CN111568878A - Method for preparing polypeptide drug microspheres based on submerged airflow spraying technology - Google Patents
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Abstract
The invention discloses a method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology, which comprises the following steps: (1) dissolving the polypeptide water-soluble drug in water to obtain an internal water phase drug solution; dissolving a carrier material in an organic solvent to obtain an oil-phase polymer solution; (2) adding the oil phase polymer solution into the inner water phase drug solution, and performing ultrasonic crushing or shearing dispersion to form a primary emulsion; (3) conveying the primary emulsion to a liquid inlet of an air flow nozzle positioned at the bottom of a liquid medium drying tank, simultaneously opening an air inlet of the air flow nozzle, atomizing the primary emulsion, spraying the primary emulsion into a surfactant-containing aqueous solution in the liquid medium drying tank from bottom to top through a nozzle opening of the air flow nozzle, simultaneously starting stirring while spraying the primary emulsion to form a multiple emulsion, and then drying in the liquid at 15-25 ℃ for 3-5 hours to remove an organic solvent to obtain the polypeptide drug microsphere. The microsphere produced by the invention has high encapsulation efficiency, high balling rate and high production efficiency.
Description
Technical Field
The invention relates to the technical field of microsphere production, in particular to a method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology.
Background
Microspheres refer to a dispersion of microparticles in which a drug is dispersed or adsorbed in a polymeric or polymeric matrix. Compared with the traditional preparation, the microsphere can reduce the administration times, improve the compliance of patients, reduce the side effect and improve the curative effect, has obvious advantages in clinic and becomes a hot spot for the research and development of new medicament forms in recent years. The microsphere preparation technology capable of realizing industrialization firstly can ensure the controllability of key quality attributes (CQAs), such as: the form, the grain diameter and the grain diameter distribution of the microspheres, the drug-loading rate, the encapsulation rate, the in vitro release degree and other CQAs are well controlled, and the microsphere has great promotion effect on the development and the technical transformation of the drug. Especially for polypeptide protein drugs, the raw material drugs are expensive, and the production scale (batch) determines the production cost of a single preparation.
For hydrophilic drugs, there are 3 typical formulation methods: multiple emulsion method, phase separation method and spray drying method. The 3 methods all have similar initial process steps, namely adding an aqueous phase solution of the medicament into an oil phase solution formed by an organic solvent and a polymer, emulsifying to form water-in-oil (W1/O) colostrum, and then respectively adopting differential medium and dispersion modes to prepare the microspheres.
Compared with a multiple emulsion method and a phase separation method, the spray drying method is most easy to realize batch amplification, is not limited by the flux (treatment capacity) of shearing equipment, and can realize continuous spray granulation. The traditional spray drying method is that the feed liquid (which can be emulsion, true solution or suspension) is sprayed into the dry inert gas flow with gradually increased temperature, the organic solvent is quickly evaporated, and the organic solvent is solidified into balls. The spray drying method does not need to use a large amount of organic reagents, is slightly influenced by the properties of high polymers, and has higher microsphere encapsulation efficiency and good batch-to-batch reproducibility. However, when the polypeptide protein microspheres are prepared by the method, polypeptide protein drugs are extremely easy to inactivate by high-temperature airflow, so that no microsphere product which is marketed by the method exists at present.
The improved spray cryosolidification method has been applied to the growth hormone (rhGH) microsphere preparation (Nutropin)
) In (1). The preparation process includes preparing rhGH into freeze dried powder with very high concentration, dispersing in polymer solution, spraying via nozzle into liquid nitrogen, and low temperature solvent extraction. During spraying, the rhGH high molecular protein and related protein are generated due to the short-term high temperature, so that the rhGH high molecular protein and related protein have strong antigenicity. In addition, due to the production conditions at ultra-low temperature, the production facilities are expensive, and finally, the variety is released from the market in 2004 due to high price and strong antigenicity.
Chinese patent CN1879606B discloses a delivery system for preparing novel drugs based on a liquid level spray method, which proposes a concept of preparing microspheres based on a liquid level air-flow spray technique, and the specific implementation is that a solution containing a carrier material is mixed or suspended with a drug or a drug solution, atomized by controlling air pressure, and sprayed into a receiving liquid to form a microparticle preparation. However, in the course of research, the inventors of the present application found that the preparation of microspheres in this way is not easy to achieve, and the biggest problem is that when a primary emulsion (greater than 1000cp) with high viscosity is sprayed onto the surface of an aqueous solution (such as PVA), a large amount of thin film is inevitably formed even under stirring, the polymer solution at the gas-liquid interface is not timely and fully surrounded by water, the surface of the microspheres is not solidified, and aggregation (similar to the dissolution phenomenon of sodium carboxymethylcellulose) is very easy to occur, resulting in a very low sphere forming rate of the microspheres.
As mentioned above, the application of the spray drying method in the industrialization of microsphere preparation needs to be greatly improved and overcome the difficulties in technical details.
Disclosure of Invention
The invention aims to provide a method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology, and the microspheres prepared by the method have high encapsulation efficiency, balling rate and production efficiency.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology comprises the following steps:
(1) dissolving the polypeptide water-soluble drug in water to obtain an internal water phase drug solution; dissolving a carrier material in an organic solvent to obtain an oil-phase polymer solution;
(2) adding the oil phase polymer solution into the inner water phase drug solution, and performing ultrasonic crushing or shearing dispersion to form a primary emulsion;
(3) conveying the primary emulsion to a liquid inlet of an airflow nozzle positioned at the bottom of a liquid medium drying tank, simultaneously opening a gas inlet of the airflow nozzle, atomizing the primary emulsion under the action of compressed air or nitrogen, spraying the primary emulsion into a surfactant-containing aqueous solution in the liquid medium drying tank from bottom to top from the liquid through a nozzle opening of the airflow nozzle, and starting stirring while spraying the primary emulsion to form multiple emulsion;
(4) and (4) drying the multiple emulsion formed in the step (3) in liquid at 15-25 ℃ for 3-5 hours to remove the organic solvent, thus obtaining the polypeptide drug microspheres.
The method is different from the method for forming the microspheres by spraying from the liquid surface to the external water phase in the prior art, and the invention develops a new way, the primary emulsion is sprayed into the external water phase from bottom to top from the liquid of the external water phase, the balling rate is high, the yield is high, the microsphere blastocyst is timely contacted with the external water phase, the aggregation cannot be generated, and a large amount of films cannot be formed. Thereby solving the technical problems that a large amount of polymer films are formed when high-concentration and high-viscosity feed liquid is sprayed into the liquid surface from top to bottom, the spheres cannot be divided, and the yield is low.
The principle of the invention is as follows: the prepared primary emulsion is fed into an air flow nozzle at a certain flow rate, the solution is dispersed into uniform micro-droplets by controlling the viscosity of the primary emulsion and the air flow rate of the air flow nozzle through the crushing action of air flowing through the nozzle opening, the micro-droplets are uniformly dispersed into an external water phase under the stirring condition when being formed, the micro-droplets are not directly contacted with the external air, the organic solvent of the formed double emulsion is removed under the stirring condition, and finally the double emulsion is solidified to form the microspheres.
Preferably, ultrasonic crushing (150W, 60-70% output power) or shear dispersion (high-speed shear, 11000-13000 rpm) is adopted, and a primary emulsion is formed after dispersion is carried out for 8-16 min.
Preferably, in the step (3), the primary emulsion is sprayed while stirring is started, the stirring speed is 250-550 rpm, and the holding time is from the beginning of the preparation of the multiple emulsion to the end of drying in the liquid.
The polypeptide water-soluble drug in the step (1) is selected from one of leuprorelin acetate, triptorelin acetate, goserelin acetate, octreotide, exenatide, lanreotide and pasireotide; the carrier material is PLA or PLGA; the organic solvent is selected from one of dichloromethane, ethyl acetate and chloroform. The PLGA has a molecular weight of 10,000-22,000Da and the terminal group is carboxyl.
The concentration of the inner water phase drug solution in the step (1) is 40-55 wt%, and the concentration of the oil phase polymer solution is 25-40 wt%. Preferably, the concentration of the inner water phase drug solution is 45-50% (w/w), and the concentration of the oil phase polymer solution is 33.3-37.5% (w/w).
In the step (2), the volume ratio of the inner water phase drug solution to the oil phase polymer solution is controlled to be 1: 11-22.
The viscosity range of the primary emulsion obtained in the step (2) is 1500-. The corresponding particle size range of colostrum is 100-200 nm. The invention is particularly suitable for preparing microspheres from high-viscosity primary emulsion, and the high-concentration primary emulsion is easy to form a large amount of films on the surfaces of the microspheres, so that the microsphere balling rate is extremely low.
In the step (3), the liquid supply speed of the primary emulsion is 10-30mL/min, the flow rate of the air flow entering the air flow nozzle is 80-150L/min, and the injection pressure of the air flow nozzle is 2-4 kg. Aiming at the specific environment of underwater spraying, the invention carries out specific improvement on the liquid supply speed of the primary emulsion, the airflow flow rate of the airflow nozzle and the spraying pressure, the parameters control the size of the particle size, the encapsulation rate of the microspheres is related to the particle size, and if the liquid supply speed, the airflow and the spraying pressure cannot be matched as required by the invention, the nozzle cannot process so much liquid in a short time, the balling rate can be reduced, namely the yield is also reduced, and a plurality of unnecessary large microspheres or substances which are adhered together can be formed.
In the step (3), the volume ratio of the dosage of the primary emulsion to the dosage of the aqueous solution containing the surfactant is 1: 80-150.
The surfactant is polyvinyl alcohol.
The concentration of the aqueous solution containing the surfactant is 0.1-1%, and the temperature of the aqueous solution containing the surfactant is controlled to 10-20 ℃.
In the step (3), the distance between the stirring paddle in the drying tank in the liquid and the airflow nozzle is 5-10 cm.
Aiming at the specific environment of underwater spraying, the distance between the stirring paddle and the airflow nozzle is specifically controlled, the distance between the stirring paddle and the airflow nozzle can influence the dispersion degree of the microsphere embryo sac just after preparation, the microsphere embryo sac can be rapidly dispersed, the balling rate and the yield can be improved, the better stirring effect is ensured, local feed liquid can be more rapidly dispersed into the whole tank body, the water-oil ratio is more uniform, and the encapsulation efficiency is more stable. This parameter is the parameter that affects the dispersion effect of the just formed microspheres, which in turn affects other parameters indirectly.
The invention has the beneficial effects that:
1. the preparation conditions of the microspheres are mild (10-25 ℃), the organic solvent is not required to be removed in a high-temperature environment, continuous production can be realized, and the equipment cost is low.
2. The encapsulation rate of the polypeptide drug microsphere prepared by the method can reach more than 93 percent, the drug release is uniform and lasting, the surface of the microsphere is smooth and has no folds, and the burst release caused by the water absorption effect cannot be increased.
3. Compared with the air flow spraying technology on the liquid surface, the method has the advantages of high balling rate and high yield, the microsphere blastocyst is contacted with external water in time, aggregation cannot be generated, a large amount of thin films cannot be formed, and the feasibility of the industrialization of the spraying technology is improved.
Drawings
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a scanning electron micrograph of the polypeptide microspheres prepared in example 1;
FIG. 3 is a scanning electron micrograph of the polypeptide microspheres prepared in comparative example 1;
FIG. 4 is a graph showing the distribution of particle sizes of the polypeptide microspheres prepared in example 1.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
General description of the embodiments
A method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology comprises the following steps:
(1) dissolving the polypeptide water-soluble drug in water to obtain an internal water phase drug solution with the concentration of 40-55 wt%; dissolving a carrier material in an organic solvent to obtain a phase polymer solution having a concentration of 25-40 wt% oil; the polypeptide water-soluble drug is selected from one of leuprorelin acetate, triptorelin acetate, goserelin acetate, octreotide, exenatide, lanreotide and pasireotide; the carrier material is PLA or PLGA; the organic solvent is selected from one of dichloromethane, ethyl acetate and chloroform.
(2) Adding the oil-phase polymer solution into the internal water-phase drug solution, wherein the volume ratio of the internal water-phase drug solution to the oil-phase polymer solution is controlled to be 1: 11-22, ultrasonic crushing or shearing dispersing to form primary emulsion, wherein the primary emulsion has a primary particle size range of 100-.
(3) Conveying the primary emulsion to a liquid inlet of an air flow nozzle positioned at the bottom of a liquid medium drying tank, simultaneously opening a gas inlet of the air flow nozzle, atomizing the primary emulsion under the action of compressed air or nitrogen, spraying the primary emulsion into a surfactant-containing aqueous solution in the liquid medium drying tank from bottom to top through a nozzle opening of the air flow nozzle, starting stirring while spraying the primary emulsion to form multiple emulsion, and then drying in the liquid at 15-25 ℃ for 3-5 hours to remove an organic solvent to obtain the polypeptide drug microsphere. The volume ratio of the dosage of the primary emulsion to the dosage of the aqueous solution containing the surfactant is 1: 80-150 parts of; the liquid supply speed of the primary emulsion is 10-30mL/min, the flow rate of the air flow entering the air flow nozzle is 80-150L/min, and the injection pressure of the air flow nozzle is 2-4 kg. The surfactant is polyvinyl alcohol, the concentration of the aqueous solution containing the surfactant is 0.1-1%, and the temperature of the aqueous solution containing the surfactant is controlled to be 10-20 ℃. The distance between the stirring paddle in the drying tank in the liquid and the air flow nozzle is 5-10 cm.
As shown in FIG. 1, the process flow of the present invention is: feeding a feed liquid (primary emulsion) into a liquid inlet of the gas flow nozzle, opening a gas inlet of the gas flow nozzle, controlling the flow rate of gas, dispersing the primary emulsion into uniform micro-droplets by utilizing the crushing effect of the gas flowing through the nozzle opening, spraying the micro-droplets into an external water phase, dispersing the micro-droplets into the external water phase under the stirring condition, drying the liquid under the stirring condition to remove an organic solvent, and solidifying to form microspheres.
The specific process is as follows:
(1) dissolving a polypeptide drug in an aqueous solution to form an internal water phase, placing the internal water phase in an internal water phase tank 1, dissolving a polymer in an organic solvent to form an oil phase, placing the oil phase in an oil phase tank 2, filtering the internal water phase in the internal water phase tank 1 and the oil phase in the oil phase tank 2 through an internal water phase injection channel 6 and an oil phase injection channel 7 respectively, injecting the filtered internal water phase and the filtered oil phase into a colostrum shearing tank 3, starting a colostrum shearing machine 31 to prepare colostrum, and shearing to form uniform colostrum;
(2) starting a compressed air storage tank 5, outputting a fixed gas flow through a gas flowmeter 10 with an adjusting valve, introducing compressed air into a primary emulsion shearing tank 3, and supplying primary emulsion into a liquid inlet 81 of an airflow nozzle 8 at a certain flow rate; the air flow nozzle is commercially available, and a Nissan-co-standing two-fluid nozzle can be selected;
(3) meanwhile, a fixed gas flow is output through a gas flow meter 10 with an adjusting valve, compressed air is introduced into a gas inlet 82 of the gas flow nozzle 8, colostrum is dispersed into uniform micro-droplets by utilizing the crushing effect of gas flowing through a top nozzle 83 through controlling the viscosity of colostrum liquid and the gas flow rate of the gas flow nozzle, the micro-droplets are sprayed into an outer water phase of the drying tank 4 in liquid after atomization, meanwhile, a stirrer 44 is started to disperse micro-spheres to form uniformly dispersed composite emulsion droplets, the drying temperature in the liquid is adjusted through a temperature control jacket 41 of the drying tank 4 in liquid, and volatilized organic solvent is discharged through an exhaust port 42 at the top of the drying tank 4 in liquid.
Example 1
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in methylene chloride to give an oil phase with a polymer concentration of 33.3%;
(2) adding the above oil phase into internal water phase (volume ratio 12.5: 1), controlling colostrum shearing temperature at 15 deg.C, shearing with high speed shearing machine at 12000rpm for 10min to form colostrum solution with viscosity of 1541cp (20.83 deg.C);
(3) setting the airflow flow rate to be 130L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed with the flow rate of 10mL/min, performing airflow crushing of compressed air, spraying 0.1% polyvinyl alcohol aqueous solution at 15 ℃ into the bottom of a liquid medium drying tank from bottom to top, forming well-dispersed double emulsion under the stirring condition of 300rpm, adjusting the liquid medium drying temperature to 20 ℃, and stirring for 4 hours to remove an organic solvent to obtain the leuprorelin acetate microspheres;
(4) sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The leuprorelin acetate microspheres prepared in this example were dispersed on a silicon wafer, subjected to a gold-spraying treatment, and the morphology of the microspheres was observed under a scanning electron microscope. The results are shown in FIG. 2, the microspheres are smooth in surface, without channels, intact in spherical shape and without adhesion.
Precisely weighing 25mg of the microspheres in the embodiment, placing the microspheres in a 25mL volumetric flask, adding DMSO, performing vortex dissolution, fixing the volume to 25mL reticle, and detecting the content of leuprorelin acetate by HPLC. The result shows that the encapsulation efficiency of the leuprolide acetate microspheres is 93.4%.
The leuprorelin acetate microspheres prepared in this example were dispersed in a 0.1% aqueous solution of tween 80, ultrasonically dispersed for 1min, and particle size was determined using a laser particle size analyzer. The results showed that the microspheres had uniform and normal particle size distribution with d10 ═ 2.34 μm, d50 ═ 10.15 μm, and d90 ═ 31.82 μm (fig. 4).
The in vitro release rate determination method is as follows: weighing 100mg of microspheres, adding the microspheres into 100mL of dissolution medium (containing 0.4% of polyvinyl alcohol, 0.1% of Tween 80 and 0.2% of lactic acid), placing the mixture in a water bath at 48 +/-1 ℃ for oscillation, taking solutions at 1h, 4h, 9h, 24h and 48h respectively, filtering the solutions, and analyzing the content of the drug, wherein the release degree method is an acceleration condition, the release degree of the acceleration condition has good linear correlation with the in-vitro long-term release degree (37 +/-1 ℃) of the microspheres, the long-term release behavior of the microspheres can be accurately predicted, the detection period is shortened, and the in-vitro release results under the acceleration condition are summarized in a table 1.
Example 2
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 37.5%;
(2) adding the above oil phase into internal water phase (volume ratio 12.5: 1), controlling colostrum shearing temperature at 17 deg.C, shearing with high speed shearing machine at 12000rpm for 10min to form colostrum solution with viscosity of 2499cp (18.94 deg.C);
(3) setting the airflow flow rate at 130L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed of 10mL/min, crushing airflow of compressed air, spraying a 0.1% polyvinyl alcohol aqueous solution at 17 ℃ into the bottom of a liquid medium drying tank from bottom to top, forming a well-dispersed double emulsion under the stirring condition of 300rpm, adjusting the liquid medium drying temperature to 20 ℃, and stirring for 4 hours to remove an organic solvent to obtain the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The encapsulation efficiency of the leuprolide acetate microspheres prepared in this example is 94.7%, the particle size d10 ═ 4.66 μm, d50 ═ 15.08 μm, and d90 ═ 44.36 μm, and the in vitro release results are summarized in table 1.
Example 3
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 33.3%;
(2) adding the above oil phase into the internal water phase (volume ratio of 12.5: 1), controlling the shearing temperature of primary emulsion at 17 deg.C, and shearing at 12000rpm for 10min with high speed shearing machine to form primary emulsion with viscosity of 1687cp (19.85 deg.C);
(3) setting the airflow flow rate to be 90L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed with the flow rate of 10mL/min, crushing airflow of compressed air, spraying 0.1% polyvinyl alcohol aqueous solution at the temperature of 17 ℃ from bottom to top at the bottom of a liquid medium drying tank, forming well-dispersed double emulsion under the stirring condition of 400rpm, adjusting the liquid medium drying temperature to 20 ℃, and stirring for 3.5 hours to remove the organic solvent, thereby obtaining the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The encapsulation efficiency of the leuprolide acetate microspheres prepared in this example is 94.2%, the particle size d10 ═ 3.08 μm, d50 ═ 14.65 μm, and d90 ═ 37.55 μm, and the in vitro release results are summarized in table 1.
Example 4
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 35.0%;
(2) adding the above oil phase into internal water phase (volume ratio of 12.5: 1), controlling colostrum shearing temperature at 15 deg.C, and shearing at 13000rpm for 10min by high speed shearing machine to form colostrum emulsion with viscosity of 1805cp (19.27 deg.C);
(3) setting the airflow flow rate at 130L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed at a flow rate of 25mL/min, crushing airflow of compressed air, spraying 0.5% polyvinyl alcohol aqueous solution at 15 ℃ into the bottom of a liquid medium drying tank from bottom to top, forming well-dispersed double emulsion under the stirring condition of 400rpm, adjusting the liquid medium drying temperature to 25 ℃, and stirring for 3.5 hours to remove the organic solvent, thereby obtaining the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The encapsulation efficiency of the leuprolide acetate microspheres prepared in this example is 95.1%, the particle size d10 ═ 4.45 μm, d50 ═ 16.94 μm, and d90 ═ 40.87 μm, and the in vitro release results are summarized in table 1.
Example 5
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 35.0%;
(2) adding the above oil phase into internal water phase (volume ratio of 12.5: 1), controlling colostrum shearing temperature at 15 deg.C, and shearing at 13000rpm with high speed shearing machine for 15min to form colostrum emulsion with colostrum liquid viscosity of 1922cp (19.05 deg.C);
(3) setting the airflow flow rate to be 90L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed with the flow rate of 25mL/min, performing airflow crushing on compressed air, spraying a 1.0% polyvinyl alcohol aqueous solution at 15 ℃ into the bottom of a liquid medium drying tank from bottom to top, forming a well-dispersed double emulsion under the stirring condition of 500rpm, adjusting the liquid medium drying temperature to 25 ℃, and stirring for 3 hours to remove an organic solvent to obtain the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The encapsulation efficiency of the leuprolide acetate microspheres prepared in this example is 96.0%, the particle size d10 ═ 8.67 μm, d50 ═ 23.25 μm, and d90 ═ 54.21 μm, and the in vitro release results are summarized in table 1.
The particle size data show that the microspheres obtained by the method have high encapsulation efficiency and good particle size distribution.
By adopting the subsurface airflow spraying technology, the primary emulsion with the viscosity of 1500-2500cp can be uniformly dispersed in the external water phase solution, the film forming phenomenon is avoided, and the microsphere yield is high.
Comparative example 1
Preparation of leuprorelin acetate microspheres by traditional multiple emulsion method
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 33.3%;
(2) adding the above oil phase into the internal water phase (volume ratio of 12.5: 1), controlling colostrum shearing temperature at 18 deg.C, and shearing at 12000rpm for 10min with high speed shearing machine to form colostrum emulsion with viscosity of 1611cp (18.39 deg.C);
(3) and (3) respectively passing the primary emulsion and the external water phase through an online shearing machine at the flow rates of 5mL/min and 600mL/min, circularly emulsifying for 2min at the rotating speed of 7000rpm to form a uniform double emulsion, adjusting the drying temperature in the solution to 20 ℃, and removing the organic solvent under stirring to obtain the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The encapsulation efficiency of the leuprolide acetate microspheres prepared in this example is 82.9%, the particle size d10 ═ 2.30 μm, d50 ═ 8.73 μm, and d90 ═ 44.8 μm, and the in vitro release results are summarized in table 1.
The leuprorelin acetate microspheres prepared in this example were dispersed on a silicon wafer, subjected to a gold-spraying treatment, and the morphology of the microspheres was observed under a scanning electron microscope. The result is shown in fig. 3, the microsphere surface has no pore channels, the sphere is complete, and no adhesion exists.
Comparative example 2
The leuprorelin acetate microspheres are prepared by adopting a CN1879606B liquid surface top spraying method
(1) Dissolving leuprorelin acetate in water to obtain an internal water phase with the drug concentration of 50%; dissolving carboxyl-terminated PLGA (7525, Mw13000Da) in dichloromethane to give an oil phase with a polymer concentration of 33.3%;
(2) adding the above oil phase into internal water phase (volume ratio 12.5: 1), controlling colostrum shearing temperature at 18 deg.C, shearing with high speed shearing machine at 12000rpm for 10min to form colostrum solution with viscosity of 1524cp (19.83 deg.C);
(3) setting the airflow rate at 130L/min, feeding the primary emulsion into a liquid inlet of an airflow nozzle at a constant speed of 10mL/min, crushing airflow of compressed air, respectively placing the airflow nozzle at different heights (10cm, 20cm and 30cm) above the liquid surface, spraying the airflow nozzle into an aqueous solution containing a surfactant (500 rpm, 400rpm and 300rpm are compared with different stirring speeds at the same time) to form a double emulsion, adjusting the drying temperature in the liquid to 20 ℃, and removing the organic solvent under stirring conditions to obtain the leuprorelin acetate microspheres.
(4) Sieving with 75 μm sieve to remove large-particle size microsphere, centrifuging at 5000rpm for 10min, collecting the microsphere, freeze drying to obtain microsphere product, and freeze drying at-5 deg.C for 20 hr, and resolution drying at 45 deg.C for 24 hr.
The high balling rate of the leuprorelin acetate microspheres prepared in the comparative example 2 cannot be obtained no matter spray granulation is carried out at different heights above the liquid surface or the stirring speed of the aqueous solution in the spraying process is adjusted, and the high-viscosity primary emulsion is treated by adopting the spray technology on the liquid surface, so that a large amount of high-molecular films appear on the liquid surface and are difficult to disperse, the yield of the microspheres is extremely low, and the yield of the microspheres is less than 20% (the upper limit of the preparation conditions).
Table 1 in vitro release results for microspheres described in examples 1-5 and comparative example 1
TABLE 2 Balling Rate (yield) of the microspheres described in examples 1-5 and comparative examples 1-2
| Example 3 | Example 4 | Example 5 | Example 6 | Example 7 | Comparative example 1 | Comparative example 2 | |
| Yield/% | 65.2％ | 68.5％ | 66.1％ | 63.0％ | 59.4％ | 56％ | <20％ |
The experimental results show that the encapsulation efficiency and release characteristics of comparative example 1 are much worse than those of examples 1-5 of the present invention; the method of comparative example 2 produced a large amount of aggregates during the preparation of microspheres, and the balling rate was very low.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (10)
1. A method for preparing polypeptide drug microspheres based on an underwater airflow spraying technology is characterized by comprising the following steps:
(1) dissolving the polypeptide water-soluble drug in water to obtain an internal water phase drug solution; dissolving a carrier material in an organic solvent to obtain an oil-phase polymer solution;
(2) adding the oil phase polymer solution into the inner water phase drug solution, and performing ultrasonic crushing or shearing dispersion to form a primary emulsion;
(3) conveying the primary emulsion to a liquid inlet of an airflow nozzle positioned at the bottom of a liquid medium drying tank, simultaneously opening a gas inlet of the airflow nozzle, atomizing the primary emulsion under the action of compressed air or nitrogen, spraying the primary emulsion into a surfactant-containing aqueous solution in the liquid medium drying tank from bottom to top from the liquid through a nozzle opening of the airflow nozzle, and starting stirring while spraying the primary emulsion to form multiple emulsion;
(4) and (4) drying the multiple emulsion formed in the step (3) in liquid at 15-25 ℃ for 3-5 hours to remove the organic solvent, thus obtaining the polypeptide drug microspheres.
2. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: the polypeptide water-soluble drug in the step (1) is selected from one of leuprorelin acetate, triptorelin acetate, goserelin acetate, octreotide, exenatide, lanreotide and pasireotide; the carrier material is PLA or PLGA; the organic solvent is selected from one of dichloromethane, ethyl acetate and chloroform.
3. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: the concentration of the inner water phase drug solution in the step (1) is 40-55 wt%, and the concentration of the oil phase polymer solution is 25-40 wt%.
4. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: in the step (2), the volume ratio of the inner water phase drug solution to the oil phase polymer solution is controlled to be 1: 11-22.
5. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: the viscosity range of the primary emulsion obtained in the step (2) is 1500-.
6. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: in the step (3), the liquid supply speed of the primary emulsion is 10-30mL/min, the flow rate of the air flow entering the air flow nozzle is 80-150L/min, and the injection pressure of the air flow nozzle is 2-4 kg.
7. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: in the step (3), the volume ratio of the dosage of the primary emulsion to the dosage of the aqueous solution containing the surfactant is 1: 80-150.
8. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to the claim 1 or 7, wherein: the surfactant is polyvinyl alcohol.
9. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 8, wherein the method comprises the following steps: the concentration of the aqueous solution containing the surfactant is 0.1-1%, and the temperature of the aqueous solution containing the surfactant is controlled to 10-20 ℃.
10. The method for preparing the polypeptide drug microsphere based on the submerged airflow spraying technology according to claim 1, wherein the method comprises the following steps: in the step (3), the distance between the stirring paddle in the drying tank in the liquid and the airflow nozzle is 5-10 cm.
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