres: Controlling the porosity and pore size distribution can not only provide more carboxyl ends but also make the internal structure of the microspheres more stable, avoiding cracking or degradation during transportation and storage, thereby improving the physical and chemical stability of the microspheres and extending the shelf life of the product.

[0056] (4) Increased biosafety: No organic solvents are used in the process of encapsulating drugs, and no plasticizers are added in the preparation of blank porous microspheres. Therefore, the final product does not require additional residue testing, which increases biosafety, reduces production costs, and facilitates industrial production.

[0057] (5) It is easy to control the performance of the final product: Since the present invention prepares blank porous microspheres first and then loads the drug, the blank porous microsphere particles can be sterilized to greatly reduce the cost of aseptic large-scale production in traditional industrial production; in addition, the properties of the polymer particles (for example, size distribution, porosity and pore size distribution, residual organic solvent) can be adjusted before drug loading, which is convenient for controlling the performance of the final product.

[0058] According to experimental verification, the encapsulation rate of the drug-loaded microspheres prepared by the present invention is above 80%, and the burst release within 1 day is below 25%. The in vitro normal-rate release shows that the cumulative release within 28 days reaches 95% or more; the average particle size of the microspheres is 30 μm, and the particle size distribution coefficient Span value is less than 1.5. In addition, the porosity of the microspheres is in the range of 78.62-81.89%, and it has been verified that the pore size of the pores inside the blank microspheres should be mostly distributed in the range of 0.010 to 5.324 μm. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figure is a schematic diagram of the process of preparing the blank microspheres and the drug-loaded microspheres according to the present invention;

[0060] Figure 2 This is an electron microscope photo of the blank porous microspheres in Example 1, with a scale of 20 μm;

[0061] Figure 3 This is an electron microscope photo of the drug-loaded microspheres after self-healing in Example 1, with a scale of 10 μm;

[0062] Figure 4 is the particle size distribution diagram of the blank porous microspheres in Example 1;

[0063] Figure 5 is the pore size distribution diagram of the blank porous microspheres in Example 1;

[0064] Figure 6 is the particle size distribution diagram of leuprorelin acetate microspheres in Example 1;

[0065] Figure 7 The in vitro release curve of leuprorelin acetate microspheres under normal release conditions in Example 1 (n=3);

[0066] Figure 8 This is a graph showing the changes in the microsphere morphology of the leuprorelin acetate microspheres released in vitro under normal release conditions in Example 1, with a scale of 10 μm;

[0067] Fig. 9 This is a graph showing the molecular weight change of leuprorelin acetate microspheres during the in vitro release process under normal release conditions in Example 1;

[0068] Figure 10-11 The pH change diagram of the incubation system of leuprolide acetate microspheres in different incubation media during the drug loading process in Example 1;

[0069] Fig.12 is the isothermal titration calorimetry curve of leuprolide acetate and PLGA under different incubation conditions in Example 1;

[0070] Fig.13 The encapsulation efficiency results of leuprolide acetate microspheres prepared under different incubation conditions in Example 1;

[0071] Fig.14 The electron microscope images of the blank porous microspheres and drug-loaded microspheres prepared under different conditions in Example 1, with a scale of 10 μm;

[0072] Fig.15 : is the pore size distribution diagram of blank porous microspheres under different preparation conditions in Example 2;

[0073] Fig.16 Graph showing pH changes of blank porous microspheres during incubation under different preparation conditions in Example 2;

[0074] Fig.17 This is an electron microscope photo of the blank porous microspheres in Example 3, with a scale of 20 μm;

[0075] Figure 18-19 This is an electron microscope photo of the drug-loaded microspheres after self-healing in Example 3, with a scale of 20 μm;

[0076] Fig. 20 This is an electron microscope photograph of the drug-loaded microspheres prepared by the traditional method in Comparative Example 1, with a scale of 20 μm. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0078] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0079] The polylactic acid-co-glycolic acid (PLGA) (75:25, Mw=14149±121.15) 2CA and polylactic acid-co-glycolic acid (PLGA) (50:50, Mw=6899±115) 1A used in the following examples represent different types of PLGA. The numbers in 2CA and 1A represent different viscosities. The larger the number, the higher the viscosity. CA and A have the same meaning, both representing that the PLGA is a carboxyl-terminated PLGA.

[0080] Example 1

[0081] A method for preparing leuprolide acetate long-acting sustained-release microspheres for injection, comprising the following steps:

[0082] The first step is to prepare the blank porous microspheres with high carboxyl density, which includes the following steps (the schematic diagram of the preparation process is shown in Figure 1 shown):

[0083] Weigh 400 mg of poly(lactic acid-co-glycolic acid) (75:25, Mw=14149±121.15) 2CA (carboxyl content determined by potentiometric titration is 203.0 mol / t), add 2 mL of dichloromethane to dissolve, and form an oil phase. 3 (w / v) is the internal water phase, 0.5 mL of the internal water phase is added to the oil phase, 12,000 rpm, homogenized for 2 min, ice bath, to prepare colostrum. The prepared colostrum is added to 30 mL of 1% (w / v) polyvinyl alcohol (PVA, Mw=85000) solution, homogenized at 6200 rpm, and homogenized for 1.5 min. Then it is transferred to 30 mL of distilled water solution for curing for 30 min, and then transferred to 500 mL of distilled water solution for curing for 4 h, and the microspheres are washed with distilled water until no foam appears. The blank porous microspheres obtained are vacuum dried for 19 h to obtain about 300 mg of PLGA porous microspheres.

[0084] The second step is to load leuprorelin acetate to obtain drug-loaded microspheres, including the following steps (the schematic diagram of its preparation process is shown in FIG. Figure 1 shown):

[0085] The prepared PLGA porous microsphere freeze-dried powder was mixed, 540 mg of microspheres were weighed into a 10 mL centrifuge tube and dispersed in 3 mL of 0.1 M HEPES buffer (pH 7.4 ± 0.4), and 3 mL of 20 mg / mL leuprorelin acetate solution was added. The leuprorelin acetate solution was also dissolved using HEPES buffer. At this time, the initial microsphere concentration was 180 mg / mL. Then, the microspheres were gently rotated and stirred at 42 ° C for 12 h. After incubation, the microspheres were centrifuged at 7012 × g for 5 min, the supernatant was removed, and the drug-loaded microspheres were washed with ddH 2 The drug-loaded microspheres were washed three times with O. The obtained drug-loaded microspheres were vacuum dried for 19 h to obtain leuprorelin acetate microspheres (LA microspheres).

[0086] Performance characterization:

[0087] (1) Scanning electron microscope

[0088] The prepared blank porous microspheres and leuprorelin acetate microspheres were redispersed in water, and the surface morphology of the microspheres (e.g. Figure 2 , 3 ). The results showed that the blank porous microspheres before self-healing were loose porous spherical structures, and after self-healing and drug loading, they showed surface wrinkles and nearly spherical microspheres with well-healed pores. The depressions and gaps on the surface of the microspheres were related to polymer rearrangement.

[0089] (2) Average particle size and particle size distribution:

[0090] The prepared blank porous microspheres and leuprolide acetate microspheres were measured for particle size and distribution using a particle size analyzer (HELOS / RODOS, PAQXOS, Germany). The test results are shown in Figure 4 and Figure 6 As shown: the particle size of the blank microspheres is 29.94 μm, and the particle size distribution coefficient span value is 1.49; the particle size of the leuprorelin acetate microspheres is 29.31 μm, and the particle size distribution coefficient span value is 1.63. It was determined that there was no significant change in the particle size of the microspheres before and after drug loading.

[0091] (3) Determination of porosity and pore size distribution:

[0092] The prepared blank porous microspheres were tested for porosity and pore size distribution using a mercury intrusion instrument (AutoPore IV 9500, America). Figure 5As shown: the mercury injection amount of the blank microspheres is 4.1124 mL / g, the porosity is 78.62%, and the pores with a pore size of 0.010 to 5.324 μm account for 88.64% of the entire pores.

[0093] (4) The encapsulation efficiency was determined by:

[0094] The drug loading and encapsulation efficiency of the microspheres were determined by high performance liquid chromatography:

[0095] 1) Chromatographic conditions

[0096] Chromatographic columns Shiseido C8 (100mm*4.6mm, 3μm) Detector DAD Detection wavelength 220nm Flow rate 1mL / min Column temperature 40℃ Sample tray temperature 4℃ Injection volume 30μL Mobile phase Acetonitrile: triethylamine phosphate buffer solution (adjusted to pH 3.00 with phosphoric acid) = 23:77, v / v

[0097] 2) Preparation of mobile phase

[0098] Add 970 mL of distilled water to a 2 L beaker, add 20.85 mL of triethylamine, stir well, adjust the pH to 3.00 with phosphoric acid, filter and degas by ultrasonication after mixing, and obtain a triethylamine phosphate buffered saline solution.

[0099] 3) Preparation of reference and test substances

[0100] Reference solution:

[0101] Weigh 10 mg of leuprolide acetate into a 50 mL volumetric flask, add 10 mL of DMSO, then dilute to scale with mobile phase, dissolve by ultrasonication, and filter (PVDF 0.22 μm), n=2.

[0102] Test solution:

[0103] Weigh 10 mg of leuprolide acetate microspheres into a 10 mL volumetric flask, add 2 mL of DMSO, dilute to scale with mobile phase, dissolve by ultrasonication, and filter (PVDF 0.22 μm), n=2.

[0104] According to the encapsulation rate formula: encapsulation rate of leuprolide acetate microspheres (EE / %) = (actual loading rate of leuprolide acetate / theoretical loading rate) × 100%; after measurement, the encapsulation rate of leuprolide acetate was 96.42% (n=3).

[0105] According to the drug loading calculation formula: drug loading (LE / %) = (total mass of microspheres × mass of microsphere drugs / total mass of microspheres) × 100%; it was determined that the drug loading of leuprorelin acetate was 9.60±0.02% (n=3).

[0106] (5) In vitro release determination method:

[0107] Set the time points to be tested (2h, 4h, 6h, 12h, 1, 3, 5, 7, 14, 21, 28, 35d, 42d) and weigh 10mg of leuprorelin acetate microspheres in a 2mL centrifuge tube, n=3, a total of 39 samples, add 1mL of release medium (the release medium is 0.01M phosphate buffer with 0.01% g / mL, pH is 7.44, and the osmotic pressure is 302m0smol / kg), put it in a water bath shaker, set the temperature to 37°C, and the speed is 120rpm. Each sample is centrifuged at 7012×g for 10 minutes, and 900μL of release buffer (supernatant) is taken for use, and it is ensured that no particles are collected. For all remaining samples, fresh release medium is replaced after sampling every week after the 7th day, and incubated to the next time point.

[0108] Sample processing:

[0109] 1) Supernatant: Sample: mobile phase = 1:5 (v / v), vortex for 1 min, centrifuge at 14000 rpm for 10 min, and take the supernatant for sampling and analysis.

[0110] 2) Microspheres: After freeze-drying the microspheres at each time point under vacuum, all the microspheres were dissolved in 2 mL of DMSO and added to a 10 mL volumetric flask, diluted to the scale with mobile phase, filtered (PVDF 0.22 μm), and then the concentration of leuprolide acetate was determined according to the "High Performance Liquid Chromatography" in the previous section to obtain the in vitro release curve.

[0111] According to the in vitro release test, the results are as follows Figure 7 As shown in the figure, the burst release of leuprolide acetate within 1 day was 12.93%, and the cumulative release of leuprolide acetate microspheres reached 99.89% after 42 days, which was completely released and reached a plateau at 28 days. Figure 8 The morphological changes of the microspheres during in vitro release were also consistent with the in vitro release curve.

[0112] (6) Determination of molecular weight changes during in vitro normal release:

[0113] Set the time points to be tested (0d, 3d, 7d, 14d, 21d, 28d, 35d, 42d) and weigh 10mg of leuprorelin acetate microspheres in a 2mL centrifuge tube, n=3, a total of 24 samples, add 1mL of release medium, place in a water bath shaker, set the temperature to 37°C, and the speed to 120rpm. Each sample was centrifuged at 7012×g for 10 minutes, the supernatant was removed and washed three times with dd H2O, and then freeze-dried for use.

[0114] Molecular weight determination using gel permeation chromatography:

[0115] 1. Preparation of standard products

[0116] Weigh 10 mg of polystyrene reference substance into a 10 mL volumetric flask, add tetrahydrofuran to dissolve (do not adjust to volume, let stand for 12 hours to dissolve and then adjust to volume and mix well), n = 1;

[0117] 2. Preparation of reference substances

[0118] Weigh 10 mg of PLGA2CA into a 10 mL volumetric flask, add tetrahydrofuran to dissolve (do not adjust the volume, let it stand for 12 hours to dissolve and then adjust the volume), n = 3;

[0119] 3. Preparation of test samples

[0120] The freeze-dried LA microsphere sample was dissolved in tetrahydrofuran and added to a 10 mL volumetric flask (not fixed to volume, left to dissolve for 12 h before fixed to volume), n = 3;

[0121] All samples were filtered through a 0.45 μm nylon filter membrane before analysis.

[0122] The molecular weight changes of leuprorelin acetate microspheres at different times under normal release in vitro Fig. 9 As shown in the figure, within 42 days of release, both Mn and Mw showed a significant downward trend, indicating that as the release progressed, the esterification reaction of PLGA was also ongoing. In addition, the PDI value also showed a downward trend, indicating that the polymer Mw was degraded faster than Mn, and the molecular weight of the long-chain PLGA decreased faster because PLGA gradually degraded into water-soluble oligomers (which could not be detected by GPC). The final degradation products were lactic acid and glycolic acid monomers, and the molecular weight distribution gradually became uniform, with PDI approaching 1.

[0123] (7) Determination of the change in pH value of the incubation system in different incubation media at different incubation time points:

[0124] In order to explore the effect of incubation medium on encapsulated drugs during the incubation process, we used the PLGA blank porous microspheres in Example 1 to load drugs and incubate them with different buffer types: Tris-HCL buffer, PBS buffer, and HEPES buffer (0.1M, pH 7.40) during the incubation and drug loading process, and measured the pH value of the system at different time points. The change in pH value can directly reflect the different deprotonation abilities of PLGA microspheres under different incubation media, because according to the peptide protonation formula: LA + +Ac - +PLGA-coo - +H + →LA + —(PLGA-COO - )+HAc, we can know that the more the peptide binds to PLGA, the more HAc there is in the system, so the pH value will decrease more. Fig.10As shown in the figure, under the three incubation media, the pH of both the PLGA blank microspheres and the leuprolide acetate-loaded microspheres during the incubation process decreased significantly. Fig.11 The pH changes of PLGA blank microspheres and LA microspheres in three different buffers were subtracted, so that it can be clearly seen in which incubation medium the protonation of PLGA is most obvious; Fig.11 As shown, the protonation of peptide drugs is most obvious under HEPES buffer, and the pH changes more obviously with time, especially at 6-12h, the change is most obvious. Therefore, during the incubation process, HEPES buffer can make the protonation degree of peptide drugs higher, so that the degree of binding between drugs and microspheres is higher, and the encapsulation efficiency is also higher.

[0125] (8) The impact of different incubation environments on drug loading and determination of incubation drug loading conditions:

[0126] The experiment was conducted by changing the incubation conditions of blank porous microspheres and leuprolide acetate ( Figures 12-13 ), and through single factor screening, the optimal incubation conditions with the highest encapsulation efficiency were determined. In order to explore the reasons, we conducted isothermal titration experiments to determine the fundamental reasons why different incubation conditions affect the encapsulation efficiency of drug loading.

[0127] First, in terms of temperature, as the temperature rises to 42°C, the encapsulation efficiency of the microspheres reaches the highest. This is mainly because the ITC results show that the dissociation constant (KD) of leuprorelin acetate and PLGA decreases with increasing temperature, indicating that higher temperatures enhance the binding affinity between the two, which is consistent with the increase in encapsulation efficiency. At the same time, the absolute values ​​of entropy change (ΔS) and Gibbs free energy change (ΔG) increase with increasing temperature, which may be due to the aggregation of hydrophobic molecules to reduce contact with water, thereby increasing the disorder of the system. The increase in temperature promotes molecular motion and strengthens hydrophobic interactions. Although the basic interaction mechanism remains unchanged, the increase in temperature affects the binding affinity and molecular dynamics changes, which in turn affects the self-healing ability and encapsulation efficiency of the microspheres. Therefore, by enhancing the hydrogen bonding between PLGA and the side chains of the peptide chain, the system stability and drug affinity can be further improved, thereby optimizing the encapsulation effect.

[0128] Secondly, the type of incubation medium. The choice of incubation medium significantly affects the binding efficiency of leuprolide acetate and PLGA. Protonation experiments showed that suitable media can improve the degree of protonation. ITC results showed that the differences in encapsulation efficiency of different media mainly originated from differences in ionic strength, charge interaction and pH stability. The moderate ionic strength of HEPES and Tris-HCl buffers helps to stabilize the binding reaction, among which the strong pH buffering capacity of HEPES further enhances the binding efficiency. In contrast, the high salt concentration in PBS will shield the charge interaction, and the phosphate ions may compete with PLGA or leuprolide acetate for binding, significantly reducing the binding efficiency.

[0129] Finally, in terms of the ion concentration of HEPES buffer, the results showed that the encapsulation efficiency of leuprolide acetate microspheres increased with increasing ionic strength, and the ITC results showed that all thermodynamic parameters were negative, among which the thermodynamic parameters (ΔH, -TΔS, ΔG and KD) decreased with increasing ionic strength, indicating that the binding affinity between PLGA and leuprolide acetate increased with increasing ionic concentration. When the ion concentration was low (0.02M), the binding process was driven by negative enthalpy change (ΔH<0), and electrostatic interaction dominated (|TΔS|<|ΔH|). At higher ionic strength (0.1M), the binding process was mainly driven by entropy (|TΔS|>|ΔH|), indicating that hydrophobic interaction and hydrogen bonding played a major role. High concentrations of free ions can weaken the electrostatic attraction between the carboxyl group of PLGA and the arginine / histidine side chain of leuprolide, and the electrostatic shielding effect shifts the binding from electrostatic interaction to hydrophobic interaction and hydrogen bonding. At the same time, high ion concentration causes water molecules to reorganize, releasing the order of water molecules and increasing system entropy, thereby promoting the formation of hydrophobic interactions and hydrogen bonds, and enhancing the binding force between leuprolide acetate and PLGA. In general, the main forces between leuprolide acetate and PLGA are hydrogen bonds and hydrophobic interactions, especially when the ion concentration is 0.1M, the binding is stronger and more stable.

[0130] Example 2

[0131] A method for preparing leuprolide acetate long-acting sustained-release microspheres for injection, comprising the following steps:

[0132] The first step is to prepare the blank porous microspheres with high carboxyl density, which includes the following steps (the schematic diagram of the preparation process is shown in Figure 1 shown):

[0133] Weigh 400 mg of poly(lactic acid-co-glycolic acid) (PLGA) (75:25, Mw = 14149 ± 121.15) 2CA and add 2 mL of dichloromethane to dissolve it to form an oil phase. 3(w / v) is the inner water phase, 0.5 mL of the inner water phase is added to the oil phase, 1,2000 rpm, homogenized for 1-3 min, ice bath, to prepare colostrum. The prepared colostrum is added to 30 mL of 1% (w / v) polyvinyl alcohol (PVA, Mw=85000) solution, 6,200 rpm, homogenized for 1.5 min. Then it is transferred to 30 mL of distilled water solution for curing for 30 min, and then transferred to 500 mL of distilled water solution for curing for 4 h, and the microspheres are washed until no foam appears. The blank porous microspheres obtained are vacuum dried for 19 h to obtain PLGA porous microspheres (such as Fig.14 superior).

[0134] The second step is to load leuprorelin acetate to obtain drug-loaded microspheres, including the following steps:

[0135] The prepared PLGA porous microsphere freeze-dried powder was mixed, 540 mg of microspheres were weighed into a 10 mL centrifuge tube and dispersed in 3 mL of 0.1 M HEPES buffer (pH 7.4 ± 0.4), and 3 mL of 20 mg / mL leuprorelin acetate solution was added. The leuprorelin acetate solution was also dissolved using HEPES buffer. At this time, the initial microsphere concentration was 180 mg / mL. Then, the microspheres were gently rotated and stirred at 42 ° C for 12 h. After incubation, the microspheres were centrifuged at 7012 × g for 5 min, the supernatant was removed, and the drug-loaded microspheres were washed with ddH 2 The drug-loaded microspheres were washed three times with water. The drug-loaded microspheres were vacuum dried for 19 h to obtain leuprorelin acetate microspheres (such as Fig.14 Down).

[0136] The following table shows the relevant parameters of blank porous microspheres obtained by mercury intrusion porosimetry and the encapsulation efficiency of drug-loaded microspheres under different preparation conditions:

[0137] Preparation conditions Drug loading / % Encapsulation rate / % Proportion Total mercury intake Porosity / % 0.5%-2min 9.60±0.02 96.03±2.51 0.8864 4.1124 78.62 0.6%-2min 9.27±0.03 92.68±3.36 0.8807 4.4313 79.98 0.5%-1.5min 8.70±0.02 87.10±2.03 0.844 4.67 83.6 0.5%-2.5min 8.34±0.02 83.42±2.60 0.8639 4.7357 84.14 0.8%-3min 8.18±0.02 81.78±2.32 0.7337 4.8554 81.89 0.5%-1min 7.80±0.04 78.21±4.39 0.3704 5.2102 82.01 0.8%-1min 7.76±0.05 77.66±5.12 0.2967 5.0646 82.29

[0138] The calculation formula for “percentage” in the above table is as follows:

[0139] like Fig.15 It can be clearly seen from the pore size distribution diagram that the main differences of the blank porous microspheres under various conditions are the distribution of pores in the pore size range of 0.010-5.324 μm and the existence and distribution of extremely small pores.

[0140] Depend on Fig.14We can also draw relevant conclusions from the above table: ① The blank porous microspheres with better encapsulation efficiency have the highest pore size ratio in the pore size range of 0.010-5.324μm, and there is no extremely small pore size and large pore size and distribution, indicating that the pore size of the microspheres most suitable for leuprorelin acetate loading is mainly in the pore size range of 0.010-5.324μm. ② The higher the pore size ratio of 0.010-5.324μm, the higher the encapsulation efficiency.

[0141] Therefore, the pore size of blank porous microspheres suitable for peptide drug delivery is mainly distributed in the pore size range of 0.010 to 5.324 μm, and the exposed terminal carboxyl content of such porous microspheres is also higher, which is more conducive to the protonation of PLGA and makes the encapsulation rate higher. Fig.16 The results of pH changes also verified this statement: Fig.16 It can be clearly seen that with the increase of incubation time, the pH value of the incubation system of all drug-loaded microspheres showed a downward trend, indicating that the protonation of PLGA and the deprotonation of polypeptides were continuously carried out during the drug loading process; and it is worth noting that the change pattern of the pH reduction of blank porous microspheres under different preparation conditions during the incubation process is: the higher the encapsulation rate of the microspheres and the higher the proportion of blank microspheres under the appropriate pore size, the more the pH will decrease, indicating that the blank porous microspheres under the appropriate pore size distribution can expose more terminal carboxyl groups to achieve more protonation of PLGA, so the combination of the two is more and the encapsulation rate is higher.

[0142] Example 3

[0143] A method for preparing leuprolide acetate long-acting sustained-release microspheres for injection, comprising the following steps:

[0144] First, the preparation process of blank microspheres includes the following steps (the preparation process diagram is shown in Figure 1 shown):

[0145] Weigh 400 mg of poly(lactic acid-co-glycolic acid) (PLGA) (50:50, Mw = 6899 ± 115) 1A, add 2 mL of dichloromethane to dissolve, and form an oil phase. 3 (w / v) is the inner water phase, 0.5 mL of the inner water phase is added to the oil phase, 12000 rpm, homogenized for 2 min, ice bath, to prepare colostrum. The prepared colostrum is added to 30 mL of 1% (w / v) polyvinyl alcohol (PVA, Mw=85000) solution, homogenized at 6200 rpm, and homogenized for 1.5 min. Then it is transferred to 30 mL of distilled water solution for curing for 30 min, and then transferred to 500 mL of distilled water solution for curing for 4 h, and the microspheres are washed until no foam appears. The blank porous microspheres obtained are vacuum dried for 19 h to obtain freeze-dried microspheres (such as Fig.17 ).

[0146] The second step is to load leuprorelin acetate to obtain drug-loaded microspheres, including the following steps:

[0147] The prepared PLGA porous microsphere freeze-dried powder was mixed, 540 mg of microspheres were weighed into a 10 mL centrifuge tube and dispersed in 3 mL of 0.1 M HEPES buffer (pH 7.4 ± 0.4), and 3 mL of 20 mg / mL leuprorelin acetate solution was added. The leuprorelin acetate solution was also dissolved using HEPES buffer. At this time, the initial microsphere concentration was 180 mg / mL. Then, the microspheres were gently rotated and stirred at 35 ° C for 12 h. After incubation, the microspheres were centrifuged at 7012 × g for 5 min, the supernatant was removed, and the drug-loaded microspheres were washed with ddH 2 The drug-loaded microspheres were washed three times with water. The drug-loaded microspheres were vacuum dried for 19 h to obtain leuprorelin acetate microspheres (such as Fig.18 ).

[0148] The drug loading and encapsulation efficiency of the microspheres were determined by high performance liquid chromatography, and the drug loading of the microspheres was determined to be 8.48%, and the encapsulation efficiency was 84.94%.

[0149] In addition, 540 mg of microspheres were weighed into a 10 mL centrifuge tube and dispersed in 3 mL of 0.1 M HEPES buffer (pH 7.4 ± 0.4), and 3 mL of 20 mg / mL leuprorelin acetate solution was added. The leuprorelin acetate solution was also dissolved using HEPES buffer. At this time, the initial microsphere concentration was 180 mg / mL. Then, the microspheres were gently rotated and stirred at 37 °C for 12 h. After the incubation was completed, the microspheres were centrifuged at 7012 × g for 5 min, the supernatant was removed, and the drug-loaded microspheres were washed with dd H 2 The drug-loaded microspheres were washed three times with water. The drug-loaded microspheres were vacuum dried for 19 h to obtain leuprorelin acetate microspheres (such as Fig.19 ).

[0150] The drug loading and encapsulation efficiency of the microspheres were determined by high performance liquid chromatography, and the drug loading of the microspheres was determined to be 9.62% and the encapsulation efficiency was 96.29%.

[0151] Comparative Example 1

[0152] Using traditional W 1 / O / W 2Leuprorelin acetate microspheres were prepared by emulsification method: 800 mg of poly(lactic acid-co-glycolic acid) (PLGA) (75:25, Mw = 14149 ± 121.15) 2CA was weighed and dissolved in 1 mL of dichloromethane to form an oil phase; 90.63 mg of leuprorelin acetate and 15.71 mg of gelatin were weighed and dissolved in 120 μL of distilled water at 60°C to form an aqueous phase; first, the oil phase was poured into the inner aqueous phase and homogenized at 10000 rpm for 1 min to obtain W 1 / O colostrum. Cool to 16°C. Then add 1.6 mL of 0.25% PVA aqueous solution to the colostrum. Homogenize at 15000 rpm for 20 seconds to obtain W 1 / O / W 2 Emulsion.

[0153] Then transfer to 500 mL of distilled water solution, solidify for 4 h, and wash the microspheres until no foam appears. The blank porous microspheres obtained were vacuum dried for 19 h to obtain freeze-dried drug-loaded microspheres (such as Fig. 20 ). The drug loading of the drug-loaded microspheres obtained by this traditional method was measured to be 6.00%, and the encapsulation efficiency was 60.04%.

[0154] In contrast, the preparation and drug loading methods and applications of the porous microsphere carrier for polypeptide drugs mentioned in the present invention significantly improve the encapsulation rate of polypeptide drugs, and can release them sustainably and completely, and also have the disadvantages of simple operation and being conducive to industrial production; they overcome the disadvantages of low encapsulation rate, complex preparation, and inconvenience in production monitoring and microsphere property control under traditional methods.

[0155] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for preparing blank porous microspheres with high carboxyl density, comprising the following steps: 1) dissolving a degradable acid-terminated polymer material having a high carboxylic acid content in an organic solvent to form an oil phase; 2) adding an internal water phase porogen to the oil phase in step 1) and homogenizing to form colostrum; 3) adding the colostrum in step 2) to the external aqueous phase and homogenizing to obtain a W1 / O / W2 type emulsion; 4) The W1 / O / W2 type emulsion of step 3) is solidified, washed and dried to obtain blank porous microspheres with high carboxyl density.

2. The preparation method according to claim 1, characterized in that: In the step 1), the organic solvent contains at least one organic solvent having a solubility in water of less than 10%. Preferably, the organic solvent includes any one or a combination of at least two of dichloromethane, chloroform, dichloroethane, ethyl acetate, butyl acetate and acetone, more preferably dichloromethane.

3. The preparation method according to claim 1 or 2, characterized in that: In the step 1), the degradable acid-terminated polymer material with a high carboxylic acid content has the following characteristics: 1) Acid-terminated degradable polymer material, 2) Polymer material with suitable molecular weight and viscosity, 3) Having more terminal carboxyl groups; Further, the polymer material includes at least one of the following: acid-terminated polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polyglutamic acid, polylactic acid-caprolactone copolymer; Furthermore, the molecular weight of the polymer material is 6 to 100 kD.

4. The preparation method according to any one of claims 1 to 3, characterized in that: In the step 1), in the oil phase, every 2 mL of organic solvent contains 200 to 400 mg of polymer material.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In the step 2), the porogen is selected from one or more of ammonium carbonate, sodium bicarbonate, sodium carbonate, sodium bicarbonate, sodium alginate, sodium chloride, zinc chloride, sucrose, and dextran; And / or, in the step 2), the porogen is added in the form of an aqueous solution with a concentration of 0.5 to 0.8% (w / v); And / or, in step 2), the volume ratio of the inner water phase to the oil phase is 1:2 to 1:5; And / or, in the step 2), the homogenization speed is 8,000-20,000 rpm; the homogenization time is 1-4 min, preferably 10,000-12,000 rpm for 1.5-2 min.

6. The preparation method according to any one of claims 1 to 5, characterized in that: In the step 3), the external aqueous phase is a 0.5-2% (w / v) polyvinyl alcohol solution, preferably a 1% (w / v) polyvinyl alcohol solution; And / or, in step 3), the volume ratio of colostrum to external aqueous phase is 1:10 to 1:15; And / or, in the step 3), the homogenization speed is 4,000-10,000 rpm; the homogenization time is 0.5-5 min, preferably 5,000-8,000 rpm for 1-2 min.

7. Blank porous microspheres with high carboxyl density prepared by the method according to any one of claims 1 to 6; Preferably, the porosity of the blank porous microspheres is in the range of 78.62% to 81.89%; the pore sizes of the pores inside the blank microspheres are mainly distributed in the range of 0.010 to 5.324 μm, and there are no extremely fine pores smaller than 0.010 μm.

8. A method for preparing microspheres that efficiently carry polypeptide drugs, comprising the following steps: a) dissolving the polypeptide drug and the blank porous microspheres with high carboxyl density as claimed in claim 7 in an incubation medium, and incubating them under mild conditions to achieve "healing" of the drug loading and the pores; b) The system obtained after incubation in step a) is centrifuged to remove the supernatant, and the obtained microspheres are washed and dried to obtain microspheres loaded with polypeptide drugs.

9. The preparation method according to claim 8, characterized in that: The polypeptide drug is a hydrophilic polypeptide drug with a pKa of ≥7 and can be protonated in an incubation medium; further, the hydrophilic polypeptide drug includes at least one of leuprolide acetate, octreotide acetate, triptorelin, exenatide, lanreotide, and pasireotide acid; the hydrophilic polypeptide drug is added in the form of a solution, and the drug concentration in the solution is 5 to 50 mg / mL; And / or, in the step a), the mass ratio of the polypeptide drug to the blank porous microspheres with a high carboxyl density is 1:3 to 1:20, preferably 1:8 to 1:15; And / or, in the step a), the incubation medium is HEPES buffer with a concentration of 0.05-0.5M, a pH of 5.8-9.8, an incubation temperature of 35-45°C, and an incubation time of 10-24h; preferably HEPES buffer with a concentration of 0.1M, a pH of 7.4, and incubation at 42°C for 12h.

10. Microspheres containing high-efficiency polypeptide drugs prepared according to the method of claim 8 or 9.

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