CN108403643B - Long-acting microsphere of protein polypeptide drug and preparation method thereof - Google Patents
Long-acting microsphere of protein polypeptide drug and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/23—Calcitonins
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
Abstract
The invention relates to a long-acting microsphere of a protein polypeptide drug and a preparation method thereof. The preparation method comprises the following steps: (1) dissolving protein or polypeptide water-soluble medicine and protein protectant in water to obtain water-phase medicine solution; dissolving a high molecular carrier material in an organic solvent to obtain an oil-phase high molecular solution; (2) adding the water-phase drug solution into the oil-phase polymer solution, and preparing into W/O emulsion by using a cell disruptor or a high-speed shearing instrument; (3) and (3) feeding the W/O emulsion into an ultrafine particle preparation system at a constant speed through a peristaltic pump to prepare the long-acting protein polypeptide drug microspheres. The preparation method has the advantages of simple prescription, mild conditions, simple preparation process, controllable parameters, good reproducibility and high production efficiency. The protein polypeptide drug microsphere prepared by the method has the advantages of high drug encapsulation rate, low burst release rate, stable and durable drug release rate, smooth surface, complete spherical shape and uniform particle size.
Description
Technical Field
The invention relates to the field of medicinal preparations, in particular to a long-acting microsphere of a protein polypeptide medicament and a preparation method thereof.
Background
Protein polypeptide drugs are the most widely used biological macromolecules at present, and play an increasingly important role in preventing, diagnosing and treating diseases due to the characteristics of high potency, good biocompatibility, low dosage and the like. Protein polypeptide drugs, which are active substances having metabolic functions in organisms, relate to various fields such as hormones, nerves, cell growth and reproduction. Compared with the traditional micromolecular chemical drugs, the protein polypeptide drug has definite internal action, small toxic and side effect, strong pharmacological action, less adverse reaction and higher safety, so the research and development work of the protein polypeptide drug becomes one of the hot spots in the field of medicine research and development.
Protein polypeptide drugs have poor stability, low oral bioavailability and short half-life in vivo, so clinically, the protein polypeptide drugs mainly take injection and freeze-dried powder injection formulations, and frequent injection administration is usually required to achieve curative effect, which results in low compliance of patients. In order to improve the bioavailability of protein polypeptide drugs and reduce the administration frequency, the development of long-acting preparations is a main direction for the research and development of new dosage forms and new technologies of drugs. After single administration, the long-acting injection microsphere preparation can maintain the effective blood concentration in vivo for several days to several months, thereby meeting the treatment requirements of different diseases. So far, protein polypeptide drug microspheres approved by FDA include leuprorelin microspheres, triptorelin microspheres, octreotide microspheres, growth hormone microspheres, and the like.
In the existing research, the W/O/W multiple emulsion method is the most common method for preparing the protein-carrying polypeptide drug microsphere preparation. The method is that water-soluble protein polypeptide medicine is dissolved in an inner water phase, carriers such as PLGA and the like are dissolved in an organic phase, the inner water phase is added into the organic phase to be emulsified to form W/O type colostrum, then the colostrum is transferred to an outer water phase containing an emulsifier to form W/O/W type multiple emulsion, and the multiple emulsion is slowly stirred in a large volume of the dispersed phase to gradually volatilize and solidify the organic phase into balls. The process has complicated steps, and the addition of the external water phase easily causes the migration of protein polypeptide drugs from the internal water phase, thus causing the defects of low encapsulation rate, high burst release rate and the like of the microspheres.
Disclosure of Invention
Based on the method, the invention provides a preparation method of the long-acting microsphere of the protein polypeptide drug, and the microsphere prepared by the method has high drug encapsulation rate, long release period and low burst release rate.
The specific technical scheme is as follows:
a preparation method of long-acting microspheres of protein polypeptide drugs comprises the following steps:
(1) dissolving protein or polypeptide water-soluble medicine and protein protectant in water to obtain water-phase medicine solution; dissolving a high molecular carrier material in an organic solvent to obtain an oil-phase high molecular solution;
(2) adding the water-phase drug solution into the oil-phase polymer solution, and preparing into W/O emulsion by using a cell disruptor or a high-speed shearing instrument;
(3) and (3) feeding the W/O emulsion into an ultrafine particle preparation system at a constant speed through a peristaltic pump to prepare the long-acting protein polypeptide drug microspheres.
In some embodiments, the protein or polypeptide water-soluble drug in step (1) is selected from one or more of bovine serum albumin, salmon calcitonin, vascular endothelial growth factor, thymopentin, and interferon.
In some embodiments, the protein protectant in step (1) is selected from one or more of mannitol, sorbitol, gelatin, sucrose, trehalose, lactose, maltose, and fructose.
In some embodiments, the polymeric carrier material in step (1) is selected from the group consisting of mPEG-PLGA, a mixture of any one or more of carboxyl-terminated PLGA and hydroxyl-terminated PLGA.
In some of these embodiments, the molecular weight of the PLGA is 20000-50000 Da; the molecular weight of the mPEG-PLGA is 30000-60000 Da.
In some embodiments, the organic solvent in step (1) is selected from one or more of dichloromethane, dimethyl carbonate and ethyl acetate.
In some of the embodiments, the organic solvent in step (1) is a mixture of dichloromethane and dimethyl carbonate in a volume ratio of 1.5-2.5: 1.
In some embodiments, the concentration of the protein or polypeptide water-soluble drug in the aqueous drug solution of step (1) is 5-30 mg/mL.
In some embodiments, the concentration of the protein or polypeptide water-soluble drug in the aqueous drug solution of step (1) is 15-25 mg/mL.
In some of these embodiments, the concentration of the protein protectant in the aqueous pharmaceutical solution of step (1) is 10-30 mg/mL.
In some of these embodiments, the concentration of the protein protectant in the aqueous pharmaceutical solution of step (1) is 15-25 mg/mL.
In some embodiments, the concentration of the polymeric carrier material in the oil-phase polymeric solution in step (1) is 20-80 mg/mL.
In some embodiments, the concentration of the polymeric carrier material in the oil-phase polymeric solution in step (1) is 30-70 mg/mL.
In some embodiments, the concentration of the polymeric carrier material in the oil-phase polymeric solution in step (1) is 40-60 mg/mL.
In some embodiments, the concentration of polymeric carrier material in the oil-phase polymeric solution in step (1) is 45-55 mg/mL.
In some embodiments, the volume ratio of the aqueous phase drug solution to the oil phase polymer solution in step (2) is 1: 10-40.
In some embodiments, the volume ratio of the aqueous phase drug solution to the oil phase polymer solution in step (2) is 1: 15-35.
In some embodiments, the volume ratio of the aqueous phase drug solution to the oil phase polymer solution in step (2) is 1: 18-25.
In some embodiments, the power of the cell disruptor in step (2) is 200-700W, and the disruption time is 30-90 s; the shear rate of the high-speed shear apparatus is 8000-12000rpm, and the shear time is 30-90 s.
In some embodiments, the power of the cell disruptor in step (2) is 300-500W.
In some embodiments, the power of the cell disruptor in step (2) is 350-450W.
In some embodiments, the liquid supply rate of the peristaltic pump in step (3) is 4-15mL/min, and the rotation speed of the spinning disk of the ultrafine particle preparation system is 7000-14000 rpm.
In some embodiments, the liquid supply rate of the peristaltic pump in step (3) is 8-12mL/min, and the rotation speed of the rotary plate of the ultrafine particle preparation system is 8000-.
In some of these embodiments, the spin-disk speed of the ultrafine particle preparation system is 8500-12500 rpm.
The invention also provides a long-acting microsphere of the protein polypeptide drug.
The specific technical scheme is as follows:
a long-acting microsphere of protein polypeptide medicine is prepared by the above preparation method.
The long-acting microsphere of the protein polypeptide drug and the preparation method thereof have the following advantages and beneficial effects:
the preparation method of the long-acting protein polypeptide drug microspheres provided by the invention is simple, mild in condition, simple in preparation process, controllable in parameters, good in reproducibility and high in production efficiency, and can realize continuous large-scale production. In the preparation method of the long-acting microsphere of the protein polypeptide drug, the aqueous solution of the protein or polypeptide drug containing a protein protective agent and the organic solution of a macromolecular carrier are directly prepared into W/O emulsion by a cell disruptor or a high-speed shearing instrument, and then the W/O emulsion is supplied to a UPPS system to be solidified to form the microsphere. Compared with the microspheres prepared by the traditional W/O/W multiple emulsion method, the method has no external water phase, can avoid the leakage of the medicine from the internal water phase to the external water phase, improve the medicine encapsulation rate, reduce the porosity, and effectively reduce the phenomenon of medicine burst release caused by the aggregation of protein or polypeptide medicines on the surface layer of the microspheres. In addition, the preparation method is mild, and the structure of the protein polypeptide drug can be protected. Therefore, the preparation method of the protein polypeptide drug injection microsphere provided by the invention can effectively solve the problems of high burst release rate, low encapsulation rate, easily damaged drug structure, uncontrollable drug release mode and the like of the traditional protein polypeptide drug microsphere preparation. The protein polypeptide drug microsphere prepared by the method has the advantages of drug encapsulation rate of over 90 percent, low burst release rate (the release rate of 3 to 6 percent in the first day), stable and durable drug release rate, sustained release period of 30 to 120 days, smooth surface, complete sphere and uniform particle size. The method is suitable for active sensitive biological medicines such as protein, polypeptide, oligopeptide, vaccine and the like, and has clinical practical application value.
The prepared long-acting microsphere of the protein polypeptide drug has higher encapsulation efficiency and lower burst release efficiency by selecting the type and molecular weight of a specific high-molecular carrier material, matching and controlling the concentration of a drug aqueous solution, the concentration of a high-molecular carrier organic solution and the ratio of the two, the specific preparation conditions of the W/O emulsion, the drug liquid supply speed of UPPS, the rotating speed of a rotary disc and other preparation parameters, and reasonably matching all the parameters within a certain range.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the long-acting injection microsphere of protein polypeptide drug of the present invention;
FIG. 2 is a scanning electron micrograph of the bovine serum albumin microspheres prepared in example 1, comparative example 1 and comparative example 2;
FIG. 3 is a graph showing the distribution of the particle size of the bovine serum albumin microspheres prepared in example 1;
FIG. 4 is a scanning electron micrograph of the bovine serum albumin microspheres prepared in example 2 and example 4;
FIG. 5 is a particle size distribution diagram of salmon calcitonin microspheres prepared in example 3;
FIG. 6 is a graph showing the distribution of the particle size of the bovine serum albumin microspheres prepared in comparative example 1;
FIG. 7 is a graph showing the distribution of the particle size of the bovine serum albumin microspheres prepared in comparative example 2;
FIG. 8 is a graph showing the comparison of the in vitro release curves of the bovine serum albumin microspheres prepared by the UPPS method and the W/O/W multiple emulsion method in example 6;
FIG. 9 is a circular dichroism chart of the secondary structure of the drug for detecting bovine serum albumin in example 7.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and with reference to the accompanying drawings.
UPPS: ultrafine particle preparation systems, device configurations and uses are described in Wen X, Peng X, Fu H, et.preparation and in vitro evaluation of bulk fibrous microspheres processing dby a novel ultra-fine processing system [ J ]. Int J Pharm,2011.416: 195-.
Principle of UPPS microsphere preparation: the solution, the suspension or the emulsion is supplied to a central nozzle at the top end of a rotary disk system through a liquid inlet system, the preparation liquid reaches the rotary disk through the nozzle, the preparation liquid is dispersed into microdroplets under the high-speed rotation of the rotary disk, the solvent of the microdroplets is volatilized in an airflow field, and the microspheres are obtained after solidification.
Example 1: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the embodiment comprises the following steps:
(1) dissolving bovine serum albumin in an aqueous solution containing 20mg/mL mannitol to obtain an aqueous phase (W) containing 20mg/mL bovine serum albumin, and dissolving a carboxyl-terminal polylactic acid-glycolic acid copolymer (PLGA, LA: GA ═ 50:50, molecular weight 40000Da) in a mixed solvent of dichloromethane and dimethyl carbonate 2:1(v: v) to obtain an oil phase (O) having a polymer concentration of 50 mg/mL;
(2) adding the prepared water phase (bovine serum albumin aqueous solution) into the oil phase (PLGA organic solution) (oil-water volume ratio is 20:1), and emulsifying the water phase and the oil phase under the conditions of ice bath, power of 400W and time of 30s by using a cell disruptor to prepare W/O emulsion;
(3) and (3) feeding the W/O emulsion into UPPS at a constant speed of 10mL/min through a peristaltic pump, shearing and atomizing under the action of a high-speed rotary disc (the rotating speed is 12000rpm) to form microdroplets, volatilizing a solvent of the microdroplets in an airflow field, and solidifying the microdroplets to obtain bovine serum albumin microspheres, wherein the microspheres are marked as microspheres 1.
The bovine serum albumin microspheres prepared in this example are placed on a metal object stage to which a conductive adhesive tape is attached, a scanning electron microscope sample is prepared by spraying fine gold, and the morphology of the microspheres is observed under a cold field Scanning Electron Microscope (SEM), as a result, the microspheres are shown in a diagram a in fig. 2, and as can be seen from the diagram a in fig. 2, the microspheres prepared in this example have smooth surfaces, complete spheres and regular particles without adhesion.
Precisely weighing 5mg of the bovine serum albumin microspheres prepared in the embodiment, placing the bovine serum albumin microspheres in a 7mL centrifuge tube, adding 2mL of lysis solution (the composition of the lysis solution is 0.1mol/L NaOH solution containing 0.5% (w/v) sodium dodecyl sulfate), mixing in a vortex manner, placing the mixture in a 37 ℃ and 100rpm gas bath oscillator, oscillating for 24h until the microspheres are dissolved, centrifuging for 5min at 12000rpm, taking supernatant, determining the content of bovine serum albumin in the microspheres according to a Micro-BCA method, and calculating the drug encapsulation rate. The experimental result shows that the encapsulation rate of the bovine serum albumin microspheres prepared in example 1 reaches 103.15 +/-2.18.
The bovine serum albumin microspheres prepared in this example were subjected to particle size and distribution measurement using a laser particle size analyzer (Mastersizer2000) (see fig. 3 for results). The results showed that the bovine serum albumin microspheres prepared in this example had uniform and normal particle sizes, and d (0.1) ═ 11.37 ± 1.42 μm, d (0.5) ═ 15.92 ± 2.01 μm, and d (0.9) ═ 23.01 ± 4.00 μm.
Example 2: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the embodiment comprises the following steps:
(1) dissolving bovine serum albumin in an aqueous solution containing 20mg/mL mannitol to obtain an aqueous phase (W) containing 20mg/mL bovine serum albumin, and dissolving polylactic acid-glycolic acid copolymer (PLGA, molecular weight 20000Da, LA: GA ═ 50:50) in a mixed solvent of dichloromethane and dimethyl carbonate 2:1(v: v) at the carboxyl terminal to obtain an oil phase (O) with a polymer concentration of 50 mg/mL;
(2) adding the prepared water phase (bovine serum albumin aqueous solution) into the oil phase (PLGA organic solution) (oil-water volume ratio is 20:1), and emulsifying the water phase and the oil phase under the conditions of ice bath, power of 400W and time of 30s by using a cell disruptor to prepare W/O emulsion;
(3) and (3) feeding the W/O emulsion into UPPS at a constant speed of 10mL/min through a peristaltic pump, shearing and atomizing under the action of a high-speed rotary disc (the rotating speed is 12000rpm) to form microdroplets, volatilizing a solvent of the microdroplets in an airflow field, and solidifying the microdroplets to obtain the serum albumin microspheres, wherein the microdroplets are marked as microspheres 2.
The scanning electron micrograph of the bovine serum albumin microspheres prepared in this example is shown as B in fig. 4 (the determination method is the same as that in example 1), and as can be seen from B in fig. 4, most of the microspheres have smooth surfaces, complete spheres and regular particles without adhesion.
Example 3: preparation of salmon calcitonin microsphere
The preparation method of the salmon calcitonin microsphere comprises the following steps:
(1) dissolving salmon calcitonin in an aqueous solution containing 20mg/mL of mannitol to obtain an aqueous phase (W) containing salmon calcitonin at a concentration of 20mg/mL, and dissolving polylactic-co-glycolic acid (PLGA, molecular weight 40000Da) in a mixed solvent of dichloromethane and dimethyl carbonate 2:1(v: v) to obtain an oil phase (O) containing a polymer at a concentration of 50 mg/mL;
(2) adding the prepared water phase (salmon calcitonin aqueous solution) into the oil phase (PLGA organic solution added) at a volume ratio of 20:1, and emulsifying the water phase and the PLGA organic solution by using a cell disruptor under the conditions of ice bath, power of 400W and time of 30s to prepare W/O emulsion;
(3) and (2) feeding the W/O emulsion into UPPS at a constant speed of 10mL/min through a peristaltic pump, shearing and atomizing under the action of a high-speed rotary disc (the rotating speed is 12000rpm) to form microdroplets, volatilizing a solvent of the microdroplets in an airflow field, and solidifying the microdroplets to obtain the salmon calcitonin microsphere.
The particle size distribution of the salmon calcitonin microspheres prepared in this example is shown in fig. 5 (the determination method is the same as that in example 1), d (0.1) ═ 10.70 ± 0.44 μm, d (0.5) ═ 125.46 ± 1.03 μm, and d (0.9) ═ 23.31 ± 1.63 μm.
Example 4: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of this example is basically the same as example 1, except that the polymer concentration in the oil phase is different, and the polymer concentrations are 20mg/mL, 40mg/mL, 60mg/mL and 80mg/mL respectively. The resulting microspheres were recorded as microspheres 3-6, respectively.
The morphology of each microsphere prepared in this example was observed under a cold field Scanning Electron Microscope (SEM) according to the method of example 1, and as a result: the microsphere 3 is irregular in shape due to too low PLGA concentration, the microsphere collapses (as shown in A in figure 4), the microspheres 4 and 5 are complete in spherical shape, the particles are regular and are not adhered, the microspheres 6 are filiform due to too high PLGA concentration, and the shape of part of the microspheres is irregular (as shown in C in figure 4).
Example 5: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the embodiment is basically the same as that of embodiment 1, except that the oil-water volume ratio is different, and the oil-water volumes are 5:1, 10:1, 30:1, 40:1 and 60:1, respectively. The resulting microspheres were designated microspheres 7-11, respectively.
The morphology of each microsphere prepared in this example was observed under a cold field Scanning Electron Microscope (SEM) according to the method of example 1, and as a result: the microspheres 7 are not completely dried due to the over-high proportion of the water phase, and are adhered among the microspheres, so that the dispersibility is poor; the surfaces of the microspheres 8, 9 and 10 are smooth, the spheres are complete, and no adhesion exists among the particles; the microspheres 11 failed to form microspheres due to the high proportion of oil phase.
Example 6: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of this example is basically the same as example 1, except that the rotation speed of the high-speed rotary disk is different, and the rotation speed of the high-speed rotary disk is 6000rpm, 9000rpm and 15000rpm respectively. The resulting microspheres are designated microspheres 12-14, respectively.
The morphology of each microsphere prepared in this example was observed under a cold field Scanning Electron Microscope (SEM) according to the method of example 1, and as a result: the surfaces of the microspheres 12 and 13 are smooth, the spheres are complete, and no adhesion exists among the particles; the microspheres 14 partially take on a flake shape due to the excessive rotation speed of the spinning disk.
Example 7: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the embodiment is basically the same as that of embodiment 1, except that the liquid supply speeds are different, and the liquid supply speeds are 3mL/min and 20mL/min respectively. The resulting microspheres were designated microspheres 15-16, respectively.
The morphology of each microsphere prepared in this example was observed under a cold field Scanning Electron Microscope (SEM) according to the method of example 1, and as a result: the surface of the microsphere 15 is smooth, the spherical shape is complete, but the particle size distribution is not uniform; the microspheres 16 are not completely volatilized due to too high liquid supply speed, are irregular in spherical shape and are aggregated in particles.
Example 8: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of this example is basically the same as that of example 1, except that the power of the cell disruptor is different, and the power of the cell disruptor is 100W, 200W, 500W and 700W respectively. The obtained microspheres are respectively marked as microspheres 17-20.
The morphology of each microsphere prepared in this example was observed under a cold field Scanning Electron Microscope (SEM) according to the method of example 1, and as a result: when the power of the cell disruptor is 100W, no formed microspheres are seen, and when the power is 200W, 500W and 700W, the surfaces of the microspheres are smooth, the spheres are complete, and no adhesion exists among the particles.
The encapsulation efficiency of the bovine serum albumin microspheres in each example was measured according to the measurement method of example 1, and the results are shown in table 1:
TABLE 1 encapsulation efficiency of bovine serum albumin microspheres
As can be seen from Table 1, the encapsulation efficiency of the BSA microspheres is mainly related to the concentration of PLGA, the volume ratio of oil to water, the rotation speed of the spinning disk and the power of the cell disruptor for forming the W/O emulsion. Within a certain range, the higher the PLGA concentration is, the higher the encapsulation efficiency of the medicine is, and when the PLGA concentration is 40-80mg/mL, the encapsulation efficiency of the microspheres can reach more than 90%. The difference of the oil-water phase volume ratio can affect the property of the W/O emulsion, and further affect the encapsulation efficiency of the microspheres, and when the volume ratio is 10:1-30:1, the encapsulation efficiency of the microspheres can reach over 90 percent. When the rotating speed of the rotary disc is 6000-12000rpm, the encapsulation efficiency of the microspheres is hardly influenced, and the encapsulation efficiency of the obtained microspheres is high, and when the rotating speed reaches 15000rpm, the encapsulation efficiency is reduced, which is caused by the breakage of the microspheres due to overhigh rotating speed. When the power of the cell disruption instrument is 200-700W, the encapsulation efficiency of the microspheres is higher, and when the power is 400-500W, the encapsulation efficiency can reach 90%.
Comparative example 1: preparation of bovine serum albumin W/O/W microspheres
(1) Bovine serum albumin was dissolved in an aqueous solution containing 20mg/mL of mannitol to obtain an aqueous phase (W1) containing 20mg/mL of bovine serum albumin, and polylactic acid-glycolic acid copolymer (PLGA, molecular weight 40000Da) was dissolved in a mixed solvent of methylene chloride and dimethyl carbonate 2:1(v: v) to obtain an oil phase having a polymer concentration of 50 mg/mL. Weighing a certain amount of polyvinyl alcohol (PVA), adding a proper amount of ultrapure water, heating and stirring at 90 ℃ until the PVA is completely dissolved, stopping heating, standing and cooling to prepare a PVA aqueous solution with the concentration of 50mg/mL, and placing the PVA aqueous solution in a refrigerator at 4 ℃ for later use as an external water phase (W2).
(2) Adding the prepared water phase (W1, bovine serum albumin aqueous solution) into an oil phase (PLGA organic solution) (oil-water volume ratio is 20:1), and emulsifying the water phase and the oil phase by using a cell disruptor under the conditions of ice bath, power of 400W and time of 30s to prepare W/O emulsion; then adding an external water phase (W2, 50mg/mL PVA aqueous solution), wherein the volume ratio of the oil phase to the external water phase is 1:10, and emulsifying the two under the conditions of ice bath, 300W of power and 30s of time by using a cell disruptor to prepare W/O/W emulsion;
(3) transferring the W/O/W emulsion into 500mL of 50mg/mL sodium chloride aqueous solution, stirring overnight at a stirring speed of 600rpm to volatilize the organic solvent and solidify the microspheres; centrifuging to collect microspheres, and washing with ultrapure water for three times; and (4) freeze-drying the obtained microspheres to obtain bovine serum albumin W/O/W microspheres, and marking as microspheres 21.
The results of scanning electron microscopy of the bovine serum albumin W/O/W microspheres prepared in this comparative example are shown in FIG. 2B (the determination method is the same as that in example 1). The microspheres prepared in the comparative example were smaller in particle size and non-uniform in size compared to the microspheres prepared in example 1. The particle size distribution of the W/O/W microspheres is shown in fig. 6 (the determination method is the same as that of example 1), and the microspheres prepared in the comparative example have a wider particle size distribution and two peaks, i.e., d (0.1) ═ 1.78 ± 0.15 μm, d (0.5) ═ 10.66 ± 1.79 μm, and d (0.9) ═ 148.62 ± 8.71 μm, compared with the microspheres prepared in example 1; the encapsulation efficiency of the medicine is 75.39 +/-0.58% (the determination method is the same as that of the example 1), and is lower than that of the bovine serum albumin microspheres prepared in the example 1.
Comparative example 2: preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the comparative example comprises the following steps:
(1) preparation of bovine serum albumin microparticles: bovine serum albumin and PEG polyethylene glycol (5: 1, w/w) were added to a phosphate buffer solution at pH 7.4 to form a mixed solution. Pre-freezing at-80 deg.C overnight, and vacuum drying to obtain solid powder. The resulting mixed solid powder was thoroughly washed with methylene chloride to remove PEG, and then dried to obtain bovine serum albumin BSA microparticles (S).
(2) Preparation of S/O suspension: a carboxyl-terminal polylactic-co-glycolic acid (PLGA, molecular weight 40000Da, LA: GA ═ 50:50) was dissolved in a mixed solvent of dichloromethane and dimethyl carbonate 2:1(v: v) to obtain an oil phase (O) having a polymer concentration of 50 mg/mL. Adding the prepared solid phase (bovine serum albumin particles) into an oil phase (PLGA organic solution) (bovine serum albumin: PLGA is 1:50, W/W), and uniformly mixing the two under the conditions of ice bath, 400W of power and 30S of time by using a cell disruptor to prepare S/O suspension;
(3) and (3) feeding the S/O suspension into UPPS at a constant speed of 10mL/min through a peristaltic pump, shearing and atomizing under the action of a high-speed rotary disc (the rotating speed is 12000rpm) to form microdroplets, volatilizing a solvent of the microdroplets in an airflow field, and solidifying the microdroplets to obtain bovine serum albumin microspheres, wherein the microspheres are marked as microspheres 22.
The results of scanning electron microscopy of the bovine serum albumin microspheres prepared in this comparative example are shown in FIG. 2C (the determination method is the same as that in example 1). Compared with the microspheres prepared in example 1, the microspheres prepared in the comparative example have collapsed spheres, unsmooth surfaces and irregular shapes. The particle size distribution of the microspheres prepared in this comparative example is shown in fig. 7 (the determination method is the same as that of example 1), and compared with the microspheres prepared in example 1, the particle size of the microspheres prepared in the comparative example is not normally distributed, and d (0.1) ═ 7.123 ± 4.58 μm, d (0.5) ═ 17.14 ± 1.56 μm, and d (0.9) ═ 27.46 ± 0.12 μm; the encapsulation efficiency of the medicine is 73.57 + -2.83 (the determination method is the same as that of the example 1), which is lower than that of the bovine serum albumin microsphere prepared in the example 1.
Example 9: in-vitro drug release curve for detecting bovine serum albumin microspheres
Precisely weighing 20mg of each of the bovine serum albumin microspheres prepared in example 1 and the microspheres prepared in comparative example 1, placing the 20mg of each of the bovine serum albumin microspheres and the microspheres in a 2mL sample centrifuge tube, adding 1mL of phosphate buffer PBS (pH 7.4), placing the mixture in an air bath oscillator, oscillating the mixture at constant temperature under the conditions of 37 ℃ and 100rpm, taking out the centrifuge tube at a preset time point, centrifuging the mixture for 5min under the condition of 10000rpm, sucking all supernatants and measuring the drug amount of the supernatants; the sample tubes were additionally supplemented with 1mL of fresh PBS (pH 7.4) and placed in an air bath shaker for continued shaking. Samples released in vitro were assayed for bovine serum albumin content using the Micro BCA kit (see figure 7 for results). Experimental results show that the sustained-release time of the microspheres prepared in example 1 is about 110 days, the first-day release amount is only 4.83 +/-0.16%, the first-day release amount of the W/O/W microspheres prepared in comparative example 1 reaches 20.01 +/-0.45%, and the burst rate is high.
The results of the in vitro release profiles of the bovine serum albumin microspheres of the other examples and comparative examples, the sustained release time, the first day release amount, and the cumulative release amount, which were measured in the same manner, are shown in table 2.
TABLE 2 in vitro drug release results for bovine serum albumin microspheres
| Microsphere numbering | Time of release (Tian) | First day release (%) | Cumulative amount of Release (%) |
| 1 | 110 | 4.83±0.16 | 90.41±5.00 |
| 2 | 85 | 3.58±0.22 | 90.95±2.17 |
| 3 | 110 | 16.91±2.37 | 86.24±2.82 |
| 4 | 110 | 5.28±1.33 | 92.53±2.56 |
| 5 | 110 | 5.89±0.97 | 96.78±8.37 |
| 6 | 110 | 3.32±0.24 | 39.43±1.01 |
| 7 | 110 | 18.43±0.74 | 93.98±0.64 |
| 8 | 110 | 12.12±0.32 | 93.81±9.33 |
| 9 | 110 | 4.19±0.14 | 83.79±0.65 |
| 10 | 110 | 3.55±0.30 | 84.06±0.64 |
| 11 | 85 | 1.67±0.70 | 46.73±2.46 |
| 12 | 110 | 22.33±0.31 | 87.22±7.79 |
| 13 | 110 | 3.75±0.35 | 91.60±2.51 |
| 14 | 110 | 15.29±0.50 | 87.74±2.46 |
| 15 | 110 | 3.64±0.31 | 90.41±2.78 |
| 16 | 110 | 5.81±0.53 | 87.45±1.74 |
| 17 | 30 | 35.52±0.42 | 89.09±1.06 |
| 18 | 110 | 8.39±0.29 | 80.69±2.56 |
| 19 | 110 | 3.81±0.26 | 82.15±0.64 |
| 20 | 110 | 3.52±0.15 | 75.14±6.99 |
| 21 | 110 | 20.01±0.45 | 94.47±2.84 |
| 22 | 110 | 30.82±1.20 | 79.82±0.98 |
As can be seen from Table 2, the release of BSA microspheres is mainly related to the PLGA concentration, the oil-water-phase volume ratio, the spinning speed and the power of the cell disruptor for forming W/O emulsion. When the PLGA concentration is 40-60mg/mL, the 24h burst rate is less than 6% and the cumulative release amount can reach more than 80%. The difference of the oil-water phase volume ratio can affect the property of the W/O emulsion and further affect the release of the microspheres, when the volume ratio is 20:1-40:1, the 24-hour burst release rate is less than 5 percent, and the cumulative release amount can reach more than 80 percent. When the rotating speed of the rotary disc is 9000-. The power of the cell disruptor can affect the properties of the W/O emulsion, such as the particle size, the stability of the emulsion, and the like, and further affect the formability of the microspheres and the release of the microspheres, wherein when the power is 400-500W, the 24h burst release rate is less than 5% and the cumulative release amount can reach more than 80%. In addition, the preparation method of the microspheres of comparative example 1 and comparative example 2 is different from that of example 1, and both the encapsulation efficiency and the release effect are far worse than those of example 1.
Example 10: circular dichroism chromatography method for detecting secondary structure of medicine in microsphere
Precisely weighing a proper amount of microspheres prepared in example 1, respectively placing the microspheres in 7mL centrifuge tubes, adding 0.5mL dichloromethane to dissolve the microspheres, adding ultrapure water, carrying out vortex mixing for 20min, fully extracting bovine serum albumin into a water phase, centrifuging at 10000rpm for 5min, transferring a supernatant into another centrifuge tube, and determining the concentration of the bovine serum albumin in the supernatant by using a Micro BCA kit; and preparing a certain amount of bovine serum albumin raw material medicine into a standard reference substance by using ultrapure water, wherein the concentration of the reference substance is consistent with that of the liquid to be detected.
Detecting the secondary structure of the protein drug in each sample by using a circular dichroism method: the samples were added to a 1mm cuvette, then the cuvette was placed in a circular dichroism spectrometer with a wavelength scan range of 180-260nm, and the circular dichroism curves of the samples were recorded, with the results shown in FIG. 9. The results show that the bovine serum albumin drug in the microsphere sample prepared in example 1 shows a circular dichroism chromatogram similar to that of a bovine serum albumin bulk drug, which indicates that the microsphere prepared in example 1 can protect the secondary structure of protein.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A preparation method of long-acting microspheres for protein polypeptide drugs is characterized by comprising the following steps:
(1) dissolving protein or polypeptide water-soluble medicine and protein protectant in water to obtain water-phase medicine solution; dissolving a high molecular carrier material in an organic solvent to obtain an oil-phase high molecular solution; the protein or polypeptide water-soluble medicine is selected from bovine serum albumin;
(2) adding the water-phase drug solution into the oil-phase polymer solution, and preparing into W/O emulsion by using a cell disruptor; the power of the cell disruption instrument is 200-700W, and the disruption time is 30-90 s;
(3) feeding the W/O emulsion into an ultrafine particle preparation system at a constant speed through a peristaltic pump to prepare the long-acting protein polypeptide drug microspheres; the rotating disc rotating speed of the superfine particle preparation system is 6000-12000 rpm; the liquid supply speed of the peristaltic pump is 10 mL/min;
wherein the protein protective agent is mannitol, and the concentration is 20 mg/mL; the concentration of the protein or polypeptide water-soluble medicine in the water-phase medicine solution is 20 mg/mL;
the polymer carrier material is PLGA with carboxyl end, the molecular weight is 20000-50000Da, and the concentration in the oil phase polymer solution is 40-60 mg/mL;
the volume ratio of the water-phase drug solution to the oil-phase polymer solution is 1: 10-40.
2. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 1,
the organic solvent in the step (1) is one or more selected from dichloromethane, dimethyl carbonate and ethyl acetate.
3. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 2,
the organic solvent in the step (1) is dichloromethane and dimethyl carbonate.
4. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 3,
the organic solvent in the step (1) is a mixture of dichloromethane and dimethyl carbonate with the volume ratio of 1.5-2.5: 1.
5. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 1,
the concentration of the polymer carrier material in the oil-phase polymer solution in the step (1) is 45-55 mg/mL.
6. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 1, wherein the volume ratio of the aqueous phase drug solution to the oil phase polymer solution in step (2) is 1: 15-35.
7. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 6, wherein the volume ratio of the aqueous phase drug solution to the oil phase polymer solution in step (2) is 1: 18-25.
8. The method for preparing long-acting microspheres of protein polypeptide drugs according to claim 1, wherein in step (3), the rotational speed of the spinning disk of the system for preparing ultrafine particles is 12000 rpm.
9. The method for preparing long-acting microspheres for protein polypeptide drugs according to claim 1, wherein in the step (3), the rotation speed of the spinning disk of the system for preparing ultrafine particles is 6000 to 9000 rpm.
10. A long-acting microsphere of a protein polypeptide drug, which is prepared by the preparation method of any one of claims 1 to 9.
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